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Sample records for ligands targeting tumour-associated

  1. Dual targeted immunotherapy via in vivo delivery of biohybrid RNAi-peptide nanoparticles to tumour-associated macrophages and cancer cells.

    Science.gov (United States)

    Conde, João; Bao, Chenchen; Tan, Yeqi; Cui, Daxiang; Edelman, Elazer R; Azevedo, Helena S; Byrne, Hugh J; Artzi, Natalie; Tian, Furong

    2015-07-15

    Lung cancer is associated with very poor prognosis and considered one of the leading causes of death worldwide. Here, we present highly potent and selective bio-hybrid RNAi-peptide nanoparticles that can induce specific and long-lasting gene therapy in inflammatory tumour associated macrophages (TAMs), via an immune modulation of the tumour milieu combined with tumour suppressor effects. Our data prove that passive gene silencing can be achieved in cancer cells using regular RNAi NPs. When combined with M2 peptide-based targeted immunotherapy that immuno-modulates TAMs cell-population, a synergistic effect and long-lived tumour eradication can be observed along with increased mice survival. Treatment with low doses of siRNA (ED50 0.0025-0.01 mg/kg) in a multi and long-term dosing system substantially reduced the recruitment of inflammatory TAMs in lung tumour tissue, reduced tumour size (∼95%) and increased animal survival (∼75%) in mice. Our results suggest that it is likely that the combination of silencing important genes in tumour cells and in their supporting immune cells in the tumour microenvironment, such as TAMs, will greatly improve cancer clinical outcomes.

  2. Tumor targeting via integrin ligands

    Directory of Open Access Journals (Sweden)

    Udaya Kiran eMarelli

    2013-08-01

    Full Text Available Selective and targeted delivery of drugs to tumors is a major challenge for an effective cancer therapy and also to overcome the side effects associated with current treatments. Overexpression of various receptors on tumor cells is a characteristic structural and biochemical aspect of tumors and distinguishes them from physiologically normal cells. This abnormal feature is therefore suitable for selectively directing anticancer molecules to tumors by using ligands that can preferentially recognize such receptors. Several subtypes of integrin receptors that are crucial for cell adhesion, cell signaling, cell viability and motility have been shown to have an upregulated expression on cancer cells. Thus, ligands that recognize specific integrin subtypes represent excellent candidates to be conjugated to drugs or drug carrier systems and be targeted to tumors. In this regard, integrins recognizing the RGD cell adhesive sequence have been extensively targeted for tumor specific drug delivery. Here we review key recent examples on the presentation of RGD-based integrin ligands by means of distinct drug delivery systems, and discuss the prospects of such therapies to specifically target tumor cells.

  3. Targeting Selectins and Their Ligands in Cancer

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    Alessandro eNatoni

    2016-04-01

    Full Text Available Aberrant glycosylation is a hallmark of cancer cells with increased evidence pointing to a role in tumor progression. In particular, aberrant sialylation of glycoproteins and glycolipids have been linked to increased immune cell evasion, drug evasion, drug resistance, tumor invasiveness, and vascular dissemination leading to metastases. Hypersialylation of cancer cells is largely the result of overexpression of sialyltransferases. Humans differentially express twenty different sialyltransferases in a tissue-specific manner, each of which catalyze the attachment of sialic acids via different glycosidic linkages (2-3; 2-6 or 2-8 to the underlying glycan chain. One important mechanism whereby overexpression of sialyltransferases contributes to an enhanced metastatic phenotype is via the generation of selectin ligands. Selectin ligand function requires the expression of sialyl-Lewis X and its structural-isomer sialyl-Lewis A, which are synthesized by the combined action of alpha 1-3-fucosyltransferases, 2-3-sialyltransferases, 1-4-galactosyltranferases, and N-acetyl--glucosaminyltransferases. The α2-3-sialyltransferases ST3Gal4 and ST3Gal6 are critical to the generation of functional E- and P-selectin ligands and overexpression of these sialyltransferases have been linked to increased risk of metastatic disease in solid tumors and poor outcome in multiple myeloma. Thus, targeting selectins and their ligands as well as the enzymes involved in their generation, in particular sialyltransferases, could be beneficial to many cancer patients. Potential strategies include sialyltransferase inhibition and the use of selectin antagonists, such as glycomimetic drugs and antibodies. Here, we review ongoing efforts to optimize the potency and selectivity of sialyltransferase inhibitors, including the potential for targeted delivery approaches, as well as evaluate the potential utility of selectin inhibitors, which are now in early clinical

  4. Design of targeting ligands in medicinal inorganic chemistry.

    Science.gov (United States)

    Storr, Tim; Thompson, Katherine H; Orvig, Chris

    2006-06-01

    This tutorial review will highlight recent advances in medicinal inorganic chemistry pertaining to the use of multifunctional ligands for enhanced effect. Ligands that adequately bind metal ions and also include specific targeting features are gaining in popularity due to their ability to enhance the efficacy of less complicated metal-based agents. Moving beyond the traditional view of ligands modifying reactivity, stabilizing specific oxidation states, and contributing to substitution inertness, we will discuss recent work involving metal complexes with multifunctional ligands that target specific tissues, membrane receptors, or endogenous molecules, including enzymes.

  5. Brain tumour-associated status epilepticus.

    Science.gov (United States)

    Goonawardena, Janindu; Marshman, Laurence A G; Drummond, Katharine J

    2015-01-01

    We have reviewed the scant literature on status epilepticus in patients with brain tumours. Patients with brain tumour-associated epilepsy (TAE) appear less likely to develop status epilepticus (TASE) than patients with epilepsy in the general population (EGP) are to develop status epilepticus (SEGP). TASE is associated with lesions in similar locations as TAE; in particular, the frontal lobes. However, in contrast to TAE, where seizures commence early in the course of the disease or at presentation, TASE is more likely to occur later in the disease course and herald tumour progression. In marked contrast to TAE, where epilepsy risk is inversely proportional to Word Health Organization tumour grade, TASE risk appears to be directly proportional to tumour grade (high grade gliomas appear singularly predisposed). Whilst anti-epileptic drug (AED) resistance is more common in TAE than EGP (with resistance directly proportional to tumour grade and frontal location), TASE appears paradoxically more responsive to simple AED regimes than either TAE or SEGP. Although some results suggest that mortality may be higher with TASE than with SEGP, it is likely that (as with SEGP) the major determinant of mortality is the underlying disease process. Because all such data have been derived from retrospective studies, because TASE and SEGP are less common than TAE and EGP, and because TASE and SEGP classification has often been inconsistent, findings can only be considered preliminary: multi-centre, prospective studies are required. Whilst preliminary, our review suggests that TASE has a distinct clinical profile compared to TAE and SEGP.

  6. Targeting Ligand-Dependent and Ligand-Independent Androgen Receptor Signaling in Prostate Cancer

    Science.gov (United States)

    2015-10-01

    Award Number: W81XWH-12-1-0288 TITLE: Targeting Ligand-Dependent and Ligand-Independent Androgen Receptor Signaling in Prostate Cancer...average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed...and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of

  7. Protecting Ligands Enhance Selective Targeting of Multivalent Nanoparticles

    CERN Document Server

    Angioletti-Uberti, Stefano

    2016-01-01

    Nanoparticles functionalized with multiple ligands can be programmed to bind biological targets, e.g. cells, depending on the receptors they express, providing a general platform for the development of different technologies, from selective drug-delivery to biosensing. In order to be highly selective ligands should exclusively bind to specific targeted receptors, since formation of bonds with other, untargeted ones would lead to non-specific binding and potentially harmful behaviour. This poses a particular problem for multivalent nanoparticles, because even very weak bonds can collectively lead to strong binding. A statistical mechanical model is presented here to describe the extent to which bond strength and nanoparticle valency can induce non-selective adsorption. The same model is used to describe a possible solution: functionalization of the nanoparticles with "protective" receptors. The latter compete with cell receptors for the targeting ligands, and can be optimized to strongly reduce the effect of u...

  8. Ligand-targeted particulate nanomedicines undergoing clinical evaluations: current status

    NARCIS (Netherlands)

    Meel, van der Roy; Vehmeijer, Laurens J.C.; Kok, Robbert J.; Storm, Gert; Gaal, van Ethlinn V.B.

    2013-01-01

    Since the introduction of Doxil® on the market nearly 20 years ago, a number of nanomedicines have become part of treatment regimens in the clinic. With the exception of antibody–drug conjugates, these nanomedicines are all devoid of targeting ligands and rely solely on their physicochemical propert

  9. MOST: most-similar ligand based approach to target prediction.

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    Huang, Tao; Mi, Hong; Lin, Cheng-Yuan; Zhao, Ling; Zhong, Linda L D; Liu, Feng-Bin; Zhang, Ge; Lu, Ai-Ping; Bian, Zhao-Xiang

    2017-03-11

    Many computational approaches have been used for target prediction, including machine learning, reverse docking, bioactivity spectra analysis, and chemical similarity searching. Recent studies have suggested that chemical similarity searching may be driven by the most-similar ligand. However, the extent of bioactivity of most-similar ligands has been oversimplified or even neglected in these studies, and this has impaired the prediction power. Here we propose the MOst-Similar ligand-based Target inference approach, namely MOST, which uses fingerprint similarity and explicit bioactivity of the most-similar ligands to predict targets of the query compound. Performance of MOST was evaluated by using combinations of different fingerprint schemes, machine learning methods, and bioactivity representations. In sevenfold cross-validation with a benchmark Ki dataset from CHEMBL release 19 containing 61,937 bioactivity data of 173 human targets, MOST achieved high average prediction accuracy (0.95 for pKi ≥ 5, and 0.87 for pKi ≥ 6). Morgan fingerprint was shown to be slightly better than FP2. Logistic Regression and Random Forest methods performed better than Naïve Bayes. In a temporal validation, the Ki dataset from CHEMBL19 were used to train models and predict the bioactivity of newly deposited ligands in CHEMBL20. MOST also performed well with high accuracy (0.90 for pKi ≥ 5, and 0.76 for pKi ≥ 6), when Logistic Regression and Morgan fingerprint were employed. Furthermore, the p values associated with explicit bioactivity were found be a robust index for removing false positive predictions. Implicit bioactivity did not offer this capability. Finally, p values generated with Logistic Regression, Morgan fingerprint and explicit activity were integrated with a false discovery rate (FDR) control procedure to reduce false positives in multiple-target prediction scenario, and the success of this strategy it was demonstrated with a case of fluanisone

  10. From Toxins Targeting Ligand Gated Ion Channels to Therapeutic Molecules

    Directory of Open Access Journals (Sweden)

    Antoine Taly

    2011-03-01

    Full Text Available Ligand-gated ion channels (LGIC play a central role in inter-cellular communication. This key function has two consequences: (i these receptor channels are major targets for drug discovery because of their potential involvement in numerous human brain diseases; (ii they are often found to be the target of plant and animal toxins. Together this makes toxin/receptor interactions important to drug discovery projects. Therefore, toxins acting on LGIC are presented and their current/potential therapeutic uses highlighted.

  11. How reliable are ligand-centric methods for Target Fishing?

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    Antonio ePeon

    2016-04-01

    Full Text Available Computational methods for Target Fishing (TF, also known as Target Prediction or Polypharmacology Prediction, can be used to discover new targets for small-molecule drugs. This may result in repositioning the drug in a new indication or improving our current understanding of its efficacy and side effects. While there is a substantial body of research on TF methods, there is still a need to improve their validation, which is often limited to a small part of the available targets and not easily interpretable by the user. Here we discuss how target-centric TF methods are inherently limited by the number of targets that can possibly predict (this number is by construction much larger in ligand-centric techniques. We also propose a new benchmark to validate TF methods, which is particularly suited to analyse how predictive performance varies with the query molecule. On average over approved drugs, we estimate that only five predicted targets will have to be tested to find two true targets with submicromolar potency (a strong variability in performance is however observed. In addition, we find that an approved drug has currently an average of eight known targets, which reinforces the notion that polypharmacology is a common and strong event. Furthermore, with the assistance of a control group of randomly-selected molecules, we show that the targets of approved drugs are generally harder to predict.

  12. Evaluation of Polymeric Nanomedicines Targeted to PSMA: Effect of Ligand on Targeting Efficiency.

    Science.gov (United States)

    Fuchs, Adrian V; Tse, Brian W C; Pearce, Amanda K; Yeh, Mei-Chun; Fletcher, Nicholas L; Huang, Steve S; Heston, Warren D; Whittaker, Andrew K; Russell, Pamela J; Thurecht, Kristofer J

    2015-10-12

    Targeted nanomedicines offer a strategy for greatly enhancing accumulation of a therapeutic within a specific tissue in animals. In this study, we report on the comparative targeting efficiency toward prostate-specific membrane antigen (PSMA) of a number of different ligands that are covalently attached by the same chemistry to a polymeric nanocarrier. The targeting ligands included a small molecule (glutamate urea), a peptide ligand, and a monoclonal antibody (J591). A hyperbranched polymer (HBP) was utilized as the nanocarrier and contained a fluorophore for tracking/analysis, whereas the pendant functional chain-ends provided a handle for ligand conjugation. Targeting efficiency of each ligand was assessed in vitro using flow cytometry and confocal microscopy to compare degree of binding and internalization of the HBPs by human prostate cancer (PCa) cell lines with different PSMA expression status (PC3-PIP (PSMA+) and PC3-FLU (PSMA-). The peptide ligand was further investigated in vivo, in which BALB/c nude mice bearing subcutaneous PC3-PIP and PC3-FLU PCa tumors were injected intravenously with the HBP-peptide conjugate and assessed by fluorescence imaging. Enhanced accumulation in the tumor tissue of PC3-PIP compared to PC3-FLU highlighted the applicability of this system as a future imaging and therapeutic delivery vehicle.

  13. Programmed death-1 & its ligands: promising targets for cancer immunotherapy.

    Science.gov (United States)

    Shrimali, Rajeev K; Janik, John E; Abu-Eid, Rasha; Mkrtichyan, Mikayel; Khleif, Samir N

    2015-01-01

    Novel strategies for cancer treatment involving blockade of immune inhibitors have shown significant progress toward understanding the molecular mechanism of tumor immune evasion. The preclinical findings and clinical responses associated with programmed death-1 (PD-1) and PD-ligand pathway blockade seem promising, making these targets highly sought for cancer immunotherapy. In fact, the anti-PD-1 antibodies, pembrolizumab and nivolumab, were recently approved by the US FDA for the treatment of unresectable and metastatic melanoma resistant to anticytotoxic T-lymphocyte antigen-4 antibody (ipilimumab) and BRAF inhibitor. Here, we discuss strategies of combining PD-1/PD-ligand interaction inhibitors with other immune checkpoint modulators and standard-of-care therapy to break immune tolerance and induce a potent antitumor activity, which is currently a research area of key scientific pursuit.

  14. Inhibition of angiopoietin 2 attenuates lumen formation of tumour-associated vessels in vivo.

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    Suzuki, Rei; Yamamoto, Hirofumi; Ngan, Chew Yee; Ohtsuka, Masahisa; Kitani, Kotaro; Uemura, Mamoru; Nishimura, Junichi; Takemasa, Ichiro; Mizushima, Tsunekazu; Sekimoto, Mitsugu; Minamoto, Toshinari; Doki, Yuichiro; Mori, Masaki

    2013-11-01

    Anti-angiogenic therapy, inhibition of a co-operative process with vascular endothelial cells and pericytes could be an effective strategy to treat malignant tumours. Apart from vascular endothelial growth factor (VEGF), angiopoietin 2 (Ang2) is a promising target of anti-angiogenic therapy. Although inhibition of Ang2 has been shown to decrease tumour size in preclinical and phase I trials, its mechanisms of action remain largely unknown. To elucidate the mechanisms of Ang2 inhibition, we have focused on differentiation of the vessels as well as on growth of the vessels, especially in vivo. L1-10, a selective Ang2 inhibitor was used. The in vitro effects of Ang2 inhibition or addition of Ang2 using HUVECs were also examined. Growth and differentiation of tumour-associated vessels were investigated in xenografts derived from a colon cancer treated by L1-10. Effects of VEGF inhibition were also examined to discriminate Ang2-specific action on the tumour-associated vessels. In vitro studies showed that VEGF enhanced proliferation and tube formation of HUVECs, and caused a significant increase in Rac1 and CDC42 expression when cultured in the collagen matrix gel, whereas neither Ang2 nor L1-10 affected in vitro behaviour of HUVECs or levels of the proteins. In vivo, on the other hand, we found that Ang2 inhibition with treatment of L1-10 dose‑dependently decreased tumour growth. Furthermore, we found that L1-10 treatment extends the tumour-associated vessels whilst it suppressed a sound lumen formation. Histological analysis on xenografts suggests that Ang2 inhibition could have disturbed in vivo vascular differentiation. Our data provide a novel aspect that Ang2 may play an essential role in in vivo vascular differentiation, thus supporting a rationale for Ang2-targeted therapy against colon cancer.

  15. Targeting Protein-Protein Interactions with Trimeric Ligands: High Affinity Inhibitors of the MAGUK Protein Family

    DEFF Research Database (Denmark)

    Nissen, Klaus B; Kedström, Linda Maria Haugaard; Wilbek, Theis S

    2015-01-01

    PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95...... of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG...

  16. Integration of screening and identifying ligand(s) from medicinal plant extracts based on target recognition by using NMR spectroscopy

    OpenAIRE

    2015-01-01

    Authors: Yalin Tang, Qian Shang, Junfeng Xiang, Qianfan Yang, Qiuju Zhou, Lin Li, Hong Zhang, Qian Li, Hongxia Sun, Aijiao Guan, Wei Jiang & Wei Gai ### Abstract This protocol presents the screening of ligand(s) from medicinal plant extracts based on target recognition by using NMR spectroscopy. A detailed description of sample preparation and analysis process is provided. NMR spectroscopies described here are 1H NMR, diffusion-ordered spectroscopy (DOSY), relaxation-edited NMR, ...

  17. Brain targeted delivery of paclitaxel using endogenous ligand

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    Sanjeev R. Acharya

    2016-06-01

    Full Text Available The objective of the present investigation was to formulate nanoparticles constructed using PLGA polymer for the effective targeted delivery to brain via nasal route. The PLGA nanoparticles were optimized using novel design of experiment technique by 23 full factorial design. Drug: polymer ratio (X1, surfactant concentration (X2 and stirring speed (X3 were identified as critical process parameters, and its impact on particle size (Y1 and % entrapment efficiency (Y2 was studied. The optimized nanoparticle formulation was conjugated with glutathione as an endogenous ligand by using carbodiimide chemistry using (1-Ethyl-3-(3-dimethylaminopropylcarbodiimide (EDAC as linker molecule. From Ellman's assay, it was found that a total of 691.27 ± 151 units of glutathione were conjugated upon each PLGA nanoparticle. The in vitro release studies as well as ex vivo studies revealed biphasic pattern of drug release with initial burst release followed by slow exponential release of drug over a period of 24 h. The in vivo biodistribution studies were conducted on rat following nasal administration of the nanoparticle formulation (conjugated and unconjugated and were compared with plain paclitaxel suspension. The results clearly demonstrated that the brain targeting efficiency was enhanced with the glutathione conjugated formulation (387.474% as compared to the unconjugated nanoparticle formulation (224.327%. Further, the in vitro in vivo correlation studies revealed good relationship (R2 > 0.95 as obtained from the levy plot. Glutathione proves to be an efficient vector for the successful transport of poor bioavailable drug to the brain.

  18. Ligands targeting the excitatory amino acid transporters (EAATs).

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    Dunlop, John; Butera, John A

    2006-01-01

    This review provides an overview of ligands for the excitatory amino acid transporters (EAATs), a family of high-affinity glutamate transporters localized to the plasma membrane of neurons and astroglial cells. Ligand development from the perspective of identifying novel and more selective tools for elucidating transporter subtype function, and the potential of transporter ligands in a therapeutic setting are discussed. Acute pharmacological modulation of EAAT activity in the form of linear and conformationally restricted glutamate and aspartate analogs is presented, in addition to recent strategies aimed more toward modulating transporter expression levels, the latter of particular significance to the development of transporter based therapeutics.

  19. Comparison of five different targeting ligands to enhance accumulation of liposomes into the brain.

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    van Rooy, Inge; Mastrobattista, Enrico; Storm, Gert; Hennink, Wim E; Schiffelers, Raymond M

    2011-02-28

    In many different studies nanocarriers modified with targeting ligands have been used to target to the brain. Many ligands have been successful, but it is difficult to compare results from different studies to determine which targeting ligand is the best. Therefore, we selected five targeting ligands (transferrin, RI7217, COG133, angiopep-2, and CRM197) and compared their ability to target liposomes to the brain in vitro and in vivo. In vitro, only CRM197-modified liposomes were able to bind to murine endothelial cells (bEnd.3). Both CRM197 and RI7217-modified liposomes associated with human endothelial cells (hCMEC/D3). In vivo, uptake of targeted liposomes was tested at 12h after iv injection. For some of the ligands, additional time points of 1 and 6h were tested. Only the RI7217 was able to significantly enhance brain uptake in vivo at all time points. Uptake in the brain capillaries was up to 10 times higher compared to untargeted liposomes, and uptake in the brain parenchyma was up to 4.3 times higher. Additionally, these results show that many targeting ligands that have been described for brain targeting, do not target to the brain in vivo when coupled to a liposomal delivery vehicle.

  20. Targeting protein-protein interactions with trimeric ligands: high affinity inhibitors of the MAGUK protein family.

    Directory of Open Access Journals (Sweden)

    Klaus B Nissen

    Full Text Available PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins.

  1. The role of tumour-associated macrophages in bone metastasis

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    Sofia Sousa

    2016-09-01

    Full Text Available This overview addresses the recent research developments in the role of tumour-associated macrophages (TAM in bone metastasis biology and management of breast and prostate cancer as well as in primary and lung metastatic osteosarcoma. Immunosuppressive M2-type TAMs have been shown to associate with poor prognosis. Throughout their life cycle, macrophages (Macs can adapt to environmental cues and influence the surroundings by secreting different cytokines and enzymes crucial to matrix remodelling, infection fighting, immune regulation and/or inflammation. In general terms, there is a broad and complex spectrum of Mac polarization statuses from M1 (classically activated/inflammatory to M2 (alternatively activated/wound healing/immune regulating Macs. Often the activation status of TAMs resembles more the M2-type. Considering the physiological functions of M2 Macs, it is no surprise that TAMs appear to have a role in metastasis, participating in almost every step of the metastatic cascade, which we review and explore in selected bone tropic cancers.

  2. Evaluation of the novel algorithm of flexible ligand docking with moveable target-protein atoms.

    Science.gov (United States)

    Sulimov, Alexey V; Zheltkov, Dmitry A; Oferkin, Igor V; Kutov, Danil C; Katkova, Ekaterina V; Tyrtyshnikov, Eugene E; Sulimov, Vladimir B

    2017-01-01

    We present the novel docking algorithm based on the Tensor Train decomposition and the TT-Cross global optimization. The algorithm is applied to the docking problem with flexible ligand and moveable protein atoms. The energy of the protein-ligand complex is calculated in the frame of the MMFF94 force field in vacuum. The grid of precalculated energy potentials of probe ligand atoms in the field of the target protein atoms is not used. The energy of the protein-ligand complex for any given configuration is computed directly with the MMFF94 force field without any fitting parameters. The conformation space of the system coordinates is formed by translations and rotations of the ligand as a whole, by the ligand torsions and also by Cartesian coordinates of the selected target protein atoms. Mobility of protein and ligand atoms is taken into account in the docking process simultaneously and equally. The algorithm is realized in the novel parallel docking SOL-P program and results of its performance for a set of 30 protein-ligand complexes are presented. Dependence of the docking positioning accuracy is investigated as a function of parameters of the docking algorithm and the number of protein moveable atoms. It is shown that mobility of the protein atoms improves docking positioning accuracy. The SOL-P program is able to perform docking of a flexible ligand into the active site of the target protein with several dozens of protein moveable atoms: the native crystallized ligand pose is correctly found as the global energy minimum in the search space with 157 dimensions using 4700 CPU ∗ h at the Lomonosov supercomputer.

  3. Identification of ligand-target pairs from combined libraries of small molecules and unpurified protein targets in cell lysates.

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    McGregor, Lynn M; Jain, Tara; Liu, David R

    2014-02-26

    We describe the development and validation of interaction determination using unpurified proteins (IDUP), a method that selectively amplifies DNA sequences identifying ligand+target pairs from a mixture of DNA-linked small molecules and unpurified protein targets in cell lysates. By operating in cell lysates, IDUP preserves native post-translational modifications and interactions with endogenous binding partners, thereby enabling the study of difficult-to-purify targets and increasing the potential biological relevance of detected interactions compared with methods that require purified proteins. In IDUP, target proteins are associated with DNA oligonucleotide tags either non-covalently using a DNA-linked antibody or covalently using a SNAP-tag. Ligand-target binding promotes hybridization of a self-priming hairpin that is extended by a DNA polymerase to create a DNA strand that contains sequences identifying both the target and its ligand. These sequences encoding ligand+target pairs are selectively amplified by PCR and revealed by high-throughput DNA sequencing. IDUP can respond to the effect of affinity-modulating adaptor proteins in cell lysates that would be absent in ligand screening or selection methods using a purified protein target. This capability was exemplified by the 100-fold amplification of DNA sequences encoding FRB+rapamycin or FKBP+rapamycin in samples overexpressing both FRB and FKBP (FRB·rapamycin+FKBP, Kd ≈ 100 fM; FKBP·rapamycin+FRB, Kd = 12 nM). In contrast, these sequences were amplified 10-fold less efficiently in samples overexpressing either FRB or FKBP alone (rapamycin+FKBP, Kd ≈ 0.2 nM; rapamcyin+FRB, Kd = 26 μM). Finally, IDUP was used to process a model library of DNA-linked small molecules and a model library of cell lysates expressing SNAP-target fusions combined in a single sample. In this library×library experiment, IDUP resulted in enrichment of sequences corresponding to five known ligand+target pairs ranging in binding

  4. Novel ligands that target the mitochondrial membrane protein mitoNEET

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    Bieganski, Robert M.; Yarmush, Martin L.

    2012-01-01

    Ligands of the thiazolidinedione (TZD) class of compounds, pioglitazone (Actos™) and rosiglitazone (Avandia™) are currently approved for treatment of type 2 diabetes and are known to bind to the PPAR-γ nuclear receptor subtype. Recent evidence suggesting PPAR-γ independent action of the TZDs led to the discovery of a novel integral outer mitochondrial membrane protein, mitoNEET. In spite of the several reported X-ray crystal structures of the unbound form of mitoNEET, the location and nature of the mitoNEET ligand binding sites (LBS) remain unknown. In this study, a molecular blind docking (BD) method was used to discover potential mitoNEET LBS and novel ligands, utilizing the program AutoDock Vina (v 1.0.2). Validation of BD was performed on the PPAR-γ receptor (PDB ID: 1ZGY) with the test compound rosiglitazone, demonstrating that the binding conformation of rosiglitazone determined by AutoDock Vina matches well with that of the cocrystallized ligand (root mean square deviation of the heavy atoms 1.45 Å). The locations and a general ligand binding interaction model for the LBS were determined, leading to the discovery of novel mitoNEET ligands. An in vitro fluorescence binding assay utilizing purified recombinant mitoNEET protein was used to determine the binding affinity of a predicted mitoNEET ligand, and the data obtained is in good agreement with AutoDock Vina results. The discovery of potential mitoNEET ligand binding sites and novel ligands, opens up the possibility for detailed structural studies of mitoNEET–ligand complexes, as well as rational design of novel ligands specifically targeted for mitoNEET. PMID:21531159

  5. Aptamers: active targeting ligands for cancer diagnosis and therapy.

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    Wu, Xu; Chen, Jiao; Wu, Min; Zhao, Julia Xiaojun

    2015-01-01

    Aptamers, including DNA, RNA and peptide aptamers, are a group of promising recognition units that can specifically bind to target molecules and cells. Due to their excellent specificity and high affinity to targets, aptamers have attracted great attention in various fields in which selective recognition units are required. They have been used in biosensing, drug delivery, disease diagnosis and therapy (especially for cancer treatment). In this review, we summarized recent applications of DNA and RNA aptamers in cancer theranostics. The specific binding ability of aptamers to cancer-related markers and cancer cells ensured their high performance for early diagnosis of cancer. Meanwhile, the efficient targeting ability of aptamers to cancer cells and tissues provided a promising way to deliver imaging agents and drugs for cancer imaging and therapy. Furthermore, with the development of nanoscience and nanotechnology, the conjugation of aptamers with functional nanomaterials paved an exciting way for the fabrication of theranostic agents for different types of cancers, which might be a powerful tool for cancer treatment.

  6. Identification of Pancreatic Cancer Specific Cell-Surface Markers for Development of Targeting Ligands

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    Morse, David L.; Hostetter, Galen; Balagurunathan, Yoganand; Gillies, Robert J.; Han, Haiyong

    2014-01-01

    Pancreatic cancer is generally detected at later stages with a poor prognosis and a high-mortality rate. Development of theranostic imaging agents that non-invasively target pancreatic cancer by gene expression and deliver therapies directly to malignant cells could greatly improve therapeutic outcomes. Small-peptide ligands that bind cell-surface proteins and are conjugated to imaging moieties have demonstrated efficacy in cancer imaging. Identification of cancer specific targets is a major bottleneck in the development of such agents. Herein, a method is presented that uses DNA microarray expression profiling of large sets of normal and cancer tissues to identify targets expressed in cancer but not expressed in relevant normal tissues. Identified targets are subsequently validated for protein expression using tissue microarray. Further validations are performed by quantifying expression in pancreatic cancer cells by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR), by immunocytochemistry and immunohistochemistry and by reviewing data and literature in public databases. Validated targets are selected for ligand development based on the existence of a known ligand or by known structure activity relationships useful for development of novel ligands. PMID:20217597

  7. Identification of ligands that target the HCV-E2 binding site on CD81

    Science.gov (United States)

    Olaby, Reem Al; Azzazy, Hassan M.; Harris, Rodney; Chromy, Brett; Vielmetter, Jost; Balhorn, Rod

    2013-04-01

    Hepatitis C is a global health problem. While many drug companies have active R&D efforts to develop new drugs for treating Hepatitis C virus (HCV), most target the viral enzymes. The HCV glycoprotein E2 has been shown to play an essential role in hepatocyte invasion by binding to CD81 and other cell surface receptors. This paper describes the use of AutoDock to identify ligand binding sites on the large extracellular loop of the open conformation of CD81 and to perform virtual screening runs to identify sets of small molecule ligands predicted to bind to two of these sites. The best sites selected by AutoLigand were located in regions identified by mutational studies to be the site of E2 binding. Thirty-six ligands predicted by AutoDock to bind to these sites were subsequently tested experimentally to determine if they bound to CD81-LEL. Binding assays conducted using surface Plasmon resonance revealed that 26 out of 36 (72 %) of the ligands bound in vitro to the recombinant CD81-LEL protein. Competition experiments performed using dual polarization interferometry showed that one of the ligands predicted to bind to the large cleft between the C and D helices was also effective in blocking E2 binding to CD81-LEL.

  8. Identification of ligands that target the HCV-E2 binding site on CD81.

    Science.gov (United States)

    Olaby, Reem Al; Azzazy, Hassan M; Harris, Rodney; Chromy, Brett; Vielmetter, Jost; Balhorn, Rod

    2013-04-01

    Hepatitis C is a global health problem. While many drug companies have active R&D efforts to develop new drugs for treating Hepatitis C virus (HCV), most target the viral enzymes. The HCV glycoprotein E2 has been shown to play an essential role in hepatocyte invasion by binding to CD81 and other cell surface receptors. This paper describes the use of AutoDock to identify ligand binding sites on the large extracellular loop of the open conformation of CD81 and to perform virtual screening runs to identify sets of small molecule ligands predicted to bind to two of these sites. The best sites selected by AutoLigand were located in regions identified by mutational studies to be the site of E2 binding. Thirty-six ligands predicted by AutoDock to bind to these sites were subsequently tested experimentally to determine if they bound to CD81-LEL. Binding assays conducted using surface Plasmon resonance revealed that 26 out of 36 (72 %) of the ligands bound in vitro to the recombinant CD81-LEL protein. Competition experiments performed using dual polarization interferometry showed that one of the ligands predicted to bind to the large cleft between the C and D helices was also effective in blocking E2 binding to CD81-LEL.

  9. [Enzyme-catalyzed synthesis of ASGPR ligand-targeted modifier in non-aqueous medium].

    Science.gov (United States)

    Cheng, Yi; Wu, Wei; Zhang, Dong-qing; Mai, Yan-zhen

    2010-09-01

    The asialoglycoprotein receptor (ASGPR) was used to mediate drug carrier for hepatic targeted drug delivery, this article showed the enzyme-catalyzed esterification of galactose and vinyl stearate and a kind of ASGPR ligand-targeted which was used to insert the surface of liposome has been synthesized. The structure of product has been confirmed by TLC, ESI-MS and 1H NMR. The factors of types and quantity of enzyme, organic solvents, molar ratio of substrate, temperature and time of reaction have been studied. Results showed when using acetone as reaction medium, the quantity of Novozym 435 immobilized lipase was 30 mg mL(-1), molar ratio of galactose to vinyl stearate was 1:5, and reacted at 60 degrees C for 12 h, the transformation of vinyl stearate reached more than 70%. This study provides a novel and efficient route to the synthesis of ligand-targeted modifier.

  10. Structure-based DNA-targeting strategies with small molecule ligands for drug discovery.

    Science.gov (United States)

    Sheng, Jia; Gan, Jianhua; Huang, Zhen

    2013-09-01

    Nucleic acids are the molecular targets of many clinical anticancer drugs. However, compared with proteins, nucleic acids have traditionally attracted much less attention as drug targets in structure-based drug design, partially because limited structural information of nucleic acids complexed with potential drugs is available. Over the past several years, enormous progresses in nucleic acid crystallization, heavy-atom derivatization, phasing, and structural biology have been made. Many complicated nucleic acid structures have been determined, providing new insights into the molecular functions and interactions of nucleic acids, especially DNAs complexed with small molecule ligands. Thus, opportunities have been created to further discover nucleic acid-targeting drugs for disease treatments. This review focuses on the structure studies of DNAs complexed with small molecule ligands for discovering lead compounds, drug candidates, and/or therapeutics.

  11. A targeting ligand enhances infectivity and cytotoxicity of an oncolytic adenovirus in human pancreatic cancer tissues.

    Science.gov (United States)

    Yamamoto, Yuki; Hiraoka, Nobuyoshi; Goto, Naoko; Rin, Yosei; Miura, Kazuki; Narumi, Kenta; Uchida, Hiroaki; Tagawa, Masatoshi; Aoki, Kazunori

    2014-10-28

    The addition of a targeting strategy is necessary to enhance oncolysis and secure safety of a conditionally replicative adenovirus (CRAd). We have constructed an adenovirus library displaying random peptides on the fiber, and have successfully identified a pancreatic cancer-targeting ligand (SYENFSA). Here, the usefulness of cancer-targeted CRAd for pancreatic cancer was examined as a preclinical study. First, we constructed a survivin promoter-regulated CRAd expressing enhanced green fluorescent protein gene (EGFP), which displayed the identified targeting ligand (AdSur-SYE). The AdSur-SYE resulted in higher gene transduction efficiency and oncolytic potency than the untargeted CRAd (AdSur) in several pancreatic cancer cell lines. An intratumoral injection of AdSur-SYE significantly suppressed the growth of subcutaneous tumors, in which AdSur-SYE effectively proliferated and spread. An ectopic infection in adjacent tissues and organs of intratumorally injected AdSur-SYE was decreased compared with AdSur. Then, to examine whether the targeting ligand actually enhanced the infectivity of CRAd in human pancreatic cancer tissues, tumor cells prepared from surgical specimens were infected with viruses. The AdSur-SYE increased gene transduction efficiency 6.4-fold higher than did AdSur in single cells derived from human pancreatic cancer, whereas the infectivity of both vectors was almost the same in the pancreas and other cancers. Immunostaining showed that most EGFP(+) cells were cytokeratin-positive in the sliced tissues, indicating that pancreatic cancer cells but not stromal cells were injected with AdSur-SYE. AdSur-SYE resulted in a stronger oncolysis in the primary pancreatic cancer cells co-cultured with mouse embryonic fibroblasts than AdSur did. CRAd in combination with a tumor-targeting ligand is promising as a next-generation of oncolytic virotherapy for pancreatic cancer.

  12. In Silico target fishing: addressing a "Big Data" problem by ligand-based similarity rankings with data fusion.

    Science.gov (United States)

    Liu, Xian; Xu, Yuan; Li, Shanshan; Wang, Yulan; Peng, Jianlong; Luo, Cheng; Luo, Xiaomin; Zheng, Mingyue; Chen, Kaixian; Jiang, Hualiang

    2014-01-01

    Ligand-based in silico target fishing can be used to identify the potential interacting target of bioactive ligands, which is useful for understanding the polypharmacology and safety profile of existing drugs. The underlying principle of the approach is that known bioactive ligands can be used as reference to predict the targets for a new compound. We tested a pipeline enabling large-scale target fishing and drug repositioning, based on simple fingerprint similarity rankings with data fusion. A large library containing 533 drug relevant targets with 179,807 active ligands was compiled, where each target was defined by its ligand set. For a given query molecule, its target profile is generated by similarity searching against the ligand sets assigned to each target, for which individual searches utilizing multiple reference structures are then fused into a single ranking list representing the potential target interaction profile of the query compound. The proposed approach was validated by 10-fold cross validation and two external tests using data from DrugBank and Therapeutic Target Database (TTD). The use of the approach was further demonstrated with some examples concerning the drug repositioning and drug side-effects prediction. The promising results suggest that the proposed method is useful for not only finding promiscuous drugs for their new usages, but also predicting some important toxic liabilities. With the rapid increasing volume and diversity of data concerning drug related targets and their ligands, the simple ligand-based target fishing approach would play an important role in assisting future drug design and discovery.

  13. A D-peptide ligand of nicotine acetylcholine receptors for brain-targeted drug delivery.

    Science.gov (United States)

    Wei, Xiaoli; Zhan, Changyou; Shen, Qing; Fu, Wei; Xie, Cao; Gao, Jie; Peng, Chunmei; Zheng, Ping; Lu, Weiyue

    2015-03-01

    Lysosomes of brain capillary endothelial cells are implicated in nicotine acetylcholine receptor (nAChR)-mediated transcytosis and act as an enzymatic barrier for the transport of peptide ligands to the brain. A D-peptide ligand of nAChRs (termed (D)CDX), which binds to nAChRs with an IC50 value of 84.5 nM, was developed by retro-inverso isomerization. (D)CDX displayed exceptional stability in lysosomal homogenate and serum, and demonstrated significantly higher transcytosis efficiency in an in vitro blood-brain barrier monolayer compared with the parent L-peptide. When modified on liposomal surface, (D)CDX facilitated significant brain-targeted delivery of liposomes. As a result, brain-targeted delivery of (D)CDX modified liposomes enhanced therapeutic efficiency of encapsulated doxorubicin for glioblastoma. This study illustrates the importance of ligand stability in nAChRs-mediated transcytosis, and paves the way for developing stable brain-targeted entities.

  14. The Notch ligand Jagged1 as a target for anti-tumour therapy

    Directory of Open Access Journals (Sweden)

    Demin eLi

    2014-09-01

    Full Text Available The Notch pathway is increasingly attracting attention as a source of therapeutic targets for cancer. Ligand-induced Notch signalling has been implicated in various aspects of cancer biology; as a consequence pan-Notch inhibitors and therapeutic antibodies targeting one or more of the Notch receptors have been investigated for cancer therapy. Alternatively, Notch ligands provide attractive options for therapy in cancer treatment due to their more restricted expression and better-defined functions, as well as their low rate of mutations in cancer. One of the Notch ligands, Jagged1 (JAG1, is overexpressed in many cancer types, and plays an important role in several aspects of tumour biology. In fact, JAG1-stimulated Notch activation is directly implicated in tumour growth through maintaining cancer stem cell populations, promoting cell survival, inhibiting apoptosis and driving cell proliferation and metastasis. In addition, JAG1 can indirectly affect cancer by influencing tumour microenvironment components such as tumour vasculature and immune cell infiltration. This article gives an overview of JAG1 and its role in tumour biology, and its potential as a therapeutic target.

  15. Immunology in the clinic review series; focus on cancer: tumour-associated macrophages: undisputed stars of the inflammatory tumour microenvironment.

    Science.gov (United States)

    Allavena, P; Mantovani, A

    2012-02-01

    Mononuclear phagocytes are cells of the innate immunity that defend the host against harmful pathogens and heal tissues after injury. Contrary to expectations, in malignancies, tumour-associated macrophages (TAM) promote disease progression by supporting cancer cell survival, proliferation and invasion. TAM and related myeloid cells [Tie2(+) monocytes and myeloid-derived suppressor cells (MDSC)] also promote tumour angiogenesis and suppress adaptive immune responses. These divergent biological activities are mediated by macrophages/myeloid cells with distinct functional polarization, which are ultimately dictated by microenvironmental cues. Clinical and experimental evidence has shown that cancer tissues with high infiltration of TAM are associated with poor patient prognosis and resistance to therapies. Targeting of macrophages in tumours is considered a promising therapeutic strategy: depletion of TAM or their 're-education' as anti-tumour effectors is under clinical investigation and will hopefully contribute to the success of conventional anti-cancer treatments.

  16. In silico target fishing for rationalized ligand discovery exemplified on constituents of Ruta graveolens.

    Science.gov (United States)

    Rollinger, Judith M; Schuster, Daniela; Danzl, Birgit; Schwaiger, Stefan; Markt, Patrick; Schmidtke, Michaela; Gertsch, Jürg; Raduner, Stefan; Wolber, Gerhard; Langer, Thierry; Stuppner, Hermann

    2009-02-01

    The identification of targets whose interaction is likely to result in the successful treatment of a disease is of growing interest for natural product scientists. In the current study we performed an exemplary application of a virtual parallel screening approach to identify potential targets for 16 secondary metabolites isolated and identified from the aerial parts of the medicinal plant RUTA GRAVEOLENS L. Low energy conformers of the isolated constituents were simultaneously screened against a set of 2208 pharmacophore models generated in-house for the IN SILICO prediction of putative biological targets, i. e., target fishing. Based on the predicted ligand-target interactions, we focused on three biological targets, namely acetylcholinesterase (AChE), the human rhinovirus (HRV) coat protein and the cannabinoid receptor type-2 (CB (2)). For a critical evaluation of the applied parallel screening approach, virtual hits and non-hits were assayed on the respective targets. For AChE the highest scoring virtual hit, arborinine, showed the best inhibitory IN VITRO activity on AChE (IC (50) 34.7 muM). Determination of the anti-HRV-2 effect revealed 6,7,8-trimethoxycoumarin and arborinine to be the most active antiviral constituents with IC (50) values of 11.98 muM and 3.19 muM, respectively. Of these, arborinine was predicted virtually. Of all the molecules subjected to parallel screening, one virtual CB (2) ligand was obtained, i. e., rutamarin. Interestingly, in experimental studies only this compound showed a selective activity to the CB (2) receptor ( Ki of 7.4 muM) by using a radioligand displacement assay. The applied parallel screening paradigm with constituents of R. GRAVEOLENS on three different proteins has shown promise as an IN SILICO tool for rational target fishing and pharmacological profiling of extracts and single chemical entities in natural product research.

  17. Prolonged signaling at the parathyroid hormone receptor by peptide ligands targeted to a specific receptor conformation.

    Science.gov (United States)

    Okazaki, Makoto; Ferrandon, Sebastien; Vilardaga, Jean-Pierre; Bouxsein, Mary L; Potts, John T; Gardella, Thomas J

    2008-10-28

    The parathyroid hormone receptor (PTHR) is a class B G protein-coupled receptor that plays critical roles in bone and mineral ion metabolism. Ligand binding to the PTHR involves interactions to both the amino-terminal extracellular (N) domain, and transmembrane/extracellular loop, or juxtamembrane (J) regions of the receptor. Recently, we found that PTH(1-34), but not PTH-related protein, PTHrP(1-36), or M-PTH(1-14) (M = Ala/Aib(1),Aib(3),Gln(10),Har(11),Ala(12),Trp(14),Arg(19)), binds to the PTHR in a largely GTPgammaS-resistant fashion, suggesting selective binding to a novel, high-affinity conformation (R(0)), distinct from the GTPgammaS-sensitive conformation (RG). We examined the effects in vitro and in vivo of introducing the M substitutions, which enhance interaction to the J domain, into PTH analogs extended C-terminally to incorporate residues involved in the N domain interaction. As compared with PTH(1-34), M-PTH(1-28) and M-PTH(1-34) bound to R(0) with higher affinity, produced more sustained cAMP responses in cells, formed more stable complexes with the PTHR in FRET and subcellular localization assays, and induced more prolonged calcemic and phosphate responses in mice. Moreover, after 2 weeks of daily injection in mice, M-PTH(1-34) induced larger increases in trabecular bone volume and greater increases in cortical bone turnover, than did PTH(1-34). Thus, the putative R(0) PTHR conformation can form highly stable complexes with certain PTH ligand analogs and thereby mediate surprisingly prolonged signaling responses in bone and/or kidney PTH target cells. Controlling, via ligand analog design, the selectivity with which a PTH ligand binds to R(0), versus RG, may be a strategy for optimizing signaling duration time, and hence therapeutic efficacy, of PTHR agonist ligands.

  18. Generalization of a targeted library design protocol: application to 5-HT7 receptor ligands.

    Science.gov (United States)

    Nordling, Erik; Homan, Evert

    2004-01-01

    Herein a general concept for the design of targeted libraries for proteins with binding sites that are divided into subsites is laid out, including several practical aspects and their solutions. The design is based on a chemogenomic classification of the subsites followed by collection of bioactive molecular fragments and virtual library generation. The general process is outlined and applied to the assembly of a library of 500 molecules targeting the serotonin type 7 (5-HT7) receptor, a class A G-Protein Coupled Receptor (GPCR). Utilizing commercially available building blocks of similar size and composition, a reference library was created. Control sets of known ligands for the 5-HT7 receptor, other GPCRs, and nuclear receptors were collected from literature sources. Principal component analysis of molecular descriptors for the two libraries and the literature sets, displayed a focusing of the targeted library to the region in the chemical space defined by the literature actives, suggesting a denser coverage of the bioactive region than for the more diverse reference library. Additional computational validations, including PCA class predictions, 3D pharmacophore modeling, and docking calculations all indicated an enrichment factor of 5-HT7 ligand-like molecules in the range of 2-4 for the targeted library compared to the reference library.

  19. Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms.

    Science.gov (United States)

    Akinc, Akin; Querbes, William; De, Soma; Qin, June; Frank-Kamenetsky, Maria; Jayaprakash, K Narayanannair; Jayaraman, Muthusamy; Rajeev, Kallanthottathil G; Cantley, William L; Dorkin, J Robert; Butler, James S; Qin, Liuliang; Racie, Timothy; Sprague, Andrew; Fava, Eugenio; Zeigerer, Anja; Hope, Michael J; Zerial, Marino; Sah, Dinah W Y; Fitzgerald, Kevin; Tracy, Mark A; Manoharan, Muthiah; Koteliansky, Victor; Fougerolles, Antonin de; Maier, Martin A

    2010-07-01

    Lipid nanoparticles (LNPs) have proven to be highly efficient carriers of short-interfering RNAs (siRNAs) to hepatocytes in vivo; however, the precise mechanism by which this efficient delivery occurs has yet to be elucidated. We found that apolipoprotein E (apoE), which plays a major role in the clearance and hepatocellular uptake of physiological lipoproteins, also acts as an endogenous targeting ligand for ionizable LNPs (iLNPs), but not cationic LNPs (cLNPs). The role of apoE was investigated using both in vitro studies employing recombinant apoE and in vivo studies in wild-type and apoE(-/-) mice. Receptor dependence was explored in vitro and in vivo using low-density lipoprotein receptor (LDLR(-/-))-deficient mice. As an alternative to endogenous apoE-based targeting, we developed a targeting approach using an exogenous ligand containing a multivalent N-acetylgalactosamine (GalNAc)-cluster, which binds with high affinity to the asialoglycoprotein receptor (ASGPR) expressed on hepatocytes. Both apoE-based endogenous and GalNAc-based exogenous targeting appear to be highly effective strategies for the delivery of iLNPs to liver.

  20. Identification and characterization of a novel peptide ligand of Tie2 for targeting gene therapy

    Institute of Scientific and Technical Information of China (English)

    Xianghua Wu; Jianren Gu; Zonghai Li; Ming Yao; Huamao Wang; Sumin Qu; Xianlian Chen; Jinjun Li; Ye Sun; Yuhong Xu

    2008-01-01

    Tyrosine kinase with immunoglobulin and epidermal growth factor homology domain-2 (Tie2) has been considered as a rational target for gene therapy in solid tumors. In order to identify a novel peptide ligand of Tie2 for targeted gene therapy, we screened a phage display peptide library and identified a candidate peptide ligand NSLSNASEFRAPY(designated GA5).Binding assays and Scatchard analysis revealed that GA5 could specifically bind to Tie2 with a dissociation constant of 2.1×10-8M.In addition,we showed that GA5 was internalized into tumor cells highly expressing Tie2.In the biodistribution assay.125I-GA5 was mainly accumniated in SPC-A1 xenograft tumors that express Tie2.Ingene delivery studies,GA5-conjugated polyethylenimine vector could achieve greater transgene transduction than non-targeted vectors both in vitro and in vivo.Tumor growth inhibition was observed in SPC-A1 xenograft-bearing mice that received eight intratumoral injections of GA5 polyethylenimine/p53 complexes in 3 weeks.The difference in tumor volume between the experiment and control groups was significant(P<0.05).Our results showed that GA5 is a potentially efficient targeting element for cancer gene or molecular therapy.

  1. Glycans as biofunctional ligands for gold nanorods: stability and targeting in protein-rich media.

    Science.gov (United States)

    García, Isabel; Sánchez-Iglesias, Ana; Henriksen-Lacey, Malou; Grzelczak, Marek; Penadés, Soledad; Liz-Marzán, Luis M

    2015-03-18

    Poly(ethylene glycol) (PEG) has become the gold standard for stabilization of plasmonic nanoparticles (NPs) in biofluids, because it prevents aggregation while minimizing unspecific interactions with proteins. Application of Au NPs in biological environments requires the use of ligands that can target selected receptors, even in the presence of protein-rich media. We demonstrate here the stabilizing effect of low-molecular-weight glycans on both spherical and rod-like plasmonic NPs under physiological conditions, as bench-marked against the well-established PEG ligands. Glycan-coated NPs are resistant to adsorption of proteins from serum-containing media and avoid phagocytosis by macrophage-like cells, but retain selectivity toward carbohydrate-binding proteins in protein-rich biological media. These results open the way toward the design of efficient therapeutic/diagnostic glycan-decorated plasmonic nanotools for specific biological applications.

  2. Targeting the thyroid-stimulating hormone receptor with small molecule ligands and antibodies

    Science.gov (United States)

    Davies, Terry F; Latif, Rauf

    2015-01-01

    Introduction The thyroid-stimulating hormone receptor (TSHR) is the essential molecule for thyroid growth and thyroid hormone production. Since it is also a key autoantigen in Graves’ disease and is involved in thyroid cancer pathophysiology, the targeting of the TSHR offers a logical model for disease control. Areas covered We review the structure and function of the TSHR and the progress in both small molecule ligands and TSHR antibodies for their therapeutic potential. Expert opinion Stabilization of a preferential conformation for the TSHR by allosteric ligands and TSHR antibodies with selective modulation of the signaling pathways is now possible. These tools may be the next generation of therapeutics for controlling the pathophysiological consequences mediated by the effects of the TSHR in the thyroid and other extrathyroidal tissues. PMID:25768836

  3. Eph family receptors and ligands in vascular cell targeting and assembly.

    Science.gov (United States)

    Stein, E; Schoecklmann, H; Daniel, T O

    1997-11-01

    Members of the Eph family of receptor tyrosine kinases determine neural cell aggregation and targeting behavior, functions that are also critical in vascular assembly and remodeling. Among this class of diverse receptors, EphA2 (Eck) and EphB1 (ELK) represent prototypes for two receptor subfamilies distinguished by high-affinity interaction with either glycerophosphatidylinositol (GPI)-linked or transmembrane ligands, respectively. EphA2 participates in angiogenic responses to tumor necrosis factor (TNF) through an autocrine loop affecting endothelial cell migration. EphB1 and its ligand Ephrin-B1 (LERK-2) are important determinants of assembly of endothelial cells from the microvasculature of the kidney, where both are expressed in endothelial progenitors and in glomerular microvascular endothelial cells. Ephrin-B1 activation of EphB1 promotes assembly of these cells into capillary-like structures. Interaction trap approaches have identified downstream signaling proteins that complex with ligand-activated EphA2 or EphB1, including nonreceptor tyrosine kinases and SH2 domain-containing adapter proteins. The Grb 10 adapter is one of a subset that binds activated EphB1, but not EphA2, defining distinct signaling mechanisms for these related endothelial receptors. On the basis of observations in vascular endothelial cells and recent results defining Eph receptor and ligand roles in neural cell targeting, we propose that these receptors direct cell-cell recognition events that are critical in vasculogenesis and angiogenesis. (Trends Cardiovasc Med 1997;7:329-334). © 1997, Elsevier Science Inc.

  4. Clustered Integrin Ligands as a Novel Approach for the Targeting of Non-Viral Vectors

    Science.gov (United States)

    Ng, Quinn Kwan Tai

    Gene transfer or gene delivery is described as the process in which foreign DNA is introduced into cells. Over the years, gene delivery has gained the attention of many researchers and has been developed as powerful tools for use in biotechnology and medicine. With the completion of the Human Genome Project, such advances in technology allowed for the identification of diseases ranging from hereditary disorders to acquired ones (cancer) which were thought to be incurable. Gene therapy provides the means necessary to treat or eliminate genetic diseases from its origin, unlike traditional medicine which only treat symptoms. With ongoing clinical trials for gene therapy increasing, the greatest difficulty still lies in developing safe systems which can target cells of interest to provide efficient delivery. Nature, over millions of years of evolution, has provided an example of one of the most efficient delivery systems: viruses. Although the use of viruses for gene delivery has been well studied, the safety issues involving immunogenicity, insertional mutagenesis, high cost, and poor reproducibility has provided problems for their clinical application. From understanding viruses, we gain insight to designing new systems for non-viral gene delivery. One of these techniques utilized by adenoviruses is the clustering of ligands on its surface through the use of a protein called a penton base. Through the use of nanotechnology we can mimic this basic concept in non-viral gene delivery systems. This dissertation research is focused on developing and applying a novel system for displaying the integrin binding ligand (RGD) in a constrained manner to form a clustered integrin ligand binding platform to be used to enhance the targeting and efficiency of non-viral gene delivery vectors. Peptide mixed monolayer protected gold nanoparticles provides a suitable surface for ligand clustering. A relationship between the peptide ratios in the reaction solution used to form these

  5. Liposomes for targeting hepatocellular carcinoma: use of conjugated arabinogalactan as targeting ligand.

    Science.gov (United States)

    Shah, Sanket M; Goel, Peeyush N; Jain, Ankitkumar S; Pathak, Pankaj O; Padhye, Sameer G; Govindarajan, Srinath; Ghosh, Sandipto S; Chaudhari, Pradip R; Gude, Rajiv P; Gopal, Vijaya; Nagarsenker, Mangal S

    2014-12-30

    Present study investigates the potential of chemically modified (Shah et al., 2013) palmitoylated arabinogalactan (PAG) in guiding liposomal delivery system and targeting asialoglycoprotein receptors (ASGPR) which are expressed in hepatocellular carcinoma (HCC). PAG was incorporated in liposomes during preparation and doxorubicin hydrochloride was actively loaded in preformed liposomes with and without PAG. The liposomal systems with or without PAG were evaluated for in vitro release, in vitro cytotoxicity, in vitro cell uptake on ASGPR(+) cells, in vivo pharmacokinetic study, in vivo biodistribution study, and in vivo efficacy study in immunocompromised mice. The particle size for all the liposomal systems was below 200 nm with a negative zeta potential. Doxorubicin loaded PAG liposomes released significantly higher amount of doxorubicin at pH 5.5 as compared to pH 7.4, providing advantage for targeted tumor therapy. Doxorubicin in PAG liposomes showed superior cytotoxicity on ASGPR(+) HepG2 cells as compared to ASGPR(-), MCF7, A549, and HT29 cells. Superior uptake of doxorubicin loaded PAG liposomes as compared to doxorubicin loaded conventional liposomes was evident in confocal microscopy studies. Higher AUC in pharmacokinetic study and higher deposition in liver was observed for PAG liposomes compared to conventional liposomes. Significantly higher tumor suppression was noted in immunocompromised mice for mice treated with PAG liposomes as compared to the conventional liposomes. Targeting ability and superior activity of PAG liposomes is established pre-clinically suggesting potential of targeted delivery system for improved treatment of HCC.

  6. Identification of novel human WW domain-containing proteins by cloning of ligand targets.

    Science.gov (United States)

    Pirozzi, G; McConnell, S J; Uveges, A J; Carter, J M; Sparks, A B; Kay, B K; Fowlkes, D M

    1997-06-06

    A recently described protein module consisting of 35-40 semiconserved residues, termed the WW domain, has been identified in a number of diverse proteins including dystrophin and Yes-associated protein (YAP). Two putative ligands of YAP, termed WBP-1 and WBP-2, have been found previously to contain several short peptide regions consisting of PPPPY residues (PY motif) that mediate binding to the WW domain of YAP. Although the function(s) of the WW domain remain to be elucidated, these observations strongly support a role for the WW domain in protein-protein interactions. Here we report the isolation of three novel human cDNAs encoding a total of nine WW domains, using a newly developed approach termed COLT (cloning of ligand targets), in which the rapid cloning of modular protein domains is accomplished by screening cDNA expression libraries with specific peptide ligands. Two of the new genes identified appear to be members of a family of proteins, including Rsp5 and Nedd-4, which have ubiquitin-protein ligase activity. In addition, we demonstrate that peptides corresponding to PY and PY-like motifs present in several known signaling or regulatory proteins, including RasGAP, AP-2, p53BP-2 (p53-binding protein-2), interleukin-6 receptor-alpha, chloride channel CLCN5, and epithelial sodium channel ENaC, can selectively bind to certain of these novel WW domains.

  7. Efficient Liver Targeting by Polyvalent Display of a Compact Ligand for the Asialoglycoprotein Receptor.

    Science.gov (United States)

    Sanhueza, Carlos A; Baksh, Michael M; Thuma, Benjamin; Roy, Marc D; Dutta, Sanjay; Préville, Cathy; Chrunyk, Boris A; Beaumont, Kevin; Dullea, Robert; Ammirati, Mark; Liu, Shenping; Gebhard, David; Finley, James E; Salatto, Christopher T; King-Ahmad, Amanda; Stock, Ingrid; Atkinson, Karen; Reidich, Benjamin; Lin, Wen; Kumar, Rajesh; Tu, Meihua; Menhaji-Klotz, Elnaz; Price, David A; Liras, Spiros; Finn, M G; Mascitti, Vincent

    2017-03-08

    A compact and stable bicyclic bridged ketal was developed as a ligand for the asialoglycoprotein receptor (ASGPR). This compound showed excellent ligand efficiency, and the molecular details of binding were revealed by the first X-ray crystal structures of ligand-bound ASGPR. This analogue was used to make potent di- and trivalent binders of ASGPR. Extensive characterization of the function of these compounds showed rapid ASGPR-dependent cellular uptake in vitro and high levels of liver/plasma selectivity in vivo. Assessment of the biodistribution in rodents of a prototypical Alexa647-labeled trivalent conjugate showed selective hepatocyte targeting with no detectable distribution in nonparenchymal cells. This molecule also exhibited increased ASGPR-directed hepatocellular uptake and prolonged retention compared to a similar GalNAc derived trimer conjugate. Selective release in the liver of a passively permeable small-molecule cargo was achieved by retro-Diels-Alder cleavage of an oxanorbornadiene linkage, presumably upon encountering intracellular thiol. Therefore, the multicomponent construct described here represents a highly efficient delivery vehicle to hepatocytes.

  8. Selective recognition and stabilization of new ligands targeting the potassium form of the human telomeric G-quadruplex DNA

    Science.gov (United States)

    Lin, Yi-Hwa; Chuang, Show-Mei; Wu, Pei-Ching; Chen, Chun-Liang; Jeyachandran, Sivakamavalli; Lo, Shou-Chen; Huang, Hsu-Shan; Hou, Ming-Hon

    2016-08-01

    The development of a ligand that is capable of distinguishing among the wide variety of G-quadruplex structures and targeting telomeres to treat cancer is particularly challenging. In this study, the ability of two anthraquinone telomerase inhibitors (NSC749235 and NSC764638) to target telomeric G-quadruplex DNA was probed. We found that these ligands specifically target the potassium form of telomeric G-quadruplex DNA over the DNA counterpart. The characteristic interaction with the telomeric G-quadruplex DNA and the anticancer activities of these ligands were also explored. The results of this present work emphasize our understanding of the binding selectivity of anthraquinone derivatives to G-quadruplex DNA and assists in future drug development for G-quadruplex-specific ligands.

  9. Targeting bladder tumor cells in voided urine of Chinese patients with FITC-CSNRDARRC peptide ligand

    Directory of Open Access Journals (Sweden)

    Jia XY

    2012-05-01

    . The advantage was maintained in terms of the detection of invasive tumors between the FITC-CSNRDARRC ligand and UC (90.48% versus 23.81%, P = 0.001 as well as between FISH and UC (85.71% versus 23.81%, P = 0.003. The specificities for the FITC-CSNRDARRC ligand, UC, and FISH were 100%.Conclusion: Results show that the FITC-CSNRDARRC ligand is a promising noninvasive tool for diagnosis and surveillance in patients suspected of having a new bladder tumor.Keywords: bladder tumor, tumor-targeting, FITC-CSNRDARRC ligand, fluorescent probe

  10. Fluorescence assays for monitoring RNA-ligand interactions and riboswitch-targeted drug discovery screening.

    Science.gov (United States)

    Liu, J; Zeng, C; Zhou, S; Means, J A; Hines, J V

    2015-01-01

    Riboswitches and other noncoding regulatory RNA are intriguing targets for the development of therapeutic agents. A significant challenge in the drug discovery process, however, is the identification of potent compounds that bind the target RNA specifically and disrupt its function. Essential to this process is an effectively designed cascade of screening assays. A screening cascade for identifying compounds that target the T box riboswitch antiterminator element is described. In the primary assays, moderate to higher throughput screening of compound libraries is achieved by combining the sensitivity of fluorescence techniques with functionally relevant assays. Active compounds are then validated and the binding to target RNA further characterized in secondary assays. The cascade of assays monitor ligand-induced changes in the steady-state fluorescence of an attached dye or internally incorporated 2-aminopurine; the fluorescence anisotropy of an RNA complex; and, the thermal denaturation fluorescence profile of a fluorophore-quencher labeled RNA. While the assays described have been developed for T box riboswitch-targeted drug discovery, the fluorescence methods and screening cascade design principles can be applied to drug discovery efforts targeted toward other medicinally relevant noncoding RNA.

  11. Optimization of a novel peptide ligand targeting human carbonic anhydrase IX.

    Directory of Open Access Journals (Sweden)

    Shoaib Rana

    Full Text Available BACKGROUND: Carbonic anhydrase IX (CA IX is a hypoxia-regulated transmembrane protein over-expressed in various types of human cancer. Recently, a new peptide with affinity for human carbonic anhydrase IX (CaIX-P1 was identified using the phage display technology. Aim of the present study is to characterize the binding site in the sequence of CaIX-P1, in order to optimize the binding and metabolic properties and use it for targeting purposes. METHODOLOGY/PRINCIPAL FINDINGS: Various fragments of CaIX-P1 were synthesized on solid support using Fmoc chemistry. Alanine scanning was performed for identification of the amino acids crucial for target binding. Derivatives with increased binding affinity were radiolabeled and in vitro studies were carried out on the CA IX positive human renal cell carcinoma cell line SKRC 52 and the CA IX negative human pancreatic carcinoma cell line BxPC3. Metabolic stability was investigated in cell culture medium and human serum. Organ distribution and planar scintigraphy studies were performed in Balb/c nu/nu mice carrying subcutaneously transplanted SKRC 52 tumors. The results of our studies clearly identified amino acids that are important for target binding. Among various fragments and derivatives the ligand CaIX-P1-4-10 (NHVPLSPy was found to possess increased binding potential in SKRC 52 cells, whereas no binding capacity for BxPC3 cells was observed. Binding of radiolabeled CaIX-P1-4-10 on CA IX positive cells could be inhibited by both the unlabeled and the native CaIX-P1 peptide but not by control peptides. Stability experiments indicated the degradation site in the sequence of CaIX-P1-4-10. Biodistribution studies showed a higher in vivo accumulation in the tumor than in most healthy tissues. CONCLUSIONS: Our data reveal modifications in the sequence of the CA IX affine ligand CaIX-P1 that might be favorable for improvement of target affinity and metabolic stability, which are necessary prior to the use of

  12. Molecular investigations of protriptyline as a multi-target directed ligand in Alzheimer's disease.

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    Sneha B Bansode

    Full Text Available Alzheimer's disease (AD is a complex neurodegenerative disorder involving multiple cellular and molecular processes. The discovery of drug molecules capable of targeting multiple factors involved in AD pathogenesis would greatly facilitate in improving therapeutic strategies. The repositioning of existing non-toxic drugs could dramatically reduce the time and costs involved in developmental and clinical trial stages. In this study, preliminary screening of 140 FDA approved nervous system drugs by docking suggested the viability of the tricyclic group of antidepressants against three major AD targets, viz. Acetylcholinesterase (AChE, β-secretase (BACE-1, and amyloid β (Aβ aggregation, with one member, protriptyline, showing highest inhibitory activity. Detailed biophysical assays, together with isothermal calorimetry, fluorescence quenching experiments, kinetic studies and atomic force microscopy established the strong inhibitory activity of protriptyline against all three major targets. The molecular basis of inhibition was supported with comprehensive molecular dynamics simulations. Further, the drug inhibited glycation induced amyloid aggregation, another important causal factor in AD progression. This study has led to the discovery of protriptyline as a potent multi target directed ligand and established its viability as a promising candidate for AD treatment.

  13. The Prelude on Novel Receptor and Ligand Targets Involved in the Treatment of Diabetes Mellitus

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    Venu Gopal Jonnalagadda

    2014-05-01

    Full Text Available Metabolic disorders are a group of disorders, due to the disruption of the normal metabolic process at a cellular level. Diabetes Mellitus and Tyrosinaemia are the majorly reported metabolic disorders. Among them, Diabetes Mellitus is a one of the leading metabolic syndrome, affecting 5 to 7 % of the population worldwide and mainly characterised by elevated levels of glucose and is associated with two types of physiological event disturbances such as impaired insulin secretion and insulin resistance. Up to now, various treatment strategies are like insulin, alphaglucosidase inhibitors, biguanides, incretins were being followed. Concurrently, various novel therapeutic strategies are required to advance the therapy of Diabetes mellitus. For the last few decades, there has been an extensive research in understanding the metabolic pathways involved in Diabetes Mellitus at the cellular level and having the profound knowledge on cell-growth, cell-cycle, and apoptosis at a molecular level provides new targets for the treatment of Diabetes Mellitus. Receptor signalling has been involved in these mechanisms, to translate the information coming from outside. To understand the various receptors involved in these pathways, we must have a sound knowledge on receptors and ligands involved in it. This review mainly summarises the receptors and ligands which are involved the Diabetes Mellitus. Finally, researchers have to develop the alternative chemical moieties that retain their affinity to receptors and efficacy. Diabetes Mellitus being a metabolic disorder due to the glucose surfeit, demands the need for regular exercise along with dietary changes.

  14. Methods for Identifying Ligands that Target Nucleic Acid Molecules and Nucleic Acid Structural Motifs

    Science.gov (United States)

    Disney, Matthew D. (Inventor); Childs-Disney, Jessica L. (Inventor)

    2017-01-01

    Disclosed are methods for identifying a nucleic acid (e.g., RNA, DNA, etc.) motif which interacts with a ligand. The method includes providing a plurality of ligands immobilized on a support, wherein each particular ligand is immobilized at a discrete location on the support; contacting the plurality of immobilized ligands with a nucleic acid motif library under conditions effective for one or more members of the nucleic acid motif library to bind with the immobilized ligands; and identifying members of the nucleic acid motif library that are bound to a particular immobilized ligand. Also disclosed are methods for selecting, from a plurality of candidate ligands, one or more ligands that have increased likelihood of binding to a nucleic acid molecule comprising a particular nucleic acid motif, as well as methods for identifying a nucleic acid which interacts with a ligand.

  15. A new peptide ligand for targeting human carbonic anhydrase IX, identified through the phage display technology.

    Directory of Open Access Journals (Sweden)

    Vasileios Askoxylakis

    Full Text Available UNLABELLED: Carbonic anhydrase IX (CAIX is a transmembrane enzyme found to be overexpressed in various tumors and associated with tumor hypoxia. Ligands binding this target may be used to visualize hypoxia, tumor manifestation or treat tumors by endoradiotherapy. METHODS: Phage display was performed with a 12 amino acid phage display library by panning against a recombinant extracellular domain of human carbonic anhydrase IX. The identified peptide CaIX-P1 was chemically synthesized and tested in vitro on various cell lines and in vivo in Balb/c nu/nu mice carrying subcutaneously transplanted tumors. Binding, kinetic and competition studies were performed on the CAIX positive human renal cell carcinoma cell line SKRC 52, the CAIX negative human renal cell carcinoma cell line CaKi 2, the human colorectal carcinoma cell line HCT 116 and on human umbilical vein endothelial cells (HUVEC. Organ distribution studies were carried out in mice, carrying SKRC 52 tumors. RNA expression of CAIX in HCT 116 and HUVEC cells was investigated by quantitative real time PCR. RESULTS: In vitro binding experiments of (125I-labeled-CaIX-P1 revealed an increased uptake of the radioligand in the CAIX positive renal cell carcinoma cell line SKRC 52. Binding of the radioligand in the colorectal carcinoma cell line HCT 116 increased with increasing cell density and correlated with the mRNA expression of CAIX. Radioligand uptake was inhibited up to 90% by the unlabeled CaIX-P1 peptide, but not by the negative control peptide octreotide at the same concentration. No binding was demonstrated in CAIX negative CaKi 2 and HUVEC cells. Organ distribution studies revealed a higher accumulation in SKRC 52 tumors than in heart, spleen, liver, muscle, intestinum and brain, but a lower uptake compared to blood and kidney. CONCLUSIONS: These data indicate that CaIX-P1 is a promising candidate for the development of new ligands targeting human carbonic anhydrase IX.

  16. Multivalent display of quinic acid based ligands for targeting E-selectin expressing cells.

    Science.gov (United States)

    Shamay, Yosi; Paulin, Denise; Ashkenasy, Gonen; David, Ayelet

    2009-10-08

    The site-specific expression of molecular markers on endothelial cells of blood vessels during inflammatory response and angiogenesis provides an opportunity to target drugs and imaging molecules to the vascular endothelium of diseased tissues. This paper describes an innovative strategy for selective delivery of polymer conjugates to E- and P-selectin expressing cells using a series of quinic acid (Qa) based non-carbohydrate analogues of the natural ligand sialyl Lewis(x) (sLe(x)) as targeting moieties. We demonstrate that such analogues antagonize the adhesion of sLe(x) expressing HL-60 cells to both E- and P-selectin. Significantly, the apparent avidity of polymer conjugates carrying multiple Qa copies has increased by 3 orders of magnitude relative to their monomeric forms. Furthermore, we found that the major mechanism of copolymer entry and delivery into E-selectin expressing cells is endocytosis. These selectin-targetable copolymers provide the foundation to support controlled delivery of anticancer drugs and imaging agents to tumor vasculature for therapeutic and diagnostic applications.

  17. Multi-ligand nanoparticles for targeted drug delivery to the injured vascular wall

    Science.gov (United States)

    Kona, Soujanya

    Pathological conditions like coronary artery disease, acute myocardial infarction, stroke, and peripheral artery diseases as well as cardiovascular interventions used in the treatment of coronary artery diseases such as angioplasty and stenting damage/injure the blood vessel wall, leading to inflamed or activated endothelial cells that have been implicated in events leading to thrombosis, inflammation, and restenosis. Oral administration of anti-coagulant and anti-inflammatory drugs causes systemic toxicity, bleeding, patient incompliance, and inadequate amounts of drugs at the injured area. Though drug-eluting stents have shown therapeutic benefits, complications such as in-stent restenosis and late thrombosis still remain and are a cause for concern. Rapid growth in the field of nanotechnology and nanoscience in recent years has paved the way for new targeted and controlled drug delivery strategies. In this perspective, the development of biodegradable nanoparticles for targeted intracellular drug delivery to the inflamed endothelial cells may offer an improved avenue for treatment of cardiovascular diseases. The major objective of this research was to develop "novel multi-ligand nanoparticles," as drug carriers that can efficiently target and deliver therapeutic agents to the injured/inflamed vascular cells under dynamic flow conditions. Our approach mimics the natural binding ability of platelets to injured/activated endothelial cells through glycoprotein Ib (GPIb) bound to P-selectin expressed on inflamed endothelial cells and to the subendothelium through GPIb binding to von Willebrand factor (vWF) deposited onto the injured vascular wall. Our design also exploits the natural cell membrane translocation ability of the internalizing cell peptide - trans-activating transcriptor (TAT) to enhance the nanoparticle uptake by the targeted cells. Our hypothesis is that these multi-ligand nanoparticles would show an increased accumulation at the injury site since GPIb

  18. Optimized hydrophobic interactions and hydrogen bonding at the target-ligand interface leads the pathways of drug-designing.

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    Rohan Patil

    Full Text Available BACKGROUND: Weak intermolecular interactions such as hydrogen bonding and hydrophobic interactions are key players in stabilizing energetically-favored ligands, in an open conformational environment of protein structures. However, it is still poorly understood how the binding parameters associated with these interactions facilitate a drug-lead to recognize a specific target and improve drugs efficacy. To understand this, comprehensive analysis of hydrophobic interactions, hydrogen bonding and binding affinity have been analyzed at the interface of c-Src and c-Abl kinases and 4-amino substituted 1H-pyrazolo [3, 4-d] pyrimidine compounds. METHODOLOGY: In-silico docking studies were performed, using Discovery Studio software modules LigandFit, CDOCKER and ZDOCK, to investigate the role of ligand binding affinity at the hydrophobic pocket of c-Src and c-Abl kinase. Hydrophobic and hydrogen bonding interactions of docked molecules were compared using LigPlot program. Furthermore, 3D-QSAR and MFA calculations were scrutinized to quantify the role of weak interactions in binding affinity and drug efficacy. CONCLUSIONS: The in-silico method has enabled us to reveal that a multi-targeted small molecule binds with low affinity to its respective targets. But its binding affinity can be altered by integrating the conformationally favored functional groups at the active site of the ligand-target interface. Docking studies of 4-amino-substituted molecules at the bioactive cascade of the c-Src and c-Abl have concluded that 3D structural folding at the protein-ligand groove is also a hallmark for molecular recognition of multi-targeted compounds and for predicting their biological activity. The results presented here demonstrate that hydrogen bonding and optimized hydrophobic interactions both stabilize the ligands at the target site, and help alter binding affinity and drug efficacy.

  19. Near-infrared optical imaging in glioblastoma xenograft with ligand-targeting {alpha}3 integrin

    Energy Technology Data Exchange (ETDEWEB)

    Xiao, Wenwu; Yao, Nianhuan; Peng, Li; Liu, Ruiwu; Lam, Kit S. [University of California Davis, Division of Hematology and Oncology, Department of Internal Medicine, UC Davis Cancer Center, Sacramento, CA (United States)

    2009-01-15

    Patients with glioblastoma usually have a very poor prognosis. Even with a combination of radiotherapy plus temozolomide, the median survival of these patients is only 14.6 months. New treatment approaches to this cancer are needed. Our purpose is to develop new cell surface-binding ligands for glioblastoma cells and use them as targeted imaging and therapeutic agents for this deadly disease. One-bead one-compound combinatorial cyclic peptide libraries were screened with live human glioblastoma U-87MG cells. The binding affinity and targeting specificity of peptides identified were tested with in vitro experiments on cells and in vivo and ex vivo experiments on U-87MG xenograft mouse model. A cyclic peptide, LXY1, was identified and shown to be binding to the {alpha}3 integrin of U-87MG cells with moderately high affinity (K{sub d} = 0.5 {+-} 0.1 {mu}M) and high specificity. Biotinylated LXY1, when complexed with streptavidin-Cy5.5 (SA-Cy5.5) conjugate, targeted both subcutaneous and orthotopic U-87MG xenograft implants in nude mice. The in vivo targeting specificity was further verified by strong inhibition of tumor uptake of LXY1-biotin-SA-Cy5.5 complex when intravenously injecting the animals with anti-{alpha}3 integrin antibody or excess unlabeled LXY1 prior to administrating the imaging probe. The smaller univalent LXY1-Cy5.5 conjugate (2,279 Da) was found to have a faster accumulation in the U-87MG tumor and shorter retention time compared with the larger tetravalent LXY1-biotin-SA-Cy5.5 complex (approximately 64 kDa). Collectively, the data reveals that LXY1 has the potential to be developed into an effective imaging and therapeutic targeting agent for human glioblastoma. (orig.)

  20. A novel small peptide as an epidermal growth factor receptor targeting ligand for nanodelivery in vitro

    Directory of Open Access Journals (Sweden)

    Han CY

    2013-04-01

    Full Text Available Cui-yan Han,1,2 Li-ling Yue,2 Ling-yu Tai,1 Li Zhou,2 Xue-yan Li,2 Gui-hua Xing,2 Xing-gang Yang,1 Ming-shuang Sun,1 Wei-san Pan1 1School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, People’s Republic of China; 2Qiqihar Medical University, Qiqihar, People’s Republic of China Abstract: The epidermal growth factor receptor (EGFR serves an important function in the proliferation of tumors in humans and is an effective target for the treatment of cancer. In this paper, we studied the targeting characteristics of small peptides (AEYLR, EYINQ, and PDYQQD that were derived from three major autophosphorylation sites of the EGFR C-terminus domain in vitro. These small peptides were labeled with fluorescein isothiocyanate (FITC and used the peptide LARLLT as a positive control, which bound to putative EGFR selected from a virtual peptide library by computer-aided design, and the independent peptide RALEL as a negative control. Analyses with flow cytometry and an internalization assay using NCI-H1299 and K562 with high EGFR and no EGFR expression, respectively, indicated that FITC-AEYLR had high EGFR targeting activity. Biotin-AEYLR that was specifically bound to human EGFR proteins demonstrated a high affinity for human non-small-cell lung tumors. We found that AEYLR peptide-conjugated, nanostructured lipid carriers enhanced specific cellular uptake in vitro during a process that was apparently mediated by tumor cells with high-expression EGFR. Analysis of the MTT assay indicated that the AEYLR peptide did not significantly stimulate or inhibit the growth activity of the cells. These findings suggest that, when mediated by EGFR, AEYLR may be a potentially safe and efficient delivery ligand for targeted chemotherapy, radiotherapy, and gene therapy. Keywords: EGFR, small peptide, tumor targeting, lung cancer, NLC

  1. Combining on-chip synthesis of a focused combinatorial library with computational target prediction reveals imidazopyridine GPCR ligands.

    Science.gov (United States)

    Reutlinger, Michael; Rodrigues, Tiago; Schneider, Petra; Schneider, Gisbert

    2014-01-07

    Using the example of the Ugi three-component reaction we report a fast and efficient microfluidic-assisted entry into the imidazopyridine scaffold, where building block prioritization was coupled to a new computational method for predicting ligand-target associations. We identified an innovative GPCR-modulating combinatorial chemotype featuring ligand-efficient adenosine A1/2B and adrenergic α1A/B receptor antagonists. Our results suggest the tight integration of microfluidics-assisted synthesis with computer-based target prediction as a viable approach to rapidly generate bioactivity-focused combinatorial compound libraries with high success rates.

  2. Chemically modified peptide scaffolds target the CFTR-associated ligand PDZ domain.

    Directory of Open Access Journals (Sweden)

    Jeanine F Amacher

    Full Text Available PDZ domains are protein-protein interaction modules that coordinate multiple signaling and trafficking pathways in the cell and that include active therapeutic targets for diseases such as cancer, cystic fibrosis, and addiction. Our previous work characterized a PDZ interaction that restricts the apical membrane half-life of the cystic fibrosis transmembrane conductance regulator (CFTR. Using iterative cycles of peptide-array and solution-binding analysis, we targeted the PDZ domain of the CFTR-Associated Ligand (CAL, and showed that an engineered peptide inhibitor rescues cell-surface expression of the most common CFTR disease mutation ΔF508. Here, we present a series of scaffolds containing chemically modifiable side chains at all non-motif positions along the CAL PDZ domain binding cleft. Concordant equilibrium dissociation constants were determined in parallel by fluorescence polarization, isothermal titration calorimetry, and surface plasmon resonance techniques, confirming robust affinity for each scaffold and revealing an enthalpically driven mode of inhibitor binding. Structural studies demonstrate a conserved binding mode for each peptide, opening the possibility of combinatorial modification. Finally, we diversified one of our peptide scaffolds with halogenated substituents that yielded modest increases in binding affinity. Overall, this work validates our approach and provides a stereochemical foundation for further CAL inhibitor design and screening.

  3. Chemically modified peptide scaffolds target the CFTR-associated ligand PDZ domain.

    Science.gov (United States)

    Amacher, Jeanine F; Zhao, Ruizhi; Spaller, Mark R; Madden, Dean R

    2014-01-01

    PDZ domains are protein-protein interaction modules that coordinate multiple signaling and trafficking pathways in the cell and that include active therapeutic targets for diseases such as cancer, cystic fibrosis, and addiction. Our previous work characterized a PDZ interaction that restricts the apical membrane half-life of the cystic fibrosis transmembrane conductance regulator (CFTR). Using iterative cycles of peptide-array and solution-binding analysis, we targeted the PDZ domain of the CFTR-Associated Ligand (CAL), and showed that an engineered peptide inhibitor rescues cell-surface expression of the most common CFTR disease mutation ΔF508. Here, we present a series of scaffolds containing chemically modifiable side chains at all non-motif positions along the CAL PDZ domain binding cleft. Concordant equilibrium dissociation constants were determined in parallel by fluorescence polarization, isothermal titration calorimetry, and surface plasmon resonance techniques, confirming robust affinity for each scaffold and revealing an enthalpically driven mode of inhibitor binding. Structural studies demonstrate a conserved binding mode for each peptide, opening the possibility of combinatorial modification. Finally, we diversified one of our peptide scaffolds with halogenated substituents that yielded modest increases in binding affinity. Overall, this work validates our approach and provides a stereochemical foundation for further CAL inhibitor design and screening.

  4. Ligand efficiency-based support vector regression models for predicting bioactivities of ligands to drug target proteins.

    Science.gov (United States)

    Sugaya, Nobuyoshi

    2014-10-27

    The concept of ligand efficiency (LE) indices is widely accepted throughout the drug design community and is frequently used in a retrospective manner in the process of drug development. For example, LE indices are used to investigate LE optimization processes of already-approved drugs and to re-evaluate hit compounds obtained from structure-based virtual screening methods and/or high-throughput experimental assays. However, LE indices could also be applied in a prospective manner to explore drug candidates. Here, we describe the construction of machine learning-based regression models in which LE indices are adopted as an end point and show that LE-based regression models can outperform regression models based on pIC50 values. In addition to pIC50 values traditionally used in machine learning studies based on chemogenomics data, three representative LE indices (ligand lipophilicity efficiency (LLE), binding efficiency index (BEI), and surface efficiency index (SEI)) were adopted, then used to create four types of training data. We constructed regression models by applying a support vector regression (SVR) method to the training data. In cross-validation tests of the SVR models, the LE-based SVR models showed higher correlations between the observed and predicted values than the pIC50-based models. Application tests to new data displayed that, generally, the predictive performance of SVR models follows the order SEI > BEI > LLE > pIC50. Close examination of the distributions of the activity values (pIC50, LLE, BEI, and SEI) in the training and validation data implied that the performance order of the SVR models may be ascribed to the much higher diversity of the LE-based training and validation data. In the application tests, the LE-based SVR models can offer better predictive performance of compound-protein pairs with a wider range of ligand potencies than the pIC50-based models. This finding strongly suggests that LE-based SVR models are better than pIC50-based

  5. Targeting breast to brain metastatic tumours with death receptor ligand expressing therapeutic stem cells.

    Science.gov (United States)

    Bagci-Onder, Tugba; Du, Wanlu; Figueiredo, Jose-Luiz; Martinez-Quintanilla, Jordi; Shah, Khalid

    2015-06-01

    Characterizing clinically relevant brain metastasis models and assessing the therapeutic efficacy in such models are fundamental for the development of novel therapies for metastatic brain cancers. In this study, we have developed an in vivo imageable breast-to-brain metastasis mouse model. Using real time in vivo imaging and subsequent composite fluorescence imaging, we show a widespread distribution of micro- and macro-metastasis in different stages of metastatic progression. We also show extravasation of tumour cells and the close association of tumour cells with blood vessels in the brain thus mimicking the multi-foci metastases observed in the clinics. Next, we explored the ability of engineered adult stem cells to track metastatic deposits in this model and show that engineered stem cells either implanted or injected via circulation efficiently home to metastatic tumour deposits in the brain. Based on the recent findings that metastatic tumour cells adopt unique mechanisms of evading apoptosis to successfully colonize in the brain, we reasoned that TNF receptor superfamily member 10A/10B apoptosis-inducing ligand (TRAIL) based pro-apoptotic therapies that induce death receptor signalling within the metastatic tumour cells might be a favourable therapeutic approach. We engineered stem cells to express a tumour selective, potent and secretable variant of a TRAIL, S-TRAIL, and show that these cells significantly suppressed metastatic tumour growth and prolonged the survival of mice bearing metastatic breast tumours. Furthermore, the incorporation of pro-drug converting enzyme, herpes simplex virus thymidine kinase, into therapeutic S-TRAIL secreting stem cells allowed their eradication post-tumour treatment. These studies are the first of their kind that provide insight into targeting brain metastasis with stem-cell mediated delivery of pro-apoptotic ligands and have important clinical implications.

  6. Multi-target directed donepezil-like ligands for Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Mercedes eUnzeta

    2016-05-01

    Full Text Available Alzheimer's disease (AD, the most common form of adult onset dementia, is an age-related neurodegenerative disorder characterized by progressive memory loss, decline in language skills and other cognitive impairments. Although its etiology is not completely known, several factors including deficits of acetylcholine, β-amyloid deposits, τ-protein phosphorylation, oxidative stress and neuroinflammation are considered to play significant roles in the pathophysiology of this disease. For a long time, AD patients have been treated with acetylcholinesterase inhibitors such as donepezil (Aricept® but with limited therapeutic success. This might be due to the complex multifactorial nature of AD, a fact that has prompted the design of new Multi-Target-Directed Ligands (MTDL based on the one molecule, multiple targets paradigm. Thus, in this context, different series of novel multifunctional molecules with antioxidant, anti-amyloid, anti-inflammatory and metal-chelating properties able to interact with multiple enzymes of therapeutic interest in AD pathology including acetylcholinesterase, butyrylcholinesterase and monoamine oxidases A and B have been designed and assessed biologically. This review describes the multiple targets, the design rationale and an in-house MTDL library, bearing the N-benzylpiperidine motif present in donepezil, linked to different heterocyclic ring systems (indole, pyridine or 8-hydroxyquinoline with special emphasis on compound ASS234, an N-propargylindole derivative. The description of the in vitro biological properties of the compounds and discussion of the corresponding structure-activity-relationships allows us to highlight new issues for the identification of more efficient MTDL for use in AD therapy.

  7. Identification of a novel peptide ligand targeting visceral adipose tissue via transdermal route by in vivo phage display.

    Science.gov (United States)

    Lee, Nam Kyung; Kim, Hong Shin; Kim, Kyung Hyun; Kim, Eun-Bae; Cho, Chong Su; Kang, Sang Kee; Choi, Yun Jaie

    2011-11-01

    To find novel peptide ligands targeting visceral adipose tissue (visceral fat) via transdermal route, in vivo phage display screening was conducted by dermal administration of a phage-peptide library to rats and a peptide sequence, CGLHPAFQC (designated as TDA1), was identified as a targeting ligand to visceral adipose tissue through the consecutive transdermal biopannings. Adipocyte-specific affinity and transdermal activity of the TDA1 were validated in vitro and targeting ability of the dermally administered TDA1 to visceral adipose tissue was also confirmed in vivo. TDA1 was effectively translocated into systemic circulation after dermal administration and selectively targeted visceral adipose tissue without any preference to other organs tested. Fluorescent microscopic analysis revealed that the TDA1 could be specifically localized in the hair follicles of the skin, as well as in the visceral adipose tissue. Thus, we inferred that dermally administered TDA1 would first access systemic circulation via hair follicles as its transdermal route and then could target visceral fat effectively. The overall results suggest that the TDA1 peptide could be potentially applied as a homing moiety for delivery of anti-obesity therapeutics to visceral fat through the convenient transdermal pathway.

  8. Screening of Small Molecule Microarrays for Ligands Targeted to the Extracellular Epitopes of Living Cells

    Directory of Open Access Journals (Sweden)

    Jeong Heon Lee

    2015-02-01

    Full Text Available The screening of living cells using high-throughput microarrays is technically challenging. Great care must be taken in the chemical presentation of potential ligands and the number of collisions that cells make with them. To overcome these issues, we have developed a glass slide-based microarray system to discover small molecule ligands that preferentially bind to one cell type over another, including when the cells differ by only a single receptor. Chemical spots of 300 ± 10 µm in diameter are conjugated covalently to glass slides using an arraying robot, and novel near-infrared fluorophores with peak emission at 700 nm and 800 nm are used to label two different cell types. By carefully optimizing incubation conditions, including cell density, motion, kinetics, detection, etc. we demonstrate that cell-ligand binding occurs, and that the number of cells bound per chemical spot correlates with ligand affinity and specificity. This screening system lays the foundation for high-throughput discovery of novel ligands to the cell surface.

  9. Development of a novel microbubble-liposome complex conjugated with peptide ligands targeting IL4R on brain tumor cells.

    Science.gov (United States)

    Park, See-Hyoung; Yoon, Young Ii; Moon, Hyoungwon; Lee, Ga-Hyun; Lee, Byung-Heon; Yoon, Tae-Jong; Lee, Hak Jong

    2016-07-01

    Gas (SF6)-filled microbubbles (MBs) were prepared by emulsion and solvent-evaporation method. The prepared MBs were further conjugated with doxorubicin (Dox)-loaded nano-sized liposome and peptide ligands to interleukin-4 receptor (IL4R) for targeting brain tumor cells. The final MB-liposome (Dox)-IL4R targeting peptide ligand [MB-Lipo (Dox)-IL4RTP] had a spherical structure with the mean size of 1,500 nm. The MB-Lipo (Dox)‑IL4RTP exhibited cellular uptake in U87MG brain tumor cells (a brain tumor cell line expressing strongly IL4R) with frequency ultrasound energy suggesting that MB-Lipo (Dox)‑IL4RTP provided effective targeting ability for brain tumor cells. In addition, WST-1 assay results showed that MB-Lipo (Dox)‑IL4RTP inhibited the proliferation of U87MG cells IL4R‑dependently. This was confirmed by western blotting of γH2AX, phospho (Ser15)-p53, p53 and p21 which are signal transduction proteins involved in DNA damage response and cell cycle arrest. Taken together, these results indicate that MB-Lipo (Dox)-IL4RTP represents a promising ultrasonic contrast agent for tumor-targeting ultrasonic imaging.

  10. IFPTarget: A Customized Virtual Target Identification Method Based on Protein-Ligand Interaction Fingerprinting Analyses.

    Science.gov (United States)

    Li, Guo-Bo; Yu, Zhu-Jun; Liu, Sha; Huang, Lu-Yi; Yang, Ling-Ling; Lohans, Christopher T; Yang, Sheng-Yong

    2017-07-24

    Small-molecule target identification is an important and challenging task for chemical biology and drug discovery. Structure-based virtual target identification has been widely used, which infers and prioritizes potential protein targets for the molecule of interest (MOI) principally via a scoring function. However, current "universal" scoring functions may not always accurately identify targets to which the MOI binds from the retrieved target database, in part due to a lack of consideration of the important binding features for an individual target. Here, we present IFPTarget, a customized virtual target identification method, which uses an interaction fingerprinting (IFP) method for target-specific interaction analyses and a comprehensive index (Cvalue) for target ranking. Evaluation results indicate that the IFP method enables substantially improved binding pose prediction, and Cvalue has an excellent performance in target ranking for the test set. When applied to screen against our established target library that contains 11,863 protein structures covering 2842 unique targets, IFPTarget could retrieve known targets within the top-ranked list and identified new potential targets for chemically diverse drugs. IFPTarget prediction led to the identification of the metallo-β-lactamase VIM-2 as a target for quercetin as validated by enzymatic inhibition assays. This study provides a new in silico target identification tool and will aid future efforts to develop new target-customized methods for target identification.

  11. Epidermal growth factor receptor ligands as new extracellular targets for the metastasis-promoting S100A4 protein

    DEFF Research Database (Denmark)

    Klingelhöfer, Jörg; Møller, Henrik D.; Sumer, Eren U

    2009-01-01

    The function of S100A4, a member of the calcium-binding S100 protein family, has been associated with tumor invasion and metastasis. Although an essential pro-metastatic role of extracellular S100A4 in tumor progression has been demonstrated, the identification of the precise underlying mechanisms...... and protein partners (receptors) has remained elusive. To identify putative targets for extracellular S100A4, we screened a phage display peptide library using S100A4 as bait. We identified three independent peptide motifs with varying affinities for the S100A4 protein. Sequence analyses indicated...... that the most abundant peptide mimicked the F/YCC motif present in the epidermal growth factor domain of ErbB receptor ligands. S100A4 selectively interacted with a number of epidermal growth factor receptor (EGFR) ligands, demonstrating highest affinity for amphiregulin. Importantly, we found that S100A4...

  12. Ligand-Directed Functional Selectivity at the Mu Opioid Receptor Revealed by Label-Free Integrative Pharmacology On-Target

    Science.gov (United States)

    Morse, Megan; Tran, Elizabeth; Sun, Haiyan; Levenson, Robert; Fang, Ye

    2011-01-01

    Development of new opioid drugs that provide analgesia without producing dependence is important for pain treatment. Opioid agonist drugs exert their analgesia effects primarily by acting at the mu opioid receptor (MOR) sites. High-resolution differentiation of opioid ligands is crucial for the development of new lead drug candidates with better tolerance profiles. Here, we use a label-free integrative pharmacology on-target (iPOT) approach to characterize the functional selectivity of a library of known opioid ligands for the MOR. This approach is based on the ability to detect dynamic mass redistribution (DMR) arising from the activation of the MOR in living cells. DMR assays were performed in HEK-MOR cells with and without preconditioning with probe molecules using label-free resonant waveguide grating biosensors, wherein the probe molecules were used to modify the activity of specific signaling proteins downstream the MOR. DMR signals obtained were then translated into high resolution heat maps using similarity analysis based on a numerical matrix of DMR parameters. Our data indicate that the iPOT approach clearly differentiates functional selectivity for distinct MOR signaling pathways among different opioid ligands, thus opening new avenues to discover and quantify the functional selectivity of currently used and novel opioid receptor drugs. PMID:22003401

  13. Ligand-directed functional selectivity at the mu opioid receptor revealed by label-free integrative pharmacology on-target.

    Directory of Open Access Journals (Sweden)

    Megan Morse

    Full Text Available Development of new opioid drugs that provide analgesia without producing dependence is important for pain treatment. Opioid agonist drugs exert their analgesia effects primarily by acting at the mu opioid receptor (MOR sites. High-resolution differentiation of opioid ligands is crucial for the development of new lead drug candidates with better tolerance profiles. Here, we use a label-free integrative pharmacology on-target (iPOT approach to characterize the functional selectivity of a library of known opioid ligands for the MOR. This approach is based on the ability to detect dynamic mass redistribution (DMR arising from the activation of the MOR in living cells. DMR assays were performed in HEK-MOR cells with and without preconditioning with probe molecules using label-free resonant waveguide grating biosensors, wherein the probe molecules were used to modify the activity of specific signaling proteins downstream the MOR. DMR signals obtained were then translated into high resolution heat maps using similarity analysis based on a numerical matrix of DMR parameters. Our data indicate that the iPOT approach clearly differentiates functional selectivity for distinct MOR signaling pathways among different opioid ligands, thus opening new avenues to discover and quantify the functional selectivity of currently used and novel opioid receptor drugs.

  14. MHC-I Ligand Discovery Using Targeted Database Searches of Mass Spectrometry Data: Implications for T-Cell Immunotherapies

    DEFF Research Database (Denmark)

    Murphy, J. Patrick; Konda, Prathyusha; Kowalewski, Daniel J.

    2017-01-01

    we offer a solution to this problem whereby we developed a targeted database search approach and accompanying tool SpectMHC, that is based on a priori-predicted MHC-I peptides. We first validated the approach using MS data from two different allotype-specific immunoprecipitates for the C57BL/6 mouse......-I peptides, wherein MS spectra are compared against a reference proteome. Unfortunately, matching these spectra to reference proteome databases is hindered by inflated search spaces attributed to a lack of enzyme restriction in the searches, limiting the efficiency with which MHC ligands are discovered. Here...... background. We then developed allotype-specific HLA databases to search previously published MS data sets of human peripheral blood mononuclear cells (PBMCs). This targeted search strategy improved peptide identifications for both mouse and human ligandomes by greater than 2-fold and is superior...

  15. Magnetic ligand fishing as a targeting tool for HPLC-HRMS-SPE-NMR: α-glucosidase inhibitory ligands and alkylresorcinol glycosides from Eugenia catharinae

    DEFF Research Database (Denmark)

    Wubshet, Sileshi Gizachew; Brighente, Inês M. C.; Moaddel, Ruin

    2015-01-01

    A bioanalytical platform combining magnetic ligand fishing for α-glucosidase inhibition profiling and HPLC-HRMS-SPE-NMR for structural identification of α-glucosidase inhibitory ligands, both directly from crude plant extracts, is presented. Magnetic beads with N-terminus-coupled α-glucosidase we...

  16. The IUPHAR/BPS Guide to PHARMACOLOGY: an expert-driven knowledgebase of drug targets and their ligands

    Science.gov (United States)

    Pawson, Adam J.; Sharman, Joanna L.; Benson, Helen E.; Faccenda, Elena; Alexander, Stephen P.H.; Buneman, O. Peter; Davenport, Anthony P.; McGrath, John C.; Peters, John A.; Southan, Christopher; Spedding, Michael; Yu, Wenyuan; Harmar, Anthony J.

    2014-01-01

    The International Union of Basic and Clinical Pharmacology/British Pharmacological Society (IUPHAR/BPS) Guide to PHARMACOLOGY (http://www.guidetopharmacology.org) is a new open access resource providing pharmacological, chemical, genetic, functional and pathophysiological data on the targets of approved and experimental drugs. Created under the auspices of the IUPHAR and the BPS, the portal provides concise, peer-reviewed overviews of the key properties of a wide range of established and potential drug targets, with in-depth information for a subset of important targets. The resource is the result of curation and integration of data from the IUPHAR Database (IUPHAR-DB) and the published BPS ‘Guide to Receptors and Channels’ (GRAC) compendium. The data are derived from a global network of expert contributors, and the information is extensively linked to relevant databases, including ChEMBL, DrugBank, Ensembl, PubChem, UniProt and PubMed. Each of the ∼6000 small molecule and peptide ligands is annotated with manually curated 2D chemical structures or amino acid sequences, nomenclature and database links. Future expansion of the resource will complete the coverage of all the targets of currently approved drugs and future candidate targets, alongside educational resources to guide scientists and students in pharmacological principles and techniques. PMID:24234439

  17. Using consensus-shape clustering to identify promiscuous ligands and protein targets and to choose the right query for shape-based virtual screening.

    Science.gov (United States)

    Pérez-Nueno, Violeta I; Ritchie, David W

    2011-06-27

    Ligand-based shape matching approaches have become established as important and popular virtual screening (VS) techniques. However, despite their relative success, many authors have discussed how best to choose the initial query compounds and which of their conformations should be used. Furthermore, it is increasingly the case that pharmaceutical companies have multiple ligands for a given target and these may bind in different ways to the same pocket. Conversely, a given ligand can sometimes bind to multiple targets, and this is clearly of great importance when considering drug side-effects. We recently introduced the notion of spherical harmonic-based "consensus shapes" to help deal with these questions. Here, we apply a consensus shape clustering approach to the 40 protein-ligand targets in the DUD data set using PARASURF/PARAFIT. Results from clustering show that in some cases the ligands for a given target are split into two subgroups which could suggest they bind to different subsites of the same target. In other cases, our clustering approach sometimes groups together ligands from different targets, and this suggests that those ligands could bind to the same targets. Hence spherical harmonic-based clustering can rapidly give cross-docking information while avoiding the expense of performing all-against-all docking calculations. We also report on the effect of the query conformation on the performance of shape-based screening of the DUD data set and the potential gain in screening performance by using consensus shapes calculated in different ways. We provide details of our analysis of shape-based screening using both PARASURF/PARAFIT and ROCS, and we compare the results obtained with shape-based and conventional docking approaches using MSSH/SHEF and GOLD. The utility of each type of query is analyzed using commonly reported statistics such as enrichment factors (EF) and receiver-operator-characteristic (ROC) plots as well as other early performance metrics.

  18. Liposomal Tumor Targeting in Drug Delivery Utilizing MMP-2- and MMP-9-Binding Ligands

    Directory of Open Access Journals (Sweden)

    Oula Penate Medina

    2011-01-01

    Full Text Available Nanotechnology offers an alternative to conventional treatment options by enabling different drug delivery and controlled-release delivery strategies. Liposomes being especially biodegradable and in most cases essentially nontoxic offer a versatile platform for several different delivery approaches that can potentially enhance the delivery and targeting of therapies to tumors. Liposomes penetrate tumors spontaneously as a result of fenestrated blood vessels within tumors, leading to known enhanced permeability and subsequent drug retention effects. In addition, liposomes can be used to carry radioactive moieties, such as radiotracers, which can be bound at multiple locations within liposomes, making them attractive carriers for molecular imaging applications. Phage display is a technique that can deliver various high-affinity and selectivity peptides to different targets. In this study, gelatinase-binding peptides, found by phage display, were attached to liposomes by covalent peptide-PEG-PE anchor creating a targeted drug delivery vehicle. Gelatinases as extracellular targets for tumor targeting offer a viable alternative for tumor targeting. Our findings show that targeted drug delivery is more efficient than non-targeted drug delivery.

  19. Radiosynthesis of a new PSMA targeting ligand ([{sup 18}F]FPy-DUPA-Pep)

    Energy Technology Data Exchange (ETDEWEB)

    Malik, Noeen, E-mail: noeen.malik@uniklinik-ulm.d [Clinic for Nuclear Medicine, University Hospital, Ulm (Germany); Machulla, Hans-Juergen; Solbach, Christoph; Winter, Gordon; Reske, Sven N.; Zlatopolskiy, Boris [Clinic for Nuclear Medicine, University Hospital, Ulm (Germany)

    2011-07-15

    Due to the specificity of expression of PSMA (prostate specific membrane antigen) particularly in prostate cancer cells (e.g. LNCaP), numerous PSMA ligands have been synthesized until now. In the current study, we synthesized DUPA-Pep having 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA) linked via 8-aminooctanoic acid to two phenylalanine residues and chose 6-[{sup 18}F]fluoronicotinic acid 2,3,5,6-tetrafluorophenyl ester [{sup 18}F]FPy-TFP as a prosthetic group for coupling. [{sup 18}F]FPy-DUPA-Pep was obtained in a radiochemical yield of 48{+-}0.9% (decay uncorrected) within 50 min with a chemical purity of >98%.

  20. Identification of Target Ligands of CORYNE in Arabidopsis by Phage Display Library

    Institute of Scientific and Technical Information of China (English)

    Heng Zhao; Shuzhen Li; Jiping Sheng; Lin Shen; Yuhui Yang; Bin Yao

    2011-01-01

    CORYNE (CRN) plays important roles in stem cell division and differentiation of shoot apical meristem (SAM) in Arabidopsis thaliana. The cytoplasmic kinase domain of CRN has been cloned and expressed in Escherichia coil, and further purified by two consecutive steps of affinity chromatography. By using this purified CRN as a ligand, a 12-mer random-peptide library was used to determine the specific amino acid sequences binding with the recombinant CRN molecule. After four rounds of biopanning, positive phage clones were isolated and sequenced, and further tested by enzyme linked immunosorbent assay for their binding ability and specificity. Two positive clones that specifically bind to the intracellular protein kinase domain of CRN have been identified. Alignment of these peptides and the kinase-associated protein phosphatase (KAPP) shows high similarity, indicating that KAPP might interact with the cytoplasmic kinase domain of CRN and negatively regulate the CLV signal. Our current study would be helpful to better understand the CLV3 signal pathway.

  1. Bacterial ligands generated in a phagosome are targets of the cytosolic innate immune system.

    Directory of Open Access Journals (Sweden)

    Anat A Herskovits

    2007-03-01

    Full Text Available Macrophages are permissive hosts to intracellular pathogens, but upon activation become microbiocidal effectors of innate and cell-mediated immunity. How the fate of internalized microorganisms is monitored by macrophages, and how that information is integrated to stimulate specific immune responses is not understood. Activation of macrophages with interferon (IFN-gamma leads to rapid killing and degradation of Listeria monocytogenes in a phagosome, thus preventing escape of bacteria to the cytosol. Here, we show that activated macrophages induce a specific gene expression program to L. monocytogenes degraded in the phago-lysosome. In addition to activation of Toll-like receptor (TLR signaling pathways, degraded bacteria also activated a TLR-independent transcriptional response that was similar to the response induced by cytosolic L. monocytogenes. More specifically, degraded bacteria induced a TLR-independent IFN-beta response that was previously shown to be specific to cytosolic bacteria and not to intact bacteria localized to the phagosome. This response required the generation of bacterial ligands in the phago-lysosome and was largely dependent on nucleotide-binding oligomerization domain 2 (NOD2, a cytosolic receptor known to respond to bacterial peptidoglycan fragments. The NOD2-dependent response to degraded bacteria required the phagosomal membrane potential and the activity of lysosomal proteases. The NOD2-dependent IFN-beta production resulted from synergism with other cytosolic microbial sensors. This study supports the hypothesis that in activated macrophages, cytosolic innate immune receptors are activated by bacterial ligands generated in the phagosome and transported to the cytosol.

  2. Smart ligand: aptamer-mediated targeted delivery of chemotherapeutic drugs and siRNA for cancer therapy.

    Science.gov (United States)

    Li, Xin; Zhao, Qinghe; Qiu, Liyan

    2013-10-28

    Aptamers are a class of oligonucleotides that can specifically bind to different targets with high affinity. Since their discovery in 1980s, aptamers have attracted considerable interests in medical applications. So far, initial research using aptamers as delivery systems has produced exciting results. In this review, we summarize recent progress in aptamer-mediated chemotherapeutic drug and siRNA delivery systems in tumor treatment. With regard to chemotherapeutic drugs, the 2 main methods for targeted delivery using aptamers are as follows: aptamer-drug systems (in which aptamers directly deliver the drug both as a carrier and as a ligand) and aptamer-nanoparticles systems (in which nanoparticles function together with aptamers for targeted delivery of drugs). For delivery of siRNA, aptamers can be utilized by the following ways to facilitate targeting: (1) linked by a connector; (2) form a chimera; and (3) combined with nanoparticles. In co-delivery system, the advantages associated with the use of aptamers are beginning to become apparent also. Here, the challenges and new perspectives in the field of aptamer-mediated delivery have been discussed.

  3. Ligand peptide-grafted PEGylated liposomes using HER2 targeted peptide-lipid derivatives for targeted delivery in breast cancer cells: The effect of serine-glycine repeated peptides as a spacer.

    Science.gov (United States)

    Suga, Tadaharu; Fuchigami, Yuki; Hagimori, Masayori; Kawakami, Shigeru

    2017-02-22

    Ligand peptide-grafted PEGylated liposomes have been widely studied for targeted drug delivery systems. Because ligand peptides are commonly grafted using PEG as a spacer on the surface of PEGylated liposomes, the interaction between ligand peptides and their corresponding receptors can be interrupted by steric hindrance of the PEG layer. Therefore, we aimed to develop ligand peptide-lipid derivatives to enhance the targeting efficiency of ligand peptide-grafted PEGylated liposomes, and designed a new ligand peptide-lipid derivatives having serine-glycine repeats (SG)n as a spacer based on the peptide length calculated by PyMol (v0.99). We selected KCCYSL (KCC) as the ligand peptide for binding to human epidermal growth factor receptor-2 (HER2). We synthesized new KCC-(SG)n-lipid derivatives (n=3, 5, 7) and evaluated their cellular association in breast cancer cells. KCC-(SG)n/PEGylated liposomes dramatically increased cellular association on HER2-positive breast cancer cells. The results suggest that KCC can be grafted on the surface of KCC-(SG)n/PEGylated liposomes prepared from KCC-(SG)n-lipid derivatives (n=3, 5, 7). In summary, we succeeded in developing KCC-(SG)n-lipid derivatives for the preparation of ligand peptide-grafted PEGylated liposomes.

  4. Bivalent ligands that target μ opioid (MOP) and cannabinoid1 (CB1) receptors are potent analgesics devoid of tolerance.

    Science.gov (United States)

    Le Naour, Morgan; Akgün, Eyup; Yekkirala, Ajay; Lunzer, Mary M; Powers, Mike D; Kalyuzhny, Alexander E; Portoghese, Philip S

    2013-07-11

    Given that μ opioid (MOP) and canabinoid (CB1) receptors are colocalized in various regions of the central nervous system and have been reported to associate as heteromer (MOP-CB1) in cultured cells, the possibility of functional, endogenous MOP-CB1 in nociception and other pharmacologic effects has been raised. As a first step in investigating this possibility, we have synthesized a series of bivalent ligands 1-5 that contain both μ agonist and CB1 antagonist pharmacophores for use as tools to study the functional interaction between MOP and CB1 receptors in vivo. Immunofluorescent studies on HEK293 cells coexpressing both receptors suggested 5 (20-atom spacer) to be the only member of the series that bridges the protomers of the heteromer. Antinociceptive testing in mice revealed 5 to be the most potent member of the series. As neither a mixture of monovalent ligands 9 + 10 nor bivalents 2-5 produced tolerance in mice, MOR-CB1 apparently is not an important target for reducing tolerance.

  5. Synthesis and evaluation of ligand targeting the somatostatin receptor for drug delivery to tumor cell

    Energy Technology Data Exchange (ETDEWEB)

    Lee, So Young; Hong, Young Don; Jung, Sung Hee; Choi, Sun Ju [Radioisotope Research Division, Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2015-12-15

    Most of targeted therapies block the action of certain enzymes, proteins, or other molecules involved in the growth and spread of cancer cells to produce its cytotoxic effect. Either small molecule drugs or monoclonal antibodies are mostly used in targeted therapies. Unfortunately, targeted therapy has a certain degree of unwanted side effect like other cytotoxicity inducing chemotherapies. To overcome and to reduce unwanted side effects during a cancer therapy, recently radiopeptide therapies has got the worlds' attraction for the tumor targeting modalities due to its beneficial effect on less side effect compared to cytotoxic chemotherapies. Among radiopeptide therapies, {sup 177}Lu-DOTATATE is a major modality as an effective one invented so far in treating neuroendocrine tumor (NET) and it has been in clinical trials at least one decade. Although it does have rather effective therapeutic effect on NET, it has less effective in rather large solid tumor. There are many ways to improve or increase therapeutic effect of radiopeptide are a finding the potent small molecules to target the tumor site selectively, or a labeling with radioisotope of emitting high energy, or an improving its biological half-life by introducing different moieties to increase lipophilicity. Present study was focus to increase a biological halflife of radio somatostatin which will target the somatostatin receptor by altering the bifunctional chelator (BFCA) by introducing lipophilic moiety to the somatostatin, which would make the labeled peptide stay longer in the tumor site and thus it can intensify the therapeutic effect on tumor cell itself and around tissues.

  6. Ligand-specific targeting of microspheres to phagocytes by surface modification with poly(L-lysine)-grafted poly(ethylene glycol) conjugate.

    Science.gov (United States)

    Faraasen, Sofia; Vörös, János; Csúcs, Gábor; Textor, Marcus; Merkle, Hans P; Walter, Elke

    2003-02-01

    The purpose of this study was to demonstrate specific receptor-mediated targeting of phagocytes by functional surface coatings of microparticles, shielding from nonspecific phagocytosis and allowing ligand-specific interactions via molecular recognition. Coatings of the comb polymer poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) were investigated for potential to inhibit 1) nonspecific spreading of human blood-derived macrophages (MOs) and dendritic cells (DCs) on glass and 2) nonspecific phagocytosis of PLL-g-PEG-coated, carboxylated polystyrene (PS) or biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres. Coating was performed by adsorption of positively charged PLL-g-PEG on negatively charged microparticles or plasma-cleaned glass through electrostatic interaction. The feasibility of ligand-specific interactions was tested with a model ligand, RGD, conjugated to PEG chains of PLL-g-PEG to form PLL-g-PEG-RGD and compared with inactive ligand conjugate, PLL-g-PEG-RDG. Coatings with PLL-g-PEG largely impaired the adherence and spreading of MOs and DCs on glass. The repellent character of PLL-g-PEG coatings drastically reduced phagocytosis of coated PS and PLGA microparticles to 10% in presence of serum. With both MOs and DCs, we observed ligand-specific interactions with PLL-g-PEG-RGD coatings on glass and PS and PLGA microspheres. Ligand specificity was abolished when using inactive ligand conjugate PLL-g-PEG-RDG, whereas repellency of coating was maintained. Coatings of PLL-g-PEG-ligand conjugates provide a novel technology for ligand specific targeting of microspheres to MOs and DCs while reducing nonspecific phagocytosis.

  7. TGF-beta receptor 2 downregulation in tumour-associated stroma worsens prognosis and high-grade tumours show more tumour-associated macrophages and lower TGF-beta1 expression in colon carcinoma: a retrospective study

    Directory of Open Access Journals (Sweden)

    Papadopoulos Thomas

    2007-08-01

    Full Text Available Abstract Background Histological phenotype and clinical behaviour of malignant tumours are not only dependent on alterations in the epithelial cell compartment, but are affected by their interaction with inflammatory cells and tumour-associated stroma. Studies in animal models have shown influence of tumour-associated macrophages (TAM on histological grade of differentiation in colon carcinoma. Disruption of transforming growth factor beta (TGF-beta signalling in tumour cells is related to more aggressive clinical behaviour. Expression data of components of this pathway in tumour-associated stroma is limited. Methods Tissue micro arrays of 310 colon carcinomas from curatively resected patients in UICC stage II and III were established. In a first step we quantified amount of CD68 positive TAMs and expression of components of TGF-beta signalling (TGF-beta1, TGF-beta receptors type 1 and 2, Smad 3 and 4 in tumour and associated stroma. Further we analyzed correlation to histological and clinical parameters (histological grade of differentiation (low-grade (i.e. grade 1 and 2 vs. high-grade (i.e. grade 3 and 4, lymph node metastasis, distant metastasis, 5 year cancer related survival using Chi-square or Fisher's exact test, when appropriate, to compare frequencies, Kaplan-Meier method to calculate 5-year rates of distant metastases and cancer-related survival and log rank test to compare the rates of distant metastases and survival. To identify independent prognostic factors Cox regression analysis including lymph node status and grading was performed. Results High-grade tumours and those with lymph node metastases showed higher rates of TAMs and lower expression of TGF-beta1. Loss of nuclear Smad4 expression in tumor was associated with presence of lymph node metastasis, but no influence on prognosis could be demonstrated. Decrease of both TGF-beta receptors in tumour-associated stroma was associated with increased lymph node metastasis and

  8. Design, synthesis and preliminary bio-evaluation of glucose-cholesterol derivatives as ligands for brain targeting liposomes

    Institute of Scientific and Technical Information of China (English)

    Fan Lei; Wei Fan; Xian Kun Li; Shan Wang; Li Hai; Yong Wu

    2011-01-01

    A series of glucose-cholesterol derivatives 8a-8e as ligands for brain targeting liposomes were synthesized. The preparation of compound 6 involved temporary protection of glucose with chlorotrimethylsilicane and hexamethyldisilazane followed by selectively hydrolyzed. The known cholesteryl tosylate 1 were coupled to ethylene glycols to afford alcohol 2a-2e. Substitution and deprotection of alcohol 2a-2e furnished the acids 4a-4e, which was condensed with compound 6 to get compounds 7a-7e, and then was deprotected in tetrahydrofuran with TEA to obtain the title compounds. As a model drug, tegafur was entrapped by liposomes coupled with 8b, and preliminary in vivo evaluation shown 8b could enhance the ability of liposomes delivering tegafur across the blood brain barrier.

  9. Structural basis for small molecule targeting of the programmed death ligand 1 (PD-L1)

    NARCIS (Netherlands)

    Zak, Krzysztof M.; Grudnik, Przemyslaw; Guzik, Katarzyna; Zieba, Bartosz J.; Musielak, Bogdan; Dömling, Alexander; Dubin, Grzegorz; Holak, Tad A.

    2016-01-01

    Targeting the PD-1/PD-L1 immunologic checkpoint with monoclonal antibodies has provided unprecedented results in cancer treatment in the recent years. Development of chemical inhibitors for this pathway lags the antibody development because of insufficient structural information. The first

  10. Structural basis for small molecule targeting of the programmed death ligand 1 (PD-L1)

    NARCIS (Netherlands)

    Zak, Krzysztof M.; Grudnik, Przemyslaw; Guzik, Katarzyna; Zieba, Bartosz J.; Musielak, Bogdan; Dömling, Alexander; Dubin, Grzegorz; Holak, Tad A.

    2016-01-01

    Targeting the PD-1/PD-L1 immunologic checkpoint with monoclonal antibodies has provided unprecedented results in cancer treatment in the recent years. Development of chemical inhibitors for this pathway lags the antibody development because of insufficient structural information. The first nonpeptid

  11. Theranostic Value of Multimers: Lessons Learned from Trimerization of Neurotensin Receptor Ligands and Other Targeting Vectors

    Directory of Open Access Journals (Sweden)

    Simone Maschauer

    2017-03-01

    Full Text Available Neurotensin receptor 1 (NTS1 is overexpressed on a variety of cancer entities; for example, prostate cancer, ductal pancreatic adenocarcinoma, and breast cancer. Therefore, it represents an interesting target for the diagnosis of these cancers types by positron emission tomography (PET [...

  12. Peroxisome Proliferator Activated Receptor A Ligands as Anticancer Drugs Targeting Mitochondrial Metabolism

    OpenAIRE

    Grabacka, Maja; Pierzchalska, Malgorzata; Reiss, Krzysztof

    2013-01-01

    Tumor cells show metabolic features distinctive from normal tissues, with characteristically enhanced aerobic glycolysis, glutaminolysis and lipid synthesis. Peroxisome proliferator activated receptor α (PPAR α) is activated by nutrients (fatty acids and their derivatives) and influences these metabolic pathways acting antagonistically to oncogenic Akt and c-Myc. Therefore PPAR α can be regarded as a candidate target molecule in supplementary anticancer pharmacotherapy as well as dietary ther...

  13. Identification of novel RHPS4-derivative ligands with improved toxicological profiles and telomere-targeting activities.

    Science.gov (United States)

    Rizzo, Angela; Iachettini, Sara; Zizza, Pasquale; Cingolani, Chiara; Porru, Manuela; Artuso, Simona; Stevens, Malcolm; Hummersone, Marc; Biroccio, Annamaria; Salvati, Erica; Leonetti, Carlo

    2014-10-06

    The pentacyclic acridinium salt RHPS4 (3,11-difluoro-6,8,13-trimethyl-8H-quino [4,3,2-kl] acridinium methosulfate, compound 1) is one of the most interesting DNA G-quadruplex binding molecules due to its high efficacy in tumor cell growth inhibition both in in vitro models and in vivo against human tumor xenografts in combination with conventional chemotherapeutics. Despite compound 1 having desirable chemical and pharmaceutical properties, its potential as a therapeutic agent is compromised by off-target effects on cardiovascular physiology. In this paper we report a new series of structurally-related compounds which were developed in an attempt to minimize its off-target profile, but maintaining the same favorable chemical and pharmacological features of the lead compound. By performing a comparative analysis it was possible to identify which derivatives had the following properties: (i) to show a reduced capacity in respect to compound 1 to inhibit the hERG tail current tested in a patch clamp assay and/or to interact with the human recombinant β2 receptor; (ii) to maintain both a good G4-binding affinity and cancer cell selectivity; and (iii) to trigger DNA damage with specific telomere uncapping. These studies allowed us to identify a novel G4-stabilizing molecule, compound 8, being characterized by reduced off-target effects and potent telomere on-target properties compared to the prototypic compound 1. Moreover, compound 8 shares with compound 1 the same molecular mode of action and an anti-tumour activity specifically restricted to replicating cells, as evident with its particularly efficient activity in combination therapy with a topoisomerase I inhibitor. In conclusion, we have identified a new pentacyclic derivative 8 having suitable properties to be the focus of further investigations as a clinical candidate for cancer therapy.

  14. Rapid Discovery of Functional Small Molecule Ligands against Proteomic Targets through Library-Against-Library Screening.

    Science.gov (United States)

    Wu, Chun-Yi; Wang, Don-Hong; Wang, Xiaobing; Dixon, Seth M; Meng, Liping; Ahadi, Sara; Enter, Daniel H; Chen, Chao-Yu; Kato, Jason; Leon, Leonardo J; Ramirez, Laura M; Maeda, Yoshiko; Reis, Carolina F; Ribeiro, Brianna; Weems, Brittany; Kung, Hsing-Jien; Lam, Kit S

    2016-06-13

    Identifying "druggable" targets and their corresponding therapeutic agents are two fundamental challenges in drug discovery research. The one-bead-one-compound (OBOC) combinatorial library method has been developed to discover peptides or small molecules that bind to a specific target protein or elicit a specific cellular response. The phage display cDNA expression proteome library method has been employed to identify target proteins that interact with specific compounds. Here, we combined these two high-throughput approaches, efficiently interrogated approximately 10(13) possible molecular interactions, and identified 91 small molecule compound beads that interacted strongly with the phage library. Of 19 compounds resynthesized, 4 were cytotoxic against cancer cells; one of these compounds was found to interact with EIF5B and inhibit protein translation. As more binding pairs are confirmed and evaluated, the "library-against-library" screening approach and the resulting small molecule-protein domain interaction database may serve as a valuable tool for basic research and drug development.

  15. Properties of a general PK/PD model of antibody-ligand interactions for therapeutic antibodies that bind to soluble endogenous targets.

    Science.gov (United States)

    Davda, Jasmine P; Hansen, Ryan J

    2010-01-01

    Antibodies that target endogenous soluble ligands are an important class of biotherapeutic agents. While much focus has been placed on characterization of antibody pharmacokinetics, less emphasis has been given to characterization of antibody effects on their soluble targets. We describe here the properties of a generalized mechanism-based PK/PD model used to characterize the in vivo interaction of an antibody and an endogenous soluble ligand. The assumptions and properties of the model are explored, and situations are described when deviations from the basic assumptions may be necessary. This model is most useful for in vivo situations where both antibody and ligand levels are available following drug administration. For a given antibody exposure, the extent and duration of suppression of free ligand is impacted by the apparent affinity of the interaction, as well as by the rate of ligand turnover. The applicability of the general equilibrium model of in vivo antibody-ligand interaction is demonstrated with an anti-Aß antibody.

  16. Rational design of ligands targeting triplet repeating transcripts that cause RNA dominant disease: application to myotonic muscular dystrophy type 1 and spinocerebellar ataxia type 3.

    Science.gov (United States)

    Pushechnikov, Alexei; Lee, Melissa M; Childs-Disney, Jessica L; Sobczak, Krzysztof; French, Jonathan M; Thornton, Charles A; Disney, Matthew D

    2009-07-22

    Herein, we describe the design of high affinity ligands that bind expanded rCUG and rCAG repeat RNAs expressed in myotonic dystrophy type 1 (DM1) and spinocerebellar ataxia type 3. These ligands also inhibit, with nanomolar IC(50) values, the formation of RNA-protein complexes that are implicated in both disorders. The expanded rCUG and rCAG repeats form stable RNA hairpins with regularly repeating internal loops in the stem and have deleterious effects on cell function. The ligands that bind the repeats display a derivative of the bisbenzimidazole Hoechst 33258, which was identified by searching known RNA-ligand interactions for ligands that bind the internal loop displayed in these hairpins. A series of 13 modularly assembled ligands with defined valencies and distances between ligand modules was synthesized to target multiple motifs in these RNAs simultaneously. The most avid binder, a pentamer, binds the rCUG repeat hairpin with a K(d) of 13 nM. When compared to a series of related RNAs, the pentamer binds to rCUG repeats with 4.4- to >200-fold specificity. Furthermore, the affinity of binding to rCUG repeats shows incremental gains with increasing valency, while the background binding to genomic DNA is correspondingly reduced. Then, it was determined whether the modularly assembled ligands inhibit the recognition of RNA repeats by Muscleblind-like 1 (MBNL1) protein, the expanded-rCUG binding protein whose sequestration leads to splicing defects in DM1. Among several compounds with nanomolar IC(50) values, the most potent inhibitor is the pentamer, which also inhibits the formation of rCAG repeat-MBNL1 complexes. Comparison of the binding data for the designed synthetic ligands and MBNL1 to repeating RNAs shows that the synthetic ligand is 23-fold higher affinity and more specific to DM1 RNAs than MBNL1. Further studies show that the designed ligands are cell permeable to mouse myoblasts. Thus, cell permeable ligands that bind repetitive RNAs have been designed

  17. Atorvastatin-loaded micelles with bone-targeted ligand for the treatment of osteoporosis.

    Science.gov (United States)

    Xie, Yonghui; Tan, Xueying; Huang, Jian; Huang, Hongwei; Zou, Ping; Hu, Jingbo

    2017-11-01

    Osteoporosis is a common bone disorder where the declined bone mass is far more than normal physiological status and usually associated with enhanced fracture risk, reduced bone strength and even deteriorated quality of life. Recent studies showed that statins could exert beneficial effects on bones via promoting osteoblastic activity mediated by increased expression of bone morphogenetic protein 2 and also by suppressing osteoclast proliferation. In this study, we developed atorvastatin-loaded tetracycline-poly (ethylene glycol)-poly(lactic-co-glycolic acid) (TC-PEG-PLGA/ATO) micelles for the targeted treatment of osteoporosis. The TC-PEG-PLGA was synthesized under the action of coupling reagents and then ATO was encapsulated through solvent diffusion method with encapsulation efficiency and drug loading of 89.32 ± 2.48% and 8.20 ± 0.53%, respectively. The release of ATO from micelles could be maintained for more than 48 h in pH 7.4 PBS. Pharmacokinetic results further demonstrated that TC-PEG-PLGA micelles could effectively shield ATO leakage from micelles and prolong their circulation time. Benefiting from TC specifically binding to hydroxyapatite (HAp), TC-PEG-PLGA/ATO micelles exerted good bone-targeted ability, as demonstrated by in vitro HAp affinity assay and biodistribution. Pharmacodynamic studies showed that TC-PEG-PLGA/ATO micelles could effectively improve bone mineral density and bone mechanical strength in osteoporotic rats. These results suggest that TC-PEG-PLGA/ATO micelles hold significant promise for the targeted treatment of osteoporosis.

  18. The Research Progress of Ligand-targeted Particulate Nanomedicines against Tumor%配体靶向纳米药物抗肿瘤的研究进展

    Institute of Scientific and Technical Information of China (English)

    黄晓宇; 贾晓燕; 李蕾; 夏秀梅; 鲁斌

    2016-01-01

    There are 13 kinds of ligand-targeted particulate nanomedicines (PNMs) which have stepped into clinical tri-als so far, including delivery forms based on liposomes, polymers, retrovirus vectors and bacteria-derived cells and so on. This review emphasized the research progress about 13 kinds of ligand-targeted PNMs. Main contents included:(1) the preliminary clinical trials of ligand-targeted PNMs;(2) the application of ligand-targeted PNMs in malignant tumor;(3) the role of ligands in PNMs;(4) the acting positions of ligand-targeted PNMs in target tissue are not subject to ligands. This advantage will help for location, and then improve their specificity. In the end, we talked about the prospect development of ligand-targeted PNMs.%目前,已有13种靶向配体纳米微粒(PNMs)进入临床试验阶段,包括基于脂质体和基于聚合物以及逆转录病毒载体和细菌衍生型细胞等运载形式。本文主要从以下方面分别对此13种靶向配体纳米药物的研究进展进行综述:(1)配体靶向PNMs的前期临床试验;(2)配体靶向PNMs在恶性肿瘤治疗中的应用;(3)靶向配体对于靶向PNMs药物所起的作用;(4)PNMs作用于靶组织中的位置不会受到相关靶向配体的影响,有利于定位于靶细胞,提升其特异性。并就其靶向配体PNMs未来的发展方向进行展望。

  19. ‘Living’ PEGylation on gold nanoparticles to optimize cancer cell uptake by controlling targeting ligand and charge densities

    Science.gov (United States)

    Chen, Hongwei; Paholak, Hayley; Ito, Masayuki; Sansanaphongpricha, Kanokwan; Qian, Wei; Che, Yong; Sun, Duxin

    2013-09-01

    We report and demonstrate biomedical applications of a new technique—‘living’ PEGylation—that allows control of the density and composition of heterobifunctional PEG (HS-PEG-R; thiol-terminated poly(ethylene glycol)) on gold nanoparticles (AuNPs). We first establish ‘living’ PEGylation by incubating HS-PEG5000-COOH with AuNPs (˜20 nm) at increasing molar ratios from zero to 2000. This causes the hydrodynamic layer thickness to differentially increase up to 26 nm. The controlled, gradual increase in PEG-COOH density is revealed after centrifugation, based on the ability to re-suspend the pellet and increase the AuNP absorption. Using a fluorescamine-based assay we quantify differential HS-PEG5000-NH2 binding to AuNPs, revealing that it is highly efficient until AuNP saturation is reached. Furthermore, the zeta potential incrementally changes from -44.9 to +52.2 mV and becomes constant upon saturation. Using ‘living’ PEGylation we prepare AuNPs with different ratios of HS-PEG-RGD (RGD: Arg-Gly-Asp) and incubate them with U-87 MG (malignant glioblastoma) and non-target cells, demonstrating that targeting ligand density is critical to maximizing the efficiency of targeting of AuNPs to cancer cells. We also sequentially control the HS-PEG-R density to develop multifunctional nanoparticles, conjugating positively charged HS-PEG-NH2 at increasing ratios to AuNPs containing negatively charged HS-PEG-COOH to reduce uptake by macrophage cells. This ability to minimize non-specific binding/uptake by healthy cells could further improve targeted nanoparticle efficacy.

  20. Targeting P-selectin glycoprotein ligand-1/P-selectin interactions as a novel therapy for metabolic syndrome.

    Science.gov (United States)

    Patel, Madhukar S; Miranda-Nieves, David; Chen, Jiaxuan; Haller, Carolyn A; Chaikof, Elliot L

    2016-12-09

    Obesity-induced insulin resistance and metabolic syndrome continue to pose an important public health challenge worldwide as they significantly increase the risk of type 2 diabetes and atherosclerotic cardiovascular disease. Advances in the pathophysiologic understanding of this process has identified that chronic inflammation plays a pivotal role. In this regard, given that both animal models and human studies have demonstrated that the interaction of P-selectin glycoprotein ligand-1 (PSGL-1) with P-selectin is not only critical for normal immune response but also is upregulated in the setting of metabolic syndrome, PSGL-1/P-selectin interactions provide a novel target for preventing and treating resultant disease. Current approaches of interfering with PSGL-1/P-selectin interactions include targeted antibodies, recombinant immunoglobulins that competitively bind P-selectin, and synthetic molecular therapies. Experimental models as well as clinical trials assessing the role of these modalities in a variety of diseases have continued to contribute to the understanding of PSGL-1/P-selectin interactions and have demonstrated the difficulty in creating clinically relevant therapeutics. Most recently, however, computational simulations have further enhanced our understanding of the structural features of PSGL-1 and related glycomimetics, which are responsible for high-affinity selectin interactions. Leveraging these insights for the design of next generation agents has thus led to development of a promising synthetic method for generating PSGL-1 glycosulfopeptide mimetics for the treatment of metabolic syndrome.

  1. Targeting of RAGE-ligand signaling impairs breast cancer cell invasion and metastasis.

    Science.gov (United States)

    Kwak, T; Drews-Elger, K; Ergonul, A; Miller, P C; Braley, A; Hwang, G H; Zhao, D; Besser, A; Yamamoto, Y; Yamamoto, H; El-Ashry, D; Slingerland, J M; Lippman, M E; Hudson, B I

    2017-03-01

    The receptor for advanced glycation end products (RAGE) is highly expressed in various cancers and is correlated with poorer outcome in breast and other cancers. Here we tested the role of targeting RAGE by multiple approaches in the tumor and tumor microenvironment, to inhibit the metastatic process. We first tested how RAGE impacts tumor cell-intrinsic mechanisms using either RAGE overexpression or knockdown with short hairpin RNAs (shRNAs). RAGE ectopic overexpression in breast cancer cells increased MEK-EMT (MEK-epithelial-to-mesenchymal transition) signaling, transwell invasion and soft agar colony formation, and in vivo promoted lung metastasis independent of tumor growth. RAGE knockdown with multiple independent shRNAs in breast cancer cells led to decreased transwell invasion and soft agar colony formation, without affecting proliferation. In vivo, targeting RAGE shRNA knockdown in human and mouse breast cancer cells, decreased orthotopic tumor growth, reduced tumor angiogenesis and recruitment of inflammatory cells, and markedly decreased metastasis to the lung and liver in multiple xenograft and syngeneic mouse models. To test the non-tumor cell microenvironment role of RAGE, we performed syngeneic studies with orthotopically injected breast cancer cells in wild-type and RAGE-knockout C57BL6 mice. RAGE-knockout mice displayed striking impairment of tumor cell growth compared with wild-type mice, along with decreased mitogen-activated protein kinase signaling, tumor angiogenesis and inflammatory cell recruitment. To test the combined inhibition of RAGE in both tumor cell-intrinsic and non-tumor cells of the microenvironment, we performed in vivo treatment of xenografted tumors with FPS-ZM1 (1 mg/kg, two times per week). Compared with vehicle, FPS-ZM1 inhibited primary tumor growth, inhibited tumor angiogenesis and inflammatory cell recruitment and, most importantly, prevented metastasis to the lung and liver. These data demonstrate that RAGE drives tumor

  2. Neuropeptides and neuropeptide receptors: drug targets, and peptide and non-peptide ligands: a tribute to Prof. Dieter Seebach.

    Science.gov (United States)

    Hoyer, Daniel; Bartfai, Tamas

    2012-11-01

    The number of neuropeptides and their corresponding receptors has increased steadily over the last fourty years: initially, peptides were isolated from gut or brain (e.g., Substance P, somatostatin), then by targeted mining in specific regions (e.g., cortistatin, orexin in the brain), or by deorphanization of G-protein-coupled receptors (GPCRs; orexin, ghrelin receptors) and through the completion the Human Genome Project. Neuropeptides (and their receptors) have regionally restricted distributions in the central and peripheral nervous system. The neuropeptide signaling is somewhat more distinct spatially than signaling with classical, low-molecular-weight neurotransmitters that are more widely expressed, and, therefore, one assumes that drugs acting at neuropeptide receptors may have more selective pharmacological actions with possibly fewer side effects than drugs acting on glutamatergic, GABAergic, monoaminergic, or cholinergic systems. Neuropeptide receptors, which may have a few or multiple subtypes and splice variants, belong almost exclusively to the GPCR family also known as seven-transmembrane receptors (7TM), a favorite class of drug targets in the pharmaceutical industry. Most neuropeptides are co-stored and co-released with classic neurotransmitters, albeit often only at higher frequencies of stimulation or at bursting activity, thus restricting the neuropeptide signaling to specific circumstances, another reason to assume that neuropeptide drug mimics may have less side effects. Neuropeptides possess a wide spectrum of functions from neurohormone, neurotransmitter to growth factor, but also as key inflammatory mediators. Neuropeptides become 'active' when the nervous system is challenged, e.g., by stress, injury, drug abuse, or neuropsychiatric disorders with genetic, epigenetic, and/or environmental components. The unsuspected number of true neuropeptides and their cognate receptors provides opportunities to identify novel targets for the treatment of

  3. Nasal immunization with M cell-targeting ligand-conjugated ApxIIA toxin fragment induces protective immunity against Actinobacillus pleuropneumoniae infection in a murine model.

    Science.gov (United States)

    Park, Jisang; Seo, Ki-Weon; Kim, Sae-Hae; Lee, Ha-Yan; Kim, Bumseok; Lim, Chae Woong; Kim, Jin-Hee; Yoo, Han Sang; Jang, Yong-Suk

    2015-05-15

    Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia and severe economic loss in the swine industry has been caused by the infection. Therefore, the development of an effective vaccine against the bacteria is necessary. ApxII toxin, among several virulence factors expressed by the bacteria, is considered to be a promising vaccine candidate because ApxII toxin not only accompanies cytotoxic and hemolytic activities, but is also expressed in all 15 serotypes of bacteria except serotypes 10 and 14. In this study, we identified the peptide ligand capable of targeting the ligand-conjugated ApxIIA #5 fragment antigen to nasopharynx-associated lymphoid tissue. It was found that nasal immunization with ligand-conjugated ApxIIA #5 induced efficient mucosal and systemic immune responses measured at the levels of antigen-specific antibodies, cytokine-secreting cells after antigen exposure, and antigen-specific lymphocyte proliferation. More importantly, the nasal immunization induced protective immunity against nasal challenge infection of the bacteria, which was confirmed by histopathological studies and bacterial clearance after challenge infection. Collectively, we confirmed that the ligand capable of targeting the ligand-conjugated antigen to nasopharynx-associated lymphoid tissue can be used as an effective nasal vaccine adjuvant to induce protective immunity against A. pleuropneumoniae infection. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Peptide ligand and PEG-mediated long-circulating liposome targeted to FGFR overexpressing tumor in vivo

    Directory of Open Access Journals (Sweden)

    Cai L

    2012-08-01

    Full Text Available Lulu Cai,1,3,† Xianhuo Wang,4,† Wenwen Wang,1,† Neng Qiu,1 Jiaolin Wen,1 Xingmei Duan,1 Xia Li,1 Xiang Chen,1 Li Yang,1 Zhiyong Qian,1 Yuquan Wei,1 Lijuan Chen,1,2 1State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, 2State Key Laboratory Breeding Base of Systematic Research, Development and Utilization of Chinese Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, 3Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital, Sichuan Chengdu, China; 4Tianjin Medical University Cancer Institute and Hospital, Department of Lymphoma, Sino-US Center for Lymphoma and Leukemia, Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University, Tianjin, China†These authors equally contributed to this researchBackground and methods: Paclitaxel, a widely used antitumor agent, has limited clinical application due to its hydrophobicity and systemic toxicity. To achieve sustained and targeted delivery of paclitaxel to tumor sites, liposomes composed of egg phosphatidylcholine, cholesterol, and distearolyphosphatidyl ethanolamine-N-poly(ethylene glycol (PEG2000 were prepared by a lipid film method. In addition, the liposomes also contained truncated fibroblast growth factor fragment-PEG-cholesterol as a ligand targeting the tumor marker fibroblast growth factor receptor. Physicochemical characteristics, such as particle size, zeta potential, entrapment efficiency, and release profiles were investigated. Pharmacokinetics and biodistribution were evaluated in C57BL/6 J mice bearing B16 melanoma after intravenous injection of paclitaxel formulated in Cremophor EL (free paclitaxel, conventional liposomes (CL-PTX, or in targeted PEGylated liposomes (TL-PTX.Results: Compared with CL-PTX and free paclitaxel, TL-PTX prolonged the half-life of paclitaxel by 2.01-fold and 3.40-fold, respectively, in plasma and improved the AUC0→t values of paclitaxel by

  5. Enhancement of the priming efficacy of DNA vaccines encoding dendritic cell-targeted antigens by synergistic toll-like receptor ligands

    Directory of Open Access Journals (Sweden)

    Kornbluth Richard S

    2009-08-01

    Full Text Available Abstract Background Targeting of protein antigens to dendritic cells (DC via the DEC205 receptor enhances presentation of antigen-derived peptides on MHC-I and MHC-II molecules and, in the presence of costimulatory signals, antigen-specific immune responses. The immunogenicity and efficacy of DNA vaccination can also be enhanced by fusing the encoded antigen to single chain antibodies directed against DEC205. To further improve this strategy, we evaluated different toll-like receptor ligands (TLR and CD40 ligands (CD40L as adjuvants for DNA vaccines encoding a DEC205-single-chain antibody fused to the ovalbumin model antigen or HIV-1 Gag and assessed the priming efficacy of DNA in a DNA prime adenoviral vector boost immunization regimen. Results Mice were primed with the adjuvanted DEC-205 targeted DNA vaccines and boosted with adenoviral vectors encoding the same antigens. CD8+ T cell responses were determined after the adenoviral booster immunization, to determine how well the different DNA immunization regimens prime for the adenoviral boost. In the absence of adjuvants, targeting of DNA-encoded ovalbumin to DCs suppressed CD8+ T-cell responses after the adenoviral booster immunization. CD8+ T-cell responses to the DEC205 targeted DNA vaccines increased only slightly by adding either the TLR-9 ligand CpG, the TLR-3 ligand Poly I:C, or CD40 ligand expression plasmids. However, the combination of both TLR-ligands led to a strong enhancement of CD8+ T-cell responses compared to a non-targeted DNA vaccine. This finding was confirmed using HIV Gag as antigen. Conclusion Although DNA prime adenoviral vector boost immunizations belong to the strongest inducers of cytotoxic T cell responses in different animal models and humans, the CD8+ T cell responses can be further improved by targeting the DNA encoded antigen to DEC205 in the presence of synergistic TLR ligands CpG and Poly I:C.

  6. Toward the Rational Design of Galactosylated Glycoclusters That Target Pseudomonas aeruginosa Lectin A (LecA): Influence of Linker Arms That Lead to Low-Nanomolar Multivalent Ligands.

    Science.gov (United States)

    Wang, Shuai; Dupin, Lucie; Noël, Mathieu; Carroux, Cindy J; Renaud, Louis; Géhin, Thomas; Meyer, Albert; Souteyrand, Eliane; Vasseur, Jean-Jacques; Vergoten, Gérard; Chevolot, Yann; Morvan, François; Vidal, Sébastien

    2016-08-01

    Anti-infectious strategies against pathogen infections can be achieved through antiadhesive strategies by using multivalent ligands of bacterial virulence factors. LecA and LecB are lectins of Pseudomonas aeruginosa implicated in biofilm formation. A series of 27 LecA-targeting glycoclusters have been synthesized. Nine aromatic galactose aglycons were investigated with three different linker arms that connect the central mannopyranoside core. A low-nanomolar (Kd =19 nm, microarray) ligand with a tyrosine-based linker arm could be identified in a structure-activity relationship study. Molecular modeling of the glycoclusters bound to the lectin tetramer was also used to rationalize the binding properties observed.

  7. Synthesis and evaluation of multi-target-directed ligands against Alzheimer's disease based on the fusion of donepezil and ebselen.

    Science.gov (United States)

    Luo, Zonghua; Sheng, Jianfei; Sun, Yang; Lu, Chuanjun; Yan, Jun; Liu, Anqiu; Luo, Hai-Bin; Huang, Ling; Li, Xingshu

    2013-11-27

    A novel series of compounds obtained by fusing the cholinesterase inhibitor donepezil and the antioxidant ebselen were designed as multi-target-directed ligands against Alzheimer's disease. An in vitro assay showed that some of these molecules did not exhibit highly potent cholinesterase inhibitory activity but did have various other ebselen-related pharmacological effects. Among the molecules, compound 7d, one of the most potent acetylcholinesterase inhibitors (IC50 values of 0.042 μM for Electrophorus electricus acetylcholinesterase and 0.097 μM for human acetylcholinesterase), was found to be a strong butyrylcholinesterase inhibitor (IC50 = 1.586 μM), to possess rapid H2O2 and peroxynitrite scavenging activity and glutathione peroxidase-like activity (ν0 = 123.5 μM min(-1)), and to be a substrate of mammalian TrxR. A toxicity test in mice showed no acute toxicity at doses of up to 2000 mg/kg. According to an in vitro blood-brain barrier model, 7d is able to penetrate the central nervous system.

  8. Targeting essential Eimeria ninakohlyakimovae sporozoite ligands for caprine host endothelial cell invasion with a phage display peptide library.

    Science.gov (United States)

    Ruiz, A; Pérez, D; Muñoz, M C; Molina, J M; Taubert, A; Jacobs-Lorena, M; Vega-Rodríguez, J; López, A M; Hermosilla, C

    2015-11-01

    Eimeria ninakohlyakimovae is an important coccidian parasite of goats which causes severe diarrhoea in young animals. Specific molecules that mediate E. ninakohlyakimovae host interactions and molecular mechanisms involved in the pathogenesis are still unknown. Although strong circumstantial evidence indicates that E. ninakohlyakimovae sporozoite interactions with caprine endothelial host cells (ECs) are specific, hardly any information is available about the interacting molecules that confer host cell specificity. In this study, we describe a novel method to identify surface proteins of caprine umbilical vein endothelial cells (CUVEC) using a phage display library. After several panning rounds, we identified a number of peptides that specifically bind to the surface of CUVEC. Importantly, caprine endothelial cell peptide 2 (PCEC2) and PCEC5 selectively reduced the infection rate by E. ninakohlyakimovae sporozoites. These preliminary data give new insight for the molecular identification of ligands involved in the interaction between E. ninakohlyakimovae sporozoites and host ECs. Further studies using this phage approach might be useful to identify new potential target molecules for the development of anti-coccidial drugs or even new vaccine strategies.

  9. Carboxymethyl chitosan nanoparticles coupled with CD59-specific ligand peptide for targeted delivery of C-phycocyanin to HeLa cells.

    Science.gov (United States)

    Yang, Peng; Li, Bing; Yin, Qi-Feng; Wang, Yu-Juan

    2017-03-01

    The combination of nanotechnology and medicine will be the next generation of vehicles for targeted drug delivery. Carboxymethyl chitosan loaded with the anticancer drug C-phycocyanin and the CD59-specific ligand peptide for cancer cell targeting were used to create C-phycocyanin/carboxymethyl chitosan-CD59-specific ligand peptide nanoparticles using the ionic-gelation method. Optimal synthesis conditions, selected by response surface methodology, comprised the ratio carboxymethyl chitosan:C-phycocyanin = 3:1, and carboxymethyl chitosan and CaCl2 concentrations of 2.0 and 1.0 mg/mL, respectively. The resulting nanoparticles were spherical, with diameters of approximately 200 nm; the entrapment efficient was about 65%; and the drug loading was about 20%. The release of C-phycocyanin from C-phycocyanin/carboxymethyl chitosan nanoparticles was pH sensitive and had a sustainable effect in vitro. Guided by the CD59-specific ligand peptide, the nanoparticles efficiently targeted the surface of HeLa cells and had an obvious inhibitory effect on HeLa cell proliferation as determined by methyl thiazolyl tetrazolium assays. The nanoparticles were hemocompatible and induced apoptosis by upregulation of cleaved caspase-3 and cleaved polyADP-ribose polymerase proteins, and downregulation of Bcl-2 proteins. Our study provides a novel approach to the research and development of marine drugs, and support for targeted therapy using anticancer drugs.

  10. Response of SCP-2L domain of human MFE-2 to ligand removal: binding site closure and burial of peroxisomal targeting signal.

    Science.gov (United States)

    Lensink, M F; Haapalainen, A M; Hiltunen, J K; Glumoff, T; Juffer, A H

    2002-10-11

    In the study of the structure and function relationship of human MFE-2, we have investigated the dynamics of human MFE-2SCP-2L (hSCP-2L) and its response to ligand removal. A comparison was made with homologous rabbit SCP-2. Breathing and a closing motion are found, identifiable with an adjustment in size and a closing off of the binding pocket. Crucial residues for structural integrity have been identified. Particularly mobile areas of the protein are loop 1 that is connecting helices A and C in space, and helix D, next to the entrance of the pocket. In hSCP-2L, the binding pocket gets occupied by Phe93, which is making a tight hydrophobic contact with Trp36. In addition, it is found that the C-terminal peroxisomal targeting signal (PTS1) that is solvent exposed in the complexed structure becomes buried when no ligand is present. Moreover, an anti-correlation exists between burial of PTS1 and the size of the binding pocket. The results are in accordance with plant nsLTPs, where a similar accommodation of binding pocket size was found after ligand binding/removal. Furthermore, the calculations support the suggestion of a ligand-assisted targeting mechanism.

  11. Identification of mismatch repair gene mutations in young patients with colorectal cancer and in patients with multiple tumours associated with hereditary non-polyposis colorectal cancer

    NARCIS (Netherlands)

    Niessen, R C; Berends, M J W; Wu, Y; Sijmons, R H; Hollema, H; Ligtenberg, M J L; de Walle, H E K; de Vries, E G E; Karrenbeld, A; Buys, C H C M; van der Zee, A G J; Hofstra, R M W; Kleibeuker, J H

    2006-01-01

    Background: Patients with early-onset colorectal cancer (CRC) or those with multiple tumours associated with hereditary non-polyposis colorectal cancer (HNPCC) raise suspicion of the presence of germline DNA mismatch repair (MMR) gene mutations. Aim: To analyse the value of family history,

  12. Identification of mismatch repair gene mutations in young patients with colorectal cancer and in patients with multiple tumours associated with hereditary non-polyposis colorectal cancer.

    NARCIS (Netherlands)

    Niessen, R.C.; Berends, M.J.; Wu, Y.; Sijmons, R.H.; Hollema, H.; Ligtenberg, M.J.L.; Walle, H.E. de; Vries, E.G.F. de; Karrenbeld, A.; Buys, C.H.C.M.; Zee, A.G. van der; Hofstra, R.M.; Kleibeuker, J.H.

    2006-01-01

    BACKGROUND: Patients with early-onset colorectal cancer (CRC) or those with multiple tumours associated with hereditary non-polyposis colorectal cancer (HNPCC) raise suspicion of the presence of germline DNA mismatch repair (MMR) gene mutations. AIM: To analyse the value of family history,

  13. The PDGF-BB-SOX7 axis-modulated IL-33 in pericytes and stromal cells promotes metastasis through tumour-associated macrophages

    DEFF Research Database (Denmark)

    Yang, Yunlong; Andersson, Patrik; Hosaka, Kayoko

    2016-01-01

    Signalling molecules and pathways that mediate crosstalk between various tumour cellular compartments in cancer metastasis remain largely unknown. We report a mechanism of the interaction between perivascular cells and tumour-associated macrophages (TAMs) in promoting metastasis through the IL-33...

  14. Target and resistance-related proteins of recombinant mutant human tumor necrosis factor-related apoptosis-inducing ligand on myeloma cell lines

    OpenAIRE

    JIAN, YUAN; Chen, Yuling; GENG, CHUANYING; Liu, Nian; YANG, GUANGZHONG; Liu, Jinwei; Li, Xin; Deng, Haiteng; CHEN, WENMING

    2016-01-01

    Recombinant mutant human tumor necrosis factor-related apoptosis-inducing ligand (rmhTRAIL) has become a potential therapeutic drug for multiple myeloma (MM). However, the exact targets and resistance mechanisms of rmhTRAIL on MM cells remain to be elucidated. The present study aimed to investigate the target and resistance-related proteins of rmhTRAIL on myeloma cell lines. A TRAIL-sensitive myeloma cell line, RPMI 8226, and a TRAIL-resistance one, U266, were chosen and the differentially ex...

  15. Pan-HER - an antibody mixture targeting EGFR, HER2, and HER3 abrogates preformed and ligand-induced EGFR homo- and heterodimers

    DEFF Research Database (Denmark)

    Ellebaek, Sofie; Pedersen, Susanne Brix; Grandal, Michael

    2016-01-01

    Abs is development of acquired resistance through mechanisms such as alterated receptor dimerization patterns and dependencies. Pan-HER is a mixture of six mAbs simultaneously targeting epidermal growth factor receptor (EGFR), HER2, and HER3 with two mAbs against each receptor. Pan-HER has previously demonstrated....... The effect of Pan-HER on cell proliferation and HER-family receptor degradation was superior to treatment with single mAbs targeting either single receptor, and similar to targeting a single receptor with two non-overlapping antibodies. Furthermore, changes in EGFR-dimerization patterns after treatment......-HER and the EGFR-targeting mAb mixture also blocked EGF-binding and thereby ligand-induced changes in EGFR-dimerization levels. These results suggest that Pan-HER reduces the cellular capability to switch HER-dependency and dimerization pattern in response to treatment and thus hold promise for future clinical...

  16. An Algorithm to Identify Target-Selective Ligands – A Case Study of 5-HT7/5-HT1A Receptor Selectivity

    Science.gov (United States)

    Kurczab, Rafał; Canale, Vittorio; Zajdel, Paweł; Bojarski, Andrzej J.

    2016-01-01

    A computational procedure to search for selective ligands for structurally related protein targets was developed and verified for serotonergic 5-HT7/5-HT1A receptor ligands. Starting from a set of compounds with annotated activity at both targets (grouped into four classes according to their activity: selective toward each target, not-selective and not-selective but active) and with an additional set of decoys (prepared using DUD methodology), the SVM (Support Vector Machines) models were constructed using a selective subset as positive examples and four remaining classes as negative training examples. Based on these four component models, the consensus classifier was then constructed using a data fusion approach. The combination of two approaches of data representation (molecular fingerprints vs. structural interaction fingerprints), different training set sizes and selection of the best SVM component models for consensus model generation, were evaluated to determine the optimal settings for the developed algorithm. The results showed that consensus models with molecular fingerprints, a larger training set and the selection of component models based on MCC maximization provided the best predictive performance. PMID:27271158

  17. An Algorithm to Identify Target-Selective Ligands - A Case Study of 5-HT7/5-HT1A Receptor Selectivity.

    Directory of Open Access Journals (Sweden)

    Rafał Kurczab

    Full Text Available A computational procedure to search for selective ligands for structurally related protein targets was developed and verified for serotonergic 5-HT7/5-HT1A receptor ligands. Starting from a set of compounds with annotated activity at both targets (grouped into four classes according to their activity: selective toward each target, not-selective and not-selective but active and with an additional set of decoys (prepared using DUD methodology, the SVM (Support Vector Machines models were constructed using a selective subset as positive examples and four remaining classes as negative training examples. Based on these four component models, the consensus classifier was then constructed using a data fusion approach. The combination of two approaches of data representation (molecular fingerprints vs. structural interaction fingerprints, different training set sizes and selection of the best SVM component models for consensus model generation, were evaluated to determine the optimal settings for the developed algorithm. The results showed that consensus models with molecular fingerprints, a larger training set and the selection of component models based on MCC maximization provided the best predictive performance.

  18. Ultrasound Molecular Imaging of the Breast Cancer Neovasculature using Engineered Fibronectin Scaffold Ligands: A Novel Class of Targeted Contrast Ultrasound Agent.

    Science.gov (United States)

    Abou-Elkacem, Lotfi; Wilson, Katheryne E; Johnson, Sadie M; Chowdhury, Sayan M; Bachawal, Sunitha; Hackel, Benjamin J; Tian, Lu; Willmann, Jürgen K

    2016-01-01

    Molecularly-targeted microbubbles (MBs) are increasingly being recognized as promising contrast agents for oncological molecular imaging with ultrasound. With the detection and validation of new molecular imaging targets, novel binding ligands are needed that bind to molecular imaging targets with high affinity and specificity. In this study we assessed a novel class of potentially clinically translatable MBs using an engineered 10(th) type III domain of human-fibronectin (MB-FN3VEGFR2) scaffold-ligand to image VEGFR2 on the neovasculature of cancer. The in vitro binding of MB-FN3VEGFR2 to a soluble VEGFR2 was assessed by flow-cytometry (FACS) and binding to VEGFR2-expressing cells was assessed by flow-chamber cell attachment studies under flow shear stress conditions. In vivo binding of MB-FN3VEGFR2 was tested in a transgenic mouse model (FVB/N Tg(MMTV/PyMT634Mul) of breast cancer and control litter mates with normal mammary glands. In vitro FACS and flow-chamber cell attachment studies showed significantly (P<0.01) higher binding to VEGFR2 using MB-FN3VEGFR2 than control agents. In vivo ultrasound molecular imaging (USMI) studies using MB-FN3VEGFR2 demonstrated specific binding to VEGFR2 and was significantly higher (P<0.01) in breast cancer compared to normal breast tissue. Ex vivo immunofluorescence-analysis showed significantly (P<0.01) increased VEGFR2-expression in breast cancer compared to normal mammary tissue. Our results suggest that MBs coupled to FN3-scaffolds can be designed and used for USMI of breast cancer neoangiogenesis. Due to their small size, stability, solubility, the lack of glycosylation and disulfide bonds, FN3-scaffolds can be recombinantly produced with the advantage of generating small, high affinity ligands in a cost efficient way for USMI.

  19. Tumour-associated antigens in systemic sclerosis patients with interstitial lung disease: association with lung involvement and cancer risk.

    Science.gov (United States)

    De Luca, Giacomo; Bosello, Silvia L; Berardi, Giorgia; Rucco, Manuela; Canestrari, Giovanni; Correra, Miriam; Mirone, Luisa; Forni, Franca; Di Mario, Clara; Danza, Francesco M; Pirronti, Tommaso; Ferraccioli, Gianfranco

    2015-11-01

    To evaluate the serum levels of tumour-associated antigens (TAAs) in patients with SSc and interstitial lung disease (ILD) and to define whether their levels mirror the severity and the progression of lung damage. Data from 80 SSc patients with ILD were collected at baseline and after 2 years as well as from 40 SSc controls without ILD. The occurrence of any malignancy was recorded. At baseline, an increase of at least one TAA was present in 35 SSc patients with ILD compared with 6 SSc patients without ILD (P < 0.0001); this was associated with lower forced vital capacity (FVC) and higher interstitial and alveolar scores. Levels of carbohydrate antigen 15-3 and carcinoembryonic antigen inversely correlated with FVC and directly correlated with alveolar and interstitial scores and their levels were higher in patients who presented a progression of lung damage after 2 years. During 4 years of follow-up, a malignancy was detected in seven patients who already had an increase of at least one TAA. Values of TAAs increased over time in patients who developed cancer, while their trend remained stable in the others. At multivariate analysis, to have three or more TAAs emerged as a strong independent predictor of the development of malignancies [relative risk 24.1 (95% CI 1.8, 315.0), P = 0.02]. TAAs can be elevated in the sera of SSc patients and correlate with the degree of lung damage, suggesting a role as severity biomarkers. Close follow-up is necessary in SSc patients because of the increased cancer risk overall in patients with increased TAAs. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  20. The prognostic impact of tumour-associated macrophages and Reed-Sternberg cells in paediatric Hodgkin lymphoma.

    Science.gov (United States)

    Gupta, Sumit; Yeh, Stacy; Chami, Rose; Punnett, Angela; Chung, Catherine

    2013-10-01

    Tumour-associated macrophages (TAM) are associated with treatment failure in adults with Hodgkin lymphoma (HL). Equivalent data in paediatric HL are sparse. We aimed to determine the prognostic significance of TAM and Reed-Sternberg (RS) cells in paediatric HL. All children aged 0-18 with HL between 1980 and 2009 with available diagnostic biopsy material were identified. A treatment failure-enriched cohort was assembled. Demographic, disease and outcome data were abstracted. Tissue microarrays with duplicate cores were constructed from diagnostic biopsy material and stained with immunohistochemical markers for TAM (CD68, CD163) and RS (CD30). A high score was defined as >5% positive cells relative to overall cellularity in any core. The association of candidate variables with event-free survival (EFS) was determined using Cox proportional hazards. The final study cohort comprised 96 patients with a median age of 14 years (interquartile range 11-15). Agreement on scores between cores from the same biopsy revealed weighted kappas of 0.60, 0.68 and 0.73 for CD30, CD68 and CD163 respectively, indicating moderate tumour heterogeneity. In univariate analysis, a high CD30 score was significantly associated with treatment failure (hazard ratio (HR) 2.27; 95th confidence interval 1.01-5.11; p<0.05). High CD68 and CD163 scores were not associated with EFS. Unlike adult HL, a higher percentage of RS cells was associated with poor outcome, while a higher percentage of TAM was not. Adult HL findings may not extend to paediatric HL. Cooperative group trials of paediatric HL should prospectively determine the association of different components of the tumour microenvironment with outcome. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Ligand-binding mass spectrometry to study biotransformation of fusion protein drugs and guide immunoassay development: strategic approach and application to peptibodies targeting the thrombopoietin receptor.

    Science.gov (United States)

    Hall, Michael P; Gegg, Colin; Walker, Kenneth; Spahr, Christopher; Ortiz, Robert; Patel, Vimal; Yu, Steven; Zhang, Liana; Lu, Hsieng; DeSilva, Binodh; Lee, Jean W

    2010-12-01

    The knowledge of in vivo biotransformation (e.g., proteolysis) of protein therapeutic candidates reveals structural liabilities that impact stability. This information aids the development and confirmation of ligand-binding assays with the required specificity for bioactive moieties (including intact molecule and metabolites) for appropriate PK profiling. Furthermore, the information can be used for re-engineering of constructs to remove in vivo liabilities in order to design the most stable candidates. We have developed a strategic approach of ligand-binding mass spectrometry (LBMS) to study biotransformation of fusion proteins of peptides fused to human Fc ("peptibodies") using anti-human Fc immunoaffinity capture followed by tiered mass spectrometric interrogation. LBMS offers the combined power of selectivity of ligand capture with the specificity and detailed molecular-level information of mass spectrometry. In this paper, we demonstrate the preclinical application of LBMS to three peptibodies, AMG531 (romiplostim), AMG195(linear), and AMG195(loop), that target the thrombopoietin receptor. The data show that ligand capture offers excellent sample cleanup and concentration of intact peptibodies and metabolites for subsequent query by matrix-assisted laser desorption ionization time-of-flight mass spectrometry for identification of in vivo proteolytic points. Additional higher-resolution analysis by nanoscale liquid chromatography interfaced with electrospray ionization mass spectrometry is required for identification of heterogeneous metabolites. Five proteolytic points are accurately identified for AMG531 and two for AMG195(linear), while AMG195(loop) is the most stable construct in rats. We recommend the use of LBMS to assess biotransformation and in vivo stability during early preclinical phase development for all novel fusion proteins.

  2. Quinine binding by the cocaine-binding aptamer. Thermodynamic and hydrodynamic analysis of high-affinity binding of an off-target ligand.

    Science.gov (United States)

    Reinstein, Oren; Yoo, Mina; Han, Chris; Palmo, Tsering; Beckham, Simone A; Wilce, Matthew C J; Johnson, Philip E

    2013-12-03

    The cocaine-binding aptamer is unusual in that it tightly binds molecules other than the ligand it was selected for. Here, we study the interaction of the cocaine-binding aptamer with one of these off-target ligands, quinine. Isothermal titration calorimetry was used to quantify the quinine-binding affinity and thermodynamics of a set of sequence variants of the cocaine-binding aptamer. We find that the affinity of the cocaine-binding aptamer for quinine is 30-40 times stronger than it is for cocaine. Competitive-binding studies demonstrate that both quinine and cocaine bind at the same site on the aptamer. The ligand-induced structural-switching binding mechanism of an aptamer variant that contains three base pairs in stem 1 is retained with quinine as a ligand. The short stem 1 aptamer is unfolded or loosely folded in the free form and becomes folded when bound to quinine. This folding is confirmed by NMR spectroscopy and by the short stem 1 construct having a more negative change in heat capacity of quinine binding than is seen when stem 1 has six base pairs. Small-angle X-ray scattering (SAXS) studies of the free aptamer and both the quinine- and the cocaine-bound forms show that, for the long stem 1 aptamers, the three forms display similar hydrodynamic properties, and the ab initio shape reconstruction structures are very similar. For the short stem 1 aptamer there is a greater variation among the SAXS-derived ab initio shape reconstruction structures, consistent with the changes expected with its structural-switching binding mechanism.

  3. Pan-HER-An antibody mixture targeting EGFR, HER2 and HER3 abrogates preformed and ligand-induced EGFR homo- and heterodimers.

    Science.gov (United States)

    Ellebaek, Sofie; Brix, Susanne; Grandal, Michael; Lantto, Johan; Horak, Ivan D; Kragh, Michael; Poulsen, Thomas Tuxen

    2016-11-01

    The human epidermal growth factor receptor (HER)-family is involved in development of many epithelial cancers. Therefore, HER-family members constitute important targets for anti-cancer therapeutics such as monoclonal antibodies (mAbs). A limitation to the success of single HER-targeting mAbs is development of acquired resistance through mechanisms such as alterted receptor dimerization patterns and dependencies. Pan-HER is a mixture of six mAbs simultaneously targeting epidermal growth factor receptor (EGFR), HER2 and HER3 with two mAbs against each receptor. Pan-HER has previously demonstrated broader efficacy than targeting single or dual receptor combinations also in resistant settings. In light of this broad efficacy, we decided to investigate the effect of Pan-HER compared with single HER-targeting with single and dual mAbs on HER-family cross-talk and dimerization focusing on EGFR. The effect of Pan-HER on cell proliferation and HER-family receptor degradation was superior to treatment with single mAbs targeting either single receptor, and similar to targeting a single receptor with two non-overlapping antibodies. Furthermore, changes in EGFR-dimerization patterns after treatment with Pan-HER were investigated by in situ proximity ligation assay and co-immunoprecipitation, demonstrating that Pan-HER and the EGFR-targeting mAb mixture efficiently down-regulate basal EGFR homo- and heterodimerization in two tested cell lines, whereas single mAbs had limited effects. Pan-HER and the EGFR-targeting mAb mixture also blocked EGF-binding and thereby ligand-induced changes in EGFR-dimerization levels. These results suggest that Pan-HER reduces the cellular capability to switch HER-dependency and dimerization pattern in response to treatment and thus hold promise for future clinical development of Pan-HER in resistant settings.

  4. Templated Formation of Discrete RNA and DNA:RNA Hybrid G-Quadruplexes and Their Interactions with Targeting Ligands.

    Science.gov (United States)

    Bonnat, Laureen; Dejeu, Jérôme; Bonnet, Hugues; Génnaro, Béatrice; Jarjayes, Olivier; Thomas, Fabrice; Lavergne, Thomas; Defrancq, Eric

    2016-02-24

    G-rich RNA and DNA oligonucleotides derived from the human telomeric sequence were assembled onto addressable cyclopeptide platforms through oxime ligations and copper-catalyzed azide-alkyne cycloaddition (CuAAc) reactions. The resulting conjugates were able to fold into highly stable RNA and DNA:RNA hybrid G-quadruplex (G4) architectures as demonstrated by UV, circular dichroism (CD), and NMR spectroscopic analysis. Whereas rationally designed parallel RNA and DNA:RNA hybrid G4 topologies could be obtained, we could not force the formation of an antiparallel RNA G4 structure, thus supporting the idea that this topology is strongly disfavored. The binding affinities of four representative G4 ligands toward the discrete RNA and DNA:RNA hybrid G4 topologies were compared to the one obtained with the corresponding DNA G4 structure. Surface plasmon resonance (SPR) binding analysis suggests that the accessibility to G4 recognition elements is different among the three structures and supports the idea that G4 ligands might be shaped to achieve structure selectivity in a biological context.

  5. Transcriptional control of Flt3 ligand targeted by fluorouracil-induced Egr-1 promoter in hematopoietic damage

    Directory of Open Access Journals (Sweden)

    Fu Yan

    2009-09-01

    Full Text Available Abstract Background Ionizing radiation (IR activate the early growth response-1 (Egr-1 promoter by production of radical oxygen intermediates (ROIs. Egr-EF, an expression vector pCIneo containing Egr-1 promoter cloned upstream of the cDNA for Flt3 ligand, was used to treat hematopoietic damage. 5-fluorouracil, a commonly used chemotherapeutic agent, cause tumor cell death by producing DNA damage and generating ROIs. We therefore hypothesized that clinically employed chemotherapeutic agents that increase ROIs could also be employed to activate Egr-EF in a chemoinducible gene therapy strategy. The goal of this study was to explore the effect of Flt3 Ligand gene transcription regulated by fluorouracil-induced Egr-1 promoter on hematopoietic recovery. Methods Human Flt3 Ligand (FL cDNA and enhanced green fluorescent protein (EGFP cDNA were linked together with IRES and inserted into the expression vector pCI-neo under control of the Egr-1 promoter (Egr-EF. The vector was transfected into the HFCL human bone marrow stromal cell line, and these cells were exposed to 5-FU, a chemotherapeutic drug. Expression of FL by HFCL/EF cells after 5-FU treatment was determined with ELISA, western blot and RT-PCR assays. In addition, the effect of FL from HFCL/EF cell culture supernatants on growth of CD34+ cells from cord blood was also studied. HFCL/EF cells were injected into CB-17 combined immunodeficient (SCID mice with B16 melanoma. 5-FU was given three days after injection of the HFCL/EF cells. In the recipient mice, white blood cell levels in peripheral blood and expression of EGFP and FL in human stromal cells were measured. Tumor volumes in tumor-bearing mice were also measured. Results 5-FU treatment increased EGFP levels and secreted FL levels in HFCL/EF cells. Supernatants from HFCL/EF cell cultures treated with 5-FU increased CD34+ cell growth significantly. HFCL/EF exhibited an increase in the number of white blood cells after chemotherapy

  6. Targeted Cancer Therapy Systems: An In Silico Study of Radiohalogenated Ligands in the Estrogen Receptor and the Synthesis of a Molecular Toolkit for the Fabrication of Customizable Nanoparticles

    Science.gov (United States)

    Barnsley, Kelton K.

    Chemotherapy is often limited by off-target toxicity and the development of multi-drug resistance in response to treatment. Strategies which reduce off-target toxicity by passively or actively targeting cancer cells may improve the efficacy of chemotherapy. Herein, two projects relating to targeted therapy are described. In the first project, the binding modes of 1,1-bis(4-hydroxyphenyl)-2-phenylethylenes (THPEs), a class of synthetic estrogens previously developed by our group, in the human estrogen receptor alpha-ligand binding domain were studied using molecular modeling programs YASARA AutoDock and Schrodinger Glide. The results were internally consistent and supported the observation that a bromine or iodine atom at the 2-position of the THPEs contributes positively to their binding in the estrogen receptor. In the second project, a "molecular toolkit" approach to the synthesis of multifunctional nanoparticles was envisioned. Our hypothesis was that the physical and chemical properties of the final product could be defined by controlling the types and relative amounts of prefunctionalized polymer units (PPUs) as well as the emulsification conditions. The design and syntheses of heterobifunctional linkers and other components for a preliminary molecular toolkit are reported, and the literature on select heterobifunctional aliphatic linkers is examined.

  7. MHC-I Ligand Discovery Using Targeted Database Searches of Mass Spectrometry Data: Implications for T-Cell Immunotherapies

    DEFF Research Database (Denmark)

    Murphy, J. Patrick; Konda, Prathyusha; Kowalewski, Daniel J.

    2017-01-01

    we offer a solution to this problem whereby we developed a targeted database search approach and accompanying tool SpectMHC, that is based on a priori-predicted MHC-I peptides. We first validated the approach using MS data from two different allotype-specific immunoprecipitates for the C57BL/6 mouse...... background. We then developed allotype-specific HLA databases to search previously published MS data sets of human peripheral blood mononuclear cells (PBMCs). This targeted search strategy improved peptide identifications for both mouse and human ligandomes by greater than 2-fold and is superior...... to traditional “no enzyme” searches of reference proteomes. Our targeted database search promises to uncover otherwise missed novel T-cell epitopes of therapeutic potential....

  8. Smuggling Drugs into the Brain: An Overview of Ligands Targeting Transcytosis for Drug Delivery across the Blood-Brain Barrier

    NARCIS (Netherlands)

    Zuhorn, Inge; Georgieva, Julia V.; Hoekstra, Dick

    2015-01-01

    The blood-brain barrier acts as a physical barrier that prevents free entry of blood-derived substances, including those intended for therapeutic applications. The development of molecular Trojan horses is a promising drug targeting technology that allows for non-invasive delivery of therapeutics in

  9. Synthesis of liver-targeting dual-ligand modified GCGA/5-FU nanoparticles and their characteristics in vitro and in vivo.

    Science.gov (United States)

    Cheng, Mingrong; Gao, Xiaoyan; Wang, Yong; Chen, Houxiang; He, Bing; Li, Yingchun; Han, Jiang; Zhang, Zhiping

    2013-01-01

    Nanoparticle drug delivery systems using polymers hold promise for clinical applications. We synthesized dual-ligand modified chitosan (GCGA) nanoparticles using lactic acid, glycyrrhetinic acid, and chitosan to target the liver in our previous studies. We then synthesized the GCGA/5-FU nanoparticles by conjugating 5-fluorouracil (5-FU) onto the GCGA nanomaterial, which had a mean particle size of 239.9 nm, a polydispersity index of 0.040, a zeta potential of +21.2 mV, and a drug loading of 3.90%. GCGA/5-FU nanoparticles had good slow release properties, and the release process could be divided into five phases: small burst release, gentle release, second burst release, steady release, and slow release. Inhibitory effects of GCGA/5-FU on tumor cells targeted the liver, and were time and dose dependent. GCGA nanoparticles significantly prolonged the efficacy of 5-FU on tumor cells, and alleviated the resistance of tumor cells to 5-FU. GCGA/5-FU nanoparticles were mostly concentrated in the liver, indicating that the GCGA nanoparticles were liver targeting. GCGA/5-FU nanoparticles significantly suppressed tumor growth in orthotopic liver transplantation mouse model, and improved mouse survival.

  10. A Fusion Protein between Streptavidin and the Endogenous TLR4 Ligand EDA Targets Biotinylated Antigens to Dendritic Cells and Induces T Cell Responses In Vivo

    Directory of Open Access Journals (Sweden)

    Laura Arribillaga

    2013-01-01

    Full Text Available The development of tools for efficient targeting of antigens to antigen presenting cells is of great importance for vaccine development. We have previously shown that fusion proteins containing antigens fused to the extra domain A from fibronectin (EDA, an endogenous TLR4 ligand, which targets antigens to TLR4-expressing dendritic cells (DC, are highly immunogenic. To facilitate the procedure of joining EDA to any antigen of choice, we have prepared the fusion protein EDAvidin by linking EDA to the N terminus of streptavidin, allowing its conjugation with biotinylated antigens. We found that EDAvidin, as streptavidin, forms tetramers and binds biotin or biotinylated proteins with a Kd ~ 2.6 × 10−14 mol/L. EDAvidin favours the uptake of biotinylated green fluorescent protein by DC. Moreover, EDAvidin retains the proinflammatory properties of EDA, inducing NF-κβ by TLR4-expressing cells, as well as the production of TNF-α by the human monocyte cell line THP1 and IL-12 by DC. More importantly, immunization of mice with EDAvidin conjugated with the biotinylated nonstructural NS3 protein from hepatitis C virus induces a strong anti-NS3 T cell immune response. These results open a new way to use the EDA-based delivery tool to target any antigen of choice to DC for vaccination against infectious diseases and cancer.

  11. Ligand-conjugated mesoporous silica nanorattles based on enzyme targeted prodrug delivery system for effective lung cancer therapy

    Energy Technology Data Exchange (ETDEWEB)

    Sundarraj, Shenbagamoorthy, E-mail: sundarrajbu09@gmail.com [Proteomics and Molecular Cell Physiology Laboratory, Department of Zoology, Bharathiar University, Coimbatore 641 046, TN (India); Thangam, Ramar [Proteomics and Molecular Cell Physiology Laboratory, Department of Zoology, Bharathiar University, Coimbatore 641 046, TN (India); Department of Virology, King Institute of Preventive Medicine and Research, Guindy, Chennai 600 032, TN (India); Sujitha, Mohanan V.; Vimala, Karuppaiya [Proteomics and Molecular Cell Physiology Laboratory, Department of Zoology, Bharathiar University, Coimbatore 641 046, TN (India); Kannan, Soundarapandian, E-mail: skperiyaruniv@gmail.com [Proteomics and Molecular Cell Physiology Laboratory, Department of Zoology, Bharathiar University, Coimbatore 641 046, TN (India); Department of Zoology, Periyar University, Salem 636 011, TN (India)

    2014-03-15

    Epidermal growth factor receptor antibody (EGFRAb) conjugated silica nanorattles (SNs) were synthesized and used to develop receptor mediated endocytosis for targeted drug delivery strategies for cancer therapy. The present study determined that the rate of internalization of silica nanorattles was found to be high in lung cancer cells when compared with the normal lung cells. EGFRAb can specifically bind to EGFR, a receptor that is highly expressed in lung cancer cells, but is expressed at low levels in other normal cells. Furthermore, in vitro studies clearly substantiated that the cPLA{sub 2}α activity, arachidonic acid release and cell proliferation were considerably reduced by pyrrolidine-2 loaded EGFRAb-SN in H460 cells. The cytotoxicity, cell cycle arrest and apoptosis were significantly induced by the treatment of pyrrolidine-2 loaded EGFRAb-SN when compared with free pyrrolidine-2 and pyrrolidine-2 loaded SNs in human non-small cell lung cancer cells. An in vivo toxicity assessment showed that silica nanorattles and EGFRAb-SN-pyrrolidine-2 exhibited low systemic toxicity in healthy Balb/c mice. The EGFRAb-SN-pyrrolidine-2 showed a much better antitumor activity (38%) with enhanced tumor inhibition rate than the pyrrolidine-2 on the non-small cell lung carcinoma subcutaneous model. Thus, the present findings validated the low toxicity and high therapeutic potentials of EGFRAb-SN-pyrrolidine-2, which may provide a convincing evidence of the silica nanorattles as new potential carriers for targeted drug delivery systems. - Highlights: • EGFRAb-SN developed for receptor-mediated Drug delivery system (DDS). • EGFRAb-SN-pyrrolidine-2 targeted DDS for cPLA2α inhibition in NSLC. • Study indicates EGFRAb-SN-pyrrolidine-2 as an efficient in target dug delivery carrier. • Study explains entire efficiency of EGFRAb-SN-pyrrolidine-2 in vitro and in vivo models.

  12. The Plasmodium falciparum erythrocyte invasion ligand Pfrh4 as a target of functional and protective human antibodies against malaria.

    Directory of Open Access Journals (Sweden)

    Linda Reiling

    Full Text Available BACKGROUND: Acquired antibodies are important in human immunity to malaria, but key targets remain largely unknown. Plasmodium falciparum reticulocyte-binding-homologue-4 (PfRh4 is important for invasion of human erythrocytes and may therefore be a target of protective immunity. METHODS: IgG and IgG subclass-specific responses against different regions of PfRh4 were determined in a longitudinal cohort of 206 children in Papua New Guinea (PNG. Human PfRh4 antibodies were tested for functional invasion-inhibitory activity, and expression of PfRh4 by P. falciparum isolates and sequence polymorphisms were determined. RESULTS: Antibodies to PfRh4 were acquired by children exposed to P. falciparum malaria, were predominantly comprised of IgG1 and IgG3 subclasses, and were associated with increasing age and active parasitemia. High levels of antibodies, particularly IgG3, were strongly predictive of protection against clinical malaria and high-density parasitemia. Human affinity-purified antibodies to the binding region of PfRh4 effectively inhibited erythrocyte invasion by P. falciparum merozoites and antibody levels in protected children were at functionally-active concentrations. Although expression of PfRh4 can vary, PfRh4 protein was expressed by most isolates derived from the cohort and showed limited sequence polymorphism. CONCLUSIONS: Evidence suggests that PfRh4 is a target of antibodies that contribute to protective immunity to malaria by inhibiting erythrocyte invasion and preventing high density parasitemia. These findings advance our understanding of the targets and mechanisms of human immunity and evaluating the potential of PfRh4 as a component of candidate malaria vaccines.

  13. Comprehensive optimization of a single-chain variable domain antibody fragment as a targeting ligand for a cytotoxic nanoparticle.

    Science.gov (United States)

    Zhang, Kathy; Geddie, Melissa L; Kohli, Neeraj; Kornaga, Tad; Kirpotin, Dmitri B; Jiao, Yang; Rennard, Rachel; Drummond, Daryl C; Nielsen, Ulrik B; Xu, Lihui; Lugovskoy, Alexey A

    2015-01-01

    Antibody-targeted nanoparticles have the potential to significantly increase the therapeutic index of cytotoxic anti-cancer therapies by directing them to tumor cells. Using antibodies or their fragments requires careful engineering because multiple parameters, including affinity, internalization rate and stability, all need to be optimized. Here, we present a case study of the iterative engineering of a single chain variable fragment (scFv) for use as a targeting arm of a liposomal cytotoxic nanoparticle. We describe the effect of the orientation of variable domains, the length and composition of the interdomain protein linker that connects VH and VL, and stabilizing mutations in both the framework and complementarity-determining regions (CDRs) on the molecular properties of the scFv. We show that variable domain orientation can alter cross-reactivity to murine antigen while maintaining affinity to the human antigen. We demonstrate that tyrosine residues in the CDRs make diverse contributions to the binding affinity and biophysical properties, and that replacement of non-essential tyrosines can improve the stability and bioactivity of the scFv. Our studies demonstrate that a comprehensive engineering strategy may be required to identify a scFv with optimal characteristics for nanoparticle targeting.

  14. Suppressive IL-17A(+)Foxp3(+) and ex-Th17 IL-17A(neg)Foxp3(+) Treg cells are a source of tumour-associated Treg cells.

    Science.gov (United States)

    Downs-Canner, Stephanie; Berkey, Sara; Delgoffe, Greg M; Edwards, Robert P; Curiel, Tyler; Odunsi, Kunle; Bartlett, David L; Obermajer, Nataša

    2017-03-14

    Th17 and regulatory T (Treg) cells are integral in maintaining immune homeostasis and Th17-Treg imbalance is associated with inflammatory immunosuppression in cancer. Here we show that Th17 cells are a source of tumour-induced Foxp3(+) cells. In addition to natural (n)Treg and induced (i)Treg cells that develop from naive precursors, suppressive IL-17A(+)Foxp3(+) and ex-Th17 Foxp3(+) cells are converted from IL-17A(+)Foxp3(neg) cells in tumour-bearing mice. Metabolic phenotyping of Foxp3-expressing IL-17A(+), ex-Th17 and iTreg cells demonstrates the dissociation between the metabolic fitness and the suppressive function of Foxp3-expressing Treg cell subsets. Although all Foxp3-expressing subsets are immunosuppressive, glycolysis is a prominent metabolic pathway exerted only by IL-17A(+)Foxp3(+) cells. Transcriptome analysis and flow cytometry of IL-17A(+)Foxp3(+) cells indicate that Folr4, GARP, Itgb8, Pglyrp1, Il1rl1, Itgae, TIGIT and ICOS are Th17-to-Treg cell transdifferentiation-associated markers. Tumour-associated Th17-to-Treg cell conversion identified here provides insights for targeting the dynamism of Th17-Treg cells in cancer immunotherapy.

  15. Synthesis of liver-targeting dual-ligand modified GCGA/5-FU nanoparticles and their characteristics in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Chen M

    2013-11-01

    Full Text Available Mingrong Cheng,1,2,* Xiaoyan Gao,3,* Yong Wang,4,* Houxiang Chen,5 Bing He,6 Yingchun Li,2 Jiang Han,1 Zhiping Zhang11Department of General Surgery, Pudong New Area District Zhoupu Hospital, Shanghai, People’s Republic of China; 2Department of Endoscopy, 3Department of Plastic Surgery, Pudong New Area District Zhoupu Hospital, Shanghai, People's Republic of China; 4School of Materials Science and Engineering, Wuhan University of Technology, Wuhan, People’s Republic of China; 5Zhejiang Huafon Fiber Research Institute, Zhejiang Huafon Spandex Co, Ltd, Wenzhou, People’s Republic of China; 6Department of General Surgery, Shanghai Fifth People’s Hospital, Fudan University, Shanghai, People's Republic of China *These authors equally contributed to this research Abstract: Nanoparticle drug delivery systems using polymers hold promise for clinical applications. We synthesized dual-ligand modified chitosan (GCGA nanoparticles using lactic acid, glycyrrhetinic acid, and chitosan to target the liver in our previous studies. We then synthesized the GCGA/5-FU nanoparticles by conjugating 5-fluorouracil (5-FU onto the GCGA nanomaterial, which had a mean particle size of 239.9 nm, a polydispersity index of 0.040, a zeta potential of +21.2 mV, and a drug loading of 3.90%. GCGA/5-FU nanoparticles had good slow release properties, and the release process could be divided into five phases: small burst release, gentle release, second burst release, steady release, and slow release. Inhibitory effects of GCGA/5-FU on tumor cells targeted the liver, and were time and dose dependent. GCGA nanoparticles significantly prolonged the efficacy of 5-FU on tumor cells, and alleviated the resistance of tumor cells to 5-FU. GCGA/5-FU nanoparticles were mostly concentrated in the liver, indicating that the GCGA nanoparticles were liver targeting. GCGA/5-FU nanoparticles significantly suppressed tumor growth in orthotopic liver transplantation mouse model, and improved

  16. A transferrin variant as the targeting ligand for polymeric nanoparticles incorporated in 3-D PLGA porous scaffolds.

    Science.gov (United States)

    Lopes, André M; Chen, Kevin Y; Kamei, Daniel T

    2017-04-01

    We have developed doxorubicin (DOX)-loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (DP) conjugated with polyethylene glycol (PEG) and transferrin (Tf) to form Tf-PEG-DPs (TPDPs), and incorporated these TPDPs into three-dimensional (3-D) PLGA porous scaffolds to form a controlled delivery system. To our knowledge, this represents the first use of a Tf variant (oxalate Tf) to improve the targeted delivery of drug-encapsulated nanoparticles (NPs) in PLGA scaffolds to PC3 prostate cancer cells. The PLGA scaffolds with TPDPs incorporated have been shown to release drugs for sustained delivery and provided a continuous release of DOX. The MTS assay was also performed to determine the potency of native and oxalate TPDPs, and a 3.0-fold decrease in IC50 values were observed between the native and oxalate TPDPs. The lower IC50 value for the oxalate version signifies greater potency compared to the native version, since a lower concentration of drug was required to achieve the same therapeutic effect. These results suggest that this technology has potential to become a new implantable polymeric device to improve the controlled and targeted drug delivery of Tf-conjugated NPs for cancer therapy.

  17. Decitabine immunosensitizes human gliomas to NY-ESO-1 specific T lymphocyte targeting through the Fas/Fas Ligand pathway

    Science.gov (United States)

    2011-01-01

    Background The lack of effective treatments for gliomas makes them a significant health problem and highlights the need for the development of novel and innovative treatment approaches. Immunotherapy is an appealing strategy because of the potential ability for immune cells to traffic to and destroy infiltrating tumor cells. However, the absence of well-characterized, highly immunogenic tumor-rejection antigens (TRA) in gliomas has limited the implementation of targeted immune-based therapies. Methods We hypothesized that treatment with the demethylating agent, decitabine, would upregulate the expression of TRA on tumor cells, thereby facilitating enhanced surveillance by TRA-specific T cells. Results and Discussion Treatment of human glioma cells with decitabine increased the expression of NY-ESO-1 and other well characterized cancer testes antigens. The upregulation of NY-ESO-1 made these tumors susceptible to NY-ESO-1-specific T-cell recognition and lysis. Interestingly, decitabine treatment of T98 glioma cells also sensitized them to Fas-dependent apoptosis with an agonistic antibody, while a Fas blocking antibody could largely prevent the enhanced functional recognition by NY-ESO-1 specific T cells. Thus, decitabine treatment transformed a non-immunogenic glioma cell into an immunogenic target that was efficiently recognized by NY-ESO-1--specific T cells. Conclusions Such data supports the hypothesis that agents which alter epigenetic cellular processes may "immunosensitize" tumor cells to tumor-specific T cell-mediated lysis. PMID:22060015

  18. Decitabine immunosensitizes human gliomas to NY-ESO-1 specific T lymphocyte targeting through the Fas/Fas Ligand pathway

    Directory of Open Access Journals (Sweden)

    Konkankit Veerauo V

    2011-11-01

    Full Text Available Abstract Background The lack of effective treatments for gliomas makes them a significant health problem and highlights the need for the development of novel and innovative treatment approaches. Immunotherapy is an appealing strategy because of the potential ability for immune cells to traffic to and destroy infiltrating tumor cells. However, the absence of well-characterized, highly immunogenic tumor-rejection antigens (TRA in gliomas has limited the implementation of targeted immune-based therapies. Methods We hypothesized that treatment with the demethylating agent, decitabine, would upregulate the expression of TRA on tumor cells, thereby facilitating enhanced surveillance by TRA-specific T cells. Results and Discussion Treatment of human glioma cells with decitabine increased the expression of NY-ESO-1 and other well characterized cancer testes antigens. The upregulation of NY-ESO-1 made these tumors susceptible to NY-ESO-1-specific T-cell recognition and lysis. Interestingly, decitabine treatment of T98 glioma cells also sensitized them to Fas-dependent apoptosis with an agonistic antibody, while a Fas blocking antibody could largely prevent the enhanced functional recognition by NY-ESO-1 specific T cells. Thus, decitabine treatment transformed a non-immunogenic glioma cell into an immunogenic target that was efficiently recognized by NY-ESO-1--specific T cells. Conclusions Such data supports the hypothesis that agents which alter epigenetic cellular processes may "immunosensitize" tumor cells to tumor-specific T cell-mediated lysis.

  19. A combined ligand-based and target-based drug design approach for G-protein coupled receptors: application to salvinorin A, a selective kappa opioid receptor agonist

    Science.gov (United States)

    Singh, Nidhi; Chevé, Gwénaël; Ferguson, David M.; McCurdy, Christopher R.

    2006-08-01

    Combined ligand-based and target-based drug design approaches provide a synergistic advantage over either method individually. Therefore, we set out to develop a powerful virtual screening model to identify novel molecular scaffolds as potential leads for the human KOP (hKOP) receptor employing a combined approach. Utilizing a set of recently reported derivatives of salvinorin A, a structurally unique KOP receptor agonist, a pharmacophore model was developed that consisted of two hydrogen bond acceptor and three hydrophobic features. The model was cross-validated by randomizing the data using the CatScramble technique. Further validation was carried out using a test set that performed well in classifying active and inactive molecules correctly. Simultaneously, a bovine rhodopsin based "agonist-bound" hKOP receptor model was also generated. The model provided more accurate information about the putative binding site of salvinorin A based ligands. Several protein structure-checking programs were used to validate the model. In addition, this model was in agreement with the mutation experiments carried out on KOP receptor. The predictive ability of the model was evaluated by docking a set of known KOP receptor agonists into the active site of this model. The docked scores correlated reasonably well with experimental p K i values. It is hypothesized that the integration of these two independently generated models would enable a swift and reliable identification of new lead compounds that could reduce time and cost of hit finding within the drug discovery and development process, particularly in the case of GPCRs.

  20. MiR-34a targeting of Notch ligand delta-like 1 impairs CD15+/CD133+ tumor-propagating cells and supports neural differentiation in medulloblastoma.

    Directory of Open Access Journals (Sweden)

    Pasqualino de Antonellis

    Full Text Available BACKGROUND: Through negative regulation of gene expression, microRNAs (miRNAs can function as oncosuppressors in cancers, and can themselves show altered expression in various tumor types. Here, we have investigated medulloblastoma tumors (MBs, which arise from an early impairment of developmental processes in the cerebellum, where Notch signaling is involved in many of the cell-fate-determining stages. Notch regulates a subset of MB cells that have stem-cell-like properties and can promote tumor growth. On the basis of this evidence, we hypothesized that miRNAs targeting the Notch pathway can regulate these phenomena, and can be used in anti-cancer therapies. METHODOLOGY/PRINCIPAL FINDINGS: In a screening of potential targets within Notch signaling, miR-34a was seen to be a regulator of the Notch pathway through its targeting of Notch ligand Delta-like 1 (Dll1. Down-regulation of Dll1 expression by miR-34a negatively regulates cell proliferation, and induces apoptosis and neural differentiation in MB cells. Using an inducible tetracycline on-off model of miR-34a expression, we show that in Daoy MB cells, Dll1 is the first target that is regulated in MB, as compared to the other targets analyzed here: Cyclin D1, cMyc and CDK4. MiR-34a expression negatively affects CD133(+/CD15(+ tumor-propagating cells, then we assay through reverse-phase proteomic arrays, Akt and Stat3 signaling hypo-phosphorylation. Adenoviruses carrying the precursor miR-34a induce neurogenesis of tumor spheres derived from a genetic animal model of MB (Patch1(+/- p53(-/-, thus providing further evidence that the miR-34a/Dll1 axis controls both autonomous and non autonomous signaling of Notch. In vivo, miR-34a overexpression carried by adenoviruses reduces tumor burden in cerebellum xenografts of athymic mice, thus demonstrating an anti-tumorigenic role of miR-34a in vivo. CONCLUSIONS/SIGNIFICANCE: Despite advances in our understanding of the pathogenesis of MB, one-third of

  1. Target and resistance-related proteins of recombinant mutant human tumor necrosis factor-related apoptosis-inducing ligand on myeloma cell lines.

    Science.gov (United States)

    Jian, Yuan; Chen, Yuling; Geng, Chuanying; Liu, Nian; Yang, Guangzhong; Liu, Jinwei; Li, Xin; Deng, Haiteng; Chen, Wenming

    2016-06-01

    Recombinant mutant human tumor necrosis factor-related apoptosis-inducing ligand (rmhTRAIL) has become a potential therapeutic drug for multiple myeloma (MM). However, the exact targets and resistance mechanisms of rmhTRAIL on MM cells remain to be elucidated. The present study aimed to investigate the target and resistance-related proteins of rmhTRAIL on myeloma cell lines. A TRAIL-sensitive myeloma cell line, RPMI 8226, and a TRAIL-resistance one, U266, were chosen and the differentially expressed proteins between the two cell lines were analyzed prior and subsequent to rmhTRAIL administration by a liquid chromatography-tandem mass spectrometry method. The results showed that following TRAIL treatment, 6 apoptosis-related proteins, calpain small subunit 1 (CPNS1), peflin (PEF1), B-cell receptor-associated protein 31 (BAP31), apoptosis-associated speck-like protein containing CARD (ASC), BAG family molecular chaperone regulator 2 (BAG2) and chromobox protein homolog 3 (CBX3), were upregulated in RPMI 8226 cells while no change was identified in the U266 cells. Furthermore, small ubiquitin-related modifier 1 and several other ubiquitin proteasome pathway (UPP)-related proteins expressed higher levels in TRAIL-resistant cells U266 compared to the RPMI-8226 cells prior and subsequent to rmhTRAIL treatment. These results suggested that CPNS1, PEF1, BAP31, ASC, BAG2 and CBX3 were possibly target proteins of rmhTRAIL on RPMI 8226 cells, while UPP may have a vital role in mediating TRAIL-resistance in U266 cells.

  2. Linker-extended native cyanovirin-N facilitates PEGylation and potently inhibits HIV-1 by targeting the glycan ligand.

    Directory of Open Access Journals (Sweden)

    Jia Chen

    Full Text Available Cyanovirin-N (CVN potently inhibits human immunodeficiency virus type 1 (HIV-1 infection, but both cytotoxicity and immunogenicity have hindered the translation of this protein into a viable therapeutic. A molecular docking analysis suggested that up to 12 residues were involved in the interaction of the reverse parallel CVN dimer with the oligosaccharide targets, among which Leu-1 was the most prominent hot spot residue. This finding provided a possible explanation for the lack of anti-HIV-1 activity observed with N-terminal PEGylated CVN. Therefore, linker-CVN (LCVN was designed as a CVN derivative with a flexible and hydrophilic linker (Gly4Ser3 at the N-terminus. The N-terminal α-amine of LCVN was PEGylated to create 10 K PEG-aldehyde (ALD-LCVN. LCVN and 10 K PEG-ALD-LCVN retained the specificity and affinity of CVN for high mannose N-glycans. Moreover, LCVN exhibited significant anti-HIV-1 activity with attenuated cytotoxicity in the HaCaT keratinocyte cell line and MT-4 T lymphocyte cell lines. 10 K PEG-ALD-LCVN also efficiently inactivated HIV-1 with remarkably decreased cytotoxicity and pronounced cell-to-cell fusion inhibitory activity in vitro. The linker-extended CVN and the mono-PEGylated derivative were determined to be promising candidates for the development of an anti-HIV-1 agent. This derivatization approach provided a model for the PEGylation of biologic candidates without introducing point mutations.

  3. Linker-extended native cyanovirin-N facilitates PEGylation and potently inhibits HIV-1 by targeting the glycan ligand.

    Science.gov (United States)

    Chen, Jia; Huang, Dane; Chen, Wei; Guo, Chaowan; Wei, Bo; Wu, Chongchao; Peng, Zhou; Fan, Jun; Hou, Zhibo; Fang, Yongsheng; Wang, Yifei; Kitazato, Kaio; Yu, Guoying; Zou, Chunbin; Qian, Chuiwen; Xiong, Sheng

    2014-01-01

    Cyanovirin-N (CVN) potently inhibits human immunodeficiency virus type 1 (HIV-1) infection, but both cytotoxicity and immunogenicity have hindered the translation of this protein into a viable therapeutic. A molecular docking analysis suggested that up to 12 residues were involved in the interaction of the reverse parallel CVN dimer with the oligosaccharide targets, among which Leu-1 was the most prominent hot spot residue. This finding provided a possible explanation for the lack of anti-HIV-1 activity observed with N-terminal PEGylated CVN. Therefore, linker-CVN (LCVN) was designed as a CVN derivative with a flexible and hydrophilic linker (Gly4Ser)3 at the N-terminus. The N-terminal α-amine of LCVN was PEGylated to create 10 K PEG-aldehyde (ALD)-LCVN. LCVN and 10 K PEG-ALD-LCVN retained the specificity and affinity of CVN for high mannose N-glycans. Moreover, LCVN exhibited significant anti-HIV-1 activity with attenuated cytotoxicity in the HaCaT keratinocyte cell line and MT-4 T lymphocyte cell lines. 10 K PEG-ALD-LCVN also efficiently inactivated HIV-1 with remarkably decreased cytotoxicity and pronounced cell-to-cell fusion inhibitory activity in vitro. The linker-extended CVN and the mono-PEGylated derivative were determined to be promising candidates for the development of an anti-HIV-1 agent. This derivatization approach provided a model for the PEGylation of biologic candidates without introducing point mutations.

  4. Apoptosis Induction by Targeting Interferon Gamma Receptor 2 (IFNgammaR2) in Prostate Cancer: Ligand (IFNgamma)-Independent Novel Function of IFNgammaR2 as a Bax Inhibitor

    Science.gov (United States)

    2015-08-01

    AWARD NUMBER: W81XWH-12-1-0331 TITLE: Apoptosis Induction by Targeting Interferon Gamma Receptor 2 (IFNgammaR2) in Prostate Cancer: Ligand...DATE August 2015 2. REPORT TYPE Annual 3. DATES COVERED 1Aug2014 - 31Jul2015 4. TITLE AND SUBTITLE Apoptosis Induction by Targeting Interferon ...Introduction In our preliminary study, we identified interferon γ receptor 2 (IFNγR2) as a Bax suppressor using yeast-based functional screening of Bax

  5. Controlling the specificity of modularly assembled small molecules for RNA via ligand module spacing: targeting the RNAs that cause myotonic muscular dystrophy.

    Science.gov (United States)

    Lee, Melissa M; Childs-Disney, Jessica L; Pushechnikov, Alexei; French, Jonathan M; Sobczak, Krzysztof; Thornton, Charles A; Disney, Matthew D

    2009-12-02

    tetramer also bind approximately 13- and approximately 63-fold more tightly to DM1 RNAs than does MBNL1. The modularly assembled compounds are cell permeable and nontoxic as determined by flow cytometry. The results establish that for these two systems: (i) a programmable modular assembly approach can provide synthetic ligands for RNA with affinities and specificities that exceed those of natural proteins; and, (ii) the spacing of ligand modules can be used to tune specificity for one RNA target over another.

  6. Ligand modeling and design

    Energy Technology Data Exchange (ETDEWEB)

    Hay, B.P. [Pacific Northwest National Lab., Richland, WA (United States)

    1997-10-01

    The purpose of this work is to develop and implement a molecular design basis for selecting organic ligands that would be used in the cost-effective removal of specific radionuclides from nuclear waste streams. Organic ligands with metal ion specificity are critical components in the development of solvent extraction and ion exchange processes that are highly selective for targeted radionuclides. The traditional approach to the development of such ligands involves lengthy programs of organic synthesis and testing, which in the absence of reliable methods for screening compounds before synthesis, results in wasted research effort. The author`s approach breaks down and simplifies this costly process with the aid of computer-based molecular modeling techniques. Commercial software for organic molecular modeling is being configured to examine the interactions between organic ligands and metal ions, yielding an inexpensive, commercially or readily available computational tool that can be used to predict the structures and energies of ligand-metal complexes. Users will be able to correlate the large body of existing experimental data on structure, solution binding affinity, and metal ion selectivity to develop structural design criteria. These criteria will provide a basis for selecting ligands that can be implemented in separations technologies through collaboration with other DOE national laboratories and private industry. The initial focus will be to select ether-based ligands that can be applied to the recovery and concentration of the alkali and alkaline earth metal ions including cesium, strontium, and radium.

  7. Insight into the role of dual-ligand modification in low molecular weight heparin based nanocarrier for targeted delivery of doxorubicin.

    Science.gov (United States)

    Du, Hongliang; Liu, Mengrui; Yu, Aihua; Ji, Jianbo; Zhai, Guangxi

    2017-05-15

    Low molecular weight heparin nanoparticles (LMWH) modified by glycyrrhetinic acid (GA) (LMWH-GA) and further decorated by lactobionic acid (LA) (LA-LMWH-GA) were reported as novel hepatocellular carcinoma (HPC)-targeted carriers to overcome multidrug resistance (MDR) of doxorubicin (DOX). The drug-loaded nanoparticles had negative charge of around -25mV and average size range of 70-170nm. These nanoparticles performed sustained drug release in vitro and prolonged DOX residence time in blood circulation in vivo. Compared to free DOX, DOX-loaded nanoparticles demonstrated increased DOX accumulation in drug-resistance HepG2/ADR cells and enhanced in vitro therapeutic efficacy. However, DOX/LA-LMWH-GA with dual ligands didn't show higher cellular uptake and cytotoxicity than single GA modified DOX/LMWH-GA, although both GA-mediated and LA-mediated endocytosis were involved in their cell internalization. Uptake pathway inhibition study revealed the less efficacy of DOX/LA-LMWH-GA in cellular level could be attributed to the reduced effect of micropinocytosis and caveolae-mediated endocytosis in cellular uptake. Interestingly, the DOX-loaded nanoparticles developed from lower drug/carrier feeding ratio possessed higher performance in cell internalization and in vitro efficacy compared to those developed from higher drug/carrier feeding ratio, which could highlight the role of carrier in drug delivery process. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Clinical Significance of Folate Receptor-positive Circulating Tumor Cells Detected by Ligand-targeted Polymerase Chain Reaction in Lung Cancer

    Science.gov (United States)

    Wang, Lin; Wu, Chuanyong; Qiao, Lihua; Yu, Wenjun; Guo, Qiaomei; Zhao, Mingna; Yang, Guohua; Zhao, Hang; Lou, Jiatao

    2017-01-01

    Background: As the heterogeneity of CTCs is becoming increasingly better understood, it is clear that identifying particular subtypes of CTCs would be more relevant. Methods: We detected folate receptor (FR)-positive circulating tumor cells (FR+-CTCs) by a novel ligand-targeted polymerase chain reaction (LT-PCR) detection technique. Results: In the none-dynamic study, FR+-CTC levels of patients with lung cancer were significantly higher than controls (patients with benign lung diseases and healthy controls). With a threshold of 8.7 CTC units, FR+-CTC showed a sensitivity of 77.7% and specificity of 89.5% in the diagnosis of lung cancer. When compared with established clinical biomarkers including carcinoembryonic antigen (CEA), cytokeratin 19 fragment (CYFRA21-1), and neuron-specific enolase (NSE), FR+-CTC showed the highest diagnostic efficiency. Notably, the combination of FR+-CTC, CEA, NSE, and CYFRA21-1 could significantly improve the diagnostic efficacy in differentiating patients with lung cancer from benign lung disease. In our dynamic surveillance study, the CTC levels of 62 non-small cell lung cancer (NSCLC) patients decreased significantly after tumor resection. Conclusion: We established a LT-PCR-based FR+-CTC detection platform for patients with lung cancer that exhibits high sensitivity and specificity. This platform would be clinical useful in lung cancer diagnosis and treatment response assessment.

  9. PoSSuM v.2.0: data update and a new function for investigating ligand analogs and target proteins of small-molecule drugs.

    Science.gov (United States)

    Ito, Jun-ichi; Ikeda, Kazuyoshi; Yamada, Kazunori; Mizuguchi, Kenji; Tomii, Kentaro

    2015-01-01

    PoSSuM (http://possum.cbrc.jp/PoSSuM/) is a database for detecting similar small-molecule binding sites on proteins. Since its initial release in 2011, PoSSuM has grown to provide information related to 49 million pairs of similar binding sites discovered among 5.5 million known and putative binding sites. This enlargement of the database is expected to enhance opportunities for biological and pharmaceutical applications, such as predictions of new functions and drug discovery. In this release, we have provided a new service named PoSSuM drug search (PoSSuMds) at http://possum.cbrc.jp/PoSSuM/drug_search/, in which we selected 194 approved drug compounds retrieved from ChEMBL, and detected their known binding pockets and pockets that are similar to them. Users can access and download all of the search results via a new web interface, which is useful for finding ligand analogs as well as potential target proteins. Furthermore, PoSSuMds enables users to explore the binding pocket universe within PoSSuM. Additionally, we have improved the web interface with new functions, including sortable tables and a viewer for visualizing and downloading superimposed pockets.

  10. Inhibition of MicroRNA-149-5p Induces Apoptosis of Acute Myeloid Leukemia Cell Line THP-1 by Targeting Fas Ligand (FASLG)

    Science.gov (United States)

    Tian, Peijun; Yan, Li

    2016-01-01

    Background This study was aimed to reveal the role of miR-149-5p in acute myeloid leukemia (AML) cells apoptosis and the possible mechanism involved. Material/Methods The expression of miR-149-5p in leukemia cell lines, as well as the blood and bone marrow (BM) samples from leukemia patients, were monitored by reverse-transcription polymerase chain reaction (RT-PCR). AML cell line THP-1 was transfected with miR-149-5p mimic or inhibitor, and then cell apoptosis was determined using the APO Percentage assay kit. The target of miR-149-5p was predicted by using the microRNA.org database, and verified by RT-PCR, Western blot, and Dual-Luciferase reporter assays. Further, small interfering RNA (siRNA) against the target gene was co-transfected with miR-149-5p inhibitor, and then the cell apoptosis and the expression of apoptosis-related proteins were assessed. Results MiR-149-5p was significantly up-regulated in leukemia cell lines and samples from leukemia patients (P<0.01 or P<0.001), especially in THP-1 cells and samples from AML patients. Cell apoptosis was significantly decreased by miR-149-5p overexpression (P<0.01) and increased by miR-149-5p suppression (P<0.05). Fas Ligand (FASLG) was a direct target of miR-149-5p, and was negatively regulated by miR-149-5p. More importantly, the inductive effects of miR-149-5p suppression on cell apoptosis were abrogated by si-FASLG (P<0.01). Furthermore, the up-regulative effects of miR-149-5p suppression on the phosphorylated form of Fas-associated via death domain (p-FADD), caspase-8, caspase-2, caspase-3, and the cleaved forms of these caspases were abrogated by si-FASLG. Conclusions Inhibition of miR-149-5p can induce apoptosis in THP-1 cells. These inductive effects might be via targeting FASLG and activating FADD and caspases. PMID:28013316

  11. Identification of Candidate Target Cyp Genes for microRNAs Whose Expression Is Altered by PCN and TCPOBOP, Representative Ligands of PXR and CAR.

    Science.gov (United States)

    Moriya, Nozomu; Kataoka, Hiromi; Nishikawa, Jun-Ichi; Kugawa, Fumihiko

    2016-08-01

    MicroRNAs (miRNAs) are small non-coding RNAs that are involved in mRNA post-transcriptional regulation. The deregulation of miRNAs affects the expression of drug-metabolizing enzymes, drug transporters, and nuclear receptors, all of which are important in regulating drug metabolism. miRNA expression can be altered by several endogenous or exogenous agents, such as steroid hormones, carcinogens, and therapeutic drugs. However, it is unclear whether hepatic miRNA expression is regulated by nuclear receptors, such as pregnane X receptor (PXR) and constitutive androstane receptor (CAR), which are indispensable for the expression of the CYPs. Here we investigated the effects of the mouse PXR and CAR ligands pregnenolone-16α-carbonitrile (PCN) and 1,4-bis[(3,5-dichloropyridin-2-yl)oxy]benzene (TCPOBOP) on hepatic miRNA expression in mice. We found that the expression of 9 miRNAs was increased (>2-fold) and of 4 miRNAs was decreased (>50%) in response to PCN, while TCPOBOP treatment led to the up-regulation of 8 miRNAs and down-regulation of 6 miRNAs. Using several miRNA target prediction algorithms, we found that the predicted target genes included several lesser known Cyp genes (Cyp1a1, Cyp1b1, Cyp2b10, Cyp2c38, Cyp2u1, Cyp4a12a/b, Cyp4v3, Cyp17a1, Cyp39a1, and Cyp51). We analyzed the expression of these genes in response to PCN and TCPOBOP and found changes in their mRNA levels, some of which were negatively correlated with the expression of their corresponding miRNAs, suggesting that miRNAs may play a role in regulating Cyp enzyme expression. Further studies will be required to fully elucidate the miRNA regulatory mechanisms that contribute to modulating CYP expression.

  12. Development of a novel DNA aptamer ligand targeting to primary cultured tumor endothelial cells by a cell-based SELEX method.

    Directory of Open Access Journals (Sweden)

    Mst Naznin Ara

    Full Text Available The present study used a spontaneous cell-based SELEX method (Systemic Evolution of Ligands by EXponential Enrichment to produce DNA aptamers that specifically bind to cell surface proteins or biomarkers produced by primary cultured mouse tumor endothelial cells (mTECs. In solid tumors, new blood vessels are formed through an angiogenesis process, and this plays a critical role in cancer development as well as metastasis. To combat angiogenesis, an appropriate diagnosis and a molecular-level understanding of the different cancer types are now a high priority. The novel DNA aptamer AraHH001, developed in this study, binds specifically to mTECs with high affinity in the nano-molar range, but does not bind to normal skin endothelial cells (skin-ECs. The selected DNA aptamer was also found to bind to cultured human tumor endothelial cells (hTECs, isolated from a clinical patient with a renal carcinoma. The aptamer AraHH001 showed significant anti-angiogenesis activity by inhibiting tube formation by mTECs on matrigel. Interestingly, a confocal laser scanning microscopy examination of in vitro cellular uptake revealed that AraHH001 was assimilated by mTECs, and became co-localized in acidic compartments, as detected by labeling with Lysotracker Red. Therefore, the development of a specific DNA aptamer that binds to mTECs, as reported here for the first time, holds great promise not only as a therapeutic aptamer but also as a targeted molecular probe that appears to play a major role in angiogenesis, and for the development of a targeted new drug delivery system.

  13. Tumour-associated macrophages are related to progression in patients with      metastatic melanoma following interleukin-2 based immunotherapy

    DEFF Research Database (Denmark)

    Hansen, Bettina Dencker; Schmidt, Henrik; Maase, Hans von der

    2006-01-01

    The aim of the present study was to analyze whether leukocyte subsets in       peripheral blood and tumour biopsies obtained before treatment were able       to predict response or survival in patients with metastatic melanoma       following Interleukin-2 (IL-2) based immunotherapy. Flow cytometry......-associated       CD64(+) macrophages in tumour biopsies were statistically significantly       associated with poor response to treatment. Our data suggest that       tumour-associated macrophages may correlate negatively with response,       which may be of biological importance for IL-2 based immunotherapy...... of       malignant melanoma...

  14. LigandRNA: computational predictor of RNA-ligand interactions.

    Science.gov (United States)

    Philips, Anna; Milanowska, Kaja; Lach, Grzegorz; Bujnicki, Janusz M

    2013-12-01

    RNA molecules have recently become attractive as potential drug targets due to the increased awareness of their importance in key biological processes. The increase of the number of experimentally determined RNA 3D structures enabled structure-based searches for small molecules that can specifically bind to defined sites in RNA molecules, thereby blocking or otherwise modulating their function. However, as of yet, computational methods for structure-based docking of small molecule ligands to RNA molecules are not as well established as analogous methods for protein-ligand docking. This motivated us to create LigandRNA, a scoring function for the prediction of RNA-small molecule interactions. Our method employs a grid-based algorithm and a knowledge-based potential derived from ligand-binding sites in the experimentally solved RNA-ligand complexes. As an input, LigandRNA takes an RNA receptor file and a file with ligand poses. As an output, it returns a ranking of the poses according to their score. The predictive power of LigandRNA favorably compares to five other publicly available methods. We found that the combination of LigandRNA and Dock6 into a "meta-predictor" leads to further improvement in the identification of near-native ligand poses. The LigandRNA program is available free of charge as a web server at http://ligandrna.genesilico.pl.

  15. Evaluation of [(111/114m)In]CHX-A''-DTPA-ZHER2:342, an affibody ligand coniugate for targeting of HER2-expressing malignant tumors.

    Science.gov (United States)

    Orlova, A; Rosik, D; Sandström, M; Lundqvist, H; Einarsson, L; Tolmachev, V

    2007-12-01

    Radionuclide imaging of the HER2 receptor, which is a target for trastuzumab therapy, can provide important diagnostic information. Further, targeting radionuclide therapy might be an option for treatment of HER2 expressing tumors. The phage-display selected Affibody ligand Z(HER2:342), which binds to HER2 with an affinity of 22 pM, may here play an important role. The small size of the Z(HER2:342), 7.5 kDa, enables quick tumor localization and fast blood clearance. Earlier, successful targeting of HER2-expressing xenografts using Z(HER2:342) labeled using [(111)In]benzyl-DTPA was reported. By changing to the CHX-A''-DTPA chelator, the stability and labeling kinetics of the radiometal-Z(HER2:342) conjugate can be improved. The aim of this study was to evaluate the labeling of the CHX-A''-DTPA-Z(HER2:342) conjugate with (111)In for diagnostic imaging and with (114m)In for locoregional radionuclide therapy. The isothiocyanate derivative of CHX-A''-DTPA was coupled to Z(HER2:342) in alkaline conditions at 37 degrees C. The conjugate was labeled with both (111)In and (114m)In and evaluated in vitro and in vivo. Labeling with (111)In and (114m)In provided >95% yield after 30 min at RT. Specific radioactivity was 0.5 and 12 MBq/nmol, for (114m)In and (111)In, respectively. The radiolabeled conjugates demonstrated specific binding to HER2 expressing SKOV-3 cells. In mice bearing SKOV-3 xenografts, the tumor uptake of [(111)In]CHX-A''-DTPA-Z(HER2:342) 4 h postinjection was 10.3+/-3.6% IA/g and tumor-to-blood ratio about 190. [(111)In]CHX-A''-DTPA-Z(HER2:342) is a promising candidate for the visualization of HER2 expression in malignant tumors. Labeled with (114m)In it could also be used for locoregional treatment of HER2 expressing tumors.

  16. Highly Selective Targeting of Hepatic Stellate Cells for Liver Fibrosis Treatment Using a d-Enantiomeric Peptide Ligand of Fn14 Identified by Mirror-Image mRNA Display.

    Science.gov (United States)

    Huang, Luying; Xie, Jing; Bi, Qiuyan; Li, Zhuoxuan; Liu, Sha; Shen, Qing; Li, Chong

    2017-05-01

    Although liver fibrosis is a major public health issue, there is still no effective drug therapy in the clinic. Fibroblast growth factor-inducible 14 (Fn14), a membrane receptor highly specifically expressed in activated hepatic stellate cells (HSCs), is the key driver of liver fibrosis, and thus, it has a great potential as a novel target for the development of effective treatment. Here, we identified a d-enantiomeric peptide ligand of Fn14 through mirror-image mRNA display. This included the chemical synthesis of a d-enantiomer of the target protein (extracellular domain of Fn14), identification of an l-peptide ligand of d-Fn14 using a constructed mRNA peptide library, and identification of a d-enantiomer of the l-peptide, which is a ligand of the natural Fn14 for reasons of symmetry. The obtained d-peptide ligand showed strong binding to Fn14 while maintaining high proteolytic resistance. As a targeting moiety, this d-peptide successfully mediated high selectivity of activated HSCs for liposomal vehicles compared to that of other major cell types in the liver and significantly enhanced the accumulation of liposomes in the liver fibrosis region of a carbon tetrachloride-induced mouse model. Moreover, in combination with curcumin as an encapsulated load, a liposomal formulation conjugated with this d-peptide showed powerful inhibition of the proliferation of activated HSCs and reduced the liver fibrosis to a significant extent in vivo. This Fn14-targeting strategy may represent a promising approach to targeted drug delivery for liver fibrosis treatment. Meanwhile, the mirror-image mRNA display can provide a new arsenal for the development of d-peptide-based therapeutics against a variety of human diseases.

  17. Structure-based identification of CaMKIIα-interacting MUPP1 PDZ domains and rational design of peptide ligands to target such interaction in human fertilization.

    Science.gov (United States)

    Zhang, Yi-Le; Han, Zhao-Feng; Sun, Ying-Pu

    2016-06-01

    The recognition and association between Ca(2+)/calmodulin-activated protein kinase II-α (CaMKIIα) and multi-PDZ domain protein 1 (MUPP1) plays an important role in sperm acrosome reaction and human fertilization, which is mediated by the binding of CaMKIIα's C-terminal tail to one or more PDZ domains of the scaffolding protein MUPP1. In this study, we attempt to identify the CaMKIIα-interacting MUPP1 PDZ domains and to design peptide ligands that can potently target and then competitively disrupt such interaction. Here, a synthetic biology approach was proposed to systematically characterize the structural basis, energetic property, dynamic behavior and biological implication underlying the intermolecular interactions between the C-terminal peptide of CaMKIIα and all the 13 PDZ domains of MUPP1. These domains can be grouped into four clusters in terms of their sequence, structure and physiochemical profile; different clusters appear to recognize different classes of PDZ-binding motifs. The cluster 3 includes two members, i.e. MUPP1 PDZ 5 and 11 domains, which were suggested to bind class II motif Φ-X-Φ(-COOH) of the C-terminal peptide SGAPSV(-COOH) of CaMKIIα. Subsequently, the two domains were experimentally measured as the moderate- and high-affinity binders of the peptide by using fluorescence titration (dissociation constants K d = 25.2 ± 4.6 and 0.47 ± 0.08 µM for peptide binding to PDZ 5 and 11, respectively), which was in line with theoretical prediction (binding free energies ΔG total = -7.6 and -9.2 kcal/mol for peptide binding to PDZ 5 and 11, respectively). A systematic mutation of SGAPSV(-COOH) residues suggested few favorable amino acids at different residue positions of the peptide, which were then combined to generate a number of potent peptide mutants for PDZ 11 domain. Consequently, two peptides (SIAPNV(-COOH) and SIVMNV(-COOH)) were identified to have considerably improved affinity with K d increase by ~tenfold relative to

  18. Synthesis and pharmacological evaluation of dual acting ligands targeting the adenosine A2A and dopamine D2 receptors for the potential treatment of Parkinson's disease.

    Science.gov (United States)

    Jörg, Manuela; May, Lauren T; Mak, Frankie S; Lee, Kiew Ching K; Miller, Neil D; Scammells, Peter J; Capuano, Ben

    2015-01-22

    A relatively new strategy in drug discovery is the development of dual acting ligands. These molecules are potentially able to interact at two orthosteric binding sites of a heterodimer simultaneously, possibly resulting in enhanced subtype selectivity, higher affinity, enhanced or modified physiological response, and reduced reliance on multiple drug administration regimens. In this study, we have successfully synthesized a series of classical heterobivalent ligands as well as a series of more integrated and "drug-like" dual acting molecules, incorporating ropinirole as a dopamine D2 receptor agonist and ZM 241385 as an adenosine A2A receptor antagonist. The best compounds of our series maintained the potency of the original pharmacophores at both receptors (adenosine A2A and dopamine D2). In addition, the integrated dual acting ligands also showed promising results in preliminary blood-brain barrier permeability tests, whereas the classical heterobivalent ligands are potentially more suited as pharmacological tools.

  19. The next chapter in MOF pillaring strategies: Trigonal heterofunctional ligands to access targeted high-connected three dimensional nets, isoreticular platforms

    KAUST Repository

    Eubank, Jarrod F.

    2011-11-09

    A new pillaring strategy, based on a ligand-to-axial approach that combines the two previous common techniques, axial-to-axial and ligand-to-ligand, and permits design, access, and construction of higher dimensional MOFs, is introduced and validated. Trigonal heterofunctional ligands, in this case isophthalic acid cores functionalized at the 5-position with N-donor (e.g., pyridyl- or triazolyl-type) moieties, are designed and utilized to pillar pretargeted two-dimensional layers (supermolecular building layers, SBLs). These SBLs, based on edge transitive Kagomé and square lattices, are cross-linked into predicted three-dimensional MOFs with tunable large cavities, resulting in isoreticular platforms. © 2011 American Chemical Society.

  20. TARGET:?

    National Research Council Canada - National Science Library

    James M Acton

    2014-01-01

      By 2003. as military planners had become worried that the country's long-range conventional weapons, such as cruise missiles, might be too slow to reach hypothetical distant targets that needed to be struck urgently...

  1. Expression of tumour necrosis factor-related apoptosis-inducing ligand death receptors in sporadic and hereditary colorectal tumours : Potential targets for apoptosis induction

    NARCIS (Netherlands)

    Koornstra, JJ; Jalving, M; Rijcken, FEM; Westra, Jantine; Zwart, N; Hollema, H; de Vries, EGE; Hofstra, RWM; Plukker, JTM; de Jong, S; Kleibeuker, JH

    2005-01-01

    Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and antibodies against TRAIL receptors death receptor 4 (DR4) and death receptor 5 (DR5) are under investigation for cancer therapy. To study the potential application of these agents, the expression of DR4 and DR5 were studied immunoh

  2. Radioprotection of bone marrow stem cell subsets by interleukin-1 and kit-ligand : Implications for CFU-S as the responsible target cell population

    NARCIS (Netherlands)

    van Os, Ronald; Lamont, G; Witsell, A; Mauch, PM

    1997-01-01

    Various cytokines have been reported to have radioprotective effects on the bone marrow. Of these, c-kit-ligand (KL) and interleukin-1 (IL-1) have the most dramatic effect when given prior to total body irradiation (TBI). Given simultaneously, KL and IL-1 demonstrated a strong effect on increasing t

  3. Ligand placement based on prior structures: the guided ligand-replacement method

    Energy Technology Data Exchange (ETDEWEB)

    Klei, Herbert E. [Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Bristol-Myers Squibb, Princeton, NJ 08543-4000 (United States); Moriarty, Nigel W., E-mail: nwmoriarty@lbl.gov; Echols, Nathaniel [Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Terwilliger, Thomas C. [Los Alamos National Laboratory, Los Alamos, NM 87545-0001 (United States); Baldwin, Eric T. [Bristol-Myers Squibb, Princeton, NJ 08543-4000 (United States); Natural Discovery LLC, Princeton, NJ 08542-0096 (United States); Pokross, Matt; Posy, Shana [Bristol-Myers Squibb, Princeton, NJ 08543-4000 (United States); Adams, Paul D. [Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); University of California at Berkeley, Berkeley, CA 94720-1762 (United States)

    2014-01-01

    A new module, Guided Ligand Replacement (GLR), has been developed in Phenix to increase the ease and success rate of ligand placement when prior protein-ligand complexes are available. The process of iterative structure-based drug design involves the X-ray crystal structure determination of upwards of 100 ligands with the same general scaffold (i.e. chemotype) complexed with very similar, if not identical, protein targets. In conjunction with insights from computational models and assays, this collection of crystal structures is analyzed to improve potency, to achieve better selectivity and to reduce liabilities such as absorption, distribution, metabolism, excretion and toxicology. Current methods for modeling ligands into electron-density maps typically do not utilize information on how similar ligands bound in related structures. Even if the electron density is of sufficient quality and resolution to allow de novo placement, the process can take considerable time as the size, complexity and torsional degrees of freedom of the ligands increase. A new module, Guided Ligand Replacement (GLR), was developed in Phenix to increase the ease and success rate of ligand placement when prior protein–ligand complexes are available. At the heart of GLR is an algorithm based on graph theory that associates atoms in the target ligand with analogous atoms in the reference ligand. Based on this correspondence, a set of coordinates is generated for the target ligand. GLR is especially useful in two situations: (i) modeling a series of large, flexible, complicated or macrocyclic ligands in successive structures and (ii) modeling ligands as part of a refinement pipeline that can automatically select a reference structure. Even in those cases for which no reference structure is available, if there are multiple copies of the bound ligand per asymmetric unit GLR offers an efficient way to complete the model after the first ligand has been placed. In all of these applications, GLR

  4. Primary breast cancer tumours contain high amounts of IgA1 immunoglobulin: an immunohistochemical analysis of a possible carrier of the tumour-associated Tn antigen.

    Directory of Open Access Journals (Sweden)

    Charlotte Welinder

    Full Text Available The Tn antigen (GalNAc alpha-O-Ser/Thr as defined by the binding of the lectin, helix pomatia agglutinin (HPA or anti-Tn monoclonal antibodies, is known to be exposed in a majority of cancers, and it has also been shown to correlate positively with the metastatic capacity in breast carcinoma. The short O-glycan that forms the antigen is carried by a number of different proteins. One potential carrier of the Tn antigen is immunoglobulin A1 (IgA1, which we surprisingly found in tumour cells of the invasive parts of primary breast carcinoma. Conventional immunohistochemical analysis of paraffin-embedded sections from primary breast cancers showed IgA1 to be present in the cytoplasm and plasma membrane of 35 out of 36 individual primary tumours. The immunohistochemical staining of HPA and anti-Tn antibody (GOD3-2C4 did to some extent overlap with the presence of IgA1 in the tumours, but differences were seen in the percentage of stained cells and in the staining pattern in the different breast cancers analysed. Anti-Tn antibody and HPA were also shown to specifically bind to a number of possible constellations of the Tn antigen in the hinge region of IgA1. Both reagents could also detect the presence of Tn positive IgA in serum. On average 51% of the tumour cells in the individual breast cancer tumour sections showed staining for IgA1. The overall amount of staining in the invasive part of the tumour with the anti Tn antibody was 67%, and 93% with HPA. The intra-expression or uptake of IgA1 in breast cancer makes it a new potential carrier of the tumour associated and immunogenic Tn antigen.

  5. PPAR-γ ligands repress TGFβ-induced myofibroblast differentiation by targeting the PI3K/Akt pathway: implications for therapy of fibrosis.

    Directory of Open Access Journals (Sweden)

    Ajit A Kulkarni

    Full Text Available Transforming growth factor beta (TGFβ induced differentiation of human lung fibroblasts to myofibroblasts is a key event in the pathogenesis of pulmonary fibrosis. Although the typical TGFβ signaling pathway involves the Smad family of transcription factors, we have previously reported that peroxisome proliferator-activated receptor-γ (PPAR-γ ligands inhibit TGFβ-mediated differentiation of human lung fibroblasts to myofibroblasts via a Smad-independent pathway. TGFβ also activates the phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt pathway leading to phosphorylation of Akt(S473. Here, we report that PPAR-γ ligands, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO and 15-deoxy-(12,14-15d-prostaglandin J(2 (15d-PGJ(2, inhibit human myofibroblast differentiation of normal and idiopathic pulmonary fibrotic (IPF fibroblasts, by blocking Akt phosphorylation at Ser473 by a PPAR-γ-independent mechanism. The PI3K inhibitor LY294002 and a dominant-negative inactive kinase-domain mutant of Akt both inhibited TGFβ-stimulated myofibroblast differentiation, as determined by Western blotting for α-smooth muscle actin and calponin. Prostaglandin A(1 (PGA(1, a structural analogue of 15d-PGJ(2 with an electrophilic center, also reduced TGFβ-driven phosphorylation of Akt, while CAY10410, another analogue that lacks an electrophilic center, did not; implying that the activity of 15d-PGJ(2 and CDDO is dependent on their electrophilic properties. PPAR-γ ligands inhibited TGFβ-induced Akt phosphorylation via both post-translational and post-transcriptional mechanisms. This inhibition is independent of MAPK-p38 and PTEN but is dependent on TGFβ-induced phosphorylation of FAK, a kinase that acts upstream of Akt. Thus, PPAR-γ ligands inhibit TGFβ signaling by affecting two pro-survival pathways that culminate in myofibroblast differentiation. Further studies of PPAR-γ ligands and small electrophilic molecules may lead to a new generation of

  6. Prospective Preliminary In Vitro Investigation of a Magnetic Iron Oxide Nanoparticle Conjugated with Ligand CD80 and VEGF Antibody As a Targeted Drug Delivery System for the Induction of Cell Death in Rodent Osteosarcoma Cells.

    Science.gov (United States)

    Kovach, AnneMarie Kay; Gambino, Jen M; Nguyen, Vina; Nelson, Zach; Szasz, Taylor; Liao, Jun; Williams, Lakiesha; Bulla, Sandra; Prabhu, Raj

    2016-01-01

    Target drug deliveries using nanotechnology are a novel consideration in the treatment of cancer. We present herein an in vitro mouse model for the preliminary investigation of the efficacy of an iron oxide nanoparticle complex conjugated to vascular endothelial growth factor (VEGF) antibody and ligand cluster of differentiation 80 (CD80) for the purpose of eventual translational applications in the treatment of human osteosarcoma (OSA). The 35 nm diameter iron oxide magnetic nanoparticles are functionalized with an n-hydroxysuccinimide biocompatible coating and are conjugated on the surface to proteins VEGF antibody and ligand CD80. Combined, these proteins have the ability to target OSA cells and induce apoptosis. The proposed system was tested on a cancerous rodent osteoblast cell line (ATCCTM(NPO) CRL-2836) at four different concentrations (0.1, 1.0, 10.0, and 100.0 μg/mL) of ligand CD80 alone, VEGF antibody alone, and a combination thereof (CD80+VEGF). Systems were implemented every 24 h over different sequential treatment timelines: 24, 48, and 72 h, to find the optimal protein concentration required for a reduction in cell proliferation. Results demonstrated that a combination of ligand CD80 and VEGF antibody was consistently most effective at reducing aberrant osteoblastic proliferation for both the 24- and 72-h timelines. At 48 h, however, an increase in cell proliferation was documented for the 0.1 and 1 μg/mL groups. For the 24- and 72-h tests, concentrations of 1.0 μg/mL of CD80+VEGF and 0.1 μg/mL of VEGF antibody were most effective. Concentrations of 10.0 and 100.0 μg/mL of CD80+VEGF reduced cell proliferation, but not as remarkably as the 1.0 μg/mL concentration. In addition, cell proliferation data showed that multiple treatments (72-h test) induced cell death in the osteoblasts better than a single treatment. Future targeted drug delivery system research includes trials in OSA cell lines from greater phylum species having

  7. Prospective Preliminary In Vitro Investigation of a Magnetic Iron Oxide Nanoparticle Conjugated with Ligand CD80 and VEGF Antibody As a Targeted Drug Delivery System for the Induction of Cell Death in Rodent Osteosarcoma Cells

    Directory of Open Access Journals (Sweden)

    Anne Marie Kay Kovach

    2016-10-01

    Full Text Available Target drug deliveries using nanotechnology are a novel consideration in the treatment of cancer. We present herein an in vitro mouse model for the preliminary investigation of the efficacy of an iron oxide nanoparticle complex conjugated to vascular endothelial growth factor (VEGF antibody and ligand cluster of differentiation 80 (CD80 for the purpose of eventual translational applications in the treatment of human osteosarcoma (OSA. The 35 nm diameter iron oxide magnetic nanoparticles are functionalized with an n-hydroxysuccinimide biocompatible coating and are conjugated on the surface to proteins VEGF antibody and ligand CD80. Combined, these proteins have the ability to target OSA cells and induce apoptosis. The proposed system was tested on a cancerous rodent osteoblast cell line (ATCCTMNPO CRL-2836 at four different concentrations (0.1, 1.0, 10.0, and 100.0 μg/mL of ligand CD80 alone, VEGF antibody alone, and a combination thereof (CD80+VEGF. Systems were implemented every 24 h over different sequential treatment timelines: 24, 48, and 72 h, to find the optimal protein concentration required for a reduction in cell proliferation. Results demonstrated that a combination of ligand CD80 and VEGF antibody was consistently most effective at reducing aberrant osteoblastic proliferation for both the 24- and 72-h timelines. At 48 h, however, an increase in cell proliferation was documented for the 0.1 and 1 μg/mL groups. For the 24- and 72-h tests, concentrations of 1.0 μg/mL of CD80+VEGF and 0.1 μg/mL of VEGF antibody were most effective. Concentrations of 10.0 and 100.0 μg/mL of CD80+VEGF reduced cell proliferation, but not as remarkably as the 1.0 μg/mL concentration. In addition, cell proliferation data showed that multiple treatments (72-h test induced cell death in the osteoblasts better than a single treatment. Future targeted drug delivery system research includes trials in OSA cell lines from greater phylum

  8. Highly potent extranuclear-targeted luminescent iridium(iii) antitumor agents containing benzimidazole-based ligands with a handle for functionalization.

    Science.gov (United States)

    Yellol, Jyoti; Pérez, Sergio A; Yellol, Gorakh; Zajac, Juraj; Donaire, Antonio; Vigueras, Gloria; Novohradsky, Vojtech; Janiak, Christoph; Brabec, Viktor; Ruiz, José

    2016-12-01

    A series of 6 substitutionally inert and luminescent iridium(iii) antitumor agents of the type [Ir(C(∧)N)2(N(∧)N)][PF6] containing a benzimidazole N(∧)N ligand with an ester group as a handle for further functionalization has been prepared. They exhibit IC50 values in the high nanomolar range in some ovarian and breast cancer cell lines (approximately 100× more cytotoxic than cisplatin (CDDP) in MDA-MB-231) and are located in the actin cortex predominantly as shown by confocal luminescence microscopy. This discovery could open the door to a new large family of drug bioconjugates with diverse and simultaneous functions.

  9. Pan-HER - an antibody mixture targeting EGFR, HER2, and HER3 abrogates preformed and ligand-induced EGFR homo- and heterodimers

    DEFF Research Database (Denmark)

    Ellebaek, Sofie; Pedersen, Susanne Brix; Grandal, Michael

    2016-01-01

    The human epidermal growth factor receptor (HER)-family is involved in development of many epithelial cancers. Therefore, HER-family members constitute important targets for anti-cancer therapeutics such as monoclonal antibodies (mAbs). A limitation to the success of single HER-targeting m...... broader efficacy than targeting single or dual receptor combinations also in resistant settings. In light of this broad efficacy, we decided to investigate the effect of Pan-HER compared with single HER-targeting with single and dual mAbs on HER-family cross-talk and dimerization focusing on EGFR....... The effect of Pan-HER on cell proliferation and HER-family receptor degradation was superior to treatment with single mAbs targeting either single receptor, and similar to targeting a single receptor with two non-overlapping antibodies. Furthermore, changes in EGFR-dimerization patterns after treatment...

  10. Automated design of ligands to polypharmacological profiles

    Science.gov (United States)

    Besnard, Jérémy; Ruda, Gian Filippo; Setola, Vincent; Abecassis, Keren; Rodriguiz, Ramona M.; Huang, Xi-Ping; Norval, Suzanne; Sassano, Maria F.; Shin, Antony I.; Webster, Lauren A.; Simeons, Frederick R.C.; Stojanovski, Laste; Prat, Annik; Seidah, Nabil G.; Constam, Daniel B.; Bickerton, G. Richard; Read, Kevin D.; Wetsel, William C.; Gilbert, Ian H.; Roth, Bryan L.; Hopkins, Andrew L.

    2012-01-01

    The clinical efficacy and safety of a drug is determined by its activity profile across multiple proteins in the proteome. However, designing drugs with a specific multi-target profile is both complex and difficult. Therefore methods to rationally design drugs a priori against profiles of multiple proteins would have immense value in drug discovery. We describe a new approach for the automated design of ligands against profiles of multiple drug targets. The method is demonstrated by the evolution of an approved acetylcholinesterase inhibitor drug into brain penetrable ligands with either specific polypharmacology or exquisite selectivity profiles for G-protein coupled receptors. Overall, 800 ligand-target predictions of prospectively designed ligands were tested experimentally, of which 75% were confirmed correct. We also demonstrate target engagement in vivo. The approach can be a useful source of drug leads where multi-target profiles are required to achieve either selectivity over other drug targets or a desired polypharmacology. PMID:23235874

  11. The IMiDs targets IKZF-1/3 and IRF4 as novel negative regulators of NK cell-activating ligands expression in multiple myeloma.

    Science.gov (United States)

    Fionda, Cinzia; Abruzzese, Maria Pia; Zingoni, Alessandra; Cecere, Francesca; Vulpis, Elisabetta; Peruzzi, Giovanna; Soriani, Alessandra; Molfetta, Rosa; Paolini, Rossella; Ricciardi, Maria Rosaria; Petrucci, Maria Teresa; Santoni, Angela; Cippitelli, Marco

    2015-09-15

    Immunomodulatory drugs (IMiDs) have potent anti-tumor activities in multiple myeloma (MM) and are able to enhance the cytotoxic function of natural killer (NK) cells, important effectors of the immune response against MM. Here, we show that these drugs can enhance the expression of the NKG2D and DNAM-1 activating receptor ligands MICA and PVR/CD155 in human MM cell lines and primary malignant plasma cells. Depletion of cereblon (CRBN) by shRNA interference strongly impaired upregulation of these ligands and, more interestingly, IMiDs/CRBN-mediated downregulation of the transcription factors Ikaros (IKZF1), Aiolos (IKZF3) and IRF4 was critical for these regulatory mechanisms. Indeed, shRNA knockdown of IKZF1 or IKZF3 expression was both necessary and sufficient for the upregulation of MICA and PVR/CD155 expression, suggesting that these transcription factors can repress these genes; accordingly, the direct interaction and the negative role of IKZF1 and IKZF3 proteins on MICA and PVR/CD155 promoters were demonstrated. Finally, MICA expression was enhanced in IRF4-silenced cells, indicating a specific suppressive role of this transcription factor on MICA gene expression in MM cells.Taken together, these findings describe novel molecular pathways involved in the regulation of MICA and PVR/CD155 gene expression and identify the transcription factors IKZF-1/IKZF-3 and IRF4 as repressors of these genes in MM cells.

  12. Periodicity in tumor vasculature targeting kinetics of ligand-functionalized nanoparticles studied by dynamic contrast enhanced magnetic resonance imaging and intravital microscopy.

    Science.gov (United States)

    Hak, Sjoerd; Cebulla, Jana; Huuse, Else Marie; Davies, Catharina de L; Mulder, Willem J M; Larsson, Henrik B W; Haraldseth, Olav

    2014-01-01

    In the past two decades advances in the development of targeted nanoparticles have facilitated their application as molecular imaging agents and targeted drug delivery vehicles. Nanoparticle-enhanced molecular imaging of the angiogenic tumor vasculature has been of particular interest. Not only because angiogenesis plays an important role in various pathologies, but also since endothelial cell surface receptors are directly accessible for relatively large circulating nanoparticles. Typically, nanoparticle targeting towards these receptors is studied by analyzing the contrast distribution on tumor images acquired before and at set time points after administration. Although several exciting proof-of-concept studies demonstrated qualitative assessment of relative target concentration and distribution, these studies did not provide quantitative information on the nanoparticle targeting kinetics. These kinetics will not only depend on nanoparticle characteristics, but also on receptor binding and recycling. In this study, we monitored the in vivo targeting kinetics of αvβ3-integrin specific nanoparticles with intravital microscopy and dynamic contrast enhanced magnetic resonance imaging, and using compartment modeling we were able to quantify nanoparticle targeting rates. As such, this approach can facilitate optimization of targeted nanoparticle design and it holds promise for providing more quantitative information on in vivo receptor levels. Interestingly, we also observed a periodicity in the accumulation kinetics of αvβ3-integrin targeted nanoparticles and hypothesize that this periodicity is caused by receptor binding, internalization and recycling dynamics. Taken together, this demonstrates that our experimental approach provides new insights in in vivo nanoparticle targeting, which may proof useful for vascular targeting in general.

  13. Periodicity in tumor vasculature targeting kinetics of ligand-functionalized nanoparticles studied by dynamic contrast enhanced magnetic resonance imaging and intravital microscopy

    DEFF Research Database (Denmark)

    Hak, Sjoerd; Cebulla, Jana; Huuse, Else Marie

    2014-01-01

    kinetics. These kinetics will not only depend on nanoparticle characteristics, but also on receptor binding and recycling. In this study, we monitored the in vivo targeting kinetics of αvβ3-integrin specific nanoparticles with intravital microscopy and dynamic contrast enhanced magnetic resonance imaging...... in the accumulation kinetics of αvβ3-integrin targeted nanoparticles and hypothesize that this periodicity is caused by receptor binding, internalization and recycling dynamics. Taken together, this demonstrates that our experimental approach provides new insights in in vivo nanoparticle targeting, which may proof...

  14. Reducing toxicity of 4–1BB costimulation: targeting 4–1BB ligands to the tumor stroma with bi-specific aptamer conjugates

    Science.gov (United States)

    Schrand, B; Berezhnoy, A; Brenneman, R; Williams, A; Levay, A; Gilboa, E

    2015-01-01

    Systemic administration of immune modulatory antibodies to cancer patients is associated with autoimmune pathologies. We have developed a clinically feasible and broadly applicable approach to limit immune stimulation to disseminated tumor lesions using a bi-specific agonistic 4–1BB oligonucleotide aptamer targeted to a broadly expressed stromal product (e.g., VEGF or osteopontin). The stroma-targeted aptamer conjugates engendered potent antitumor immunity against unrelated tumors and exhibited a superior therapeutic index compared to non-targeted agonistic 4–1BB antibody. PMID:25949891

  15. Imidazoline receptors ligands

    Directory of Open Access Journals (Sweden)

    Agbaba Danica

    2012-01-01

    Full Text Available Extensive biochemical and pharmacological studies have determined three different subtypes of imidazoline receptors: I1-imidazoline receptors (I1-IR involved in central inhibition of sympathicus that produce hypotensive effect; I2-imidazoline receptors (I2-IR modulate monoamine oxidase B activity (MAO-B; I3-imidazoline receptors (I3-IR regulate insulin secretion from pancreatic β-cells. Therefore, the I1/I2/I3 imidazoline receptors are selected as new, interesting targets for drug design and discovery. Novel selective I1/I2/I3 agonists and antagonists have been recently developed. In the present review, we provide a brief update to the field of imidazoline research, highlighting some of the chemical diversity and progress made in the 2D-QSAR, 3D-QSAR and quantitative pharmacophore development studies of I1-IR and I2-IR imidazoline receptor ligands. Theoretical studies of I3-IR ligands are not yet performed because of insufficient number of synthesized I3-IR ligands.

  16. Inhibition of acetylcholinesterase, beta-amyloid aggregation, and NMDA receptors in Alzheimer's disease: a promising direction for the multi-target-directed ligands gold rush.

    Science.gov (United States)

    Rosini, Michela; Simoni, Elena; Bartolini, Manuela; Cavalli, Andrea; Ceccarini, Luisa; Pascu, Nicoleta; McClymont, David W; Tarozzi, Andrea; Bolognesi, Maria L; Minarini, Anna; Tumiatti, Vincenzo; Andrisano, Vincenza; Mellor, Ian R; Melchiorre, Carlo

    2008-08-14

    Alzheimer's disease (AD) is a multifactorial syndrome with several target proteins contributing to its etiology. To confront AD, an innovative strategy is to design single chemical entities able to simultaneously modulate more than one target. Here, we present compounds that inhibit acetylcholinesterase and NMDA receptor activity. Furthermore, these compounds inhibit AChE-induced Abeta aggregation and display antioxidant properties, emerging as lead candidates for treating AD.

  17. Dissecting the Binding Mode of Low Affinity Phage Display Peptide Ligands to Protein Targets by Hydrogen/Deuterium Exchange Coupled to Mass Spectrometry

    DEFF Research Database (Denmark)

    Leurs, Ulrike; Lohse, Brian; Ming, Shonoi A;

    2014-01-01

    Phage display (PD) is frequently used to discover peptides capable of binding to biological protein targets. The structural characterization of peptide-protein complexes is often challenging due to their low binding affinities and high structural flexibility. Here, we investigate the use of hydro......Phage display (PD) is frequently used to discover peptides capable of binding to biological protein targets. The structural characterization of peptide-protein complexes is often challenging due to their low binding affinities and high structural flexibility. Here, we investigate the use...

  18. [Lys40(Ahx-DTPA-111In)NH2]exendin-4, a very promising ligand for glucagon-like peptide-1 (GLP-1) receptor targeting.

    NARCIS (Netherlands)

    Wild, D.; Behe, M.; Wicki, A.; Storch, D.; Waser, B.; Gotthardt, M.; Keil, B.; Christofori, G.; Reubi, J.C.; Macke, H.R.

    2006-01-01

    High levels of glucagon-like peptide-1 (GLP-1) receptor expression in human insulinomas and gastrinomas provide an attractive target for imaging, therapy, and intraoperative tumor localization, using receptor-avid radioligands. The goal of this study was to establish a tumor model for GLP-1 receptor

  19. Research progress on target therapeutic agents of HER-2 extracellular ligand-binding domain in breast cancer%乳腺癌HER-2胞外配体结合区靶点治疗的研究进展*

    Institute of Scientific and Technical Information of China (English)

    钟锦绣; 李亚梅(综述); 关晏星(审校)

    2013-01-01

    The target therapeutic agents of HER-2 extracellular ligand-binding domain have become the core of breast cancer research. A small peptide molecule and an anti-HER2 extracellular domain monoclonal antibody conjugated with protein toxins, radioisotopes, and chemotherapeutic drugs (immunoconjugate) can improve efficacy and reduce systemic toxicity. Vaccines based on HER-2 extracellular region should protect patients from HER-2-overexpressing breast cancer growth. In this review, studies on targeted-block therapies of the HER-2 extracellular ligand-binding domain in breast cancer were discussed to provide references for clinical applications.%针对乳腺癌HER-2受体胞外结合区的靶点治疗成为当今研究的热点。小分子多肽、HER-2胞外结合区的单抗药物及其与蛋白毒素、放射性核素,化疗药物的偶联物即免疫偶联物既能增强药物的有效性,又可减少对正常组织的毒害。HER-2胞外区肽疫苗可有效预防HER-2高表达乳腺癌的生长。本文将对乳腺癌HER-2胞外区靶向阻断治疗的研究进行综述,为相应的临床应用提供参考。

  20. Biological activity of sporolides A and B from Salinispora tropica: in silico target prediction using ligand-based pharmacophore mapping and in vitro activity validation on HIV-1 reverse transcriptase.

    Science.gov (United States)

    Dineshkumar, Kesavan; Aparna, Vasudevan; Madhuri, Kantilal Z; Hopper, Waheeta

    2014-03-01

    Sporolides A and B are novel polycyclic macrolides from the obligate marine actinomycetes, Salinispora tropica. The unique and novel structure of sporolides makes them interesting candidates for targeting diverse biological activities. Biological target prediction of sporolides was carried out using ligand-based pharmacophore screening against known inhibitors and drugs. Validation of pharmacophore screening was carried out for the identified hits. New biological targets predicted for sporolides using this method were HIV-1 reverse transcriptase, adenosine A3 receptor, endothelin receptor ET-A, oxytocin receptor, voltage-gated L-type calcium channel α-1C subunit/calcium channel α/Δ subunit 1. Drug-likeness properties were predicted for the selected compounds using QikProp module. Sporolides A and B showed maximum docking score with HIV-1 reverse transcriptase. Structural interaction fingerprints analysis indicated similar binding pattern of the sporolides with the HIV-1 reverse transcriptase. Sporolide B exhibited good inhibitory activity against HIV-1 reverse transcriptase in in vitro fluorescent assay.

  1. The Recognition of Identical Ligands by Unrelated Proteins.

    Science.gov (United States)

    Barelier, Sarah; Sterling, Teague; O'Meara, Matthew J; Shoichet, Brian K

    2015-12-18

    The binding of drugs and reagents to off-targets is well-known. Whereas many off-targets are related to the primary target by sequence and fold, many ligands bind to unrelated pairs of proteins, and these are harder to anticipate. If the binding site in the off-target can be related to that of the primary target, this challenge resolves into aligning the two pockets. However, other cases are possible: the ligand might interact with entirely different residues and environments in the off-target, or wholly different ligand atoms may be implicated in the two complexes. To investigate these scenarios at atomic resolution, the structures of 59 ligands in 116 complexes (62 pairs in total), where the protein pairs were unrelated by fold but bound an identical ligand, were examined. In almost half of the pairs, the ligand interacted with unrelated residues in the two proteins (29 pairs), and in 14 of the pairs wholly different ligand moieties were implicated in each complex. Even in those 19 pairs of complexes that presented similar environments to the ligand, ligand superposition rarely resulted in the overlap of related residues. There appears to be no single pattern-matching "code" for identifying binding sites in unrelated proteins that bind identical ligands, though modeling suggests that there might be a limited number of different patterns that suffice to recognize different ligand functional groups.

  2. Tenfibgen ligand nanoencapsulation delivers bi-functional anti-CK2 RNAi oligomer to key sites for prostate cancer targeting using human xenograft tumors in mice.

    Directory of Open Access Journals (Sweden)

    Janeen H Trembley

    Full Text Available Protected and specific delivery of nucleic acids to malignant cells remains a highly desirable approach for cancer therapy. Here we present data on the physical and chemical characteristics, mechanism of action, and pilot therapeutic efficacy of a tenfibgen (TBG-shell nanocapsule technology for tumor-directed delivery of single stranded DNA/RNA chimeric oligomers targeting CK2αα' to xenograft tumors in mice. The sub-50 nm size TBG nanocapsule (s50-TBG is a slightly negatively charged, uniform particle of 15 - 20 nm size which confers protection to the nucleic acid cargo. The DNA/RNA chimeric oligomer (RNAi-CK2 functions to decrease CK2αα' expression levels via both siRNA and antisense mechanisms. Systemic delivery of s50-TBG-RNAi-CK2 specifically targets malignant cells, including tumor cells in bone, and at low doses reduces size and CK2-related signals in orthotopic primary and metastatic xenograft prostate cancer tumors. In conclusion, the s50-TBG nanoencapsulation technology together with the chimeric oligomer targeting CK2αα' offer significant promise for systemic treatment of prostate malignancy.

  3. The ligand binding domain controls glucocorticoid receptor dynamics independent of ligand release.

    Science.gov (United States)

    Meijsing, Sebastiaan H; Elbi, Cem; Luecke, Hans F; Hager, Gordon L; Yamamoto, Keith R

    2007-04-01

    Ligand binding to the glucocorticoid receptor (GR) results in receptor binding to glucocorticoid response elements (GREs) and the formation of transcriptional regulatory complexes. Equally important, these complexes are continuously disassembled, with active processes driving GR off GREs. We found that co-chaperone p23-dependent disruption of GR-driven transcription depended on the ligand binding domain (LBD). Next, we examined the importance of the LBD and of ligand dissociation in GR-GRE dissociation in living cells. We showed in fluorescence recovery after photobleaching studies that dissociation of GR from GREs is faster in the absence of the LBD. Furthermore, GR interaction with a target promoter revealed ligand-specific exchange rates. However, using covalently binding ligands, we demonstrated that ligand dissociation is not required for receptor dissociation from GREs. Overall, these studies showed that activities impinging on the LBD regulate GR exchange with GREs but that the dissociation of GR from GREs is independent from ligand dissociation.

  4. The natural product Aristolactam Allla as a new ligand targeting the polo-box domain of polo-like kinase 1 potently inhibits cancer cell proliferation

    Institute of Scientific and Technical Information of China (English)

    Li LI; Xu WANG; Jing CHEN; Hong DING; Yu ZHANG; Tian-cen HU; Li-hong HU; Hua-liang JIANG; Xu SHEN

    2009-01-01

    Aim:To search for novel inhibitors of human polo-like kinase 1 (Plkl), which plays important roles in various aspects of mitotic progres-sion and is believed as a promising anti-cancer drug target, and further investigate the potential inhibition mechanism of active com-pounds against Plk1, thus developing potent anti-tumor lead compounds.Methods: Surface plasmon resonance (SPR) technology-based assay and enzymatic inhibition assay were used to screen Plk1 inhibi-tors. Sulphorhodamine B (SRB)-based assay, flow cytometry, confocal microscopy and Western blotting were used to further identify the potent Plk1 inhibitor. To investigate the inhibitory mechanism of the active compound against Plk1, enzymatic inhibition assay, SPR and yeast two-hybrid technology-based assays were used.Results: Aristolactam AIlla was identified as a new type of Plk1 inhibitors, targeting the Polo Box domain (PBD) which is another effi-cient tactic for exploring Plk1 inhibitors. Further studies indicated that it could block the proliferations of HeLa, A549, HGC and the HCT-8/V cells (clinical Navelbine-resistant cancer cell), induce mitotic arrest of HeLa cells at G2/M phase with spindle abnormalities and promote apoptosis in HeLa cells. The results from SPR and yeast two-hybrid technology-based assays suggested that it could tar-get both the catalytic domain of Plk1 (CD) and PBD and enhance the CD/PBD interaction.Conclusion: Our current work is expected to shed light on the potential anti-tumor mechanism of Aristolactam AIlla, and this natural product might be possibly used as a lead compound for further developing anti-tumor drugs.

  5. High-Affinity DNA Aptamer Generation Targeting von Willebrand Factor A1-Domain by Genetic Alphabet Expansion for Systematic Evolution of Ligands by Exponential Enrichment Using Two Types of Libraries Composed of Five Different Bases.

    Science.gov (United States)

    Matsunaga, Ken-Ichiro; Kimoto, Michiko; Hirao, Ichiro

    2017-01-11

    The novel evolutionary engineering method ExSELEX (genetic alphabet expansion for systematic evolution of ligands by exponential enrichment) provides high-affinity DNA aptamers that specifically bind to target molecules, by introducing an artificial hydrophobic base analogue as a fifth component into DNA aptamers. Here, we present a newer version of ExSELEX, using a library with completely randomized sequences consisting of five components: four natural bases and one unnatural hydrophobic base, 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds). In contrast to the limited number of Ds-containing sequence combinations in our previous library, the increased complexity of the new randomized library could improve the success rates of high-affinity aptamer generation. To this end, we developed a sequencing method for each clone in the enriched library after several rounds of selection. Using the improved library, we generated a Ds-containing DNA aptamer targeting von Willebrand factor A1-domain (vWF) with significantly higher affinity (KD = 75 pM), relative to those generated by the initial version of ExSELEX, as well as that of the known DNA aptamer consisting of only the natural bases. In addition, the Ds-containing DNA aptamer was stabilized by introducing a mini-hairpin DNA resistant to nucleases, without any loss of affinity (KD = 61 pM). This new version is expected to consistently produce high-affinity DNA aptamers.

  6. Tacrine-Trolox Hybrids: A Novel Class of Centrally Active, Nonhepatotoxic Multi-Target-Directed Ligands Exerting Anticholinesterase and Antioxidant Activities with Low In Vivo Toxicity.

    Science.gov (United States)

    Nepovimova, Eugenie; Korabecny, Jan; Dolezal, Rafael; Babkova, Katerina; Ondrejicek, Ales; Jun, Daniel; Sepsova, Vendula; Horova, Anna; Hrabinova, Martina; Soukup, Ondrej; Bukum, Neslihan; Jost, Petr; Muckova, Lubica; Kassa, Jiri; Malinak, David; Andrs, Martin; Kuca, Kamil

    2015-11-25

    Coupling of two distinct pharmacophores, tacrine and trolox, endowed with different biological properties, afforded 21 hybrid compounds as novel multifunctional candidates against Alzheimer's disease. Several of them showed improved inhibitory properties toward acetylcholinesterase (AChE) in relation to tacrine. These hybrids also scavenged free radicals. Molecular modeling studies in tandem with kinetic analysis exhibited that these hybrids target both catalytic active site as well as peripheral anionic site of AChE. In addition, incorporation of the moiety bearing antioxidant abilities displayed negligible toxicity on human hepatic cells. This striking effect was explained by formation of nontoxic metabolites after 1 h incubation in human liver microsomes system. Finally, tacrine-trolox hybrids exhibited low in vivo toxicity after im administration in rats and potential to penetrate across blood-brain barrier. All of these outstanding in vitro results in combination with promising in vivo outcomes highlighted derivative 7u as the lead structure worthy of further investigation.

  7. Synthesis and pharmacological evaluation of 8- and 9-substituted benzolactam-v8 derivatives as potent ligands for protein kinase C, a therapeutic target for Alzheimer's disease.

    Science.gov (United States)

    Mach, Ulrich R; Lewin, Nancy E; Blumberg, Peter M; Kozikowski, Alan P

    2006-03-01

    A central element in the pathophysiology of Alzheimer's disease (AD) is the formation of amyloid plaques, which result from abnormal processing of the amyloid precursor protein (APP). The processing of APP is largely provided by three key enzymes, namely the alpha-, beta-, and gamma-secretases. As the latter two contribute to the formation of neurotoxic Abeta fragments while alpha-secretase does not, a decrease in the amyloidogenic products can be brought about either by inhibition of the beta- and gamma-secretases or through the activation of alpha-secretase. It is now known that the activation of protein kinase C (PKC) enhances alpha-secretase activity and therefore represents a possible target for the development of agents urgently needed for the treatment of this devastating neurodegenerative disorder. In the present study, new benzolactam-V8-based PKC activators were synthesized and tested for their binding affinity toward PKCalpha. All compounds tested showed binding values in the nanomolar concentration range. In accordance with previous publications, 9-substitution dramatically increased PKC binding affinity in comparison with the corresponding 8-substituted analogues. In addition to the location of the side chain on the aromatic ring, the binding affinities of these benzolactams were found to depend on the orientation, length, and electronic properties of this appendage. An interesting decrease in binding affinity was found for the 9-thienyl analogue 13, suggesting adverse electronic interactions of the sulfur atom with PKC or parts of the cellular membrane.

  8. Bexarotene ligand pharmaceuticals.

    Science.gov (United States)

    Hurst, R E

    2000-12-01

    target rates; side effects were primarily limited to local skin reactions [349982]. Ligand has worldwide rights to market bexarotene capsules, and will market the drug in the US, Canada and selected European markets. In Spain, Portugal, Greece and Central and South America, Ferrer Internacional will market and distribute the drug. As of December 1999, Ligand was seeking additional distribution partners for select European and Asian markets [351604]. In January 2000, Alfa Wassermann signed an agreement with Ligand to exclusively market and distribute Targretin gel and capsules in Italy. Alfa paid US $0.75 million on signing with additional amounts up to an aggregate total of US $1.0 million on achievement of certain registration milestones, which are expected to be met in 2000 [351882].

  9. Synthesis and biological evaluation of new [Tc(N)(PS)]-based mixed-ligand compounds useful in the design of target-specific radiopharmaceuticals: the 2-methoxyphenylpiperazine dithiocarbamate derivatives as an example.

    Science.gov (United States)

    Bolzati, Cristina; Salvarese, Nicola; Carta, Davide; Refosco, Fiorenzo; Dolmella, Alessandro; Pietzsch, Hans Jürgen; Bergmann, Ralf; Bandoli, Giuliano

    2011-01-01

    This study presents the first application of a general procedure based on the use of the [Tc(N)Cl(PS)(PPh(3))] species (PS is an alkyl phosphinothiolate ligand) for the preparation of Tc(N) target-specific compounds. [Tc(N)Cl(PS)(PPh(3))] selectively reacts with an appropriate dithiocarbamate ligand (S(∧)Y) to give [Tc(N)(PS)(S(∧)Y)] compounds. 1-(2-Methoxyphenyl)piperazine, which displays a potent and specific affinity for 5HT(1A) receptors, was selected as a functional group and conjugated to the dithiocarbamate unit through different spacers (L( n )). [(99m)Tc(N)(PS)(L( n ))] complexes were prepared in high yield (more than 90%). The chemical identity of (99m)Tc complexes was determined by high performance liquid chromatography comparison with the corresponding (99g)Tc complexes. All complexes were found to be inert toward transchelation with an excess of glutathione and cysteine. No notable biotransformation of the native compound into different species by the in vitro action of the serum and liver enzymes was shown. Nanomolar affinity for the 5HT(1A) receptor was obtained for [(99m)Tc(N)(PSiso)L(3)] (IC(50) = 1.5 nM); a reduction of the affinity was observed for the other complexes as a function of the shortening of the alkyl chain interposed between the dithiocarbamate and the pharmacophore. Negligible brain uptake was found from in vivo distribution data of [(99m)Tc(N)(PSiso)L(3)]. The key finding of this study is that the complexes maintained good affinity and selectivity for 5HT(1A) receptors, and the IC(50) value for [(99g)Tc(N)(PSiso)L(3)] being comparable to the IC(50) value found for WAY 100635. This result confirmed the possibility of preparing [(99m)Tc(N)(PS)]-based target-specific compounds without affecting the affinity and selectivity of the bioactive molecules for the corresponding receptors.

  10. An inhalable β₂-adrenoceptor ligand-directed guanidinylated chitosan carrier for targeted delivery of siRNA to lung.

    Science.gov (United States)

    Luo, Yongfeng; Zhai, Xinyun; Ma, Chaonan; Sun, Peng; Fu, Zhiping; Liu, Wenguang; Xu, Jun

    2012-08-20

    SiRNA-based strategies appear to be an exciting new approach for the treatment of respiratory diseases. To extrapolate siRNA-mediated interventions from bench to bedside in this area, several aspects have to be jointly considered, including a safe and efficient gene carrier with pulmonary deposition efficiency, as well as in vivo method for siRNA/nanoparticles delivery. Accordingly, in this work, (i) a non-viral DNA vector, guanidinylated chitosan (GCS) that has been developed in our previous study [X.Y. Zhai, P. Sun, Y.F. Luo, C.N. Ma, J. Xu, W.G. Liu, 2011], was tested for siRNA delivery. We demonstrated that GCS was able to completely condense siRNA at weight ratio 40:1, forming nanosize particles of diameter ~100 nm, 15 mV in surface potential. Guanidinylation of chitosan not only decreased the cytotoxicity but also facilitated cellular internalization of siRNA nanoparticles, leading to an enhanced gene-silencing efficiency compared to the pristine chitosan (CS). (ii) We chemically coupled salbutamol, a β(2)-adrenoceptor agonist, to GCS (SGCS), which successfully improved targeting specificity of the green fluorescent protein (GFP)-siRNA carrier to lung cells harbored with β(2)-adrenergic receptor, and remarkably enhanced the efficacy of gene silence in vitro and in the lung of enhanced green fluorescent protein (EGFP)-transgenic mice in vivo. (iii) It was proved that this chitosan-based polymer was able to provide both the pDNA and siRNA with the protection against destructive shear forces generated by the mesh-based nebulizers. Aerosol treatment improved the nanoparticle size distribution, which should be in favor of enhancing the transfection efficiency. We suggest a potential application of the chitosan-derived nanodelivery vehicle (SGCS) in RNA interference therapy for lung diseases via aerosol inhalation.

  11. Searching for the Multi-Target-Directed Ligands against Alzheimer's disease: discovery of quinoxaline-based hybrid compounds with AChE, H₃R and BACE 1 inhibitory activities.

    Science.gov (United States)

    Huang, Wenhai; Tang, Li; Shi, Ying; Huang, Shufang; Xu, Lei; Sheng, Rong; Wu, Peng; Li, Jia; Zhou, Naiming; Hu, Yongzhou

    2011-12-01

    A novel series of quinoxaline derivatives, as Multi-Target-Directed Ligands (MTDLs) for AD treatment, were designed by lending the core structural elements required for H(3)R antagonists and hybridizing BACE 1 inhibitor 1 with AChE inhibitor BYYT-25. A virtual database consisting of quinoxaline derivatives was first screened on a pharmacophore model of BACE 1 inhibitors, and then filtered by a molecular docking model of AChE. Seventeen quinoxaline derivatives with high score values were picked out, synthesized and evaluated for their biological activities. Compound 11a, the most effective MTDL, showed the potent activity to H(3)R/AChE/BACE 1 (H(3)R antagonism, IC(50)=280.0 ± 98.0 nM; H(3)R inverse agonism, IC(50)=189.3 ± 95.7 nM; AChE, IC(50)=483 ± 5 nM; BACE 1, 46.64±2.55% inhibitory rate at 20 μM) and high selectivity over H(1)R/H(2)R/H(4)R. Furthermore, the protein binding patterns between 11a and AChE/BACE 1 showed that it makes several essential interactions with the enzymes.

  12. Predicting protein-ligand affinity with a random matrix framework.

    Science.gov (United States)

    Lee, Alpha A; Brenner, Michael P; Colwell, Lucy J

    2016-11-29

    Rapid determination of whether a candidate compound will bind to a particular target receptor remains a stumbling block in drug discovery. We use an approach inspired by random matrix theory to decompose the known ligand set of a target in terms of orthogonal "signals" of salient chemical features, and distinguish these from the much larger set of ligand chemical features that are not relevant for binding to that particular target receptor. After removing the noise caused by finite sampling, we show that the similarity of an unknown ligand to the remaining, cleaned chemical features is a robust predictor of ligand-target affinity, performing as well or better than any algorithm in the published literature. We interpret our algorithm as deriving a model for the binding energy between a target receptor and the set of known ligands, where the underlying binding energy model is related to the classic Ising model in statistical physics.

  13. A retinoid X receptor (RXR)-selective retinoid reveals that RXR-alpha is potentially a therapeutic target in breast cancer cell lines, and that it potentiates antiproliferative and apoptotic responses to peroxisome proliferator-activated receptor ligands.

    Science.gov (United States)

    Crowe, David L; Chandraratna, Roshantha A S

    2004-01-01

    Certain lipids have been shown to be ligands for a subgroup of the nuclear hormone receptor superfamily known as the peroxisome proliferator-activated receptors (PPARs). Ligands for these transcription factors have been used in experimental cancer therapies. PPARs heterodimerize and bind DNA with retinoid X receptors (RXRs), which have homology to other members of the nuclear receptor superfamily. Retinoids have been found to be effective in treating many types of cancer. However, many breast cancers become resistant to the chemotherapeutic effects of these drugs. Recently, RXR-selective ligands were discovered that inhibited proliferation of all-trans retinoic acid resistant breast cancer cells in vitro and caused regression of the disease in animal models. There are few published studies on the efficacy of combined therapy using PPAR and RXR ligands for breast cancer prevention or treatment. We determined the effects of selective PPAR and RXR ligands on established human breast cancer cell lines in vitro. PPAR-alpha and PPAR-gamma ligands induced apoptotic and antiproliferative responses in human breast cancer cell lines, respectively, which were associated with specific changes in gene expression. These responses were potentiated by the RXR-selective ligand AGN194204. Interestingly, RXR-alpha-overexpressing retinoic acid resistant breast cancer cell lines were more sensitive to the effects of the RXR-selective compound. RXR-selective retinoids can potentiate the antiproliferative and apoptotic responses of breast cancer cell lines to PPAR ligands.

  14. A ligand's view of target similarity

    DEFF Research Database (Denmark)

    Garland, Stephen L; Gloriam, David E

    2011-01-01

    GPCR binding site-directed techniques are rapidly evolving into powerful tools for modern drug discovery. Many of these approaches bridge chemistry and biology, which are inseparable concepts in nature but are often treated as separate worlds in drug discovery and science in general. This review ...

  15. Predicting protein-ligand affinity with a random matrix framework

    OpenAIRE

    Lee, Alpha Albert; Brenner, MP; Colwell, Lucy Jane

    2016-01-01

    Rapid determination of whether a candidate compound will bind to a particular target receptor remains a stumbling block in drug discovery. We use an approach inspired by random matrix theory to decompose the known ligand set of a target in terms of orthogonal "signals" of salient chemical features, and distinguish these from the much larger set of ligand chemical features that are not relevant for binding to that particular target receptor. After removing the noise caused by finite sampling, ...

  16. Polypharmacology: in silico methods of ligand design and development.

    Science.gov (United States)

    McKie, Samuel A

    2016-04-01

    How to design a ligand to bind multiple targets, rather than to a single target, is the focus of this review. Rational polypharmacology draws on knowledge that is both broad ranging and hierarchical. Computer-aided multitarget ligand design methods are described according to their nested knowledge level. Ligand-only and then receptor-ligand strategies are first described; followed by the metabolic network viewpoint. Subsequently strategies that view infectious diseases as multigenomic targets are discussed, and finally the disease level interpretation of medicinal therapy is considered. As yet there is no consensus on how best to proceed in designing a multitarget ligand. The current methodologies are bought together in an attempt to give a practical overview of how polypharmacology design might be best initiated.

  17. Designer TGFβ superfamily ligands with diversified functionality.

    Directory of Open Access Journals (Sweden)

    George P Allendorph

    Full Text Available Transforming Growth Factor--beta (TGFβ superfamily ligands, including Activins, Growth and Differentiation Factors (GDFs, and Bone Morphogenetic Proteins (BMPs, are excellent targets for protein-based therapeutics because of their pervasiveness in numerous developmental and cellular processes. We developed a strategy termed RASCH (Random Assembly of Segmental Chimera and Heteromer, to engineer chemically-refoldable TGFβ superfamily ligands with unique signaling properties. One of these engineered ligands, AB208, created from Activin-βA and BMP-2 sequences, exhibits the refolding characteristics of BMP-2 while possessing Activin-like signaling attributes. Further, we find several additional ligands, AB204, AB211, and AB215, which initiate the intracellular Smad1-mediated signaling pathways more strongly than BMP-2 but show no sensitivity to the natural BMP antagonist Noggin unlike natural BMP-2. In another design, incorporation of a short N-terminal segment from BMP-2 was sufficient to enable chemical refolding of BMP-9, without which was never produced nor refolded. Our studies show that the RASCH strategy enables us to expand the functional repertoire of TGFβ superfamily ligands through development of novel chimeric TGFβ ligands with diverse biological and clinical values.

  18. Ligand modeling and design

    Energy Technology Data Exchange (ETDEWEB)

    Hay, B. [Pacific Northwest Lab., Richland, WA (United States)

    1996-10-01

    The purpose of this work is to develop and implement a molecular design basis for selecting organic ligands that would be used tin applications for the cost-effective removal of specific radionuclides from nuclear waste streams.

  19. CLiBE: a database of computed ligand binding energy for ligand-receptor complexes.

    Science.gov (United States)

    Chen, X; Ji, Z L; Zhi, D G; Chen, Y Z

    2002-11-01

    Consideration of binding competitiveness of a drug candidate against natural ligands and other drugs that bind to the same receptor site may facilitate the rational development of a candidate into a potent drug. A strategy that can be applied to computer-aided drug design is to evaluate ligand-receptor interaction energy or other scoring functions of a designed drug with that of the relevant ligands known to bind to the same binding site. As a tool to facilitate such a strategy, a database of ligand-receptor interaction energy is developed from known ligand-receptor 3D structural entries in the Protein Databank (PDB). The Energy is computed based on a molecular mechanics force field that has been used in the prediction of therapeutic and toxicity targets of drugs. This database also contains information about ligand function and other properties and it can be accessed at http://xin.cz3.nus.edu.sg/group/CLiBE.asp. The computed energy components may facilitate the probing of the mode of action and other profiles of binding. A number of computed energies of some PDB ligand-receptor complexes in this database are studied and compared to experimental binding affinity. A certain degree of correlation between the computed energy and experimental binding affinity is found, which suggests that the computed energy may be useful in facilitating a qualitative analysis of drug binding competitiveness.

  20. Ligands of Therapeutic Utility for the Liver X Receptors

    Directory of Open Access Journals (Sweden)

    Rajesh Komati

    2017-01-01

    Full Text Available Liver X receptors (LXRs have been increasingly recognized as a potential therapeutic target to treat pathological conditions ranging from vascular and metabolic diseases, neurological degeneration, to cancers that are driven by lipid metabolism. Amidst intensifying efforts to discover ligands that act through LXRs to achieve the sought-after pharmacological outcomes, several lead compounds are already being tested in clinical trials for a variety of disease interventions. While more potent and selective LXR ligands continue to emerge from screening of small molecule libraries, rational design, and empirical medicinal chemistry approaches, challenges remain in minimizing undesirable effects of LXR activation on lipid metabolism. This review provides a summary of known endogenous, naturally occurring, and synthetic ligands. The review also offers considerations from a molecular modeling perspective with which to design more specific LXRβ ligands based on the interaction energies of ligands and the important amino acid residues in the LXRβ ligand binding domain.

  1. Ligand prediction from protein sequence and small molecule information using support vector machines and fingerprint descriptors.

    Science.gov (United States)

    Geppert, Hanna; Humrich, Jens; Stumpfe, Dagmar; Gärtner, Thomas; Bajorath, Jürgen

    2009-04-01

    Support vector machine (SVM) database search strategies are presented that aim at the identification of small molecule ligands for targets for which no ligand information is currently available. In pharmaceutical research and chemical biology, this situation is faced, for example, when studying orphan targets or newly identified members of protein families. To investigate methods for de novo ligand identification in the absence of known three-dimensional target structures or active molecules, we have focused on combining sequence and ligand information for closely and distantly related proteins. To provide a basis for these investigations, a set of 11 protease targets from different families was assembled together with more than 2000 inhibitors directed against individual proteases. We have compared SVM approaches that combine protein sequence and ligand information in different ways and utilize 2D fingerprints as ligand descriptors. These methodologies were applied to search for inhibitors of individual proteases not taken into account during learning. A target sequence-ligand kernel and, in particular, a linear combination of multiple target-directed SVMs consistently identified inhibitors with high accuracy including test cases where homology-based similarity searching using data fusion and conventional SVM ranking nearly or completely failed. The SVM linear combination and target-ligand kernel methods described herein are intuitive and straightforward to adopt for ligand prediction against other targets.

  2. Coarse-grained molecular dynamics simulations of protein-ligand binding.

    Science.gov (United States)

    Negami, Tatsuki; Shimizu, Kentaro; Terada, Tohru

    2014-09-30

    Coarse-grained molecular dynamics (CGMD) simulations with the MARTINI force field were performed to reproduce the protein-ligand binding processes. We chose two protein-ligand systems, the levansucrase-sugar (glucose or sucrose), and LinB-1,2-dichloroethane systems, as target systems that differ in terms of the size and shape of the ligand-binding pocket and the physicochemical properties of the pocket and the ligand. Spatial distributions of the Coarse-grained (CG) ligand molecules revealed potential ligand-binding sites on the protein surfaces other than the real ligand-binding sites. The ligands bound most strongly to the real ligand-binding sites. The binding and unbinding rate constants obtained from the CGMD simulation of the levansucrase-sucrose system were approximately 10 times greater than the experimental values; this is mainly due to faster diffusion of the CG ligand in the CG water model. We could obtain dissociation constants close to the experimental values for both systems. Analysis of the ligand fluxes demonstrated that the CG ligand molecules entered the ligand-binding pockets through specific pathways. The ligands tended to move through grooves on the protein surface. Thus, the CGMD simulations produced reasonable results for the two different systems overall and are useful for studying the protein-ligand binding processes.

  3. CB receptor ligands from plants.

    Science.gov (United States)

    Woelkart, Karin; Salo-Ahen, Outi M H; Bauer, Rudolf

    2008-01-01

    Advances in understanding the physiology and pharmacology of the endogenous cannabinoid system have potentiated the interest of cannabinoid receptors as potential therapeutic targets. Cannabinoids have been shown to modulate a variety of immune cell functions and have therapeutic implications on central nervous system (CNS) inflammation, chronic inflammatory conditions such as arthritis, and may be therapeutically useful in treating autoimmune conditions such as multiple sclerosis. Many of these drug effects occur through cannabinoid receptor signalling mechanisms and the modulation of cytokines and other gene products. Further, endocannabinoids have been found to have many physiological and patho-physiological functions, including mood alteration and analgesia, control of energy balance, gut motility, motor and co-ordination activities, as well as alleviation of neurological, psychiatric and eating disorders. Plants offer a wide range of chemical diversity and have been a growing domain in the search for effective cannabinoid ligands. Cannabis sativa L. with the known plant cannabinoid, Delta(9-)tetrahydrocannabinol (THC) and Echinacea species with the cannabinoid (CB) receptor-binding lipophilic alkamides are the best known herbal cannabimimetics. This review focuses on the state of the art in CB ligands from plants, as well their possible therapeutic and immunomodulatory effects.

  4. Notch信号在增生性瘢痕表皮中的表达%Expression of Notch receptors, ligands and downstream target genes in epidermis of hypertrophic scar

    Institute of Scientific and Technical Information of China (English)

    夏炜; 潘宝华; 刘宾; 张曦; 马福成; 王映梅; 杨晓婷; 刘丹; 郭树忠

    2009-01-01

    Objective To study the expression of Notch receptors, ligands and downstream target genes in hypertrophic scar and normal skin, and to investigate its role in the development of hypertrophic scar. Methods By immunohistochemistry, the expression of epidermal differentiation markers-β1 integrin, keratin 14 (K14) and keratin 19 (K19), as well as Notch 1-4 and Jagged1 were examined in hypertrophic scars and normal skins. The expression of Notch downstream genes- P21 and P63 was analyzed with real-time quantitative PCR and immtmohistechemistry staining. Results Histological analysis revealed a significant epidermal thickening in the hypertrophic scars, with excessive cell layers above the basal layer. Compared to the normal epidermis, the expression of β1 integrin, K19 and K14 decreased in hypertrophic scars (P<0.05). Positive expression rate of Notch1 and Jagged1 in keratinocytes was significantly higher in hypertrophic scar than in normal skin (P<0.05), while there was no difference in Notch2 and 3 positive expression rate. Furthermore, the expression of P21 was significantly up-regulated, while the expression of P63 was down-regulated in keratinocytes of hypertrophic scar (P<0.05). Conclusions Notch signal may play an important role in hypertrophic scar pathogenesis. Over-defferentiation of Keratinocytes in hypertrophic scar may be related to the overexpression of Notch1 and Jagged1, up-regalation of P21 gene and down-regulation of P63 gene.%目的 研究Notch信号相关分子在增生性瘢痕表皮中的表达情况,探讨其是否参与增生性瘢痕的形成. 方法 收集年龄、性别、部位互为对照的增生性瘢痕和健康皮肤组织各8例.行免疫组织化学检测:①表皮分化标志物,包括整合素β1、角蛋白14(K14)和19(K19);②Notch受体1~4以及配体Jagged1.行Real-time PCR和免疫组织化学检测Notch下游基因P21和P63的表达以及定位. 结果 组织学检测发现增生性瘢痕表皮较健康表皮明显增厚,

  5. Ligand fitting with CCP4

    Science.gov (United States)

    2017-01-01

    Crystal structures of protein–ligand complexes are often used to infer biology and inform structure-based drug discovery. Hence, it is important to build accurate, reliable models of ligands that give confidence in the interpretation of the respective protein–ligand complex. This paper discusses key stages in the ligand-fitting process, including ligand binding-site identification, ligand description and conformer generation, ligand fitting, refinement and subsequent validation. The CCP4 suite contains a number of software tools that facilitate this task: AceDRG for the creation of ligand descriptions and conformers, Lidia and JLigand for two-dimensional and three-dimensional ligand editing and visual analysis, Coot for density interpretation, ligand fitting, analysis and validation, and REFMAC5 for macromolecular refinement. In addition to recent advancements in automatic carbohydrate building in Coot (LO/Carb) and ligand-validation tools (FLEV), the release of the CCP4i2 GUI provides an integrated solution that streamlines the ligand-fitting workflow, seamlessly passing results from one program to the next. The ligand-fitting process is illustrated using instructive practical examples, including problematic cases such as post-translational modifications, highlighting the need for careful analysis and rigorous validation. PMID:28177312

  6. Synthesis and enzymatic cleavage of dual-ligand quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Sewell, Sarah L. [Department of Biomedical Engineering, Vanderbilt University, Nashville, TN (United States); Giorgio, Todd D., E-mail: todd.d.giorgio@vanderbilt.edu [Department of Biomedical Engineering, Vanderbilt University, Nashville, TN (United States); Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN (United States)

    2009-05-05

    Site directed therapy promises to minimize treatment-limiting systemic effects associated with cytotoxic agents that have no specificity for pathologic tissues. One general strategy is to target cell surface receptors uniquely presented on particular tissues. Highly specific in vivo targeting of an emerging neoplasm through a single molecular recognition mechanism has not generally been successful. Nonspecific binding and specific binding to non-target cells compromise the therapeutic index of small molecule, ubiquitous cancer targeting ligands. In this work, we have designed and fabricated a nanoparticle (NP) construct that could potentially overcome the current limitations of targeted in vivo delivery. Quantum dots (QDs) were functionalized with a poly(ethylene glycol) (PEG) modified to enable specific cleavage by matrix metalloprotease-7 (MMP-7). The QDs were further functionalized with folic acid, a ligand for a cell surface receptor that is overexpressed in many tumors, but also expressed in some normal tissues. The nanomolecular construct is designed so that the PEG initially conceals the folate ligand and construct binding to cells is inhibited. MMP-7 activated peptide cleavage and subsequent unmasking of the folate ligand occurs only near tumor tissue, resulting in a proximity activated (PA) targeting system. QDs functionalized with both the MMP-7 cleavable substrate and folic acid were successfully synthesized and characterized. The proteolytic capability of the dual ligand QD construct was quantitatively assessed by fluorometric analysis and compared to a QD construct functionalized with only the PA ligand. The dual ligand PA nanoparticles studied here exhibit significant susceptibility to cleavage by MMP-7 at physiologically relevant conditions. The capacity to autonomously convert a biopassivated nanostructure to a tissue-specific targeted delivery agent in vivo represents a paradigm change for site-directed therapies.

  7. Targeted therapies and immune checkpoint inhibitors in the treatment of metastatic melanoma patients: a guide and update for pathologists.

    Science.gov (United States)

    Kakavand, Hojabr; Wilmott, James S; Long, Georgina V; Scolyer, Richard A

    2016-02-01

    The previously dismal prospects for patients with advanced stage metastatic melanoma have greatly improved in recent years. Enhanced understanding of both the pathogenesis of melanoma and its molecular drivers, as well as the importance and regulation of anti-tumour immune responses, have provided new therapeutic opportunities for melanoma patients. There are two major distinct categories of systemic treatments with activity for patients with metastatic melanoma: (1) targeted therapies, which act to inhibit the oncogenes that drive the aberrant growth and dissemination of the tumour; and (2) immune checkpoint inhibitor therapies, which act to enhance anti-tumour immune responses by blocking negative regulators of immunity. Pathologists play a critical and expanding role in the selection of the most appropriate treatment for individual metastatic melanoma patients in the modern era of personalised/precision medicine. The molecular pathology testing of melanoma tumour tissue for the presence of targetable oncogenic mutations is already part of routine practice in many institutions. In addition, other potential oncogenic therapeutic targets continue to be identified and pathology testing techniques must readily adapt to this rapidly changing field. Recent research findings suggest that pathological assessment of tumour associated immune cells and immunosuppressive ligand expression of the tumour are likely to be important in identifying patients most likely to benefit from immune checkpoint inhibitors. Similarly, pathological and molecular observations of on-treatment tumour tissue biopsies taken from patients on targeted therapies have provided new insights into the mechanisms of action of targeted molecular therapies, have contributed to the identification of resistance mechanisms to these novel therapies and may be of higher value for selecting patients most likely to benefit from therapies. These data have already provided a rational biological basis for the

  8. Targeting of Antibodies using Aptamers

    OpenAIRE

    2003-01-01

    The chapter presents a methodology for the rapid selection of aptamers against antibody targets. It is a detailed account of the various methodological steps that describe the selection of aptamers, including PCR steps, buffers to be used, target immobilisation, partitioning and amplification of aptamers, clonning and sequencing, to results in high affinity and specificity ligands for the chosen target antibody.

  9. Analysis of macromolecules, ligands and macromolecule-ligand complexes

    Science.gov (United States)

    Von Dreele, Robert B [Los Alamos, NM

    2008-12-23

    A method for determining atomic level structures of macromolecule-ligand complexes through high-resolution powder diffraction analysis and a method for providing suitable microcrystalline powder for diffraction analysis are provided. In one embodiment, powder diffraction data is collected from samples of polycrystalline macromolecule and macromolecule-ligand complex and the refined structure of the macromolecule is used as an approximate model for a combined Rietveld and stereochemical restraint refinement of the macromolecule-ligand complex. A difference Fourier map is calculated and the ligand position and points of interaction between the atoms of the macromolecule and the atoms of the ligand can be deduced and visualized. A suitable polycrystalline sample of macromolecule-ligand complex can be produced by physically agitating a mixture of lyophilized macromolecule, ligand and a solvent.

  10. Ligand-Receptor Interactions

    CERN Document Server

    Bongrand, Pierre

    2008-01-01

    The formation and dissociation of specific noncovalent interactions between a variety of macromolecules play a crucial role in the function of biological systems. During the last few years, three main lines of research led to a dramatic improvement of our understanding of these important phenomena. First, combination of genetic engineering and X ray cristallography made available a simultaneous knowledg of the precise structure and affinity of series or related ligand-receptor systems differing by a few well-defined atoms. Second, improvement of computer power and simulation techniques allowed extended exploration of the interaction of realistic macromolecules. Third, simultaneous development of a variety of techniques based on atomic force microscopy, hydrodynamic flow, biomembrane probes, optical tweezers, magnetic fields or flexible transducers yielded direct experimental information of the behavior of single ligand receptor bonds. At the same time, investigation of well defined cellular models raised the ...

  11. Therapeutic androgen receptor ligands

    OpenAIRE

    Allan, George F.; Sui, Zhihua

    2003-01-01

    In the past several years, the concept of tissue-selective nuclear receptor ligands has emerged. This concept has come to fruition with estrogens, with the successful marketing of drugs such as raloxifene. The discovery of raloxifene and other selective estrogen receptor modulators (SERMs) has raised the possibility of generating selective compounds for other pathways, including androgens (that is, selective androgen receptor modulators, or SARMs).

  12. KLIFS: a knowledge-based structural database to navigate kinase-ligand interaction space.

    Science.gov (United States)

    van Linden, Oscar P J; Kooistra, Albert J; Leurs, Rob; de Esch, Iwan J P; de Graaf, Chris

    2014-01-23

    Protein kinases regulate the majority of signal transduction pathways in cells and have become important targets for the development of designer drugs. We present a systematic analysis of kinase-ligand interactions in all regions of the catalytic cleft of all 1252 human kinase-ligand cocrystal structures present in the Protein Data Bank (PDB). The kinase-ligand interaction fingerprints and structure database (KLIFS) contains a consistent alignment of 85 kinase ligand binding site residues that enables the identification of family specific interaction features and classification of ligands according to their binding modes. We illustrate how systematic mining of kinase-ligand interaction space gives new insights into how conserved and selective kinase interaction hot spots can accommodate the large diversity of chemical scaffolds in kinase ligands. These analyses lead to an improved understanding of the structural requirements of kinase binding that will be useful in ligand discovery and design studies.

  13. Ligands recognizing the minor groove of DNA: development and applications.

    Science.gov (United States)

    Wemmer, D E

    Polyamide ligands comprised of pyrrole, imidazole and hydroxypyrrole rings have been developed over the past decade which can be used to target many different, predetermined DNA sequences through recognition of functional groups in the minor groove. The design principles for these ligands are described with a description of the characterization of their binding. Variations containing linked recognition modules have been described which allow high affinity and specificity recognition of DNA sequences of over 15 base pairs. Recent applications of these ligands in affecting biological response through competition with proteins for DNA binding sites are reviewed. Copyright 2001 John Wiley & Sons, Inc.

  14. Tumour-associated and non-tumour-associated microbiota in colorectal cancer

    Science.gov (United States)

    Flemer, Burkhardt; Lynch, Denise B; Brown, Jillian M R; Jeffery, Ian B; Ryan, Feargal J; Claesson, Marcus J; O'Riordain, Micheal; Shanahan, Fergus; O'Toole, Paul W

    2017-01-01

    Objective A signature that unifies the colorectal cancer (CRC) microbiota across multiple studies has not been identified. In addition to methodological variance, heterogeneity may be caused by both microbial and host response differences, which was addressed in this study. Design We prospectively studied the colonic microbiota and the expression of specific host response genes using faecal and mucosal samples (‘ON’ and ‘OFF’ the tumour, proximal and distal) from 59 patients undergoing surgery for CRC, 21 individuals with polyps and 56 healthy controls. Microbiota composition was determined by 16S rRNA amplicon sequencing; expression of host genes involved in CRC progression and immune response was quantified by real-time quantitative PCR. Results The microbiota of patients with CRC differed from that of controls, but alterations were not restricted to the cancerous tissue. Differences between distal and proximal cancers were detected and faecal microbiota only partially reflected mucosal microbiota in CRC. Patients with CRC can be stratified based on higher level structures of mucosal-associated bacterial co-abundance groups (CAGs) that resemble the previously formulated concept of enterotypes. Of these, Bacteroidetes Cluster 1 and Firmicutes Cluster 1 were in decreased abundance in CRC mucosa, whereas Bacteroidetes Cluster 2, Firmicutes Cluster 2, Pathogen Cluster and Prevotella Cluster showed increased abundance in CRC mucosa. CRC-associated CAGs were differentially correlated with the expression of host immunoinflammatory response genes. Conclusions CRC-associated microbiota profiles differ from those in healthy subjects and are linked with distinct mucosal gene-expression profiles. Compositional alterations in the microbiota are not restricted to cancerous tissue and differ between distal and proximal cancers. PMID:26992426

  15. GPCR biased ligands as novel heart failure therapeutics.

    Science.gov (United States)

    Violin, Jonathan D; Soergel, David G; Boerrigter, Guido; Burnett, John C; Lark, Michael W

    2013-10-01

    G protein-coupled receptors have been successfully targeted by numerous therapeutics including drugs that have transformed the management of cardiovascular disease. However, many GPCRs, when activated or blocked by drugs, elicit both beneficial and adverse pharmacology. Recent work has demonstrated that in some cases, the salutary and deleterious signals linked to a specific GPCR can be selectively targeted by "biased ligands" that entrain subsets of a receptor's normal pharmacology. This review briefly summarizes the advances and current state of the biased ligand field, focusing on an example: biased ligands targeting the angiotensin II type 1 receptor. These compounds exhibit unique pharmacology, distinct from classic agonists or antagonists, and one such molecule is now in clinical development for the treatment of acute heart failure.

  16. Ru[(bpy)₂(dppz)]²⁺ and Rh[(bpy)₂(chrysi)]³⁺ targeting double strand DNA: the shape of the intercalating ligand tunes the free energy landscape of deintercalation.

    Science.gov (United States)

    Franco, Duvan; Vargiu, Attilio V; Magistrato, Alessandra

    2014-08-04

    Octahedral metal complexes can bind to double strand (ds) DNA either by intercalation or by insertion, this latter mechanism being observed in the case of mismatched base pairs (bps). In this work we modeled the process of deintercalation from the major groove for Δ-Ru[(bpy)2(dppz)](2+) (1) and Δ-Rh[(bpy)2(chrysi)](3+) (2), prototypical examples of metallo-intercalators and metallo-insertors, respectively. By using advanced sampling techniques, we show that the two complexes have comparable deintercalation barriers and that in both systems the main cost of deintercalation is due to disruption of π-π stacking interactions between the intercalating moiety and the bps flanking the binding site. A striking difference between dppz and chrysi is found in their intercalation modes, being their longest axes, respectively, perpendicular and parallel to the P-P direction between opposite DNA strands. This leads the two ligands to deintercalate from the DNA through different mechanisms. Compound 1 goes through the formation of a metastable short-lived intermediate, with an overall free energy barrier of ~14.5 kcal/mol, in line with experimental findings. Due to the length of the dppz intercalating moiety, an extended plateau appears in the free energy landscape at ~3 kcal/mol above the most stable minimum. Compound 2 must cross a similar barrier (~15.5 kcal/mol), but does not form intermediates along the deintercalation path, and the deintercalation profile is steeper than that found for 1. Thus, the shape of the intercalating moiety affects the deintercalation mechanism of these inorganic molecules. This work is a first step to rationalize from a computational perspective the factors tuning the preferential binding mode of inorganic molecules (such as diagnostic probes, therapeutic agents, or regulators of DNA expression) to ds DNA.

  17. Comprehensive assessment of flexible-ligand docking algorithms: current effectiveness and challenges.

    Science.gov (United States)

    Huang, Sheng-You

    2017-03-14

    Protein-ligand docking has been playing an important role in modern drug discovery. To model drug-target binding in real systems, a number of flexible-ligand docking algorithms with different sampling strategies and scoring methods have been subsequently developed over the past three decades, while rigid-ligand docking is still being used because of its compelling computational efficiency. Here, a comprehensive assessment has been conducted to investigate the effectiveness of flexible-ligand docking versus rigid-ligand docking for three representative docking algorithms (global optimization, incremental construction and multi-conformer docking) in virtual screening and pose prediction on the Directory of Useful Decoys. It was found that overall flexible-ligand docking did not achieve a statistically significant improvement in enrichments over rigid-ligand docking in virtual screening, but all docking programs significantly improved the success rates when considering ligand flexibility in pose prediction. The worse effectiveness of flexible-ligand docking in virtual screening than in pose prediction suggests that the challenges of current docking algorithms exist in ranking more than docking, although the use of flexible-ligand docking in virtual screening was justified by its better effectiveness for more flexible ligand in virtual screening. Challenges for scoring, including internal energy, charge polarization, entropy and flexibility, were investigated and discussed. An empirical way was also proposed to consider loss of ligand conformational entropy for virtual screening. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. Identification of Soft Matter Binding Peptide Ligands Using Phage Display.

    Science.gov (United States)

    Günay, Kemal Arda; Klok, Harm-Anton

    2015-10-21

    Phage display is a powerful tool for the selection of highly affine, short peptide ligands. While originally primarily used for the identification of ligands to proteins, the scope of this technique has significantly expanded over the past two decades. Phage display nowadays is also increasingly applied to identify ligands that selectively bind with high affinity to a broad range of other substrates including natural and biological polymers as well as a variety of low-molecular-weight organic molecules. Such peptides are of interest for various reasons. The ability to selectively and with high affinity bind to the substrate of interest allows the conjugation or immobilization of, e.g., nanoparticles or biomolecules, or generally, facilitates interactions at materials interfaces. On the other hand, presentation of peptide ligands that selectively bind to low-molecular-weight organic materials is of interest for the development of sensor surfaces. The aim of this article is to highlight the opportunities provided by phage display for the identification of peptide ligands that bind to synthetic or natural polymer substrates or to small organic molecules. The article will first provide an overview of the different peptide ligands that have been identified by phage display that bind to these "soft matter" targets. The second part of the article will discuss the different characterization techniques that allow the determination of the affinity of the identified ligands to the respective substrates.

  19. Melatonin: functions and ligands.

    Science.gov (United States)

    Singh, Mahaveer; Jadhav, Hemant R

    2014-09-01

    Melatonin is a chronobiotic substance that acts as synchronizer by stabilizing bodily rhythms. Its synthesis occurs in various locations throughout the body, including the pineal gland, skin, lymphocytes and gastrointestinal tract (GIT). Its synthesis and secretion is controlled by light and dark conditions, whereby light decreases and darkness increases its production. Thus, melatonin is also known as the 'hormone of darkness'. Melatonin and analogs that bind to the melatonin receptors are important because of their role in the management of depression, insomnia, epilepsy, Alzheimer's disease (AD), diabetes, obesity, alopecia, migraine, cancer, and immune and cardiac disorders. In this review, we discuss the mechanism of action of melatonin in these disorders, which could aid in the design of novel melatonin receptor ligands.

  20. Consensus virtual screening approaches to predict protein ligands.

    Science.gov (United States)

    Kukol, Andreas

    2011-09-01

    In order to exploit the advantages of receptor-based virtual screening, namely time/cost saving and specificity, it is important to rely on algorithms that predict a high number of active ligands at the top ranks of a small molecule database. Towards that goal consensus methods combining the results of several docking algorithms were developed and compared against the individual algorithms. Furthermore, a recently proposed rescoring method based on drug efficiency indices was evaluated. Among AutoDock Vina 1.0, AutoDock 4.2 and GemDock, AutoDock Vina was the best performing single method in predicting high affinity ligands from a database of known ligands and decoys. The rescoring of predicted binding energies with the water/octanol partition coefficient did not lead to an improvement averaged over ten receptor targets. Various consensus algorithms were investigated and a simple combination of AutoDock and AutoDock Vina results gave the most consistent performance that showed early enrichment of known ligands for all receptor targets investigated. In case a number of ligands is known for a specific target, every method proposed in this study should be evaluated. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  1. Apoptosis Induction by Targeting Interferon Gamma Receptor 2 (IFNgammaR2) in Prostate Cancer: Ligand (IFNgamma) Independent Novel Function of IFNgammaR2 as a Bax Inhibitor

    Science.gov (United States)

    2016-10-01

    Accomplished experiments 10 NFkB inhibitor ( Parthenolide ) suppressed IFNγR2 expression To develop technologies targeting IFNγR2, we proposed...inhibitor. Parthenolide is a plant-derived compound which is known to inhibit NFkB activity[9, 10]. In our preliminary study, we found that parthenolide ...line (Fig.12. We found that IFNγR2 expression was suppressed by parthenolide (from 5 uM) within 1 day after the treatment (Fig. 12 shows the result of

  2. Apoptosis Induction by Targeting Interferon Gamma Receptor 2 (IFNgammaR2) in Prostate Cancer: Ligand (IFNgamma)-Independent Novel Function of IFNgammaR2 as a Bax Inhibitor

    Science.gov (United States)

    2015-08-01

    inhibitor of Bax. Bax is a key mediator of apoptosis. We found that IFNγR2 is overexpressed in prostate cancer, and we hypothesize that abnormally high...We found that IFNγR2 levels are abnormally elevated in prostate cancer cell lines. Short hairpin (sh) RNA- mediated knockdown of IFNγR2 was able to... enhances Bax activation. (Months 1-24) Task 2: To identify the subtype of prostate cancer that can be effectively treated by IFNγR2-targeting

  3. Solution NMR Structure of a Ligand/Hybrid-2-G-Quadruplex Complex Reveals Rearrangements that Affect Ligand Binding.

    Science.gov (United States)

    Wirmer-Bartoschek, Julia; Bendel, Lars Erik; Jonker, Hendrik R A; Grün, J Tassilo; Papi, Francesco; Bazzicalupi, Carla; Messori, Luigi; Gratteri, Paola; Schwalbe, Harald

    2017-06-12

    Telomeric G-quadruplexes have recently emerged as drug targets in cancer research. Herein, we present the first NMR structure of a telomeric DNA G-quadruplex that adopts the biologically relevant hybrid-2 conformation in a ligand-bound state. We solved the complex with a metalorganic gold(III) ligand that stabilizes G-quadruplexes. Analysis of the free and bound structures reveals structural changes in the capping region of the G-quadruplex. The ligand is sandwiched between one terminal G-tetrad and a flanking nucleotide. This complex structure involves a major structural rearrangement compared to the free G-quadruplex structure as observed for other G-quadruplexes in different conformations, invalidating simple docking approaches to ligand-G-quadruplex structure determination. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. The therapeutic potential of allosteric ligands for free fatty acid sensitive GPCRs

    OpenAIRE

    Hudson, Brian D.; Ulven, Trond; Milligan, Graeme

    2013-01-01

    G protein coupled receptors (GPCRs) are the most historically successful therapeutic targets. Despite this success there are many important aspects of GPCR pharmacology and function that have yet to be exploited to their full therapeutic potential. One in particular that has been gaining attention in recent times is that of GPCR ligands that bind to allosteric sites on the receptor distinct from the orthosteric site of the endogenous ligand. As therapeutics, allosteric ligands possess many th...

  5. Macrocyclic G-quadruplex ligands

    DEFF Research Database (Denmark)

    Nielsen, M C; Ulven, Trond

    2010-01-01

    G-quadruplex stabilizing compounds have recently received increased interest due to their potential application as anticancer therapeutics. A significant number of structurally diverse G-quadruplex ligands have been developed. Some of the most potent and selective ligands currently known are macr...

  6. Ligand-based virtual screening under partial shape constraints

    Science.gov (United States)

    von Behren, Mathias M.; Rarey, Matthias

    2017-03-01

    Ligand-based virtual screening has proven to be a viable technology during the search for new lead structures in drug discovery. Despite the rapidly increasing number of published methods, meaningful shape matching as well as ligand and target flexibility still remain open challenges. In this work, we analyze the influence of knowledge-based sterical constraints on the performance of the recently published ligand-based virtual screening method mRAISE. We introduce the concept of partial shape matching enabling a more differentiated view on chemical structure. The new method is integrated into the LBVS tool mRAISE providing multiple options for such constraints. The applied constraints can either be derived automatically from a protein-ligand complex structure or by manual selection of ligand atoms. In this way, the descriptor directly encodes the fit of a ligand into the binding site. Furthermore, the conservation of close contacts between the binding site surface and the query ligand can be enforced. We validated our new method on the DUD and DUD-E datasets. Although the statistical performance remains on the same level, detailed analysis reveal that for certain and especially very flexible targets a significant improvement can be achieved. This is further highlighted looking at the quality of calculated molecular alignments using the recently introduced mRAISE dataset. The new partial shape constraints improved the overall quality of molecular alignments especially for difficult targets with highly flexible or different sized molecules. The software tool mRAISE is freely available on Linux operating systems for evaluation purposes and academic use (see http://www.zbh.uni-hamburg.de/raise).

  7. Ligand-based virtual screening under partial shape constraints

    Science.gov (United States)

    von Behren, Mathias M.; Rarey, Matthias

    2017-04-01

    Ligand-based virtual screening has proven to be a viable technology during the search for new lead structures in drug discovery. Despite the rapidly increasing number of published methods, meaningful shape matching as well as ligand and target flexibility still remain open challenges. In this work, we analyze the influence of knowledge-based sterical constraints on the performance of the recently published ligand-based virtual screening method mRAISE. We introduce the concept of partial shape matching enabling a more differentiated view on chemical structure. The new method is integrated into the LBVS tool mRAISE providing multiple options for such constraints. The applied constraints can either be derived automatically from a protein-ligand complex structure or by manual selection of ligand atoms. In this way, the descriptor directly encodes the fit of a ligand into the binding site. Furthermore, the conservation of close contacts between the binding site surface and the query ligand can be enforced. We validated our new method on the DUD and DUD-E datasets. Although the statistical performance remains on the same level, detailed analysis reveal that for certain and especially very flexible targets a significant improvement can be achieved. This is further highlighted looking at the quality of calculated molecular alignments using the recently introduced mRAISE dataset. The new partial shape constraints improved the overall quality of molecular alignments especially for difficult targets with highly flexible or different sized molecules. The software tool mRAISE is freely available on Linux operating systems for evaluation purposes and academic use (see http://www.zbh.uni-hamburg.de/raise).

  8. RAGE and its ligands in retinal disease.

    Science.gov (United States)

    Barile, Gaetano R; Schmidt, Ann M

    2007-12-01

    RAGE, the receptor for advanced glycation endproducts (AGEs), is a multiligand signal transduction receptor of the immunoglobulin superfamily of cell surface molecules that has been implicated in the pathogenesis of diabetic complications, neurodegenerative diseases, inflammatory disorders, and cancer. These diverse biologic disorders reflect the multiplicity of ligands capable of cellular interaction via RAGE that include, in addition to AGEs, amyloid-beta (Abeta) peptide, the S100/calgranulin family of proinflammatory cytokines, and amphoterin, a member of the High Mobility Group Box (HMGB) DNA-binding proteins. In the retina, RAGE expression is present in neural cells, the vasculature, and RPE cells, and it has also been detected in pathologic cellular retinal responses including epiretinal and neovascular membrane formation. Ligands for RAGE, in particular AGEs, have emerged as relevant to the pathogenesis of diabetic retinopathy and age-related macular disease. While the understanding of RAGE and its role in retinal dysfunction with aging, diabetes mellitus, and/or activation of pro-inflammatory pathways is less complete compared to other organ systems, increasing evidence indicates that RAGE can initiate and sustain significant cellular perturbations in the inner and outer retina. For these reasons, antagonism of RAGE interactions with its ligands may be a worthwhile therapeutic target in such seemingly disparate, visually threatening retinal diseases as diabetic retinopathy, age-related macular degeneration, and proliferative vitreoretinopathy.

  9. A library screening approach identifies naturally occurring RNA sequences for a G-quadruplex binding ligand.

    Science.gov (United States)

    Mirihana Arachchilage, Gayan; Morris, Mark J; Basu, Soumitra

    2014-02-07

    An RNA G-quadruplex library was synthesised and screened against kanamycin A as the ligand. Naturally occurring G-quadruplex forming sequences that differentially bind to kanamycin A were identified and characterized. This provides a simple and effective strategy for identification of potential intracellular G-quadruplex targets for a ligand.

  10. An ontology for pharmaceutical ligands and its application for in silico screening and library design.

    Science.gov (United States)

    Schuffenhauer, Ansgar; Zimmermann, Jürg; Stoop, Ruedi; van der Vyver, Jan-Jan; Lecchini, Steffano; Jacoby, Edgar

    2002-01-01

    Annotation efforts in biosciences have focused in past years mainly on the annotation of genomic sequences. Only very limited effort has been put into annotation schemes for pharmaceutical ligands. Here we propose annotation schemes for the ligands of four major target classes, enzymes, G protein-coupled receptors (GPCRs), nuclear receptors (NRs), and ligand-gated ion channels (LGICs), and outline their usage for in silico screening and combinatorial library design. The proposed schemes cover ligand functionality and hierarchical levels of target classification. The classification schemes are based on those established by the EC, GPCRDB, NuclearDB, and LGICDB. The ligands of the MDL Drug Data Report (MDDR) database serve as a reference data set of known pharmacologically active compounds. All ligands were annotated according to the schemes when attribution was possible based on the activity classification provided by the reference database. The purpose of the ligand-target classification schemes is to allow annotation-based searching of the ligand database. In addition, the biological sequence information of the target is directly linkable to the ligand, hereby allowing sequence similarity-based identification of ligands of next homologous receptors. Ligands of specified levels can easily be retrieved to serve as comprehensive reference sets for cheminformatics-based similarity searches and for design of target class focused compound libraries. Retrospective in silico screening experiments within the MDDR01.1 database, searching for structures binding to dopamine D2, all dopamine receptors and all amine-binding class A GPCRs using known dopamine D2 binding compounds as a reference set, have shown that such reference sets are in particular useful for the identification of ligands binding to receptors closely related to the reference system. The potential for ligand identification drops with increasing phylogenetic distance. The analysis of the focus of a tertiary

  11. Automated docking of flexible ligands: applications of AutoDock.

    Science.gov (United States)

    Goodsell, D S; Morris, G M; Olson, A J

    1996-01-01

    AutoDock is a suite of C programs used to predict the bound conformations of a small, flexible ligand to a macromolecular target of known structure. The technique combines simulated annealing for conformation searching with a rapid grid-based method of energy evaluation. This paper reviews recent applications of the technique and describes the enhancements included in the current release.

  12. Glutamate receptor ligands

    DEFF Research Database (Denmark)

    Guldbrandt, Mette; Johansen, Tommy N; Frydenvang, Karla Andrea;

    2002-01-01

    Homologation and substitution on the carbon backbone of (S)-glutamic acid [(S)-Glu, 1], as well as absolute stereochemistry, are structural parameters of key importance for the pharmacological profile of (S)-Glu receptor ligands. We describe a series of methyl-substituted 2-aminoadipic acid (AA......-ray crystallographic analyses, chemical correlation, and CD spectral analyses. The effects of the individual stereoisomers at ionotropic and metabotropic (S)-Glu receptors (iGluRs and mGluRs) were characterized. Compounds with S-configuration at the alpha-carbon generally showed mGluR2 agonist activity of similar...... limited effect on pharmacology. Structure-activity relationships at iGluRs in the rat cortical wedge preparation showed a complex pattern, some compounds being NMDA receptor agonists [e.g., EC(50) =110 microM for (2S,5RS)-5-methyl-AA (6a,b)] and some compounds showing NMDA receptor antagonist effects [e...

  13. Targeting Platinum Compounds: synthesis and biological activity

    OpenAIRE

    VAN ZUTPHEN, Steven

    2005-01-01

    Inspired by cisplatin, the inorganic drug discovered by Barnett Rosenberg in 1965, the research described in this thesis uses targeting ligands, or ligands varied in a combinatorial fashion, to find platinum complexes with more specific modes of action. These studies have lead to the development of novel (solid-phase) synthetic methods and to the discovery of several compounds with promising biological properties.

  14. Targeting Platinum Compounds : synthesis and biological activity

    NARCIS (Netherlands)

    Zutphen, Steven van

    2005-01-01

    Inspired by cisplatin, the inorganic drug discovered by Barnett Rosenberg in 1965, the research described in this thesis uses targeting ligands, or ligands varied in a combinatorial fashion, to find platinum complexes with more specific modes of action. These studies have lead to the development of

  15. Orphan receptor ligand discovery by pickpocketing pharmacological neighbors.

    Science.gov (United States)

    Ngo, Tony; Ilatovskiy, Andrey V; Stewart, Alastair G; Coleman, James L J; McRobb, Fiona M; Riek, R Peter; Graham, Robert M; Abagyan, Ruben; Kufareva, Irina; Smith, Nicola J

    2017-02-01

    Understanding the pharmacological similarity of G protein-coupled receptors (GPCRs) is paramount for predicting ligand off-target effects, drug repurposing, and ligand discovery for orphan receptors. Phylogenetic relationships do not always correctly capture pharmacological similarity. Previous family-wide attempts to define pharmacological relationships were based on three-dimensional structures and/or known receptor-ligand pairings, both unavailable for orphan GPCRs. Here, we present GPCR-CoINPocket, a novel contact-informed neighboring pocket metric of GPCR binding-site similarity that is informed by patterns of ligand-residue interactions observed in crystallographically characterized GPCRs. GPCR-CoINPocket is applicable to receptors with unknown structure or ligands and accurately captures known pharmacological relationships between GPCRs, even those undetected by phylogeny. When applied to orphan receptor GPR37L1, GPCR-CoINPocket identified its pharmacological neighbors, and transfer of their pharmacology aided in discovery of the first surrogate ligands for this orphan with a 30% success rate. Although primarily designed for GPCRs, the method is easily transferable to other protein families.

  16. Riboswitch structure in the ligand-free state.

    Science.gov (United States)

    Liberman, Joseph A; Wedekind, Joseph E

    2012-01-01

    Molecular investigations of riboswitches bound to small-molecule effectors have produced a wealth of information on how these molecules achieve high affinity and specificity for a target ligand. X-ray crystal structures have been determined for the ligand-free state for representatives of the preQ₁-I, S-adenosylmethionine I, lysine, and glycine aptamer classes. These structures in conjunction with complimentary techniques, such as in-line probing, NMR spectroscopy, Förster resonance energy transfer, small-angle scattering, and computational simulations, have demonstrated that riboswitches adopt multiple conformations in the absence of ligand. Despite a number of investigations that support ligand-dependent folding, mounting evidence suggests that free-state riboswitches interact with their effectors in the sub-populations of largely prefolded states as embodied by the principle of conformational selection, which has been documented extensively for protein-mediated ligand interactions. Fundamental riboswitch investigations of the bound and free states have advanced our understanding of RNA folding, ligand recognition, and how these factors culminate in communication between an aptamer and its expression platform. An understanding of these topics is essential to comprehend riboswitch gene regulation at the molecular level, which has already provided a basis to understand the mechanism of action of natural antimicrobials.

  17. Identification of inhibitors against the potential ligandable sites in the active cholera toxin.

    Science.gov (United States)

    Gangopadhyay, Aditi; Datta, Abhijit

    2015-04-01

    The active cholera toxin responsible for the massive loss of water and ions in cholera patients via its ADP ribosylation activity is a heterodimer of the A1 subunit of the bacterial holotoxin and the human cytosolic ARF6 (ADP Ribosylation Factor 6). The active toxin is a potential target for the design of inhibitors against cholera. In this study we identified the potential ligandable sites of the active cholera toxin which can serve as binding sites for drug-like molecules. By employing an energy-based approach to identify ligand binding sites, and comparison with the results of computational solvent mapping, we identified two potential ligandable sites in the active toxin which can be targeted during structure-based drug design against cholera. Based on the probe affinities of the identified ligandable regions, docking-based virtual screening was employed to identify probable inhibitors against these sites. Several indole-based alkaloids and phosphates showed strong interactions to the important residues of the ligandable region at the A1 active site. On the other hand, 26 top scoring hits were identified against the ligandable region at the A1 ARF6 interface which showed strong hydrogen bonding interactions, including guanidines, phosphates, Leucopterin and Aristolochic acid VIa. This study has important implications in the application of hybrid structure-based and ligand-based methods against the identified ligandable sites using the identified inhibitors as reference ligands, for drug design against the active cholera toxin.

  18. Inside job: ligand-receptor pharmacology beneath the plasma membrane.

    Science.gov (United States)

    Babcock, Joseph J; Li, Min

    2013-07-01

    Most drugs acting on the cell surface receptors are membrane permeable and thus able to engage their target proteins in different subcellular compartments. However, these drugs' effects on cell surface receptors have historically been studied on the plasma membrane alone. Increasing evidence suggests that small molecules may also modulate their targeted receptors through membrane trafficking or organelle-localized signaling inside the cell. These additional modes of interaction have been reported for functionally diverse ligands of GPCRs, ion channels, and transporters. Such intracellular drug-target engagements affect cell surface expression. Concurrent intracellular and cell surface signaling may also increase the complexity and therapeutic opportunities of small molecule modulation. Here we discuss examples of ligand-receptor interactions that are present in both intra- and extracellular sites, and the potential therapeutic opportunities presented by this phenomenon.

  19. Inside job: ligand-receptor pharmacology beneath the plasma membrane

    Institute of Scientific and Technical Information of China (English)

    Joseph J BABCOCK; Min LI

    2013-01-01

    Most drugs acting on the cell surface receptors are membrane permeable and thus able to engage their target proteins in different subcellular compartments.However,these drugs' effects on cell surface receptors have historically been studied on the plasma membrane alone.Increasing evidence suggests that small molecules may also modulate their targeted receptors through membrane trafficking or organelle-localized signaling inside the cell.These additional modes of interaction have been reported for functionally diverse ligands of GPCRs,ion channels,and transporters.Such intracellular drug-target engagements affect cell surface expression.Concurrent intracellular and cell surface signaling may also increase the complexity and therapeutic opportunities of small molecule modulation.Here we discuss examples of ligand-receptor interactions that are present in both intra- and extracellular sites,and the potential therapeutic opportunities presented by this phenomenon.

  20. Advances Towards The Discovery of GPR55 Ligands.

    Science.gov (United States)

    Morales, Paula; Jagerovic, Nadine

    2016-01-01

    The G-protein-coupled receptor 55 (GPR55) was identified in 1999. It was proposed as a novel member of the endocannabinoid system due to the fact that some endogenous, plant-derived and synthetic cannabinoid ligands act on GPR55. However, the complexity of the cellular downstream signaling pathways related to GPR55 activation delayed the discovery of selective GPR55 ligands. It was only a few years ago that the high throughput screening of libraries of pharmaceutical companies and governmental organizations allowed to identify selective GPR55 agonists and antagonists. Since then, several GPR55 modulator scaffolds have been reported. The relevance of GPR55 has been explored in diverse physiological and pathological processes revealing its role in inflammation, neuropathic pain, bone physiology, diabetes and cancer. Considering GPR55 as a new promising therapeutic target, there is a clear need for new selective and potent GPR55 modulators. This review will address a current structural update of GPR55 ligands.

  1. Activation of Neuropeptide FF Receptors by Kisspeptin Receptor Ligands.

    Science.gov (United States)

    Oishi, Shinya; Misu, Ryosuke; Tomita, Kenji; Setsuda, Shohei; Masuda, Ryo; Ohno, Hiroaki; Naniwa, Yousuke; Ieda, Nahoko; Inoue, Naoko; Ohkura, Satoshi; Uenoyama, Yoshihisa; Tsukamura, Hiroko; Maeda, Kei-Ichiro; Hirasawa, Akira; Tsujimoto, Gozoh; Fujii, Nobutaka

    2011-01-13

    Kisspeptin is a member of the RFamide neuropeptide family that is implicated in gonadotropin secretion. Because kisspeptin-GPR54 signaling is implicated in the neuroendocrine regulation of reproduction, GPR54 ligands represent promising therapeutic agents against endocrine secretion disorders. In the present study, the selectivity profiles of GPR54 agonist peptides were investigated for several GPCRs, including RFamide receptors. Kisspeptin-10 exhibited potent binding and activation of neuropeptide FF receptors (NPFFR1 and NPFFR2). In contrast, short peptide agonists bound with much lower affinity to NPFFRs while showing relatively high selectivity toward GPR54. The possible localization of secondary kisspeptin targets was also demonstrated by variation in the levels of GnRH release from the median eminence and the type of GPR54 agonists used. Negligible affinity of the reported NPFFR ligands to GPR54 was observed and indicates the unidirectional cross-reactivity between both ligands.

  2. DINC: a new AutoDock-based protocol for docking large ligands.

    Science.gov (United States)

    Dhanik, Ankur; McMurray, John S; Kavraki, Lydia E

    2013-01-01

    Using the popular program AutoDock, computer-aided docking of small ligands with 6 or fewer rotatable bonds, is reasonably fast and accurate. However, docking large ligands using AutoDock's recommended standard docking protocol is less accurate and computationally slow. In our earlier work, we presented a novel AutoDock-based incremental protocol (DINC) that addresses the limitations of AutoDock's standard protocol by enabling improved docking of large ligands. Instead of docking a large ligand to a target protein in one single step as done in the standard protocol, our protocol docks the large ligand in increments. In this paper, we present three detailed examples of docking using DINC and compare the docking results with those obtained using AutoDock's standard protocol. We summarize the docking results from an extended docking study that was done on 73 protein-ligand complexes comprised of large ligands. We demonstrate not only that DINC is up to 2 orders of magnitude faster than AutoDock's standard protocol, but that it also achieves the speed-up without sacrificing docking accuracy. We also show that positional restraints can be applied to the large ligand using DINC: this is useful when computing a docked conformation of the ligand. Finally, we introduce a webserver for docking large ligands using DINC. Docking large ligands using DINC is significantly faster than AutoDock's standard protocol without any loss of accuracy. Therefore, DINC could be used as an alternative protocol for docking large ligands. DINC has been implemented as a webserver and is available at http://dinc.kavrakilab.org. Applications such as therapeutic drug design, rational vaccine design, and others involving large ligands could benefit from DINC and its webserver implementation.

  3. Visualization of Metal-to-Ligand and Ligand-to-Ligand Charge Transfer in Metal-Ligand Complexes

    Institute of Scientific and Technical Information of China (English)

    Yong Ding; Jian-xiu Guo; Xiang-si Wang; Sha-sha Liu; Feng-cai Ma

    2009-01-01

    Three methods including the atomic resolved density of state, charge difference density, and the transition density matrix are used to visualize metal to ligand charge transfer (MLCT) in ruthenium(Ⅱ) ammine complex. The atomic resolved density of state shows that there is density of Ru on the HOMOs. All the density is localized on the ammine, which reveals that the excited electrons in the Ru complex are delocalized over the ammine ligand. The charge difference density shows that all the holes are localized on the Ru and the electrons on the ammine. The localization explains the MLCT on excitation. The transition density matrix shows that there is electron-hole coherence between Ru and ammine. These methods are also used to examine the MLCT in Os(bpy)(p0p)Cl ("Osp0p"; bpy=2,2'-bipyridyl; p0p=4,4'-bipyridyl) and the ligand-to-ligand charge transfer (LLCT) in Alq3. The calculated results show that these methods are powerful to examine MLCT and LLCT in the metal-ligand system.

  4. Molecular Recognition and Ligand Association

    Science.gov (United States)

    Baron, Riccardo; McCammon, J. Andrew

    2013-04-01

    We review recent developments in our understanding of molecular recognition and ligand association, focusing on two major viewpoints: (a) studies that highlight new physical insight into the molecular recognition process and the driving forces determining thermodynamic signatures of binding and (b) recent methodological advances in applications to protein-ligand binding. In particular, we highlight the challenges posed by compensating enthalpic and entropic terms, competing solute and solvent contributions, and the relevance of complex configurational ensembles comprising multiple protein, ligand, and solvent intermediate states. As more complete physics is taken into account, computational approaches increase their ability to complement experimental measurements, by providing a microscopic, dynamic view of ensemble-averaged experimental observables. Physics-based approaches are increasingly expanding their power in pharmacology applications.

  5. Why mercury prefers soft ligands

    Energy Technology Data Exchange (ETDEWEB)

    Riccardi, Demian M [ORNL; Guo, Hao-Bo [ORNL; Gu, Baohua [ORNL; Parks, Jerry M [ORNL; Summers, Anne [University of Georgia, Athens, GA; Miller, S [University of California, San Francisco; Liang, Liyuan [ORNL; Smith, Jeremy C [ORNL

    2013-01-01

    Mercury (Hg) is a major global pollutant arising from both natural and anthropogenic sources. Defining the factors that determine the relative affinities of different ligands for the mercuric ion, Hg2+, is critical to understanding its speciation, transformation, and bioaccumulation in the environment. Here, we use quantum chemistry to dissect the relative binding free energies for a series of inorganic anion complexes of Hg2+. Comparison of Hg2+ ligand interactions in the gaseous and aqueous phases shows that differences in interactions with a few, local water molecules led to a clear periodic trend within the chalcogenide and halide groups and resulted in the well-known experimentally observed preference of Hg2+ for soft ligands such as thiols. Our approach establishes a basis for understanding Hg speciation in the biosphere.

  6. A modified fluorescent intercalator displacement assay for RNA ligand discovery.

    Science.gov (United States)

    Asare-Okai, Papa Nii; Chow, Christine S

    2011-01-15

    Fluorescent intercalator displacement (FID) is a convenient and practical tool for identifying new nucleic acid-binding ligands. The success of FID is based on the fact that it can be fashioned into a versatile screening assay for assessing the relative binding affinities of compounds to nucleic acids. FID is a tagless approach; the target RNAs and the ligands or small molecules under investigation do not need to be modified in order to be examined. In this study, a modified FID assay for screening RNA-binding ligands was established using 3-methyl-2-((1-(3-(trimethylammonio)propyl)-4-quinolinylidene)methyl)benzothiazolium (TO-PRO) as the fluorescent indicator. Electrospray ionization mass spectrometry (ESI-MS) results provide direct evidence that correlates the reduction in fluorescence intensity observed in the FID assay with displacement of the dye molecule from RNA. The assay was successfully applied to screen a variety of RNA-binding ligands with a set of small hairpin RNAs. Ligands that bind with moderate affinity to the chosen RNA constructs (A-site, TAR [transactivation response element], h31 [helix 31], and H69 [helix 69] were identified. Copyright © 2010 Elsevier Inc. All rights reserved.

  7. Continuous microfluidic assortment of interactive ligands (CMAIL)

    Science.gov (United States)

    Hsiao, Yi-Hsing; Huang, Chao-Yang; Hu, Chih-Yung; Wu, Yen-Yu; Wu, Chung-Hsiun; Hsu, Chia-Hsien; Chen, Chihchen

    2016-08-01

    Finding an interactive ligand-receptor pair is crucial to many applications, including the development of monoclonal antibodies. Biopanning, a commonly used technique for affinity screening, involves a series of washing steps and is lengthy and tedious. Here we present an approach termed continuous microfluidic assortment of interactive ligands, or CMAIL, for the screening and sorting of antigen-binding single-chain variable antibody fragments (scFv) displayed on bacteriophages (phages). Phages carrying native negative charges on their coat proteins were electrophoresed through a hydrogel matrix functionalized with target antigens under two alternating orthogonal electric fields. During the weak horizontal electric field phase, phages were differentially swept laterally depending on their affinity for the antigen, and all phages were electrophoresed down to be collected during the strong vertical electric field phase. Phages of different affinity were spatially separated, allowing the continuous operation. More than 105 CFU (colony forming unit) antigen-interacting phages were isolated with ~100% specificity from a phage library containing 3 × 109 individual members within 40 minutes of sorting using CMAIL. CMAIL is rapid, sensitive, specific, and does not employ washing, elution or magnetic beads. In conclusion, we have developed an efficient and cost-effective method for isolating and sorting affinity reagents involving phage display.

  8. Computational design of nanoparticle drug delivery systems for selective targeting.

    Science.gov (United States)

    Duncan, Gregg A; Bevan, Michael A

    2015-10-01

    Ligand-functionalized nanoparticles capable of selectively binding to diseased versus healthy cell populations are attractive for improved efficacy of nanoparticle-based drug and gene therapies. However, nanoparticles functionalized with high affinity targeting ligands may lead to undesired off-target binding to healthy cells. In this work, Monte Carlo simulations were used to quantitatively determine net surface interactions, binding valency, and selectivity between targeted nanoparticles and cell surfaces. Dissociation constant, KD, and target membrane protein density, ρR, are explored over a range representative of healthy and cancerous cell surfaces. Our findings show highly selective binding to diseased cell surfaces can be achieved with multiple, weaker affinity targeting ligands that can be further optimized by varying the targeting ligand density, ρL. Using the approach developed in this work, nanomedicines can be optimally designed for exclusively targeting diseased cells and tissues.

  9. Development and evaluation of targeting ligands surface modified paclitaxel nanocrystals.

    Science.gov (United States)

    Sohn, Jeong Sun; Yoon, Doo-Soo; Sohn, Jun Youn; Park, Jeong-Sook; Choi, Jin-Seok

    2017-03-01

    To overcome the toxicity of excipient or blank nanoparticles for drug delivery nano-system, the surface modified paclitaxel nanocrystals (PTX-NC) have been developed. PTX-NCs were prepared by nano-precipitation method. The surface of PTX-NCs were modified by grafting with apo-transferrin (Tf) or hyaluronic acid (HA). The physical properties of PTX-NCs were evaluated by field emission scanning electron microscope (FE-SEM), zeta-sizer, zeta-potential, differential scanning calorimetry (DSC) and Fourier transform infrared (FT-IR) spectrometry. In vitro drug release study was performed in phosphate buffered saline (PBS) with or without 0.5% (w/v) Tween 80 for 24h. Cellular uptake was studied at time intervals of 0.5, 1, and 2h in MCF-7 cells, and cell growth inhibition study was performed for 24h using MCF-7 cells (cancer cells), and HaCaT cells (normal cells). Three different types of PTX-NCs with a mean size of 236.0±100.6nm (PTX-NC), 302.0±152.0nm (Tf-PTX-NC) and 339±180.6nm (HA-PTX-NC) were successfully prepared. The drug release profiles showed 29.1%/6.9% (PTX (pure)), 40.7%/23.9% (PTX-NC), 50.5%/25.1% (Tf-PTX-NC) and 46.8/24.8% (HA-PTX-NC) in PBS with/without 0.5% (w/v) Tween 80 for 24h, respectively. As per the results, the drug release of PTX-NCs showed the faster release as compared to that of PTX (pure). Surface modified PTX-NCs exhibited higher values for cell permeability than unmodified PTX-NC in the cellular uptake study. Surface modified PTX-NCs inhibited the cell growth approximately to 60% in MCF-7 cells, however effect of surface modified PTX-NCs on normal cell line was lower than the PTX-NC and PTX (pure). In conclusion, biological macromolecules (Tf or HA) surface modified PTX-NC enhanced the cellular uptake and the cell growth inhibition.

  10. Targeting Ligand Dependent and Ligand Independent Androgen Receptor Signaling in Prostate Cancer

    Science.gov (United States)

    2014-10-01

    transient PPIs. Identifying binding pocket hotspots—small areas of bumps and holes that largely determine binding— can become complicated with respect to...Dricot A, Li N, et al. Towards a proteome-scale map of the human protein-protein interaction network. Nature 2005;437:1173–8. 11. Stelzl U, Worm U

  11. A Tunable Coarse-Grained Model for Ligand-Receptor Interaction

    Science.gov (United States)

    Guantes, Raúl; Miguez, David G.

    2013-01-01

    Cell-surface receptors are the most common target for therapeutic drugs. The design and optimization of next generation synthetic drugs require a detailed understanding of the interaction with their corresponding receptors. Mathematical approximations to study ligand-receptor systems based on reaction kinetics strongly simplify the spatial constraints of the interaction, while full atomistic ligand-receptor models do not allow for a statistical many-particle analysis, due to their high computational requirements. Here we present a generic coarse-grained model for ligand-receptor systems that accounts for the essential spatial characteristics of the interaction, while allowing statistical analysis. The model captures the main features of ligand-receptor kinetics, such as diffusion dependence of affinity and dissociation rates. Our model is used to characterize chimeric compounds, designed to take advantage of the receptor over-expression phenotype of certain diseases to selectively target unhealthy cells. Molecular dynamics simulations of chimeric ligands are used to study how selectivity can be optimized based on receptor abundance, ligand-receptor affinity and length of the linker between both ligand subunits. Overall, this coarse-grained model is a useful approximation in the study of systems with complex ligand-receptor interactions or spatial constraints. PMID:24244115

  12. Lead Generation and Optimization Based on Protein-Ligand Complementarity

    Directory of Open Access Journals (Sweden)

    Koji Ogata

    2010-06-01

    Full Text Available This work proposes a computational procedure for structure-based lead generation and optimization, which relies on the complementarity of the protein-ligand interactions. This procedure takes as input the known structure of a protein-ligand complex. Retaining the positions of the ligand heavy atoms in the protein binding site it designs structurally similar compounds considering all possible combinations of atomic species (N, C, O, CH3, NH,etc. Compounds are ranked based on a score which incorporates energetic contributions evaluated using molecular mechanics force fields. This procedure was used to design new inhibitor molecules for three serine/threonine protein kinases (p38 MAP kinase, p42 MAP kinase (ERK2, and c-Jun N-terminal kinase 3 (JNK3. For each enzyme, the calculations produce a set of potential inhibitors whose scores are in agreement with IC50 data and Ki values. Furthermore, the native ligands for each protein target, scored within the five top-ranking compounds predicted by our method, one of the top-ranking compounds predicted to inhibit JNK3 was synthesized and his inhibitory activity confirmed against ATP hydrolysis. Our computational procedure is therefore deemed to be a useful tool for generating chemically diverse molecules active against known target proteins.

  13. An approach to rational ligand-design based on a thermodynamic analysis.

    Science.gov (United States)

    Ui, Mihoko; Tsumoto, Kouhei

    2010-11-01

    Thermodynamic analysis is an effective tool in screening of lead-compounds for development of potential drug candidates. In most cases, a ligand achieve high affinity and specificity to a target protein by means of both favorable enthalpy and entropy terms, which can be reflected in binding profiles of Isothermal Titration Calorimetry (ITC). A favorable enthalpy change suggests the contribution of noncovalent contacts such as hydrogen bonding and van der Waals interaction between a ligand and its target protein. In general, optimization of binding enthalpy is more difficult than that of entropies in ligand-design; therefore, it is desirable to choose firstly a lead-compound based on its binding enthalpic gain. In this paper, we demonstrate the utility of thermodynamic approach to ligand screening using anti-ciguatoxin antibody 10C9 as a model of a target protein which possesses a large hydrophobic pocket. As a result of this screening, we have identified three compounds that could bind to the antigen-binding pocket of 10C9 with a few kcal/mol of favorable binding enthalpy. Comparison of their structure with the proper antigen ciguatoxin CTX3C revealed that 10C9 rigorously identifies their cyclic structure and a characteristic hydroxyl group. ITC measurement might be useful and powerful for a rational ligand screening and the optimization of the ligand; the enthalpic gain is an effective index for ligand-design studies.

  14. Novel radioiodinated neuroreceptor ligands

    Energy Technology Data Exchange (ETDEWEB)

    Musachio, J.L.

    1993-01-01

    Since many bioactive compounds do not readily undergo direct labeling with radioisotopes of iodine, the novel prosthetic groups, p-toluenesulfonate esters of (E)- and (Z)-3-(tri-n-butylstannyl)prop-2-en-1-ol, were designed to complement existing methods for radioiodine incorporation. The preparation and synthetic utility of these bifunctional reagents are described. These vinylstannylated alkylating agents were coupled with nucleophilic functionalities (amide nitrogen, secondary amine, tertiary alcohol) in acceptable to excellent yields. Regio- and stereospecific radioiododestannylation with retention of configuration occurred under mild, no-carrier-added conditions to give the corresponding radiolabeled N- or O-iodoallyl analogs in good radiochemical yields with high specific radioactivities. The methodology is versatile and well-suited to selective labeling of small molecules with radioisotopes of iodine. Of particular importance are the N-iodoallyl analogs of spiperone and the O-iodoallyl analog of diprenorphine for in vitro and in vivo studies of dopamine D[sub 2] and opioid receptors. For in vivo studies of central serotonin 5-HT[sub 2] receptors via single photon emission computed tomography (SPECT), novel radioiodinated N1-alkyl-2-iodo-LSD derivatives were synthesized. These target radioligands were prepared in moderate radiochemical yields. D-(+)-N1-ethyl-2-iodo-LSD, EIL, was identified as the most promising candidate of this series. [[sup 125]I]-EIL binds to central 5-HT[sub 2] receptors with high affinity and selectivity in vitro and labels 5-HT[sub 2] receptors in vivo with high specificity. For preparation of EIL labeled with [sup 123]I, an optimized procedure was developed that gave [[sup 123]I]-EIL in acceptable yields. This radioligand allowed visualization of serotonin 5-HT[sub 2] sites in living baboon brain via SPECT. [[sup 123]I]-EIL may serve as an agent for tomographic studies of human cerebral 5-HT[sub 2] receptors in normal and disease states.

  15. A solvated ligand rotamer approach and its application in computational protein design.

    Science.gov (United States)

    Huang, Xiaoqiang; Yang, Ji; Zhu, Yushan

    2013-03-01

    The structure-based design of protein-ligand interfaces with respect to different small molecules is of great significance in the discovery of functional proteins. By statistical analysis of a set of protein-ligand complex structures, it was determined that water-mediated hydrogen bonding at the protein-ligand interface plays a crucial role in governing the binding between the protein and the ligand. Based on the novel statistic results, a solvated ligand rotamer approach was developed to explicitly describe the key water molecules at the protein-ligand interface and a water-mediated hydrogen bonding model was applied in the computational protein design context to complement the continuum solvent model. The solvated ligand rotamer approach produces only one additional solvated rotamer for each rotamer in the ligand rotamer library and does not change the number of side-chain rotamers at each protein design site. This has greatly reduced the total combinatorial number in sequence selection for protein design, and the accuracy of the model was confirmed by two tests. For the water placement test, 61% of the crystal water molecules were predicted correctly in five protein-ligand complex structures. For the sequence recapitulation test, 44.7% of the amino acid identities were recovered using the solvated ligand rotamer approach and the water-mediated hydrogen bonding model, while only 30.4% were recovered when the explicitly bound waters were removed. These results indicated that the developed solvated ligand rotamer approach is promising for functional protein design targeting novel protein-ligand interactions.

  16. A race for RAGE ligands.

    Science.gov (United States)

    Schleicher, Erwin D

    2010-08-01

    In experimental animals a causal involvement of the multiligand receptor for advanced glycation end products (RAGE) in the development of diabetic vascular complications has been demonstrated. However, the nature of RAGE ligands present in patients with diabetic nephropathy has not yet been defined; this leaves open the relevance of the RAGE system to the human disease.

  17. 去唾液酸糖蛋白受体介导的肝靶向脂质体配体的酶促催化合成研究%Study on the Enzyme-Catalyzed Synthesis of Ligands for ASGPR-mediated Liver Targeting Liposomes

    Institute of Scientific and Technical Information of China (English)

    郭波红; 程怡; 林绿萍; 吴卫; 林德晖

    2012-01-01

    目的 用酶促催化乳糖酸与十八胺合成一种可用于镶嵌脂质体表面的去唾液酸糖蛋白靶向配体修饰物.方法 通过红外光谱(IR)、质谱(ESI-MS)和核磁共振(1H-NMR)对产物结构进行确证,并对酶种类、反应介质、酶的加入量、底物摩尔比、反应温度等影响因素进行考察.结果 二甲基亚砜作为反应介质;Novozym 435固定化脂肪酶作为催化剂、酶加入量为400U·mL-1、乳糖酸和十八胺的摩尔比为2:1、40℃下反应24 h,十八胺的转化率可达99%以上.结论 酶促催化法可用来合成肝靶向脂质体配体.%OBJECTIVE To synthesize asialoglycoprotein receptor ligand-targeted modifier which is used to insert the surface of liposome by enzyme-catalyzed amidation of lactobionic acid and stearamine. METHODS The structure of the product was confirmed-by IR, ESI-MS and ' H-NMR. The effects of types and quantity of enzyme, organic solvents, molar ratio of substrate and temperature of reaction were studied. RESULTS When using DMSO as reaction medium, Novozym 435 immobilized lipase at 400 U ? mL ~ , molar ratio of lactobionic acid to stearamine at 2- 1, and reacting at 40 °C for 24 h, the transformation of stearamine reached more than 99%. CONCLUSION The enzyme catalysis is useful for synthesizing liver targeting liposomes.

  18. Rational design of metal coordination compounds with azomethine ligands

    Energy Technology Data Exchange (ETDEWEB)

    Garnovskii, Alexander D; Vasil' chenko, Igor S [Institute of Physical and Organic Chemistry, Rostov State University, Rostov-on-Don (Russian Federation)

    2002-11-30

    This review surveys the state of art in the coordination chemistry of chelating azomethine systems, viz., amino(hydroxy)-azomethines, {beta}-aminovinyl ketones, {beta}-aminovinylimines and their sulfur- and selenium-containing analogues. Variations in the fine structure of azomethine ligands allow one to perform the targeted synthesis of chelate and molecular, mono-, bi- and polynuclear, homo- and heterometallic structures. The bibliography includes 425 reference000.

  19. Ligand pose and orientational sampling in molecular docking.

    Directory of Open Access Journals (Sweden)

    Ryan G Coleman

    Full Text Available Molecular docking remains an important tool for structure-based screening to find new ligands and chemical probes. As docking ambitions grow to include new scoring function terms, and to address ever more targets, the reliability and extendability of the orientation sampling, and the throughput of the method, become pressing. Here we explore sampling techniques that eliminate stochastic behavior in DOCK3.6, allowing us to optimize the method for regularly variable sampling of orientations. This also enabled a focused effort to optimize the code for efficiency, with a three-fold increase in the speed of the program. This, in turn, facilitated extensive testing of the method on the 102 targets, 22,805 ligands and 1,411,214 decoys of the Directory of Useful Decoys-Enhanced (DUD-E benchmarking set, at multiple levels of sampling. Encouragingly, we observe that as sampling increases from 50 to 500 to 2000 to 5000 to 20,000 molecular orientations in the binding site (and so from about 1×10(10 to 4×10(10 to 1×10(11 to 2×10(11 to 5×10(11 mean atoms scored per target, since multiple conformations are sampled per orientation, the enrichment of ligands over decoys monotonically increases for most DUD-E targets. Meanwhile, including internal electrostatics in the evaluation ligand conformational energies, and restricting aromatic hydroxyls to low energy rotamers, further improved enrichment values. Several of the strategies used here to improve the efficiency of the code are broadly applicable in the field.

  20. Cassia obtusifolia MetE as a cytosolic target for potassium isolespedezate, a leaf-opening factor of Cassia plants: target exploration by a compact molecular-probe strategy.

    Science.gov (United States)

    Ueda, Minoru; Manabe, Yoshiyuki; Otsuka, Yuki; Kanzawa, Nobuyuki

    2011-12-01

    Affinity chromatography by using ligand-immobilized bead technology is generally the first choice for target exploration of a bioactive ligand. However, when a ligand has comparatively low affinity against its target, serious difficulties will be raised in affinity-based target detection. We report here that the use of compact molecular probes (CMP) will be advantageous in such cases; it enables the retention of moderate affinity between the ligand and its target in contrast to immobilizing the ligand on affinity beads that will cause a serious drop in affinity to preclude target detection. In the CMP strategy, a CMP containing an azide handle is used for an initial affinity-based labeling of target, and subsequent tagging by CuAAC with a large FLAG tag will give a tagged target protein. By using the CMP strategy, we succeeded in the identification of Cassia obtusifolia MetE as a cytosolic target protein of potassium isolespedezate (1), a moderately bioactive ligand.

  1. Using RosettaLigand for small molecule docking into comparative models.

    Directory of Open Access Journals (Sweden)

    Kristian W Kaufmann

    Full Text Available Computational small molecule docking into comparative models of proteins is widely used to query protein function and in the development of small molecule therapeutics. We benchmark RosettaLigand docking into comparative models for nine proteins built during CASP8 that contain ligands. We supplement the study with 21 additional protein/ligand complexes to cover a wider space of chemotypes. During a full docking run in 21 of the 30 cases, RosettaLigand successfully found a native-like binding mode among the top ten scoring binding modes. From the benchmark cases we find that careful template selection based on ligand occupancy provides the best chance of success while overall sequence identity between template and target do not appear to improve results. We also find that binding energy normalized by atom number is often less than -0.4 in native-like binding modes.

  2. Revealing a steroid receptor ligand as a unique PPAR[gamma] agonist

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Shengchen; Han, Ying; Shi, Yuzhe; Rong, Hui; Zheng, Songyang; Jin, Shikan; Lin, Shu-Yong; Lin, Sheng-Cai; Li, Yong (Pitt); (Xiamen)

    2012-06-28

    Peroxisome proliferator-activated receptor gamma (PPAR{gamma}) regulates metabolic homeostasis and is a molecular target for anti-diabetic drugs. We report here the identification of a steroid receptor ligand, RU-486, as an unexpected PPAR{gamma} agonist, thereby uncovering a novel signaling route for this steroid drug. Similar to rosiglitazone, RU-486 modulates the expression of key PPAR{gamma} target genes and promotes adipocyte differentiation, but with a lower adipogenic activity. Structural and functional studies of receptor-ligand interactions reveal the molecular basis for a unique binding mode for RU-486 in the PPAR{gamma} ligand-binding pocket with distinctive properties and epitopes, providing the molecular mechanisms for the discrimination of RU-486 from thiazolidinediones (TZDs) drugs. Our findings together indicate that steroid compounds may represent an alternative approach for designing non-TZD PPAR{gamma} ligands in the treatment of insulin resistance.

  3. The therapeutic potential of allosteric ligands for free fatty acid sensitive GPCRs

    DEFF Research Database (Denmark)

    Hudson, Brian D; Ulven, Trond; Milligan, Graeme

    2013-01-01

    G protein coupled receptors (GPCRs) are the most historically successful therapeutic targets. Despite this success there are many important aspects of GPCR pharmacology and function that have yet to be exploited to their full therapeutic potential. One in particular that has been gaining attention...... in recent times is that of GPCR ligands that bind to allosteric sites on the receptor distinct from the orthosteric site of the endogenous ligand. As therapeutics, allosteric ligands possess many theoretical advantages over their orthosteric counterparts, including more complex modes of action, improved...... safety, more physiologically appropriate responses, better target selectivity, and reduced likelihood of desensitisation and tachyphylaxis. Despite these advantages, the development of allosteric ligands is often difficult from a medicinal chemistry standpoint due to the more complex challenge...

  4. Glucagon-like peptide-1 receptor ligand interactions: structural cross talk between ligands and the extracellular domain.

    Directory of Open Access Journals (Sweden)

    Graham M West

    Full Text Available Activation of the glucagon-like peptide-1 receptor (GLP-1R in pancreatic β-cells potentiates insulin production and is a current therapeutic target for the treatment of type 2 diabetes mellitus (T2DM. Like other class B G protein-coupled receptors (GPCRs, the GLP-1R contains an N-terminal extracellular ligand binding domain. N-terminal truncations on the peptide agonist generate antagonists capable of binding to the extracellular domain, but not capable of activating full length receptor. The main objective of this study was to use Hydrogen/deuterium exchange (HDX to identify how the amide hydrogen bonding network of peptide ligands and the extracellular domain of GLP-1R (nGLP-1R were altered by binding interactions and to then use this platform to validate direct binding events for putative GLP-1R small molecule ligands. The HDX studies presented here for two glucagon-like peptide-1 receptor (GLP-1R peptide ligands indicates that the antagonist exendin-4[9-39] is significantly destabilized in the presence of nonionic detergents as compared to the agonist exendin-4. Furthermore, HDX can detect stabilization of exendin-4 and exendin-4[9-39] hydrogen bonding networks at the N-terminal helix [Val19 to Lys27] upon binding to the N-terminal extracellular domain of GLP-1R (nGLP-1R. In addition we show hydrogen bonding network stabilization on nGLP-1R in response to ligand binding, and validate direct binding events with the extracellular domain of the receptor for putative GLP-1R small molecule ligands.

  5. Predicting Electrophoretic Mobility of Protein-Ligand Complexes for Ligands from DNA-Encoded Libraries of Small Molecules.

    Science.gov (United States)

    Bao, Jiayin; Krylova, Svetlana M; Cherney, Leonid T; Hale, Robert L; Belyanskaya, Svetlana L; Chiu, Cynthia H; Shaginian, Alex; Arico-Muendel, Christopher C; Krylov, Sergey N

    2016-05-17

    Selection of target-binding ligands from DNA-encoded libraries of small molecules (DELSMs) is a rapidly developing approach in drug-lead discovery. Methods of kinetic capillary electrophoresis (KCE) may facilitate highly efficient homogeneous selection of ligands from DELSMs. However, KCE methods require accurate prediction of electrophoretic mobilities of protein-ligand complexes. Such prediction, in turn, requires a theory that would be applicable to DNA tags of different structures used in different DELSMs. Here we present such a theory. It utilizes a model of a globular protein connected, through a single point (small molecule), to a linear DNA tag containing a combination of alternating double-stranded and single-stranded DNA (dsDNA and ssDNA) regions of varying lengths. The theory links the unknown electrophoretic mobility of protein-DNA complex with experimentally determined electrophoretic mobilities of the protein and DNA. Mobility prediction was initially tested by using a protein interacting with 18 ligands of various combinations of dsDNA and ssDNA regions, which mimicked different DELSMs. For all studied ligands, deviation of the predicted mobility from the experimentally determined value was within 11%. Finally, the prediction was tested for two proteins and two ligands with a DNA tag identical to those of DELSM manufactured by GlaxoSmithKline. Deviation between the predicted and experimentally determined mobilities did not exceed 5%. These results confirm the accuracy and robustness of our model, which makes KCE methods one step closer to their practical use in selection of drug leads, and diagnostic probes from DELSMs.

  6. Controlled-deactivation cannabinergic ligands.

    Science.gov (United States)

    Sharma, Rishi; Nikas, Spyros P; Paronis, Carol A; Wood, Jodianne T; Halikhedkar, Aneetha; Guo, Jason Jianxin; Thakur, Ganesh A; Kulkarni, Shashank; Benchama, Othman; Raghav, Jimit Girish; Gifford, Roger S; Järbe, Torbjörn U C; Bergman, Jack; Makriyannis, Alexandros

    2013-12-27

    We report an approach for obtaining novel cannabinoid analogues with controllable deactivation and improved druggability. Our design involves the incorporation of a metabolically labile ester group at the 2'-position on a series of (-)-Δ(8)-THC analogues. We have sought to introduce benzylic substituents α to the ester group which affect the half-lives of deactivation through enzymatic activity while enhancing the affinities and efficacies of individual ligands for the CB1 and CB2 receptors. The 1'-(S)-methyl, 1'-gem-dimethyl, and 1'-cyclobutyl analogues exhibit remarkably high affinities for both CB receptors. The novel ligands are susceptible to enzymatic hydrolysis by plasma esterases in a controllable manner, while their metabolites are inactive at the CB receptors. In further in vitro and in vivo experiments key analogues were shown to be potent CB1 receptor agonists and to exhibit CB1-mediated hypothermic and analgesic effects.

  7. Privileged chiral ligands and catalysts

    CERN Document Server

    Zhou, Qi-Lin

    2011-01-01

    This ultimate ""must have"" and long awaited reference for every chemist working in the field of asymmetric catalysis starts with the core structure of the catalysts, explaining why a certain ligand or catalyst is so successful. It describes in detail the history, the basic structural characteristics, and the applications of these ""privileged catalysts"". A novel concept that gives readers a much deeper insight into the topic.

  8. Outcome of the First wwPDB/CCDC/D3R Ligand Validation Workshop.

    Science.gov (United States)

    Adams, Paul D; Aertgeerts, Kathleen; Bauer, Cary; Bell, Jeffrey A; Berman, Helen M; Bhat, Talapady N; Blaney, Jeff M; Bolton, Evan; Bricogne, Gerard; Brown, David; Burley, Stephen K; Case, David A; Clark, Kirk L; Darden, Tom; Emsley, Paul; Feher, Victoria A; Feng, Zukang; Groom, Colin R; Harris, Seth F; Hendle, Jorg; Holder, Thomas; Joachimiak, Andrzej; Kleywegt, Gerard J; Krojer, Tobias; Marcotrigiano, Joseph; Mark, Alan E; Markley, John L; Miller, Matthew; Minor, Wladek; Montelione, Gaetano T; Murshudov, Garib; Nakagawa, Atsushi; Nakamura, Haruki; Nicholls, Anthony; Nicklaus, Marc; Nolte, Robert T; Padyana, Anil K; Peishoff, Catherine E; Pieniazek, Susan; Read, Randy J; Shao, Chenghua; Sheriff, Steven; Smart, Oliver; Soisson, Stephen; Spurlino, John; Stouch, Terry; Svobodova, Radka; Tempel, Wolfram; Terwilliger, Thomas C; Tronrud, Dale; Velankar, Sameer; Ward, Suzanna C; Warren, Gregory L; Westbrook, John D; Williams, Pamela; Yang, Huanwang; Young, Jasmine

    2016-04-05

    Crystallographic studies of ligands bound to biological macromolecules (proteins and nucleic acids) represent an important source of information concerning drug-target interactions, providing atomic level insights into the physical chemistry of complex formation between macromolecules and ligands. Of the more than 115,000 entries extant in the Protein Data Bank (PDB) archive, ∼75% include at least one non-polymeric ligand. Ligand geometrical and stereochemical quality, the suitability of ligand models for in silico drug discovery and design, and the goodness-of-fit of ligand models to electron-density maps vary widely across the archive. We describe the proceedings and conclusions from the first Worldwide PDB/Cambridge Crystallographic Data Center/Drug Design Data Resource (wwPDB/CCDC/D3R) Ligand Validation Workshop held at the Research Collaboratory for Structural Bioinformatics at Rutgers University on July 30-31, 2015. Experts in protein crystallography from academe and industry came together with non-profit and for-profit software providers for crystallography and with experts in computational chemistry and data archiving to discuss and make recommendations on best practices, as framed by a series of questions central to structural studies of macromolecule-ligand complexes. What data concerning bound ligands should be archived in the PDB? How should the ligands be best represented? How should structural models of macromolecule-ligand complexes be validated? What supplementary information should accompany publications of structural studies of biological macromolecules? Consensus recommendations on best practices developed in response to each of these questions are provided, together with some details regarding implementation. Important issues addressed but not resolved at the workshop are also enumerated. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Targeted nanoparticles for colorectal cancer

    DEFF Research Database (Denmark)

    Cisterna, Bruno A.; Kamaly, Nazila; Choi, Won Il

    2016-01-01

    Colorectal cancer (CRC) is highly prevalent worldwide, and despite notable progress in treatment still leads to significant morbidity and mortality. The use of nanoparticles as a drug delivery system has become one of the most promising strategies for cancer therapy. Targeted nanoparticles could...... take advantage of differentially expressed molecules on the surface of tumor cells, providing effective release of cytotoxic drugs. Several efforts have recently reported the use of diverse molecules as ligands on the surface of nanoparticles to interact with the tumor cells, enabling the effective...... delivery of antitumor agents. Here, we present recent advances in targeted nanoparticles against CRC and discuss the promising use of ligands and cellular targets in potential strategies for the treatment of CRCs....

  10. Human cytomegalovirus UL141 promotes efficient downregulation of the natural killer cell activating ligand CD112

    OpenAIRE

    Prod'homme, Virginie; Sugrue, Daniel M.; Stanton, Richard J.; Nomoto, Akio; Davies, James; Rickards, Carole R.; Cochrane, Daniel; Moore, Melanie; Wilkinson, Gavin W. G.; Tomasec, Peter

    2010-01-01

    Human cytomegalovirus (HCMV) UL141 induces protection against natural killer cell-mediated cytolysis by downregulating cell surface expression of CD155 (nectin-like molecule 5; poliovirus receptor), a ligand for the activating receptor DNAM-1 (CD226). However, DNAM-1 is also recognized to bind a second ligand, CD112 (nectin-2). We now show that HCMV targets CD112 for proteasome-mediated degradation by 48 h post-infection, thus removing both activating ligands for DNAM-1 from the cell surface ...

  11. A Novel Strategy for Proteome-wide Ligand Screening Using Cross-linked Phage Matrices*

    OpenAIRE

    Qian, Chen; LIU, Jian-ning; Tang, Fengyuan; Yuan, Dawen; Guo, Zhigang; Zhang, Jing

    2010-01-01

    To find a suitable ligand from a complex antigen system is still a mission to be accomplished. Here we have explored a novel “library against proteome” panning strategy for ligand screening and antigen purification from a complex system using phage-displayed antibody technology. Human plasma proteome was targeted for phage library panning. During the process, the panning was carried out in solution, using a biotin/streptavidin beads separation system, for three rounds. Nine monoclonal phages,...

  12. The molecular basis of ligand interaction at free fatty acid receptor 4 (FFA4/GPR120)

    OpenAIRE

    Hudson, Brian D.; Shimpukade, Bharat; Milligan, Graeme; Ulven, Trond

    2014-01-01

    The long-chain fatty acid receptor FFA4(previously GPR120) is receiving substantial interest as a novel target for the treatment of metabolic and inflammatory disease. The current study examines for the first time the detailed mode of binding of both a long-chain fatty acid and synthetic agonist ligands at FFA4 by integrating molecular modeling, receptor mutagenesis, and ligand structure-activity relationship approaches in an iterative format. In doing so, residues required for binding of fat...

  13. Discriminating agonist and antagonist ligands of the nuclear receptors using 3D-pharmacophores.

    Science.gov (United States)

    Lagarde, Nathalie; Delahaye, Solenne; Zagury, Jean-François; Montes, Matthieu

    2016-01-01

    Nuclear receptors (NRs) constitute an important class of therapeutic targets. We evaluated the performance of 3D structure-based and ligand-based pharmacophore models in predicting the pharmacological profile of NRs ligands using the NRLiSt BDB database. We could generate selective pharmacophores for agonist and antagonist ligands and we found that the best performances were obtained by combining the structure-based and the ligand-based approaches. The combination of pharmacophores that were generated allowed to cover most of the chemical space of the NRLiSt BDB datasets. By screening the whole NRLiSt BDB on our 3D pharmacophores, we demonstrated their selectivity towards their dedicated NRs ligands. The 3D pharmacophores herein presented can thus be used as a predictor of the pharmacological activity of NRs ligands.Graphical AbstractUsing a combination of structure-based and ligand-based pharmacophores, agonist and antagonist ligands of the Nuclear Receptors included in the NRLiSt BDB database could be separated.

  14. Role of Receptor Tyrosine Kinases and Their Ligands in Glioblastoma

    Directory of Open Access Journals (Sweden)

    Estefanía Carrasco-García

    2014-04-01

    Full Text Available Glioblastoma multiforme is the most frequent, aggressive and fatal type of brain tumor. Glioblastomas are characterized by their infiltrating nature, high proliferation rate and resistance to chemotherapy and radiation. Recently, oncologic therapy experienced a rapid evolution towards “targeted therapy,” which is the employment of drugs directed against particular targets that play essential roles in proliferation, survival and invasiveness of cancer cells. A number of molecules involved in signal transduction pathways are used as molecular targets for the treatment of various tumors. In fact, inhibitors of these molecules have already entered the clinic or are undergoing clinical trials. Cellular receptors are clear examples of such targets and in the case of glioblastoma multiforme, some of these receptors and their ligands have become relevant. In this review, the importance of glioblastoma multiforme in signaling pathways initiated by extracellular tyrosine kinase receptors such as EGFR, PDGFR and IGF-1R will be discussed. We will describe their ligands, family members, structure, activation mechanism, downstream molecules, as well as the interaction among these pathways. Lastly, we will provide an up-to-date review of the current targeted therapies in cancer, in particular glioblastoma that employ inhibitors of these pathways and their benefits.

  15. The imidazoline receptors and ligands in pain modulation

    Directory of Open Access Journals (Sweden)

    Nurcan Bektas

    2015-01-01

    Full Text Available Pain is an unpleasant experience and effects daily routine negatively. Although there are various drugs, many of them are not entirely successful in relieving pain, since pain modulation is a complex process involving numerous mediators and receptors. Therefore, it is a rational approach to identify the factors involved in the complex process and develop new agents that act on these pain producing mechanisms. In this respect, the involvement of the imidazoline receptors in pain modulation has drawn attention in recent years. In this review, it is aimed to focus on the imidazoline receptors and their ligands which contribute to the pain modulation. It is demonstrated that imidazoline-2 (I2 receptors are steady new drug targets for analgesics. Even if the mechanism of I2receptor is not well known in the modulation of pain, it is known that it plays a role in tonic and chronic pain but not in acute phasic pain. Moreover, the I2receptor ligands increase the analgesic effects of opioids in both acute and chronic pain and prevent the development of opioid tolerance. So, they are valuable for the chronic pain treatment and also therapeutic coadjuvants in the management of chronic pain with opiate drugs due to the attenuation of opioid tolerance and addiction. Thus, the use of the ligands which bind to the imidazoline receptors is an effective strategy for relieving pain. This educational forum exhibits the role of imidazoline receptors and ligands in pain process by utilizing experimental studies.

  16. Structural Analysis Uncovers Lipid-Binding Properties of Notch Ligands

    Directory of Open Access Journals (Sweden)

    Chandramouli R. Chillakuri

    2013-11-01

    Full Text Available The Notch pathway is a core cell-cell signaling system in metazoan organisms with key roles in cell-fate determination, stem cell maintenance, immune system activation, and angiogenesis. Signals are initiated by extracellular interactions of the Notch receptor with Delta/Serrate/Lag-2 (DSL ligands, whose structure is highly conserved throughout evolution. To date, no structure or activity has been associated with the extreme N termini of the ligands, even though numerous mutations in this region of Jagged-1 ligand lead to human disease. Here, we demonstrate that the N terminus of human Jagged-1 is a C2 phospholipid recognition domain that binds phospholipid bilayers in a calcium-dependent fashion. Furthermore, we show that this activity is shared by a member of the other class of Notch ligands, human Delta-like-1, and the evolutionary distant Drosophila Serrate. Targeted mutagenesis of Jagged-1 C2 domain residues implicated in calcium-dependent phospholipid binding leaves Notch interactions intact but can reduce Notch activation. These results reveal an important and previously unsuspected role for phospholipid recognition in control of this key signaling system.

  17. Fluorescent ligands for studying neuropeptide receptors by confocal microscopy

    Directory of Open Access Journals (Sweden)

    Beaudet A.

    1998-01-01

    Full Text Available This paper reviews the use of confocal microscopy as it pertains to the identification of G-protein coupled receptors and the study of their dynamic properties in cell cultures and in mammalian brain following their tagging with specific fluorescent ligands. Principles that should guide the choice of suitable ligands and fluorophores are discussed. Examples are provided from the work carried out in the authors' laboratory using custom synthetized fluoresceinylated or BODIPY-tagged bioactive peptides. The results show that confocal microscopic detection of specifically bound fluorescent ligands permits high resolution appraisal of neuropeptide receptor distribution both in cell culture and in brain sections. Within the framework of time course experiments, it also allows for a dynamic assessment of the internalization and subsequent intracellular trafficking of bound fluorescent molecules. Thus, it was found that neurotensin, somatostatin and mu- and delta-selective opioid peptides are internalized in a receptor-dependent fashion and according to receptor-specific patterns into their target cells. In the case of neurotensin, this internalization process was found to be clathrin-mediated, to proceed through classical endosomal pathways and, in neurons, to result in a mobilization of newly formed endosomes from neural processes to nerve cell bodies and from the periphery of cell bodies towards the perinuclear zone. These mechanisms are likely to play an important role for ligand inactivation, receptor regulation and perhaps also transmembrane signaling.

  18. Supervised Machine Learning Methods Applied to Predict Ligand- Binding Affinity.

    Science.gov (United States)

    Heck, Gabriela S; Pintro, Val O; Pereira, Richard R; de Ávila, Mauricio B; Levin, Nayara M B; de Azevedo, Walter F

    2017-01-01

    Calculation of ligand-binding affinity is an open problem in computational medicinal chemistry. The ability to computationally predict affinities has a beneficial impact in the early stages of drug development, since it allows a mathematical model to assess protein-ligand interactions. Due to the availability of structural and binding information, machine learning methods have been applied to generate scoring functions with good predictive power. Our goal here is to review recent developments in the application of machine learning methods to predict ligand-binding affinity. We focus our review on the application of computational methods to predict binding affinity for protein targets. In addition, we also describe the major available databases for experimental binding constants and protein structures. Furthermore, we explain the most successful methods to evaluate the predictive power of scoring functions. Association of structural information with ligand-binding affinity makes it possible to generate scoring functions targeted to a specific biological system. Through regression analysis, this data can be used as a base to generate mathematical models to predict ligandbinding affinities, such as inhibition constant, dissociation constant and binding energy. Experimental biophysical techniques were able to determine the structures of over 120,000 macromolecules. Considering also the evolution of binding affinity information, we may say that we have a promising scenario for development of scoring functions, making use of machine learning techniques. Recent developments in this area indicate that building scoring functions targeted to the biological systems of interest shows superior predictive performance, when compared with other approaches. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  19. Exploring Ramachandran and chi space: conformationally constrained amino acids and peptides in the design of bioactive polypeptide ligands.

    Science.gov (United States)

    Cowell, S M; Lee, Y S; Cain, J P; Hruby, V J

    2004-11-01

    Ligand binding and concomitant changes in receptor structure provide the means to target signal transduction pathways. With appropriate refinement of the ligand's interaction with the "receptor," one in theory could produce ligands that have greater therapeutic benefits. This review will discuss how, when these ligands are amino acids and peptides, the introduction of appropriate conformational constraints provides a powerful strategy for improved drug design. This review will discuss how various constraints on amino acids can provide a powerful tool for ligand design, determination of the three dimensional pharmacophore and new insights into receptor systems and information transduction. Through the use of constrained ligands, new information regarding their interaction with their "receptor" systems, and further refinement of the use of constraints, scientists can produce more beneficial drugs for mankind.

  20. Multiple ligand detection and affinity measurement by ultrafiltration and mass spectrometry analysis applied to fragment mixture screening.

    Science.gov (United States)

    Qin, Shanshan; Ren, Yiran; Fu, Xu; Shen, Jie; Chen, Xin; Wang, Quan; Bi, Xin; Liu, Wenjing; Li, Lixin; Liang, Guangxin; Yang, Cheng; Shui, Wenqing

    2015-07-30

    Binding affinity of a small molecule drug candidate to a therapeutically relevant biomolecular target is regarded the first determinant of the candidate's efficacy. Although the ultrafiltration-LC/MS (UF-LC/MS) assay enables efficient ligand discovery for a specific target from a mixed pool of compounds, most previous analysis allowed for relative affinity ranking of different ligands. Moreover, the reliability of affinity measurement for multiple ligands with UF-LC/MS has hardly been strictly evaluated. In this study, we examined the accuracy of K(d) determination through UF-LC/MS by comparison with classical ITC measurement. A single-point K(d) calculation method was found to be suitable for affinity measurement of multiple ligands bound to the same target when binding competition is minimized. A second workflow based on analysis of the unbound fraction of compounds was then developed, which simplified sample preparation as well as warranted reliable ligand discovery. The new workflow implemented in a fragment mixture screen afforded rapid and sensitive detection of low-affinity ligands selectively bound to the RNA polymerase NS5B of hepatitis C virus. More importantly, ligand identification and affinity measurement for mixture-based fragment screens by UF-LC/MS were in good accordance with single ligand evaluation by conventional SPR analysis. This new approach is expected to become a valuable addition to the arsenal of high-throughput screening techniques for fragment-based drug discovery.

  1. Predicting the Reliability of Drug-target Interaction Predictions with Maximum Coverage of Target Space.

    Science.gov (United States)

    Peón, Antonio; Naulaerts, Stefan; Ballester, Pedro J

    2017-06-19

    Many computational methods to predict the macromolecular targets of small organic molecules have been presented to date. Despite progress, target prediction methods still have important limitations. For example, the most accurate methods implicitly restrict their predictions to a relatively small number of targets, are not systematically validated on drugs (whose targets are harder to predict than those of non-drug molecules) and often lack a reliability score associated with each predicted target. Here we present a systematic validation of ligand-centric target prediction methods on a set of clinical drugs. These methods exploit a knowledge-base covering 887,435 known ligand-target associations between 504,755 molecules and 4,167 targets. Based on this dataset, we provide a new estimate of the polypharmacology of drugs, which on average have 11.5 targets below IC50 10 µM. The average performance achieved across clinical drugs is remarkable (0.348 precision and 0.423 recall, with large drug-dependent variability), especially given the unusually large coverage of the target space. Furthermore, we show how a sparse ligand-target bioactivity matrix to retrospectively validate target prediction methods could underestimate prospective performance. Lastly, we present and validate a first-in-kind score capable of accurately predicting the reliability of target predictions.

  2. The Foundations of Protein-Ligand Interaction

    Science.gov (United States)

    Klebe, Gerhard

    For the specific design of a drug we must first answer the question: How does a drug achieve its activity? An active ingredient must, in order to develop its action, bind to a particular target molecule in the body. Usually this is a protein, but also nucleic acids in the form of RNA and DNA can be target structures for active agents. The most important condition for binding is at first that the active agent exhibits the correct size and shape in order to optimally fit into a cavity exposed to the surface of the protein, the "bindingpocket". It is further necessary for the surface properties of the ligand and protein to be mutually compatible to form specific interactions. In 1894 Emil Fischer compared the exact fit of a substrate for the catalytic centre of an enzyme with the picture of a "lock-and-key". Paul Ehrlich coined in 1913 "Corpora non agunt nisi fixata", literally "bodies do not work when they are not bound". He wanted to imply that active agents that are meant to kill bacteria or parasites must be "fixed" by them, i.e. linked to their structures. Both concepts form the starting point for any rational concept in the development of active pharmaceutical ingredients. In many respects they still apply today. A drug must, after being administered, reach its target and interact with a biological macromolecule. Specific agents have a large affinity and sufficient selectivity to bind to the macromolecule's active site. This is the only way they can develop the desired biological activity without side-effects.

  3. PHOSPHATURIC MESENCHYMAL TUMOUR ASSOCIATED WITH OSTEOMALACIA : A CASE REPORT

    Directory of Open Access Journals (Sweden)

    Veena

    2015-02-01

    Full Text Available Phosphaturic mesenchymal tumour is a tumour that can involve bone or soft tissue. This is a rare tumour and is known to be associated with osteomalasia. This is caused by tumour induced expression of fibroblastic growth factor (FGF23. We present a case of PMT in a 72 year old female patient who was diagnosed with osteomalasia due to nutritional deficiency of vitamin D and was appropriately treated but later presented with a mass in her foot.

  4. Aptamers for Targeted Drug Delivery

    Directory of Open Access Journals (Sweden)

    Partha Ray

    2010-05-01

    Full Text Available Aptamers are a class of therapeutic oligonucleotides that form specific three-dimensional structures that are dictated by their sequences. They are typically generated by an iterative screening process of complex nucleic acid libraries employing a process termed Systemic Evolution of Ligands by Exponential Enrichment (SELEX. SELEX has traditionally been performed using purified proteins, and cell surface receptors may be challenging to purify in their properly folded and modified conformations. Therefore, relatively few aptamers have been generated that bind cell surface receptors. However, improvements in recombinant fusion protein technology have increased the availability of receptor extracellular domains as purified protein targets, and the development of cell-based selection techniques has allowed selection against surface proteins in their native configuration on the cell surface. With cell-based selection, a specific protein target is not always chosen, but selection is performed against a target cell type with the goal of letting the aptamer choose the target. Several studies have demonstrated that aptamers that bind cell surface receptors may have functions other than just blocking receptor-ligand interactions. All cell surface proteins cycle intracellularly to some extent, and many surface receptors are actively internalized in response to ligand binding. Therefore, aptamers that bind cell surface receptors have been exploited for the delivery of a variety of cargoes into cells. This review focuses on recent progress and current challenges in the field of aptamer-mediated delivery.

  5. Computational protein-ligand docking and virtual drug screening with the AutoDock suite.

    Science.gov (United States)

    Forli, Stefano; Huey, Ruth; Pique, Michael E; Sanner, Michel F; Goodsell, David S; Olson, Arthur J

    2016-05-01

    Computational docking can be used to predict bound conformations and free energies of binding for small-molecule ligands to macromolecular targets. Docking is widely used for the study of biomolecular interactions and mechanisms, and it is applied to structure-based drug design. The methods are fast enough to allow virtual screening of ligand libraries containing tens of thousands of compounds. This protocol covers the docking and virtual screening methods provided by the AutoDock suite of programs, including a basic docking of a drug molecule with an anticancer target, a virtual screen of this target with a small ligand library, docking with selective receptor flexibility, active site prediction and docking with explicit hydration. The entire protocol will require ∼5 h.

  6. Modulation of actin dynamics as potential macrophage subtype-targeting anti-tumour strategy

    Science.gov (United States)

    Pergola, Carlo; Schubert, Katrin; Pace, Simona; Ziereisen, Jana; Nikels, Felix; Scherer, Olga; Hüttel, Stephan; Zahler, Stefan; Vollmar, Angelika M.; Weinigel, Christina; Rummler, Silke; Müller, Rolf; Raasch, Martin; Mosig, Alexander; Koeberle, Andreas; Werz, Oliver

    2017-01-01

    Tumour-associated macrophages mainly comprise immunosuppressive M2 phenotypes that promote tumour progression besides anti-tumoural M1 subsets. Selective depletion or reprogramming of M2 may represent an innovative anti-cancer strategy. The actin cytoskeleton is central for cellular homeostasis and is targeted for anti-cancer chemotherapy. Here, we show that targeting G-actin nucleation using chondramide A (ChA) predominantly depletes human M2 while promoting the tumour-suppressive M1 phenotype. ChA reduced the viability of M2, with minor effects on M1, but increased tumour necrosis factor (TNF)α release from M1. Interestingly, ChA caused rapid disruption of dynamic F-actin filaments and polymerization of G-actin, followed by reduction of cell size, binucleation and cell division, without cellular collapse. In M1, but not in M2, ChA caused marked activation of SAPK/JNK and NFκB, with slight or no effects on Akt, STAT-1/-3, ERK-1/2, and p38 MAPK, seemingly accounting for the better survival of M1 and TNFα secretion. In a microfluidically-supported human tumour biochip model, circulating ChA-treated M1 markedly reduced tumour cell viability through enhanced release of TNFα. Together, ChA may cause an anti-tumoural microenvironment by depletion of M2 and activation of M1, suggesting induction of G-actin nucleation as potential strategy to target tumour-associated macrophages in addition to neoplastic cells. PMID:28134280

  7. Structural Dynamics of the Cereblon Ligand Binding Domain

    Science.gov (United States)

    Hartmann, Marcus D.; Boichenko, Iuliia; Coles, Murray; Lupas, Andrei N.; Hernandez Alvarez, Birte

    2015-01-01

    Cereblon, a primary target of thalidomide and its derivatives, has been characterized structurally from both bacteria and animals. Especially well studied is the thalidomide binding domain, CULT, which shows an invariable structure across different organisms and in complex with different ligands. Here, based on a series of crystal structures of a bacterial representative, we reveal the conformational flexibility and structural dynamics of this domain. In particular, we follow the unfolding of large fractions of the domain upon release of thalidomide in the crystalline state. Our results imply that a third of the domain, including the thalidomide binding pocket, only folds upon ligand binding. We further characterize the structural effect of the C-terminal truncation resulting from the mental-retardation linked R419X nonsense mutation in vitro and offer a mechanistic hypothesis for its irresponsiveness to thalidomide. At 1.2Å resolution, our data provide a view of thalidomide binding at atomic resolution. PMID:26024445

  8. Structural dynamics of the cereblon ligand binding domain.

    Directory of Open Access Journals (Sweden)

    Marcus D Hartmann

    Full Text Available Cereblon, a primary target of thalidomide and its derivatives, has been characterized structurally from both bacteria and animals. Especially well studied is the thalidomide binding domain, CULT, which shows an invariable structure across different organisms and in complex with different ligands. Here, based on a series of crystal structures of a bacterial representative, we reveal the conformational flexibility and structural dynamics of this domain. In particular, we follow the unfolding of large fractions of the domain upon release of thalidomide in the crystalline state. Our results imply that a third of the domain, including the thalidomide binding pocket, only folds upon ligand binding. We further characterize the structural effect of the C-terminal truncation resulting from the mental-retardation linked R419X nonsense mutation in vitro and offer a mechanistic hypothesis for its irresponsiveness to thalidomide. At 1.2Å resolution, our data provide a view of thalidomide binding at atomic resolution.

  9. Radiochemical studies of {sup 99m}Tc complexes of modified cysteine ligands and bifunctional chelating agents

    Energy Technology Data Exchange (ETDEWEB)

    Pillai, M.R.A. E-mail: ambi@magnum.barc.ernet.in; Kothari, Kanchan; Banerjee, Sharmila; Samuel, Grace; Suresh, Manju; Sarma, H.D.; Jurisson, S

    1999-07-01

    The synthesis of four novel ligands using the amino-acid cysteine and its ethyl carboxylate derivative is described. The synthetic method involves a two-step procedure, wherein the intermediate Schiff base formed by the condensation of the amino group of the cysteine substrate and salicylaldehyde is reduced to give the target ligands. The intermediates and the final products were characterized by high resolution nuclear magnetic resonance spectroscopy. Complexation studies of the ligands with {sup 99m}Tc were optimized using stannous tartrate as the reducing agent under varying reaction conditions. The complexes were characterized using standard quality control techniques such as thin layer chromatography, paper electrophoresis, and paper chromatography. Lipophilicities of the complexes were estimated by solvent extraction into chloroform. Substantial changes in net charge and lipophilicity of the {sup 99m}Tc complexes were observed on substituting the carboxylic acid functionality in ligands I and II with the ethyl carboxylate groups (ligands III and IV). All the ligands formed {sup 99m}Tc complexes in high yield. Whereas the complexes with ligands I and II were observed to be hydrophilic in nature and not extractable into CHCl{sub 3}, ligands III and IV resulted in neutral and lipophilic {sup 99m}Tc complexes. The {sup 99m}Tc complex with ligand III was not stable and on storage formed a hydrophilic and nonextractable species. The biodistribution of the complexes of ligands I and II showed that they cleared predominantly through the kidneys, whereas the complexes with ligands III and IV were excreted primarily through the hepatobiliary system. No significant brain uptake was observed with the {sup 99m}Tc complexes with ligands III and IV despite their favorable properties of neutrality, lipophilicity, and conversion into a hydrophilic species. These ligands offer potential for use as bifunctional chelating agents.

  10. Ligand photo-isomerization triggers conformational changes in iGluR2 ligand binding domain.

    Directory of Open Access Journals (Sweden)

    Tino Wolter

    Full Text Available Neurological glutamate receptors bind a variety of artificial ligands, both agonistic and antagonistic, in addition to glutamate. Studying their small molecule binding properties increases our understanding of the central nervous system and a variety of associated pathologies. The large, oligomeric multidomain membrane protein contains a large and flexible ligand binding domains which undergoes large conformational changes upon binding different ligands. A recent application of glutamate receptors is their activation or inhibition via photo-switchable ligands, making them key systems in the emerging field of optochemical genetics. In this work, we present a theoretical study on the binding mode and complex stability of a novel photo-switchable ligand, ATA-3, which reversibly binds to glutamate receptors ligand binding domains (LBDs. We propose two possible binding modes for this ligand based on flexible ligand docking calculations and show one of them to be analogues to the binding mode of a similar ligand, 2-BnTetAMPA. In long MD simulations, it was observed that transitions between both binding poses involve breaking and reforming the T686-E402 protein hydrogen bond. Simulating the ligand photo-isomerization process shows that the two possible configurations of the ligand azo-group have markedly different complex stabilities and equilibrium binding modes. A strong but slow protein response is observed after ligand configuration changes. This provides a microscopic foundation for the observed difference in ligand activity upon light-switching.

  11. Target Laboratory

    Data.gov (United States)

    Federal Laboratory Consortium — [Part of the ATLAS user facility.] The Physics Division operates a target development laboratory that produces targets and foils of various thickness and substrates,...

  12. Target Laboratory

    Data.gov (United States)

    Federal Laboratory Consortium — [Part of the ATLAS user facility.] The Physics Division operates a target development laboratory that produces targets and foils of various thickness and substrates,...

  13. Measurement of protein-ligand complex formation.

    Science.gov (United States)

    Lowe, Peter N; Vaughan, Cara K; Daviter, Tina

    2013-01-01

    Experimental approaches to detect, measure, and quantify protein-ligand binding, along with their theoretical bases, are described. A range of methods for detection of protein-ligand interactions is summarized. Specific protocols are provided for a nonequilibrium procedure pull-down assay, for an equilibrium direct binding method and its modification into a competition-based measurement and for steady-state measurements based on the effects of ligands on enzyme catalysis.

  14. Thermodynamics of ligand binding to histone deacetylase like amidohydrolase from Bordetella/Alcaligenes.

    Science.gov (United States)

    Meyners, Christian; Baud, Matthias G J; Fuchter, Matthew J; Meyer-Almes, Franz-Josef

    2014-03-01

    Thermodynamic studies on ligand-protein binding have become increasingly important in the process of drug design. In combination with structural data and molecular dynamics simulations, thermodynamic studies provide relevant information about the mode of interaction between compounds and their target proteins and therefore build a sound basis for further drug optimization. Using the example of histone deacetylases (HDACs), particularly the histone deacetylase like amidohydrolase (HDAH) from Bordetella/Alcaligenes, a novel sensitive competitive fluorescence resonance energy transfer-based binding assay was developed and the thermodynamics of interaction of both fluorescent ligands and inhibitors to histone deacetylase like amidohydrolase were investigated. The assay consumes only small amounts of valuable target proteins and is suitable for fast kinetic and mechanistic studies as well as high throughput screening applications. Binding affinity increased with increasing length of aliphatic spacers (n = 4-7) between the hydroxamate moiety and the dansyl head group of ligand probes. Van't Hoff plots revealed an optimum in enthalpy contribution to the free energy of binding for the dansyl-ligand with hexyl spacer. The selectivity in the series of dansyl-ligands against human class I HDAC1 but not class II HDACs 4 and 6 increased with the ratio of ΔH(0)/ΔG(0). The data clearly emphasize the importance of thermodynamic signatures as useful general guidance for the optimization of ligands or rational drug design.

  15. Physics-based scoring of protein-ligand interactions: explicit polarizability, quantum mechanics and free energies.

    Science.gov (United States)

    Bryce, Richard A

    2011-04-01

    The ability to accurately predict the interaction of a ligand with its receptor is a key limitation in computer-aided drug design approaches such as virtual screening and de novo design. In this article, we examine current strategies for a physics-based approach to scoring of protein-ligand affinity, as well as outlining recent developments in force fields and quantum chemical techniques. We also consider advances in the development and application of simulation-based free energy methods to study protein-ligand interactions. Fuelled by recent advances in computational algorithms and hardware, there is the opportunity for increased integration of physics-based scoring approaches at earlier stages in computationally guided drug discovery. Specifically, we envisage increased use of implicit solvent models and simulation-based scoring methods as tools for computing the affinities of large virtual ligand libraries. Approaches based on end point simulations and reference potentials allow the application of more advanced potential energy functions to prediction of protein-ligand binding affinities. Comprehensive evaluation of polarizable force fields and quantum mechanical (QM)/molecular mechanical and QM methods in scoring of protein-ligand interactions is required, particularly in their ability to address challenging targets such as metalloproteins and other proteins that make highly polar interactions. Finally, we anticipate increasingly quantitative free energy perturbation and thermodynamic integration methods that are practical for optimization of hits obtained from screened ligand libraries.

  16. VS-APPLE: A Virtual Screening Algorithm Using Promiscuous Protein-Ligand Complexes.

    Science.gov (United States)

    Okuno, Tatsuya; Kato, Koya; Terada, Tomoki P; Sasai, Masaki; Chikenji, George

    2015-06-22

    As the number of structurally resolved protein-ligand complexes increases, the ligand-binding pockets of many proteins have been found to accommodate multiple different compounds. Effective use of these structural data is important for developing virtual screening (VS) methods that identify bioactive compounds. Here, we introduce a VS method, VS-APPLE (Virtual Screening Algorithm using Promiscuous Protein-Ligand complExes), based on promiscuous protein-ligand binding structures. In VS-APPLE, multiple ligands bound to a pocket are combined into a query template for screening. Both the structural match between a test compound and the multiple-ligand template and the possible collisions between the test compound and the target protein are evaluated by an efficient geometric hashing method. The performance of VS-APPLE was examined on a filtered, clustered version of the Directory of Useful Decoys data set. In Area Under the Curve analyses of this data set, VS-APPLE outperformed several popular screening programs. Judging from the performance of VS-APPLE, the structural data of promiscuous protein-ligand bindings could be further analyzed and exploited for developing VS methods.

  17. Computational protocol for predicting the binding affinities of zinc containing metalloprotein-ligand complexes.

    Science.gov (United States)

    Jain, Tarun; Jayaram, B

    2007-06-01

    Zinc is one of the most important metal ions found in proteins performing specific functions associated with life processes. Coordination geometry of the zinc ion in the active site of the metalloprotein-ligand complexes poses a challenge in determining ligand binding affinities accurately in structure-based drug design. We report here an all atom force field based computational protocol for estimating rapidly the binding affinities of zinc containing metalloprotein-ligand complexes, considering electrostatics, van der Waals, hydrophobicity, and loss in conformational entropy of protein side chains upon ligand binding along with a nonbonded approach to model the interactions of the zinc ion with all the other atoms of the complex. We examined the sensitivity of the binding affinity predictions to the choice of Lennard-Jones parameters, partial atomic charges, and dielectric treatments adopted for system preparation and scoring. The highest correlation obtained was R2 = 0.77 (r = 0.88) for the predicted binding affinity against the experiment on a heterogenous dataset of 90 zinc containing metalloprotein-ligand complexes consisting of five unique protein targets. Model validation and parameter analysis studies underscore the robustness and predictive ability of the scoring function. The high correlation obtained suggests the potential applicability of the methodology in designing novel ligands for zinc-metalloproteins. The scoring function has been web enabled for free access at www.scfbio-iitd.res.in/software/drugdesign/bapplz.jsp as BAPPL-Z server (Binding Affinity Prediction of Protein-Ligand complexes containing Zinc metal ions).

  18. Theranostics Targeting Metastatic Breast Cancer

    Science.gov (United States)

    2016-10-01

    2016. Active Targeting of Cancer Cells, Masaryk University, CZECH REPUBLIC, May 2016. Websites or other Internet sites none Technologies or... trafficking , thus impacting the efficacy of receptor-mediated drug delivery for cancer therapy. These factors include the following: (i) the rate of ligand...The V, Labrie C, Belanger A, Simard J, Lin SX, Pelletier G. Endocrine and intracrine sources of androgens in women : Inhibition of breast cancer and

  19. Nanomedicine: Perspective and promises with ligand-directed molecular imaging

    Energy Technology Data Exchange (ETDEWEB)

    Pan Dipanjan [Department of Medicine, Washington University Medical School, St. Louis, MO (United States)], E-mail: dipanjan@wustl.edu; Lanza, Gregory M.; Wickline, Samuel A. [Department of Medicine, Washington University Medical School, St. Louis, MO (United States); Caruthers, Shelton D. [Department of Medicine, Washington University Medical School, St. Louis, MO (United States); Philips Healthcare, Andover, MA (United States)], E-mail: scaruthers@cmrl.wustl.edu

    2009-05-15

    Molecular imaging and targeted drug delivery play an important role toward personalized medicine, which is the future of patient management. Of late, nanoparticle-based molecular imaging has emerged as an interdisciplinary area, which shows promises to understand the components, processes, dynamics and therapies of a disease at a molecular level. The unprecedented potential of nanoplatforms for early detection, diagnosis and personalized treatment of diseases, have found application in every biomedical imaging modality. Biological and biophysical barriers are overcome by the integration of targeting ligands, imaging agents and therapeutics into the nanoplatform which allow for theranostic applications. In this article, we have discussed the opportunities and potential of targeted molecular imaging with various modalities putting a particular emphasis on perfluorocarbon nanoemulsion-based platform technology.

  20. Phenanthroline-2,9-bistriazoles as selective G-quadruplex ligands

    DEFF Research Database (Denmark)

    Nielsen, Mads Corvinius; Larsen, Anders Foller; Abdikadir, Faisal Hussein

    2014-01-01

    G-quadruplex (G4) ligands are currently receiving considerable attention as potential anticancer therapeutics. A series of phenanthroline-2,9-bistriazoles carrying tethered positive end groups has been synthesized and evaluated as G4 stabilizers. The compounds were efficiently assembled by copper......(I)-catalyzed azide-alkyne cycloaddition (CuAAC) in CH2Cl2 and water in the presence of a complexing agent. Characterization of the target compounds on telomeric and c-KIT G4 sequences led to the identification of guanidinium-substituted compounds as potent G4 DNA ligands with high selectivity over duplex DNA....... The diisopropylguanidium ligands exhibited high selectivity for the proto-oncogenic sequence c-KIT over the human telomeric sequence in the surface plasmon resonance (SPR) assay, whereas the compounds appeared potent on both G4 structures in the FRET melting temperature assay. The phenanthroline-2,9-bistriazole ligands...

  1. Design and synthesis of dual 5-HT1A and 5-HT7 receptor ligands.

    Science.gov (United States)

    Ofori, Edward; Zhu, Xue Y; Etukala, Jagan R; Peprah, Kwakye; Jordan, Kamanski R; Adkins, Adia A; Bricker, Barbara A; Kang, Hye J; Huang, Xi-Ping; Roth, Bryan L; Ablordeppey, Seth Y

    2016-08-15

    5-HT1A and 5-HT7 receptors have been at the center of discussions recently due in part to their major role in the etiology of major central nervous system diseases such as depression, sleep disorders, and schizophrenia. As part of our search to identify dual targeting ligands for these receptors, we have carried out a systematic modification of a selective 5HT7 receptor ligand culminating in the identification of several dual 5-HT1A and 5-HT7 receptor ligands. Compound 16, a butyrophenone derivative of tetrahydroisoquinoline (THIQ), was identified as the most potent agent with low nanomolar binding affinities to both receptors. Interestingly, compound 16 also displayed moderate affinity to other clinically relevant dopamine receptors. Thus, it is anticipated that compound 16 may serve as a lead for further exploitation in our quest to identify new ligands with the potential to treat diseases of CNS origin.

  2. Tools and techniques to study ligand-receptor interactions and receptor activation by TNF superfamily members.

    Science.gov (United States)

    Schneider, Pascal; Willen, Laure; Smulski, Cristian R

    2014-01-01

    Ligands and receptors of the TNF superfamily are therapeutically relevant targets in a wide range of human diseases. This chapter describes assays based on ELISA, immunoprecipitation, FACS, and reporter cell lines to monitor interactions of tagged receptors and ligands in both soluble and membrane-bound forms using unified detection techniques. A reporter cell assay that is sensitive to ligand oligomerization can identify ligands with high probability of being active on endogenous receptors. Several assays are also suitable to measure the activity of agonist or antagonist antibodies, or to detect interactions with proteoglycans. Finally, self-interaction of membrane-bound receptors can be evidenced using a FRET-based assay. This panel of methods provides a large degree of flexibility to address questions related to the specificity, activation, or inhibition of TNF-TNF receptor interactions in independent assay systems, but does not substitute for further tests in physiologically relevant conditions.

  3. Structural determinants for the inhibitory ligands of orotidine-5′-monophosphate decarboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Meza-Avina, Maria Elena; Wei, Lianhu; Liu, Yan; Poduch, Ewa; Bello, Angelica M.; Mishra, Ram K.; Pai, Emil F.; Kotra, Lakshmi P. (TGRI); (Toronto)

    2010-06-14

    In recent years, orotidine-5{prime}-monophosphate decarboxylase (ODCase) has gained renewed attention as a drug target. As a part of continuing efforts to design novel inhibitors of ODCase, we undertook a comprehensive study of potent, structurally diverse ligands of ODCase and analyzed their structural interactions in the active site of ODCase. These ligands comprise of pyrazole or pyrimidine nucleotides including the mononucleotide derivatives of pyrazofurin, barbiturate ribonucleoside, and 5-cyanouridine, as well as, in a computational approach, 1,4-dihydropyridine-based non-nucleoside inhibitors such as nifedipine and nimodipine. All these ligands bind in the active site of ODCase exhibiting distinct interactions paving the way to design novel inhibitors against this interesting enzyme. We propose an empirical model for the ligand structure for rational modifications in new drug design and potentially new lead structures.

  4. Structural basis for PPAR partial or full activation revealed by a novel ligand binding mode

    Science.gov (United States)

    Capelli, Davide; Cerchia, Carmen; Montanari, Roberta; Loiodice, Fulvio; Tortorella, Paolo; Laghezza, Antonio; Cervoni, Laura; Pochetti, Giorgio; Lavecchia, Antonio

    2016-10-01

    The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors involved in the regulation of the metabolic homeostasis and therefore represent valuable therapeutic targets for the treatment of metabolic diseases. The development of more balanced drugs interacting with PPARs, devoid of the side-effects showed by the currently marketed PPARγ full agonists, is considered the major challenge for the pharmaceutical companies. Here we present a structure-based virtual screening approach that let us identify a novel PPAR pan-agonist with a very attractive activity profile and its crystal structure in the complex with PPARα and PPARγ, respectively. In PPARα this ligand occupies a new pocket whose filling is allowed by the ligand-induced switching of the F273 side chain from a closed to an open conformation. The comparison between this pocket and the corresponding cavity in PPARγ provides a rationale for the different activation of the ligand towards PPARα and PPARγ, suggesting a novel basis for ligand design.

  5. ONRLDB--manually curated database of experimentally validated ligands for orphan nuclear receptors: insights into new drug discovery.

    Science.gov (United States)

    Nanduri, Ravikanth; Bhutani, Isha; Somavarapu, Arun Kumar; Mahajan, Sahil; Parkesh, Raman; Gupta, Pawan

    2015-01-01

    Orphan nuclear receptors are potential therapeutic targets. The Orphan Nuclear Receptor Ligand Binding Database (ONRLDB) is an interactive, comprehensive and manually curated database of small molecule ligands targeting orphan nuclear receptors. Currently, ONRLDB consists of ∼11,000 ligands, of which ∼6500 are unique. All entries include information for the ligand, such as EC50 and IC50, number of aromatic rings and rotatable bonds, XlogP, hydrogen donor and acceptor count, molecular weight (MW) and structure. ONRLDB is a cross-platform database, where either the cognate small molecule modulators of a receptor or the cognate receptors to a ligand can be searched. The database can be searched using three methods: text search, advanced search or similarity search. Substructure search, cataloguing tools, and clustering tools can be used to perform advanced analysis of the ligand based on chemical similarity fingerprints, hierarchical clustering, binning partition and multidimensional scaling. These tools, together with the Tree function provided, deliver an interactive platform and a comprehensive resource for identification of common and unique scaffolds. As demonstrated, ONRLDB is designed to allow selection of ligands based on various properties and for designing novel ligands or to improve the existing ones. Database URL: http://www.onrldb.org/.

  6. Architecture effects on multivalent interactions by polypeptide-based multivalent ligands

    Science.gov (United States)

    Liu, Shuang

    protein materials, including structural as well as functional proteins. Therefore, polypeptide-based multivalent scaffolds are used to display ligands to assess the contribution of different architectural parameters to the multivalent binding events. In this work, a family of alanine-rich alpha-helical glycopolypeptides was designed and synthesized by a combination of protein engineering and chemical coupling, to display two types of saccharide ligands for two different multivalent binding systems. The valencies, chain length and spacing between adjacent ligands of these multivalent ligands were designed in order to study architecture effects on multivalent interactions. The polypeptides and their glycoconjugates were characterized via various methods, including SDS-PAGE, NMR, HPLC, amino acid analysis (AAA), MALDI, circular dichroism (CD) and GPC. In the first multivalent binding system, cholera toxin B pentamer (CT B5) was chosen to be the protein receptor due to its well-characterized structure, lack of significant steric interference of binding to multiple binding sites, and requirement of only simple monosaccharide as ligands. Galactopyranoside was incorporated into polypeptide scaffolds through amine-carboxylic acid coupling to the side chains of glutamic acid residues. The inhibition and binding to CT B5 of these glycopolypeptide ligands were evaluated by direct enzyme-linked assay (DELA). As a complement method, weak affinity chromatography (WAC) was also used to evaluate glycopolypeptides binding to a CT B5 immobilized column. The architecture effects on CT B 5 inhibition are discussed. In the second system, cell surface receptor L-selectin was targeted by polypeptide-based multivalent ligands containing disulfated galactopyranoside ligands, due to its important roles in various immunological activities. The effects of glycopolypeptide architectural variables L-selectin shedding were evaluated via ELISA-based assays. These polypeptide-based multivalent ligands

  7. PL-PatchSurfer: A Novel Molecular Local Surface-Based Method for Exploring Protein-Ligand Interactions

    Directory of Open Access Journals (Sweden)

    Bingjie Hu

    2014-08-01

    Full Text Available Structure-based computational methods have been widely used in exploring protein-ligand interactions, including predicting the binding ligands of a given protein based on their structural complementarity. Compared to other protein and ligand representations, the advantages of a surface representation include reduced sensitivity to subtle changes in the pocket and ligand conformation and fast search speed. Here we developed a novel method named PL-PatchSurfer (Protein-Ligand PatchSurfer. PL-PatchSurfer represents the protein binding pocket and the ligand molecular surface as a combination of segmented surface patches. Each patch is characterized by its geometrical shape and the electrostatic potential, which are represented using the 3D Zernike descriptor (3DZD. We first tested PL-PatchSurfer on binding ligand prediction and found it outperformed the pocket-similarity based ligand prediction program. We then optimized the search algorithm of PL-PatchSurfer using the PDBbind dataset. Finally, we explored the utility of applying PL-PatchSurfer to a larger and more diverse dataset and showed that PL-PatchSurfer was able to provide a high early enrichment for most of the targets. To the best of our knowledge, PL-PatchSurfer is the first surface patch-based method that treats ligand complementarity at protein binding sites. We believe that using a surface patch approach to better understand protein-ligand interactions has the potential to significantly enhance the design of new ligands for a wide array of drug-targets.

  8. Exploiting novel molecular targets in gastrointestinal cancers

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Novel molecular targets are being discovered as we learn more about the aberrant processes underlying various cancers. Efforts to translate this knowledge are starting to impact on the care of patients with gastrointestinal cancers. The epidermal growth factor receptor (EGFR) pathway and angiogenesis have been targeted successfully in colorectal cancer with cetuximab, panitunumab and bevacizumab. Similarly, EGFR-targeting with erlotinib yielded significant survival benefit in pancreatic cancer when combined with gemcitabine. The multi-targeting approach with sorafenib has made it the first agent to achieve significant survival benefit in hepatocellular carcinoma. Efforts to exploit the dysregulated Akt/mTOR pathway in GI cancer therapy are ongoing. These molecular targets can be disrupted by various approaches, including the use of monoclonal antibody to intercept extracellular ligands and disrupt receptor-ligand binding, and small molecule inhibitors that interrupt the activation of intracellular kinases.

  9. Improved Targeting of Cancers with Nanotherapeutics

    DEFF Research Database (Denmark)

    Foster, Christian; Watson, Andre; Kaplinsky, Joseph John;

    2017-01-01

    Targeted cancer nanotherapeutics offers numerous opportunities for the selective uptake of toxic chemotherapies within tumors and cancer cells. The unique properties of nanoparticles, such as their small size, large surface-to-volume ratios, and the ability to achieve multivalency of targeting...... ligands on their surface, provide superior advantages for nanoparticle-based drug delivery to a variety of cancers. This review highlights various key concepts in the design of targeted nanotherapeutics for cancer therapy, and discusses physicochemical parameters affecting nanoparticle targeting, along...... with recent developments for cancer-targeted nanomedicines....

  10. Galactose as Broad Ligand for Multiple Tumor Imaging and Therapy.

    Science.gov (United States)

    Ma, Yuxiang; Chen, Haiyan; Su, Shanyuhan; Wang, Tong; Zhang, Congying; Fida, Guissi; Cui, Sisi; Zhao, Juan; Gu, Yueqing

    2015-01-01

    Galactose residues could be specifically recognized by the asialoglycoprotein receptor (ASGPR) which is highly exhibited on liver tissues. However, ASGPR has not been widely investigated on different tumor cell lines except for hepatoma carcinoma cells, which motivates us to investigate the possibility of galactose serving as a board tumor ligand. In this study, a galactose (Gal)-based probe conjugated with fluorescence dye MPA (Gal-MPA) was constructed for the evaluation of tumor affinities/targeted ability on different tumor cell lines. In the vitro cell study, it was indicated that the fluorescence probe Gal-MPA displayed higher cell affinity to tumor cells (HepG2, MCF-7 and A549) than that of the normal liver cells l02. In the vivo dynamic study of Gal-MPA in tumor-bearing mice (HepG2, MCF-7, A549, HCT116, U87, MDA-MB-231 and S180), it was shown that its high tumor targeted ability with the maximal tumor/normal tissue ratio reached up to 6.8. Meanwhile, the fast tumor-targeted ability within 2 hours and long retention on tumor site up to 120 hours were observed. Our results demonstrated that galactose should be a promising broad ligand for multiple tumor imaging and targeted therapy. Subsequently, Gal was covalently conjugated to doxorubicin (DOX) to form prodrug Gal-DOX for tumor targeted therapy. The therapeutic results of Gal-DOX than DOX being better suggested that galactosylated prodrugs might have the prospective potential in tumor targeted therapy.

  11. Targeting Nuclear Receptors with Marine Natural Products

    Directory of Open Access Journals (Sweden)

    Chunyan Yang

    2014-01-01

    Full Text Available Nuclear receptors (NRs are important pharmaceutical targets because they are key regulators of many metabolic and inflammatory diseases, including diabetes, dyslipidemia, cirrhosis, and fibrosis. As ligands play a pivotal role in modulating nuclear receptor activity, the discovery of novel ligands for nuclear receptors represents an interesting and promising therapeutic approach. The search for novel NR agonists and antagonists with enhanced selectivities prompted the exploration of the extraordinary chemical diversity associated with natural products. Recent studies involving nuclear receptors have disclosed a number of natural products as nuclear receptor ligands, serving to re-emphasize the translational possibilities of natural products in drug discovery. In this review, the natural ligands of nuclear receptors will be described with an emphasis on their mechanisms of action and their therapeutic potentials, as well as on strategies to determine potential marine natural products as nuclear receptor modulators.

  12. PoLi: A Virtual Screening Pipeline Based on Template Pocket and Ligand Similarity.

    Science.gov (United States)

    Roy, Ambrish; Srinivasan, Bharath; Skolnick, Jeffrey

    2015-08-24

    Often in pharmaceutical research the goal is to identify small molecules that can interact with and appropriately modify the biological behavior of a new protein target. Unfortunately, most proteins lack both known structures and small molecule binders, prerequisites of many virtual screening, VS, approaches. For such proteins, ligand homology modeling, LHM, that copies ligands from homologous and perhaps evolutionarily distant template proteins, has been shown to be a powerful VS approach to identify possible binding ligands. However, if we want to target a specific pocket for which there is no homologous holo template protein structure, then LHM will not work. To address this issue, in a new pocket-based approach, PoLi, we generalize LHM by exploiting the fact that the number of distinct small molecule ligand-binding pockets in proteins is small. PoLi identifies similar ligand-binding pockets in a holo template protein library, selectively copies relevant parts of template ligands, and uses them for VS. In practice, PoLi is a hybrid structure and ligand-based VS algorithm that integrates 2D fingerprint-based and 3D shape-based similarity metrics for improved virtual screening performance. On standard DUD and DUD-E benchmark databases, using modeled receptor structures, PoLi achieves an average enrichment factor of 13.4 and 9.6, respectively, in the top 1% of the screened library. In contrast, traditional docking-based VS using AutoDock Vina and homology-based VS using FINDSITE(filt) have an average enrichment of 1.6 (3.0) and 9.0 (7.9) on the DUD (DUD-E) sets, respectively. Experimental validation of PoLi predictions on dihydrofolate reductase, DHFR, using differential scanning fluorimetry, DSF, identifies multiple ligands with diverse molecular scaffolds, thus demonstrating the advantage of PoLi over current state-of-the-art VS methods.

  13. 01-ERD-111 - The Development of Synthetic High Affinity Ligands

    Energy Technology Data Exchange (ETDEWEB)

    Perkins, J; Balhorn, R; Cosman, M; Lightstone, F; Zeller, L

    2004-02-05

    The aim of this project was to develop Synthetic High-Affinity Ligands (SHALs), which bind with high affinity and specificity to proteins of interest for national security and cancer therapy applications. The aim of producing synthetic ligands for sensory devices as an alternative to antibody-based detection assays and therapeutic agents is to overcome the drawbacks associated with antibody-based in next-generation sensors and systems. The focus area of the project was the chemical synthesis of the SHALs. The project concentrated on two different protein targets. (a) The C fragment of tetanus and botulinum toxin, potential biowarfare agents. A SHAL for tetanus or botulinum toxin would be incorporated into a sensory device for the toxins. (b) HLA-DR10, a protein found in high abundance on the surface of Non-Hodgkins Lymphoma. A SHAL specific to a tumor marker, labeled with a radionuclide, would enable the targeted delivery of radiation therapy to metastatic disease. The technical approach used to develop a SHAL for each protein target will be described in more detail below. However, in general, the development of a SHAL requires a combination of computational modeling techniques, modern nuclear magnetic resonance spectroscopy (NMR) and synthetic chemistry.

  14. Cofactor-Controlled Chirality of Tropoisomeric Ligand

    NARCIS (Netherlands)

    Théveau, L.; Bellini, R.; Dydio, P.; Szabo, Z.; van der Werf, A.; Sander, R.A.; Reek, J.N.H.; Moberg, C.

    2016-01-01

    A new tropos ligand with an integrated anion receptor receptor site has been prepared. Chiral carboxylate and phosphate anions that bind in the anion receptor unit proved capable of stabilizing chiral conformations of the achiral flexible bidentate biaryl phosphite ligand, as shown by variable

  15. Flexible Ligand Docking Using Evolutionary Algorithms

    DEFF Research Database (Denmark)

    Thomsen, Rene

    2003-01-01

    The docking of ligands to proteins can be formulated as a computational problem where the task is to find the most favorable energetic conformation among the large space of possible protein–ligand complexes. Stochastic search methods such as evolutionary algorithms (EAs) can be used to sample large...

  16. Flexible Ligand Docking Using Differential Evolution

    DEFF Research Database (Denmark)

    Thomsen, René

    2003-01-01

    the most favorable energetic conformation among the large space of possible protein-ligand complexes. Stochastic search methods, such as evolutionary algorithms (EAs), can be used to sample large search spaces effectively and is one of the preferred methods for flexible ligand docking. The differential...

  17. Rhodium olefin complexes of diiminate type ligands

    NARCIS (Netherlands)

    Willems, Sander Theodorus Hermanus

    2003-01-01

    The mono-anionic beta-diiminate ligand (ArNC(CH3)CHC(CH3)NAr) on several previous occasions proved useful in stabilising low coordination numbers for both early and late transition metals. In this thesis the reactivity of the rhodium olefin complexes of one of these beta-diiminate ligands (Ar = 2,6-

  18. Ligand sphere conversions in terminal carbide complexes

    DEFF Research Database (Denmark)

    Morsing, Thorbjørn Juul; Reinholdt, Anders; Sauer, Stephan P. A.

    2016-01-01

    Metathesis is introduced as a preparative route to terminal carbide complexes. The chloride ligands of the terminal carbide complex [RuC(Cl)2(PCy3)2] (RuC) can be exchanged, paving the way for a systematic variation of the ligand sphere. A series of substituted complexes, including the first exam...

  19. Directed evolution of estrogen receptor proteins with altered ligand-binding specificities.

    Science.gov (United States)

    Islam, Kazi Mohammed Didarul; Dilcher, Meik; Thurow, Corinna; Vock, Carsten; Krimmelbein, Ilga Kristine; Tietze, Lutz Friedjan; Gonzalez, Victor; Zhao, Huimin; Gatz, Christiane

    2009-01-01

    Transcriptional activators that respond to ligands with no cellular targets are powerful tools that can confer regulated expression of a transgene in almost all biological systems. In this study, we altered the ligand-binding specificity of the human estrogen receptor alpha (hER alpha) so that it would recognize a non-steroidal synthetic compound with structural similarities to the phytoestrogen resveratrol. For this purpose, we performed iterative rounds of site-specific saturation mutagenesis of a fixed set of ligand-contacting residues and subsequent random mutagenesis of the entire ligand-binding domain. Selection of the receptor mutants and quantification of the interaction were carried out by exploiting a yeast two-hybrid system that reports the ligand-dependent interaction between hER alpha and steroid receptor coactivator-1 (SRC-1). The screen was performed with a synthetic ligand (CV3320) that promoted growth of the reporter yeast strain to half maximal levels at a concentration of 3.7 microM. The optimized receptor mutant (L384F/L387M/Y537S) showed a 67-fold increased activity to the synthetic ligand CV3320 (half maximal yeast growth at 0.055 microM) and a 10-fold decreased activity to 17beta-estradiol (E2; half maximal yeast growth at 4 nM). The novel receptor-ligand pair partially fulfills the requirements for a specific 'gene switch' as it responds to concentrations of the synthetic ligand which do not activate the wildtype receptor. Due to its residual responsiveness to E2 at concentrations (4 nM) that might occur in vivo, further improvements have to be performed to render the system applicable in organisms with endogenous E2 synthesis.

  20. Androgen Receptor Promotes Ligand-Independent Prostate Cancer Progression through c-Myc Upregulation

    Science.gov (United States)

    Gao, Lina; Schwartzman, Jacob; Gibbs, Angela; Lisac, Robert; Kleinschmidt, Richard; Wilmot, Beth; Bottomly, Daniel; Coleman, Ilsa; Nelson, Peter; McWeeney, Shannon; Alumkal, Joshi

    2013-01-01

    The androgen receptor (AR) is the principal therapeutic target in prostate cancer. For the past 70 years, androgen deprivation therapy (ADT) has been the major therapeutic focus. However, some patients do not benefit, and those tumors that do initially respond to ADT eventually progress. One recently described mechanism of such an effect is growth and survival-promoting effects of the AR that are exerted independently of the AR ligands, testosterone and dihydrotestosterone. However, specific ligand-independent AR target genes that account for this effect were not well characterized. We show here that c-Myc, which is a key mediator of ligand-independent prostate cancer growth, is a key ligand-independent AR target gene. Using microarray analysis, we found that c-Myc and AR expression levels strongly correlated with each other in tumors from patients with castration-resistant prostate cancer (CRPC) progressing despite ADT. We confirmed that AR directly regulates c-Myc transcription in a ligand-independent manner, that AR and c-Myc suppression reduces ligand-independent prostate cancer cell growth, and that ectopic expression of c-Myc attenuates the anti-growth effects of AR suppression. Importantly, treatment with the bromodomain inhibitor JQ1 suppressed c-Myc function and suppressed ligand-independent prostate cancer cell survival. Our results define a new link between two critical proteins in prostate cancer – AR and c-Myc – and demonstrate the potential of AR and c-Myc-directed therapies to improve prostate cancer control. PMID:23704919

  1. Asymmetric catalysis based on tropos ligands.

    Science.gov (United States)

    Aikawa, Kohsuke; Mikami, Koichi

    2012-11-21

    All enantiopure atropisomeric (atropos) ligands essentially require enantiomeric resolution or synthetic transformation from a chiral pool. In sharp contrast, the use of tropos (chirally flexible) ligands, which are highly modular, versatile, and easy to synthesize without enantiomeric resolution, has recently been the topic of much interest in asymmetric catalysis. Racemic catalysts bearing tropos ligands can be applied to asymmetric catalysis through enantiomeric discrimination by the addition of a chiral source, which preferentially transforms one catalyst enantiomer into a highly activated catalyst enantiomer. Additionally, racemic catalysts bearing tropos ligands can also be utilized as atropos enantiopure catalysts obtained via the control of chirality by a chiral source followed by the memory of chirality. In this feature article, our results on the asymmetric catalysis via the combination of various central metals and tropos ligands are summarized.

  2. Ligand-Gated Ion Channels: Permeation and Activation1

    Science.gov (United States)

    Lynch, Joseph W.; Barry, Peter H.

    Ligand-gated ion channels (LGICs) are fast-responding channels in which the receptor, which binds the activating molecule (the ligand), and the ion channel are part of the same nanomolecular protein complex. This chapter will describe the properties and functions of the nicotinic acetylcholine LGIC superfamily, which play a critical role in the fast chemical transmission of electrical signals between nerve cells at synapses and between nerve and muscle cells at endplates. All the processing functions of the brain and the resulting behavioral output depend on chemical transmission across such neuronal interconnections. To describe the properties of the channels of this LGIC superfamily,we will mainly use two examples of this family of channels: the excitatory nicotinic acetylcholine receptor (nAChR) and the inhibitory glycine receptor (GlyR) channels. In the chemical transmission of electrical signals, the arrival of an electrical signal at the synaptic terminal of a nerve causes the release of a chemical signal—a neurotransmitter molecule (the ligand, also referred to as the agonist). The neurotransmitter rapidly diffuses across the very narrow 20-40 nm synaptic gap between the cells and binds to the LGIC receptors in the membrane of the target (postsynaptic) cell and generates a new electrical signal in that cell (e.g., Kandel et al., 2000). How this chemical signal is converted into an electrical one depends on the fundamental properties of LGICs and the ionic composition of the postsynaptic cell and its external solution.

  3. Electrophilic Metal Alkyl Chemistry in New Ligand Environments

    Energy Technology Data Exchange (ETDEWEB)

    Jordan, Richard F. [University of Chicago

    2013-06-30

    The goals of this project were to design new electrophilic metal alkyl complexes and to exploit these systems in fundamental studies of olefin polymerization and other important and new catalytic reactions. A key target reaction is insertion copolymerization of olefins and polar CH2=CHX vinyl monomers such as vinyl halides and vinyl ethers. During the period covered by this report we (i) investigated the properties of ortho-alkoxy-arylphosphine ligands in Ni-based olefin polymerization catalysts, (ii) studied the synthesis of double-end-capped polyethylene using group 4 metal catalysts that contain tris-pyrazolylborate ligands, (iii) explored the ethylene insertion reactivity of group 4 metal tris-pyrazolyl-borate complexes, (iv) showed that (α-diimine)PdMe{sup +} species undergo multiple insertion of silyl vinyl ethers, (v) synthesized and explored the reactivity of base-free Ni benzyl complexes that contain ortho-phosphino-arene sulfonate ligands, (vi) established the mechanism of the reaction of vinyl chloride with (α-diimine)PdMe{sup +} catalysts, (vii) explored the role of cationic polymerization and insertion chemistry in the reactions of vinyl ethers with (α-diimine)PdMe{sup +} species, (viii) discovered a new class of self-assembled tetranuclear Pd catalysts that produce high molecular weight linear polyethylene and copolymerize ethylene and vinyl fluoride, and (ix) developed model systems that enabled investigation of cis-trans isomerization of {phosphine-sulfonate}Pd(II) complexes.

  4. Ligand binding mechanics of maltose binding protein.

    Science.gov (United States)

    Bertz, Morten; Rief, Matthias

    2009-11-13

    In the past decade, single-molecule force spectroscopy has provided new insights into the key interactions stabilizing folded proteins. A few recent studies probing the effects of ligand binding on mechanical protein stability have come to quite different conclusions. While some proteins seem to be stabilized considerably by a bound ligand, others appear to be unaffected. Since force acts as a vector in space, it is conceivable that mechanical stabilization by ligand binding is dependent on the direction of force application. In this study, we vary the direction of the force to investigate the effect of ligand binding on the stability of maltose binding protein (MBP). MBP consists of two lobes connected by a hinge region that move from an open to a closed conformation when the ligand maltose binds. Previous mechanical experiments, where load was applied to the N and C termini, have demonstrated that MBP is built up of four building blocks (unfoldons) that sequentially detach from the folded structure. In this study, we design the pulling direction so that force application moves the two MBP lobes apart along the hinge axis. Mechanical unfolding in this geometry proceeds via an intermediate state whose boundaries coincide with previously reported MBP unfoldons. We find that in contrast to N-C-terminal pulling experiments, the mechanical stability of MBP is increased by ligand binding when load is applied to the two lobes and force breaks the protein-ligand interactions directly. Contour length measurements indicate that MBP is forced into an open conformation before unfolding even if ligand is bound. Using mutagenesis experiments, we demonstrate that the mechanical stabilization effect is due to only a few key interactions of the protein with its ligand. This work illustrates how varying the direction of the applied force allows revealing important details about the ligand binding mechanics of a large protein.

  5. Integrin Targeted Delivery of Chemotherapeutics

    Directory of Open Access Journals (Sweden)

    Kai Chen, Xiaoyuan Chen

    2011-01-01

    Full Text Available Targeted delivery of chemotherapeutics is defined in the sense, that is, to maximize the therapeutic index of a chemotherapeutic agent by strictly localizing its pharmacological activity to the site or tissue of action. Integrins are a family of heterodimeric transmembrane glycoproteins involved in a wide range of cell-to-extracellular matrix (ECM and cell-to-cell interactions. As cell surface receptors, integrins readily interact with extracellular ligands and play a vital role in angiogenesis, leukocytes function and tumor development, which sets up integrins as an excellent target for chemotherapy treatment. The peptide ligands containing the arginine-glycine-aspartic acid (RGD, which displays a strong binding affinity and selectivity to integrins, particularly to integrin αvβ3, have been developed to conjugate with various conventional chemotherapeutic agents, such as small molecules, peptides and proteins, and nanoparticle-carried drugs for integtrin targeted therapeutic studies. This review highlights the recent advances in integrin targeted delivery of chemotherapeutic agents with emphasis on target of integrin αvβ3, and describes the considerations for the design of the diverse RGD peptide-chemotherapeutics conjugates and their major applications.

  6. Tuning the topology and functionality of metal-organic frameworks by ligand design.

    Science.gov (United States)

    Zhao, Dan; Timmons, Daren J; Yuan, Daqiang; Zhou, Hong-Cai

    2011-02-15

    Metal-organic frameworks (MOFs)-highly crystalline hybrid materials that combine metal ions with rigid organic ligands-have emerged as an important class of porous materials. The organic ligands add flexibility and diversity to the chemical structures and functions of these materials. In this Account, we summarize our laboratory's experience in tuning the topology and functionality of MOFs by ligand design. These investigations have led to new materials with interesting properties. By using a ligand that can adopt different symmetry conformations through free internal bond rotation, we have obtained two MOFs that are supramolecular stereoisomers of each other at different reaction temperatures. In another case, where the dimerized ligands function as a D(3)-Piedfort unit spacer, we achieve chiral (10,3)-a networks. In the design of MOF-based materials for hydrogen and methane storage, we focused on increasing the gas affinity of frameworks by using ligands with different geometries to control the pore size and effectively introduce unsaturated metal centers (UMCs) into the framework. Framework interpenetration in PCN-6 (PCN stands for porous coordination network) can lead to higher hydrogen uptake. Because of the proper alignment of the UMCs, PCN-12 holds the record for uptake of hydrogen at 77 K/760 Torr. In the case of methane storage, PCN-14 with anthracene-derived ligand achieves breakthrough storage capacity, at a level 28% higher than the U.S. Department of Energy target. Selective gas adsorption requires a pore size comparable to that of the target gas molecules; therefore, we use bulky ligands and network interpenetration to reduce the pore size. In addition, with the help of an amphiphilic ligand, we were able to use temperature to continuously change pore size in a 2D layer MOF. Adding charge to an organic ligand can also stabilize frameworks. By ionizing the amine group within mesoMOF-1, the resulting electronic repulsion keeps the network from

  7. Carcinoma-Associated Fibroblasts Are a Promising Therapeutic Target

    Energy Technology Data Exchange (ETDEWEB)

    Togo, Shinsaku, E-mail: shinsaku@juntendo.ac.jp [Department of Respiratory Medicine, Juntendo University School of Medicine, Tokyo 113-8412 (Japan); Polanska, Urszula M. [CR-UK Stromal-Tumour Interaction Group, Paterson Institute for Cancer Research, The University of Manchester, Wilmslow Road, Manchester M20 4BX (United Kingdom); Horimoto, Yoshiya [Department of Pathology and Oncology, Juntendo University School of Medicine, Tokyo 113-8412 (Japan); Atopy Research Centre, Juntendo University School of Medicine, Tokyo 113-8412 (Japan); Department of Breast Oncology, Juntendo University School of Medicine, Tokyo 113-8412 (Japan); Orimo, Akira, E-mail: shinsaku@juntendo.ac.jp [CR-UK Stromal-Tumour Interaction Group, Paterson Institute for Cancer Research, The University of Manchester, Wilmslow Road, Manchester M20 4BX (United Kingdom); Department of Pathology and Oncology, Juntendo University School of Medicine, Tokyo 113-8412 (Japan); Atopy Research Centre, Juntendo University School of Medicine, Tokyo 113-8412 (Japan)

    2013-01-31

    Human carcinomas frequently exhibit significant stromal reactions such as the so-called “desmoplastic stroma” or “reactive stroma”, which is characterised by the existence of large numbers of stromal cells and extracellular matrix proteins. Carcinoma-associated fibroblasts (CAFs), which are rich in activated fibroblast populations exemplified by myofibroblasts, are among the predominant cell types present within the tumour-associated stroma. Increased numbers of stromal myofibroblasts are often associated with high-grade malignancies with poor prognoses in humans. CAF myofibroblasts possess abilities to promote primary tumour development, growth and progression by stimulating the processes of neoangiogenesis as well as tumour cell proliferation, survival, migration and invasion. Moreover, it has been demonstrated that CAFs serve as a niche supporting the metastatic colonisation of disseminated carcinoma cells in distant organs. Their contribution to primary and secondary malignancies makes these fibroblasts a potential therapeutic target and they also appear to be relevant to the development of drug resistance and tumour recurrence. This review summarises our current knowledge of tumour-promoting CAFs and discusses the therapeutic feasibility of targeting these cells as well as disrupting heterotypic interactions with other cell types in tumours that may improve the efficacy of current anti-tumour therapies.

  8. Liver-targeting macromolecular MRI contrast agents

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Macromolecular ligands with liver-targeting group (pyridoxamine, PM) PHEA-DTPA-PM and PAEA-DTPA-PM were prepared by the incorporation of different amount of diethylenetriaminepentaacetic acid monopyridoxamine group (DTPA-PM) into poly-a, b-[N-(2-hydroxyethyl)-L- aspartamide] (PHEA) and poly-a, b-[N-(2-aminoethyl)-L-aspartamide] (PAEA). The macromolecular ligands thus obtained were further complexed with gadolinium chloride to give macromolecular MRI contrast agents with different Gd(Ⅲ) contents. These macromolecular ligands and their gadolinium complexes were characterized by 1H NMR, IR, UV and elementary analysis. Relaxivity studies showed that these polyaspartamide gadolinium complexes possess higher relaxation effectiveness than that of the clinically used Gd-DTPA. Magnetic resonance imaging of the liver in rats and experimental data of biodistribution in mice indicate that these macromolecular MRI contrast agents containing pyridoxamine exhibit liver-targeting property.

  9. Correcting ligands, metabolites, and pathways

    Directory of Open Access Journals (Sweden)

    Vriend Gert

    2006-11-01

    Full Text Available Abstract Background A wide range of research areas in bioinformatics, molecular biology and medicinal chemistry require precise chemical structure information about molecules and reactions, e.g. drug design, ligand docking, metabolic network reconstruction, and systems biology. Most available databases, however, treat chemical structures more as illustrations than as a datafield in its own right. Lack of chemical accuracy impedes progress in the areas mentioned above. We present a database of metabolites called BioMeta that augments the existing pathway databases by explicitly assessing the validity, correctness, and completeness of chemical structure and reaction information. Description The main bulk of the data in BioMeta were obtained from the KEGG Ligand database. We developed a tool for chemical structure validation which assesses the chemical validity and stereochemical completeness of a molecule description. The validation tool was used to examine the compounds in BioMeta, showing that a relatively small number of compounds had an incorrect constitution (connectivity only, not considering stereochemistry and that a considerable number (about one third had incomplete or even incorrect stereochemistry. We made a large effort to correct the errors and to complete the structural descriptions. A total of 1468 structures were corrected and/or completed. We also established the reaction balance of the reactions in BioMeta and corrected 55% of the unbalanced (stoichiometrically incorrect reactions in an automatic procedure. The BioMeta database was implemented in PostgreSQL and provided with a web-based interface. Conclusion We demonstrate that the validation of metabolite structures and reactions is a feasible and worthwhile undertaking, and that the validation results can be used to trigger corrections and improvements to BioMeta, our metabolite database. BioMeta provides some tools for rational drug design, reaction searches, and

  10. Expression of mammalian GPCRs in C. elegans generates novel behavioural responses to human ligands

    NARCIS (Netherlands)

    M.S. Teng (Michelle); M.P.J. Dekkers (Martijn); B.L. Ng (Bee Ling); S. Rademakers (Suzanne); G. Jansen (Gert); A.G. Fraser (Andrew); J. McCafferty (Gert)

    2006-01-01

    textabstractBackground: G-protein-coupled receptors (GPCRs) play a crucial role in many biological processes and represent a major class of drug targets. However, purification of GPCRs for biochemical study is difficult and current methods of studying receptor-ligand interactions involve in vitro sy

  11. Expression of mammalian GPCRs in C. elegans generates novel behavioural responses to human ligands

    NARCIS (Netherlands)

    M.S. Teng (Michelle); M.P.J. Dekkers (Martijn); B.L. Ng (Bee Ling); S. Rademakers (Suzanne); G. Jansen (Gert); A.G. Fraser (Andrew); J. McCafferty (Gert)

    2006-01-01

    textabstractBackground: G-protein-coupled receptors (GPCRs) play a crucial role in many biological processes and represent a major class of drug targets. However, purification of GPCRs for biochemical study is difficult and current methods of studying receptor-ligand interactions involve in vitro sy

  12. The molecular basis of ligand interaction at free fatty acid receptor 4 (FFA4/GPR120)

    DEFF Research Database (Denmark)

    Hudson, Brian D; Shimpukade, Bharat; Milligan, Graeme

    2014-01-01

    The long-chain fatty acid receptor FFA4 (previously GPR120) is receiving substantial interest as a novel target for the treatment of metabolic and inflammatory disease. This study examines for the first time the detailed mode of binding of both long-chain fatty acid and synthetic agonist ligands ...

  13. Tuning of gate adsorption: modification of a flexible metal-organic framework by secondary organic ligands.

    Science.gov (United States)

    Kondo, Atsushi; Fujii, Takuro; Maeda, Kazuyuki

    2014-06-14

    For realizing selective adsorption of targeted molecules, a flexible metal-organic framework (MOF) was modified with monodentate secondary ligands. Although the modified MOF retains CO2 adsorptivities with a vertical adsorption uptake, the material also shows gate adsorptivities of a specific gas molecule that the pristine MOF does not adsorb.

  14. 1,2,3-triazolyl amino acids as AMPA receptor ligands

    DEFF Research Database (Denmark)

    Stanley, Nathan J.; Pedersen, Daniel Sejer; Nielsen, Birgitte

    2010-01-01

    The central nervous system glutamate receptors are an important target for drug discovery. Herein we report initial investigations into the synthesis and glutamate receptor activity of 1,2,3-triazolyl amino acids. Two compounds were found to be selective AMPA receptor ligands, which warrant further...

  15. Multicomponent mixtures for cryoprotection and ligand solubilization

    Directory of Open Access Journals (Sweden)

    Lidia Ciccone

    2015-09-01

    Full Text Available Mixed cryoprotectants have been developed for the solubilization of ligands for crystallization of protein–ligand complexes and for crystal soaking. Low affinity lead compounds with poor solubility are problematic for structural studies. Complete ligand solubilization is required for co-crystallization and crystal soaking experiments to obtain interpretable electron density maps for the ligand. Mixed cryo-preserving compounds are needed prior to X-ray data collection to reduce radiation damage at synchrotron sources. Here we present dual-use mixes that act as cryoprotectants and also promote the aqueous solubility of hydrophobic ligands. Unlike glycerol that increases protein solubility and can cause crystal melting the mixed solutions of cryo-preserving compounds that include precipitants and solubilizers, allow for worry-free crystal preservation while simultaneously solubilizing relatively hydrophobic ligands, typical of ligands obtained in high-throughput screening. The effectiveness of these mixture has been confirmed on a human transthyretin crystals both during crystallization and in flash freezing of crystals.

  16. Coordinate unsaturation with fluorinated ligands

    Energy Technology Data Exchange (ETDEWEB)

    Rack, J.L.; Hurlburt, P.K.; Anderson, O.P.; Strauss, S.H. [Colorado State Univ., Ft. Collins, CO (United States)

    1993-12-31

    The preparation and characterization of Zn(OTeF{sub 5}){sub 2} has resulted in a model compound with which to explore the concept of coordinative unsaturation. The coordination of solvents of varying donicity and dielectric constant to the Zn(II) ions in Zn(OTeF{sub 5}){sub 2} was studied by vapor phase monometry, NMR and IR spectroscopy, conductimetry, and X-Ray crystallography. The structures of [Zn(C{sub 6}H{sub 5}NO{sub 2}){sub 2}(OTeF{sub 5})2]2 and Zn(C{sub 6}H{sub 5}NO{sub 2}){sub 3}(OTEF{sub 5}){sub 2} demonstrate the electronic flexibility of some weakly coordinating solvents in that nitrobenzene can function as either an {eta}{sup 1}O or {eta}{sup 2}O,O`-ligand. The dependence of the number of bound solvent molecules and the degree of OTeF{sub 5}{minus} dissociation on solvent donor number and dielectric constant will be presented.

  17. International Union of Basic and Clinical Pharmacology. XC. multisite pharmacology: recommendations for the nomenclature of receptor allosterism and allosteric ligands.

    Science.gov (United States)

    Christopoulos, Arthur; Changeux, Jean-Pierre; Catterall, William A; Fabbro, Doriano; Burris, Thomas P; Cidlowski, John A; Olsen, Richard W; Peters, John A; Neubig, Richard R; Pin, Jean-Philippe; Sexton, Patrick M; Kenakin, Terry P; Ehlert, Frederick J; Spedding, Michael; Langmead, Christopher J

    2014-10-01

    Allosteric interactions play vital roles in metabolic processes and signal transduction and, more recently, have become the focus of numerous pharmacological studies because of the potential for discovering more target-selective chemical probes and therapeutic agents. In addition to classic early studies on enzymes, there are now examples of small molecule allosteric modulators for all superfamilies of receptors encoded by the genome, including ligand- and voltage-gated ion channels, G protein-coupled receptors, nuclear hormone receptors, and receptor tyrosine kinases. As a consequence, a vast array of pharmacologic behaviors has been ascribed to allosteric ligands that can vary in a target-, ligand-, and cell-/tissue-dependent manner. The current article presents an overview of allostery as applied to receptor families and approaches for detecting and validating allosteric interactions and gives recommendations for the nomenclature of allosteric ligands and their properties.

  18. Distribution of unselectively bound ligands along DNA.

    Science.gov (United States)

    Lando, Dmitri Y; Nechipurenko, Yury D

    2008-10-01

    Unselective and reversible adsorption of ligands on DNA for a model of binding proposed by Zasedatelev, Gursky, and Volkenshtein is considered. In this model, the interaction between neighboring ligands located at the distance of i binding centers is characterized by the statistical weight ai. Each ligand covers L binding centers. For this model, expressions for binding averages are represented in a new simple form. This representation is convenient for the calculation of the fraction of inter-ligand distances of i binding centers fd(i) and the fraction of binding centers included in the distances of i binding centers fbc(i) for various types of interaction between bound ligands. It is shown that, for non-cooperative binding, contact cooperativity and long-range cooperativity, the fraction of the zero inter-ligand distance fd(0) is maximal at any relative concentration of bound ligands (r). Calculations demonstrate that, at low r, fd(0) approximately r.ao, and fd(i) approximately r at 11/r-L, then fd(i) rapidly decreases with i at any r for all types of inter-ligand interaction. At high ligand concentration (r is close to rmax=L(-1)), fd(0) is close to unity and fd(i) rapidly decreases with i for any type of inter-ligand interaction. For strong contact cooperativity, fd(0) is close to unity in a much lager r interval ((0.5-1).rmax), and fd(1) approximately ao(-1) at r approximately 0.5.rmax. In the case of long-range interaction between bound ligands, the dependence fd(i) is more complex and has a maximum at i approximately (1/r-L)1/2 for anti-cooperative binding. fbc(i) is maximal at i approximately 1/r-L for all types of binding except the contact cooperativity. A strong asymmetry in the influence of contact cooperativity and anticooperativity on the ligand distribution along DNA is demonstrated.

  19. Investigations of Takeout proteins' ligand binding and release mechanism using molecular dynamics simulation.

    Science.gov (United States)

    Zhang, Huijing; Yu, Hui; Zhao, Xi; Liu, Xiaoguang; Feng, Xianli; Huang, Xuri

    2016-07-29

    Takeout (To) proteins exist in a diverse range of insect species. They are involved in many important processes of insect physiology and behaviors. As the ligand carriers, To proteins can transport the small molecule to the target tissues. However, ligand release mechanism of To proteins is unclear so far. In this contribution, the process and pathway of the ligand binding and release are revealed by conventional molecular dynamics simulation, steered molecular dynamics simulation and umbrella sampling methods. Our results show that the α4-side of the protein is the unique gate for the ligand binding and release. The structural analysis confirms that the internal cavity of the protein has high rigidity, which is in accordance with the recent experimental results. By using the potential of mean force calculations in combination with residue cross correlation calculation, we concluded that the binding between the ligand and To proteins is a process of conformational selection. Furthermore, the conformational changes of To proteins and the hydrophobic interactions both are the key factors for ligand binding and release.

  20. Enzyme-linked enzyme-binding assay for Pin1 WW domain ligands.

    Science.gov (United States)

    Mercedes-Camacho, Ana Y; Etzkorn, Felicia A

    2010-07-01

    Peptidyl prolyl cis-trans isomerase (PPIase) interacting with NIMA-1 (Pin1) catalyzes the cis-trans isomerization of pSer/pThr-Pro amide bonds. Pin1 is a two-domain protein that represents a promising target for the treatment of cancer. Both domains of Pin1 bind the pSer/pThr-Pro motif; PPIase enzymatic activity occurs in the catalytic domain, and the WW domain acts as a recognition module for the pSer/pThr-Pro motif. An assay we call an enzyme-linked enzyme-binding assay (ELEBA) was developed to measure the K(d) of ligands that bind selectively to the WW domain. A ligand specific for the WW domain of Pin1 was covalently immobilized in a 96-well plate. Commercially available Pin1 conjugated to horseradish peroxidase was used for chemiluminescent detection of ligands that block the association of the WW domain with immobilized ligand. The peptide ligands were derived from the cell cycle regulatory phosphatase, Cdc25c, residues 45-50. The K(d) values for Fmoc-VPRpTPVGGGK-NH2 and Ac-VPRpTPV-NH2 were determined to be 36+/-4 and 110+/-30 microM, respectively. The ELEBA offers a selective approach for detecting ligands that bind to the Pin1 WW domain, even in the presence of the catalytic domain. This method may be applied to any dual specificity, multidomain protein. 2010 Elsevier Inc. All rights reserved.

  1. A comparison of various optimization algorithms of protein-ligand docking programs by fitness accuracy.

    Science.gov (United States)

    Guo, Liyong; Yan, Zhiqiang; Zheng, Xiliang; Hu, Liang; Yang, Yongliang; Wang, Jin

    2014-07-01

    In protein-ligand docking, an optimization algorithm is used to find the best binding pose of a ligand against a protein target. This algorithm plays a vital role in determining the docking accuracy. To evaluate the relative performance of different optimization algorithms and provide guidance for real applications, we performed a comparative study on six efficient optimization algorithms, containing two evolutionary algorithm (EA)-based optimizers (LGA, DockDE) and four particle swarm optimization (PSO)-based optimizers (SODock, varCPSO, varCPSO-ls, FIPSDock), which were implemented into the protein-ligand docking program AutoDock. We unified the objective functions by applying the same scoring function, and built a new fitness accuracy as the evaluation criterion that incorporates optimization accuracy, robustness, and efficiency. The varCPSO and varCPSO-ls algorithms show high efficiency with fast convergence speed. However, their accuracy is not optimal, as they cannot reach very low energies. SODock has the highest accuracy and robustness. In addition, SODock shows good performance in efficiency when optimizing drug-like ligands with less than ten rotatable bonds. FIPSDock shows excellent robustness and is close to SODock in accuracy and efficiency. In general, the four PSO-based algorithms show superior performance than the two EA-based algorithms, especially for highly flexible ligands. Our method can be regarded as a reference for the validation of new optimization algorithms in protein-ligand docking.

  2. Accelerated Molecular Dynamics Simulations of Ligand Binding to a Muscarinic G-protein Coupled Receptor

    Science.gov (United States)

    Kappel, Kalli; Miao, Yinglong; McCammon, J. Andrew

    2017-01-01

    Elucidating the detailed process of ligand binding to a receptor is pharmaceutically important for identifying druggable binding sites. With the ability to provide atomistic detail, computational methods are well poised to study these processes. Here, accelerated molecular dynamics (aMD) is proposed to simulate processes of ligand binding to a G-protein coupled receptor (GPCR), in this case the M3 muscarinic receptor, which is a target for treating many human diseases, including cancer, diabetes and obesity. Long-timescale aMD simulations were performed to observe the binding of three chemically diverse ligand molecules: antagonist tiotropium (TTP), partial agonist arecoline (ARc), and full agonist acetylcholine (ACh). In comparison with earlier microsecond-timescale conventional MD simulations, aMD greatly accelerated the binding of ACh to the receptor orthosteric ligand-binding site and the binding of TTP to an extracellular vestibule. Further aMD simulations also captured binding of ARc to the receptor orthosteric site. Additionally, all three ligands were observed to bind in the extracellular vestibule during their binding pathways, suggesting that it is a metastable binding site. This study demonstrates the applicability of aMD to protein-ligand binding, especially the drug recognition of GPCRs. PMID:26537408

  3. Structure-based discovery of selective serotonin 5-HT(1B) receptor ligands.

    Science.gov (United States)

    Rodríguez, David; Brea, José; Loza, María Isabel; Carlsson, Jens

    2014-08-05

    The development of safe and effective drugs relies on the discovery of selective ligands. Serotonin (5-hydroxytryptamine [5-HT]) G protein-coupled receptors are therapeutic targets for CNS disorders but are also associated with adverse drug effects. The determination of crystal structures for the 5-HT1B and 5-HT2B receptors provided an opportunity to identify subtype selective ligands using structure-based methods. From docking screens of 1.3 million compounds, 22 molecules were predicted to be selective for the 5-HT1B receptor over the 5-HT2B subtype, a requirement for safe serotonergic drugs. Nine compounds were experimentally verified as 5-HT1B-selective ligands, with up to 300-fold higher affinities for this subtype. Three of the ligands were agonists of the G protein pathway. Analysis of state-of-the-art homology models of the two 5-HT receptors revealed that the crystal structures were critical for predicting selective ligands. Our results demonstrate that structure-based screening can guide the discovery of ligands with specific selectivity profiles.

  4. Phenanthroline-2,9-bistriazoles as selective G-quadruplex ligands.

    Science.gov (United States)

    Nielsen, Mads Corvinius; Larsen, Anders Foller; Abdikadir, Faisal Hussein; Ulven, Trond

    2014-01-24

    G-quadruplex (G4) ligands are currently receiving considerable attention as potential anticancer therapeutics. A series of phenanthroline-2,9-bistriazoles carrying tethered positive end groups has been synthesized and evaluated as G4 stabilizers. The compounds were efficiently assembled by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) in CH2Cl2 and water in the presence of a complexing agent. Characterization of the target compounds on telomeric and c-KIT G4 sequences led to the identification of guanidinium-substituted compounds as potent G4 DNA ligands with high selectivity over duplex DNA. The diisopropylguanidium ligands exhibited high selectivity for the proto-oncogenic sequence c-KIT over the human telomeric sequence in the surface plasmon resonance (SPR) assay, whereas the compounds appeared potent on both G4 structures in the FRET melting temperature assay. The phenanthroline-2,9-bistriazole ligands were thus identified as potent G4 ligands with high selectivity over duplex DNA, and preliminary results indicate that the scaffold may form basis for the development of subtype-specific G4 ligands.

  5. A Vitamin D Receptor Selectively Activated by Gemini Analogs Reveals Ligand Dependent and Independent Effects

    Directory of Open Access Journals (Sweden)

    Tiphaine Huet

    2015-02-01

    Full Text Available The bioactive form of vitamin D [1,25(OH2D3] regulates mineral and bone homeostasis and exerts potent anti-inflammatory and antiproliferative properties through binding to the vitamin D receptor (VDR. The 3D structures of the VDR ligand-binding domain with 1,25(OH2D3 or gemini analogs unveiled the molecular mechanism underlying ligand recognition. On the basis of structure-function correlations, we generated a point-mutated VDR (VDRgem that is unresponsive to 1,25(OH2D3, but the activity of which is efficiently induced by the gemini ligands. Moreover, we show that many VDR target genes are repressed by unliganded VDRgem and that mineral ion and bone homeostasis are more impaired in VDRgem mice than in VDR null mice, demonstrating that mutations abolishing VDR ligand binding result in more severe skeletal defects than VDR null mutations. As gemini ligands induce VDRgem transcriptional activity in mice and normalize their serum calcium levels, VDRgem is a powerful tool to further unravel both liganded and unliganded VDR signaling.

  6. Discovery of Aptamer Ligands for Hepatic Stellate Cells Using SELEX.

    Science.gov (United States)

    Chen, Zhijin; Liu, Hao; Jain, Akshay; Zhang, Li; Liu, Chang; Cheng, Kun

    2017-01-01

    Insulin like growth factor II receptor (IGFIIR) is a transmembrane protein overexpressed in activated hepatic stellate cells (HSCs), which are the major target for the treatment of liver fibrosis. In this study, we aim to discover an IGFIIR-specific aptamer that can be potentially used as a targeting ligand for the treatment and diagnosis of liver fibrosis. Systematic evolution of ligands by exponential enrichment (SELEX) was conducted on recombinant human IGFIIR to identify IGFIIR-specific aptamers. The binding affinity and specificity of the discovered aptamers to IGFIIR and hepatic stellate cells were studied using flow cytometry and Surface Plasmon Resonance (SPR). Aptamer-20 showed the highest affinity to recombinant human IGFIIR protein with a Kd of 35.5 nM, as determined by SPR. Aptamer-20 also has a high affinity (apparent Kd 45.12 nM) to LX-2 human hepatic stellate cells. Binding of aptamer-20 to hepatic stellate cells could be inhibited by knockdown of IGFIIR using siRNA, indicating a high specificity of the aptamer. The aptamer formed a chimera with an anti-fibrotic PCBP2 siRNA and delivered the siRNA to HSC-T6 cells to trigger silencing activity. In Vivo biodistribution study of the siRNA-aptamer chimera also demonstrated a high and specific uptake in the liver of the rats with CCl4-induced liver fibrosis. These data suggest that aptamer-20 is a high-affinity ligand for antifibrotic and diagnostic agents for liver fibrosis.

  7. LIBSA--a method for the determination of ligand-binding preference to allosteric sites on receptor ensembles.

    Science.gov (United States)

    Hocker, Harrison J; Rambahal, Nandini; Gorfe, Alemayehu A

    2014-02-24

    Incorporation of receptor flexibility into computational drug discovery through the relaxed complex scheme is well suited for screening against a single binding site. In the absence of a known pocket or if there are multiple potential binding sites, it may be necessary to do docking against the entire surface of the target (global docking). However no suitable and easy-to-use tool is currently available to rank global docking results based on the preference of a ligand for a given binding site. We have developed a protocol, termed LIBSA for LIgand Binding Specificity Analysis, that analyzes multiple docked poses against a single or ensemble of receptor conformations and returns a metric for the relative binding to a specific region of interest. By using novel filtering algorithms and the signal-to-noise ratio (SNR), the relative ligand-binding frequency at different pockets can be calculated and compared quantitatively. Ligands can then be triaged by their tendency to bind to a site instead of ranking by affinity alone. The method thus facilitates screening libraries of ligand cores against a large library of receptor conformations without prior knowledge of specific pockets, which is especially useful to search for hits that selectively target a particular site. We demonstrate the utility of LIBSA by showing that it correctly identifies known ligand binding sites and predicts the relative preference of a set of related ligands for different pockets on the same receptor.

  8. PSOVina: The hybrid particle swarm optimization algorithm for protein-ligand docking.

    Science.gov (United States)

    Ng, Marcus C K; Fong, Simon; Siu, Shirley W I

    2015-06-01

    Protein-ligand docking is an essential step in modern drug discovery process. The challenge here is to accurately predict and efficiently optimize the position and orientation of ligands in the binding pocket of a target protein. In this paper, we present a new method called PSOVina which combined the particle swarm optimization (PSO) algorithm with the efficient Broyden-Fletcher-Goldfarb-Shannon (BFGS) local search method adopted in AutoDock Vina to tackle the conformational search problem in docking. Using a diverse data set of 201 protein-ligand complexes from the PDBbind database and a full set of ligands and decoys for four representative targets from the directory of useful decoys (DUD) virtual screening data set, we assessed the docking performance of PSOVina in comparison to the original Vina program. Our results showed that PSOVina achieves a remarkable execution time reduction of 51-60% without compromising the prediction accuracies in the docking and virtual screening experiments. This improvement in time efficiency makes PSOVina a better choice of a docking tool in large-scale protein-ligand docking applications. Our work lays the foundation for the future development of swarm-based algorithms in molecular docking programs. PSOVina is freely available to non-commercial users at http://cbbio.cis.umac.mo .

  9. Novel Suicide Ligands of Tubulin Arrest Cancer Cells in S-Phase

    Directory of Open Access Journals (Sweden)

    Ashley Davis

    1999-12-01

    Full Text Available It is presently accepted that the mechanism of action for all anti-tumor tubulin ligands involves the perturbation of microtubule dynamics during the G2/M phase of cell division and subsequent entry into apoptosis 1]. In this report, we challenge the established dogma by describing a unique mechanism of action caused by a novel series of tubulin ligands, halogenated derivatives of acetamido benzoyl ethyl ester. We have developed a suicide ligand for tubulin, which covalently attaches to the target and shows potent cancericidal activity in tissue culture assays and in animal tumor models. These compounds target early S-phase at the G1/S transition rather than the G2/M phase and mitotic arrest. Bcl-2 phosphorylation, a marker of mitotic microtubule inhibition by other tubulin ligands was dramatically altered, phosphorylation was rapid and biphasic rather than a slow linear event. The halogenated ethyl ester series of derivatives thus constitute a unique set of tubulin ligands which induce a novel mechanism of apoptosis.

  10. Programmed Death Ligand 2 in Cancer-Induced Immune Suppression

    Directory of Open Access Journals (Sweden)

    Esdy N. Rozali

    2012-01-01

    Full Text Available Inhibitory molecules of the B7/CD28 family play a key role in the induction of immune tolerance in the tumor microenvironment. The programmed death-1 receptor (PD-1, with its ligands PD-L1 and PD-L2, constitutes an important member of these inhibitory pathways. The relevance of the PD-1/PD-L1 pathway in cancer has been extensively studied and therapeutic approaches targeting PD-1 and PD-L1 have been developed and are undergoing human clinical testing. However, PD-L2 has not received as much attention and its role in modulating tumor immunity is less clear. Here, we review the literature on the immunobiology of PD-L2, particularly on its possible roles in cancer-induced immune suppression and we discuss the results of recent studies targeting PD-L2 in cancer.

  11. Identifying Protein Stabilizing Ligands Using GroEL

    Science.gov (United States)

    Naik, Subhashchandra; Haque, Inamul; Degner, Nick; Kornilayev, Boris; Bomhoff, Gregory; Hodges, Jacob; Khorassani, Ara-Azad; Katayama, Hiroo; Morris, Jill; Kelly, Jeffery; Seed, John; Fisher, Mark T.

    2010-01-01

    Over the past five years, it has become increasingly apparent to researchers that the initial promise and excitement of using gene replacement therapies to ameliorate folding diseases are still far from being broadly or easily applicable. Because a large number of human diseases are protein folding diseases (~30 to 50%), many researchers now realize that more directed approaches to target and reverse the fundamental misfolding reactions preceding disease are highly feasible and offer the potential of developing more targeted drug therapies. This is also true with a large number of so called “orphan protein folding diseases”. The development of a broad-based general screening array method using the chaperonin as a detection platform will enable us to screen large chemical combinatorial libraries for specific ligands against the elusive transient, primary reactions that often lead to protein misfolding. This development will provide a highly desirable tool for the pharmaceutical, academic and medical professions. PMID:19802819

  12. Ligand conjugation to bimodal poly(ethylene glycol) brush layers on microbubbles.

    Science.gov (United States)

    Chen, Cherry C; Borden, Mark A

    2010-08-17

    Using microbubbles as model systems, we examined molecular diffusion and binding to colloidal surfaces in bimodal poly(ethylene glycol) (PEG) brush layers. A microbubble is a gaseous colloidal particle with a diameter of less than 10 mum, of which the surface comprises amphiphilic phospholipids self-assembled to form a lipid monolayer shell. Due to the compressible gas core, microbubbles provide a sensitive acoustic response and are currently used as ultrasound contrast agents. Similar to the design of long circulating liposomes, PEG chains are typically incorporated into the shell of microbubbles to form a steric barrier against coalescence and adsorption of macromolecules to the microbubble surface. We introduced a buried-ligand architecture (BLA) design where the microbubble surface was coated with a bimodal PEG brush. After microbubbles were generated, fluorescent ligands with different molecular weights were conjugated to the tethered functional groups on the shorter PEG chains, while the longer PEG chains served as a shield to protect these ligands from exposure to the surrounding environment. BLA microbubbles reduced the binding of macromolecules (>10 kDa) to the tethers due to the steric hindrance of the PEG overbrush while allowing the uninhibited attachment of small molecules (microbubbles compared to exposed-ligand architecture (ELA) microbubbles. The binding of SA-FITC to BLA microbubbles suggested a possible phase separation between the lipid species on the surface leading to populations of revealed and concealed ligands. Ligand conjugation kinetics was independent of microbubble size, regardless of ligand size or microbubble architecture. We observed, for the first time, streptavidin-induced surface structure formation for ELA microbubbles and proposed that this phenomenon may be correlated to flow cytometry scattering measurements. We therefore demonstrated the feasibility of postlabeling for small-molecule ligands to BLA microbubbles to generate

  13. Dynamics of target-mediated drug disposition.

    Science.gov (United States)

    Peletier, Lambertus A; Gabrielsson, Johan

    2009-12-08

    We present a mathematical analysis of the basic model underlying target-mediated drug disposition (TMDD) in which a ligand is supplied through an initial bolus or through a constant rate infusion and forms a complex with a receptor (target), which is supplied and removed continuously. Ligand and complex may be eliminated according to first-order processes. We assume that the total receptor pool (free and bound) is constant in time and we give a geometrical description of the evolution of the concentrations of ligand, receptor and receptor-ligand complex which offers a transparent way to compare the full model with simpler models such as the quasi-steady-state (QSS) model, the quasi-equilibrium (QE) model and the empirical Michaelis-Menten (MM) model; we also give precise conditions on the parameters in the TMDD model for the validity of these reduced models. We relate characteristic properties of time courses to parameter regimes and, in particular, we identify and explain non-monotone dependence of the time-to-steady-state on the infusion rate. Finally, we discuss how the volume of the central compartment may be overestimated because of singular initial behaviour of the time course of the ligand concentration.

  14. Ligand inducible assembly of a DNA tetrahedron.

    Science.gov (United States)

    Dohno, Chikara; Atsumi, Hiroshi; Nakatani, Kazuhiko

    2011-03-28

    Here we show that a small synthetic ligand can be used as a key building component for DNA nanofabrication. Using naphthyridinecarbamate dimer (NCD) as a molecular glue for DNA hybridization, we demonstrate NCD-triggered formation of a DNA tetrahedron.

  15. Tuning the Topology and Functionality of Metal–Organic Frameworks by Ligand Design

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Dan; Timmons, Daren J; Yuan, Daqiang; Zhou, Hong-Cai

    2011-02-15

    Metal–organic frameworks (MOFs)—highly crystalline hybrid materials that combine metal ions with rigid organic ligands—have emerged as an important class of porous materials. The organic ligands add flexibility and diversity to the chemical structures and functions of these materials. In this Account, we summarize our laboratory’s experience in tuning the topology and functionality of MOFs by ligand design. These investigations have led to new materials with interesting properties. By using a ligand that can adopt different symmetry conformations through free internal bond rotation, we have obtained two MOFs that are supramolecular stereoisomers of each other at different reaction temperatures. In another case, where the dimerized ligands function as a D₃-Piedfort unit spacer, we achieve chiral (10,3)-a networks. In the design of MOF-based materials for hydrogen and methane storage, we focused on increasing the gas affinity of frameworks by using ligands with different geometries to control the pore size and effectively introduce unsaturated metal centers (UMCs) into the framework. Framework interpenetration in PCN-6 (PCN stands for porous coordination network) can lead to higher hydrogen uptake. Because of the proper alignment of the UMCs, PCN-12 holds the record for uptake of hydrogen at 77 K/760 Torr. In the case of methane storage, PCN-14 with anthracene-derived ligand achieves breakthrough storage capacity, at a level 28% higher than the U.S. Department of Energy target. Selective gas adsorption requires a pore size comparable to that of the target gas molecules; therefore, we use bulky ligands and network interpenetration to reduce the pore size. In addition, with the help of an amphiphilic ligand, we were able to use temperature to continuously change pore size in a 2D layer MOF. Adding charge to an organic ligand can also stabilize frameworks. By ionizing the amine group within mesoMOF-1, the resulting electronic repulsion keeps the network from

  16. Nye ligander for Pt-MOF strukturer

    OpenAIRE

    Jakobsen, Søren

    2006-01-01

    Metalorganic frameworks (MOFs) are a new type of compounds which have been intensely investigated during the last few years. They have been synthesized using a wide variety of metals and ligands constructing a vast number of 1, 2 and 3 dimensional structures, some of which possess zeolite-type physics and chemistry. Our approach is to incorporate platinum metal sites into the structures making them bimetallic and potentially catalytically active. Therefore a number of N-N-type ligands (dii...

  17. SnapShot: GPCR-Ligand Interactions.

    Science.gov (United States)

    Ghosh, Eshan; Nidhi, Kumari; Shukla, Arun K

    2014-12-18

    G-protein-coupled receptors enable cells to recognize numerous external stimuli and to transmit corresponding signals across the plasma membrane to trigger appropriate cellular responses. Crystal structures of a number of these receptors have now been determined in inactive and active conformations bound to chemically and functionally distinct ligands. These crystal structures illustrate overall receptor organization and atomic details of ligand-receptor interactions. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Development of the Novel PEG-PE-Based Polymer for the Reversible Attachment of Specific Ligands to Liposomes: Synthesis and in vitro Characterization

    Science.gov (United States)

    Biswas, Swati; Dodwadkar, Namita S.; Sawant, Rupa R.; Torchilin, Vladimir P.

    2011-01-01

    Surface grafting of liposomes with the wide variety of ligands including antibodies and other proteins is a promising approach for targeted delivery of therapeutics. In this paper, we describe a simple method of synthesizing a hydrazine-functionalized polyethylene glycol-phosphatidylethanolamine (PEG-PE)-based amphiphilic polymer which can conjugate a variety of ligands via a reversible, pH-cleavable bond. In this method, the targeting ligand is attached to the distal end of the PEG chain, which facilitates its easy access to the targeted site of interaction. The reversible attachment of targeting ligands is useful especially in multifunctional liposomal systems, where after successfully performing the function of targeting to the specific site, the bulky ligands, such as proteins or antibodies, are cleaved off in response to an environmental stimulus to expose some other functionalities such as ligands for intracellular penetration or organelle-specific targeting. To investigate the applicability of the protocol, the model ligands monoclonal antinucleosome antibody 2C5 and antimyosin antibody 2G4, and glycoproteins concanavalin A (Con-A) and avidin were conjugated to the synthesized polymer and incorporated into liposomes. In vitro assays including biochemical, enzyme-linked immunosorbent, fluorescence microscopy and flow cytometry were used to confirm three key characteristics of the modified and/or liposome-attached proteins: successful conjugation of the targeting ligands to the polymer, preservation of specific activity of the ligands after the conjugation and liposome attachment, and the facile pH-sensitive ligand detachment. Monoclonal mAb 2C5 and 2G4, immobilized on the liposome surface, retained their binding affinity to corresponding antigens as confirmed by ELISA. The Con A-bearing liposomes showed significantly higher agglutination in the presence of its substrate mannan compared to plain liposomes (PL) and avidin-functionalized liposomes bound

  19. A versatile dinucleating ligand containing sulfonamide groups

    DEFF Research Database (Denmark)

    Sundberg, Jonas; Witt, Hannes; Cameron, Lisa

    2014-01-01

    Copper, iron, and gallium coordination chemistries of the new pentadentate bis-sulfonamide ligand 2,6-bis(N-2-pyridylmethylsulfonamido)-4-methylphenol (psmpH3) were investigated. PsmpH3 is capable of varying degrees of deprotonation, and notably, complexes containing the fully trideprotonated...... ligand can be prepared in aqueous solutions using only divalent metal ions. Two of the copper(II) complexes, [Cu2(psmp)(OH)] and [Cu2(psmp)(OAc)2]-, demonstrate the anticipated 1:2 ligand/metal stoichiometry and show that the dimetallic binding site created for exogenous ligands possesses high inherent...... flexibility since additional one- and three-atom bridging ligands bridge the two copper(II) ions in each complex, respectively. This gives rise to a difference of 0.4 Å in the Cu···Cu distances. Complexes with 2:3 and 2:1 ligand/metal stoichiometries for the divalent and trivalent metal ions, respectively...

  20. LigandRFs: random forest ensemble to identify ligand-binding residues from sequence information alone

    KAUST Repository

    Chen, Peng

    2014-12-03

    Background Protein-ligand binding is important for some proteins to perform their functions. Protein-ligand binding sites are the residues of proteins that physically bind to ligands. Despite of the recent advances in computational prediction for protein-ligand binding sites, the state-of-the-art methods search for similar, known structures of the query and predict the binding sites based on the solved structures. However, such structural information is not commonly available. Results In this paper, we propose a sequence-based approach to identify protein-ligand binding residues. We propose a combination technique to reduce the effects of different sliding residue windows in the process of encoding input feature vectors. Moreover, due to the highly imbalanced samples between the ligand-binding sites and non ligand-binding sites, we construct several balanced data sets, for each of which a random forest (RF)-based classifier is trained. The ensemble of these RF classifiers forms a sequence-based protein-ligand binding site predictor. Conclusions Experimental results on CASP9 and CASP8 data sets demonstrate that our method compares favorably with the state-of-the-art protein-ligand binding site prediction methods.

  1. Fully Flexible Docking of Medium Sized Ligand Libraries with RosettaLigand.

    Directory of Open Access Journals (Sweden)

    Samuel DeLuca

    Full Text Available RosettaLigand has been successfully used to predict binding poses in protein-small molecule complexes. However, the RosettaLigand docking protocol is comparatively slow in identifying an initial starting pose for the small molecule (ligand making it unfeasible for use in virtual High Throughput Screening (vHTS. To overcome this limitation, we developed a new sampling approach for placing the ligand in the protein binding site during the initial 'low-resolution' docking step. It combines the translational and rotational adjustments to the ligand pose in a single transformation step. The new algorithm is both more accurate and more time-efficient. The docking success rate is improved by 10-15% in a benchmark set of 43 protein/ligand complexes, reducing the number of models that typically need to be generated from 1000 to 150. The average time to generate a model is reduced from 50 seconds to 10 seconds. As a result we observe an effective 30-fold speed increase, making RosettaLigand appropriate for docking medium sized ligand libraries. We demonstrate that this improved initial placement of the ligand is critical for successful prediction of an accurate binding position in the 'high-resolution' full atom refinement step.

  2. Mac-2 binding protein is a novel E-selectin ligand expressed by breast cancer cells.

    Science.gov (United States)

    Shirure, Venktesh S; Reynolds, Nathan M; Burdick, Monica M

    2012-01-01

    fluorescence microscopy, underscoring the possible role of Mac-2BP as an E-selectin ligand. In summary, breast cancer cells express Mac-2BP as a novel E-selectin ligand, potentially revealing a new prognostic and therapeutic target for breast cancer.

  3. Consensus of sample-balanced classifiers for identifying ligand-binding residue by co-evolutionary physicochemical characteristics of amino acids

    KAUST Repository

    Chen, Peng

    2013-01-01

    Protein-ligand binding is an important mechanism for some proteins to perform their functions, and those binding sites are the residues of proteins that physically bind to ligands. So far, the state-of-the-art methods search for similar, known structures of the query and predict the binding sites based on the solved structures. However, such structural information is not commonly available. In this paper, we propose a sequence-based approach to identify protein-ligand binding residues. Due to the highly imbalanced samples between the ligand-binding sites and non ligand-binding sites, we constructed several balanced data sets, for each of which a random forest (RF)-based classifier was trained. The ensemble of these RF classifiers formed a sequence-based protein-ligand binding site predictor. Experimental results on CASP9 targets demonstrated that our method compared favorably with the state-of-the-art. © Springer-Verlag Berlin Heidelberg 2013.

  4. Asialoglycoprotein receptor mediated hepatocyte targeting - strategies and applications.

    Science.gov (United States)

    D'Souza, Anisha A; Devarajan, Padma V

    2015-04-10

    Hepatocyte resident afflictions continue to affect the human population unabated. The asialoglycoprotein receptor (ASGPR) is primarily expressed on hepatocytes and minimally on extra-hepatic cells. This makes it specifically attractive for receptor-mediated drug delivery with minimum concerns of toxicity. ASGPR facilitates internalization by clathrin-mediated endocytosis and exhibits high affinity for carbohydrates specifically galactose, N-acetylgalactosamine and glucose. Isomeric forms of sugar, galactose density and branching, spatial geometry and galactose linkages are key factors influencing ligand-receptor binding. Popular ligands for ASGPR mediated targeting are carbohydrate polymers, arabinogalactan and pullulan. Other ligands include galactose-bearing glycoproteins, glycopeptides and galactose modified polymers and lipids. Drug-ligand conjugates provide a viable strategy; nevertheless ligand-anchored nanocarriers provide an attractive option for ASGPR targeted delivery and are widely explored. The present review details various ligands and nanocarriers exploited for ASGPR mediated delivery of drugs to hepatocytes. Nanocarrier properties affecting ASGPR mediated uptake are discussed at length. The review also highlights the clinical relevance of ASGPR mediated targeting and applications in diagnostics. ASGPR mediated hepatocyte targeting provides great promise for improved therapy of hepatic afflictions.

  5. Production, purification, and characterization of scFv TNF ligand fusion proteins.

    Science.gov (United States)

    Fick, Andrea; Wyzgol, Agnes; Wajant, Harald

    2012-01-01

    Single-chain variable fragments (scFvs) specific for tumor-associated cell surface antigens are the most broadly used reagents to direct therapeutic or diagnostic effector molecules, such as toxins, radioisotopes, and CD3-stimulating scFvs, to tumors. One novel class of effector molecules that can be targeted to tumors by scFvs are ligands of the tumor necrosis factor (TNF) family. Typically, these molecules have apoptosis inducing and/or immune stimulating properties and are therefore highly attractive for cancer treatment. N-terminal fusion of scFvs does not interfere with the receptor binding capabilities of TNF ligands and thus allows the straightforward generation of scFv TNF ligand fusion proteins. We report here a protocol for the purification of eukaryotically produced scFv TNF ligand fusion proteins based on affinity chromatography on anti-Flag agarose and further describe assays for the determination of the targeting index of this type of scFv-targeted proteins.

  6. Exploring the Ligand-Protein Networks in Traditional Chinese Medicine: Current Databases, Methods, and Applications

    Directory of Open Access Journals (Sweden)

    Mingzhu Zhao

    2013-01-01

    Full Text Available The traditional Chinese medicine (TCM, which has thousands of years of clinical application among China and other Asian countries, is the pioneer of the “multicomponent-multitarget” and network pharmacology. Although there is no doubt of the efficacy, it is difficult to elucidate convincing underlying mechanism of TCM due to its complex composition and unclear pharmacology. The use of ligand-protein networks has been gaining significant value in the history of drug discovery while its application in TCM is still in its early stage. This paper firstly surveys TCM databases for virtual screening that have been greatly expanded in size and data diversity in recent years. On that basis, different screening methods and strategies for identifying active ingredients and targets of TCM are outlined based on the amount of network information available, both on sides of ligand bioactivity and the protein structures. Furthermore, applications of successful in silico target identification attempts are discussed in detail along with experiments in exploring the ligand-protein networks of TCM. Finally, it will be concluded that the prospective application of ligand-protein networks can be used not only to predict protein targets of a small molecule, but also to explore the mode of action of TCM.

  7. Synthesis and characterization of mixed ligand chiral nanoclusters

    KAUST Repository

    Guven, Zekiye P.

    2016-06-22

    Chiral mixed ligand silver nanoclusters were synthesized in the presence of a chiral and an achiral ligand. While the chiral ligand led mostly to the formation of nanoparticles, the presence of the achiral ligand drastically increased the yield of nanoclusters with enhanced chiral properties. © 2016 The Royal Society of Chemistry.

  8. A Fluorescence Displacement Assay for Antidepressant Drug Discovery Based on Ligand-Conjugated Quantum Dots

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Jerry [Vanderbilt University; Tomlinson, Ian [Oak Ridge National Laboratory (ORNL); Warnement, Michael [Vanderbilt University; Iwamoto, Hideki [Vanderbilt University

    2011-01-01

    The serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) protein plays a central role in terminating 5-HT neurotransmission and is the most important therapeutic target for the treatment of major depression and anxiety disorders. We report an innovative, versatile, and target-selective quantum dot (QD) labeling approach for SERT in single Xenopus oocytes that can be adopted as a drug-screening platform. Our labeling approach employs a custom-made, QD-tagged indoleamine derivative ligand, IDT318, that is structurally similar to 5-HT and accesses the primary binding site with enhanced human SERT selectivity. Incubating QD-labeled oocytes with paroxetine (Paxil), a high-affinity SERT-specific inhibitor, showed a concentration- and time-dependent decrease in QD fluorescence, demonstrating the utility of our approach for the identification of SERT modulators. Furthermore, with the development of ligands aimed at other pharmacologically relevant targets, our approach may potentially form the basis for a multitarget drug discovery platform.

  9. PSMA Ligands for Radionuclide Imaging and Therapy of Prostate Cancer: Clinical Status.

    Science.gov (United States)

    Lütje, Susanne; Heskamp, Sandra; Cornelissen, Alexander S; Poeppel, Thorsten D; van den Broek, Sebastiaan A M W; Rosenbaum-Krumme, Sandra; Bockisch, Andreas; Gotthardt, Martin; Rijpkema, Mark; Boerman, Otto C

    2015-01-01

    Prostate cancer (PCa) is the most common malignancy in men worldwide, leading to substantial morbidity and mortality. At present, imaging of PCa has become increasingly important for staging, restaging, and treatment selection. Until recently, choline-based positron emission tomography/computed tomography (PET/CT) represented the state-of-the-art radionuclide imaging technique for these purposes. However, its application is limited to patients with high PSA levels and Gleason scores. Prostate-specific membrane antigen (PSMA) is a promising new target for specific imaging of PCa, because it is upregulated in the majority of PCa. Moreover, PSMA can serve as a target for therapeutic applications. Currently, several small-molecule PSMA ligands with excellent in vivo tumor targeting characteristics are being investigated for their potential in theranostic applications in PCa. Here, a review of the recent developments in PSMA-based diagnostic imaging and therapy in patients with PCa with radiolabeled PSMA ligands is provided.

  10. Antibiotic drugs targeting bacterial RNAs

    Directory of Open Access Journals (Sweden)

    Weiling Hong

    2014-08-01

    Full Text Available RNAs have diverse structures that include bulges and internal loops able to form tertiary contacts or serve as ligand binding sites. The recent increase in structural and functional information related to RNAs has put them in the limelight as a drug target for small molecule therapy. In addition, the recognition of the marked difference between prokaryotic and eukaryotic rRNA has led to the development of antibiotics that specifically target bacterial rRNA, reduce protein translation and thereby inhibit bacterial growth. To facilitate the development of new antibiotics targeting RNA, we here review the literature concerning such antibiotics, mRNA, riboswitch and tRNA and the key methodologies used for their screening.

  11. Comparison of ligand- and structure-based virtual screening on the DUD data set.

    Science.gov (United States)

    von Korff, Modest; Freyss, Joel; Sander, Thomas

    2009-02-01

    Several in-house developed descriptors and our in-house docking tool ActDock were compared with virtual screening on the data set of useful decoys (DUD). The results were compared with the chemical fingerprint descriptor from ChemAxon and with the docking results of the original DUD publication. The DUD is the first published data set providing active molecules, decoys, and references for crystal structures of ligand-target complexes. The DUD was designed for the purpose of evaluating docking programs. It contains 2950 active compounds against a total of 40 target proteins. Furthermore, for every ligand the data set contains 36 structurally dissimilar decoy compounds with similar physicochemical properties. We extracted the ligands from the target proteins to extend the applicability of the data set to include ligand based virtual screening. From the 40 target proteins, 37 contained ligands that we used as query molecules for virtual screening evaluation. With this data set a large comparison was done between four different chemical fingerprints, a topological pharmacophore descriptor, the Flexophore descriptor, and ActDock. The Actelion docking tool relies on a MM2 forcefield and a pharmacophore point interaction statistic for scoring; the details are described in this publication. In terms of enrichment rates the chemical fingerprint descriptors performed better than the Flexophore and the docking tool. After removing molecules chemically similar to the query molecules the Flexophore descriptor outperformed the chemical descriptors and the topological pharmacophore descriptors. With the similarity matrix calculations used in this study it was shown that the Flexophore is well suited to find new chemical entities via "scaffold hopping". The Flexophore descriptor can be explored with a Java applet at http://www.cheminformatics.ch in the submenu Tools-->Flexophore. Its usage is free of charge and does not require registration.

  12. Molecular Dynamics Investigation of gluazo, a Photo-Switchable Ligand for the Glutamate Receptor GluK2.

    Directory of Open Access Journals (Sweden)

    Yanan Guo

    Full Text Available Photochromic ligands (PCLs, defined as photoswitchable molecules that are able to endow native receptors with a sensitivity towards light, have become a promising photopharmacological tool for various applications in biology. In general, PCLs consist of a ligand of the target receptor covalently linked to an azobenzene, which can be reversibly switched between two configurations upon light illumination. Gluazo, as a PCL that targets excitatory amino acid receptors, in its dark-adapted trans iso-form was characterized to be a partial agonist of the kainate glutamate receptor GluK2. Application of UV light leads to the formation of the cis form, with remarkedly reduced affinity towards GluK2. The mechanism of the change of ligand affinity induced by the photoisomerization was unresolved. The presented computational study explains how the isomerization of such a PCL affects the structural changes in the target receptor that lead to its activation.

  13. Protozoacidal Trojan-Horse: use of a ligand-lytic peptide for selective destruction of symbiotic protozoa within termite guts.

    Science.gov (United States)

    Sethi, Amit; Delatte, Jennifer; Foil, Lane; Husseneder, Claudia

    2014-01-01

    For novel biotechnology-based termite control, we developed a cellulose bait containing freeze-dried genetically engineered yeast which expresses a protozoacidal lytic peptide attached to a protozoa-recognizing ligand. The yeast acts as a 'Trojan-Horse' that kills the cellulose-digesting protozoa in the termite gut, which leads to the death of termites, presumably due to inefficient cellulose digestion. The ligand targets the lytic peptide specifically to protozoa, thereby increasing its protozoacidal efficiency while protecting non-target organisms. After ingestion of the bait, the yeast propagates in the termite's gut and is spread throughout the termite colony via social interactions. This novel paratransgenesis-based strategy could be a good supplement for current termite control using fortified biological control agents in addition to chemical insecticides. Moreover, this ligand-lytic peptide system could be used for drug development to selectively target disease-causing protozoa in humans or other vertebrates.

  14. High-throughput identification of telomere-binding ligands based on the fluorescence regulation of DNA-copper nanoparticles.

    Science.gov (United States)

    Yang, Luzhu; Wang, Yanjun; Li, Baoxin; Jin, Yan

    2017-01-15

    Formation of the G-quadruplex in the human telomeric DNA is an effective way to inhibit telomerase activity. Therefore, screening ligands of G-quadruplex has potential applications in the treatment of cancer by inhibit telomerase activity. Although several techniques have been explored for screening of telomeric G-quadruplexes ligands, high-throughput screening method for fast screening telomere-binding ligands from the large compound library is still urgently needed. Herein, a label-free fluorescence strategy has been proposed for high-throughput screening telomere-binding ligands by using DNA-copper nanoparticles (DNA-CuNPs) as a signal probe. In the absence of ligands, human telomeric DNA (GDNA) hybridized with its complementary DNA (cDNA) to form double stranded DNA (dsDNA) which can act as an efficient template for the formation of DNA-CuNPs, leading to the high fluorescence of DNA-CuNPs. In the presence of ligands, GDNA folded into G-quadruplex. Single-strdanded cDNA does not support the formation of DNA-CuNP, resulting in low fluorescence of DNA-CuNPs. Therefore, telomere-binding ligands can be high-throughput screened by monitoring the change in the fluorescence of DNA-CuNPs. Thirteen traditional chinese medicines were screened. Circular dichroism (CD) measurements demonstrated that the selected ligands could induce single-stranded telomeric DNA to form G-quadruplex. The telomere repeat amplification protocol (TRAP) assay demonstrated that the selected ligands can effectively inhibit telomerase activity. Therefore, it offers a cost-effective, label-free and reliable high-throughput way to identify G-quadruplex ligands, which holds great potential in discovering telomerase-targeted anticancer drugs. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Characterization of the Raf kinase inhibitory protein (RKIP binding pocket: NMR-based screening identifies small-molecule ligands.

    Directory of Open Access Journals (Sweden)

    Anne N Shemon

    Full Text Available BACKGROUND: Raf kinase inhibitory protein (RKIP, also known as phoshaptidylethanolamine binding protein (PEBP, has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE. In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized. METHODS/FINDINGS: In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity. CONCLUSIONS/SIGNIFICANCE: This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential.

  16. Positron emission tomography evaluation of somatostatin receptor targeted (64)Cu-TATE-liposomes in a human neuroendocrine carcinoma mouse model

    DEFF Research Database (Denmark)

    Petersen, Anncatrine Luisa; Binderup, Tina; Jølck, Rasmus Irming

    2012-01-01

    Targeted therapeutic and diagnostic nanocarriers functionalized with antibodies, peptides or other targeting ligands that recognize over-expressed receptors or antigens on tumor cells have potential in the diagnosis and therapy of cancer. Somatostatin receptors (SSTRs) are over-expressed in a var......Targeted therapeutic and diagnostic nanocarriers functionalized with antibodies, peptides or other targeting ligands that recognize over-expressed receptors or antigens on tumor cells have potential in the diagnosis and therapy of cancer. Somatostatin receptors (SSTRs) are over...

  17. New polypyridine anchoring ligands for coordination complexes and surface functionalization

    OpenAIRE

    Müller, Steffen

    2015-01-01

    This PhD thesis focuses on the synthesis of new polypyridine anchoring ligands and several dfferent applications. The ligands consist of a coordinating part, a flexible linker and an anchoring group. Due to the fact that different anchoring groups were used, the ligands can be applied for several types of surface-materials. Using these anchoring ligands, several coordination complexes were synthesized. Ruthenium-based complexes, bearing an ion-sensitive ligand, were tested towards...

  18. Antiproton Target

    CERN Multimedia

    1980-01-01

    Antiproton target used for the AA (antiproton accumulator). The first type of antiproton production target used from 1980 to 1982 comprised a rod of copper 3mm diameter and 120mm long embedded in a graphite cylinder that was itself pressed into a finned aluminium container. This assembly was air-cooled and it was used in conjunction with the Van der Meer magnetic horn. In 1983 Fermilab provided us with lithium lenses to replace the horn with a view to increasing the antiproton yield by about 30%. These lenses needed a much shorter target made of heavy metal - iridium was chosen for this purpose. The 50 mm iridium rod was housed in an extension to the original finned target container so that it could be brought very close to the entrance to the lithium lens. Picture 1 shows this target assembly and Picture 2 shows it mounted together with the lithium lens. These target containers had a short lifetime due to a combination of beam heating and radiation damage. This led to the design of the water-cooled target in...

  19. Multiple ligand simultaneous docking: orchestrated dancing of ligands in binding sites of protein.

    Science.gov (United States)

    Li, Huameng; Li, Chenglong

    2010-07-30

    Present docking methodologies simulate only one single ligand at a time during docking process. In reality, the molecular recognition process always involves multiple molecular species. Typical protein-ligand interactions are, for example, substrate and cofactor in catalytic cycle; metal ion coordination together with ligand(s); and ligand binding with water molecules. To simulate the real molecular binding processes, we propose a novel multiple ligand simultaneous docking (MLSD) strategy, which can deal with all the above processes, vastly improving docking sampling and binding free energy scoring. The work also compares two search strategies: Lamarckian genetic algorithm and particle swarm optimization, which have respective advantages depending on the specific systems. The methodology proves robust through systematic testing against several diverse model systems: E. coli purine nucleoside phosphorylase (PNP) complex with two substrates, SHP2NSH2 complex with two peptides and Bcl-xL complex with ABT-737 fragments. In all cases, the final correct docking poses and relative binding free energies were obtained. In PNP case, the simulations also capture the binding intermediates and reveal the binding dynamics during the recognition processes, which are consistent with the proposed enzymatic mechanism. In the other two cases, conventional single-ligand docking fails due to energetic and dynamic coupling among ligands, whereas MLSD results in the correct binding modes. These three cases also represent potential applications in the areas of exploring enzymatic mechanism, interpreting noisy X-ray crystallographic maps, and aiding fragment-based drug design, respectively.

  20. Immobilisation of ligands by radio-derivatized polymers; Immobilisering av ligander med radioderiverte polymerer

    Energy Technology Data Exchange (ETDEWEB)

    Varga, J.M.; Fritsch, P.

    1995-01-30

    The invention relates to radio-derivatized polymers and a method of producing them by contacting non-polymerizable conjugands with radiolysable polymers in the presence of irradiation. The resulting radio-derivatized polymers can be further linked with ligand of organic or inorganic nature to immobilize such ligands. 2 figs., 5 tabs.

  1. Human cytomegalovirus UL141 promotes efficient downregulation of the natural killer cell activating ligand CD112

    Science.gov (United States)

    Prod'homme, Virginie; Sugrue, Daniel M.; Stanton, Richard J.; Nomoto, Akio; Davies, James; Rickards, Carole R.; Cochrane, Daniel; Moore, Melanie; Wilkinson, Gavin W. G.; Tomasec, Peter

    2010-01-01

    Human cytomegalovirus (HCMV) UL141 induces protection against natural killer cell-mediated cytolysis by downregulating cell surface expression of CD155 (nectin-like molecule 5; poliovirus receptor), a ligand for the activating receptor DNAM-1 (CD226). However, DNAM-1 is also recognized to bind a second ligand, CD112 (nectin-2). We now show that HCMV targets CD112 for proteasome-mediated degradation by 48 h post-infection, thus removing both activating ligands for DNAM-1 from the cell surface during productive infection. Significantly, cell surface expression of both CD112 and CD155 was restored when UL141 was deleted from the HCMV genome. While gpUL141 alone is sufficient to mediate retention of CD155 in the endoplasmic reticulum, UL141 requires assistance from additional HCMV-encoded functions to suppress expression of CD112. PMID:20410314

  2. Modulation of estrogen receptor α levels by endogenous and exogenous ligands

    Directory of Open Access Journals (Sweden)

    P. La Rosa

    2011-01-01

    Full Text Available ERα is a ligand-activated transcription factor, member of the nuclear receptor superfamily. Regulation of ERα levels is intrinsically required for its transcriptional activity and thus for the modulation of the physiological actions of the cognate hormone 17β-estradiol (E2. Indeed, ERα exogenous ligands that target this molecular circuitry are used as drugs in clinical practice. Interestingly, some natural and synthetic molecules, which human beings are commonly exposed to, interfere with the endocrine system and operate through ERα by selectively modifying its signalling. In addition, these molecules may also modulate ERα cellular content. Here, we report the recent advances in our understanding of how exogenous ERα ligands impact on receptor levels and change the physiological E2-dipendent modulation of specific cellular function.

  3. In Pursuit of Fully Flexible Protein-Ligand Docking: Modeling the Bilateral Mechanism of Binding.

    Science.gov (United States)

    Henzler, Angela M; Rarey, Matthias

    2010-03-15

    Modern structure-based drug design aims at accounting for the intrinsic flexibility of therapeutic relevant targets. Over the last few years a considerable amount of docking approaches that encounter this challenging problem has emerged. Here we provide the readership with an overview of established methods for fully flexible protein-ligand docking and current developments in the field. All methods are based on one of two fundamental models which describe the dynamic behavior of proteins upon ligand binding. Methods for ensemble docking (ED) model the protein conformational change before the ligand is placed, whereas induced-fit docking (IFD) optimizes the protein structure afterwards. A third category of docking approaches is formed by recent approaches that follow both concepts. This categorization allows to comprehensively discover strengths and weaknesses of the individual processes and to extract information for their applicability in real world docking scenarios.

  4. Peptides identify multiple hotspots within the ligand binding domain of the TNF receptor 2

    Directory of Open Access Journals (Sweden)

    Lennick Michael

    2003-01-01

    Full Text Available Abstract Background Hotspots are defined as the minimal functional domains involved in protein:protein interactions and sufficient to induce a biological response. Results Here we describe the use of complex and high diversity phage display libraries to isolate peptides (called Hotspot Ligands or HSPLs which sub-divide the ligand binding domain of the tumor necrosis factor receptor 2 (TNFR2; p75 into multiple hotspots. We have shown that these libraries could generate HSPLs which not only subdivide hotspots on protein and non-protein targets but act as agonists or antagonists. Using this approach, we generated peptides which were specific for human TNFR2, could be competed by the natural ligands, TNFα and TNFβ and induced an unexpected biological response in a TNFR2-specific manner. Conclusions To our knowledge, this is the first report describing the dissection of the TNFR2 into biologically active hotspots with the concomitant identification of a novel and unexpected biological activity.

  5. Organotellurium ligands - designing and complexation reactions

    Indian Academy of Sciences (India)

    Ajai K Singh

    2002-08-01

    A variety of tellurium ligands has been designed and studied for their complexation reactions in the last decade. Of these hybrid telluroethers, halotellurium ligands and polytellurides are the most notable ones. RTe- and polytelluride ions have also been used to design clusters. Ligation of ditelluroethers and several hybrid telluroethers is extensively studied in our laboratories. The ditelluroether ligand RTeCH2TeR (where R = 4-MeOC6H4) (1), similar to dppm [1,2-bis(diphenylphosphino) methane], has been synthesized in good yield (∼80 %) by reacting CHCl3 with RTe- (generated in situ by borohydride reduction of R2Te2). Iodine reacts with 1 to give tetra-iodo derivative, which has intermolecular Te$\\cdots$I interactions resulting in a macro structure containing rectangular Te-I$\\cdots$Te bridges. 1 readily forms four membered rings with Pd(II) and Ru(II). On the formation of this chelate ring, the signal in 125Te NMR spectra shifts significantly upfield (50-60 ppm). The bridging mode of 1 has been shown in [Ru(-cymene)Cl2](-1)[Ru(-cymene)Cl2]. The hybrid telluroether ligands explored are of the types (Te, S), (Te, N) and (Te, O). The tellurium donor site has strong trans influence, which is manifested more strongly in square planar complexes of palladium(II). The morpholine N-donor site has been found to have weaker donor characteristics in (Te, N) ligands than pyridine and alkylamine donor sites of analogous ligands. The singlet oxygen readily oxidises the coordinated Te. This oxidation follows first order kinetics. The complexation reaction of RuCl3.H2O with N-[2-(4-methoxyphenyltelluro)ethyl]phthalimide (2) results in a novel (Te, N, O)-heterocycle, Te-chloro,Te-anisyl-1a-aza-4-oxa-3-tellura-1H, 2H, 4aH-9 fluorenone. The (Te, O) ligands can be used as hemilabile ligands, the oxygen atom temporarily protects the vacant coordination site before the arrival of the substrate. The chelate shifts observed in 125Te NMR spectra of metal complexes of Te-ligands have

  6. Dockomatic - automated ligand creation and docking

    Directory of Open Access Journals (Sweden)

    Hampikian Greg

    2010-11-01

    Full Text Available Abstract Background The application of computational modeling to rationally design drugs and characterize macro biomolecular receptors has proven increasingly useful due to the accessibility of computing clusters and clouds. AutoDock is a well-known and powerful software program used to model ligand to receptor binding interactions. In its current version, AutoDock requires significant amounts of user time to setup and run jobs, and collect results. This paper presents DockoMatic, a user friendly Graphical User Interface (GUI application that eases and automates the creation and management of AutoDock jobs for high throughput screening of ligand to receptor interactions. Results DockoMatic allows the user to invoke and manage AutoDock jobs on a single computer or cluster, including jobs for evaluating secondary ligand interactions. It also automates the process of collecting, summarizing, and viewing results. In addition, DockoMatic automates creation of peptide ligand .pdb files from strings of single-letter amino acid abbreviations. Conclusions DockoMatic significantly reduces the complexity of managing multiple AutoDock jobs by facilitating ligand and AutoDock job creation and management.

  7. Dockomatic - automated ligand creation and docking.

    Science.gov (United States)

    Bullock, Casey W; Jacob, Reed B; McDougal, Owen M; Hampikian, Greg; Andersen, Tim

    2010-11-08

    The application of computational modeling to rationally design drugs and characterize macro biomolecular receptors has proven increasingly useful due to the accessibility of computing clusters and clouds. AutoDock is a well-known and powerful software program used to model ligand to receptor binding interactions. In its current version, AutoDock requires significant amounts of user time to setup and run jobs, and collect results. This paper presents DockoMatic, a user friendly Graphical User Interface (GUI) application that eases and automates the creation and management of AutoDock jobs for high throughput screening of ligand to receptor interactions. DockoMatic allows the user to invoke and manage AutoDock jobs on a single computer or cluster, including jobs for evaluating secondary ligand interactions. It also automates the process of collecting, summarizing, and viewing results. In addition, DockoMatic automates creation of peptide ligand .pdb files from strings of single-letter amino acid abbreviations. DockoMatic significantly reduces the complexity of managing multiple AutoDock jobs by facilitating ligand and AutoDock job creation and management.

  8. Sliding tethered ligands add topological interactions to the toolbox of ligand-receptor design

    Science.gov (United States)

    Bauer, Martin; Kékicheff, Patrick; Iss, Jean; Fajolles, Christophe; Charitat, Thierry; Daillant, Jean; Marques, Carlos M.

    2015-09-01

    Adhesion in the biological realm is mediated by specific lock-and-key interactions between ligand-receptor pairs. These complementary moieties are ubiquitously anchored to substrates by tethers that control the interaction range and the mobility of the ligands and receptors, thus tuning the kinetics and strength of the binding events. Here we add sliding anchoring to the toolbox of ligand-receptor design by developing a family of tethered ligands for which the spacer can slide at the anchoring point. Our results show that this additional sliding degree of freedom changes the nature of the adhesive contact by extending the spatial range over which binding may sustain a significant force. By introducing sliding tethered ligands with self-regulating length, this work paves the way for the development of versatile and reusable bio-adhesive substrates with potential applications for drug delivery and tissue engineering.

  9. A new class of PN3-pincer ligands for metal–ligand cooperative catalysis

    KAUST Repository

    Li, Huaifeng

    2014-12-01

    Work on a new class of PN3-pincer ligands for metal-ligand cooperative catalysis is reviewed. While the field of the pyridine-based PN3-transition metal pincer complexes is still relatively young, many important applications of these complexes have already emerged. In several cases, the PN3-pincer complexes for metal-ligand cooperative catalysis result in significantly improved or unprecedented activities. The synthesis and coordination chemistry of PN3-pincer ligands are briefly summarized first to cover the synthetic routes for their preparation, followed by a focus review on their applications in catalysis. A specific emphasis is placed on the later section about the role of PN3-pincer ligands\\' dearomatization-rearomatization steps during the catalytic cycles. The mechanistic insights from density functional theory (DFT) calculations are also discussed.

  10. Targeted toxins in brain tumor therapy.

    Science.gov (United States)

    Li, Yan Michael; Hall, Walter A

    2010-11-01

    Targeted toxins, also known as immunotoxins or cytotoxins, are recombinant molecules that specifically bind to cell surface receptors that are overexpressed in cancer and the toxin component kills the cell. These recombinant proteins consist of a specific antibody or ligand coupled to a protein toxin. The targeted toxins bind to a surface antigen or receptor overexpressed in tumors, such as the epidermal growth factor receptor or interleukin-13 receptor. The toxin part of the molecule in all clinically used toxins is modified from bacterial or plant toxins, fused to an antibody or carrier ligand. Targeted toxins are very effective against cancer cells resistant to radiation and chemotherapy. They are far more potent than any known chemotherapy drug. Targeted toxins have shown an acceptable profile of toxicity and safety in early clinical studies and have demonstrated evidence of a tumor response. Currently, clinical trials with some targeted toxins are complete and the final results are pending. This review summarizes the characteristics of targeted toxins and the key findings of the important clinical studies with targeted toxins in malignant brain tumor patients. Obstacles to successful treatment of malignant brain tumors include poor penetration into tumor masses, the immune response to the toxin component and cancer heterogeneity. Strategies to overcome these limitations are being pursued in the current generation of targeted toxins.

  11. Targeted Toxins in Brain Tumor Therapy

    Directory of Open Access Journals (Sweden)

    Walter A. Hall

    2010-11-01

    Full Text Available Targeted toxins, also known as immunotoxins or cytotoxins, are recombinant molecules that specifically bind to cell surface receptors that are overexpressed in cancer and the toxin component kills the cell. These recombinant proteins consist of a specific antibody or ligand coupled to a protein toxin. The targeted toxins bind to a surface antigen or receptor overexpressed in tumors, such as the epidermal growth factor receptor or interleukin-13 receptor. The toxin part of the molecule in all clinically used toxins is modified from bacterial or plant toxins, fused to an antibody or carrier ligand. Targeted toxins are very effective against cancer cells resistant to radiation and chemotherapy. They are far more potent than any known chemotherapy drug. Targeted toxins have shown an acceptable profile of toxicity and safety in early clinical studies and have demonstrated evidence of a tumor response. Currently, clinical trials with some targeted toxins are complete and the final results are pending. This review summarizes the characteristics of targeted toxins and the key findings of the important clinical studies with targeted toxins in malignant brain tumor patients. Obstacles to successful treatment of malignant brain tumors include poor penetration into tumor masses, the immune response to the toxin component and cancer heterogeneity. Strategies to overcome these limitations are being pursued in the current generation of targeted toxins.

  12. Anticonvulsant properties of histamine H3 receptor ligands belonging to N-substituted carbamates of imidazopropanol.

    Science.gov (United States)

    Sadek, Bassem; Shehab, Safa; Więcek, Małgorzata; Subramanian, Dhanasekaran; Shafiullah, Mohamed; Kieć-Kononowicz, Katarzyna; Adem, Abdu

    2013-09-01

    Ligands targeting central histamine H3 receptors (H3Rs) for epilepsy might be a promising therapeutic approach. Therefore, the previously described and structurally strongly related imidazole-based derivatives belonging to carbamate class with high H3R in vitro affinity, in-vivo antagonist potency, and H3R selectivity profile were investigated on their anticonvulsant activity in maximal electroshock (MES)-induced and pentylenetetrazole (PTZ)-kindled seizure models in Wistar rats. The effects of systemic injection of H3R ligands 1-13 on MES-induced and PTZ-kindled seizures were screened and evaluated against the reference antiepileptic drug (AED) Phenytoin (PHT) and the standard histamine H3R inverse agonist/antagonist Thioperamide (THP) to determine their potential as new antiepileptic drugs. Following administration of the H3R ligands 1-13 (5, 10 and 15 mg/kg, ip) there was a significant dose dependent reduction in MES-induced seizure duration. The protective action observed for the pentenyl carbamate derivative 4, the most protective H3R ligand among 1-13, was significantly higher (P histamine (RAMH) (10mg/kg), or with the CNS penetrant H1R antagonist Pyrilamine (PYR) (10mg/kg). In addition, subeffective dose of H3R ligand 4 (5mg/kg, ip) significantly potentiated the protective action in rats pretreated with PHT (5mg/kg, ip), a dose without appreciable protective effect when given alone. In contrast, pretreatment with H3R ligand 4 (10mg/kg ip) failed to modify PTZ-kindled convulsion, whereas the reference drug PHT was found to fully protect PTZ-induced seizure. These results indicate that some of the investigated imidazole-based H3R ligands 1-13 may be of future therapeutic value in epilepsy.

  13. Conotoxins: Molecular and Therapeutic Targets

    Science.gov (United States)

    Lewis, Richard J.

    Marine molluscs known as cone snails produce beautiful shells and a complex array of over 50,000 venom peptides evolved for prey capture and defence. Many of these peptides selectively modulate ion channels and transporters, making them a valuable source of new ligands for studying the role these targets play in normal and disease physiology. A number of conopeptides reduce pain in animal models, and several are now in pre-clinical and clinical development for the treatment of severe pain often associated with diseases such as cancer. Less than 1% of cone snail venom peptides are pharmacologically characterised.

  14. Assessing protein kinase target similarity

    DEFF Research Database (Denmark)

    Gani, Osman A; Thakkar, Balmukund; Narayanan, Dilip

    2015-01-01

    : focussed chemical libraries, drug repurposing, polypharmacological design, to name a few. Protein kinase target similarity is easily quantified by sequence, and its relevance to ligand design includes broad classification by key binding sites, evaluation of resistance mutations, and the use of surrogate......" of sequence and crystal structure information, with statistical methods able to identify key correlates to activity but also here, "the devil is in the details." Examples from specific repurposing and polypharmacology applications illustrate these points. This article is part of a Special Issue entitled...

  15. AutoDock-GIST: Incorporating Thermodynamics of Active-Site Water into Scoring Function for Accurate Protein-Ligand Docking.

    Science.gov (United States)

    Uehara, Shota; Tanaka, Shigenori

    2016-11-23

    Water plays a significant role in the binding process between protein and ligand. However, the thermodynamics of water molecules are often underestimated, or even ignored, in protein-ligand docking. Usually, the free energies of active-site water molecules are substantially different from those of waters in the bulk region. The binding of a ligand to a protein causes a displacement of these waters from an active site to bulk, and this displacement process substantially contributes to the free energy change of protein-ligand binding. The free energy of active-site water molecules can be calculated by grid inhomogeneous solvation theory (GIST), using molecular dynamics (MD) and the trajectory of a target protein and water molecules. Here, we show a case study of the combination of GIST and a docking program and discuss the effectiveness of the displacing gain of unfavorable water in protein-ligand docking. We combined the GIST-based desolvation function with the scoring function of AutoDock4, which is called AutoDock-GIST. The proposed scoring function was assessed employing 51 ligands of coagulation factor Xa (FXa), and results showed that both scoring accuracy and docking success rate were improved. We also evaluated virtual screening performance of AutoDock-GIST using FXa ligands in the directory of useful decoys-enhanced (DUD-E), thus finding that the displacing gain of unfavorable water is effective for a successful docking campaign.

  16. Ligand binding reduces SUMOylation of the peroxisome proliferator-activated receptor γ (PPARγ activation function 1 (AF1 domain.

    Directory of Open Access Journals (Sweden)

    Rolf Diezko

    Full Text Available Peroxisome proliferator-activated receptor gamma (PPARγ is a ligand-activated nuclear receptor regulating adipogenesis, glucose homeostasis and inflammatory responses. The activity of PPARγ is controlled by post-translational modifications including SUMOylation and phosphorylation that affects its biological and molecular functions. Several important aspects of PPARγ SUMOylation including SUMO isoform-specificity and the impact of ligand binding on SUMOylation remain unresolved or contradictory. Here, we present a comprehensive study of PPARγ1 SUMOylation. We show that PPARγ1 can be modified by SUMO1 and SUMO2. Mutational analyses revealed that SUMOylation occurs exclusively within the N-terminal activation function 1 (AF1 domain predominantly at lysines 33 and 77. Ligand binding to the C-terminal ligand-binding domain (LBD of PPARγ1 reduces SUMOylation of lysine 33 but not of lysine 77. SUMOylation of lysine 33 and lysine 77 represses basal and ligand-induced activation by PPARγ1. We further show that lysine 365 within the LBD is not a target for SUMOylation as suggested in a previous report, but it is essential for full LBD activity. Our results suggest that PPARγ ligands negatively affect SUMOylation by interdomain communication between the C-terminal LBD and the N-terminal AF1 domain. The ability of the LBD to regulate the AF1 domain may have important implications for the evaluation and mechanism of action of therapeutic ligands that bind PPARγ.

  17. Cationic ruthenium alkylidene catalysts bearing phosphine ligands.

    Science.gov (United States)

    Endo, Koji; Grubbs, Robert H

    2016-02-28

    The discovery of highly active catalysts and the success of ionic liquid immobilized systems have accelerated attention to a new class of cationic metathesis catalysts. We herein report the facile syntheses of cationic ruthenium catalysts bearing bulky phosphine ligands. Simple ligand exchange using silver(i) salts of non-coordinating or weakly coordinating anions provided either PPh3 or chelating Ph2P(CH2)nPPh2 (n = 2 or 3) ligated cationic catalysts. The structures of these newly reported catalysts feature unique geometries caused by ligation of the bulky phosphine ligands. Their activities and selectivities in standard metathesis reactions were also investigated. These cationic ruthenium alkylidene catalysts reported here showed moderate activity and very similar stereoselectivity when compared to the second generation ruthenium dichloride catalyst in ring-closing metathesis, cross metathesis, and ring-opening metathesis polymerization assays.

  18. Flexible Ligand Docking Using Evolutionary Algorithms

    DEFF Research Database (Denmark)

    Thomsen, Rene

    2003-01-01

    The docking of ligands to proteins can be formulated as a computational problem where the task is to find the most favorable energetic conformation among the large space of possible protein–ligand complexes. Stochastic search methods such as evolutionary algorithms (EAs) can be used to sample large...... search spaces effectively and is one of the commonly used methods for flexible ligand docking. During the last decade, several EAs using different variation operators have been introduced, such as the ones provided with the AutoDock program. In this paper we evaluate the performance of different EA...... settings such as choice of variation operators, population size, and usage of local search. The comparison is performed on a suite of six docking problems previously used to evaluate the performance of search algorithms provided with the AutoDock program package. The results from our investigation confirm...

  19. "Molecular Switches" on mGluR Allosteric Ligands That Modulate Modes of Pharmacology

    Science.gov (United States)

    Wood, Michael R.; Hopkins, Corey R.; Brogan, John T.; Conn, P. Jeffrey; Lindsley, Craig W.

    2013-01-01

    G-Protein-coupled receptors (GPCRs) represent the largest class of drug targets, accounting for more than 40% of marketed drugs; however, discovery efforts for many GPCRs have failed to provide viable drug candidates. Historically, drug discovery efforts have focused on developing ligands that act at the orthosteric site of the endogenous agonist. Recently, efforts have focused on functional assay paradigms and the discovery of ligands that act at allosteric sites to modulate receptor function in either a positive, negative, or neutral manner. Allosteric modulators have numerous advantages over orthosteric ligands, including high subtype selectivity; the ability to mimic physiological conditions; the lack of densensitization, downregulation, and internalization; and reduced side effects. Despite these virtues, challenging issues have now arisen for allosteric modulators of metabotropic glutamate receptors (mGluRs): shallow SAR, ligand-directed trafficking, and the identification of subtle “molecular switches” that modulate the modes of pharmacology. Here, we will discuss the impact of modest structural changes to multiple mGluR allosteric ligands scaffolds that unexpectedly modulate pharmacology and raise concerns over metabolism and the pharmacology of metabolites. PMID:21341760

  20. Theory and Normal Mode Analysis of Change in Protein Vibrational Dynamics on Ligand Binding

    Energy Technology Data Exchange (ETDEWEB)

    Mortisugu, Kei [RIKEN, Japan; Njunda, Brigitte [Computational Molecular Biophysics, Interdisciplinary Center for Scientific Computing (IWR); Smith, Jeremy C [ORNL

    2009-12-01

    The change of protein vibrations on ligand binding is of functional and thermodynamic importance. Here, this process is characterized using a simple analytical 'ball-and-spring' model and all-atom normal-mode analysis (NMA) of the binding of the cancer drug, methotrexate (MTX) to its target, dihydrofolate reductase (DHFR). The analytical model predicts that the coupling between protein vibrations and ligand external motion generates entropy-rich, low-frequency vibrations in the complex. This is consistent with the atomistic NMA which reveals vibrational softening in forming the DHFR-MTX complex, a result also in qualitative agreement with neutron-scattering experiments. Energy minimization of the atomistic bound-state (B) structure while gradually decreasing the ligand interaction to zero allows the generation of a hypothetical 'intermediate' (I) state, without the ligand force field but with a structure similar to that of B. In going from I to B, it is found that the vibrational entropies of both the protein and MTX decrease while the complex structure becomes enthalpically stabilized. However, the relatively weak DHFR:MTX interaction energy results in the net entropy gain arising from coupling between the protein and MTX external motion being larger than the loss of vibrational entropy on complex formation. This, together with the I structure being more flexible than the unbound structure, results in the observed vibrational softening on ligand binding.

  1. Protein-protein interface analysis and hot spots identification for chemical ligand design.

    Science.gov (United States)

    Chen, Jing; Ma, Xiaomin; Yuan, Yaxia; Pei, Jianfeng; Lai, Luhua

    2014-01-01

    Rational design for chemical compounds targeting protein-protein interactions has grown from a dream to reality after a decade of efforts. There are an increasing number of successful examples, though major challenges remain in the field. In this paper, we will first give a brief review of the available methods that can be used to analyze protein-protein interface and predict hot spots for chemical ligand design. New developments of binding sites detection, ligandability and hot spots prediction from the author's group will also be described. Pocket V.3 is an improved program for identifying hot spots in protein-protein interface using only an apo protein structure. It has been developed based on Pocket V.2 that can derive receptor-based pharmacophore model for ligand binding cavity. Given similarities and differences between the essence of pharmacophore and hot spots for guiding design of chemical compounds, not only energetic but also spatial properties of protein-protein interface are used in Pocket V.3 for dealing with protein-protein interface. In order to illustrate the capability of Pocket V.3, two datasets have been used. One is taken from ASEdb and BID having experimental alanine scanning results for testing hot spots prediction. The other is taken from the 2P2I database containing complex structures of protein-ligand binding at the original protein-protein interface for testing hot spots application in ligand design.

  2. Ligand Dependent Switch from RXR Homo- to RXR-NURR1 Heterodimerization.

    Science.gov (United States)

    Scheepstra, Marcel; Andrei, Sebastian A; de Vries, Rens M J M; Meijer, Femke A; Ma, Jian-Nong; Burstein, Ethan S; Olsson, Roger; Ottmann, Christian; Milroy, Lech-Gustav; Brunsveld, Luc

    2017-09-20

    Retinoid X receptors (RXRs) play key roles in many physiological processes in both the periphery and central nervous system. In addition, RXRs form heterodimers with other nuclear receptors to exert their physiological effects. The nuclear receptor related 1 protein (NURR1) is particularly interesting because of its role in promoting differentiation and survival of dopamine neurons. However, only a small number of RXR-heterodimer selective modulators are available, with limited chemical diversity. This work describes the synthesis, biochemical evaluation, and structural elucidation of a novel series of RXR ligands with strongly biased interactions with RXRα-NURR1 heterodimers. Targeted modifications to the small molecule biaryl scaffold caused local RXRα side-chain disturbances and displacement of secondary structural elements upon ligand binding. This resulted in the repositioning of protein helices in the heterodimer interface of RXRα, alterations in homo- versus heterodimer formation, and modulation of activation function 2 (AF2). The data provide a rationale for the design of RXR ligands consisting of a highly conserved hydrophilic region, strongly contributing to the ligand affinity, and a variable hydrophobic region, which efficiently probes the effects of structural changes at the level of the ligand on co-regulator recruitment or the RXRα-NURR1 dimerization interface.

  3. Human NKG2D-ligands: cell biology strategies to ensure immune recognition

    Directory of Open Access Journals (Sweden)

    Lola eFernández-Messina

    2012-09-01

    Full Text Available Immune recognition mediated by the activating receptor NKG2D plays an important role for the elimination of stressed cells, including tumours and virus-infected cells. On the other hand, the ligands for NKG2D can also be shed into the sera of cancer patients where they weaken the immune response by downmodulating the receptor on effector cells, mainly NK and T cells. Although both families of NKG2D-ligands, MICA/B and ULBPs, are related to MHC molecules and their expression is increased after stress, many differences are observed in terms of their biochemical properties and cell trafficking. In this paper, we summarise the variety of NKG2D-ligands and propose that selection pressure has driven evolution of diversity in their trafficking and shedding, but not receptor binding affinity. However, it is also possible to identify functional properties common to individual ULBP molecules and MICA/B alleles, but not generally conserved within the MIC or ULBP families. These characteristics likely represent examples of convergent evolution for efficient immune recognition, but are also attractive targets for pathogen immune evasion strategies. Categorization of NKG2D-ligands according to their biological features, rather than their genetic family, may help to achieve a better understanding of NKG2D-ligand association with disease.

  4. Direct identification of ligand-receptor interactions on living cells and tissues.

    Science.gov (United States)

    Frei, Andreas P; Jeon, Ock-Youm; Kilcher, Samuel; Moest, Hansjoerg; Henning, Lisa M; Jost, Christian; Plückthun, Andreas; Mercer, Jason; Aebersold, Ruedi; Carreira, Erick M; Wollscheid, Bernd

    2012-10-01

    Many cellular responses are triggered by proteins, drugs or pathogens binding to cell-surface receptors, but it can be challenging to identify which receptors are bound by a given ligand. Here we describe TRICEPS, a chemoproteomic reagent with three moieties--one that binds ligands containing an amino group, a second that binds glycosylated receptors on living cells and a biotin tag for purifying the receptor peptides for identification by quantitative mass spectrometry. We validated this ligand-based, receptor-capture (LRC) technology using insulin, transferrin, apelin, epidermal growth factor, the therapeutic antibody trastuzumab and two DARPins targeting ErbB2. In some cases, we could also determine the approximate ligand-binding sites on the receptors. Using TRICEPS to label intact mature vaccinia viruses, we identified the cell surface proteins AXL, M6PR, DAG1, CSPG4 and CDH13 as binding factors on human cells. This technology enables the identification of receptors for many types of ligands under near-physiological conditions and without the need for genetic manipulations.

  5. A novel ligand-mapping method based on molecular liquid theory.

    Science.gov (United States)

    Imai, Takashi

    2011-01-01

    The recent development of a novel ligand-mapping method is reviewed. The method is based on a statistical-mechanical molecular theory of solvation, known as the three-dimensional reference interaction site model (3D-RISM). In the 3D-RISM-based ligand mapping (3D-RISM-LM) method, using the all-atom model for a target protein immersed in a ligand-water mixture solvent, the 3D-spatial distributions of the ligand atomic sites around the protein are first obtained, and then the most probable binding modes of the ligand molecule are constructed from the distributions. Unlike conventional docking simulations, 3D-RISM-LM can incorporate the effect of water from the atomic to thermodynamic level into the binding affinity through statistical mechanics. It has been demonstrated that 3D-RISM-LM can sensitively detect even weak binding modes of small molecules over the entire surface of protein. Therefore, this approach is expected to be particularly useful in fragment-based drug design.

  6. TSPO PIGA Ligands Promote Neurosteroidogenesis and Human Astrocyte Well-Being

    Directory of Open Access Journals (Sweden)

    Eleonora Da Pozzo

    2016-06-01

    Full Text Available The steroidogenic 18 kDa translocator protein (TSPO is an emerging, attractive therapeutic tool for several pathological conditions of the nervous system. Here, 13 high affinity TSPO ligands belonging to our previously described N,N-dialkyl-2-phenylindol-3-ylglyoxylamide (PIGA class were evaluated for their potential ability to affect the cellular Oxidative Metabolism Activity/Proliferation index, which is used as a measure of astrocyte well-being. The most active PIGA ligands were also assessed for steroidogenic activity in terms of pregnenolone production, and the values were related to the metabolic index in rat and human models. The results showed a positive correlation between the increase in the Oxidative Metabolism Activity/Proliferation index and the pharmacologically induced stimulation of steroidogenesis. The specific involvement of steroid molecules in mediating the metabolic effects of the PIGA ligands was demonstrated using aminoglutethimide, a specific inhibitor of the first step of steroid biosynthesis. The most promising steroidogenic PIGA ligands were the 2-naphthyl derivatives that showed a long residence time to the target, in agreement with our previous data. In conclusion, TSPO ligand-induced neurosteroidogenesis was involved in astrocyte well-being.

  7. CHARMM Force Field Parameterization of Peroxisome Proliferator-Activated Receptor γ Ligands

    Science.gov (United States)

    Mottin, Melina; Souza, Paulo C. T.; Ricci, Clarisse G.; Skaf, Munir S.

    2016-01-01

    The peroxisome proliferator-activated receptor γ (PPARγ) ligands are important therapeutic drugs for the treatment of type 2 diabetes, obesity and cardiovascular diseases. In particular, partial agonists and non-agonists are interesting targets to reduce glucose levels, presenting few side effects in comparison to full agonists. In this work, we present a set of CHARMM-based parameters of a molecular mechanics force field for two PPARγ ligands, GQ16 and SR1664. GQ16 belongs to the thiazolidinedione class of drugs and it is a PPARγ partial agonist that has been shown to promote the “browning” of white adipose tissue. SR1664 is the precursor of the PPARγ non-agonist class of ligands that activates PPARγ in a non-classical manner. Here, we use quantum chemical calculations consistent with the CHARMM protocol to obtain bonded and non-bonded parameters, including partial atomic charges and effective torsion potentials for both molecules. The newly parameterized models were evaluated by examining the behavior of GQ16 and SR1664 free in water and bound to the ligand binding pocket of PPARγ using molecular dynamics simulations. The potential parameters derived here are readily transferable to a variety of pharmaceutical compounds and similar PPARγ ligands. PMID:28025495

  8. GalaxyDock BP2 score: a hybrid scoring function for accurate protein-ligand docking

    Science.gov (United States)

    Baek, Minkyung; Shin, Woong-Hee; Chung, Hwan Won; Seok, Chaok

    2017-07-01

    Protein-ligand docking is a useful tool for providing atomic-level understanding of protein functions in nature and design principles for artificial ligands or proteins with desired properties. The ability to identify the true binding pose of a ligand to a target protein among numerous possible candidate poses is an essential requirement for successful protein-ligand docking. Many previously developed docking scoring functions were trained to reproduce experimental binding affinities and were also used for scoring binding poses. However, in this study, we developed a new docking scoring function, called GalaxyDock BP2 Score, by directly training the scoring power of binding poses. This function is a hybrid of physics-based, empirical, and knowledge-based score terms that are balanced to strengthen the advantages of each component. The performance of the new scoring function exhibits significant improvement over existing scoring functions in decoy pose discrimination tests. In addition, when the score is used with the GalaxyDock2 protein-ligand docking program, it outperformed other state-of-the-art docking programs in docking tests on the Astex diverse set, the Cross2009 benchmark set, and the Astex non-native set. GalaxyDock BP2 Score and GalaxyDock2 with this score are freely available at http://galaxy.seoklab.org/softwares/galaxydock.html.

  9. Probing interaction of a fluorescent ligand with HIV TAR RNA

    Science.gov (United States)

    Qi, Liang; Zhang, Jing; He, Tian; Huo, Yuan; Zhang, Zhi-Qi

    2017-02-01

    Trans-activator of Transcription (Tat) antagonists could block the interaction between Tat protein and its target, trans-activation responsive region (TAR) RNA, to inhibit Tat function and prevent human immunodeficiency virus type 1 (HIV-1) replication. For the first time, a small fluorescence ligand, ICR 191, was found to interact with TAR RNA at the Tat binding site and compete with Tat. It was also observed that the fluorescence of ICR 191 could be quenched when binding to TAR RNA and recovered when discharged via competition with Tat peptide or a well-known Tat inhibitor, neomycin B. The binding parameters of ICR 191 to TAR RNA were determined through theoretical calculations. Mass spectrometry, circular dichroism and molecular docking were used to further confirm the interaction of ICR 191 with TAR RNA. Inspired by these discoveries, a primary fluorescence model for the discovery of Tat antagonists was built using ICR 191 as a fluorescence indicator and the feasibility of this model was evaluated. This ligand-RNA interaction could provide a new strategy for research aimed at discovering Tat antagonists.

  10. Pharmacokinetic Steady-States Highlight Interesting Target-Mediated Disposition Properties.

    Science.gov (United States)

    Gabrielsson, Johan; Peletier, Lambertus A

    2017-05-01

    In this paper, we derive explicit expressions for the concentrations of ligand L, target R and ligand-target complex RL at steady state for the classical model describing target-mediated drug disposition, in the presence of a constant-rate infusion of ligand. We demonstrate that graphing the steady-state values of ligand, target and ligand-target complex, we obtain striking and often singular patterns, which yield a great deal of insight and understanding about the underlying processes. Deriving explicit expressions for the dependence of L, R and RL on the infusion rate, and displaying graphs of the relations between L, R and RL, we give qualitative and quantitive information for the experimentalist about the processes involved. Understanding target turnover is pivotal for optimising these processes when target-mediated drug disposition (TMDD) prevails. By a combination of mathematical analysis and simulations, we also show that the evolution of the three concentration profiles towards their respective steady-states can be quite complex, especially for lower infusion rates. We also show how parameter estimates obtained from iv bolus studies can be used to derive steady-state concentrations of ligand, target and complex. The latter may serve as a template for future experimental designs.

  11. Ligand Intermediates in Metal-Catalyzed Reactions

    Energy Technology Data Exchange (ETDEWEB)

    Gladysz, John A.

    1999-07-31

    The longest-running goal of this project has been the synthesis, isolation, and physical chemical characterization of homogeneous transition metal complexes containing ligand types believed to be intermediates in the metal-catalyzed conversion of CO/H{sub 2}, CO{sub 2}, CH{sub 4}, and similar raw materials to organic fuels, feedstocks, etc. In the current project period, complexes that contain unusual new types of C{sub x}(carbide) and C{sub x}O{sub y} (carbon oxide) ligands have been emphasized. A new program in homogeneous fluorous phase catalysis has been launched as described in the final report.

  12. Efficient chemoenzymatic synthesis of chiral pincer ligands.

    Science.gov (United States)

    Felluga, Fulvia; Baratta, Walter; Fanfoni, Lidia; Pitacco, Giuliana; Rigo, Pierluigi; Benedetti, Fabio

    2009-05-01

    Chiral, nonracemic pincer ligands based on the 6-phenyl-2-aminomethylpyridine and 2-aminomethylbenzo[h]quinoline scaffolds were obtained by a chemoenzymatic approach starting from 2-pyridyl and 2-benzoquinolyl ethanone. In the enantiodifferentiating step, secondary alcohols of opposite absolute configuration were obtained by a baker's yeast reduction of the ketones and by lipase-mediated dynamic kinetic resolution of the racemic alcohols. Their transformation into homochiral 1-methyl-1-heteroarylethanamines occurred without loss of optical purity, giving access to pincer ligands used in enantioselective catalysis.

  13. GPR35 as a novel therapeutic target

    Directory of Open Access Journals (Sweden)

    Amanda eMacKenzie

    2011-11-01

    Full Text Available G protein-coupled receptors (GPCRs remain the best studied class of cell surface receptors and the most tractable family of proteins for novel small molecule drug discovery. Despite this, a considerable number of GPCRs remain poorly characterised and in a significant number of cases, endogenous ligand(s that activate them remain undefined or of questionable physiological relevance. GPR35 was initially discovered over a decade ago but has remained an ‘orphan’ receptor. Recent publications have highlighted novel ligands, both endogenously produced and synthetic, which demonstrate significant potency at this receptor. Furthermore, evidence is accumulating which highlights potential roles for GPR35 in disease and therefore, efforts to characterise GPR35 more fully and develop it as a novel therapeutic target in conditions that range from diabetes, hypertension to asthma are increasing. Recently identified ligands have shown marked species selective properties, indicating major challenges for future drug development. As we begin to understand these issues, the continuing efforts to identify novel agonist and antagonist ligands for GPR35 will help to decipher its true physiological relevance; translating multiple assay systems in vitro, to animal disease systems in vivo and finally to man.

  14. Protein ligand-tethered synthetic calcium indicator for localization control and spatiotemporal calcium imaging in plant cells.

    Science.gov (United States)

    Takaoka, Yousuke; Shigenaga, Miyuki; Imai, Masaki; Nukadzuka, Yuuki; Ishimaru, Yasuhiro; Saito, Kei; Yokoyama, Ryusuke; Nishitani, Kazuhiko; Ueda, Minoru

    2016-01-01

    In plant biology, calcium ions are involved in a variety of intriguing biological phenomena as a secondary messenger. However, most conventional calcium indicators are not applicable for plant cells because of the difficulty with their localization control in plant cells. We here introduce a method to monitor spatiotemporal Ca(2+) dynamics in living plant cells based on linking the synthetic calcium indicator Calcium Green-1 to a natural product-based protein ligand. In a proof-of-concept study using cultured BY-2 cells overexpressing the target protein for the ligand, the ligand-tethered probe accumulated in the cytosol and nucleus, and enabled real-time monitoring of the cytosolic and nucleus Ca(2+) dynamics under the physiological condition. The present strategy using ligand-tethered fluorescent sensors may be successfully applied to reveal the spatiotemporal dynamics of calcium ions in living plant cells.

  15. Multifunctional ligands in medicinal inorganic chemistry--current trends and future directions.

    Science.gov (United States)

    Chiang, Linus; Jones, Michael R; Ferreira, Cara L; Storr, Tim

    2012-01-01

    This review will highlight recent advances in ligand design for innovative applications in medicinal inorganic chemistry. Ligands that effectively bind metal ions and also include specific features to enhance targeting, reporting, and overall efficacy are driving innovation in areas of disease diagnosis and therapy. Increasing the potency of therapeutic compounds, while limiting side-effects, is a common goal in medicinal chemistry. In an effort to achieve this goal, compounds are being developed that either target a disease site, or are activated by a disease specific biological process. Metal complexes containing targeting functions and/or bioactive ligands, as well as agents that are activated by specific enzymes, or changes in pH and pO2, provide new avenues for drug development. Radiodiagnostic compounds, magnetic resonance imaging agents, and optical probes containing transition metals offer versatility unavailable to organic imaging agents. In certain cases, dual modality agents have been developed, and will be highlighted. Finally, we will discuss targeted metal binding compounds for treatment of metal overload disorders, and the recent application to neurodegenerative disease.

  16. The novel sigma-2 receptor ligand SW43 stabilizes pancreas cancer progression in combination with gemcitabine

    Directory of Open Access Journals (Sweden)

    Goedegebuure Peter

    2010-11-01

    Full Text Available Abstract Background Sigma-2 receptors are over-expressed in proliferating cancer cells, making an attractive target for the targeted treatment of pancreatic cancer. In this study, we investigated the role of the novel sigma-2 receptor ligand SW43 to induce apoptosis and augment standard chemotherapy. Results The binding affinity for sigma-2 ligands is high in pancreas cancer, and they induce apoptosis with a rank order of SV119 in vitro. Combining these compounds with gemcitabine further increased apoptosis and decreased viability. Our in vivo model showed that sigma-2 ligand treatment decreased tumor volume to the same extent as gemcitabine. However, SW43 combination treatment with gemcitabine was superior to the other compounds and resulted in stabilization of tumor volume during treatment, with minimal toxicities. Conclusions This study shows that the sigma-2 ligand SW43 has the greatest capacity to augment gemcitabine in a pre-clinical model of pancreas cancer and has provided us with the rationale to move this compound forward with clinical investigations for patients with pancreatic cancer.

  17. Microfluidic-based G-quadruplex ligand displacement assay for alkaloid anticancer drug screening.

    Science.gov (United States)

    Shen, Haihui; Zhang, Bo; Xu, Huiyan; Sun, Yue; Wu, Qiwang; Shen, Hong; Liu, Yingchun

    2017-02-05

    Some natural heterocyclic alkaloids containing planar group show potential to complex with specific promoter region of protooncogene for stabilizing the G-quadruplex (G4) structure which nowadays promises to be a target in anticancer drug design. However, in view of the polymorphic characteristics and structural complexity of heterocyclic alkaloids, it is desirable to develop high-throughput and low-consumption approach for anticancer drug screening. In this paper, an intensive study on alkaloid ligand/G4 DNA interaction has been conducted, demonstrating that the end-stacking interaction is the favorable binding mode between the oncogene-related Pu22 G4 DNA and the heterocyclic alkaloid ligand. Based on structural feasibility and energy minimization, a ligand displacement assay for screening alkaloid ligand in stabilizing the oncogene target G4 has been developed, which also helps to facilitate the assessment of drug specificity. Coupled with microfluidic-based DNAzyme-catalytic chemiluminescence detection, the approach showed the advantages of high sensitivity, high throughput with low sample and reagent consumptions.

  18. The Molecular Basis of Ligand Interaction at Free Fatty Acid Receptor 4 (FFA4/GPR120)*

    Science.gov (United States)

    Hudson, Brian D.; Shimpukade, Bharat; Milligan, Graeme; Ulven, Trond

    2014-01-01

    The long-chain fatty acid receptor FFA4 (previously GPR120) is receiving substantial interest as a novel target for the treatment of metabolic and inflammatory disease. This study examines for the first time the detailed mode of binding of both long-chain fatty acid and synthetic agonist ligands at FFA4 by integrating molecular modeling, receptor mutagenesis, and ligand structure-activity relationship approaches in an iterative format. In doing so, residues required for binding of fatty acid and synthetic agonists to FFA4 have been identified. This has allowed for the refinement of a well validated model of the mode of ligand-FFA4 interaction that will be invaluable in the identification of novel ligands and the future development of this receptor as a therapeutic target. The model reliably predicted the effects of substituent variations on agonist potency, and it was also able to predict the qualitative effect of binding site mutations in the majority of cases. PMID:24860101

  19. Differential effects of EGFR ligands on endocytic sorting of the receptor.

    Science.gov (United States)

    Roepstorff, Kirstine; Grandal, Michael Vibo; Henriksen, Lasse; Knudsen, Stine Louise Jeppe; Lerdrup, Mads; Grøvdal, Lene; Willumsen, Berthe Marie; van Deurs, Bo

    2009-08-01

    Endocytic downregulation is a pivotal mechanism turning off signalling from the EGF receptor (EGFR). It is well established that whereas EGF binding leads to lysosomal degradation of EGFR, transforming growth factor (TGF)-alpha causes receptor recycling. TGF-alpha therefore leads to continuous signalling and is a more potent mitogen than EGF. In addition to EGF and TGF-alpha, five EGFR ligands have been identified. Although many of these ligands are upregulated in cancers, very little is known about their effect on EGFR trafficking. We have compared the effect of six different ligands on endocytic trafficking of EGFR. We find that, whereas they all stimulate receptor internalization, they have very diverse effects on endocytic sorting. Heparin-binding EGF-like growth factor and Betacellulin target all EGFRs for lysosomal degradation. In contrast, TGF-alpha and epiregulin lead to complete receptor recycling. EGF leads to lysosomal degradation of the majority but not all EGFRs. Amphiregulin does not target EGFR for lysosomal degradation but causes fast as well as slow EGFR recycling. The Cbl ubiquitin ligases, especially c-Cbl, are responsible for EGFR ubiquitination after stimulation with all ligands, and persistent EGFR phosphorylation and ubiquitination largely correlate with receptor degradation.

  20. Identifying high-affinity aptamer ligands with defined cross-reactivity using high-throughput guided systematic evolution of ligands by exponential enrichment.

    Science.gov (United States)

    Levay, Agata; Brenneman, Randall; Hoinka, Jan; Sant, David; Cardone, Marco; Trinchieri, Giorgio; Przytycka, Teresa M; Berezhnoy, Alexey

    2015-07-13

    Oligonucleotide aptamers represent a novel platform for creating ligands with desired specificity, and they offer many potentially significant advantages over monoclonal antibodies in terms of feasibility, cost, and clinical applicability. However, the isolation of high-affinity aptamer ligands from random oligonucleotide pools has been challenging. Although high-throughput sequencing (HTS) promises to significantly facilitate systematic evolution of ligands by exponential enrichment (SELEX) analysis, the enormous datasets generated in the process pose new challenges for identifying those rare, high-affinity aptamers present in a given pool. We show that emulsion PCR preserves library diversity, preventing the loss of rare high-affinity aptamers that are difficult to amplify. We also demonstrate the importance of using reference targets to eliminate binding candidates with reduced specificity. Using a combination of bioinformatics and functional analyses, we show that the rate of amplification is more predictive than prevalence with respect to binding affinity and that the mutational landscape within a cluster of related aptamers can guide the identification of high-affinity aptamer ligands. Finally, we demonstrate the power of this selection process for identifying cross-species aptamers that can bind human receptors and cross-react with their murine orthologs.

  1. Method of detecting luminescent target ions with modified magnetic microspheres

    Science.gov (United States)

    Shkrob, Ilya A; Kaminski, Michael D

    2014-05-13

    This invention provides methods of using modified magnetic microspheres to extract target ions from a sample in order to detect their presence in a microfluidic environment. In one or more embodiments, the microspheres are modified with molecules on the surface that allow the target ions in the sample to form complexes with specific ligand molecules on the microsphere surface. In one or more embodiments, the microspheres are modified with molecules that sequester the target ions from the sample, but specific ligand molecules in solution subsequently re-extract the target ions from the microspheres into the solution, where the complexes form independent of the microsphere surface. Once the complexes form, they are exposed to an excitation wavelength light source suitable for exciting the target ion to emit a luminescent signal pattern. Detection of the luminescent signal pattern allows for determination of the presence of the target ions in the sample.

  2. In Vivo Imaging of Molecularly Targeted Phage

    Directory of Open Access Journals (Sweden)

    Kimberly A. Kelly

    2006-12-01

    Full Text Available Rapid identification of in vivo affinity ligands would have far-reaching applications for imaging specific molecular targets, in vivo systems imaging, and medical use. We have developed a high-throughput method for identifying and optimizing ligands to map and image biologic targets of interest in vivo. We directly labeled viable phage clones with far-red fluorochromes and comparatively imaged them in vivo by multichannel fluorescence ratio imaging. Using Secreted Protein Acidic and Rich in Cysteine (osteonectin and vascular cell adhesion molecule-1 as model targets, we show that: 1 fluorescently labeled phage retains target specificity on labeling; 2 in vivo distribution can be quantitated (detection thresholds of ~ 300 phage/mm3 tissue throughout the entire depth of the tumor using fluorescent tomographic imaging; and 3 fluorescently labeled phage itself can serve as a replenishable molecular imaging agent. The described method should find widespread application in the rapid in vivo discovery and validation of affinity ligands and, importantly, in the use of fluorochrome-labeled phage clones as in vivo imaging agents.

  3. Use of ligand-modified micellar-enhanced ultrafiltration to selectively separate copper ions from wastewater streams

    Energy Technology Data Exchange (ETDEWEB)

    Shadizadeh, S.B.

    1992-12-31

    The selective removal of target ions from an aqueous solution containing ions of like charge by ligand-modified micellar-enhanced ultrafiltration (LM-MEUF), is presented. In LM-MEUF, surfactant and specially tailored ligand are added to the contaminated stream. The surfactant forms aggregates called micelles, the hydrocarbon core of which the ligand complexed with the target species will solubilize. The surfactant is chosen to have the same charge type as the target ion; therefore, other ions (with similar charge) will not associate with the micelle, which makes the separation of the target ion selective. The solution is then processed by ultrafiltration, using a membrane with pore size small enough to block the passage of the micelles. In this study the divalent copper is the target ion in the solution containing divalent calcium. The surfactant is cetylpyridinium chloride (CPC) and the ligand is 4-hexadecyloxybenzyliminodiacetic acid (C{sub 16}BIDA). Experiments were conducted with batch stirred cells and the results have been compared to separation that take place under a variety of conditions in the LM-MEUF process. Rejections of copper of up to 99.8% are observed, with almost no rejection of calcium, showing that LM-MEUF has excellent selectivity and separation efficiency.

  4. PEG-PE micelles loaded with paclitaxel and surface-modified by a PBR-ligand: synergistic anticancer effect

    Science.gov (United States)

    Musacchio, Tiziana; Laquintana, Valentino; Latrofa, Andrea; Trapani, Giuseppe; Torchilin, Vladimir P.

    2009-01-01

    Selective ligands to the Peripheral Benzodiazepine Receptor (PBR) may induce apoptosis and cell cycle arrest. An over-expression of PBR in certain cancers allowed us to consider the use of highly selective ligands to PBR for receptor-mediated drug targeting to tumors. With this in mind, we prepared PBR-targeted nanoparticulate drug delivery systems (PEG-PE micelles) loaded with the anticancer drug paclitaxel (PCL) to test possible synergistic anticancer effects. PEG2k-PE-based polymeric micelles with and without PCL were prepared in HBS, pH 7.5, and conjugated with a PBR-ligand (CB86) in 0.45% of DMSO. The cytotoxic effect of such micelles against the LN 18 human glioblastoma cell line was studied in cell culture. The micelles maintained their size and size distribution and remained intact without drug release after the PBR-ligand conjugation. The PCL-loaded PBR-targeted micelles showed a significantly enhanced toxicity against human glioblastoma LN 18 cancer cells in vitro. Thus, PBR-targeted nanopreparations may potentially serve as a new nanomedicine for targeted cancer therapy. PMID:19718800

  5. An unbiased method to build benchmarking sets for ligand-based virtual screening and its application to GPCRs.

    Science.gov (United States)

    Xia, Jie; Jin, Hongwei; Liu, Zhenming; Zhang, Liangren; Wang, Xiang Simon

    2014-05-27

    Benchmarking data sets have become common in recent years for the purpose of virtual screening, though the main focus had been placed on the structure-based virtual screening (SBVS) approaches. Due to the lack of crystal structures, there is great need for unbiased benchmarking sets to evaluate various ligand-based virtual screening (LBVS) methods for important drug targets such as G protein-coupled receptors (GPCRs). To date these ready-to-apply data sets for LBVS are fairly limited, and the direct usage of benchmarking sets designed for SBVS could bring the biases to the evaluation of LBVS. Herein, we propose an unbiased method to build benchmarking sets for LBVS and validate it on a multitude of GPCRs targets. To be more specific, our methods can (1) ensure chemical diversity of ligands, (2) maintain the physicochemical similarity between ligands and decoys, (3) make the decoys dissimilar in chemical topology to all ligands to avoid false negatives, and (4) maximize spatial random distribution of ligands and decoys. We evaluated the quality of our Unbiased Ligand Set (ULS) and Unbiased Decoy Set (UDS) using three common LBVS approaches, with Leave-One-Out (LOO) Cross-Validation (CV) and a metric of average AUC of the ROC curves. Our method has greatly reduced the "artificial enrichment" and "analogue bias" of a published GPCRs benchmarking set, i.e., GPCR Ligand Library (GLL)/GPCR Decoy Database (GDD). In addition, we addressed an important issue about the ratio of decoys per ligand and found that for a range of 30 to 100 it does not affect the quality of the benchmarking set, so we kept the original ratio of 39 from the GLL/GDD.

  6. Nitrosamines as nicotinic receptor ligands.

    Science.gov (United States)

    Schuller, Hildegard M

    2007-05-30

    Nitrosamines are carcinogens formed in the mammalian organism from amine precursors contained in food, beverages, cosmetics and drugs. The potent carcinogen, NNK, and the weaker carcinogen, NNN, are nitrosamines formed from nicotine. Metabolites of the nitrosamines react with DNA to form adducts responsible for genotoxic effects. We have identified NNK as a high affinity agonist for the alpha7 nicotinic acetylcholine receptor (alpha7nAChR) whereas NNN bound with high affinity to epibatidine-sensitive nAChRs. Diethylnitrosamine (DEN) bound to both receptors but with lower affinity. High levels of the alpha7nAChR were expressed in human small cell lung cancer (SCLC) cell lines and in hamster pulmonary neuroendocrine cells (PNECs), which serve as a model for the cell of origin of human SCLC. Exposure of SCLC or PNECs to NNK or nicotine increased expression of the alpha7nAChR and caused influx of Ca(2+), activation of PKC, Raf-1, ERK1/2, and c-myc, resulting in the stimulation of cell proliferation. Signaling via the alpha7nAChR was enhanced when cells were maintained in an environment of 10-15% CO(2) similar to that in the diseased lung. Hamsters with hyperoxia-induced pulmonary fibrosis developed neuroendocrine lung carcinomas similar to human SCLC when treated with NNK, DEN, or nicotine. The development of the NNK-induced tumors was prevented by green tea or theophylline. The beta-adrenergic receptor agonist, isoproterenol or theophylline blocked NNK-induced cell proliferation in vitro. NNK and nicotine-induced hyperactivity of the alpha7nAChR/RAF/ERK1/2 pathway thus appears to play a crucial role in the development of SCLC in smokers and could be targeted for cancer prevention.

  7. Targeted phototherapy

    Directory of Open Access Journals (Sweden)

    Zonun Sanga

    2015-03-01

    Full Text Available Conventional phototherapy uses a whole body cabinet or body part machines for the hand, foot or scalp. It has many disadvantages, due to which new phototherapy techniques have been developed. These new techniques are called targeted phototherapy. They include excimer laser, the intense pulse light (IPL system, photodynamic therapy, and an ultraviolet (UV light source with a sophisticated delivery system which is easy to operate by hand. The mechanisms of action of targeted phototherapy systems are similar to those in conventional UVB/UVA therapy. They have many advantages including lower risk of side effects, avoidance of exposure of unnecessary sites, faster response, and shorter duration of treatment. But they also have disadvantages such as high costs and inability to use them for extensive areas. This review article discusses targeted phototherapy, its mechanisms of action, and advantages and disadvantages in comparison to conventional phototherapy.

  8. Targeted Learning

    CERN Document Server

    van der Laan, Mark J

    2011-01-01

    The statistics profession is at a unique point in history. The need for valid statistical tools is greater than ever; data sets are massive, often measuring hundreds of thousands of measurements for a single subject. The field is ready to move towards clear objective benchmarks under which tools can be evaluated. Targeted learning allows (1) the full generalization and utilization of cross-validation as an estimator selection tool so that the subjective choices made by humans are now made by the machine, and (2) targeting the fitting of the probability distribution of the data toward the targe

  9. [Functional selectivity of opioid receptors ligands].

    Science.gov (United States)

    Audet, Nicolas; Archer-Lahlou, Elodie; Richard-Lalonde, Mélissa; Piñeyro-Filpo, Graciela

    2010-01-01

    Opiates are the most effective analgesics available for the treatment of severe pain. However, their clinical use is restricted by unwanted side effects such as tolerance, physical dependence and respiratory depression. The strategy to develop new opiates with reduced side effects has mainly focused on the study and production of ligands that specifically bind to different opiate receptors subtypes. However, this strategy has not allowed the production of novel therapeutic ligands with a better side effects profile. Thus, other research strategies need to be explored. One which is receiving increasing attention is the possibility of exploiting ligand ability to stabilize different receptor conformations with distinct signalling profiles. This newly described property, termed functional selectivity, provides a potential means of directing the stimulus generated by an activated receptor towards a specific cellular response. Here we summarize evidence supporting the existence of ligand-specific active conformations for two opioid receptors subtypes (delta and mu), and analyze how functional selectivity may contribute in the production of longer lasting, better tolerated opiate analgesics. double dagger.

  10. Ligand Exchange Kinetics of Environmentally Relevant Metals

    Energy Technology Data Exchange (ETDEWEB)

    Panasci, Adele Frances [Univ. of California, Davis, CA (United States)

    2014-07-15

    The interactions of ground water with minerals and contaminants are of broad interest for geochemists but are not well understood. Experiments on the molecular scale can determine reaction parameters (i.e. rates of ligand exchange, activation entropy, activation entropy, and activation volume) that can be used in computations to gain insight into reactions that occur in natural groundwaters. Experiments to determine the rate of isotopic ligand exchange for three environmentally relevant metals, rhodium (Rh), iron (Fe), and neptunium (Np), are described. Many environmental transformations of metals (e.g. reduction) in soil occur at trivalent centers, Fe(III) in particular. Contaminant ions absorb to mineral surfaces via ligand exchange, and the reversal of this reaction can be dangerous, releasing contaminants into the environment. Ferric iron is difficult to study spectroscopically because most of its complexes are paramagnetic and are generally reactive toward ligand exchange; therefore, Rh(III), which is diamagnetic and less reactive, was used to study substitution reactions that are analogous to those that occur on mineral oxide surfaces. Studies on both Np(V) and Np(VI) are important in their own right, as 237Np is a radioactive transuranic element with a half-life of 2 million years.

  11. Ligand iron catalysts for selective hydrogenation

    Science.gov (United States)

    Casey, Charles P.; Guan, Hairong

    2010-11-16

    Disclosed are iron ligand catalysts for selective hydrogenation of aldehydes, ketones and imines. A catalyst such as dicarbonyl iron hydride hydroxycyclopentadiene) complex uses the OH on the five member ring and hydrogen linked to the iron to facilitate hydrogenation reactions, particularly in the presence of hydrogen gas.

  12. Supramolecular architectures constructed using angular bipyridyl ligands

    CERN Document Server

    Barnett, S A

    2003-01-01

    This work details the synthesis and characterization of a series of coordination frameworks that are formed using bidentate angular N-donor ligands. Pyrimidine was reacted with metal(ll) nitrate salts. Reactions using Cd(NO sub 3) sub 2 receive particular focus and the analogous reactions using the linear ligand, pyrazine, were studied for comparison. In all cases, two-dimensional coordination networks were prepared. Structural diversity is observed for the Cd(ll) centres including metal-nitrate bridging. In contrast, first row transition metal nitrates form isostructural one-dimensional chains with only the bridging N-donor ligands generating polymeric propagation. The angular ligand, 2,4-bis(4-pyridyl)-1,3,5-triazine (dpt), was reacted with Cd(NO sub 3) sub 2 and Zn(NO sub 3) sub 2. Whereas Zn(NO sub 3) sub 2 compounds exhibit solvent mediated polymorphism, a range of structures were obtained for the reactions with Cd(NO sub 3) sub 2 , including the first example of a doubly parallel interpenetrated 4.8 sup...

  13. Receptor Binding Ligands to Image Infection

    NARCIS (Netherlands)

    Chianelli, M.; Boerman, O. C.; Malviya, G.; Galli, F.; Oyen, W. J. G.; Signore, A.

    2008-01-01

    The current gold standard for imaging infection is radiolabeled white blood cells. For reasons of safety, simplicity and cost, it would be desirable to have a receptor-specific ligand that could be used for imaging infection and that would allow a differential diagnosis between sterile and septic in

  14. Novel thermo-responsive fucose binding ligands for glycoprotein purification by affinity precipitation.

    Science.gov (United States)

    Arnold, Lindsay; Chen, Rachel

    2014-02-01

    Novel thermo-responsive affinity sugar binders were developed by fusing a bacterial fucose lectin with a thermo-responsive polypeptide. These designer affinity ligand fusions were produced using an Escherichia coli system capable of extracellular secretion of recombinant proteins and were isolated with a high recovery yield (95%) directly from growth medium by Inverse Temperature Cycling (ITC). With horse radish peroxidase (HRP) as a model protein, we demonstrate here that the designer thermo-responsive ligands are capable of interacting with glycans on a glycoprotein, a property that was used to develop a novel affinity precipitation method for glycoprotein purification. The method, requiring only simple process steps, affords full recovery of a target glycoprotein, and is effective at a target glycoprotein concentration as low as 1.4 pM in the presence of large amounts of contaminants. By developing other sugar binders in the similar fashion, the method should be highly useful for glycoprotein purification and detection.

  15. Composition of Overlapping Protein-Protein and Protein-Ligand Interfaces.

    Directory of Open Access Journals (Sweden)

    Ruzianisra Mohamed

    Full Text Available Protein-protein interactions (PPIs play a major role in many biological processes and they represent an important class of targets for therapeutic intervention. However, targeting PPIs is challenging because often no convenient natural substrates are available as starting point for small-molecule design. Here, we explored the characteristics of protein interfaces in five non-redundant datasets of 174 protein-protein (PP complexes, and 161 protein-ligand (PL complexes from the ABC database, 436 PP complexes, and 196 PL complexes from the PIBASE database and a dataset of 89 PL complexes from the Timbal database. In all cases, the small molecule ligands must bind at the respective PP interface. We observed similar amino acid frequencies in all three datasets. Remarkably, also the characteristics of PP contacts and overlapping PL contacts are highly similar.

  16. Novel FXa Inhibitor Identification through Integration of Ligand- and Structure-Based Approaches

    Directory of Open Access Journals (Sweden)

    Carlos F. Lagos

    2017-09-01

    Full Text Available Factor Xa (FXa, a vitamin K-dependent serine protease plays a pivotal role in the coagulation cascade, one of the most interesting targets for the development of new anticoagulants. In the present work, we performed a virtual screening campaign based on ligand-based shape and electrostatic similarity search and protein-ligand docking to discover novel FXa-targeted scaffolds for further development of inhibitors. From an initial set of 260,000 compounds from the NCI Open database, 30 potential FXa inhibitors were identified and selected for in vitro biological evaluation. Compound 5 (NSC635393, 4-(3-methyl-4H-1,4-benzothiazin-2-yl-2,4-dioxo-N-phenylbutanamide displayed an IC50 value of 2.02 nM against human FXa. The identified compound may serve as starting point for the development of novel FXa inhibitors.

  17. A comparative study examining the cytotoxicity of inducible gene expression system ligands in different cell types.

    Science.gov (United States)

    Xie, Jinger; Nair, Ayyappan; Hermiston, Terry W

    2008-02-01

    Inducible gene expression systems are being used in many in vitro and in vivo applications for target discovery, target validation and as components in exploratory therapeutic agents. Ideally, the ligands, which activate the systems, are benign so that the effects can be strictly attributed to the induced protein. As a first step to defining the potential effects of these inducers, we tested three of them, doxycycline, muristerone A and mifepristone (for tet-, ecdysone- and progesterone antagonist-inducible systems respectively), for toxicity across a panel of normal cells and cancer cell lines. In contrast to both muristerone A and mifepristone that showed no significant toxicity on any of the tested cells, we observed that doxycycline induced cell death in selected cancer and primary cell lines. The different susceptibility of cell lines to the ligands commonly used in these inducible systems suggests that it is important to consider the effects of the inducers prior to their use in experimental in vitro cell culture systems.

  18. Nonlinear scoring functions for similarity-based ligand docking and binding affinity prediction.

    Science.gov (United States)

    Brylinski, Michal

    2013-11-25

    A common strategy for virtual screening considers a systematic docking of a large library of organic compounds into the target sites in protein receptors with promising leads selected based on favorable intermolecular interactions. Despite a continuous progress in the modeling of protein-ligand interactions for pharmaceutical design, important challenges still remain, thus the development of novel techniques is required. In this communication, we describe eSimDock, a new approach to ligand docking and binding affinity prediction. eSimDock employs nonlinear machine learning-based scoring functions to improve the accuracy of ligand ranking and similarity-based binding pose prediction, and to increase the tolerance to structural imperfections in the target structures. In large-scale benchmarking using the Astex/CCDC data set, we show that 53.9% (67.9%) of the predicted ligand poses have RMSD of <2 Å (<3 Å). Moreover, using binding sites predicted by recently developed eFindSite, eSimDock models ligand binding poses with an RMSD of 4 Å for 50.0-39.7% of the complexes at the protein homology level limited to 80-40%. Simulations against non-native receptor structures, whose mean backbone rearrangements vary from 0.5 to 5.0 Å Cα-RMSD, show that the ratio of docking accuracy and the estimated upper bound is at a constant level of ∼0.65. Pearson correlation coefficient between experimental and predicted by eSimDock Ki values for a large data set of the crystal structures of protein-ligand complexes from BindingDB is 0.58, which decreases only to 0.46 when target structures distorted to 3.0 Å Cα-RMSD are used. Finally, two case studies demonstrate that eSimDock can be customized to specific applications as well. These encouraging results show that the performance of eSimDock is largely unaffected by the deformations of ligand binding regions, thus it represents a practical strategy for across-proteome virtual screening using protein models. eSimDock is freely

  19. Dissociation of Multisubunit Protein-Ligand Complexes in the Gas Phase. Evidence for Ligand Migration

    Science.gov (United States)

    Zhang, Yixuan; Deng, Lu; Kitova, Elena N.; Klassen, John S.

    2013-10-01

    The results of collision-induced dissociation (CID) experiments performed on gaseous protonated and deprotonated ions of complexes of cholera toxin B subunit homopentamer (CTB5) with the pentasaccharide (β-D-Gal p-(1→3)-β-D-Gal pNAc-(1→4)[α-D-Neu5Ac-(2→3)]-β-D-Gal p-(1→4)-β-D-Glc p (GM1)) and corresponding glycosphingolipid (β-D-Gal p-(1→3)-β-D-Gal pNAc-(1→4)[α-D-Neu5Ac-(2→3)]-β-D-Gal p-(1→4)-β-D-Glc p-Cer (GM1-Cer)) ligands, and the homotetramer streptavidin (S4) with biotin (B) and 1,2-dipalmitoyl- sn-glycero-3-phosphoethanolamine-N-(biotinyl) (Btl), are reported. The protonated (CTB5 + 5GM1)n+ ions dissociated predominantly by the loss of a single subunit, with the concomitant migration of ligand to another subunit. The simultaneous loss of ligand and subunit was observed as a minor pathway. In contrast, the deprotonated (CTB5 + 5GM1)n- ions dissociated preferentially by the loss of deprotonated ligand; the loss of ligand-bound and ligand-free subunit were minor pathways. The presence of ceramide (Cer) promoted ligand migration and the loss of subunit. The main dissociation pathway for the protonated and deprotonated (S4 + 4B)n+/- ions, as well as for deprotonated (S4 + 4Btl)n- ions, was loss of the ligand. However, subunit loss from the (S4 + 4B)n+ ions was observed as a minor pathway. The (S4 + 4Btl)n+ ions dissociated predominantly by the loss of free and ligand-bound subunit. The charge state of the complex and the collision energy were found to have little effect on the relative contribution of the different dissociation channels. Thermally-driven ligand migration between subunits was captured in the results of molecular dynamics simulations performed on protonated (CTB5 + 5GM1)15+ ions (with a range of charge configurations) at 800 K. Notably, the migration pathway was found to be highly dependent on the charge configuration of the ion. The main conclusion of this study is that the dissociation pathways of multisubunit protein-ligand

  20. Quantum.Ligand.Dock: protein-ligand docking with quantum entanglement refinement on a GPU system.

    Science.gov (United States)

    Kantardjiev, Alexander A

    2012-07-01

    Quantum.Ligand.Dock (protein-ligand docking with graphic processing unit (GPU) quantum entanglement refinement on a GPU system) is an original modern method for in silico prediction of protein-ligand interactions via high-performance docking code. The main flavour of our approach is a combination of fast search with a special account for overlooked physical interactions. On the one hand, we take care of self-consistency and proton equilibria mutual effects of docking partners. On the other hand, Quantum.Ligand.Dock is the the only docking server offering such a subtle supplement to protein docking algorithms as quantum entanglement contributions. The motivation for development and proposition of the method to the community hinges upon two arguments-the fundamental importance of quantum entanglement contribution in molecular interaction and the realistic possibility to implement it by the availability of supercomputing power. The implementation of sophisticated quantum methods is made possible by parallelization at several bottlenecks on a GPU supercomputer. The high-performance implementation will be of use for large-scale virtual screening projects, structural bioinformatics, systems biology and fundamental research in understanding protein-ligand recognition. The design of the interface is focused on feasibility and ease of use. Protein and ligand molecule structures are supposed to be submitted as atomic coordinate files in PDB format. A customization section is offered for addition of user-specified charges, extra ionogenic groups with intrinsic pK(a) values or fixed ions. Final predicted complexes are ranked according to obtained scores and provided in PDB format as well as interactive visualization in a molecular viewer. Quantum.Ligand.Dock server can be accessed at http://87.116.85.141/LigandDock.html.

  1. [Novel targets for antibiotics discovery: riboswitches].

    Science.gov (United States)

    Jia, Dong-fang; Jia, Dong-fang; Jia, Dong-fang

    2013-09-01

    Riboswitches are cis-acting domains located in mRNA sequences that could regulate gene expression by sensing small molecules without employing protein. Most known riboswitches in bacteria have naturally evolved to bind essential metabolite ligands and are involved in the regulation of critical genes that are responsible for the biosynthesis or transport of the cognate ligand. The riboswitch-mediated gene expression could be repressed by metabolite analogs, which caused bacterial growth inhibition or even death. A number of leading compounds targeting riboswitches have been discovered. A promising avenue for the development of new class of riboswitch-based antibiotics has been opened. Herein we reviewed the current findings of riboswitches that served as targets for antibacterial drug development and the underlying mechanisms. The development of high-throughput methods and rational drug design for riboswitch-specific drug discovery are relevant challenges are discussed. summarized.

  2. Structure-based Drug Screening and Ligand-Based Drug Screening Toward Protein-Compound Network

    Science.gov (United States)

    Fukunishi, Yoshifumi

    2007-12-01

    We developed two new methods to improve the accuracy of molecular interaction data using a protein-compound affinity matrix calculated by a protein-compound docking software. One method is a structure-based in silico drug screening method and another method is a ligand-based in silico drug screening method. These methods were applied to enhance the database enrichment of in silico drug screening and in silico target protein screening.

  3. Ligand-based receptor tyrosine kinase partial agonists: New paradigm for cancer drug discovery?

    Science.gov (United States)

    Riese, David J.

    2010-01-01

    Introduction Receptor tyrosine kinases (RTKs) are validated targets for oncology drug discovery and several RTK antagonists have been approved for the treatment of human malignancies. Nonetheless, the discovery and development of RTK antagonists has lagged behind the discovery and development of agents that target G-protein coupled receptors. In part, this is because it has been difficult to discover analogs of naturally-occurring RTK agonists that function as antagonists. Areas covered Here we describe ligands of ErbB receptors that function as partial agonists for these receptors, thereby enabling these ligands to antagonize the activity of full agonists for these receptors. We provide insights into the mechanisms by which these ligands function as antagonists. We discuss how information concerning these mechanisms can be translated into screens for novel small molecule- and antibody-based antagonists of ErbB receptors and how such antagonists hold great potential as targeted cancer chemotherapeutics. Expert opinion While there have been a number of important key findings into this field, the identification of the structural basis of ligand functional specificity is still of the greatest importance. While it is true that, with some notable exceptions, peptide hormones and growth factors have not proven to be good platforms for oncology drug discovery; addressing the fundamental issues of antagonistic partial agonists for receptor tyrosine kinases has the potential to steer oncology drug discovery in new directions. Mechanism based approaches are now emerging to enable the discovery of RTK partial agonists that may antagonize both agonist-dependent and –independent RTK signaling and may hold tremendous promise as targeted cancer chemotherapeutics. PMID:21532939

  4. Low Frequency of Programmed Death Ligand 1 Expression in Pediatric Cancers

    OpenAIRE

    2016-01-01

    Programmed death 1 (PD‐1)/programmed death ligand 1 (PD‐L1) pathway blockade has become a promising therapeutic target in adult cancers. We evaluated PD‐L1 expression and tumor‐infiltrating CD8+ T cells in formalin‐fixed, paraffin‐embedded tumor specimens from 53 untreated pediatric patients with eight cancer types: neuroblastoma, extracranial malignant germ cell tumor, hepatoblastoma, germinoma, medulloblastoma, renal tumor, rhabdomyosarcoma, and atypical teratoid/rhabdoid tumor. One rhabdom...

  5. Fusion of ligand-coated nanoparticles with lipid bilayers: effect of ligand flexibility.

    Science.gov (United States)

    Van Lehn, Reid C; Alexander-Katz, Alfredo

    2014-08-07

    Amphiphilic, monolayer-protected gold nanoparticles (AuNPs) have recently been shown to insert into and fuse with lipid bilayers, driven by the hydrophobic effect. The inserted transmembrane state is stabilized by the "snorkeling" of charged ligand end groups out of the bilayer interior. This snorkeling process is facilitated by the backbone flexibility of the alkanethiol ligands that comprise the monolayer. In this work, we show that fusion is favorable even in the absence of backbone flexibility by modeling the ligands as rigid rods. For rigid ligands, snorkeling is still accommodated by rotations of the ligand with respect to the grafting point, but the process incurs a more significant free energy penalty than if the backbone were fully flexible. We show that the rigid rod model predicts similar trends in the free energy change for insertion as the previous flexible model when the size of the AuNPs is varied. However, the rigidity of the ligand backbone reduces the overall magnitude of the free energy change compared to that of the flexible model. These results thus generalize previous findings to systems with hindered backbone flexibility due to either structural constraints or low temperature.

  6. Enhanced antitumor effect of novel dual-targeted paclitaxel liposomes

    Energy Technology Data Exchange (ETDEWEB)

    Meng Shuyan; Su Bo; Li Wei; Ding Yongmei; Tang Liang; Zhou Wei; Song Yin; Li Heyan; Zhou Caicun, E-mail: caicunzhou@yahoo.com.cn [Cancer Institute of Tongji University School of Medicine, Shanghai Pulmonary Hospital, 507 Zhengmin Road, Shanghai (China)

    2010-10-15

    A novel dual-targeted peptide containing an alpha V integrins specific ligand and a neuropilin-1 specific motif was developed which showed an increased specific targeting affinity to tumors. Active dual-targeted liposomes were then produced with this peptide and exhibited greater binding activity than single-targeted liposomes in vitro. Paclitaxel entrapped in this formulation greatly increased the uptake of paclitaxel in the targeting cells and significantly suppressed the growth of HUVEC and A549 cells compared with general paclitaxel injections (Taxol) and single-targeted paclitaxel liposomes. The treatment of tumor xenograft models with dual-targeted paclitaxel liposomes also resulted in better tumor growth inhibition than any other treatment groups. Therefore, the dual-targeted paclitaxel liposomes prepared in the present study might be a more promising drug for cancer treatment. Furthermore, the dual-targeting approach may produce synergistic effects that can be applied in the development of new targeted drug delivery systems.

  7. Targeted anticancer therapy: overexpressed receptors and nanotechnology.

    Science.gov (United States)

    Akhtar, Mohd Javed; Ahamed, Maqusood; Alhadlaq, Hisham A; Alrokayan, Salman A; Kumar, Sudhir

    2014-09-25

    Targeted delivery of anticancer drugs to cancer cells and tissues is a promising field due to its potential to spare unaffected cells and tissues, but it has been a major challenge to achieve success in these therapeutic approaches. Several innovative approaches to targeted drug delivery have been devised based on available knowledge in cancer biology and on technological advancements. To achieve the desired selectivity of drug delivery, nanotechnology has enabled researchers to design nanoparticles (NPs) to incorporate anticancer drugs and act as nanocarriers. Recently, many receptor molecules known to be overexpressed in cancer have been explored as docking sites for the targeting of anticancer drugs. In principle, anticancer drugs can be concentrated specifically in cancer cells and tissues by conjugating drug-containing nanocarriers with ligands against these receptors. Several mechanisms can be employed to induce triggered drug release in response to either endogenous trigger or exogenous trigger so that the anticancer drug is only released upon reaching and preferentially accumulating in the tumor tissue. This review focuses on overexpressed receptors exploited in targeting drugs to cancerous tissues and the tumor microenvironment. We briefly evaluate the structure and function of these receptor molecules, emphasizing the elegant mechanisms by which certain characteristics of cancer can be exploited in cancer treatment. After this discussion of receptors, we review their respective ligands and then the anticancer drugs delivered by nanotechnology in preclinical models of cancer. Ligand-functionalized nanocarriers have delivered significantly higher amounts of anticancer drugs in many in vitro and in vivo models of cancer compared to cancer models lacking such receptors or drug carrying nanocarriers devoid of ligand. This increased concentration of anticancer drug in the tumor site enabled by nanotechnology could have a major impact on the efficiency of cancer

  8. Antitumor and antiparasitic activity of novel ruthenium compounds with polycyclic aromatic ligands.

    Science.gov (United States)

    Miserachs, Helena Guiset; Cipriani, Micaella; Grau, Jordi; Vilaseca, Marta; Lorenzo, Julia; Medeiros, Andrea; Comini, Marcelo A; Gambino, Dinorah; Otero, Lucía; Moreno, Virtudes

    2015-09-01

    Five novel ruthenium(II)-arene complexes with polycyclic aromatic ligands were synthesized, comprising three compounds of the formula [RuCl(η(6)-p-cym)(L)][PF6], where p-cym = 1-isopropyl-4-methylbenzene and L are the bidentate aromatic ligands 1,10-phenanthroline-5,6-dione, 1, 5-amine-1,10-phenanthroline, 4, or 5,6-epoxy-5,6-dihydro-phenanthroline, 5. In the other two complexes [RuCl2(η(6)-p-cym)(L')], the metal is coordinated to a monodentate ligand L', where L' is phenanthridine, 2, or 9-carbonylanthracene, 3. All compounds were fully characterized by mass spectrometry and elemental analysis, as well as NMR and IR spectroscopic techniques. Obtained ruthenium compounds as well as their respective ligands were tested for their antiparasitic and antitumoral activities. Even though all compounds showed lower Trypanosoma brucei activity than the free ligands, they also resulted less toxic on mammalian cells. Cytotoxicity assays on HL60 cells showed a moderate antitumoral activity for all ruthenium compounds. Compound 1 was the most potent antitumoral (IC50 = 1.26±0.78 μM) and antiparasitic (IC50 = 0.19 ± 0.05 μM) agent, showing high selectivity towards the parasites (selectivity index >100). As complex 1 was the most promising antitumoral compound, its interaction with ubiquitin as potential target was also studied. In addition, obtained ruthenium compounds were found to bind DNA, and they are thought to interact with this macromolecule mainly through intercalation of the aromatic ligand.

  9. Identification and functional analysis of ligands for natural killer cell activating receptors in colon carcinoma.

    Science.gov (United States)

    Zhang, Zhang; Su, Tao; He, Liang; Wang, Hongtao; Ji, Gang; Liu, Xiaonan; Zhang, Yun; Dong, Guanglong

    2012-01-01

    Natural killer (NK) cells play important roles in the immune defense against tumor cells. The function of NK cells is determined by a balance between activating and inhibitory signals. DNAX accessory molecule-1 (DNAM-1) and NK group 2 member D (NKG2D) are major NK cell activating receptors, which transduce activating signals after binding their ligands CD155, CD112 and major histocompatibility complex class I-related chains A and B (MICA/B). However, the expression and functions of these ligands in colon carcinoma are still elusive. Here, we show the higher expression of CD155, CD112 and MICA/B in colon carcinoma tissues, although no correlations between the ligands expression and patient clinicopathological parameters were found. The subsequent cytotoxicity assay indicated that NK cells effectively kill colon carcinoma cells. Functional blocking of these ligands and/or receptors with antibodies led to significant inhibition of NK cell cytotoxicity. Importantly, expression of DNAM-1 and NKG2D was reduced in NK cells of colon cancer patients, and this reduction could directly suppress the activation of NK cells. Moreover, colon cancer patients have higher serum concentrations of sCD155 and sMICA/B (soluble ligands, secreted or shed from cells) than those in healthy donors (sCD155, 127.82 ± 44.12 vs. 63.67 ± 22.30 ng/ml; sMICA, 331.51 ± 65.23 vs. 246.74 ± 20.76 pg/ml; and sMICB, 349.42 ± 81.69 vs. 52.61 ± 17.56 pg/ml). The up-regulation of these soluble ligands may down-regulate DNAM-1 and NKG2D on NK cells, ultimately leading to the inhibition of NK cytotoxicity. Colon cancer might be a promising target for NK cell-based adoptive immunotherapy.

  10. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

    Energy Technology Data Exchange (ETDEWEB)

    Bokoch, Michael P.; Zou, Yaozhong; Rasmussen, Søren G.F.; Liu, Corey W.; Nygaard, Rie; Rosenbaum, Daniel M.; Fung, Juan José; Choi, Hee-Jung; Thian, Foon Sun; Kobilka, Tong Sun; Puglisi, Joseph D.; Weis, William I.; Pardo, Leonardo; Prosser, R. Scott; Mueller, Luciano; Kobilka, Brian K. (Stanford-MED); (Toronto); (BMS); (UAB, Spain)

    2010-01-14

    G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the {beta}{sub 2} adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.

  11. Target Space $\

    CERN Document Server

    Huggett, Nick

    2015-01-01

    This paper investigates the significance of T-duality in string theory: the indistinguishability with respect to all observables, of models attributing radically different radii to space -- larger than the observable universe, or far smaller than the Planck length, say. Two interpretational branch points are identified and discussed. First, whether duals are physically equivalent or not: by considering a duality of the familiar simple harmonic oscillator, I argue that they are. Unlike the oscillator, there are no measurements 'outside' string theory that could distinguish the duals. Second, whether duals agree or disagree on the radius of 'target space', the space in which strings evolve according to string theory. I argue for the latter position, because the alternative leaves it unknown what the radius is. Since duals are physically equivalent yet disagree on the radius of target space, it follows that the radius is indeterminate between them. Using an analysis of Brandenberger and Vafa (1989), I explain wh...

  12. Multivalent ligands control stem cell behaviour in vitro and in vivo

    Science.gov (United States)

    Conway, Anthony; Vazin, Tandis; Spelke, Dawn P.; Rode, Nikhil A.; Healy, Kevin E.; Kane, Ravi S.; Schaffer, David V.

    2013-11-01

    There is broad interest in designing nanostructured materials that can interact with cells and regulate key downstream functions. In particular, materials with nanoscale features may enable control over multivalent interactions, which involve the simultaneous binding of multiple ligands on one entity to multiple receptors on another and are ubiquitous throughout biology. Cellular signal transduction of growth factor and morphogen cues (which have critical roles in regulating cell function and fate) often begins with such multivalent binding of ligands, either secreted or cell-surface-tethered to target cell receptors, leading to receptor clustering. Cellular mechanisms that orchestrate ligand-receptor oligomerization are complex, however, so the capacity to control multivalent interactions and thereby modulate key signalling events within living systems is currently very limited. Here, we demonstrate the design of potent multivalent conjugates that can organize stem cell receptors into nanoscale clusters and control stem cell behaviour in vitro and in vivo. The ectodomain of ephrin-B2, normally an integral membrane protein ligand, was conjugated to a soluble biopolymer to yield multivalent nanoscale conjugates that potently induce signalling in neural stem cells and promote their neuronal differentiation both in culture and within the brain. Super-resolution microscopy analysis yielded insights into the organization of the receptor-ligand clusters at the nanoscale. We also found that synthetic multivalent conjugates of ephrin-B1 strongly enhance human embryonic and induced pluripotent stem cell differentiation into functional dopaminergic neurons. Multivalent bioconjugates are therefore powerful tools and potential nanoscale therapeutics for controlling the behaviour of target stem cells in vitro and in vivo.

  13. The CXCL12/CXCR4 axis promotes ligand-independent activation of the androgen receptor.

    Science.gov (United States)

    Kasina, Sathish; Macoska, Jill A

    2012-04-01

    The molecular mechanisms responsible for the transition of some prostate cancers from androgen ligand-dependent to androgen ligand-independent are incompletely established. Molecules that are ligands for G protein coupled receptors (GPCRs) have been implicated in ligand-independent androgen receptor (AR) activation. The purpose of this study was to examine whether CXCL12, the ligand for the GPCR, CXCR4, might mediate prostate cancer cell proliferation through AR-dependent mechanisms involving functional transactivation of the AR in the absence of androgen. The results of these studies showed that activation of the CXCL12/CXCR4 axis promoted: The nuclear accumulation of both wild-type and mutant AR in several prostate epithelial cell lines; AR-dependent proliferative responses; nuclear accumulation of the AR co-regulator SRC-1 protein; SRC-1:AR protein:protein association; co-localization of AR and SRC-1 on the promoters of AR-regulated genes; AR- and SRC-1 dependent transcription of AR-regulated genes; AR-dependent secretion of the AR-regulated PSA protein; P13K-dependent phosphorylation of AR; MAPK-dependent phosphorylation of SRC-1, and both MAPK- and P13K-dependent secretion of the PSA protein, in the absence of androgen. Taken together, these studies identify CXCL12 as a novel, non-steroidal growth factor that promotes the growth of prostate epithelial cells through AR-dependent mechanisms in the absence of steroid hormones. These findings support the development of novel therapeutics targeting the CXCL12/CXCR4 axis as an ancillary to those targeting the androgen/AR axis to effectively treat castration resistant/recurrent prostate tumors.

  14. A biased ligand for OXE-R uncouples Gα and Gβγ signaling within a heterotrimer

    DEFF Research Database (Denmark)

    Blättermann, Stefanie; Peters, Lucas; Ottersbach, Philipp Aaron;

    2012-01-01

    Differential targeting of heterotrimeric G protein versus β-arrestin signaling are emerging concepts in G protein-coupled receptor (GPCR) research and drug discovery, and biased engagement by GPCR ligands of either β-arrestin or G protein pathways has been disclosed. Herein we report on a new mec...

  15. Evaluation of 3-Ethyl-3-(phenylpiperazinylbutyl)oxindoles as PET Ligands for the Serotonin 5-HT7 Receptor

    DEFF Research Database (Denmark)

    Herth, Matthias M; Andersen, Valdemar L; Hansen, Hanne D;

    2015-01-01

    We have investigated several oxindole derivatives in the pursuit of a 5-HT7 receptor PET ligand. Herein the synthesis, chiral separation, and pharmacological profiling of two possible PET candidates toward a wide selection of CNS-targets are detailed. Subsequent (11)C-labeling and in vivo...

  16. Amino Acids in Nine Ligand-Prefer Ramachandran Regions

    Directory of Open Access Journals (Sweden)

    Chen Cao

    2015-01-01

    Full Text Available Several secondary structures, such as π-helix and left-handed helix, have been frequently identified at protein ligand-binding sites. A secondary structure is considered to be constrained to a specific region of dihedral angles. However, a comprehensive analysis of the correlation between main chain dihedral angles and ligand-binding sites has not been performed. We undertook an extensive analysis of the relationship between dihedral angles in proteins and their distance to ligand-binding sites, frequency of occurrence, molecular potential energy, amino acid composition, van der Waals contacts, and hydrogen bonds with ligands. The results showed that the values of dihedral angles have a strong preference for ligand-binding sites at certain regions in the Ramachandran plot. We discovered that amino acids preceding the ligand-prefer ϕ/ψ box residues are exposed more to solvents, whereas amino acids following ligand-prefer ϕ/ψ box residues form more hydrogen bonds and van der Waals contacts with ligands. Our method exhibited a similar performance compared with the program Ligsite-csc for both ligand-bound structures and ligand-free structures when just one ligand-binding site was predicted. These results should be useful for the prediction of protein ligand-binding sites and for analysing the relationship between structure and function.

  17. Open-shell organometallics: reactivity at the ligand

    NARCIS (Netherlands)

    W.I. Dzik; B. de Bruin

    2011-01-01

    The purpose of this review is to show that (cooperative) ligand radical reactivity can be effectively employed in synthetic organometallic chemistry and catalysis to achieve selectivity in radical-type transformations. The ‘redox non-innocence’ of ligands, and the controlled reactivity of ‘ligand ra

  18. Triple Bioaffinity Mass Spectrometry Concept for Thyroid Transporter Ligands

    NARCIS (Netherlands)

    Aqai, P.; Fryganas, C.; Mizuguchi, M.; Haasnoot, W.; Nielen, M.W.F.

    2012-01-01

    For the analysis of thyroid transporter ligands, a triple bioaffinity mass spectrometry (BioMS) concept was developed, with the aim at three different analytical objectives: rapid screening of any ligand, confirmation of known ligands in accordance with legislative requirements, and identification o

  19. Protein-protein interactions as druggable targets: recent technological advances.

    Science.gov (United States)

    Higueruelo, Alicia P; Jubb, Harry; Blundell, Tom L

    2013-10-01

    Classical target-based drug discovery, where large chemical libraries are screened using inhibitory assays for a single target, has struggled to find ligands that inhibit protein-protein interactions (PPI). Nevertheless, in the past decade there have been successes that have demonstrated that PPI can be useful drug targets, and the field is now evolving fast. This review focuses on the new approaches and concepts that are being developed to tackle these challenging targets: the use of fragment based methods to explore the chemical space, stapled peptides to regulate intracellular PPI, alternatives to competitive inhibition and the use of antibodies to enable small molecule discovery for these targets.

  20. Molecular Systems Pharmacology: Isoelectric Focusing Signature of Protein Kinase Cδ Provides an Integrated Measure of Its Modulation in Response to Ligands

    Science.gov (United States)

    2015-01-01

    Protein kinase C (PKC), a validated therapeutic target for cancer chemotherapy, provides a paradigm for assessing structure–activity relations, where ligand binding has multiple consequences for a target. For PKC, ligand binding controls not only PKC activation and multiple phosphorylations but also subcellular localization, affecting subsequent signaling. Using a capillary isoelectric focusing immunoassay system, we could visualize a high resolution isoelectric focusing signature of PKCδ upon stimulation by ligands of the phorbol ester and bryostatin classes. Derivatives that possessed different physicochemical characteristics and induced different patterns of biological response generated different signatures. Consistent with different patterns of PKCδ localization as one factor linked to these different signatures, we found different signatures for activated PKCδ from the nuclear and non-nuclear fractions. We conclude that the capillary isoelectric focusing immunoassay system may provide a window into the integrated consequences of ligand binding and thus afford a powerful platform for compound development. PMID:24906106

  1. Structural studies of P-type ATPase–ligand complexes using an X-ray free-electron laser

    Energy Technology Data Exchange (ETDEWEB)

    Bublitz, Maike; Nass, Karol; Drachmann, Nikolaj D.; Markvardsen, Anders J.; Gutmann, Matthias J.; Barends, Thomas R. M.; Mattle, Daniel; Shoeman, Robert L.; Doak, R. Bruce; Boutet, Sébastien; Messerschmidt, Marc; Seibert, Marvin M.; Williams, Garth J.; Foucar, Lutz; Reinhard, Linda; Sitsel, Oleg; Gregersen, Jonas L.; Clausen, Johannes D.; Boesen, Thomas; Gotfryd, Kamil; Wang, Kai-Tuo; Olesen, Claus; Møller, Jesper V.; Nissen, Poul; Schlichting, Ilme

    2015-06-11

    Membrane proteins are key players in biological systems, mediating signalling events and the specific transport ofe.g.ions and metabolites. Consequently, membrane proteins are targeted by a large number of currently approved drugs. Understanding their functions and molecular mechanisms is greatly dependent on structural information, not least on complexes with functionally or medically important ligands. Structure determination, however, is hampered by the difficulty of obtaining well diffracting, macroscopic crystals. Here, the feasibility of X-ray free-electron-laser-based serial femtosecond crystallography (SFX) for the structure determination of membrane protein–ligand complexes using microcrystals of various native-source and recombinant P-type ATPase complexes is demonstrated. The data reveal the binding sites of a variety of ligands, including lipids and inhibitors such as the hallmark P-type ATPase inhibitor orthovanadate. By analyzing the resolution dependence of ligand densities and overall model qualities, SFX data quality metrics as well as suitable refinement procedures are discussed. Even at relatively low resolution and multiplicity, the identification of ligands can be demonstrated. This makes SFX a useful tool for ligand screening and thus for unravelling the molecular mechanisms of biologically active proteins.

  2. Structural studies of P-type ATPase–ligand complexes using an X-ray free-electron laser

    Directory of Open Access Journals (Sweden)

    Maike Bublitz

    2015-07-01

    Full Text Available Membrane proteins are key players in biological systems, mediating signalling events and the specific transport of e.g. ions and metabolites. Consequently, membrane proteins are targeted by a large number of currently approved drugs. Understanding their functions and molecular mechanisms is greatly dependent on structural information, not least on complexes with functionally or medically important ligands. Structure determination, however, is hampered by the difficulty of obtaining well diffracting, macroscopic crystals. Here, the feasibility of X-ray free-electron-laser-based serial femtosecond crystallography (SFX for the structure determination of membrane protein–ligand complexes using microcrystals of various native-source and recombinant P-type ATPase complexes is demonstrated. The data reveal the binding sites of a variety of ligands, including lipids and inhibitors such as the hallmark P-type ATPase inhibitor orthovanadate. By analyzing the resolution dependence of ligand densities and overall model qualities, SFX data quality metrics as well as suitable refinement procedures are discussed. Even at relatively low resolution and multiplicity, the identification of ligands can be demonstrated. This makes SFX a useful tool for ligand screening and thus for unravelling the molecular mechanisms of biologically active proteins.

  3. Glycomimetic ligands for the human asialoglycoprotein receptor.

    Science.gov (United States)

    Mamidyala, Sreeman K; Dutta, Sanjay; Chrunyk, Boris A; Préville, Cathy; Wang, Hong; Withka, Jane M; McColl, Alexander; Subashi, Timothy A; Hawrylik, Steven J; Griffor, Matthew C; Kim, Sung; Pfefferkorn, Jeffrey A; Price, David A; Menhaji-Klotz, Elnaz; Mascitti, Vincent; Finn, M G

    2012-02-01

    The asialoglycoprotein receptor (ASGPR) is a high-capacity galactose-binding receptor expressed on hepatocytes that binds its native substrates with low affinity. More potent ligands are of interest for hepatic delivery of therapeutic agents. We report several classes of galactosyl analogues with varied substitution at the anomeric, C2-, C5-, and C6-positions. Significant increases in binding affinity were noted for several trifluoromethylacetamide derivatives without covalent attachment to the protein. A variety of new ligands were obtained with affinity for ASGPR as good as or better than that of the parent N-acetylgalactosamine, showing that modification on either side of the key C3,C4-diol moiety is well tolerated, consistent with previous models of a shallow binding pocket. The galactosyl pyranose motif therefore offers many opportunities for the attachment of other functional units or payloads while retaining low-micromolar or better affinity for the ASGPR.

  4. Leaching behavior of butanedionedioxime as gold ligand

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Butanedionedioxime, a small organic compound with low-toxicity and good chemical stability, has been proposed as an effective gold ligand in gold extraction. The result of experiment shows that: 1) highly effective gold lixiviantcan be composed of butanedionedioxime (BDM) with many oxidants, especially potassium permanganate; 2)in the leaching system of BD M- K M nO4 the suitable Ox/Lig(ratio of oxidants to gold ligands) tange is 0.20 ~ 0. 50, optimally 0.25 ~0.45 at the pH range of 7 ~ 11; 3) BDM-KMnO4 extraction of gold from an oxide ore is similar to cyanide(cyanide-O2)extraction, but the leaching rate of gold by BDM-KMnO4 is faster than that by cyanide-O2; 4) gold may readily be recov-ered by carbon adsorption and zinc precipitation

  5. Mechanics, thermodynamics, and kinetics of ligand binding to biopolymers.

    Science.gov (United States)

    Jarillo, Javier; Morín, José A; Beltrán-Heredia, Elena; Villaluenga, Juan P G; Ibarra, Borja; Cao, Francisco J

    2017-01-01

    Ligands binding to polymers regulate polymer functions by changing their physical and chemical properties. This ligand regulation plays a key role in many biological processes. We propose here a model to explain the mechanical, thermodynamic, and kinetic properties of the process of binding of small ligands to long biopolymers. These properties can now be measured at the single molecule level using force spectroscopy techniques. Our model performs an effective decomposition of the ligand-polymer system on its covered and uncovered regions, showing that the elastic properties of the ligand-polymer depend explicitly on the ligand coverage of the polymer (i.e., the fraction of the polymer covered by the ligand). The equilibrium coverage that minimizes the free energy of the ligand-polymer system is computed as a function of the applied force. We show how ligands tune the mechanical properties of a polymer, in particular its length and stiffness, in a force dependent manner. In addition, it is shown how ligand binding can be regulated applying mechanical tension on the polymer. Moreover, the binding kinetics study shows that, in the case where the ligand binds and organizes the polymer in different modes, the binding process can present transient shortening or lengthening of the polymer, caused by changes in the relative coverage by the different ligand modes. Our model will be useful to understand ligand-binding regulation of biological processes, such as the metabolism of nucleic acid. In particular, this model allows estimating the coverage fraction and the ligand mode characteristics from the force extension curves of a ligand-polymer system.

  6. Predicting targets of compounds against neurological diseases using cheminformatic methodology.

    Science.gov (United States)

    Nikolic, Katarina; Mavridis, Lazaros; Bautista-Aguilera, Oscar M; Marco-Contelles, José; Stark, Holger; do Carmo Carreiras, Maria; Rossi, Ilaria; Massarelli, Paola; Agbaba, Danica; Ramsay, Rona R; Mitchell, John B O

    2015-02-01

    Recently developed multi-targeted ligands are novel drug candidates able to interact with monoamine oxidase A and B; acetylcholinesterase and butyrylcholinesterase; or with histamine N-methyltransferase and histamine H3-receptor (H3R). These proteins are drug targets in the treatment of depression, Alzheimer's disease, obsessive disorders, and Parkinson's disease. A probabilistic method, the Parzen-Rosenblatt window approach, was used to build a "predictor" model using data collected from the ChEMBL database. The model can be used to predict both the primary pharmaceutical target and off-targets of a compound based on its structure. Molecular structures were represented based on the circular fingerprint methodology. The same approach was used to build a "predictor" model from the DrugBank dataset to determine the main pharmacological groups of the compound. The study of off-target interactions is now recognised as crucial to the understanding of both drug action and toxicology. Primary pharmaceutical targets and off-targets for the novel multi-target ligands were examined by use of the developed cheminformatic method. Several multi-target ligands were selected for further study, as compounds with possible additional beneficial pharmacological activities. The cheminformatic targets identifications were in agreement with four 3D-QSAR (H3R/D1R/D2R/5-HT2aR) models and by in vitro assays for serotonin 5-HT1a and 5-HT2a receptor binding of the most promising ligand (71/MBA-VEG8).

  7. Predicting targets of compounds against neurological diseases using cheminformatic methodology

    Science.gov (United States)

    Nikolic, Katarina; Mavridis, Lazaros; Bautista-Aguilera, Oscar M.; Marco-Contelles, José; Stark, Holger; do Carmo Carreiras, Maria; Rossi, Ilaria; Massarelli, Paola; Agbaba, Danica; Ramsay, Rona R.; Mitchell, John B. O.

    2015-02-01

    Recently developed multi-targeted ligands are novel drug candidates able to interact with monoamine oxidase A and B; acetylcholinesterase and butyrylcholinesterase; or with histamine N-methyltransferase and histamine H3-receptor (H3R). These proteins are drug targets in the treatment of depression, Alzheimer's disease, obsessive disorders, and Parkinson's disease. A probabilistic method, the Parzen-Rosenblatt window approach, was used to build a "predictor" model using data collected from the ChEMBL database. The model can be used to predict both the primary pharmaceutical target and off-targets of a compound based on its structure. Molecular structures were represented based on the circular fingerprint methodology. The same approach was used to build a "predictor" model from the DrugBank dataset to determine the main pharmacological groups of the compound. The study of off-target interactions is now recognised as crucial to the understanding of both drug action and toxicology. Primary pharmaceutical targets and off-targets for the novel multi-target ligands were examined by use of the developed cheminformatic method. Several multi-target ligands were selected for further study, as compounds with possible additional beneficial pharmacological activities. The cheminformatic targets identifications were in agreement with four 3D-QSAR (H3R/D1R/D2R/5-HT2aR) models and by in vitro assays for serotonin 5-HT1a and 5-HT2a receptor binding of the most promising ligand ( 71/MBA-VEG8).

  8. Dynamics of target-mediated drug disposition: characteristic profiles and parameter identification.

    Science.gov (United States)

    Peletier, Lambertus A; Gabrielsson, Johan

    2012-10-01

    In this paper we present a mathematical analysis of the basic model for target mediated drug disposition (TMDD). Assuming high affinity of ligand to target, we give a qualitative characterisation of ligand versus time graphs for different dosing regimes and derive accurate analytic approximations of different phases in the temporal behaviour of the system. These approximations are used to estimate model parameters, give analytical approximations of such quantities as area under the ligand curve and clearance. We formulate conditions under which a suitably chosen Michaelis-Menten model provides a good approximation of the full TMDD-model over a specified time interval.

  9. Metal-ligand cooperative activation of nitriles by a ruthenium complex with a de-aromatized PNN pincer ligand

    NARCIS (Netherlands)

    Eijsink, Linda E; Perdriau, Sébastien C P; de Vries, Johannes G; Otten, Edwin

    2016-01-01

    The pincer complex (PNN)RuH(CO), with a de-aromatized pyridine in the ligand backbone, is shown to react with nitriles in a metal-ligand cooperative manner. This leads to the formation of a series of complexes with new Ru-N(nitrile) and C(ligand)-C(nitrile) bonds. The initial nitrile cycloaddition

  10. Metal-ligand cooperative activation of nitriles by a ruthenium complex with a de-aromatized PNN pincer ligand

    NARCIS (Netherlands)

    Eijsink, Linda E; Perdriau, Sébastien C P; de Vries, Johannes G; Otten, Edwin

    2016-01-01

    The pincer complex (PNN)RuH(CO), with a de-aromatized pyridine in the ligand backbone, is shown to react with nitriles in a metal-ligand cooperative manner. This leads to the formation of a series of complexes with new Ru-N(nitrile) and C(ligand)-C(nitrile) bonds. The initial nitrile cycloaddition p

  11. Protein-ligand binding affinity determination by the waterLOGSY method: An optimised approach considering ligand rebinding

    Science.gov (United States)

    Huang, Renjie; Bonnichon, Arnaud; Claridge, Timothy D. W.; Leung, Ivanhoe K. H.

    2017-03-01

    WaterLOGSY is a popular ligand-observed NMR technique to screen for protein-ligand interactions, yet when applied to measure dissociation constants (KD) through ligand titration, the results were found to be strongly dependent on sample conditions. Herein, we show that accurate KDs can be obtained by waterLOGSY with optimised experimental setup.

  12. The ligands of CXCR4 in vascularization

    OpenAIRE

    Tuchscheerer, Nancy

    2012-01-01

    The formation of a functional and integrated vascular network is a basic process in the growth and maintenance of tissues and can be established by two forms of blood vessel growth in adults: angiogenesis and arteriogenesis. In this study, the ligands of the chemokine receptor CXCR4 and its role in angiogenesis (represented by the experimental myocardial infarction) and arteriogenesis (represented by the murine hind limb ischemia model) was investigated. The first approach identified the CXCL...

  13. Dockomatic - automated ligand creation and docking

    OpenAIRE

    Hampikian Greg; McDougal Owen M; Jacob Reed B; Bullock Casey W; Andersen Tim

    2010-01-01

    Abstract Background The application of computational modeling to rationally design drugs and characterize macro biomolecular receptors has proven increasingly useful due to the accessibility of computing clusters and clouds. AutoDock is a well-known and powerful software program used to model ligand to receptor binding interactions. In its current version, AutoDock requires significant amounts of user time to setup and run jobs, and collect results. This paper presents DockoMatic, a user frie...

  14. Selective oxoanion separation using a tripodal ligand

    Energy Technology Data Exchange (ETDEWEB)

    Custelcean, Radu; Moyer, Bruce A.; Rajbanshi, Arbin

    2016-02-16

    The present invention relates to urea-functionalized crystalline capsules self-assembled by sodium or potassium cation coordination and by hydrogen-bonding water bridges to selectively encapsulate tetrahedral divalent oxoanions from highly competitive aqueous alkaline solutions and methods using this system for selective anion separations from industrial solutions. The method involves competitive crystallizations using a tripodal tris(urea) functionalized ligand and, in particular, provides a viable approach to sulfate separation from nuclear wastes.

  15. galectin-3 ligand — EDRN Public Portal

    Science.gov (United States)

    Galectin-3 is an endogenous lectin that binds glycan epitopes of cell membrane and some extracellular glycoproteins such as integrins and laminin. Galectin-3 is involved in several biological activities including regulation of cellular cycle, modulation of adhesion and tumor progression and metastasis. Serum galectin-3 ligands have been shown to modulate the immune reaction against tumors and viruses and their level increases in sera of several neoplastic diseases.

  16. Determining the magnitude and direction of photoinduced ligand field switching in photochromic metal-organic complexes: molybdenum-tetracarbonyl spirooxazine complexes.

    Science.gov (United States)

    Paquette, Michelle M; Patrick, Brian O; Frank, Natia L

    2011-07-06

    The ability to optically switch or tune the intrinsic properties of transition metals (e.g., redox potentials, emission/absorption energies, and spin states) with photochromic metal-ligand complexes is an important strategy for developing "smart" materials. We have described a methodology for using metal-carbonyl complexes as spectroscopic probes of ligand field changes associated with light-induced isomerization of photochromic ligands. Changes in ligand field between the ring-closed spirooxazine (SO) and ring-opened photomerocyanine (PMC) forms of photochromic azahomoadamantyl and indolyl phenanthroline-spirooxazine ligands are demonstrated through FT-IR, (13)C NMR, and computational studies of their molybdenum-tetracarbonyl complexes. The frontier molecular orbitals (MOs) of the SO and PMC forms differ considerably in both electron density distributions and energies. Of the multiple π* MOs in the SO and PMC forms of the ligands, the LUMO+1, a pseudo-b(1)-symmetry phenanthroline-based MO, mixes primarily with the Mo(CO)(4) fragment and provides the major pathway for Mo(d)→phen(π*) backbonding. The LUMO+1 is found to be 0.2-0.3 eV lower in energy in the SO form relative to the PMC form, suggesting that the SO form is a better π-acceptor. Light-induced isomerization of the photochromic ligands was therefore found to lead to changes in the energies of their frontier MOs, which in turn leads to changes in π-acceptor ability and ligand field strength. Ligand field changes associated with photoisomerizable ligands allow tuning of excited-state and ground-state energies that dictate energy/electron transfer, optical/electrical properties, and spin states of a metal center upon photoisomerization, positioning photochromic ligand-metal complexes as promising targets for smart materials.

  17. Design of a multi-dopamine-modified polymer ligand optimally suited for interfacing magnetic nanoparticles with biological systems.

    Science.gov (United States)

    Wang, Wentao; Ji, Xin; Na, Hyon Bin; Safi, Malak; Smith, Alexandra; Palui, Goutam; Perez, J Manuel; Mattoussi, Hedi

    2014-06-03

    We have designed a set of multifunctional and multicoordinating polymer ligands that are optimally suited for surface functionalizing iron oxide and potentially other magnetic nanoparticles (NPs) and promoting their integration into biological systems. The amphiphilic polymers are prepared by coupling (via nucleophilic addition) several amine-terminated dopamine anchoring groups, poly(ethylene glycol) moieties, and reactive groups onto a poly(isobutylene-alt-maleic anhydride) (PIMA) chain. This design greatly benefits from the highly efficient and reagent-free one-step reaction of maleic anhydride groups with amine-containing molecules. The availability of several dopamine groups in the same ligand greatly enhances the ligand affinity, via multiple coordination, to the magnetic NPs, while the hydrophilic and reactive groups promote colloidal stability in buffer media and allow subsequent conjugation with target biomolecules. Iron oxide nanoparticles ligand exchanged with these polymer ligands have a compact hydrodynamic size and exhibit enhanced long-term colloidal stability over the pH range of 4-12 and in the presence of excess electrolytes. Nanoparticles ligated with terminally reactive polymers have been easily coupled to target dyes and tested in live cell imaging with no measurable cytotoxicity. Finally, the resulting hydrophilic nanoparticles exhibit large and size-dependent r2 relaxivity values.

  18. Fluorescence intercalator displacement assay for screening G4 ligands towards a variety of G-quadruplex structures.

    Science.gov (United States)

    Tran, Phong Lan Thao; Largy, Eric; Hamon, Florian; Teulade-Fichou, Marie-Paule; Mergny, Jean-Louis

    2011-08-01

    The potential formation of G-quadruplexes in many regions of the genome makes them an attractive target for drug design. A large number of small molecules synthesized in recent years display an ability to selectively target and stabilize G-quadruplexes. To screen for G4 ligands, we modified a G4-FID (G-quadruplex Fluorescent Intercalator Displacement) assay. This test is based on the displacement of an "on/off" fluorescence probe, Thiazole Orange (TO), from quadruplex or duplex DNA matrices by increasing amounts of a putative ligand. Selectivity measurements can easily be achieved by comparing the ability of the ligand to displace TO from various quadruplex and duplex structures. G4-FID requires neither modified oligonucleotides nor specific equipment and is an isothermal experiment. This test was adapted for high throughput screening onto 96-well plates allowing the comparison of more than twenty different structures. Fifteen different known G4 ligands belonging to different families were tested. Most compounds showed a good G4 vs duplex selectivity but exhibited little, if any, specificity for one quadruplex sequence over the others. The quest for the "perfect" specific G4 ligand is not over yet! Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  19. Analysing the effect of I1 imidazoline receptor ligands on DSS-induced acute colitis in mice.

    Science.gov (United States)

    Fehér, Ágnes; Tóth, Viktória E; Al-Khrasani, Mahmoud; Balogh, Mihály; Lázár, Bernadette; Helyes, Zsuzsanna; Gyires, Klára; Zádori, Zoltán S

    2017-02-01

    Imidazoline receptors (IRs) have been recognized as promising targets in the treatment of numerous diseases; and moxonidine and rilmenidine, agonists of I1-IRs, are widely used as antihypertensive agents. Some evidence suggests that IR ligands may induce anti-inflammatory effects acting on I1-IRs or other molecular targets, which could be beneficial in patients with inflammatory bowel disease (IBD). On the other hand, several IR ligands may stimulate also alpha2-adrenoceptors, which were earlier shown to inhibit, but in more recent studies to rather aggravate colitis. Hence, this study aimed to analyse for the first time the effect of various I1-IR ligands on intestinal inflammation. Colitis was induced in C57BL/6 mice by adding dextran sulphate sodium (DSS) to the drinking water for 7 days. Mice were treated daily with different IR ligands: moxonidine and rilmenidine (I1-IR agonists), AGN 192403 (highly selective I1-IR ligand, putative antagonist), efaroxan (I1-IR antagonist), as well as with the endogenous IR agonists agmatine and harmane. It was found that moxonidine and rilmenidine at clinically relevant doses, similarly to the other IR ligands, do not have a significant impact on the macroscopic and histological signs of DSS-evoked inflammation. Likewise, colonic myeloperoxidase and serum interleukin-6 levels remained unchanged in response to these agents. Thus, our study demonstrates that imidazoline ligands do not influence significantly the severity of DSS-colitis in mice and suggest that they probably neither affect the course of IBD in humans. However, the translational value of these findings needs to be verified with other experimental colitis models and human studies.

  20. EGF receptor ligands: recent advances [version 1; referees: 3 approved

    Directory of Open Access Journals (Sweden)

    Bhuminder Singh

    2016-09-01

    Full Text Available Seven ligands bind to and activate the mammalian epidermal growth factor (EGF receptor (EGFR/ERBB1/HER1: EGF, transforming growth factor-alpha (TGFA, heparin-binding EGF-like growth factor (HBEGF, betacellulin (BTC, amphiregulin (AREG, epiregulin (EREG, and epigen (EPGN. Of these, EGF, TGFA, HBEGF, and BTC are thought to be high-affinity ligands, whereas AREG, EREG, and EPGN constitute low-affinity ligands. This focused review is meant to highlight recent studies related to actions of the individual EGFR ligands, the interesting biology that has been uncovered, and relevant advances related to ligand interactions with the EGFR.

  1. EXPRESSION OF Fas LIGAND IN HUMAN COLON CANCER CELL LINES

    Institute of Scientific and Technical Information of China (English)

    张建军; 丁尔迅; 王强; 陈学云; 付志仁

    2001-01-01

    To investigate the expression of Fas ligand in human colon carcinoma cell lines. Methods: A total of six human colon cancer cell lines were examined for the expression of Fas ligand mRNA and cell surface protein by using RT-PCR and flow cytometry respectively. Results: The results showed that Fas ligand mRNA was expressed in all of the six cancer cell lines and Fas ligand cell surface protein was expressed in part of them. Conclusion: These data suggest that Fas ligand was expressed, at least in part, in human colon cancer cell lines and might facilitate to escape from immune surveillance of the host.

  2. Synthesis and characterization of 6,6’-bis(2-hydroxyphenyl)-2,2’-bipyridine ligand and its interaction with ct-DNA

    Energy Technology Data Exchange (ETDEWEB)

    Selamat, Norhidayah; Heng, Lee Yook; Hassan, Nurul Izzaty; Karim, Nurul Huda Abd [School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43650 Bangi, Selangor (Malaysia)

    2015-09-25

    The tetradentate ligand with four donor atoms OONN was synthesized. Bis(phenoxy)bipyridine ligand was prepared by Suzuki coupling reaction between 6,6’-dibromo-2,2’-bipyridyl and 2-hydroxyphenylboronic acid with presence of palladium (II) acetate. Bis(phenoxy)bipyridine ligand was also synthesized by demethylating of 6,6’-bis(2-methoxyphenyl)-2,2’-bipyridyl ligand through solvent free reaction using pyridine hydrocloride. The formation of both phenoxy and methoxy ligands was confirmed by {sup 1}H, 2D cosy and {sup 13}C NMR spectroscopy, ESI-MS spectrometry, FTIR spectroscopy. The purity of the ligand was confirmed by melting point. Binding studies of small molecules with DNA are useful to understand the reaction mechanism and to provide guidance for the application and design of new and more efficient drugs targeted to DNA. In this study, the binding interaction between the synthesized ligand with calf thymus-DNA (ct-DNA) has been investigated by UV/Vis DNA titration study. From the UV/Vis DNA study, it shows that bis(phenoxy)bipyridine ligand bind with ct-DNA via outside binding with binding contant K{sub b} = 1.19 × 10{sup 3} ± 0.08 M{sup −1}.

  3. M13-templated magnetic nanoparticles for targeted in vivo imaging of prostate cancer

    Science.gov (United States)

    Ghosh, Debadyuti; Lee, Youjin; Thomas, Stephanie; Kohli, Aditya G.; Yun, Dong Soo; Belcher, Angela M.; Kelly, Kimberly A.

    2012-10-01

    Molecular imaging allows clinicians to visualize the progression of tumours and obtain relevant information for patient diagnosis and treatment. Owing to their intrinsic optical, electrical and magnetic properties, nanoparticles are promising contrast agents for imaging dynamic molecular and cellular processes such as protein-protein interactions, enzyme activity or gene expression. Until now, nanoparticles have been engineered with targeting ligands such as antibodies and peptides to improve tumour specificity and uptake. However, excessive loading of ligands can reduce the targeting capabilities of the ligand and reduce the ability of the nanoparticle to bind to a finite number of receptors on cells. Increasing the number of nanoparticles delivered to cells by each targeting molecule would lead to higher signal-to-noise ratios and would improve image contrast. Here, we show that M13 filamentous bacteriophage can be used as a scaffold to display targeting ligands and multiple nanoparticles for magnetic resonance imaging of cancer cells and tumours in mice. Monodisperse iron oxide magnetic nanoparticles assemble along the M13 coat, and its distal end is engineered to display a peptide that targets SPARC glycoprotein, which is overexpressed in various cancers. Compared with nanoparticles that are directly functionalized with targeting peptides, our approach improves contrast because each SPARC-targeting molecule delivers a large number of nanoparticles into the cells. Moreover, the targeting ligand and nanoparticles could be easily exchanged for others, making this platform attractive for in vivo high-throughput screening and molecular detection.

  4. M13-templated magnetic nanoparticles for targeted in vivo imaging of prostate cancer.

    Science.gov (United States)

    Ghosh, Debadyuti; Lee, Youjin; Thomas, Stephanie; Kohli, Aditya G; Yun, Dong Soo; Belcher, Angela M; Kelly, Kimberly A

    2012-10-01

    Molecular imaging allows clinicians to visualize the progression of tumours and obtain relevant information for patient diagnosis and treatment. Owing to their intrinsic optical, electrical and magnetic properties, nanoparticles are promising contrast agents for imaging dynamic molecular and cellular processes such as protein-protein interactions, enzyme activity or gene expression. Until now, nanoparticles have been engineered with targeting ligands such as antibodies and peptides to improve tumour specificity and uptake. However, excessive loading of ligands can reduce the targeting capabilities of the ligand and reduce the ability of the nanoparticle to bind to a finite number of receptors on cells. Increasing the number of nanoparticles delivered to cells by each targeting molecule would lead to higher signal-to-noise ratios and would improve image contrast. Here, we show that M13 filamentous bacteriophage can be used as a scaffold to display targeting ligands and multiple nanoparticles for magnetic resonance imaging of cancer cells and tumours in mice. Monodisperse iron oxide magnetic nanoparticles assemble along the M13 coat, and its distal end is engineered to display a peptide that targets SPARC glycoprotein, which is overexpressed in various cancers. Compared with nanoparticles that are directly functionalized with targeting peptides, our approach improves contrast because each SPARC-targeting molecule delivers a large number of nanoparticles into the cells. Moreover, the targeting ligand and nanoparticles could be easily exchanged for others, making this platform attractive for in vivo high-throughput screening and molecular detection.

  5. AsteriX: a Web server to automatically extract ligand coordinates from figures in PDF articles.

    Science.gov (United States)

    Lounnas, V; Vriend, G

    2012-02-27

    Coordinates describing the chemical structures of small molecules that are potential ligands for pharmaceutical targets are used at many stages of the drug design process. The coordinates of the vast majority of ligands can be obtained from either publicly accessible or commercial databases. However, interesting ligands sometimes are only available from the scientific literature, in which case their coordinates need to be reconstructed manually--a process that consists of a series of time-consuming steps. We present a Web server that helps reconstruct the three-dimensional (3D) coordinates of ligands for which a two-dimensional (2D) picture is available in a PDF file. The software, called AsteriX, analyses every picture contained in the PDF file and attempts to determine automatically whether or not it contains ligands. Areas in pictures that may contain molecular structures are processed to extract connectivity and atom type information that allow coordinates to be subsequently reconstructed. The AsteriX Web server was tested on a series of articles containing a large diversity in graphical representations. In total, 88% of 3249 ligand structures present in the test set were identified as chemical diagrams. Of these, about half were interpreted correctly as 3D structures, and a further one-third required only minor manual corrections. It is principally impossible to always correctly reconstruct 3D coordinates from pictures because there are many different protocols for drawing a 2D image of a ligand, but more importantly a wide variety of semantic annotations are possible. The AsteriX Web server therefore includes facilities that allow the users to augment partial or partially correct 3D reconstructions. All 3D reconstructions are submitted, checked, and corrected by the users domain at the server and are freely available for everybody. The coordinates of the reconstructed ligands are made available in a series of formats commonly used in drug design research. The

  6. Molecular Dynamics Simulations Reveal the Mechanisms of Allosteric Activation of Hsp90 by Designed Ligands.

    Science.gov (United States)

    Vettoretti, Gerolamo; Moroni, Elisabetta; Sattin, Sara; Tao, Jiahui; Agard, David A; Bernardi, Anna; Colombo, Giorgio

    2016-04-01

    Controlling biochemical pathways through chemically designed modulators may provide novel opportunities to develop therapeutic drugs and chemical tools. The underlying challenge is to design new molecular entities able to act as allosteric chemical switches that selectively turn on/off functions by modulating the conformational dynamics of their target protein. We examine the origins of the stimulation of ATPase and closure kinetics in the molecular chaperone Hsp90 by allosteric modulators through atomistic molecular dynamics (MD) simulations and analysis of protein-ligand interactions. In particular, we focus on the cross-talk between allosteric ligands and protein conformations and its effect on the dynamic properties of the chaperone's active state. We examine the impact of different allosteric modulators on the stability, structural and internal dynamics properties of Hsp90 closed state. A critical aspect of this study is the development of a quantitative model that correlates Hsp90 activation to the presence of a certain compound, making use of information on the dynamic adaptation of protein conformations to the presence of the ligand, which allows to capture conformational states relevant in the activation process. We discuss the implications of considering the conformational dialogue between allosteric ligands and protein conformations for the design of new functional modulators.

  7. Evaluation of magnetic particles modified with a hydrophobic charge-induction ligand for antibody capture.

    Science.gov (United States)

    Gu, Jia-Li; Tong, Hong-Fei; Lin, Dong-Qiang

    2016-08-19

    Magnetic particles modified with 5-amino-benzimidazole (ABI), a ligand for hydrophobic charge-induction chromatography, were prepared and used for antibody capture. In this study, with IgG as the model target, and bovine serum albumin (BSA) as the model impurity, the separation mechanism and process of IgG was investigated. The adsorption isotherms of IgG and BSA were measured, and the effects of pH were investigated in the range of pH 4.0-8.0. The maximum adsorption capacity of IgG on the particles was 180mg/ml at pH 7.0, while low adsorption capacity of BSA (64mg/ml) was found at pH 7.0, resulting in good selectivity. The protein-ligand interactions were elucidated by adding NaCl and glycerol. The results indicated the hydrophobic interactions were the main forces for IgG-ligand association. Moreover, the batch uptake and desorption experiments demonstrated the fast adsorption and desorption processes for IgG separation. The purity of IgG separated from mimetic serum could reach 98.6%, and the purity of monoclonal antibody (mAb) from a cell culture supernatant was 97.1%. Magnetic particles with hydrophobic charge-induction ligands showed a robust performance and could purify antibody directly from the complicated feedstock without clarification, which would improve the efficiency of antibody purification.

  8. Extreme sequence divergence but conserved ligand-binding specificity in Streptococcus pyogenes M protein.

    Directory of Open Access Journals (Sweden)

    Jenny Persson

    2006-05-01

    Full Text Available Many pathogenic microorganisms evade host immunity through extensive sequence variability in a protein region targeted by protective antibodies. In spite of the sequence variability, a variable region commonly retains an important ligand-binding function, reflected in the presence of a highly conserved sequence motif. Here, we analyze the limits of sequence divergence in a ligand-binding region by characterizing the hypervariable region (HVR of Streptococcus pyogenes M protein. Our studies were focused on HVRs that bind the human complement regulator C4b-binding protein (C4BP, a ligand that confers phagocytosis resistance. A previous comparison of C4BP-binding HVRs identified residue identities that could be part of a binding motif, but the extended analysis reported here shows that no residue identities remain when additional C4BP-binding HVRs are included. Characterization of the HVR in the M22 protein indicated that two relatively conserved Leu residues are essential for C4BP binding, but these residues are probably core residues in a coiled-coil, implying that they do not directly contribute to binding. In contrast, substitution of either of two relatively conserved Glu residues, predicted to be solvent-exposed, had no effect on C4BP binding, although each of these changes had a major effect on the antigenic properties of the HVR. Together, these findings show that HVRs of M proteins have an extraordinary capacity for sequence divergence and antigenic variability while retaining a specific ligand-binding function.

  9. Identifying ligands at orphan GPCRs: current status using structure-based approaches.

    Science.gov (United States)

    Ngo, Tony; Kufareva, Irina; Coleman, James Lj; Graham, Robert M; Abagyan, Ruben; Smith, Nicola J

    2016-10-01

    GPCRs are the most successful pharmaceutical targets in history. Nevertheless, the pharmacology of many GPCRs remains inaccessible as their endogenous or exogenous modulators have not been discovered. Tools that explore the physiological functions and pharmacological potential of these 'orphan' GPCRs, whether they are endogenous and/or surrogate ligands, are therefore of paramount importance. Rates of receptor deorphanization determined by traditional reverse pharmacology methods have slowed, indicating a need for the development of more sophisticated and efficient ligand screening approaches. Here, we discuss the use of structure-based ligand discovery approaches to identify small molecule modulators for exploring the function of orphan GPCRs. These studies have been buoyed by the growing number of GPCR crystal structures solved in the past decade, providing a broad range of template structures for homology modelling of orphans. This review discusses the methods used to establish the appropriate signalling assays to test orphan receptor activity and provides current examples of structure-based methods used to identify ligands of orphan GPCRs. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc.

  10. Fragment-based solid-phase assembly of oligonucleotide conjugates with peptide and polyethylene glycol ligands.

    Science.gov (United States)

    Dirin, Mehrdad; Urban, Ernst; Noe, Christian R; Winkler, Johannes

    2016-10-04

    Ligand conjugation to oligonucleotides is an attractive strategy for enhancing the therapeutic potential of antisense and siRNA agents by inferring properties such as improved cellular uptake or better pharmacokinetic properties. Disulfide linkages enable dissociation of ligands and oligonucleotides in reducing environments found in endosomal compartments after cellular uptake. Solution-phase fragment couplin