BcL-xL Conformational Changes upon Fragment Binding Revealed by NMR

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Aguirre, Clémentine; ten Brink, Tim; Walker, Olivier; Guillière, Florence; Davesne, Dany; Krimm, Isabelle

2013-01-01

Protein-protein interactions represent difficult but increasingly important targets for the design of therapeutic compounds able to interfere with biological processes. Recently, fragment-based strategies have been proposed as attractive approaches for the elaboration of protein-protein surface inhibitors from fragment-like molecules. One major challenge in targeting protein-protein interactions is related to the structural adaptation of the protein surface upon molecular recognition. Methods capable of identifying subtle conformational changes of proteins upon fragment binding are therefore required at the early steps of the drug design process. In this report we present a fast NMR method able to probe subtle conformational changes upon fragment binding. The approach relies on the comparison of experimental fragment-induced Chemical Shift Perturbation (CSP) of amine protons to CSP simulated for a set of docked fragment poses, considering the ring-current effect from fragment binding. We illustrate the method by the retrospective analysis of the complex between the anti-apoptotic Bcl-xL protein and the fragment 4′-fluoro-[1,1′-biphenyl]-4-carboxylic acid that was previously shown to bind one of the Bcl-xL hot spots. The CSP-based approach shows that the protein undergoes a subtle conformational rearrangement upon interaction, for residues located in helices 2, 3 and the very beginning of 5. Our observations are corroborated by residual dipolar coupling measurements performed on the free and fragment-bound forms of the Bcl-xL protein. These NMR-based results are in total agreement with previous molecular dynamic calculations that evidenced a high flexibility of Bcl-xL around the binding site. Here we show that CSP of protein amine protons are useful and reliable structural probes. Therefore, we propose to use CSP simulation to assess protein conformational changes upon ligand binding in the fragment-based drug design approach. PMID:23717610

In silico fragment-based drug discovery: setup and validation of a fragment-to-lead computational protocol using S4MPLE.

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Hoffer, Laurent; Renaud, Jean-Paul; Horvath, Dragos

2013-04-22

This paper describes the use and validation of S4MPLE in Fragment-Based Drug Design (FBDD)--a strategy to build drug-like ligands starting from small compounds called fragments. S4MPLE is a conformational sampling tool based on a hybrid genetic algorithm that is able to simulate one (conformer enumeration) or more molecules (docking). The goal of the current paper is to show that due to the judicious design of genetic operators, S4MPLE may be used without any specific adaptation as an in silico FBDD tool. Such fragment-to-lead evolution involves either growing of one or linking of several fragment-like binder(s). The native ability to specifically "dock" a substructure that is covalently anchored to its target (here, some prepositioned fragment formally part of the binding site) enables it to act like dedicated de novo builders and differentiates it from most classical docking tools, which may only cope with non-covalent interactions. Besides, S4MPLE may address growing/linking scenarios involving protein site flexibility, and it might also suggest "growth" moves by bridging the ligand to the site via water-mediated interactions if H2O molecules are simply appended to the input files. Therefore, the only development overhead required to build a virtual fragment→ligand growing/linking strategy based on S4MPLE were two chemoinformatics programs meant to provide a minimalistic management of the linker library. The first creates a duplicate-free library by fragmenting a compound database, whereas the second builds new compounds, attaching chemically compatible linkers to the starting fragments. S4MPLE is subsequently used to probe the optimal placement of the linkers within the binding site, with initial restraints on atoms from initial fragments, followed by an optimization of all kept poses after restraint removal. Ranking is mainly based on two criteria: force-field potential energy and RMSD shifts of the original fragment moieties. This strategy was applied to

Computational fragment-based screening using RosettaLigand: the SAMPL3 challenge

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Kumar, Ashutosh; Zhang, Kam Y. J.

2012-05-01

SAMPL3 fragment based virtual screening challenge provides a valuable opportunity for researchers to test their programs, methods and screening protocols in a blind testing environment. We participated in SAMPL3 challenge and evaluated our virtual fragment screening protocol, which involves RosettaLigand as the core component by screening a 500 fragments Maybridge library against bovine pancreatic trypsin. Our study reaffirmed that the real test for any virtual screening approach would be in a blind testing environment. The analyses presented in this paper also showed that virtual screening performance can be improved, if a set of known active compounds is available and parameters and methods that yield better enrichment are selected. Our study also highlighted that to achieve accurate orientation and conformation of ligands within a binding site, selecting an appropriate method to calculate partial charges is important. Another finding is that using multiple receptor ensembles in docking does not always yield better enrichment than individual receptors. On the basis of our results and retrospective analyses from SAMPL3 fragment screening challenge we anticipate that chances of success in a fragment screening process could be increased significantly with careful selection of receptor structures, protein flexibility, sufficient conformational sampling within binding pocket and accurate assignment of ligand and protein partial charges.

Identification of B. anthracis N(5)-carboxyaminoimidazole ribonucleotide mutase (PurE) active site binding compounds via fragment library screening.

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Lei, Hao; Jones, Christopher; Zhu, Tian; Patel, Kavankumar; Wolf, Nina M; Fung, Leslie W-M; Lee, Hyun; Johnson, Michael E

2016-02-15

The de novo purine biosynthesis pathway is an attractive target for antibacterial drug design, and PurE from this pathway has been identified to be crucial for Bacillus anthracis survival in serum. In this study we adopted a fragment-based hit discovery approach, using three screening methods-saturation transfer difference nucleus magnetic resonance (STD-NMR), water-ligand observed via gradient spectroscopy (WaterLOGSY) NMR, and surface plasmon resonance (SPR), against B. anthracis PurE (BaPurE) to identify active site binding fragments by initially testing 352 compounds in a Zenobia fragment library. Competition STD NMR with the BaPurE product effectively eliminated non-active site binding hits from the primary hits, selecting active site binders only. Binding affinities (dissociation constant, KD) of these compounds varied between 234 and 301μM. Based on test results from the Zenobia compounds, we subsequently developed and applied a streamlined fragment screening strategy to screen a much larger library consisting of 3000 computationally pre-selected fragments. Thirteen final fragment hits were confirmed to exhibit binding affinities varying from 14μM to 700μM, which were categorized into five different basic scaffolds. All thirteen fragment hits have ligand efficiencies higher than 0.30. We demonstrated that at least two fragments from two different scaffolds exhibit inhibitory activity against the BaPurE enzyme. Published by Elsevier Ltd.

Fragment growing induces conformational changes in acetylcholine-binding protein: A structural and thermodynamic analysis

NARCIS (Netherlands)

Edink, E.S.; Rucktooa, P.; Retra, K.; Akdemir, A.; Nahar, T.T.; Zuiderveld, O.P.; van Elk, R.; Janssen, E.; van Nierop, P.; van Muijlwijk-Koezen, J.E.; Smit, A.B.; Sixma, T.K.; Leurs, R.; de Esch, I.J.P.

2011-01-01

Optimization of fragment hits toward high-affinity lead compounds is a crucial aspect of fragment-based drug discovery (FBDD). In the current study, we have successfully optimized a fragment by growing into a ligand-inducible subpocket of the binding site of acetylcholine-binding protein (AChBP).

Computational medicinal chemistry in fragment-based drug discovery: what, how and when.

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Rabal, Obdulia; Urbano-Cuadrado, Manuel; Oyarzabal, Julen

2011-01-01

The use of fragment-based drug discovery (FBDD) has increased in the last decade due to the encouraging results obtained to date. In this scenario, computational approaches, together with experimental information, play an important role to guide and speed up the process. By default, FBDD is generally considered as a constructive approach. However, such additive behavior is not always present, therefore, simple fragment maturation will not always deliver the expected results. In this review, computational approaches utilized in FBDD are reported together with real case studies, where applicability domains are exemplified, in order to analyze them, and then, maximize their performance and reliability. Thus, a proper use of these computational tools can minimize misleading conclusions, keeping the credit on FBDD strategy, as well as achieve higher impact in the drug-discovery process. FBDD goes one step beyond a simple constructive approach. A broad set of computational tools: docking, R group quantitative structure-activity relationship, fragmentation tools, fragments management tools, patents analysis and fragment-hopping, for example, can be utilized in FBDD, providing a clear positive impact if they are utilized in the proper scenario - what, how and when. An initial assessment of additive/non-additive behavior is a critical point to define the most convenient approach for fragments elaboration.

Plasminogen fragments K 1-3 and K 5 bind to different sites in fibrin fragment DD.

Science.gov (United States)

Grinenko, T V; Kapustianenko, L G; Yatsenko, T A; Yusova, O I; Rybachuk, V N

2016-01-01

Specific plasminogen-binding sites of fibrin molecule are located in Аα148-160 regions of C-terminal domains. Plasminogen interaction with these sites initiates the activation process of proenzyme and subsequent fibrin lysis. In this study we investigated the binding of plasminogen fragments K 1-3 and K 5 with fibrin fragment DD and their effect on Glu-plasminogen interaction with DD. It was shown that the level of Glu-plasminogen binding to fibrin fragment DD is decreased by 50-60% in the presence of K 1-3 and K 5. Fragments K 1-3 and K 5 have high affinity to fibrin fragment DD (Kd is 0.02 for K 1-3 and 0.054 μМ for K 5). K 5 interaction is independent and K 1-3 is partly dependent on C-terminal lysine residues. K 1-3 interacts with complex of fragment DD-immobilized K 5 as well as K 5 with complex of fragment DD-immobilized K 1-3. The plasminogen fragments do not displace each other from binding sites located in fibrin fragment DD, but can compete for the interaction. The results indicate that fibrin fragment DD contains different binding sites for plasminogen kringle fragments K 1-3 and K 5, which can be located close to each other. The role of amino acid residues of fibrin molecule Аα148-160 region in interaction with fragments K 1-3 and K 5 is discussed.

Detection of secondary binding sites in proteins using fragment screening.

Science.gov (United States)

Ludlow, R Frederick; Verdonk, Marcel L; Saini, Harpreet K; Tickle, Ian J; Jhoti, Harren

2015-12-29

Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets.

Discovery of novel dengue virus NS5 methyltransferase non-nucleoside inhibitors by fragment-based drug design.

Science.gov (United States)

Benmansour, Fatiha; Trist, Iuni; Coutard, Bruno; Decroly, Etienne; Querat, Gilles; Brancale, Andrea; Barral, Karine

2017-01-05

With the aim to help drug discovery against dengue virus (DENV), a fragment-based drug design approach was applied to identify ligands targeting a main component of DENV replication complex: the NS5 AdoMet-dependent mRNA methyltransferase (MTase) domain, playing an essential role in the RNA capping process. Herein, we describe the identification of new inhibitors developed using fragment-based, structure-guided linking and optimization techniques. Thermal-shift assay followed by a fragment-based X-ray crystallographic screening lead to the identification of three fragment hits binding DENV MTase. We considered linking two of them, which bind to proximal sites of the AdoMet binding pocket, in order to improve their potency. X-ray crystallographic structures and computational docking were used to guide the fragment linking, ultimately leading to novel series of non-nucleoside inhibitors of flavivirus MTase, respectively N-phenyl-[(phenylcarbamoyl)amino]benzene-1-sulfonamide and phenyl [(phenylcarbamoyl)amino]benzene-1-sulfonate derivatives, that show a 10-100-fold stronger inhibition of 2'-O-MTase activity compared to the initial fragments. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

A role for fragment-based drug design in developing novel lead compounds for central nervous system targets

Directory of Open Access Journals (Sweden)

Michael J. Wasko

2015-09-01

Full Text Available Hundreds of millions of U.S. dollars are invested in the research and development of a single drug. Lead compound development is an area ripe for new design strategies. Therapeutic lead candidates have been traditionally found using high-throughput in vitro pharmacologic screening, a costly method for assaying thousands of compounds. This approach has recently been augmented by virtual screening, which employs computer models of the target protein to narrow the search for possible leads. A variant of virtual screening is fragment-based drug design, an emerging in silico lead discovery method that introduces low molecular weight fragments, rather than intact compounds, into the binding pocket of the receptor model. These fragments serve as starting points for growing the lead candidate. Current efforts in virtual fragment-based drug design within central nervous system (CNS targets are reviewed, as is a recent rule-based optimization strategy in which new molecules are generated within a 3D receptor binding pocket using the fragment as a scaffold. This process places special emphasis on creating synthesizable molecules but also exposes computational questions worth addressing. Fragment-based methods provide a viable, relatively low-cost alternative for therapeutic lead discovery and optimization that can be applied to CNS targets to augment current design strategies.

Contributions of computational chemistry and biophysical techniques to fragment-based drug discovery.

Science.gov (United States)

Gozalbes, Rafael; Carbajo, Rodrigo J; Pineda-Lucena, Antonio

2010-01-01

In the last decade, fragment-based drug discovery (FBDD) has evolved from a novel approach in the search of new hits to a valuable alternative to the high-throughput screening (HTS) campaigns of many pharmaceutical companies. The increasing relevance of FBDD in the drug discovery universe has been concomitant with an implementation of the biophysical techniques used for the detection of weak inhibitors, e.g. NMR, X-ray crystallography or surface plasmon resonance (SPR). At the same time, computational approaches have also been progressively incorporated into the FBDD process and nowadays several computational tools are available. These stretch from the filtering of huge chemical databases in order to build fragment-focused libraries comprising compounds with adequate physicochemical properties, to more evolved models based on different in silico methods such as docking, pharmacophore modelling, QSAR and virtual screening. In this paper we will review the parallel evolution and complementarities of biophysical techniques and computational methods, providing some representative examples of drug discovery success stories by using FBDD.

Fragment-based drug discovery using rational design.

Science.gov (United States)

Jhoti, H

2007-01-01

Fragment-based drug discovery (FBDD) is established as an alternative approach to high-throughput screening for generating novel small molecule drug candidates. In FBDD, relatively small libraries of low molecular weight compounds (or fragments) are screened using sensitive biophysical techniques to detect their binding to the target protein. A lower absolute affinity of binding is expected from fragments, compared to much higher molecular weight hits detected by high-throughput screening, due to their reduced size and complexity. Through the use of iterative cycles of medicinal chemistry, ideally guided by three-dimensional structural data, it is often then relatively straightforward to optimize these weak binding fragment hits into potent and selective lead compounds. As with most other lead discovery methods there are two key components of FBDD; the detection technology and the compound library. In this review I outline the two main approaches used for detecting the binding of low affinity fragments and also some of the key principles that are used to generate a fragment library. In addition, I describe an example of how FBDD has led to the generation of a drug candidate that is now being tested in clinical trials for the treatment of cancer.

Fast and Efficient Fragment-Based Lead Generation by Fully Automated Processing and Analysis of Ligand-Observed NMR Binding Data.

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Peng, Chen; Frommlet, Alexandra; Perez, Manuel; Cobas, Carlos; Blechschmidt, Anke; Dominguez, Santiago; Lingel, Andreas

2016-04-14

NMR binding assays are routinely applied in hit finding and validation during early stages of drug discovery, particularly for fragment-based lead generation. To this end, compound libraries are screened by ligand-observed NMR experiments such as STD, T1ρ, and CPMG to identify molecules interacting with a target. The analysis of a high number of complex spectra is performed largely manually and therefore represents a limiting step in hit generation campaigns. Here we report a novel integrated computational procedure that processes and analyzes ligand-observed proton and fluorine NMR binding data in a fully automated fashion. A performance evaluation comparing automated and manual analysis results on (19)F- and (1)H-detected data sets shows that the program delivers robust, high-confidence hit lists in a fraction of the time needed for manual analysis and greatly facilitates visual inspection of the associated NMR spectra. These features enable considerably higher throughput, the assessment of larger libraries, and shorter turn-around times.

Towards novel therapeutics for HIV through fragment-based screening and drug design.

Science.gov (United States)

Tiefendbrunn, Theresa; Stout, C David

2014-01-01

Fragment-based drug discovery has been applied with varying levels of success to a number of proteins involved in the HIV (Human Immunodeficiency Virus) life cycle. Fragment-based approaches have led to the discovery of novel binding sites within protease, reverse transcriptase, integrase, and gp41. Novel compounds that bind to known pockets within CCR5 have also been identified via fragment screening, and a fragment-based approach to target the TAR-Tat interaction was explored. In the context of HIV-1 reverse transcriptase (RT), fragment-based approaches have yielded fragment hits with mid-μM activity in an in vitro activity assay, as well as fragment hits that are active against drug-resistant variants of RT. Fragment-based drug discovery is a powerful method to elucidate novel binding sites within proteins, and the method has had significant success in the context of HIV proteins.

A Role for Fragment-Based Drug Design in Developing Novel Lead Compounds for Central Nervous System Targets.

Science.gov (United States)

Wasko, Michael J; Pellegrene, Kendy A; Madura, Jeffry D; Surratt, Christopher K

2015-01-01

Hundreds of millions of U.S. dollars are invested in the research and development of a single drug. Lead compound development is an area ripe for new design strategies. Therapeutic lead candidates have been traditionally found using high-throughput in vitro pharmacological screening, a costly method for assaying thousands of compounds. This approach has recently been augmented by virtual screening (VS), which employs computer models of the target protein to narrow the search for possible leads. A variant of VS is fragment-based drug design (FBDD), an emerging in silico lead discovery method that introduces low-molecular weight fragments, rather than intact compounds, into the binding pocket of the receptor model. These fragments serve as starting points for "growing" the lead candidate. Current efforts in virtual FBDD within central nervous system (CNS) targets are reviewed, as is a recent rule-based optimization strategy in which new molecules are generated within a 3D receptor-binding pocket using the fragment as a scaffold. This process not only places special emphasis on creating synthesizable molecules but also exposes computational questions worth addressing. Fragment-based methods provide a viable, relatively low-cost alternative for therapeutic lead discovery and optimization that can be applied to CNS targets to augment current design strategies.

Fragment-based drug design.

Science.gov (United States)

Feyfant, Eric; Cross, Jason B; Paris, Kevin; Tsao, Désirée H H

2011-01-01

Fragment-based drug design (FBDD), which is comprised of both fragment screening and the use of fragment hits to design leads, began more than 15 years ago and has been steadily gaining in popularity and utility. Its origin lies on the fact that the coverage of chemical space and the binding efficiency of hits are directly related to the size of the compounds screened. Nevertheless, FBDD still faces challenges, among them developing fragment screening libraries that ensure optimal coverage of chemical space, physical properties and chemical tractability. Fragment screening also requires sensitive assays, often biophysical in nature, to detect weak binders. In this chapter we will introduce the technologies used to address these challenges and outline the experimental advantages that make FBDD one of the most popular new hit-to-lead process.

Fragment approaches in structure-based drug discovery

International Nuclear Information System (INIS)

Hubbard, Roderick E.

2008-01-01

Fragment-based methods are successfully generating novel and selective drug-like inhibitors of protein targets, with a number of groups reporting compounds entering clinical trials. This paper summarizes the key features of the approach as one of the tools in structure-guided drug discovery. There has been considerable interest recently in what is known as 'fragment-based lead discovery'. The novel feature of the approach is to begin with small low-affinity compounds. The main advantage is that a larger potential chemical diversity can be sampled with fewer compounds, which is particularly important for new target classes. The approach relies on careful design of the fragment library, a method that can detect binding of the fragment to the protein target, determination of the structure of the fragment bound to the target, and the conventional use of structural information to guide compound optimization. In this article the methods are reviewed, and experiences in fragment-based discovery of lead series of compounds against kinases such as PDK1 and ATPases such as Hsp90 are discussed. The examples illustrate some of the key benefits and issues of the approach and also provide anecdotal examples of the patterns seen in selectivity and the binding mode of fragments across different protein targets

In silico fragment-based drug design.

Science.gov (United States)

Konteatis, Zenon D

2010-11-01

In silico fragment-based drug design (FBDD) is a relatively new approach inspired by the success of the biophysical fragment-based drug discovery field. Here, we review the progress made by this approach in the last decade and showcase how it complements and expands the capabilities of biophysical FBDD and structure-based drug design to generate diverse, efficient drug candidates. Advancements in several areas of research that have enabled the development of in silico FBDD and some applications in drug discovery projects are reviewed. The reader is introduced to various computational methods that are used for in silico FBDD, the fragment library composition for this technique, special applications used to identify binding sites on the surface of proteins and how to assess the druggability of these sites. In addition, the reader will gain insight into the proper application of this approach from examples of successful programs. In silico FBDD captures a much larger chemical space than high-throughput screening and biophysical FBDD increasing the probability of developing more diverse, patentable and efficient molecules that can become oral drugs. The application of in silico FBDD holds great promise for historically challenging targets such as protein-protein interactions. Future advances in force fields, scoring functions and automated methods for determining synthetic accessibility will all aid in delivering more successes with in silico FBDD.

Experiences in fragment-based drug discovery.

Science.gov (United States)

Murray, Christopher W; Verdonk, Marcel L; Rees, David C

2012-05-01

Fragment-based drug discovery (FBDD) has become established in both industry and academia as an alternative approach to high-throughput screening for the generation of chemical leads for drug targets. In FBDD, specialised detection methods are used to identify small chemical compounds (fragments) that bind to the drug target, and structural biology is usually employed to establish their binding mode and to facilitate their optimisation. In this article, we present three recent and successful case histories in FBDD. We then re-examine the key concepts and challenges of FBDD with particular emphasis on recent literature and our own experience from a substantial number of FBDD applications. Our opinion is that careful application of FBDD is living up to its promise of delivering high quality leads with good physical properties and that in future many drug molecules will be derived from fragment-based approaches. Copyright © 2012 Elsevier Ltd. All rights reserved.

Advances in fragment-based drug discovery platforms.

Science.gov (United States)

Orita, Masaya; Warizaya, Masaichi; Amano, Yasushi; Ohno, Kazuki; Niimi, Tatsuya

2009-11-01

Fragment-based drug discovery (FBDD) has been established as a powerful alternative and complement to traditional high-throughput screening techniques for identifying drug leads. At present, this technique is widely used among academic groups as well as small biotech and large pharmaceutical companies. In recent years, > 10 new compounds developed with FBDD have entered clinical development, and more and more attention in the drug discovery field is being focused on this technique. Under the FBDD approach, a fragment library of relatively small compounds (molecular mass = 100 - 300 Da) is screened by various methods and the identified fragment hits which normally weakly bind to the target are used as starting points to generate more potent drug leads. Because FBDD is still a relatively new drug discovery technology, further developments and optimizations in screening platforms and fragment exploitation can be expected. This review summarizes recent advances in FBDD platforms and discusses the factors important for the successful application of this technique. Under the FBDD approach, both identifying the starting fragment hit to be developed and generating the drug lead from that starting fragment hit are important. Integration of various techniques, such as computational technology, X-ray crystallography, NMR, surface plasmon resonance, isothermal titration calorimetry, mass spectrometry and high-concentration screening, must be applied in a situation-appropriate manner.

NMR screening in fragment-based drug design: a practical guide.

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Kim, Hai-Young; Wyss, Daniel F

2015-01-01

Fragment-based drug design (FBDD) comprises both fragment-based screening (FBS) to find hits and elaboration of these hits to lead compounds. Typical fragment hits have lower molecular weight (FBDD since it identifies and localizes the binding site of weakly interacting hits on the target protein. Here we describe ligand-based NMR methods for hit identification from fragment libraries and for functional cross-validation of primary hits.

Thermodynamic signatures of fragment binding: Validation of direct versus displacement ITC titrations.

Science.gov (United States)

Rühmann, Eggert; Betz, Michael; Fricke, Marie; Heine, Andreas; Schäfer, Martina; Klebe, Gerhard

2015-04-01

Detailed characterization of the thermodynamic signature of weak binding fragments to proteins is essential to support the decision making process which fragments to take further for the hit-to-lead optimization. Isothermal titration calorimetry (ITC) is the method of choice to record thermodynamic data, however, weak binding ligands such as fragments require the development of meaningful and reliable measuring protocols as usually sigmoidal titration curves are hardly possible to record due to limited solubility. Fragments can be titrated either directly under low c-value conditions (no sigmoidal curve) or indirectly by use of a strong binding ligand displacing the pre-incubated weak fragment from the protein. The determination of Gibbs free energy is reliable and rather independent of the applied titration protocol. Even though the displacement method achieves higher accuracy, the obtained enthalpy-entropy profile depends on the properties of the used displacement ligand. The relative enthalpy differences across different displacement experiments reveal a constant signature and can serve as a thermodynamic fingerprint for fragments. Low c-value titrations are only reliable if the final concentration of the fragment in the sample cell exceeds 2-10 fold its K(D) value. Limited solubility often prevents this strategy. The present study suggests an applicable protocol to characterize the thermodynamic signature of protein-fragment binding. It shows however, that such measurements are limited by protein and fragment solubility. Deviating profiles obtained by use of different displacement ligands indicate that changes in the solvation pattern and protein dynamics most likely take influence on the resulting overall binding signature. Copyright © 2014 Elsevier B.V. All rights reserved.

Site Identification by Ligand Competitive Saturation (SILCS) Simulations for Fragment-Based Drug Design

OpenAIRE

Faller, Christina E.; Raman, E. Prabhu; MacKerell, Alexander D.; Guvench, Olgun

2015-01-01

Fragment-based drug design (FBDD) involves screening low molecular weight molecules (“fragments”) that correspond to functional groups found in larger drug-like molecules to determine their binding to target proteins or nucleic acids. Based on the principle of thermodynamic additivity, two fragments that bind non-overlapping nearby sites on the target can be combined to yield a new molecule whose binding free energy is the sum of those of the fragments. Experimental FBDD approaches, like NMR ...

Novel approach of fragment-based lead discovery applied to renin inhibitors.

Science.gov (United States)

Tawada, Michiko; Suzuki, Shinkichi; Imaeda, Yasuhiro; Oki, Hideyuki; Snell, Gyorgy; Behnke, Craig A; Kondo, Mitsuyo; Tarui, Naoki; Tanaka, Toshimasa; Kuroita, Takanobu; Tomimoto, Masaki

2016-11-15

A novel approach was conducted for fragment-based lead discovery and applied to renin inhibitors. The biochemical screening of a fragment library against renin provided the hit fragment which showed a characteristic interaction pattern with the target protein. The hit fragment bound only to the S1, S3, and S3 SP (S3 subpocket) sites without any interactions with the catalytic aspartate residues (Asp32 and Asp215 (pepsin numbering)). Prior to making chemical modifications to the hit fragment, we first identified its essential binding sites by utilizing the hit fragment's substructures. Second, we created a new and smaller scaffold, which better occupied the identified essential S3 and S3 SP sites, by utilizing library synthesis with high-throughput chemistry. We then revisited the S1 site and efficiently explored a good building block attaching to the scaffold with library synthesis. In the library syntheses, the binding modes of each pivotal compound were determined and confirmed by X-ray crystallography and the library was strategically designed by structure-based computational approach not only to obtain a more active compound but also to obtain informative Structure Activity Relationship (SAR). As a result, we obtained a lead compound offering synthetic accessibility as well as the improved in vitro ADMET profiles. The fragments and compounds possessing a characteristic interaction pattern provided new structural insights into renin's active site and the potential to create a new generation of renin inhibitors. In addition, we demonstrated our FBDD strategy integrating highly sensitive biochemical assay, X-ray crystallography, and high-throughput synthesis and in silico library design aimed at fragment morphing at the initial stage was effective to elucidate a pocket profile and a promising lead compound. Copyright © 2016 Elsevier Ltd. All rights reserved.

Predicting Flavin and Nicotinamide Adenine Dinucleotide-Binding Sites in Proteins Using the Fragment Transformation Method

Directory of Open Access Journals (Sweden)

Chih-Hao Lu

2015-01-01

Full Text Available We developed a computational method to identify NAD- and FAD-binding sites in proteins. First, we extracted from the Protein Data Bank structures of proteins that bind to at least one of these ligands. NAD-/FAD-binding residue templates were then constructed by identifying binding residues through the ligand-binding database BioLiP. The fragment transformation method was used to identify structures within query proteins that resembled the ligand-binding templates. By comparing residue types and their relative spatial positions, potential binding sites were identified and a ligand-binding potential for each residue was calculated. Setting the false positive rate at 5%, our method predicted NAD- and FAD-binding sites at true positive rates of 67.1% and 68.4%, respectively. Our method provides excellent results for identifying FAD- and NAD-binding sites in proteins, and the most important is that the requirement of conservation of residue types and local structures in the FAD- and NAD-binding sites can be verified.

Fragment-based drug discovery and protein–protein interactions

Directory of Open Access Journals (Sweden)

Turnbull AP

2014-09-01

Full Text Available Andrew P Turnbull,1 Susan M Boyd,2 Björn Walse31CRT Discovery Laboratories, Department of Biological Sciences, Birkbeck, University of London, London, UK; 2IOTA Pharmaceuticals Ltd, Cambridge, UK; 3SARomics Biostructures AB, Lund, SwedenAbstract: Protein–protein interactions (PPIs are involved in many biological processes, with an estimated 400,000 PPIs within the human proteome. There is significant interest in exploiting the relatively unexplored potential of these interactions in drug discovery, driven by the need to find new therapeutic targets. Compared with classical drug discovery against targets with well-defined binding sites, developing small-molecule inhibitors against PPIs where the contact surfaces are frequently more extensive and comparatively flat, with most of the binding energy localized in “hot spots”, has proven far more challenging. However, despite the difficulties associated with targeting PPIs, important progress has been made in recent years with fragment-based drug discovery playing a pivotal role in improving their tractability. Computational and empirical approaches can be used to identify hot-spot regions and assess the druggability and ligandability of new targets, whilst fragment screening campaigns can detect low-affinity fragments that either directly or indirectly perturb the PPI. Once fragment hits have been identified and confirmed using biochemical and biophysical approaches, three-dimensional structural data derived from nuclear magnetic resonance or X-ray crystallography can be used to drive medicinal chemistry efforts towards the development of more potent inhibitors. A small-scale comparison presented in this review of “standard” fragments with those targeting PPIs has revealed that the latter tend to be larger, be more lipophilic, and contain more polar (acid/base functionality, whereas three-dimensional descriptor data indicate that there is little difference in their three

Hot spot analysis for driving the development of hits into leads in fragment based drug discovery

Science.gov (United States)

Hall, David R.; Ngan, Chi Ho; Zerbe, Brandon S.; Kozakov, Dima; Vajda, Sandor

2011-01-01

Fragment based drug design (FBDD) starts with finding fragment-sized compounds that are highly ligand efficient and can serve as a core moiety for developing high affinity leads. Although the core-bound structure of a protein facilitates the construction of leads, effective design is far from straightforward. We show that protein mapping, a computational method developed to find binding hot spots and implemented as the FTMap server, provides information that complements the fragment screening results and can drive the evolution of core fragments into larger leads with a minimal loss or, in some cases, even a gain in ligand efficiency. The method places small molecular probes, the size of organic solvents, on a dense grid around the protein, and identifies the hot spots as consensus clusters formed by clusters of several probes. The hot spots are ranked based on the number of probe clusters, which predicts the binding propensity of the subsites and hence their importance for drug design. Accordingly, with a single exception the main hot spot identified by FTMap binds the core compound found by fragment screening. The most useful information is provided by the neighboring secondary hot spots, indicating the regions where the core can be extended to increase its affinity. To quantify this information, we calculate the density of probes from mapping, which describes the binding propensity at each point, and show that the change in the correlation between a ligand position and the probe density upon extending or repositioning the core moiety predicts the expected change in ligand efficiency. PMID:22145575

Fragmentation based

Directory of Open Access Journals (Sweden)

Shashank Srivastava

2014-01-01

Gaining the understanding of mobile agent architecture and the security concerns, in this paper, we proposed a security protocol which addresses security with mitigated computational cost. The protocol is a combination of self decryption, co-operation and obfuscation technique. To circumvent the risk of malicious code execution in attacking environment, we have proposed fragmentation based encryption technique. Our encryption technique suits the general mobile agent size and provides hard and thorny obfuscation increasing attacker’s challenge on the same plane providing better performance with respect to computational cost as compared to existing AES encryption.

The heat is on: thermodynamic analysis in fragment-based drug discovery

NARCIS (Netherlands)

Edink, E.S.; Jansen, C.J.W.; Leurs, R.; De Esch, I.J.

2010-01-01

Thermodynamic analysis provides access to the determinants of binding affinity, enthalpy and entropy. In fragment-based drug discovery (FBDD), thermodynamic analysis provides a powerful tool to discriminate fragments based on their potential for successful optimization. The thermodynamic data

Fragment-based drug discovery and its application to challenging drug targets.

Science.gov (United States)

Price, Amanda J; Howard, Steven; Cons, Benjamin D

2017-11-08

Fragment-based drug discovery (FBDD) is a technique for identifying low molecular weight chemical starting points for drug discovery. Since its inception 20 years ago, FBDD has grown in popularity to the point where it is now an established technique in industry and academia. The approach involves the biophysical screening of proteins against collections of low molecular weight compounds (fragments). Although fragments bind to proteins with relatively low affinity, they form efficient, high quality binding interactions with the protein architecture as they have to overcome a significant entropy barrier to bind. Of the biophysical methods available for fragment screening, X-ray protein crystallography is one of the most sensitive and least prone to false positives. It also provides detailed structural information of the protein-fragment complex at the atomic level. Fragment-based screening using X-ray crystallography is therefore an efficient method for identifying binding hotspots on proteins, which can then be exploited by chemists and biologists for the discovery of new drugs. The use of FBDD is illustrated here with a recently published case study of a drug discovery programme targeting the challenging protein-protein interaction Kelch-like ECH-associated protein 1:nuclear factor erythroid 2-related factor 2. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

The ways and means of fragment-based drug design.

Science.gov (United States)

Doak, Bradley C; Norton, Raymond S; Scanlon, Martin J

2016-11-01

Fragment-based drug design (FBDD) has emerged as a mainstream approach for the rapid and efficient identification of building blocks that can be used to develop high-affinity ligands against protein targets. One of the strengths of FBDD is the relative ease and low cost of the primary screen to identify fragments that bind. However, the fragments that emerge from primary screens often have low affinities, with K D values in the high μM to mM range, and a significant challenge for FBDD is to develop the initial fragments into more potent ligands. Successful fragment elaboration often requires co-structures of the fragments bound to their target proteins, as well as a range of biophysical and biochemical assays to track potency and efficacy. These challenges have led to the development of specific chemical strategies for the elaboration of weakly-binding fragments into more potent "hits" and lead compounds. In this article we review different approaches that have been employed to meet these challenges and describe some of the strategies that have resulted in several fragment-derived compounds entering clinical trials. Copyright © 2016 Elsevier Inc. All rights reserved.

Lessons from hot spot analysis for fragment-based drug discovery

Science.gov (United States)

Hall, David R.; Vajda, Sandor

2015-01-01

Analysis of binding energy hot spots at protein surfaces can provide crucial insights into the prospects for successful application of fragment-based drug discovery (FBDD), and whether a fragment hit can be advanced into a high affinity, druglike ligand. The key factor is the strength of the top ranking hot spot, and how well a given fragment complements it. We show that published data are sufficient to provide a sophisticated and quantitative understanding of how hot spots derive from protein three-dimensional structure, and how their strength, number and spatial arrangement govern the potential for a surface site to bind to fragment-sized and larger ligands. This improved understanding provides important guidance for the effective application of FBDD in drug discovery. PMID:26538314

Site-specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced Ebola detection.

Science.gov (United States)

Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

2015-07-01

The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides. © 2015 Wiley Periodicals, Inc.

Fragment-Based Protein-Protein Interaction Antagonists of a Viral Dimeric Protease.

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Gable, Jonathan E; Lee, Gregory M; Acker, Timothy M; Hulce, Kaitlin R; Gonzalez, Eric R; Schweigler, Patrick; Melkko, Samu; Farady, Christopher J; Craik, Charles S

2016-04-19

Fragment-based drug discovery has shown promise as an approach for challenging targets such as protein-protein interfaces. We developed and applied an activity-based fragment screen against dimeric Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) using an optimized fluorogenic substrate. Dose-response determination was performed as a confirmation screen, and NMR spectroscopy was used to map fragment inhibitor binding to KSHV Pr. Kinetic assays demonstrated that several initial hits also inhibit human cytomegalovirus protease (HCMV Pr). Binding of these hits to HCMV Pr was also confirmed by NMR spectroscopy. Despite the use of a target-agnostic fragment library, more than 80 % of confirmed hits disrupted dimerization and bound to a previously reported pocket at the dimer interface of KSHV Pr, not to the active site. One class of fragments, an aminothiazole scaffold, was further explored using commercially available analogues. These compounds demonstrated greater than 100-fold improvement of inhibition. This study illustrates the power of fragment-based screening for these challenging enzymatic targets and provides an example of the potential druggability of pockets at protein-protein interfaces. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Lessons from Hot Spot Analysis for Fragment-Based Drug Discovery.

Science.gov (United States)

Hall, David R; Kozakov, Dima; Whitty, Adrian; Vajda, Sandor

2015-11-01

Analysis of binding energy hot spots at protein surfaces can provide crucial insights into the prospects for successful application of fragment-based drug discovery (FBDD), and whether a fragment hit can be advanced into a high-affinity, drug-like ligand. The key factor is the strength of the top ranking hot spot, and how well a given fragment complements it. We show that published data are sufficient to provide a sophisticated and quantitative understanding of how hot spots derive from a protein 3D structure, and how their strength, number, and spatial arrangement govern the potential for a surface site to bind to fragment-sized and larger ligands. This improved understanding provides important guidance for the effective application of FBDD in drug discovery. Copyright © 2015 Elsevier Ltd. All rights reserved.

Assessment of Dengue virus helicase and methyltransferase as targets for fragment-based drug discovery.

Science.gov (United States)

Coutard, Bruno; Decroly, Etienne; Li, Changqing; Sharff, Andrew; Lescar, Julien; Bricogne, Gérard; Barral, Karine

2014-06-01

Seasonal and pandemic flaviviruses continue to be leading global health concerns. With the view to help drug discovery against Dengue virus (DENV), a fragment-based experimental approach was applied to identify small molecule ligands targeting two main components of the flavivirus replication complex: the NS3 helicase (Hel) and the NS5 mRNA methyltransferase (MTase) domains. A library of 500 drug-like fragments was first screened by thermal-shift assay (TSA) leading to the identification of 36 and 32 fragment hits binding Hel and MTase from DENV, respectively. In a second stage, we set up a fragment-based X-ray crystallographic screening (FBS-X) in order to provide both validated fragment hits and structural binding information. No fragment hit was confirmed for DENV Hel. In contrast, a total of seven fragments were identified as DENV MTase binders and structures of MTase-fragment hit complexes were solved at resolution at least 2.0Å or better. All fragment hits identified contain either a five- or six-membered aromatic ring or both, and three novel binding sites were located on the MTase. To further characterize the fragment hits identified by TSA and FBS-X, we performed enzymatic assays to assess their inhibition effect on the N7- and 2'-O-MTase enzymatic activities: five of these fragment hits inhibit at least one of the two activities with IC50 ranging from 180μM to 9mM. This work validates the FBS-X strategy for identifying new anti-flaviviral hits targeting MTase, while Hel might not be an amenable target for fragment-based drug discovery (FBDD). This approach proved to be a fast and efficient screening method for FBDD target validation and discovery of starting hits for the development of higher affinity molecules that bind to novel allosteric sites. Copyright © 2014 Elsevier B.V. All rights reserved.

Generating "fragment-based virtual library" using pocket similarity search of ligand-receptor complexes.

Science.gov (United States)

Khashan, Raed S

2015-01-01

As the number of available ligand-receptor complexes is increasing, researchers are becoming more dedicated to mine these complexes to aid in the drug design and development process. We present free software which is developed as a tool for performing similarity search across ligand-receptor complexes for identifying binding pockets which are similar to that of a target receptor. The search is based on 3D-geometric and chemical similarity of the atoms forming the binding pocket. For each match identified, the ligand's fragment(s) corresponding to that binding pocket are extracted, thus forming a virtual library of fragments (FragVLib) that is useful for structure-based drug design. The program provides a very useful tool to explore available databases.

Does aluminium bind to histidine? An NMR investigation of amyloid β12 and amyloid β16 fragments.

Science.gov (United States)

Narayan, Priya; Krishnarjuna, Bankala; Vishwanathan, Vinaya; Jagadeesh Kumar, Dasappa; Babu, Sudhir; Ramanathan, Krishna Venkatachala; Easwaran, Kalpathy Ramaier Katchap; Nagendra, Holenarasipur Gundurao; Raghothama, Srinivasarao

2013-07-01

Aluminium and zinc are known to be the major triggering agents for aggregation of amyloid peptides leading to plaque formation in Alzheimer's disease. While zinc binding to histidine in Aβ (amyloid β) fragments has been implicated as responsible for aggregation, not much information is available on the interaction of aluminium with histidine. In the NMR study of the N-terminal Aβ fragments, DAEFRHDSGYEV (Aβ12) and DAEFRHDSGYEVHHQK (Aβ16) presented here, the interactions of the fragments with aluminium have been investigated. Significant chemical shifts were observed for few residues near the C-terminus when aluminium chloride was titrated with Aβ12 and Aβ16 peptides. Surprisingly, it is nonhistidine residues which seem to be involved in aluminium binding. Based on NMR constrained structure obtained by molecular modelling, aluminium-binding pockets in Aβ12 were around charged residues such as Asp, Glu. The results are discussed in terms of native structure propagation, and the relevance of histidine residues in the sequences for metal-binding interactions. We expect that the study of such short amyloid peptide fragments will not only provide clues for plaque formation in aggregated conditions but also facilitate design of potential drugs for these targets. © 2013 John Wiley & Sons A/S.

A collagen-binding EGFR antibody fragment targeting tumors with a collagen-rich extracellular matrix

OpenAIRE

Hui Liang; Xiaoran Li; Bin Wang; Bing Chen; Yannan Zhao; Jie Sun; Yan Zhuang; Jiajia Shi; He Shen; Zhijun Zhang; Jianwu Dai

2016-01-01

Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of ...

[Fragment-based drug discovery: concept and aim].

Science.gov (United States)

Tanaka, Daisuke

2010-03-01

Fragment-Based Drug Discovery (FBDD) has been recognized as a newly emerging lead discovery methodology that involves biophysical fragment screening and chemistry-driven fragment-to-lead stages. Although fragments, defined as structurally simple and small compounds (typically FBDD primarily turns our attention to weakly but specifically binding fragments (hit fragments) as the starting point of medicinal chemistry. Hit fragments are then promoted to more potent lead compounds through linking or merging with another hit fragment and/or attaching functional groups. Another positive aspect of FBDD is ligand efficiency. Ligand efficiency is a useful guide in screening hit selection and hit-to-lead phases to achieve lead-likeness. Owing to these features, a number of successful applications of FBDD to "undruggable targets" (where HTS and other lead identification methods failed to identify useful lead compounds) have been reported. As a result, FBDD is now expected to complement more conventional methodologies. This review, as an introduction of the following articles, will summarize the fundamental concepts of FBDD and will discuss its advantages over other conventional drug discovery approaches.

The rise of fragment-based drug discovery.

Science.gov (United States)

Murray, Christopher W; Rees, David C

2009-06-01

The search for new drugs is plagued by high attrition rates at all stages in research and development. Chemists have an opportunity to tackle this problem because attrition can be traced back, in part, to the quality of the chemical leads. Fragment-based drug discovery (FBDD) is a new approach, increasingly used in the pharmaceutical industry, for reducing attrition and providing leads for previously intractable biological targets. FBDD identifies low-molecular-weight ligands (∼150 Da) that bind to biologically important macromolecules. The three-dimensional experimental binding mode of these fragments is determined using X-ray crystallography or NMR spectroscopy, and is used to facilitate their optimization into potent molecules with drug-like properties. Compared with high-throughput-screening, the fragment approach requires fewer compounds to be screened, and, despite the lower initial potency of the screening hits, offers more efficient and fruitful optimization campaigns. Here, we review the rise of FBDD, including its application to discovering clinical candidates against targets for which other chemistry approaches have struggled.

Multi-binding site model-based curve-fitting program for the computation of RIA data

International Nuclear Information System (INIS)

Malan, P.G.; Ekins, R.P.; Cox, M.G.; Long, E.M.R.

1977-01-01

In this paper, a comparison will be made of model-based and empirical curve-fitting procedures. The implementation of a multiple binding-site curve-fitting model which will successfully fit a wide range of assay data, and which can be run on a mini-computer is described. The latter sophisticated model also provides estimates of binding site concentrations and the values of the respective equilibrium constants present: the latter have been used for refining assay conditions using computer optimisation techniques. (orig./AJ) [de

A fragment-based approach leading to the discovery of a novel binding site and the selective CK2 inhibitor CAM4066.

Science.gov (United States)

De Fusco, Claudia; Brear, Paul; Iegre, Jessica; Georgiou, Kathy Hadje; Sore, Hannah F; Hyvönen, Marko; Spring, David R

2017-07-01

Recently we reported the discovery of a potent and selective CK2α inhibitor CAM4066. This compound inhibits CK2 activity by exploiting a pocket located outside the ATP binding site (αD pocket). Here we describe in detail the journey that led to the discovery of CAM4066 using the challenging fragment linking strategy. Specifically, we aimed to develop inhibitors by linking a high-affinity fragment anchored in the αD site to a weakly binding warhead fragment occupying the ATP site. Moreover, we describe the remarkable impact that molecular modelling had on the development of this novel chemical tool. The work described herein shows potential for the development of a novel class of CK2 inhibitors. Copyright © 2017. Published by Elsevier Ltd.

Native Mass Spectrometry in Fragment-Based Drug Discovery

Directory of Open Access Journals (Sweden)

Liliana Pedro

2016-07-01

Full Text Available The advent of native mass spectrometry (MS in 1990 led to the development of new mass spectrometry instrumentation and methodologies for the analysis of noncovalent protein–ligand complexes. Native MS has matured to become a fast, simple, highly sensitive and automatable technique with well-established utility for fragment-based drug discovery (FBDD. Native MS has the capability to directly detect weak ligand binding to proteins, to determine stoichiometry, relative or absolute binding affinities and specificities. Native MS can be used to delineate ligand-binding sites, to elucidate mechanisms of cooperativity and to study the thermodynamics of binding. This review highlights key attributes of native MS for FBDD campaigns.

Native Mass Spectrometry in Fragment-Based Drug Discovery.

Science.gov (United States)

Pedro, Liliana; Quinn, Ronald J

2016-07-28

The advent of native mass spectrometry (MS) in 1990 led to the development of new mass spectrometry instrumentation and methodologies for the analysis of noncovalent protein-ligand complexes. Native MS has matured to become a fast, simple, highly sensitive and automatable technique with well-established utility for fragment-based drug discovery (FBDD). Native MS has the capability to directly detect weak ligand binding to proteins, to determine stoichiometry, relative or absolute binding affinities and specificities. Native MS can be used to delineate ligand-binding sites, to elucidate mechanisms of cooperativity and to study the thermodynamics of binding. This review highlights key attributes of native MS for FBDD campaigns.

Fragment-based quantum mechanical calculation of protein-protein binding affinities.

Science.gov (United States)

Wang, Yaqian; Liu, Jinfeng; Li, Jinjin; He, Xiao

2018-04-29

The electrostatically embedded generalized molecular fractionation with conjugate caps (EE-GMFCC) method has been successfully utilized for efficient linear-scaling quantum mechanical (QM) calculation of protein energies. In this work, we applied the EE-GMFCC method for calculation of binding affinity of Endonuclease colicin-immunity protein complex. The binding free energy changes between the wild-type and mutants of the complex calculated by EE-GMFCC are in good agreement with experimental results. The correlation coefficient (R) between the predicted binding energy changes and experimental values is 0.906 at the B3LYP/6-31G*-D level, based on the snapshot whose binding affinity is closest to the average result from the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) calculation. The inclusion of the QM effects is important for accurate prediction of protein-protein binding affinities. Moreover, the self-consistent calculation of PB solvation energy is required for accurate calculations of protein-protein binding free energies. This study demonstrates that the EE-GMFCC method is capable of providing reliable prediction of relative binding affinities for protein-protein complexes. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

Comparison of renal and osseous binding of parathyroid hormone and hormonal fragments

International Nuclear Information System (INIS)

Demay, M.; Mitchell, J.; Goltzman, D.

1985-01-01

The authors compared receptor binding and adenylate cyclase stimulation of intact bovine parathyroid hormone (bPTH)-(1-84) and the synthetic amino-terminal fragments, bPTH-(1-34) and rat PTH (rPTH)-(1-34). In both canine renal membranes and cloned rat osteosarcoma cells the amino-terminal fragments bound to a single order of sites; the affinity of rPTH-(1-34) exceeded that of bPTH-(1-34), correlating with its higher potency in stimulating adenylate cyclase. In studies with oxidized bPTH-(1--84), the middle and carboxyl regions of intact PTH were found to bind to both tissues but with higher affinity to osteosarcoma cells than to renal membranes. Our results demonstrate that rPTH-(1--34) is the most favorable probe of amino-terminal PTH binding and the most potent of the PTH peptides in stimulating renal and osseous adenylate cyclase. The results also show that midregion and carboxyl determinants within intact PTH contribute to hormone binding, which does not correlate with adenylate cyclase activation and appears more significant for skeletal than for renal binding

Generation of Polar Semi-Saturated Bicyclic Pyrazoles for Fragment-Based Drug Discovery Campaigns.

Science.gov (United States)

Luise, Nicola; Wyatt, Paul

2018-05-07

Synthesising polar semi-saturated bicyclic heterocycles can lead to better starting points for fragment-based drug discovery (FBDD) programs. This communication highlights the application of diverse chemistry to construct bicyclic systems from a common intermediate, where pyrazole, a privileged heteroaromatic able to bind effectively to biological targets, is fused to diverse saturated counterparts. The generated fragments can be further developed either after confirmation of their binding pose or early in the process, as their synthetic intermediates. Essential quality control (QC) for selection of small molecules to add to a fragment library is discussed. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

SPR-based fragment screening with neurotensin receptor 1 generates novel small molecule ligands

Science.gov (United States)

Huber, Sylwia; Casagrande, Fabio; Hug, Melanie N.; Wang, Lisha; Heine, Philipp; Kummer, Lutz; Plückthun, Andreas; Hennig, Michael

2017-01-01

The neurotensin receptor 1 represents an important drug target involved in various diseases of the central nervous system. So far, the full exploitation of potential therapeutic activities has been compromised by the lack of compounds with favorable physicochemical and pharmacokinetic properties which efficiently penetrate the blood-brain barrier. Recent progress in the generation of stabilized variants of solubilized neurotensin receptor 1 and its subsequent purification and successful structure determination presents a solid starting point to apply the approach of fragment-based screening to extend the chemical space of known neurotensin receptor 1 ligands. In this report, surface plasmon resonance was used as primary method to screen 6369 compounds. Thereby 44 hits were identified and confirmed in competition as well as dose-response experiments. Furthermore, 4 out of 8 selected hits were validated using nuclear magnetic resonance spectroscopy as orthogonal biophysical method. Computational analysis of the compound structures, taking the known crystal structure of the endogenous peptide agonist into consideration, gave insight into the potential fragment-binding location and interactions and inspires chemistry efforts for further exploration of the fragments. PMID:28510609

Efficient clustering aggregation based on data fragments.

Science.gov (United States)

Wu, Ou; Hu, Weiming; Maybank, Stephen J; Zhu, Mingliang; Li, Bing

2012-06-01

Clustering aggregation, known as clustering ensembles, has emerged as a powerful technique for combining different clustering results to obtain a single better clustering. Existing clustering aggregation algorithms are applied directly to data points, in what is referred to as the point-based approach. The algorithms are inefficient if the number of data points is large. We define an efficient approach for clustering aggregation based on data fragments. In this fragment-based approach, a data fragment is any subset of the data that is not split by any of the clustering results. To establish the theoretical bases of the proposed approach, we prove that clustering aggregation can be performed directly on data fragments under two widely used goodness measures for clustering aggregation taken from the literature. Three new clustering aggregation algorithms are described. The experimental results obtained using several public data sets show that the new algorithms have lower computational complexity than three well-known existing point-based clustering aggregation algorithms (Agglomerative, Furthest, and LocalSearch); nevertheless, the new algorithms do not sacrifice the accuracy.

A Ligand-observed Mass Spectrometry Approach Integrated into the Fragment Based Lead Discovery Pipeline

Science.gov (United States)

Chen, Xin; Qin, Shanshan; Chen, Shuai; Li, Jinlong; Li, Lixin; Wang, Zhongling; Wang, Quan; Lin, Jianping; Yang, Cheng; Shui, Wenqing

2015-01-01

In fragment-based lead discovery (FBLD), a cascade combining multiple orthogonal technologies is required for reliable detection and characterization of fragment binding to the target. Given the limitations of the mainstream screening techniques, we presented a ligand-observed mass spectrometry approach to expand the toolkits and increase the flexibility of building a FBLD pipeline especially for tough targets. In this study, this approach was integrated into a FBLD program targeting the HCV RNA polymerase NS5B. Our ligand-observed mass spectrometry analysis resulted in the discovery of 10 hits from a 384-member fragment library through two independent screens of complex cocktails and a follow-up validation assay. Moreover, this MS-based approach enabled quantitative measurement of weak binding affinities of fragments which was in general consistent with SPR analysis. Five out of the ten hits were then successfully translated to X-ray structures of fragment-bound complexes to lay a foundation for structure-based inhibitor design. With distinctive strengths in terms of high capacity and speed, minimal method development, easy sample preparation, low material consumption and quantitative capability, this MS-based assay is anticipated to be a valuable addition to the repertoire of current fragment screening techniques. PMID:25666181

Prediction of trypsin/molecular fragment binding affinities by free energy decomposition and empirical scores

Science.gov (United States)

Benson, Mark L.; Faver, John C.; Ucisik, Melek N.; Dashti, Danial S.; Zheng, Zheng; Merz, Kenneth M.

2012-05-01

Two families of binding affinity estimation methodologies are described which were utilized in the SAMPL3 trypsin/fragment binding affinity challenge. The first is a free energy decomposition scheme based on a thermodynamic cycle, which included separate contributions from enthalpy and entropy of binding as well as a solvent contribution. Enthalpic contributions were estimated with PM6-DH2 semiempirical quantum mechanical interaction energies, which were modified with a statistical error correction procedure. Entropic contributions were estimated with the rigid-rotor harmonic approximation, and solvent contributions to the free energy were estimated with several different methods. The second general methodology is the empirical score LISA, which contains several physics-based terms trained with the large PDBBind database of protein/ligand complexes. Here we also introduce LISA+, an updated version of LISA which, prior to scoring, classifies systems into one of four classes based on a ligand's hydrophobicity and molecular weight. Each version of the two methodologies (a total of 11 methods) was trained against a compiled set of known trypsin binders available in the Protein Data Bank to yield scaling parameters for linear regression models. Both raw and scaled scores were submitted to SAMPL3. Variants of LISA showed relatively low absolute errors but also low correlation with experiment, while the free energy decomposition methods had modest success when scaling factors were included. Nonetheless, re-scaled LISA yielded the best predictions in the challenge in terms of RMS error, and six of these models placed in the top ten best predictions by RMS error. This work highlights some of the difficulties of predicting binding affinities of small molecular fragments to protein receptors as well as the benefit of using training data.

Critical Evaluation of Native Electrospray Ionization Mass Spectrometry for Fragment-Based Screening.

Science.gov (United States)

Göth, Melanie; Badock, Volker; Weiske, Jörg; Pagel, Kevin; Kuropka, Benno

2017-08-08

Fragment-based screening presents a promising alternative to high-throughput screening and has gained great attention in recent years. So far, only a few studies have discussed mass spectrometry as a screening technology for fragments. Herein, we report the application of native electrospray ionization mass spectrometry (MS) for screening defined sets of fragments against four different target proteins. Fragments were selected from a primary screening conducted with a thermal shift assay (TSA) and represented different binding categories. Our data indicated that, beside specific complex formation, many fragments show extensive multiple binding and also charge-state shifts. Both of these factors complicate automated data analysis and decrease the attractiveness of native MS as a primary screening tool for fragments. A comparison of the hits identified by native MS and TSA showed good agreement for two of the proteins. Furthermore, we discuss general challenges, including the determination of an optimal fragment concentration and the question of how to rank fragment hits according to their affinity. In conclusion, we consider native MS to be a highly valuable tool for the validation and deeper investigation of promising fragment hits rather than a method for primary screening. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The importance of hydration thermodynamics in fragment-to-lead optimization.

Science.gov (United States)

Ichihara, Osamu; Shimada, Yuzo; Yoshidome, Daisuke

2014-12-01

Using a computational approach to assess changes in solvation thermodynamics upon ligand binding, we investigated the effects of water molecules on the binding energetics of over 20 fragment hits and their corresponding optimized lead compounds. Binding activity and X-ray crystallographic data of published fragment-to-lead optimization studies from various therapeutically relevant targets were studied. The analysis reveals a distinct difference between the thermodynamic profile of water molecules displaced by fragment hits and those displaced by the corresponding optimized lead compounds. Specifically, fragment hits tend to displace water molecules with notably unfavorable excess entropies-configurationally constrained water molecules-relative to those displaced by the newly added moieties of the lead compound during the course of fragment-to-lead optimization. Herein we describe the details of this analysis with the goal of providing practical guidelines for exploiting thermodynamic signatures of binding site water molecules in the context of fragment-to-lead optimization. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Surface complement C3 fragments and cellular binding of microparticles in patients with SLE

DEFF Research Database (Denmark)

Winberg, Line Kjær; Nielsen, Claus Henrik; Jacobsen, Søren

2017-01-01

Objectives: To examine microparticles (MPs) from patients with SLE and healthy controls (HCs) by determining the cellular origin of the MPs, quantifying attached fragments of complement component 3 (C3) and assessing the ability of MPs to bind to circulating phagocytes and erythrocytes. These fea......Objectives: To examine microparticles (MPs) from patients with SLE and healthy controls (HCs) by determining the cellular origin of the MPs, quantifying attached fragments of complement component 3 (C3) and assessing the ability of MPs to bind to circulating phagocytes and erythrocytes...

Targeting lysine specific demethylase 4A (KDM4A) tandem TUDOR domain - A fragment based approach.

Science.gov (United States)

Upadhyay, Anup K; Judge, Russell A; Li, Leiming; Pithawalla, Ron; Simanis, Justin; Bodelle, Pierre M; Marin, Violeta L; Henry, Rodger F; Petros, Andrew M; Sun, Chaohong

2018-06-01

The tandem TUDOR domains present in the non-catalytic C-terminal half of the KDM4A, 4B and 4C enzymes play important roles in regulating their chromatin localizations and substrate specificities. They achieve this regulatory role by binding to different tri-methylated lysine residues on histone H3 (H3-K4me3, H3-K23me3) and histone H4 (H4-K20me3) depending upon the specific chromatin environment. In this work, we have used a 2D-NMR based fragment screening approach to identify a novel fragment (1a), which binds to the KDM4A-TUDOR domain and shows modest competition with H3-K4me3 binding in biochemical as well as in vitro cell based assays. A co-crystal structure of KDM4A TUDOR domain in complex with 1a shows that the fragment binds stereo-specifically to the methyl lysine binding pocket forming a network of strong hydrogen bonds and hydrophobic interactions. We anticipate that the fragment 1a can be further developed into a novel allosteric inhibitor of the KDM4 family of enzymes through targeting their C-terminal tandem TUDOR domain. Copyright © 2018 Elsevier Ltd. All rights reserved.

Dissecting fragment-based lead discovery at the von Hippel-Lindau protein:hypoxia inducible factor 1α protein-protein interface.

Science.gov (United States)

Van Molle, Inge; Thomann, Andreas; Buckley, Dennis L; So, Ernest C; Lang, Steffen; Crews, Craig M; Ciulli, Alessio

2012-10-26

Fragment screening is widely used to identify attractive starting points for drug design. However, its potential and limitations to assess the tractability of often challenging protein:protein interfaces have been underexplored. Here, we address this question by means of a systematic deconstruction of lead-like inhibitors of the pVHL:HIF-1α interaction into their component fragments. Using biophysical techniques commonly employed for screening, we could only detect binding of fragments that violate the Rule of Three, are more complex than those typically screened against classical druggable targets, and occupy two adjacent binding subsites at the interface rather than just one. Analyses based on ligand and group lipophilicity efficiency of anchored fragments were applied to dissect the individual subsites and probe for binding hot spots. The implications of our findings for targeting protein interfaces by fragment-based approaches are discussed. Copyright © 2012 Elsevier Ltd. All rights reserved.

A collagen-binding EGFR antibody fragment targeting tumors with a collagen-rich extracellular matrix.

Science.gov (United States)

Liang, Hui; Li, Xiaoran; Wang, Bin; Chen, Bing; Zhao, Yannan; Sun, Jie; Zhuang, Yan; Shi, Jiajia; Shen, He; Zhang, Zhijun; Dai, Jianwu

2016-02-17

Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of cetuximab was fused with CBD (CBD-Fab) and expressed in Pichia pastoris. CBD-Fab maintained antigen binding and anti-tumor activity of cetuximab and obtained a collagen-binding ability in vitro. The results also showed CBD-Fab was mainly enriched in tumors and had longer retention time in tumors in A431 s.c. xenografts. Furthermore, CBD-Fab showed a similar therapeutic efficacy as cetuximab in A431 xenografts. Although CBD-Fab hasn't showed better therapeutic effects than cetuximab, its smaller molecular and special target may be applicable as antibody-drug conjugates (ADC) or immunotoxins.

Momentum characteristics of spectators in fragmentation of 4He nuclei

International Nuclear Information System (INIS)

Glagolev, V.V.; Lebedev, R.M.; Strel'tsov, V.N.

1985-01-01

Some characteristics of 2 H, 3 H, 3 He fragments from 4 Hep interaction at 8.6 GeV/c have been studied. A numerical procedure, based on Monte-Carlo method, for getting the wave function of relative motion of nucleons or groups of nucleons in nucleus is proposed to describe the fragment momentum spectra and the asymmetry in Treiman-Yang angle distribution. The agreement between computed and experimental results is sufficient if one takes into account the experimental errors and the nucleus binding energy

Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin: Fragment-Based Drug Design Facilitated by Dynamic Combinatorial Chemistry.

Science.gov (United States)

Mondal, Milon; Radeva, Nedyalka; Fanlo-Virgós, Hugo; Otto, Sijbren; Klebe, Gerhard; Hirsch, Anna K H

2016-08-01

Fragment-based drug design (FBDD) affords active compounds for biological targets. While there are numerous reports on FBDD by fragment growing/optimization, fragment linking has rarely been reported. Dynamic combinatorial chemistry (DCC) has become a powerful hit-identification strategy for biological targets. We report the synergistic combination of fragment linking and DCC to identify inhibitors of the aspartic protease endothiapepsin. Based on X-ray crystal structures of endothiapepsin in complex with fragments, we designed a library of bis-acylhydrazones and used DCC to identify potent inhibitors. The most potent inhibitor exhibits an IC50 value of 54 nm, which represents a 240-fold improvement in potency compared to the parent hits. Subsequent X-ray crystallography validated the predicted binding mode, thus demonstrating the efficiency of the combination of fragment linking and DCC as a hit-identification strategy. This approach could be applied to a range of biological targets, and holds the potential to facilitate hit-to-lead optimization. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Cloud computing for protein-ligand binding site comparison.

Science.gov (United States)

Hung, Che-Lun; Hua, Guan-Jie

2013-01-01

The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery.

Neutralisation and binding of VHS virus by monovalent antibody fragments

DEFF Research Database (Denmark)

Cupit, P.M.; Lorenzen, Niels; Strachan, G.

2001-01-01

We have previously reported the cloning and characterisation of the heavy and light chain variable domain genes encoding three monoclonal antibodies (Mabs) that bind viral haemorrhagic septicaemia virus (VHSV). Two of these antibodies, 3F1H10 and 3F1A2 both neutralised the virus though 3F1A2...... appeared to recognise a broader range of virus isolates. The variable domains of these two antibodies differ by only four residues (Lorenzen et al., 2000a. Fish Shellfish Immunol. 10, 129-142). To further study the mechanism of neutralisation, Fab fragments as well as a series of recombinant bacterial...... single chain antibody (scAb) fragments were generated from the three anti-VHSV Mabs and their variable domain genes, respectively. Fabs and scAbs derived from the neutralising Mabs were both able to neutralise the VHSV type 1 isolate DK-F1. In addition, a series of scAb fragments were produced using...

Adaptive frozen orbital treatment for the fragment molecular orbital method combined with density-functional tight-binding

Science.gov (United States)

Nishimoto, Yoshio; Fedorov, Dmitri G.

2018-02-01

The exactly analytic gradient is derived and implemented for the fragment molecular orbital (FMO) method combined with density-functional tight-binding (DFTB) using adaptive frozen orbitals. The response contributions which arise from freezing detached molecular orbitals on the border between fragments are computed by solving Z-vector equations. The accuracy of the energy, its gradient, and optimized structures is verified on a set of representative inorganic materials and polypeptides. FMO-DFTB is applied to optimize the structure of a silicon nano-wire, and the results are compared to those of density functional theory and experiment. FMO accelerates the DFTB calculation of a boron nitride nano-ring with 7872 atoms by a factor of 406. Molecular dynamics simulations using FMO-DFTB applied to a 10.7 μm chain of boron nitride nano-rings, consisting of about 1.2 × 106 atoms, reveal the rippling and twisting of nano-rings at room temperature.

Anti-fouling properties of Fab’ fragments immobilized on silane-based adlayers

International Nuclear Information System (INIS)

Crivianu-Gaita, Victor; Romaschin, Alexander; Thompson, Michael

2015-01-01

Highlights: • Simple and mixed adlayers formed with Fab’ linker and/or spacers. • Binding of Fab’ fragments through TUBTS linker resulted in oriented immobilization. • Immobilized Fab’ fragments have inherent anti-fouling character. • Up to 80% fouling reduction when Fab’ fragments introduced to surfaces. • Used the minimally fouling surfaces to detect a cancer biomarker (PTHrP) in serum. - Graphical abstract: Biosensors require surfaces that are highly specific towards the target analyte and that are minimally fouling. However, surface tuning to minimize fouling is a difficult task. The last decade has seen an increase in the use of immobilized antigen-binding antibody fragments (Fab’) in biosensors. One Fab’ linker compound S-(11-trichlorosilyl-undecanyl)-benzothiosulfonate (TUBTS) and three spacers were used to create the silane-based adlayers. The ultra-high frequency electromagnetic piezoelectric acoustic sensor (EMPAS) was used to gauge the fouling properties of the various surfaces using bovine serum albumin (BSA), goat IgG, and mouse serum. X-ray photoelectron spectroscopy (XPS), contact angle, and atomic force microscopy (AFM) were employed to characterize the surfaces. It was discovered that immobilized oriented Fab’ fragments reduced the fouling levels of surfaces up to 80% compared to the surfaces without fragments. An explanation for this phenomenon is that the antibody fragments increase the hydration of the surfaces and aid in the formation of an anti-fouling water barrier. The anti-fouling effect of the Fab’ fragments is at its maximum when there is an even distribution of fragments across the surfaces. Finally, using Fab’-covered surfaces, a cancer biomarker was detected from serum, showing the applicability of this work to the field of biodetection. - Abstract: Biosensors require surfaces that are highly specific towards the target analyte and that are minimally fouling. However, surface tuning to minimize fouling is a

Anti-fouling properties of Fab’ fragments immobilized on silane-based adlayers

Energy Technology Data Exchange (ETDEWEB)

Crivianu-Gaita, Victor [Department of Chemistry, University of Toronto, Toronto, ON M5S 3H6 (Canada); Romaschin, Alexander [Clinical Biochemistry, St. Michael' s Hospital, Toronto, ON M5B 1W8 (Canada); Thompson, Michael, E-mail: mikethom@chem.utoronto.ca [Department of Chemistry, University of Toronto, Toronto, ON M5S 3H6 (Canada)

2015-12-30

Highlights: • Simple and mixed adlayers formed with Fab’ linker and/or spacers. • Binding of Fab’ fragments through TUBTS linker resulted in oriented immobilization. • Immobilized Fab’ fragments have inherent anti-fouling character. • Up to 80% fouling reduction when Fab’ fragments introduced to surfaces. • Used the minimally fouling surfaces to detect a cancer biomarker (PTHrP) in serum. - Graphical abstract: Biosensors require surfaces that are highly specific towards the target analyte and that are minimally fouling. However, surface tuning to minimize fouling is a difficult task. The last decade has seen an increase in the use of immobilized antigen-binding antibody fragments (Fab’) in biosensors. One Fab’ linker compound S-(11-trichlorosilyl-undecanyl)-benzothiosulfonate (TUBTS) and three spacers were used to create the silane-based adlayers. The ultra-high frequency electromagnetic piezoelectric acoustic sensor (EMPAS) was used to gauge the fouling properties of the various surfaces using bovine serum albumin (BSA), goat IgG, and mouse serum. X-ray photoelectron spectroscopy (XPS), contact angle, and atomic force microscopy (AFM) were employed to characterize the surfaces. It was discovered that immobilized oriented Fab’ fragments reduced the fouling levels of surfaces up to 80% compared to the surfaces without fragments. An explanation for this phenomenon is that the antibody fragments increase the hydration of the surfaces and aid in the formation of an anti-fouling water barrier. The anti-fouling effect of the Fab’ fragments is at its maximum when there is an even distribution of fragments across the surfaces. Finally, using Fab’-covered surfaces, a cancer biomarker was detected from serum, showing the applicability of this work to the field of biodetection. - Abstract: Biosensors require surfaces that are highly specific towards the target analyte and that are minimally fouling. However, surface tuning to minimize fouling is a

Fragment-based modelling of single stranded RNA bound to RNA recognition motif containing proteins

Science.gov (United States)

de Beauchene, Isaure Chauvot; de Vries, Sjoerd J.; Zacharias, Martin

2016-01-01

Abstract Protein-RNA complexes are important for many biological processes. However, structural modeling of such complexes is hampered by the high flexibility of RNA. Particularly challenging is the docking of single-stranded RNA (ssRNA). We have developed a fragment-based approach to model the structure of ssRNA bound to a protein, based on only the protein structure, the RNA sequence and conserved contacts. The conformational diversity of each RNA fragment is sampled by an exhaustive library of trinucleotides extracted from all known experimental protein–RNA complexes. The method was applied to ssRNA with up to 12 nucleotides which bind to dimers of the RNA recognition motifs (RRMs), a highly abundant eukaryotic RNA-binding domain. The fragment based docking allows a precise de novo atomic modeling of protein-bound ssRNA chains. On a benchmark of seven experimental ssRNA–RRM complexes, near-native models (with a mean heavy-atom deviation of <3 Å from experiment) were generated for six out of seven bound RNA chains, and even more precise models (deviation < 2 Å) were obtained for five out of seven cases, a significant improvement compared to the state of the art. The method is not restricted to RRMs but was also successfully applied to Pumilio RNA binding proteins. PMID:27131381

Fragment library screening identifies hits that bind to the non-catalytic surface of Pseudomonas aeruginosa DsbA1

Science.gov (United States)

Headey, Stephen J.; Vazirani, Mansha; Shouldice, Stephen R.; Coinçon, Mathieu; Tay, Stephanie; Morton, Craig J.; Simpson, Jamie S.; Martin, Jennifer L.

2017-01-01

At a time when the antibiotic drug discovery pipeline has stalled, antibiotic resistance is accelerating with catastrophic implications for our ability to treat bacterial infections. Globally we face the prospect of a future when common infections can once again kill. Anti-virulence approaches that target the capacity of the bacterium to cause disease rather than the growth or survival of the bacterium itself offer a tantalizing prospect of novel antimicrobials. They may also reduce the propensity to induce resistance by removing the strong selection pressure imparted by bactericidal or bacteriostatic agents. In the human pathogen Pseudomonas aeruginosa, disulfide bond protein A (PaDsbA1) plays a central role in the oxidative folding of virulence factors and is therefore an attractive target for the development of new anti-virulence antimicrobials. Using a fragment-based approach we have identified small molecules that bind to PaDsbA1. The fragment hits show selective binding to PaDsbA1 over the DsbA protein from Escherichia coli, suggesting that developing species-specific narrow-spectrum inhibitors of DsbA enzymes may be feasible. Structures of a co-complex of PaDsbA1 with the highest affinity fragment identified in the screen reveal that the fragment binds on the non-catalytic surface of the protein at a domain interface. This biophysical and structural data represent a starting point in the development of higher affinity compounds, which will be assessed for their potential as selective PaDsbA1 inhibitors. PMID:28346540

Structure solution of DNA-binding proteins and complexes with ARCIMBOLDO libraries

Energy Technology Data Exchange (ETDEWEB)

Pröpper, Kevin [University of Göttingen, (Germany); Instituto de Biologia Molecular de Barcelona (IBMB-CSIC), (Spain); Meindl, Kathrin; Sammito, Massimo [Instituto de Biologia Molecular de Barcelona (IBMB-CSIC), (Spain); Dittrich, Birger; Sheldrick, George M. [University of Göttingen, (Germany); Pohl, Ehmke, E-mail: ehmke.pohl@durham.ac.uk [Durham University, (United Kingdom); Usón, Isabel, E-mail: ehmke.pohl@durham.ac.uk [Instituto de Biologia Molecular de Barcelona (IBMB-CSIC), (Spain); Institucio Catalana de Recerca i Estudis Avancats (ICREA), (Spain); University of Göttingen, (Germany)

2014-06-01

The structure solution of DNA-binding protein structures and complexes based on the combination of location of DNA-binding protein motif fragments with density modification in a multi-solution frame is described. Protein–DNA interactions play a major role in all aspects of genetic activity within an organism, such as transcription, packaging, rearrangement, replication and repair. The molecular detail of protein–DNA interactions can be best visualized through crystallography, and structures emphasizing insight into the principles of binding and base-sequence recognition are essential to understanding the subtleties of the underlying mechanisms. An increasing number of high-quality DNA-binding protein structure determinations have been witnessed despite the fact that the crystallographic particularities of nucleic acids tend to pose specific challenges to methods primarily developed for proteins. Crystallographic structure solution of protein–DNA complexes therefore remains a challenging area that is in need of optimized experimental and computational methods. The potential of the structure-solution program ARCIMBOLDO for the solution of protein–DNA complexes has therefore been assessed. The method is based on the combination of locating small, very accurate fragments using the program Phaser and density modification with the program SHELXE. Whereas for typical proteins main-chain α-helices provide the ideal, almost ubiquitous, small fragments to start searches, in the case of DNA complexes the binding motifs and DNA double helix constitute suitable search fragments. The aim of this work is to provide an effective library of search fragments as well as to determine the optimal ARCIMBOLDO strategy for the solution of this class of structures.

Oriented Immobilization of Fab Fragments by Site-Specific Biotinylation at the Conserved Nucleotide Binding Site for Enhanced Antigen Detection.

Science.gov (United States)

Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

2015-09-08

Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the nucleotide binding site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and ε-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, site-specific biotinylation of Fab fragments without structural damage or loss in antigen binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.

Binding Direction-Based Two-Dimensional Flattened Contact Area Computing Algorithm for Protein-Protein Interactions.

Science.gov (United States)

Kang, Beom Sik; Pugalendhi, GaneshKumar; Kim, Ku-Jin

2017-10-13

Interactions between protein molecules are essential for the assembly, function, and regulation of proteins. The contact region between two protein molecules in a protein complex is usually complementary in shape for both molecules and the area of the contact region can be used to estimate the binding strength between two molecules. Although the area is a value calculated from the three-dimensional surface, it cannot represent the three-dimensional shape of the surface. Therefore, we propose an original concept of two-dimensional contact area which provides further information such as the ruggedness of the contact region. We present a novel algorithm for calculating the binding direction between two molecules in a protein complex, and then suggest a method to compute the two-dimensional flattened area of the contact region between two molecules based on the binding direction.

Binding Direction-Based Two-Dimensional Flattened Contact Area Computing Algorithm for Protein–Protein Interactions

Directory of Open Access Journals (Sweden)

Beom Sik Kang

2017-10-01

Full Text Available Interactions between protein molecules are essential for the assembly, function, and regulation of proteins. The contact region between two protein molecules in a protein complex is usually complementary in shape for both molecules and the area of the contact region can be used to estimate the binding strength between two molecules. Although the area is a value calculated from the three-dimensional surface, it cannot represent the three-dimensional shape of the surface. Therefore, we propose an original concept of two-dimensional contact area which provides further information such as the ruggedness of the contact region. We present a novel algorithm for calculating the binding direction between two molecules in a protein complex, and then suggest a method to compute the two-dimensional flattened area of the contact region between two molecules based on the binding direction.

A toxin-binding alkaline phosphatase fragment synergizes Bt toxin Cry1Ac against susceptible and resistant Helicoverpa armigera.

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Wenbo Chen

Full Text Available Evolution of resistance by insects threatens the continued success of pest control using insecticidal crystal (Cry proteins from the bacterium Bacillus thuringiensis (Bt in sprays and transgenic plants. In this study, laboratory selection with Cry1Ac yielded five strains of cotton bollworm, Helicoverpa armigera, with resistance ratios at the median lethal concentration (LC50 of activated Cry1Ac ranging from 22 to 1700. Reduced activity and reduced transcription of an alkaline phosphatase protein that binds Cry1Ac was associated with resistance to Cry1Ac in the four most resistant strains. A Cry1Ac-binding fragment of alkaline phosphatase from H. armigera (HaALP1f was not toxic by itself, but it increased mortality caused by Cry1Ac in a susceptible strain and in all five resistant strains. Although synergism of Bt toxins against susceptible insects by toxin-binding fragments of cadherin and aminopeptidase N has been reported previously, the results here provide the first evidence of synergism of a Bt toxin by a toxin-binding fragment of alkaline phosphatase. The results here also provide the first evidence of synergism of a Bt toxin by any toxin-binding peptide against resistant insects.

A probabilistic fragment-based protein structure prediction algorithm.

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David Simoncini

Full Text Available Conformational sampling is one of the bottlenecks in fragment-based protein structure prediction approaches. They generally start with a coarse-grained optimization where mainchain atoms and centroids of side chains are considered, followed by a fine-grained optimization with an all-atom representation of proteins. It is during this coarse-grained phase that fragment-based methods sample intensely the conformational space. If the native-like region is sampled more, the accuracy of the final all-atom predictions may be improved accordingly. In this work we present EdaFold, a new method for fragment-based protein structure prediction based on an Estimation of Distribution Algorithm. Fragment-based approaches build protein models by assembling short fragments from known protein structures. Whereas the probability mass functions over the fragment libraries are uniform in the usual case, we propose an algorithm that learns from previously generated decoys and steers the search toward native-like regions. A comparison with Rosetta AbInitio protocol shows that EdaFold is able to generate models with lower energies and to enhance the percentage of near-native coarse-grained decoys on a benchmark of [Formula: see text] proteins. The best coarse-grained models produced by both methods were refined into all-atom models and used in molecular replacement. All atom decoys produced out of EdaFold's decoy set reach high enough accuracy to solve the crystallographic phase problem by molecular replacement for some test proteins. EdaFold showed a higher success rate in molecular replacement when compared to Rosetta. Our study suggests that improving low resolution coarse-grained decoys allows computational methods to avoid subsequent sampling issues during all-atom refinement and to produce better all-atom models. EdaFold can be downloaded from http://www.riken.jp/zhangiru/software.html [corrected].

Ninety-five- and 25-kDa fragments of the human immunodeficiency virus envelope glycoprotein gp120 bind to the CD4 receptor

International Nuclear Information System (INIS)

Nygren, A.; Bergman, T.; Matthews, T.; Joernvall, H.; Wigzell, H.

1988-01-01

Iodine-125-labeled gp120 (120-kDa envelope glycoprotein) from the BH10 isolate of human immunodeficiency virus is cleaved to a limited extend with the glutamate-specific protease from Staphylococcus aureus. After disulfide bond reduction, fragments with approximate molecular masses of 95, 60, 50, and 25 kDa are produced. Tests for binding to CD4-positive cells show that only two fragments, the 95- and 25- kDa peptides, are observed in cleavage products that retain the selective binding capacity of gp120. Radiosequence analysis of the fragments after sodium dodecyl sulfate/polyacrylamide gel electrophoresis and electroblotting demonstrates that the 95-kDa fragment lacks the N-terminal region of gp120 and starts at position 143 of the mature envelope protein. The 50-kDa fragment starts at the same position. The 25-kDa binding fragment was similarly deduced to be generated as a small fragment from a cleavage site in the C-terminal part of gp120. The identifications of these fragments demonstrate that radiosequence analysis utilizing 125 I-labeled tyrosine residues can function as a useful and reliable method for small-scale determination of cleavage sites in proteins. Combined, the data suggest domain-like subdivisions of gp120, define at least two intervening segments especially sensitive to proteolytic cleavage, and demonstrate the presence of a functional region for receptor binding in the C-terminal part of the molecule

Structures of endothiapepsin-fragment complexes from crystallographic fragment screening using a novel, diverse and affordable 96-compound fragment library.

Science.gov (United States)

Huschmann, Franziska U; Linnik, Janina; Sparta, Karine; Ühlein, Monika; Wang, Xiaojie; Metz, Alexander; Schiebel, Johannes; Heine, Andreas; Klebe, Gerhard; Weiss, Manfred S; Mueller, Uwe

2016-05-01

Crystallographic screening of the binding of small organic compounds (termed fragments) to proteins is increasingly important for medicinal chemistry-oriented drug discovery. To enable such experiments in a widespread manner, an affordable 96-compound library has been assembled for fragment screening in both academia and industry. The library is selected from already existing protein-ligand structures and is characterized by a broad ligand diversity, including buffer ingredients, carbohydrates, nucleotides, amino acids, peptide-like fragments and various drug-like organic compounds. When applied to the model protease endothiapepsin in a crystallographic screening experiment, a hit rate of nearly 10% was obtained. In comparison to other fragment libraries and considering that no pre-screening was performed, this hit rate is remarkably high. This demonstrates the general suitability of the selected compounds for an initial fragment-screening campaign. The library composition, experimental considerations and time requirements for a complete crystallographic fragment-screening campaign are discussed as well as the nine fully refined obtained endothiapepsin-fragment structures. While most of the fragments bind close to the catalytic centre of endothiapepsin in poses that have been observed previously, two fragments address new sites on the protein surface. ITC measurements show that the fragments bind to endothiapepsin with millimolar affinity.

Structures of endothiapepsin–fragment complexes from crystallographic fragment screening using a novel, diverse and affordable 96-compound fragment library

Science.gov (United States)

Huschmann, Franziska U.; Linnik, Janina; Sparta, Karine; Ühlein, Monika; Wang, Xiaojie; Metz, Alexander; Schiebel, Johannes; Heine, Andreas; Klebe, Gerhard; Weiss, Manfred S.; Mueller, Uwe

2016-01-01

Crystallographic screening of the binding of small organic compounds (termed fragments) to proteins is increasingly important for medicinal chemistry-oriented drug discovery. To enable such experiments in a widespread manner, an affordable 96-compound library has been assembled for fragment screening in both academia and industry. The library is selected from already existing protein–ligand structures and is characterized by a broad ligand diversity, including buffer ingredients, carbohydrates, nucleotides, amino acids, peptide-like fragments and various drug-like organic compounds. When applied to the model protease endothiapepsin in a crystallographic screening experiment, a hit rate of nearly 10% was obtained. In comparison to other fragment libraries and considering that no pre-screening was performed, this hit rate is remarkably high. This demonstrates the general suitability of the selected compounds for an initial fragment-screening campaign. The library composition, experimental considerations and time requirements for a complete crystallographic fragment-screening campaign are discussed as well as the nine fully refined obtained endothiapepsin–fragment structures. While most of the fragments bind close to the catalytic centre of endothiapepsin in poses that have been observed previously, two fragments address new sites on the protein surface. ITC measurements show that the fragments bind to endothiapepsin with millimolar affinity. PMID:27139825

A new crystal structure fragment-based pharmacophore method for G protein-coupled receptors

DEFF Research Database (Denmark)

Fidom, Kimberley; Isberg, Vignir; Hauser, Alexander Sebastian

2015-01-01

and receptor residue pairs, from crystal structure complexes. We describe the procedure to collect a library with more than 250 fragments covering 29 residue positions within the generic transmembrane binding pocket. We describe how the library fragments are recombined and inferred to build pharmacophores...... for new targets. A validating retrospective virtual screening of histamine H1 and H3 receptor pharmacophores yielded area-under-the-curves of 0.88 and 0.82, respectively. The fragment-based method has the unique advantage that it can be applied to targets for which no (homologous) crystal structures...... or ligands are known. 47% of the class A G protein-coupled receptors can be targeted with at least four-element pharmacophores. The fragment libraries can also be used to grow known ligands or for rotamer refinement of homology models. Researchers can download the complete fragment library or a subset...

Functional display of platelet-binding VWF fragments on filamentous bacteriophage.

Directory of Open Access Journals (Sweden)

Andrew Yee

Full Text Available von Willebrand factor (VWF tethers platelets to sites of vascular injury via interaction with the platelet surface receptor, GPIb. To further define the VWF sequences required for VWF-platelet interaction, a phage library displaying random VWF protein fragments was screened against formalin-fixed platelets. After 3 rounds of affinity selection, DNA sequencing of platelet-bound clones identified VWF peptides mapping exclusively to the A1 domain. Aligning these sequences defined a minimal, overlapping segment spanning P1254-A1461, which encompasses the C1272-C1458 cystine loop. Analysis of phage carrying a mutated A1 segment (C1272/1458A confirmed the requirement of the cystine loop for optimal binding. Four rounds of affinity maturation of a randomly mutagenized A1 phage library identified 10 and 14 unique mutants associated with enhanced platelet binding in the presence and absence of botrocetin, respectively, with 2 mutants (S1370G and I1372V common to both conditions. These results demonstrate the utility of filamentous phage for studying VWF protein structure-function and identify a minimal, contiguous peptide that bind to formalin-fixed platelets, confirming the importance of the VWF A1 domain with no evidence for another independently platelet-binding segment within VWF. These findings also point to key structural elements within the A1 domain that regulate VWF-platelet adhesion.

In silico design of fragment-based drug targeting host processing α-glucosidase i for dengue fever

Science.gov (United States)

Toepak, E. P.; Tambunan, U. S. F.

2017-02-01

Dengue is a major health problem in the tropical and sub-tropical regions. The development of antiviral that targeting dengue’s host enzyme can be more effective and efficient treatment than the viral enzyme. Host enzyme processing α-glucosidase I has an important role in the maturation process of dengue virus envelope glycoprotein. The inhibition of processing α-glucosidase I can become a promising target for dengue fever treatment. The antiviral approach using in silico fragment-based drug design can generate drug candidates with high binding affinity. In this research, 198.621 compounds were obtained from ZINC15 Biogenic Database. These compounds were screened to find the favorable fragments according to Rules of Three and pharmacological properties. The screening fragments were docked into the active site of processing α-glucosidase I. The potential fragment candidates from the molecular docking simulation were linked with castanospermine (CAST) to generate ligands with a better binding affinity. The Analysis of ligand - enzyme interaction showed ligands with code LRS 22, 28, and 47 have the better binding free energy than the standard ligand. Ligand LRS 28 (N-2-4-methyl-5-((1S,3S,6S,7R,8R,8aR)-1,6,7,8-tetrahydroxyoctahydroindolizin-3-yl) pentyl) indolin-1-yl) propionamide) itself among the other ligands has the lowest binding free energy. Pharmacological properties prediction also showed the ligands LRS 22, 28, and 47 can be promising as the dengue fever drug candidates.

Mass spectrometry for fragment screening.

Science.gov (United States)

Chan, Daniel Shiu-Hin; Whitehouse, Andrew J; Coyne, Anthony G; Abell, Chris

2017-11-08

Fragment-based approaches in chemical biology and drug discovery have been widely adopted worldwide in both academia and industry. Fragment hits tend to interact weakly with their targets, necessitating the use of sensitive biophysical techniques to detect their binding. Common fragment screening techniques include differential scanning fluorimetry (DSF) and ligand-observed NMR. Validation and characterization of hits is usually performed using a combination of protein-observed NMR, isothermal titration calorimetry (ITC) and X-ray crystallography. In this context, MS is a relatively underutilized technique in fragment screening for drug discovery. MS-based techniques have the advantage of high sensitivity, low sample consumption and being label-free. This review highlights recent examples of the emerging use of MS-based techniques in fragment screening. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Fragment virtual screening based on Bayesian categorization for discovering novel VEGFR-2 scaffolds.

Science.gov (United States)

Zhang, Yanmin; Jiao, Yu; Xiong, Xiao; Liu, Haichun; Ran, Ting; Xu, Jinxing; Lu, Shuai; Xu, Anyang; Pan, Jing; Qiao, Xin; Shi, Zhihao; Lu, Tao; Chen, Yadong

2015-11-01

The discovery of novel scaffolds against a specific target has long been one of the most significant but challengeable goals in discovering lead compounds. A scaffold that binds in important regions of the active pocket is more favorable as a starting point because scaffolds generally possess greater optimization possibilities. However, due to the lack of sufficient chemical space diversity of the databases and the ineffectiveness of the screening methods, it still remains a great challenge to discover novel active scaffolds. Since the strengths and weaknesses of both fragment-based drug design and traditional virtual screening (VS), we proposed a fragment VS concept based on Bayesian categorization for the discovery of novel scaffolds. This work investigated the proposal through an application on VEGFR-2 target. Firstly, scaffold and structural diversity of chemical space for 10 compound databases were explicitly evaluated. Simultaneously, a robust Bayesian classification model was constructed for screening not only compound databases but also their corresponding fragment databases. Although analysis of the scaffold diversity demonstrated a very unevenly distribution of scaffolds over molecules, results showed that our Bayesian model behaved better in screening fragments than molecules. Through a literature retrospective research, several generated fragments with relatively high Bayesian scores indeed exhibit VEGFR-2 biological activity, which strongly proved the effectiveness of fragment VS based on Bayesian categorization models. This investigation of Bayesian-based fragment VS can further emphasize the necessity for enrichment of compound databases employed in lead discovery by amplifying the diversity of databases with novel structures.

Fragment-based docking: development of the CHARMMing Web user interface as a platform for computer-aided drug design.

Science.gov (United States)

Pevzner, Yuri; Frugier, Emilie; Schalk, Vinushka; Caflisch, Amedeo; Woodcock, H Lee

2014-09-22

Web-based user interfaces to scientific applications are important tools that allow researchers to utilize a broad range of software packages with just an Internet connection and a browser. One such interface, CHARMMing (CHARMM interface and graphics), facilitates access to the powerful and widely used molecular software package CHARMM. CHARMMing incorporates tasks such as molecular structure analysis, dynamics, multiscale modeling, and other techniques commonly used by computational life scientists. We have extended CHARMMing's capabilities to include a fragment-based docking protocol that allows users to perform molecular docking and virtual screening calculations either directly via the CHARMMing Web server or on computing resources using the self-contained job scripts generated via the Web interface. The docking protocol was evaluated by performing a series of "re-dockings" with direct comparison to top commercial docking software. Results of this evaluation showed that CHARMMing's docking implementation is comparable to many widely used software packages and validates the use of the new CHARMM generalized force field for docking and virtual screening.

Design and synthesis of dihydroisoquinolones for fragment-based drug discovery (FBDD).

Science.gov (United States)

Palmer, Nick; Peakman, Torren M; Norton, David; Rees, David C

2016-02-07

This study describes general synthesis aspects of fragments for FBDD, as illustrated by the dihydroisoquinolones 1-3. Previous Rh(III) methodology is extended to incorporate amines, heteroatoms (N and S), and substituents (halogen, ester) as potential binding groups and/or synthetic growth points for fragment-to-lead elaboration.

Immobilization of Fab' fragments onto substrate surfaces: A survey of methods and applications.

Science.gov (United States)

Crivianu-Gaita, Victor; Thompson, Michael

2015-08-15

Antibody immobilization onto surfaces has widespread applications in many different fields. It is desirable to bind antibodies such that their fragment-antigen-binding (Fab) units are oriented away from the surface in order to maximize analyte binding. The immobilization of only Fab' fragments yields benefits over the more traditional whole antibody immobilization technique. Bound Fab' fragments display higher surface densities, yielding a higher binding capacity for the analyte. The nucleophilic sulfide of the Fab' fragments allows for specific orientations to be achieved. For biosensors, this indicates a higher sensitivity and lower detection limit for a target analyte. The last thirty years have shown tremendous progress in the immobilization of Fab' fragments onto gold, Si-based, polysaccharide-based, plastic-based, magnetic, and inorganic surfaces. This review will show the current scope of Fab' immobilization techniques available and illustrate methods employed to minimize non-specific adsorption of undesirables. Furthermore, a variety of examples will be given to show the versatility of immobilized Fab' fragments in different applications and future directions of the field will be addressed, especially regarding biosensors. Copyright © 2015 Elsevier B.V. All rights reserved.

Fragment-based approaches to anti-HIV drug discovery: state of the art and future opportunities.

Science.gov (United States)

Huang, Boshi; Kang, Dongwei; Zhan, Peng; Liu, Xinyong

2015-12-01

The search for additional drugs to treat HIV infection is a continuing effort due to the emergence and spread of HIV strains resistant to nearly all current drugs. The recent literature reveals that fragment-based drug design/discovery (FBDD) has become an effective alternative to conventional high-throughput screening strategies for drug discovery. In this critical review, the authors describe the state of the art in FBDD strategies for the discovery of anti-HIV drug-like compounds. The article focuses on fragment screening techniques, direct fragment-based design and early hit-to-lead progress. Rapid progress in biophysical detection and in silico techniques has greatly aided the application of FBDD to discover candidate agents directed at a variety of anti-HIV targets. Growing evidence suggests that structural insights on key proteins in the HIV life cycle can be applied in the early phase of drug discovery campaigns, providing valuable information on the binding modes and efficiently prompting fragment hit-to-lead progression. The combination of structural insights with improved methodologies for FBDD, including the privileged fragment-based reconstruction approach, fragment hybridization based on crystallographic overlays, fragment growth exploiting dynamic combinatorial chemistry, and high-speed fragment assembly via diversity-oriented synthesis followed by in situ screening, offers the possibility of more efficient and rapid discovery of novel drugs for HIV-1 prevention or treatment. Though the use of FBDD in anti-HIV drug discovery is still in its infancy, it is anticipated that anti-HIV agents developed via fragment-based strategies will be introduced into the clinic in the future.

A Fragment-Based Ligand Screen Against Part of a Large Protein Machine: The ND1 Domains of the AAA+ ATPase p97/VCP.

Science.gov (United States)

Chimenti, Michael S; Bulfer, Stacie L; Neitz, R Jeffrey; Renslo, Adam R; Jacobson, Matthew P; James, Thomas L; Arkin, Michelle R; Kelly, Mark J S

2015-07-01

The ubiquitous AAA+ ATPase p97 functions as a dynamic molecular machine driving several cellular processes. It is essential in regulating protein homeostasis, and it represents a potential drug target for cancer, particularly when there is a greater reliance on the endoplasmic reticulum-associated protein degradation pathway and ubiquitin-proteasome pathway to degrade an overabundance of secreted proteins. Here, we report a case study for using fragment-based ligand design approaches against this large and dynamic hexamer, which has multiple potential binding sites for small molecules. A screen of a fragment library was conducted by surface plasmon resonance (SPR) and followed up by nuclear magnetic resonance (NMR), two complementary biophysical techniques. Virtual screening was also carried out to examine possible binding sites for the experimental hits and evaluate the potential utility of fragment docking for this target. Out of this effort, 13 fragments were discovered that showed reversible binding with affinities between 140 µM and 1 mM, binding stoichiometries of 1:1 or 2:1, and good ligand efficiencies. Structural data for fragment-protein interactions were obtained with residue-specific [U-(2)H] (13)CH3-methyl-labeling NMR strategies, and these data were compared to poses from docking. The combination of virtual screening, SPR, and NMR enabled us to find and validate a number of interesting fragment hits and allowed us to gain an understanding of the structural nature of fragment binding. © 2015 Society for Laboratory Automation and Screening.

Fragment growing and linking lead to novel nanomolar lactate dehydrogenase inhibitors.

Science.gov (United States)

Kohlmann, Anna; Zech, Stephan G; Li, Feng; Zhou, Tianjun; Squillace, Rachel M; Commodore, Lois; Greenfield, Matthew T; Lu, Xiaohui; Miller, David P; Huang, Wei-Sheng; Qi, Jiwei; Thomas, R Mathew; Wang, Yihan; Zhang, Sen; Dodd, Rory; Liu, Shuangying; Xu, Rongsong; Xu, Yongjin; Miret, Juan J; Rivera, Victor; Clackson, Tim; Shakespeare, William C; Zhu, Xiaotian; Dalgarno, David C

2013-02-14

Lactate dehydrogenase A (LDH-A) catalyzes the interconversion of lactate and pyruvate in the glycolysis pathway. Cancer cells rely heavily on glycolysis instead of oxidative phosphorylation to generate ATP, a phenomenon known as the Warburg effect. The inhibition of LDH-A by small molecules is therefore of interest for potential cancer treatments. We describe the identification and optimization of LDH-A inhibitors by fragment-based drug discovery. We applied ligand based NMR screening to identify low affinity fragments binding to LDH-A. The dissociation constants (K(d)) and enzyme inhibition (IC(50)) of fragment hits were measured by surface plasmon resonance (SPR) and enzyme assays, respectively. The binding modes of selected fragments were investigated by X-ray crystallography. Fragment growing and linking, followed by chemical optimization, resulted in nanomolar LDH-A inhibitors that demonstrated stoichiometric binding to LDH-A. Selected molecules inhibited lactate production in cells, suggesting target-specific inhibition in cancer cell lines.

Location and characterization of the warfarin binding site of human serum albumin A comparative study of two large fragments

NARCIS (Netherlands)

Bos, O.J.M.; Remijn, J.P.M.; Fischer, M.J.E.; Wilting, J.; Janssen, L.H.M.

1988-01-01

The warfarin binding behaviour of a large tryptic fragment (residues 198–585 which comprise domains two and three) and of a large peptic fragment (residues 1–387 which comprise domains one and two) of human serum albumin has been studied by circular dichroism and equilibrium dialysis in order to

Deciphering the Arginine-binding preferences at the substrate-binding groove of Ser/Thr kinases by computational surface mapping.

Directory of Open Access Journals (Sweden)

Avraham Ben-Shimon

2011-11-01

Full Text Available Protein kinases are key signaling enzymes that catalyze the transfer of γ-phosphate from an ATP molecule to a phospho-accepting residue in the substrate. Unraveling the molecular features that govern the preference of kinases for particular residues flanking the phosphoacceptor is important for understanding kinase specificities toward their substrates and for designing substrate-like peptidic inhibitors. We applied ANCHORSmap, a new fragment-based computational approach for mapping amino acid side chains on protein surfaces, to predict and characterize the preference of kinases toward Arginine binding. We focus on positions P-2 and P-5, commonly occupied by Arginine (Arg in substrates of basophilic Ser/Thr kinases. The method accurately identified all the P-2/P-5 Arg binding sites previously determined by X-ray crystallography and produced Arg preferences that corresponded to those experimentally found by peptide arrays. The predicted Arg-binding positions and their associated pockets were analyzed in terms of shape, physicochemical properties, amino acid composition, and in-silico mutagenesis, providing structural rationalization for previously unexplained trends in kinase preferences toward Arg moieties. This methodology sheds light on several kinases that were described in the literature as having non-trivial preferences for Arg, and provides some surprising departures from the prevailing views regarding residues that determine kinase specificity toward Arg. In particular, we found that the preference for a P-5 Arg is not necessarily governed by the 170/230 acidic pair, as was previously assumed, but by several different pairs of acidic residues, selected from positions 133, 169, and 230 (PKA numbering. The acidic residue at position 230 serves as a pivotal element in recognizing Arg from both the P-2 and P-5 positions.

A study on antigenicity and receptor-binding ability of fragment 450-650 of the spike protein of SARS coronavirus

International Nuclear Information System (INIS)

Zhao Jincun; Wang Wei; Yuan Zhihong; Jia Rujing; Zhao Zhendong; Xu Xiaojun; Lv Ping; Zhang Yan; Jiang Chengyu; Gao Xiaoming

2007-01-01

The spike (S) protein of SARS coronavirus (SARS-CoV) is responsible for viral binding with ACE2 molecules. Its receptor-binding motif (S-RBM) is located between residues 424 and 494, which folds into 2 anti-parallel β-sheets, β5 and β6. We have previously demonstrated that fragment 450-650 of the S protein (S450-650) is predominantly recognized by convalescent sera of SARS patients. The N-terminal 60 residues (450-510) of the S450-650 fragment covers the entire β6 strand of S-RBM. In the present study, we demonstrate that patient sera predominantly recognized 2 linear epitopes outside the β6 fragment, while the mouse antisera, induced by immunization of BALB/c mice with recombinant S450-650, mainly recognized the β6 strand-containing region. Unlike patient sera, however, the mouse antisera were unable to inhibit the infectivity of S protein-expressing (SARS-CoV-S) pseudovirus. Fusion protein between green fluorescence protein (GFP) and S450-650 (S450-650-GFP) was able to stain Vero E6 cells and deletion of the β6 fragment rendered the fusion product (S511-650-GFP) unable to do so. Similarly, recombinant S450-650, but not S511-650, was able to block the infection of Vero E6 cells by the SARS-CoV-S pseudovirus. Co-precipitation experiments confirmed that S450-650 was able to specifically bind with ACE2 molecules in lysate of Vero E6 cells. However, the ability of S450-510, either alone or in fusion with GFP, to bind with ACE2 was significantly poorer compared with S450-650. Our data suggest a possibility that, although the β6 strand alone is able to bind with ACE2 with relatively high affinity, residues outside the S-RBM could also assist the receptor binding of SARS-CoV-S protein

Physics-Based Fragment Acceleration Modeling for Pressurized Tank Burst Risk Assessments

Science.gov (United States)

Manning, Ted A.; Lawrence, Scott L.

2014-01-01

As part of comprehensive efforts to develop physics-based risk assessment techniques for space systems at NASA, coupled computational fluid and rigid body dynamic simulations were carried out to investigate the flow mechanisms that accelerate tank fragments in bursting pressurized vessels. Simulations of several configurations were compared to analyses based on the industry-standard Baker explosion model, and were used to formulate an improved version of the model. The standard model, which neglects an external fluid, was found to agree best with simulation results only in configurations where the internal-to-external pressure ratio is very high and fragment curvature is small. The improved model introduces terms that accommodate an external fluid and better account for variations based on circumferential fragment count. Physics-based analysis was critical in increasing the model's range of applicability. The improved tank burst model can be used to produce more accurate risk assessments of space vehicle failure modes that involve high-speed debris, such as exploding propellant tanks and bursting rocket engines.

Fragment-based virtual screening approach and molecular dynamics simulation studies for identification of BACE1 inhibitor leads.

Science.gov (United States)

Manoharan, Prabu; Ghoshal, Nanda

2018-05-01

Traditional structure-based virtual screening method to identify drug-like small molecules for BACE1 is so far unsuccessful. Location of BACE1, poor Blood Brain Barrier permeability and P-glycoprotein (Pgp) susceptibility of the inhibitors make it even more difficult. Fragment-based drug design method is suitable for efficient optimization of initial hit molecules for target like BACE1. We have developed a fragment-based virtual screening approach to identify/optimize the fragment molecules as a starting point. This method combines the shape, electrostatic, and pharmacophoric features of known fragment molecules, bound to protein conjugate crystal structure, and aims to identify both chemically and energetically feasible small fragment ligands that bind to BACE1 active site. The two top-ranked fragment hits were subjected for a 53 ns MD simulation. Principle component analysis and free energy landscape analysis reveal that the new ligands show the characteristic features of established BACE1 inhibitors. The potent method employed in this study may serve for the development of potential lead molecules for BACE1-directed Alzheimer's disease therapeutics.

When fragments link: a bibliometric perspective on the development of fragment-based drug discovery.

Science.gov (United States)

Romasanta, Angelo K S; van der Sijde, Peter; Hellsten, Iina; Hubbard, Roderick E; Keseru, Gyorgy M; van Muijlwijk-Koezen, Jacqueline; de Esch, Iwan J P

2018-05-05

Fragment-based drug discovery (FBDD) is a highly interdisciplinary field, rich in ideas integrated from pharmaceutical sciences, chemistry, biology, and physics, among others. To enrich our understanding of the development of the field, we used bibliometric techniques to analyze 3642 publications in FBDD, complementing accounts by key practitioners. Mapping its core papers, we found the transfer of knowledge from academia to industry. Co-authorship analysis showed that university-industry collaboration has grown over time. Moreover, we show how ideas from other scientific disciplines have been integrated into the FBDD paradigm. Keyword analysis showed that the field is organized into four interconnected practices: library design, fragment screening, computational methods, and optimization. This study highlights the importance of interactions among various individuals and institutions from diverse disciplines in newly emerging scientific fields. Copyright © 2018. Published by Elsevier Ltd.

Effective Fragment Potential Method for H-Bonding: How To Obtain Parameters for Nonrigid Fragments.

Science.gov (United States)

Dubinets, Nikita; Slipchenko, Lyudmila V

2017-07-20

Accuracy of the effective fragment potential (EFP) method was explored for describing intermolecular interaction energies in three dimers with strong H-bonded interactions, formic acid, formamide, and formamidine dimers, which are a part of HBC6 database of noncovalent interactions. Monomer geometries in these dimers change significantly as a function of intermonomer separation. Several EFP schemes were considered, in which fragment parameters were prepared for a fragment in its gas-phase geometry or recomputed for each unique fragment geometry. Additionally, a scheme in which gas-phase fragment parameters are shifted according to relaxed fragment geometries is introduced and tested. EFP data are compared against the coupled cluster with single, double, and perturbative triple excitations (CCSD(T)) method in a complete basis set (CBS) and the symmetry adapted perturbation theory (SAPT). All considered EFP schemes provide a good agreement with CCSD(T)/CBS for binding energies at equilibrium separations, with discrepancies not exceeding 2 kcal/mol. However, only the schemes that utilize relaxed fragment geometries remain qualitatively correct at shorter than equilibrium intermolecular distances. The EFP scheme with shifted parameters behaves quantitatively similar to the scheme in which parameters are recomputed for each monomer geometry and thus is recommended as a computationally efficient approach for large-scale EFP simulations of flexible systems.

Development of tight-binding based GW algorithm and its computational implementation for graphene

Energy Technology Data Exchange (ETDEWEB)

Majidi, Muhammad Aziz [Departemen Fisika, FMIPA, Universitas Indonesia, Kampus UI Depok (Indonesia); NUSNNI-NanoCore, Department of Physics, National University of Singapore (NUS), Singapore 117576 (Singapore); Singapore Synchrotron Light Source (SSLS), National University of Singapore (NUS), 5 Research Link, Singapore 117603 (Singapore); Naradipa, Muhammad Avicenna, E-mail: muhammad.avicenna11@ui.ac.id; Phan, Wileam Yonatan; Syahroni, Ahmad [Departemen Fisika, FMIPA, Universitas Indonesia, Kampus UI Depok (Indonesia); Rusydi, Andrivo [NUSNNI-NanoCore, Department of Physics, National University of Singapore (NUS), Singapore 117576 (Singapore); Singapore Synchrotron Light Source (SSLS), National University of Singapore (NUS), 5 Research Link, Singapore 117603 (Singapore)

2016-04-19

Graphene has been a hot subject of research in the last decade as it holds a promise for various applications. One interesting issue is whether or not graphene should be classified into a strongly or weakly correlated system, as the optical properties may change upon several factors, such as the substrate, voltage bias, adatoms, etc. As the Coulomb repulsive interactions among electrons can generate the correlation effects that may modify the single-particle spectra (density of states) and the two-particle spectra (optical conductivity) of graphene, we aim to explore such interactions in this study. The understanding of such correlation effects is important because eventually they play an important role in inducing the effective attractive interactions between electrons and holes that bind them into excitons. We do this study theoretically by developing a GW method implemented on the basis of the tight-binding (TB) model Hamiltonian. Unlike the well-known GW method developed within density functional theory (DFT) framework, our TB-based GW implementation may serve as an alternative technique suitable for systems which Hamiltonian is to be constructed through a tight-binding based or similar models. This study includes theoretical formulation of the Green’s function G, the renormalized interaction function W from random phase approximation (RPA), and the corresponding self energy derived from Feynman diagrams, as well as the development of the algorithm to compute those quantities. As an evaluation of the method, we perform calculations of the density of states and the optical conductivity of graphene, and analyze the results.

Development of tight-binding based GW algorithm and its computational implementation for graphene

International Nuclear Information System (INIS)

Majidi, Muhammad Aziz; Naradipa, Muhammad Avicenna; Phan, Wileam Yonatan; Syahroni, Ahmad; Rusydi, Andrivo

2016-01-01

Graphene has been a hot subject of research in the last decade as it holds a promise for various applications. One interesting issue is whether or not graphene should be classified into a strongly or weakly correlated system, as the optical properties may change upon several factors, such as the substrate, voltage bias, adatoms, etc. As the Coulomb repulsive interactions among electrons can generate the correlation effects that may modify the single-particle spectra (density of states) and the two-particle spectra (optical conductivity) of graphene, we aim to explore such interactions in this study. The understanding of such correlation effects is important because eventually they play an important role in inducing the effective attractive interactions between electrons and holes that bind them into excitons. We do this study theoretically by developing a GW method implemented on the basis of the tight-binding (TB) model Hamiltonian. Unlike the well-known GW method developed within density functional theory (DFT) framework, our TB-based GW implementation may serve as an alternative technique suitable for systems which Hamiltonian is to be constructed through a tight-binding based or similar models. This study includes theoretical formulation of the Green’s function G, the renormalized interaction function W from random phase approximation (RPA), and the corresponding self energy derived from Feynman diagrams, as well as the development of the algorithm to compute those quantities. As an evaluation of the method, we perform calculations of the density of states and the optical conductivity of graphene, and analyze the results.

Reconstruction of Banknote Fragments Based on Keypoint Matching Method.

Science.gov (United States)

Gwo, Chih-Ying; Wei, Chia-Hung; Li, Yue; Chiu, Nan-Hsing

2015-07-01

Banknotes may be shredded by a scrap machine, ripped up by hand, or damaged in accidents. This study proposes an image registration method for reconstruction of multiple sheets of banknotes. The proposed method first constructs different scale spaces to identify keypoints in the underlying banknote fragments. Next, the features of those keypoints are extracted to represent their local patterns around keypoints. Then, similarity is computed to find the keypoint pairs between the fragment and the reference banknote. The banknote fragments can determine the coordinate and amend the orientation. Finally, an assembly strategy is proposed to piece multiple sheets of banknote fragments together. Experimental results show that the proposed method causes, on average, a deviation of 0.12457 ± 0.12810° for each fragment while the SIFT method deviates 1.16893 ± 2.35254° on average. The proposed method not only reconstructs the banknotes but also decreases the computing cost. Furthermore, the proposed method can estimate relatively precisely the orientation of the banknote fragments to assemble. © 2015 American Academy of Forensic Sciences.

Computational Approaches to the Chemical Equilibrium Constant in Protein-ligand Binding.

Science.gov (United States)

Montalvo-Acosta, Joel José; Cecchini, Marco

2016-12-01

The physiological role played by protein-ligand recognition has motivated the development of several computational approaches to the ligand binding affinity. Some of them, termed rigorous, have a strong theoretical foundation but involve too much computation to be generally useful. Some others alleviate the computational burden by introducing strong approximations and/or empirical calibrations, which also limit their general use. Most importantly, there is no straightforward correlation between the predictive power and the level of approximation introduced. Here, we present a general framework for the quantitative interpretation of protein-ligand binding based on statistical mechanics. Within this framework, we re-derive self-consistently the fundamental equations of some popular approaches to the binding constant and pinpoint the inherent approximations. Our analysis represents a first step towards the development of variants with optimum accuracy/efficiency ratio for each stage of the drug discovery pipeline. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dynamics of fragments and associated phenomena in heavy-ion collisions using a modified secondary algorithm

Energy Technology Data Exchange (ETDEWEB)

Kumar, Rohit [Department of Physics, Panjab University, Chandigarh-160014 (India)

2016-05-06

We discuss the stability of fragments identified by secondary algorithms used to construct fragments within quantum molecular dynamics model. For this purpose we employ three different algorithms for fragment identification. 1) The conventional minimum spanning tree (MST) method based on the spatial correlations, 2) an improved version of MST with additional binding energy constraints of cold nuclear matter, 3) and that of hot matter. We find significant role of thermal binding energies over cold matter binding energies. Significant role is observed for fragment multiplicities and stopping of fragments. Whereas insignificant effect is observed on fragment’s flow.

AlphaSpace: Fragment-Centric Topographical Mapping To Target Protein–Protein Interaction Interfaces

Science.gov (United States)

2016-01-01

Inhibition of protein–protein interactions (PPIs) is emerging as a promising therapeutic strategy despite the difficulty in targeting such interfaces with drug-like small molecules. PPIs generally feature large and flat binding surfaces as compared to typical drug targets. These features pose a challenge for structural characterization of the surface using geometry-based pocket-detection methods. An attractive mapping strategy—that builds on the principles of fragment-based drug discovery (FBDD)—is to detect the fragment-centric modularity at the protein surface and then characterize the large PPI interface as a set of localized, fragment-targetable interaction regions. Here, we introduce AlphaSpace, a computational analysis tool designed for fragment-centric topographical mapping (FCTM) of PPI interfaces. Our approach uses the alpha sphere construct, a geometric feature of a protein’s Voronoi diagram, to map out concave interaction space at the protein surface. We introduce two new features—alpha-atom and alpha-space—and the concept of the alpha-atom/alpha-space pair to rank pockets for fragment-targetability and to facilitate the evaluation of pocket/fragment complementarity. The resulting high-resolution interfacial map of targetable pocket space can be used to guide the rational design and optimization of small molecule or biomimetic PPI inhibitors. PMID:26225450

Fragment-based quantitative structure-activity relationship (FB-QSAR) for fragment-based drug design.

Science.gov (United States)

Du, Qi-Shi; Huang, Ri-Bo; Wei, Yu-Tuo; Pang, Zong-Wen; Du, Li-Qin; Chou, Kuo-Chen

2009-01-30

In cooperation with the fragment-based design a new drug design method, the so-called "fragment-based quantitative structure-activity relationship" (FB-QSAR) is proposed. The essence of the new method is that the molecular framework in a family of drug candidates are divided into several fragments according to their substitutes being investigated. The bioactivities of molecules are correlated with the physicochemical properties of the molecular fragments through two sets of coefficients in the linear free energy equations. One coefficient set is for the physicochemical properties and the other for the weight factors of the molecular fragments. Meanwhile, an iterative double least square (IDLS) technique is developed to solve the two sets of coefficients in a training data set alternately and iteratively. The IDLS technique is a feedback procedure with machine learning ability. The standard Two-dimensional quantitative structure-activity relationship (2D-QSAR) is a special case, in the FB-QSAR, when the whole molecule is treated as one entity. The FB-QSAR approach can remarkably enhance the predictive power and provide more structural insights into rational drug design. As an example, the FB-QSAR is applied to build a predictive model of neuraminidase inhibitors for drug development against H5N1 influenza virus. (c) 2008 Wiley Periodicals, Inc.

Label free fragment screening using surface plasmon resonance as a tool for fragment finding - analyzing parkin, a difficult CNS target.

Directory of Open Access Journals (Sweden)

Karin Regnström

Full Text Available Surface Plasmon Resonance (SPR is rarely used as a primary High-throughput Screening (HTS tool in fragment-based approaches. With SPR instruments becoming increasingly high-throughput it is now possible to use SPR as a primary tool for fragment finding. SPR becomes, therefore, a valuable tool in the screening of difficult targets such as the ubiquitin E3 ligase Parkin. As a prerequisite for the screen, a large number of SPR tests were performed to characterize and validate the active form of Parkin. A set of compounds was designed and used to define optimal SPR assay conditions for this fragment screen. Using these conditions, more than 5000 pre-selected fragments from our in-house library were screened for binding to Parkin. Additionally, all fragments were simultaneously screened for binding to two off target proteins to exclude promiscuous binding compounds. A low hit rate was observed that is in line with hit rates usually obtained by other HTS screening assays. All hits were further tested in dose responses on the target protein by SPR for confirmation before channeling the hits into Nuclear Magnetic Resonance (NMR and other hit-confirmation assays.

Anticoagulant and calcium-binding properties of high molecular weight derivatives of human fibrinogen (plasmin fragments Y)

NARCIS (Netherlands)

Nieuwenhuizen, W.; Voskuilen, M.; Hermans, J.

1982-01-01

The present study was undertaken as a step to delineate further the localization of the calcium-binding sites in fibrinogen and to assess the anticlotting properties of fibrinogen degradation products. To this purpose, fragments Y were prepared by plasmin digestion of human fibrinogen in the

Introduction to fragment-based drug discovery.

Science.gov (United States)

Erlanson, Daniel A

2012-01-01

Fragment-based drug discovery (FBDD) has emerged in the past decade as a powerful tool for discovering drug leads. The approach first identifies starting points: very small molecules (fragments) that are about half the size of typical drugs. These fragments are then expanded or linked together to generate drug leads. Although the origins of the technique date back some 30 years, it was only in the mid-1990s that experimental techniques became sufficiently sensitive and rapid for the concept to be become practical. Since that time, the field has exploded: FBDD has played a role in discovery of at least 18 drugs that have entered the clinic, and practitioners of FBDD can be found throughout the world in both academia and industry. Literally dozens of reviews have been published on various aspects of FBDD or on the field as a whole, as have three books (Jahnke and Erlanson, Fragment-based approaches in drug discovery, 2006; Zartler and Shapiro, Fragment-based drug discovery: a practical approach, 2008; Kuo, Fragment based drug design: tools, practical approaches, and examples, 2011). However, this chapter will assume that the reader is approaching the field with little prior knowledge. It will introduce some of the key concepts, set the stage for the chapters to follow, and demonstrate how X-ray crystallography plays a central role in fragment identification and advancement.

Papain cleavage of the 38,000-dalton fragment inhibits the binding of 4, 4'-diisothiocyanostilbene-2, 2'-disulfonate to lys-539 on the 60,000-dalton fragment in human band 3.

Science.gov (United States)

Yamaguchi, Takeo; Kojima, Hideaki; Kawaguchi, Shiori; Shimada, Maiko; Aso, Haruka

2017-08-01

Human band 3 is a 98-kDa transmembrane (TM) protein comprising 14 TM segments. Papain cleavages band 3 into 38- and 60-kDa fragments. Under vigorous conditions, the cleavage of the loop region between the TM 7 of gate domain and the TM 8 of core domain in the 38-kDa fragment produces 7- and 31-kDa fragments. Conformational changes of the TM 5 segment containing Lys-539 by cleavage of the 38-kDa fragment remain unclear. Pressure-induced haemolysis of erythrocytes was suppressed by binding of 4, 4'-diisothiocyanostilbene-2, 2'-disulfonate (DIDS) to Lys-539. Such effect of DIDS was not observed upon cleavage of the 38-kDa fragment, because of inhibition of DIDS binding to Lys-539. Using fluorescence of DIDS labelled to Lys-539, conformational changes of band 3 were examined. Fluorescence spectra demonstrated that the molecular motion of DIDS is more restricted upon digestion of the 38-kDa fragment. Interestingly, the quenching of DIDS fluorescence showed that Hg2+ is less accessible to DIDS upon digestion of the 38-kDa fragment. Taken together, we propose that the conformational changes of the TM 5 segment characterized by the sequestration and restricted motion of Lys-539 are induced by the cleavage of the loop region between the TM 7 and the TM 8. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

An endogenously produced fragment of cardiac myosin-binding protein C is pathogenic and can lead to heart failure.

Science.gov (United States)

Razzaque, Md Abdur; Gupta, Manish; Osinska, Hanna; Gulick, James; Blaxall, Burns C; Robbins, Jeffrey

2013-08-16

A stable 40-kDa fragment is produced from cardiac myosin-binding protein C when the heart is stressed using a stimulus, such as ischemia-reperfusion injury. Elevated levels of the fragment can be detected in the diseased mouse and human heart, but its ability to interfere with normal cardiac function in the intact animal is unexplored. To understand the potential pathogenicity of the 40-kDa fragment in vivo and to investigate the molecular pathways that could be targeted for potential therapeutic intervention. We generated cardiac myocyte-specific transgenic mice using a Tet-Off inducible system to permit controlled expression of the 40-kDa fragment in cardiomyocytes. When expression of the 40-kDa protein is induced by crossing the responder animals with tetracycline transactivator mice under conditions in which substantial quantities approximating those observed in diseased hearts are reached, the double-transgenic mice subsequently experience development of sarcomere dysgenesis and altered cardiac geometry, and the heart fails between 12 and 17 weeks of age. The induced double-transgenic mice had development of cardiac hypertrophy with myofibrillar disarray and fibrosis, in addition to activation of pathogenic MEK-ERK pathways. Inhibition of MEK-ERK signaling was achieved by injection of the mitogen-activated protein kinase (MAPK)/ERK inhibitor U0126. The drug effectively improved cardiac function, normalized heart size, and increased probability of survival. These results suggest that the 40-kDa cardiac myosin-binding protein C fragment, which is produced at elevated levels during human cardiac disease, is a pathogenic fragment that is sufficient to cause hypertrophic cardiomyopathy and heart failure.

Fragment-based approaches to TB drugs.

Science.gov (United States)

Marchetti, Chiara; Chan, Daniel S H; Coyne, Anthony G; Abell, Chris

2018-02-01

Tuberculosis is an infectious disease associated with significant mortality and morbidity worldwide, particularly in developing countries. The rise of antibiotic resistance in Mycobacterium tuberculosis (Mtb) urgently demands the development of new drug leads to tackle resistant strains. Fragment-based methods have recently emerged at the forefront of pharmaceutical development as a means to generate more effective lead structures, via the identification of fragment molecules that form weak but high quality interactions with the target biomolecule and subsequent fragment optimization. This review highlights a number of novel inhibitors of Mtb targets that have been developed through fragment-based approaches in recent years.

Synthesis and NMR of {sup 15}N-labeled DNA fragments

Energy Technology Data Exchange (ETDEWEB)

Jones, R.A. [Rutgers, The State Univ. of New Jersey, Piscataway, NJ (United States)

1994-12-01

DNA fragments labeled with {sup 15}N at the ring nitrogens and at the exocyclic amino groups can be used to obtain novel insight into interactions such as base pairing, hydration, drug binding, and protein binding. A number of synthetic routes to {sup 15}N-labeled pyrimidine nucleosides, purines, and purine nucleosides have been reported. Moreover, many of these labeled bases or monomers have been incorporated into nucleic acids, either by chemical synthesis or by biosynthetic procedures. The focus of this chapter will be on the preparation of {sup 15}N-labeled purine 2{prime}-deoxynucleosides, their incorporation into DNA fragments by chemical synthesis, and the results of NMR studies using these labeled DNA fragments.

Binding of HIV-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (Fab and single chain variable fragment (ScFv antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations.

Directory of Open Access Journals (Sweden)

John M Louis

Full Text Available We previously reported a series of antibodies, in fragment antigen binding domain (Fab formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066 and non-neutralizing (8062 antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥ 150-fold in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

Morphological characteristics of the posterior malleolar fragment according to ankle fracture patterns: a computed tomography-based study.

Science.gov (United States)

Yi, Young; Chun, Dong-Il; Won, Sung Hun; Park, Suyeon; Lee, Sanghyeon; Cho, Jaeho

2018-02-13

The posterior malleolar fragment (PMF) of an ankle fracture can have various shapes depending on the injury mechanism. The purpose of this study was to evaluate the morphological characteristics of the PMF according to the ankle fracture pattern described in the Lauge-Hansen classification by using computed tomography (CT) images. We retrospectively analyzed CT data of 107 patients (107 ankles) who underwent surgery for trimalleolar fracture from January 2012 to December 2014. The patients were divided into two groups: 76 ankles in the supination-external rotation (SER) stage IV group and 31 ankles in the pronation-external rotation (PER) stage IV group. The PMF type of the two groups was assessed using the Haraguchi and Jan Bartonicek classification. The cross angle (α), fragment length ratio (FLR), fragment area ratio (FAR), sagittal angle (θ), and fragment height (FH) were measured to assess the morphological characteristics of the PMF. The PMF in the SER group mainly had a posterolateral shape, whereas that in the PER group mainly had a posteromedial two-part shape or a large posterolateral triangular shape (P = 0.02). The average cross angle was not significantly different between the two groups (SER group = 19.4°, PER group = 17.6°). The mean FLR and FH were significantly larger in the PER group than in the SER group (P = 0.024, P = 0.006). The mean fragment sagittal angle in the PER group was significantly smaller than that in the SER group (P = 0.017). With regard to the articular involvement, volume, and vertical nature, the SER-type fracture tends to have a smaller fragment due to the rotational force, whereas the PER-type fracture tends to have a larger fragment due to the combination of rotational and axial forces.

Target immobilization as a strategy for NMR-based fragment screening: comparison of TINS, STD, and SPR for fragment hit identification.

Science.gov (United States)

Kobayashi, Masakazu; Retra, Kim; Figaroa, Francis; Hollander, Johan G; Ab, Eiso; Heetebrij, Robert J; Irth, Hubertus; Siegal, Gregg

2010-09-01

Fragment-based drug discovery (FBDD) has become a widely accepted tool that is complementary to high-throughput screening (HTS) in developing small-molecule inhibitors of pharmaceutical targets. Because a fragment campaign can only be as successful as the hit matter found, it is critical that the first stage of the process be optimized. Here the authors compare the 3 most commonly used methods for hit discovery in FBDD: high concentration screening (HCS), solution ligand-observed nuclear magnetic resonance (NMR), and surface plasmon resonance (SPR). They selected the commonly used saturation transfer difference (STD) NMR spectroscopy and the proprietary target immobilized NMR screening (TINS) as representative of the array of possible NMR methods. Using a target typical of FBDD campaigns, the authors find that HCS and TINS are the most sensitive to weak interactions. They also find a good correlation between TINS and STD for tighter binding ligands, but the ability of STD to detect ligands with affinity weaker than 1 mM K(D) is limited. Similarly, they find that SPR detection is most suited to ligands that bind with K(D) better than 1 mM. However, the good correlation between SPR and potency in a bioassay makes this a good method for hit validation and characterization studies.

Fragment-Based Discovery of Pyrimido[1,2-b]indazole PDE10A Inhibitors.

Science.gov (United States)

Chino, Ayaka; Seo, Ryushi; Amano, Yasushi; Namatame, Ichiji; Hamaguchi, Wataru; Honbou, Kazuya; Mihara, Takuma; Yamazaki, Mayako; Tomishima, Masaki; Masuda, Naoyuki

2018-01-01

In this study, we report the identification of potent pyrimidoindazoles as phosphodiesterase10A (PDE10A) inhibitors by using the method of fragment-based drug discovery (FBDD). The pyrazolopyridine derivative 2 was found to be a fragment hit compound which could occupy a part of the binding site of PDE10A enzyme by using the method of the X-ray co-crystal structure analysis. On the basis of the crystal structure of compound 2 and PDE10A protein, a number of compounds were synthesized and evaluated, by means of structure-activity relationship (SAR) studies, which culminated in the discovery of a novel pyrimidoindazole derivative 13 having good physicochemical properties.

Dissecting Orthosteric Contacts for a Reverse-Fragment-Based Ligand Design.

Science.gov (United States)

Chandramohan, Arun; Tulsian, Nikhil K; Anand, Ganesh S

2017-08-01

Orthosteric sites on proteins are formed typically from noncontiguous interacting sites in three-dimensional space where the composite binding interaction of a biological ligand is mediated by multiple synergistic interactions of its constituent functional groups. Through these multiple interactions, ligands stabilize both the ligand binding site and the local secondary structure. However, relative energetic contributions of the individual contacts in these protein-ligand interactions are difficult to resolve. Deconvolution of the contributions of these various functional groups in natural inhibitors/ligand would greatly aid in iterative fragment-based drug discovery (FBDD). In this study, we describe an approach of progressive unfolding of a target protein using a gradient of denaturant urea to reveal the individual energetic contributions of various ligand-functional groups to the affinity of the entire ligand. Through calibrated unfolding of two protein-ligand systems: cAMP-bound regulatory subunit of Protein Kinase A (RIα) and IBMX-bound phosphodiesterase8 (PDE8), monitored by amide hydrogen-deuterium exchange mass spectrometry, we show progressive disruption of individual orthosteric contacts in the ligand binding sites, allowing us to rank the energetic contributions of these individual interactions. In the two cAMP-binding sites of RIα, exocyclic phosphate oxygens of cAMP were identified to mediate stronger interactions than ribose 2'-OH in both the RIα-cAMP binding interfaces. Further, we have also ranked the relative contributions of the different functional groups of IBMX based on their interactions with the orthosteric residues of PDE8. This strategy for deconstruction of individual binding sites and identification of the strongest functional group interaction in enzyme orthosteric sites offers a rational starting point for FBDD.

Large scale meta-analysis of fragment-based screening campaigns: privileged fragments and complementary technologies.

Science.gov (United States)

Kutchukian, Peter S; Wassermann, Anne Mai; Lindvall, Mika K; Wright, S Kirk; Ottl, Johannes; Jacob, Jaison; Scheufler, Clemens; Marzinzik, Andreas; Brooijmans, Natasja; Glick, Meir

2015-06-01

A first step in fragment-based drug discovery (FBDD) often entails a fragment-based screen (FBS) to identify fragment "hits." However, the integration of conflicting results from orthogonal screens remains a challenge. Here we present a meta-analysis of 35 fragment-based campaigns at Novartis, which employed a generic 1400-fragment library against diverse target families using various biophysical and biochemical techniques. By statistically interrogating the multidimensional FBS data, we sought to investigate three questions: (1) What makes a fragment amenable for FBS? (2) How do hits from different fragment screening technologies and target classes compare with each other? (3) What is the best way to pair FBS assay technologies? In doing so, we identified substructures that were privileged for specific target classes, as well as fragments that were privileged for authentic activity against many targets. We also revealed some of the discrepancies between technologies. Finally, we uncovered a simple rule of thumb in screening strategy: when choosing two technologies for a campaign, pairing a biochemical and biophysical screen tends to yield the greatest coverage of authentic hits. © 2014 Society for Laboratory Automation and Screening.

Stimulation of chymosin secretion by simultaneous expression with chymosin-binding llama single-domain antibody fragments in yeast

NARCIS (Netherlands)

Harmsen, M.M.; Smits, C.B.; Geus, de B.

2002-01-01

We studied the effect of coexpression of chymosin and chymosin-binding llama single-domain antibody fragments (VHHs) on the secretion of chymosin by Saccharomyces cerevisiae cells. A VHH expression library containing chymosin-specific VHHs was obtained by immunization of a llama and coexpressed with

Relationship between hot spot residues and ligand binding hot spots in protein-protein interfaces.

Science.gov (United States)

Zerbe, Brandon S; Hall, David R; Vajda, Sandor; Whitty, Adrian; Kozakov, Dima

2012-08-27

In the context of protein-protein interactions, the term "hot spot" refers to a residue or cluster of residues that makes a major contribution to the binding free energy, as determined by alanine scanning mutagenesis. In contrast, in pharmaceutical research, a hot spot is a site on a target protein that has high propensity for ligand binding and hence is potentially important for drug discovery. Here we examine the relationship between these two hot spot concepts by comparing alanine scanning data for a set of 15 proteins with results from mapping the protein surfaces for sites that can bind fragment-sized small molecules. We find the two types of hot spots are largely complementary; the residues protruding into hot spot regions identified by computational mapping or experimental fragment screening are almost always themselves hot spot residues as defined by alanine scanning experiments. Conversely, a residue that is found by alanine scanning to contribute little to binding rarely interacts with hot spot regions on the partner protein identified by fragment mapping. In spite of the strong correlation between the two hot spot concepts, they fundamentally differ, however. In particular, while identification of a hot spot by alanine scanning establishes the potential to generate substantial interaction energy with a binding partner, there are additional topological requirements to be a hot spot for small molecule binding. Hence, only a minority of hot spots identified by alanine scanning represent sites that are potentially useful for small inhibitor binding, and it is this subset that is identified by experimental or computational fragment screening.

Bootstrap embedding: An internally consistent fragment-based method

Energy Technology Data Exchange (ETDEWEB)

Welborn, Matthew; Tsuchimochi, Takashi; Van Voorhis, Troy [Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139 (United States)

2016-08-21

Strong correlation poses a difficult problem for electronic structure theory, with computational cost scaling quickly with system size. Fragment embedding is an attractive approach to this problem. By dividing a large complicated system into smaller manageable fragments “embedded” in an approximate description of the rest of the system, we can hope to ameliorate the steep cost of correlated calculations. While appealing, these methods often converge slowly with fragment size because of small errors at the boundary between fragment and bath. We describe a new electronic embedding method, dubbed “Bootstrap Embedding,” a self-consistent wavefunction-in-wavefunction embedding theory that uses overlapping fragments to improve the description of fragment edges. We apply this method to the one dimensional Hubbard model and a translationally asymmetric variant, and find that it performs very well for energies and populations. We find Bootstrap Embedding converges rapidly with embedded fragment size, overcoming the surface-area-to-volume-ratio error typical of many embedding methods. We anticipate that this method may lead to a low-scaling, high accuracy treatment of electron correlation in large molecular systems.

An ODP computational model of a cooperative binding object

Science.gov (United States)

Logé, Christophe; Najm, Elie; Chen, Ken

1997-12-01

A next generation of systems that should appear will have to manage simultaneously several geographically distributed users. These systems belong to the class of computer-supported cooperative work systems (CSCW). The development of such complex systems requires rigorous development methods and flexible open architectures. Open distributed processing (ODP) is a standardization effort that aims at providing such architectures. ODP features appropriate abstraction levels and a clear articulation between requirements, programming and infrastructure support. ODP advocates the use of formal methods for the specification of systems and components. The computational model, an object-based model, one of the abstraction levels identified within ODP, plays a central role in the global architecture. In this model, basic objects can be composed with communication and distribution abstractions (called binding objects) to form a computational specification of distributed systems, or applications. Computational specifications can then be mapped (in a mechanism akin to compilation) onto an engineering solution. We use an ODP-inspired method to computationally specify a cooperative system. We start from a general purpose component that we progressively refine into a collection of basic and binding objects. We focus on two issues of a co-authoring application, namely, dynamic reconfiguration and multiview synchronization. We discuss solutions for these issues and formalize them using the MT-LOTOS specification language that is currently studied in the ISO standardization formal description techniques group.

Impact of a Central Scaffold on the Binding Affinity of Fragment Pairs Isolated from DNA-Encoded Self-Assembling Chemical Libraries.

Science.gov (United States)

Bigatti, Martina; Dal Corso, Alberto; Vanetti, Sara; Cazzamalli, Samuele; Rieder, Ulrike; Scheuermann, Jörg; Neri, Dario; Sladojevich, Filippo

2017-11-08

The screening of encoded self-assembling chemical libraries allows the identification of fragment pairs that bind to adjacent pockets on target proteins of interest. For practical applications, it is necessary to link these ligand pairs into discrete organic molecules, devoid of any nucleic acid component. Here we describe the discovery of a synergistic binding pair for acid alpha-1 glycoprotein and a chemical strategy for the identification of optimal linkers, connecting the two fragments. The procedure yielded a set of small organic ligands, the best of which exhibited a dissociation constant of 9.9 nm, as measured in solution by fluorescence polarization. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hoogsteen base pairs proximal and distal to echinomycin binding sites on DNA

International Nuclear Information System (INIS)

Mendel, D.; Dervan, P.B.

1987-01-01

Forms of the DNA double helix containing non-Watson-Crick base-pairing have been discovered recently based on x-ray diffraction analysis of quionoxaline antibiotic-oligonucleotide complexes. In an effort to find evidence for Hoogsteen base-pairing at quinoxaline-binding sites in solution, chemical footprinting (differential cleavage reactivity) of echinomycin bound to DNA restriction fragments was examined. The authors report that purines (A>G) in the first and/or fourth base-pair positions of occupied echinomycin-binding sites are hyperreactive to diethyl pyrocarbonate. The correspondence of the solid-state data and the sites of diethyl pyrocarbonate hyperreactivity suggests that diethyl pyrocarbonate may be a sensitive reagent for the detection of Hoogsteen base-pairing in solution. Moreover, a 12-base-pair segment of alternating A-T DNA, which is 6 base pairs away from the nearest strong echinomycin-binding site, is also hyperreactive to diethyl pyrocarbonate in the presence of echinomycin. This hyperreactive segment may be an altered form of right-handed DNA that is entirely Hoogsteen base-paired

Insulin-Like Growth Factor Binding Protein 4 Fragments Provide Incremental Prognostic Information on Cardiovascular Events in Patients With ST-Segment Elevation Myocardial Infarction

DEFF Research Database (Denmark)

Hjortebjerg, Rikke; Lindberg, Søren; Pedersen, Sune

2017-01-01

BACKGROUND: Fragments of insulin-like growth factor binding protein 4 (IGFBP-4) are potential new biomarkers for cardiac risk assessment. The fragments are generated on specific cleavage by pregnancy-associated plasma protein-A, which exerts proatherogenic activity. This study investigated the pr...

[Reconstruction of long polynucleotide sequences from fragments using the Iskra-226 personal computer

Science.gov (United States)

Kostetskiĭ, P V; Dobrova, I E

1988-04-01

An algorithm for reconstructing long DNA sequences, i.e. arranging all overlapping gel readings in the contigs, and the corresponding BASIC programme for personal computer "Iskra-226" (USSR) are described. The contig construction begins with the search for all fragments overlapping the basic (longest) one follower by determination of coordinates of 5' ends of the overlapping fragments. Then the gel reading with minimal 5' end coordinate and the gel reading with maximal 3' end coordinate are selected and used as basic ones at the next assembly steps. The procedure is finished when no gel reading overlapping the basic one can be found. All gel readings entered the contig are ignored at the next steps of the assembly. Finally, one or several contigs consisted of DNA fragments are obtained. Effectiveness of the algorithm was tested on a model based on the multiple assembly of the nucleotide sequence, encoding the Na, K-ATPase alpha-subunit of pig kidney. The programme does not call for user's participation and can comprise contigs up to 10,000 nucleotides long.

Fragment-Based Screening of a Natural Product Library against 62 Potential Malaria Drug Targets Employing Native Mass Spectrometry

Science.gov (United States)

2018-01-01

Natural products are well known for their biological relevance, high degree of three-dimensionality, and access to areas of largely unexplored chemical space. To shape our understanding of the interaction between natural products and protein targets in the postgenomic era, we have used native mass spectrometry to investigate 62 potential protein targets for malaria using a natural-product-based fragment library. We reveal here 96 low-molecular-weight natural products identified as binding partners of 32 of the putative malarial targets. Seventy-nine (79) fragments have direct growth inhibition on Plasmodium falciparum at concentrations that are promising for the development of fragment hits against these protein targets. This adds a fragment library to the published HTS active libraries in the public domain. PMID:29436819

Virtual screening filters for the design of type II p38 MAP kinase inhibitors: a fragment based library generation approach.

Science.gov (United States)

Badrinarayan, Preethi; Sastry, G Narahari

2012-04-01

In this work, we introduce the development and application of a three-step scoring and filtering procedure for the design of type II p38 MAP kinase leads using allosteric fragments extracted from virtual screening hits. The design of the virtual screening filters is based on a thorough evaluation of docking methods, DFG-loop conformation, binding interactions and chemotype specificity of the 138 p38 MAP kinase inhibitors from Protein Data Bank bound to DFG-in and DFG-out conformations using Glide, GOLD and CDOCKER. A 40 ns molecular dynamics simulation with the apo, type I with DFG-in and type II with DFG-out forms was carried out to delineate the effects of structural variations on inhibitor binding. The designed docking-score and sub-structure filters were first tested on a dataset of 249 potent p38 MAP kinase inhibitors from seven diverse series and 18,842 kinase inhibitors from PDB, to gauge their capacity to discriminate between kinase and non-kinase inhibitors and likewise to selectively filter-in target-specific inhibitors. The designed filters were then applied in the virtual screening of a database of ten million (10⁷) compounds resulting in the identification of 100 hits. Based on their binding modes, 98 allosteric fragments were extracted from the hits and a fragment library was generated. New type II p38 MAP kinase leads were designed by tailoring the existing type I ATP site binders with allosteric fragments using a common urea linker. Target specific virtual screening filters can thus be easily developed for other kinases based on this strategy to retrieve target selective compounds. Copyright © 2012 Elsevier Inc. All rights reserved.

Correlation between CD16a binding and immuno effector functionality of an antigen specific immunoglobulin Fc fragment (Fcab).

Science.gov (United States)

Kainer, Manuela; Antes, Bernhard; Wiederkum, Susanne; Wozniak-Knopp, Gordana; Bauer, Anton; Rüker, Florian; Woisetschläger, Max

2012-10-15

Antigen binding immunoglobulin Fc fragments (Fcab) are generated by engineering loop regions in the CH3 domain of human IgG1 Fc. Variants of an Fcab specific for Her-2 were designed to display either enhanced (S239D:A330L:I332E) or diminished (L234A:L235A) binding affinities to the Fc receptor CD16a based on mutations described previously. The two mutant Fcab proteins demonstrated the expected modulation of CD16a binding. Interaction with recombinant or cell surface expressed Her-2 was unaffected in both mutants compared to the parental Fcab. Binding affinities for CD16a correlated with the ADCC-potencies of the Fcab variants. Additional studies indicated that the L234A:L235A variant Fcab had equivalent structural features as the unmodified Fcab since their DSC profiles were similar and antigen binding after re-folding upon partial heat denaturation had not changed. Introduction of the S239D:A330L:I332E mutations resulted in a significant reduction of the CH2 domain melting temperature, a moderate decrease of the thermal transition of the CH3 domain and lower antigen binding after thermal stress compared to the parental Fcab. We conclude that the known correlation between CD16a binding affinity and ADCC potency is also valid in Fcab proteins and that antigen specific Fcab molecules can be further engineered for fine tuning of immuno effector functions. Copyright © 2012 Elsevier Inc. All rights reserved.

Investigation on energetics of ex-vessel vapor explosion based on spontaneous nucleation fragmentation

International Nuclear Information System (INIS)

Liu, Jie; Koshizuka, Seiichi; Oka, Yoshiaki

2002-01-01

A computer code PROVER-I is developed for propagation phase of vapor explosion. A new thermal fragmentation model is proposed with three kinds of time scale for modeling instant fragmentation, spontaneous nucleation fragmentation and normal boiling fragmentation. The energetics of ex-vessel vapor explosion is investigated based on different fragmentation models. A higher pressure peak and a larger mechanical energy conversion ratio are obtained by spontaneous nucleation fragmentation. A smaller energy conversion ratio results from normal boiling fragmentation. When the delay time in thermal fragmentation model is near 0.0 ms, the pressure propagation behavior tends to be analogous with that in hydrodynamic fragmentation. If the delay time is longer, pressure attenuation occurs at the shock front. The high energy conversion ratio (>4%) is obtained in a small vapor volume fraction together with spontaneous nucleation fragmentation. These results are consistent with fuel-coolant interaction experiments with alumina melt. However, in larger vapor volume fraction conditions (α υ >0.3), the vapor explosion is weak. For corium melt, a coarse mixture with void fraction of more than 30% can be generated in the pre-mixing process because of its physical properties. In the mixture with such a high void fraction the energetic vapor explosion hardly takes place. (author)

Fragment-based identification of determinants of conformational and spectroscopic change at the ricin active site

Directory of Open Access Journals (Sweden)

Soares Alexei S

2007-11-01

Full Text Available Abstract Background Ricin is a potent toxin and known bioterrorism threat with no available antidote. The ricin A-chain (RTA acts enzymatically to cleave a specific adenine base from ribosomal RNA, thereby blocking translation. To understand better the relationship between ligand binding and RTA active site conformational change, we used a fragment-based approach to find a minimal set of bonding interactions able to induce rearrangements in critical side-chain positions. Results We found that the smallest ligand stabilizing an open conformer of the RTA active site pocket was an amide group, bound weakly by only a few hydrogen bonds to the protein. Complexes with small amide-containing molecules also revealed a switch in geometry from a parallel towards a splayed arrangement of an arginine-tryptophan cation-pi interaction that was associated with an increase and red-shift in tryptophan fluorescence upon ligand binding. Using the observed fluorescence signal, we determined the thermodynamic changes of adenine binding to the RTA active site, as well as the site-specific binding of urea. Urea binding had a favorable enthalpy change and unfavorable entropy change, with a ΔH of -13 ± 2 kJ/mol and a ΔS of -0.04 ± 0.01 kJ/(K*mol. The side-chain position of residue Tyr80 in a complex with adenine was found not to involve as large an overlap of rings with the purine as previously considered, suggesting a smaller role for aromatic stacking at the RTA active site. Conclusion We found that amide ligands can bind weakly but specifically to the ricin active site, producing significant shifts in positions of the critical active site residues Arg180 and Tyr80. These results indicate that fragment-based drug discovery methods are capable of identifying minimal bonding determinants of active-site side-chain rearrangements and the mechanistic origins of spectroscopic shifts. Our results suggest that tryptophan fluorescence provides a sensitive probe for the

EDF fragment relocation model based on the displacement of rigid bodies

International Nuclear Information System (INIS)

Callu, C.; Baron, D.; Ruck, J.M.

1997-01-01

In order to release the restricting conditions imposed to the reactor operations with regards to PCMI (Pellet-Cladding Mechanical Interaction), the simulation of a fuel rod thermomechanical behavior has to be improved. The computer programming has to cope with the more and more sophisticated mathematical modellings induced by the complexity and the interdependence of the phenomena. Therefore EDF is developing a new code - CYRANO3 - since 1990 putting emphasis on its evolution capacities. Concerning more precisely the PCMI simulation, the pellet fragmentation and the fragments relocation is one of the major aspect one must account for. Thanks to recent analytical experiments, EDF developed a new modelling based on the displacement of rigid bodies and on the calculation of the interaction efforts between the fragments. This paper presents the basis of the model, its introduction within the CYRANO3 code and its calibration on a specific analytical experiment. The modelling is then tested against PWR fuel rods deformations from the EDF data base. The results are presented and discussed. (author)

Computer systems for annotation of single molecule fragments

Science.gov (United States)

Schwartz, David Charles; Severin, Jessica

2016-07-19

There are provided computer systems for visualizing and annotating single molecule images. Annotation systems in accordance with this disclosure allow a user to mark and annotate single molecules of interest and their restriction enzyme cut sites thereby determining the restriction fragments of single nucleic acid molecules. The markings and annotations may be automatically generated by the system in certain embodiments and they may be overlaid translucently onto the single molecule images. An image caching system may be implemented in the computer annotation systems to reduce image processing time. The annotation systems include one or more connectors connecting to one or more databases capable of storing single molecule data as well as other biomedical data. Such diverse array of data can be retrieved and used to validate the markings and annotations. The annotation systems may be implemented and deployed over a computer network. They may be ergonomically optimized to facilitate user interactions.

Fragment-based discovery of a potent NAMPT inhibitor.

Science.gov (United States)

Korepanova, Alla; Longenecker, Kenton L; Pratt, Steve D; Panchal, Sanjay C; Clark, Richard F; Lake, Marc; Gopalakrishnan, Sujatha M; Raich, Diana; Sun, Chaohong; Petros, Andrew M

2017-12-12

NAMPT expression is elevated in many cancers, making this protein a potential target for anticancer therapy. We have carried out both NMR based and TR-FRET based fragment screens against human NAMPT and identified six novel binders with a range of potencies. Co-crystal structures were obtained for two of the fragments bound to NAMPT while for the other four fragments force-field driven docking was employed to generate a bound pose. Based on structural insights arising from comparison of the bound fragment poses to that of bound FK866 we were able to synthetically elaborate one of the fragments into a potent NAMPT inhibitor. Copyright © 2017 Elsevier Ltd. All rights reserved.

Optimizing virtual fragment screening for GPCRs: Identification of novel ligands for the histamine H3 receptor using ligand- and structure-based molecular fingerprints

NARCIS (Netherlands)

Sirci, F.; Istyastono, E.P.; Vischer, H.F.; Nijmeijer, S.; Kuijer, M.; Kooistra, A.J.; Wijtmans, M.; Mannhold, R.; Leurs, R.; de Esch, I.J.P.; de Graaf, C.

2012-01-01

Virtual fragment screening (VFS) is a promising new method that uses computer models to identify small, fragment-like biologically active molecules as useful starting points for fragment-based drug discovery (FBDD). Training sets of true active and inactive fragment-like molecules to construct and

Developments in SPR Fragment Screening.

Science.gov (United States)

Chavanieu, Alain; Pugnière, Martine

2016-01-01

Fragment-based approaches have played an increasing role alongside high-throughput screening in drug discovery for 15 years. The label-free biosensor technology based on surface plasmon resonance (SPR) is now sensitive and informative enough to serve during primary screens and validation steps. In this review, the authors discuss the role of SPR in fragment screening. After a brief description of the underlying principles of the technique and main device developments, they evaluate the advantages and adaptations of SPR for fragment-based drug discovery. SPR can also be applied to challenging targets such as membrane receptors and enzymes. The high-level of immobilization of the protein target and its stability are key points for a relevant screening that can be optimized using oriented immobilized proteins and regenerable sensors. Furthermore, to decrease the rate of false negatives, a selectivity test may be performed in parallel on the main target bearing the binding site mutated or blocked with a low-off-rate ligand. Fragment-based drug design, integrated in a rational workflow led by SPR, will thus have a predominant role for the next wave of drug discovery which could be greatly enhanced by new improvements in SPR devices.

The alpha-fetoprotein third domain receptor binding fragment: in search of scavenger and associated receptor targets.

Science.gov (United States)

Mizejewski, G J

2015-01-01

Recent studies have demonstrated that the carboxyterminal third domain of alpha-fetoprotein (AFP-CD) binds with various ligands and receptors. Reports within the last decade have established that AFP-CD contains a large fragment of amino acids that interact with several different receptor types. Using computer software specifically designed to identify protein-to-protein interaction at amino acid sequence docking sites, the computer searches identified several types of scavenger-associated receptors and their amino acid sequence locations on the AFP-CD polypeptide chain. The scavenger receptors (SRs) identified were CD36, CD163, Stabilin, SSC5D, SRB1 and SREC; the SR-associated receptors included the mannose, low-density lipoprotein receptors, the asialoglycoprotein receptor, and the receptor for advanced glycation endproducts (RAGE). Interestingly, some SR interaction sites were localized on the AFP-derived Growth Inhibitory Peptide (GIP) segment at amino acids #480-500. Following the detection studies, a structural subdomain analysis of both the receptor and the AFP-CD revealed the presence of epidermal growth factor (EGF) repeats, extracellular matrix-like protein regions, amino acid-rich motifs and dimerization subdomains. For the first time, it was reported that EGF-like sequence repeats were identified on each of the three domains of AFP. Thereafter, the localization of receptors on specific cell types were reviewed and their functions were discussed.

Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin: Fragment‐Based Drug Design Facilitated by Dynamic Combinatorial Chemistry

Science.gov (United States)

Mondal, Milon; Radeva, Nedyalka; Fanlo‐Virgós, Hugo; Otto, Sijbren; Klebe, Gerhard

2016-01-01

Abstract Fragment‐based drug design (FBDD) affords active compounds for biological targets. While there are numerous reports on FBDD by fragment growing/optimization, fragment linking has rarely been reported. Dynamic combinatorial chemistry (DCC) has become a powerful hit‐identification strategy for biological targets. We report the synergistic combination of fragment linking and DCC to identify inhibitors of the aspartic protease endothiapepsin. Based on X‐ray crystal structures of endothiapepsin in complex with fragments, we designed a library of bis‐acylhydrazones and used DCC to identify potent inhibitors. The most potent inhibitor exhibits an IC50 value of 54 nm, which represents a 240‐fold improvement in potency compared to the parent hits. Subsequent X‐ray crystallography validated the predicted binding mode, thus demonstrating the efficiency of the combination of fragment linking and DCC as a hit‐identification strategy. This approach could be applied to a range of biological targets, and holds the potential to facilitate hit‐to‐lead optimization. PMID:27400756

Fragment-Linking Approach Using (19)F NMR Spectroscopy To Obtain Highly Potent and Selective Inhibitors of β-Secretase.

Science.gov (United States)

Jordan, John B; Whittington, Douglas A; Bartberger, Michael D; Sickmier, E Allen; Chen, Kui; Cheng, Yuan; Judd, Ted

2016-04-28

Fragment-based drug discovery (FBDD) has become a widely used tool in small-molecule drug discovery efforts. One of the most commonly used biophysical methods in detecting weak binding of fragments is nuclear magnetic resonance (NMR) spectroscopy. In particular, FBDD performed with (19)F NMR-based methods has been shown to provide several advantages over (1)H NMR using traditional magnetization-transfer and/or two-dimensional methods. Here, we demonstrate the utility and power of (19)F-based fragment screening by detailing the identification of a second-site fragment through (19)F NMR screening that binds to a specific pocket of the aspartic acid protease, β-secretase (BACE-1). The identification of this second-site fragment allowed the undertaking of a fragment-linking approach, which ultimately yielded a molecule exhibiting a more than 360-fold increase in potency while maintaining reasonable ligand efficiency and gaining much improved selectivity over cathepsin-D (CatD). X-ray crystallographic studies of the molecules demonstrated that the linked fragments exhibited binding modes consistent with those predicted from the targeted screening approach, through-space NMR data, and molecular modeling.

Prolonged in vivo residence times of llama single-domain antibody fragments in pigs by binding to porcine immunoglobulins

NARCIS (Netherlands)

Harmsen, M.M.; Solt, van C.B.; Fijten, H.P.D.; Setten, van M.C.

2005-01-01

The therapeutic parenteral application of llama single-domain antibody fragments (VHHs) is hampered by their small size, resulting in a fast elimination from the body. Here we describe a method to increase the serum half-life of VHHs in pigs by fusion to another VHH binding to porcine immunoglobulin

Partial filling affinity capillary electrophoresis as a useful tool for fragment-based drug discovery: A proof of concept on thrombin.

Science.gov (United States)

Farcaş, E; Bouckaert, C; Servais, A-C; Hanson, J; Pochet, L; Fillet, M

2017-09-01

With the emergence of more challenging targets, a relatively new approach, fragment-based drug discovery (FBDD), proved its efficacy and gained increasing importance in the pharmaceutical industry. FBDD identifies low molecular-weight (MW) ligands (fragments) that bind to biologically important macromolecules, then a structure-guided fragment growing or merging approach is performed, contributing to the quality of the lead. However, to select the appropriate fragment to be evolved, sensitive analytical screening methods must be used to measure the affinity in the μM or even mM range. In this particular context, we developed a robust and selective partial filling affinity CE (ACE) method for the direct binding screening of a small fragment library in order to identify new thrombin inhibitors. To demonstrate the accuracy of our assay, the complex dissociation constants of three known thrombin inhibitors, namely benzamidine, p-aminobenzamidine and nafamostat were determined and found to be in good concordance with the previously reported values. Finally, the screening of a small library was performed and demonstrated the high discriminatory power of our method towards weak binders compared to classical spectrophotometric activity assay, proving the interest of our method in the context of FBDD. Copyright © 2017 Elsevier B.V. All rights reserved.

Measuring the temperature of hot nuclear fragments

International Nuclear Information System (INIS)

Wuenschel, S.; Bonasera, A.; May, L.W.; Souliotis, G.A.; Tripathi, R.; Galanopoulos, S.; Kohley, Z.; Hagel, K.; Shetty, D.V.; Huseman, K.; Soisson, S.N.; Stein, B.C.; Yennello, S.J.

2010-01-01

A new thermometer based on fragment momentum fluctuations is presented. This thermometer exhibited residual contamination from the collective motion of the fragments along the beam axis. For this reason, the transverse direction has been explored. Additionally, a mass dependence was observed for this thermometer. This mass dependence may be the result of the Fermi momentum of nucleons or the different properties of the fragments (binding energy, spin, etc.) which might be more sensitive to different densities and temperatures of the exploding fragments. We expect some of these aspects to be smaller for protons (and/or neutrons); consequently, the proton transverse momentum fluctuations were used to investigate the temperature dependence of the source.

Targeting Ligandable Pockets on Plant Homeodomain (PHD) Zinc Finger Domains by a Fragment-Based Approach.

Science.gov (United States)

Amato, Anastasia; Lucas, Xavier; Bortoluzzi, Alessio; Wright, David; Ciulli, Alessio

2018-04-20

Plant homeodomain (PHD) zinc fingers are histone reader domains that are often associated with human diseases. Despite this, they constitute a poorly targeted class of readers, suggesting low ligandability. Here, we describe a successful fragment-based campaign targeting PHD fingers from the proteins BAZ2A and BAZ2B as model systems. We validated a pool of in silico fragments both biophysically and structurally and solved the first crystal structures of PHD zinc fingers in complex with fragments bound to an anchoring pocket at the histone binding site. The best-validated hits were found to displace a histone H3 tail peptide in competition assays. This work identifies new chemical scaffolds that provide suitable starting points for future ligand optimization using structure-guided approaches. The demonstrated ligandability of the PHD reader domains could pave the way for the development of chemical probes to drug this family of epigenetic readers.

Efficient Double Fragmentation ChIP-seq Provides Nucleotide Resolution Protein-DNA Binding Profiles

NARCIS (Netherlands)

Mokry, Michal; Hatzis, Pantelis; de Bruijn, Ewart; Koster, Jan; Versteeg, Rogier; Schuijers, Jurian; van de Wetering, Marc; Guryev, Victor; Clevers, Hans; Cuppen, Edwin

2010-01-01

Immunoprecipitated crosslinked protein-DNA fragments typically range in size from several hundred to several thousand base pairs, with a significant part of chromatin being much longer than the optimal length for next-generation sequencing (NGS) procedures. Because these larger fragments may be

Fragment Based Strategies for Discovery of Novel HIV-1 Reverse Transcriptase and Integrase Inhibitors.

Science.gov (United States)

Latham, Catherine F; La, Jennifer; Tinetti, Ricky N; Chalmers, David K; Tachedjian, Gilda

2016-01-01

Human immunodeficiency virus (HIV) remains a global health problem. While combined antiretroviral therapy has been successful in controlling the virus in patients, HIV can develop resistance to drugs used for treatment, rendering available drugs less effective and limiting treatment options. Initiatives to find novel drugs for HIV treatment are ongoing, although traditional drug design approaches often focus on known binding sites for inhibition of established drug targets like reverse transcriptase and integrase. These approaches tend towards generating more inhibitors in the same drug classes already used in the clinic. Lack of diversity in antiretroviral drug classes can result in limited treatment options, as cross-resistance can emerge to a whole drug class in patients treated with only one drug from that class. A fresh approach in the search for new HIV-1 drugs is fragment-based drug discovery (FBDD), a validated strategy for drug discovery based on using smaller libraries of low molecular weight molecules (FBDD is aimed at not only finding novel drug scaffolds, but also probing the target protein to find new, often allosteric, inhibitory binding sites. Several fragment-based strategies have been successful in identifying novel inhibitory sites or scaffolds for two proven drug targets for HIV-1, reverse transcriptase and integrase. While any FBDD-generated HIV-1 drugs have yet to enter the clinic, recent FBDD initiatives against these two well-characterised HIV-1 targets have reinvigorated antiretroviral drug discovery and the search for novel classes of HIV-1 drugs.

A Fragment-Based Method of Creating Small-Molecule Libraries to Target the Aggregation of Intrinsically Disordered Proteins.

Science.gov (United States)

Joshi, Priyanka; Chia, Sean; Habchi, Johnny; Knowles, Tuomas P J; Dobson, Christopher M; Vendruscolo, Michele

2016-03-14

The aggregation process of intrinsically disordered proteins (IDPs) has been associated with a wide range of neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Currently, however, no drug in clinical use targets IDP aggregation. To facilitate drug discovery programs in this important and challenging area, we describe a fragment-based approach of generating small-molecule libraries that target specific IDPs. The method is based on the use of molecular fragments extracted from compounds reported in the literature to inhibit of the aggregation of IDPs. These fragments are used to screen existing large generic libraries of small molecules to form smaller libraries specific for given IDPs. We illustrate this approach by describing three distinct small-molecule libraries to target, Aβ, tau, and α-synuclein, which are three IDPs implicated in Alzheimer's and Parkinson's diseases. The strategy described here offers novel opportunities for the identification of effective molecular scaffolds for drug discovery for neurodegenerative disorders and to provide insights into the mechanism of small-molecule binding to IDPs.

Multi-fragment visibility determination in the context of order-independent transparency rendering

OpenAIRE

Marilena Maule

2015-01-01

Multi-fragment effects, in the computer-generated imagery context, are effects that determine pixel color based on information computed from more than one fragment. In such effects, the contribution of each fragment is extracted from its visibility with respect to a point of view. Seen through a pixel’s point of view, the visibility of one fragment depends on its spatial relationship with other fragments. This relationship can be reduced to the problem of sorting multiple fragments. Therefore...

Fragment screening of cyclin G-associated kinase by weak affinity chromatography.

Science.gov (United States)

Meiby, Elinor; Knapp, Stefan; Elkins, Jonathan M; Ohlson, Sten

2012-11-01

Fragment-based drug discovery (FBDD) has become a new strategy for drug discovery where lead compounds are evolved from small molecules. These fragments form low affinity interactions (dissociation constant (K(D)) = mM - μM) with protein targets, which require fragment screening methods of sufficient sensitivity. Weak affinity chromatography (WAC) is a promising new technology for fragment screening based on selective retention of fragments by a drug target. Kinases are a major pharmaceutical target, and FBDD has been successfully applied to several of these targets. In this work, we have demonstrated the potential to use WAC in combination with mass spectrometry (MS) detection for fragment screening of a kinase target-cyclin G-associated kinase (GAK). One hundred seventy fragments were selected for WAC screening by virtual screening of a commercial fragment library against the ATP-binding site of five different proteins. GAK protein was immobilized on a capillary HPLC column, and compound binding was characterized by frontal affinity chromatography. Compounds were screened in sets of 13 or 14, in combination with MS detection for enhanced throughput. Seventy-eight fragments (46 %) with K(D) < 200 μM were detected, including a few highly efficient GAK binders (K(D) of 2 μM; ligand efficiency = 0.51). Of special interest is that chiral screening by WAC may be possible, as two stereoisomeric fragments, which both contained one chiral center, demonstrated twin peaks. This ability, in combination with the robustness, sensitivity, and simplicity of WAC makes it a new method for fragment screening of considerable potential.

Fragment-Based, Structure-Enabled Discovery of Novel Pyridones and Pyridone Macrocycles as Potent Bromodomain and Extra-Terminal Domain (BET) Family Bromodomain Inhibitors

Energy Technology Data Exchange (ETDEWEB)

Wang, Le; Pratt, John K.; Soltwedel, Todd; Sheppard, George S.; Fidanze, Steven D.; Liu, Dachun; Hasvold, Lisa A.; Mantei, Robert A.; Holms, James H.; McClellan, William J.; Wendt, Michael D.; Wada, Carol; Frey, Robin; Hansen, T.Matthew; Hubbard, Robert; Park, Chang H.; Li, Leiming; Magoc, Terrance J.; Albert, Daniel H.; Lin, Xiaoyu; Warder, Scott E.; Kovar, Peter; Huang, Xiaoli; Wilcox, Denise; Wang, Rongqi; Rajaraman, Ganesh; Petros, Andrew M.; Hutchins, Charles W.; Panchal, Sanjay C.; Sun, Chaohong; Elmore, Steven W.; Shen, Yu; Kati, Warren M.; McDaniel, Keith F. (AbbVie)

2017-03-24

Members of the BET family of bromodomain containing proteins have been identified as potential targets for blocking proliferation in a variety of cancer cell lines. A two-dimensional NMR fragment screen for binders to the bromodomains of BRD4 identified a phenylpyridazinone fragment with a weak binding affinity (1, Ki = 160 μM). SAR investigation of fragment 1, aided by X-ray structure-based design, enabled the synthesis of potent pyridone and macrocyclic pyridone inhibitors exhibiting single digit nanomolar potency in both biochemical and cell based assays. Advanced analogs in these series exhibited high oral exposures in rodent PK studies and demonstrated significant tumor growth inhibition efficacy in mouse flank xenograft models.

FragIt: a tool to prepare input files for fragment based quantum chemical calculations.

Directory of Open Access Journals (Sweden)

Casper Steinmann

Full Text Available Near linear scaling fragment based quantum chemical calculations are becoming increasingly popular for treating large systems with high accuracy and is an active field of research. However, it remains difficult to set up these calculations without expert knowledge. To facilitate the use of such methods, software tools need to be available to support these methods and help to set up reasonable input files which will lower the barrier of entry for usage by non-experts. Previous tools relies on specific annotations in structure files for automatic and successful fragmentation such as residues in PDB files. We present a general fragmentation methodology and accompanying tools called FragIt to help setup these calculations. FragIt uses the SMARTS language to locate chemically appropriate fragments in large structures and is applicable to fragmentation of any molecular system given suitable SMARTS patterns. We present SMARTS patterns of fragmentation for proteins, DNA and polysaccharides, specifically for D-galactopyranose for use in cyclodextrins. FragIt is used to prepare input files for the Fragment Molecular Orbital method in the GAMESS program package, but can be extended to other computational methods easily.

Visualizing RNA Secondary Structure Base Pair Binding Probabilities using Nested Concave Hulls

OpenAIRE

Sansen , Joris; Bourqui , Romain; Thebault , Patricia; Allali , Julien; Auber , David

2015-01-01

International audience; The challenge 1 of the BIOVIS 2015 design contest consists in designing an intuitive visual depiction of base pairs binding probabilities for secondary structure of ncRNA. Our representation depicts the potential nucleotide pairs binding using nested concave hulls over the computed MFE ncRNA secondary structure. Thus, it allows to identify regions with a high level of uncertainty in the MFE computation and the structures which seem to match to reality.

Probing conformational states of glutaryl-CoA dehydrogenase by fragment screening

Energy Technology Data Exchange (ETDEWEB)

Begley, Darren W.; Davies, Douglas R.; Hartley, Robert C.; Hewitt, Stephen N.; Rychel, Amanda L.; Myler, Peter J.; Van Voorhis, Wesley C.; Staker, Bart L.; Stewart, Lance J. (Emerald)

2014-10-02

Glutaric acidemia type 1 is an inherited metabolic disorder which can cause macrocephaly, muscular rigidity, spastic paralysis and other progressive movement disorders in humans. The defects in glutaryl-CoA dehydrogenase (GCDH) associated with this disease are thought to increase holoenzyme instability and reduce cofactor binding. Here, the first structural analysis of a GCDH enzyme in the absence of the cofactor flavin adenine dinucleotide (FAD) is reported. The apo structure of GCDH from Burkholderia pseudomallei reveals a loss of secondary structure and increased disorder in the FAD-binding pocket relative to the ternary complex of the highly homologous human GCDH. After conducting a fragment-based screen, four small molecules were identified which bind to GCDH from B. pseudomallei. Complex structures were determined for these fragments, which cause backbone and side-chain perturbations to key active-site residues. Structural insights from this investigation highlight differences from apo GCDH and the utility of small-molecular fragments as chemical probes for capturing alternative conformational states of preformed protein crystals.

Identification of potential glutaminyl cyclase inhibitors from lead-like libraries by in silico and in vitro fragment-based screening.

Science.gov (United States)

Szaszkó, Mária; Hajdú, István; Flachner, Beáta; Dobi, Krisztina; Magyar, Csaba; Simon, István; Lőrincz, Zsolt; Kapui, Zoltán; Pázmány, Tamás; Cseh, Sándor; Dormán, György

2017-02-01

A glutaminyl cyclase (QC) fragment library was in silico selected by disconnection of the structure of known QC inhibitors and by lead-like 2D virtual screening of the same set. The resulting fragment library (204 compounds) was acquired from commercial suppliers and pre-screened by differential scanning fluorimetry followed by functional in vitro assays. In this way, 10 fragment hits were identified ([Formula: see text]5 % hit rate, best inhibitory activity: 16 [Formula: see text]). The in vitro hits were then docked to the active site of QC, and the best scoring compounds were analyzed for binding interactions. Two fragments bound to different regions in a complementary manner, and thus, linking those fragments offered a rational strategy to generate novel QC inhibitors. Based on the structure of the virtual linked fragment, a 77-membered QC target focused library was selected from vendor databases and docked to the active site of QC. A PubChem search confirmed that the best scoring analogues are novel, potential QC inhibitors.

Specific labeling of the thyroxine binding site in thyroxine-binding globulin: determination of the amino acid composition of a labeled peptide fragment isolated from a proteolytic digest of the derivatized protein.

Science.gov (United States)

Tabachnick, M; Perret, V

1987-08-01

[125I] Thyroxine has been covalently bound to the thyroxine binding site in thyroxine-binding globulin by reaction with the bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. An average of 0.47 mol of [125I] thyroxine was incorporated per mol protein; nonspecific binding amounted to 8%. A labeled peptide fragment was isolated from a proteolytic digest of the derivatized protein by HPLC and its amino acid composition was determined. Comparison with the amino acid sequence of thyroxine-binding globulin indicated partial correspondence of the labeled peptide with two possible regions in the protein. These regions also coincide with part of the barrel structure present in the closely homologous protein, alpha 1-antitrypsin.

Computational prediction of binding affinity for CYP1A2-ligand complexes using empirical free energy calculations

DEFF Research Database (Denmark)

Poongavanam, Vasanthanathan; Olsen, Lars; Jørgensen, Flemming Steen

2010-01-01

, and methods based on statistical mechanics. In the present investigation, we started from an LIE model to predict the binding free energy of structurally diverse compounds of cytochrome P450 1A2 ligands, one of the important human metabolizing isoforms of the cytochrome P450 family. The data set includes both...... substrates and inhibitors. It appears that the electrostatic contribution to the binding free energy becomes negligible in this particular protein and a simple empirical model was derived, based on a training set of eight compounds. The root mean square error for the training set was 3.7 kJ/mol. Subsequent......Predicting binding affinities for receptor-ligand complexes is still one of the challenging processes in computational structure-based ligand design. Many computational methods have been developed to achieve this goal, such as docking and scoring methods, the linear interaction energy (LIE) method...

PatchSurfers: Two methods for local molecular property-based binding ligand prediction.

Science.gov (United States)

Shin, Woong-Hee; Bures, Mark Gregory; Kihara, Daisuke

2016-01-15

Protein function prediction is an active area of research in computational biology. Function prediction can help biologists make hypotheses for characterization of genes and help interpret biological assays, and thus is a productive area for collaboration between experimental and computational biologists. Among various function prediction methods, predicting binding ligand molecules for a target protein is an important class because ligand binding events for a protein are usually closely intertwined with the proteins' biological function, and also because predicted binding ligands can often be directly tested by biochemical assays. Binding ligand prediction methods can be classified into two types: those which are based on protein-protein (or pocket-pocket) comparison, and those that compare a target pocket directly to ligands. Recently, our group proposed two computational binding ligand prediction methods, Patch-Surfer, which is a pocket-pocket comparison method, and PL-PatchSurfer, which compares a pocket to ligand molecules. The two programs apply surface patch-based descriptions to calculate similarity or complementarity between molecules. A surface patch is characterized by physicochemical properties such as shape, hydrophobicity, and electrostatic potentials. These properties on the surface are represented using three-dimensional Zernike descriptors (3DZD), which are based on a series expansion of a 3 dimensional function. Utilizing 3DZD for describing the physicochemical properties has two main advantages: (1) rotational invariance and (2) fast comparison. Here, we introduce Patch-Surfer and PL-PatchSurfer with an emphasis on PL-PatchSurfer, which is more recently developed. Illustrative examples of PL-PatchSurfer performance on binding ligand prediction as well as virtual drug screening are also provided. Copyright © 2015 Elsevier Inc. All rights reserved.

Computational characteristics of valproic acid binding to histone deacetylase

International Nuclear Information System (INIS)

Abou-Zeid, Laila A.; El-Mowafy, Abdalla M.; Eikel, D.; Nau, H.; El-Mazar, M.

2007-01-01

Recently, the anticpileptic drug valproic acid (VPA) has also demonstrated efficacy in the management of cancer and bipolar disorders. These actions are largely mediated by inhibition of the HDAC enzyme/induction of certain genes. Relative to other HDAC inhibitors such as trichostatin-A (TSA), VPA offers higher selectivity on cancer cells with virtually no detrimental effects on normal cells. The molecular underpinnings of these biological profiles for VPA remain undefined. We currently propose for an attempt to identify differences in the binding of VPA and TSA to HDAC. In this paper, conformational changes and energy calculations have derived. VPA had to accomplish conformational changes in its structure for best accommodation at the HDAC binding site. Energy computations showed that VPA has a lower binding affinitythan TSA (-53.80 vs. -66.30 Kcal/mol). These findings demonstrate that VPA binding to HDAC confers catalytic, conformational and computational characteristics that are distinct from those of TSA. These findings of VPA are consistent with a moderate inhibition of HDAC, a low toxicity on normal cells, and a higher selectivity on cancer cells than TSA. Accordingly, these newly identified binding properties of VPA can state a framework strategy for the rational design of VPA-related anticancer drugs with superior cytodifferentiating-and/or safety-profiles. (author)

Molecular-Simulation-Driven Fragment Screening for the Discovery of New CXCL12 Inhibitors.

Science.gov (United States)

Martinez-Rosell, Gerard; Harvey, Matt J; De Fabritiis, Gianni

2018-03-26

Fragment-based drug discovery (FBDD) has become a mainstream approach in drug design because it allows the reduction of the chemical space and screening libraries while identifying fragments with high protein-ligand efficiency interactions that can later be grown into drug-like leads. In this work, we leverage high-throughput molecular dynamics (MD) simulations to screen a library of 129 fragments for a total of 5.85 ms against the CXCL12 monomer, a chemokine involved in inflammation and diseases such as cancer. Our in silico binding assay was able to recover binding poses, affinities, and kinetics for the selected library and was able to predict 8 mM-affinity fragments with ligand efficiencies higher than 0.3. All of the fragment hits present a similar chemical structure, with a hydrophobic core and a positively charged group, and bind to either sY7 or H1S68 pockets, where they share pharmacophoric properties with experimentally resolved natural binders. This work presents a large-scale screening assay using an exclusive combination of thousands of short MD adaptive simulations analyzed with a Markov state model (MSM) framework.

Native State Mass Spectrometry, Surface Plasmon Resonance, and X-ray Crystallography Correlate Strongly as a Fragment Screening Combination.

Science.gov (United States)

Woods, Lucy A; Dolezal, Olan; Ren, Bin; Ryan, John H; Peat, Thomas S; Poulsen, Sally-Ann

2016-03-10

Fragment-based drug discovery (FBDD) is contingent on the development of analytical methods to identify weak protein-fragment noncovalent interactions. Herein we have combined an underutilized fragment screening method, native state mass spectrometry, together with two proven and popular fragment screening methods, surface plasmon resonance and X-ray crystallography, in a fragment screening campaign against human carbonic anhydrase II (CA II). In an initial fragment screen against a 720-member fragment library (the "CSIRO Fragment Library") seven CA II binding fragments, including a selection of nonclassical CA II binding chemotypes, were identified. A further 70 compounds that comprised the initial hit chemotypes were subsequently sourced from the full CSIRO compound collection and screened. The fragment results were extremely well correlated across the three methods. Our findings demonstrate that there is a tremendous opportunity to apply native state mass spectrometry as a complementary fragment screening method to accelerate drug discovery.

Fragment-based approaches to the discovery of kinase inhibitors.

Science.gov (United States)

Mortenson, Paul N; Berdini, Valerio; O'Reilly, Marc

2014-01-01

Protein kinases are one of the most important families of drug targets, and aberrant kinase activity has been linked to a large number of disease areas. Although eminently targetable using small molecules, kinases present a number of challenges as drug targets, not least obtaining selectivity across such a large and relatively closely related target family. Fragment-based drug discovery involves screening simple, low-molecular weight compounds to generate initial hits against a target. These hits are then optimized to more potent compounds via medicinal chemistry, usually facilitated by structural biology. Here, we will present a number of recent examples of fragment-based approaches to the discovery of kinase inhibitors, detailing the construction of fragment-screening libraries, the identification and validation of fragment hits, and their optimization into potent and selective lead compounds. The advantages of fragment-based methodologies will be discussed, along with some of the challenges associated with using this route. Finally, we will present a number of key lessons derived both from our own experience running fragment screens against kinases and from a large number of published studies.

High-throughput fragment screening by affinity LC-MS.

Science.gov (United States)

Duong-Thi, Minh-Dao; Bergström, Maria; Fex, Tomas; Isaksson, Roland; Ohlson, Sten

2013-02-01

Fragment screening, an emerging approach for hit finding in drug discovery, has recently been proven effective by its first approved drug, vemurafenib, for cancer treatment. Techniques such as nuclear magnetic resonance, surface plasmon resonance, and isothemal titration calorimetry, with their own pros and cons, have been employed for screening fragment libraries. As an alternative approach, screening based on high-performance liquid chromatography separation has been developed. In this work, we present weak affinity LC/MS as a method to screen fragments under high-throughput conditions. Affinity-based capillary columns with immobilized thrombin were used to screen a collection of 590 compounds from a fragment library. The collection was divided into 11 mixtures (each containing 35 to 65 fragments) and screened by MS detection. The primary screening was performed in 3500 fragments per day). Thirty hits were defined, which subsequently entered a secondary screening using an active site-blocked thrombin column for confirmation of specificity. One hit showed selective binding to thrombin with an estimated dissociation constant (K (D)) in the 0.1 mM range. This study shows that affinity LC/MS is characterized by high throughput, ease of operation, and low consumption of target and fragments, and therefore it promises to be a valuable method for fragment screening.

Biochemical analyses indicate that binding and cleavage specificities define the ordered processing of human Okazaki fragments by Dna2 and FEN1.

Science.gov (United States)

Gloor, Jason W; Balakrishnan, Lata; Campbell, Judith L; Bambara, Robert A

2012-08-01

In eukaryotic Okazaki fragment processing, the RNA primer is displaced into a single-stranded flap prior to removal. Evidence suggests that some flaps become long before they are cleaved, and that this cleavage involves the sequential action of two nucleases. Strand displacement characteristics of the polymerase show that a short gap precedes the flap during synthesis. Using biochemical techniques, binding and cleavage assays presented here indicate that when the flap is ∼ 30 nt long the nuclease Dna2 can bind with high affinity to the flap and downstream double strand and begin cleavage. When the polymerase idles or dissociates the Dna2 can reorient for additional contacts with the upstream primer region, allowing the nuclease to remain stably bound as the flap is further shortened. The DNA can then equilibrate to a double flap that can bind Dna2 and flap endonuclease (FEN1) simultaneously. When Dna2 shortens the flap even more, FEN1 can displace the Dna2 and cleave at the flap base to make a nick for ligation.

Small molecule inhibitors of the LEDGF site of human immunodeficiency virus integrase identified by fragment screening and structure based design.

Directory of Open Access Journals (Sweden)

Thomas S Peat

Full Text Available A fragment-based screen against human immunodeficiency virus type 1 (HIV integrase led to a number of compounds that bound to the lens epithelium derived growth factor (LEDGF binding site of the integrase catalytic core domain. We determined the crystallographic structures of complexes of the HIV integrase catalytic core domain for 10 of these compounds and quantitated the binding by surface plasmon resonance. We demonstrate that the compounds inhibit the interaction of LEDGF with HIV integrase in a proximity AlphaScreen assay, an assay for the LEDGF enhancement of HIV integrase strand transfer and in a cell based assay. The compounds identified represent a potential framework for the development of a new series of HIV integrase inhibitors that do not bind to the catalytic site of the enzyme.

Analysis of fission-fragment mass distribution within the quantum-mechanical fragmentation theory

Energy Technology Data Exchange (ETDEWEB)

Singh, Pardeep; Kaur, Harjeet [Guru Nanak Dev University, Department of Physics, Amritsar (India)

2016-11-15

The fission-fragment mass distribution is analysed for the {sup 208}Pb({sup 18}O, f) reaction within the quantum-mechanical fragmentation theory (QMFT). The reaction potential has been calculated by taking the binding energies, Coulomb potential and proximity potential of all possible decay channels and a stationary Schroedinger equation has been solved numerically to calculate the fission-fragment yield. The overall results for mass distribution are compared with those obtained in experiment. Fine structure dips in yield, corresponding to fragment shell closures at Z = 50 and N=82, which are observed by Bogachev et al., are reproduced successfully in the present calculations. These calculations will help to estimate the formation probabilities of fission fragments and to understand many related phenomena occurring in the fission process. (orig.)

Analysis of Behind-Armor Debris Fragments From Physics-Based Simulations

National Research Council Canada - National Science Library

Clarke, Jerry a; Meyer, Hubert W; Mark, Eric R

2007-01-01

.... Utilizing the capabilities of the interdisciplinary computing environment, we have developed a tool that is capable of identifying individual fragments in a CTH flat mesh or adaptive mesh refinement calculation...

Definition of IgG- and albumin-binding regions of streptococcal protein G.

Science.gov (United States)

Akerström, B; Nielsen, E; Björck, L

1987-10-05

Protein G, the immunoglobin G-binding surface protein of group C and G streptococci, also binds serum albumin. The albumin-binding site on protein G is distinct from the immunoglobulin G-binding site. By mild acid hydrolysis of the papain-liberated protein G fragment (35 kDa), a 28-kDa fragment was produced which retained full immunoglobulin G-binding activity (determined by Scatchard plotting) but had lost all albumin-binding capacity. A protein G (65 kDa), isolated after cloning and expression of the protein G gene in Escherichia coli, had comparable affinity to immunoglobulin G (5-10 X 10(10)M-1), but much higher affinity to albumin than the 35- and 28-kDa protein G fragments (31, 2.6, and 0 X 10(9)M-1, respectively). The amino-terminal amino acid sequences of the 65-, 35-, and 28-kDa fragments allowed us to exactly locate the three fragments in an overall sequence map of protein G, based on the partial gene sequences published by Guss et al. (Guss, B., Eliasson, M., Olsson, A., Uhlen, M., Frej, A.-K., Jörnvall, H., Flock, J.-I., and Lindberg, M. (1986) EMBO J. 5, 1567-1575) and Fahnestock et al. (Fahnestock, S. R., Alexander, P., Nagle, J., and Filpula, D. (1986) J. Bacteriol. 167, 870-880). In this map could then be deduced the location of three homologous albumin-binding regions and three homologous immunoglobulin G-binding regions.

A Bioinorganic Approach to Fragment-Based Drug Discovery Targeting Metalloenzymes.

Science.gov (United States)

Cohen, Seth M

2017-08-15

Metal-dependent enzymes (i.e., metalloenzymes) make up a large fraction of all enzymes and are critically important in a wide range of biological processes, including DNA modification, protein homeostasis, antibiotic resistance, and many others. Consequently, metalloenzymes represent a vast and largely untapped space for drug development. The discovery of effective therapeutics that target metalloenzymes lies squarely at the interface of bioinorganic and medicinal chemistry and requires expertise, methods, and strategies from both fields to mount an effective campaign. In this Account, our research program that brings together the principles and methods of bioinorganic and medicinal chemistry are described, in an effort to bridge the gap between these fields and address an important class of medicinal targets. Fragment-based drug discovery (FBDD) is an important drug discovery approach that is particularly well suited for metalloenzyme inhibitor development. FBDD uses relatively small but diverse chemical structures that allow for the assembly of privileged molecular collections that focus on a specific feature of the target enzyme. For metalloenzyme inhibition, the specific feature is rather obvious, namely, a metal-dependent active site. Surprisingly, prior to our work, the exploration of diverse molecular fragments for binding the metal active sites of metalloenzymes was largely unexplored. By assembling a modest library of metal-binding pharmacophores (MBPs), we have been able to find lead hits for many metalloenzymes and, from these hits, develop inhibitors that act via novel mechanisms of action. A specific case study on the use of this strategy to identify a first-in-class inhibitor of zinc-dependent Rpn11 (a component of the proteasome) is highlighted. The application of FBDD for the development of metalloenzyme inhibitors has raised several other compelling questions, such as how the metalloenzyme active site influences the coordination chemistry of bound

NMR-Fragment Based Virtual Screening: A Brief Overview.

Science.gov (United States)

Singh, Meenakshi; Tam, Benjamin; Akabayov, Barak

2018-01-25

Fragment-based drug discovery (FBDD) using NMR has become a central approach over the last twenty years for development of small molecule inhibitors against biological macromolecules, to control a variety of cellular processes. Yet, several considerations should be taken into account for obtaining a therapeutically relevant agent. In this review, we aim to list the considerations that make NMR fragment screening a successful process for yielding potent inhibitors. Factors that may govern the competence of NMR in fragment based drug discovery are discussed, as well as later steps that involve optimization of hits obtained by NMR-FBDD.

NMR-Fragment Based Virtual Screening: A Brief Overview

Directory of Open Access Journals (Sweden)

Meenakshi Singh

2018-01-01

Full Text Available Fragment-based drug discovery (FBDD using NMR has become a central approach over the last twenty years for development of small molecule inhibitors against biological macromolecules, to control a variety of cellular processes. Yet, several considerations should be taken into account for obtaining a therapeutically relevant agent. In this review, we aim to list the considerations that make NMR fragment screening a successful process for yielding potent inhibitors. Factors that may govern the competence of NMR in fragment based drug discovery are discussed, as well as later steps that involve optimization of hits obtained by NMR-FBDD.

Monte Carlo simulation as a tool to predict blasting fragmentation based on the Kuz Ram model

Science.gov (United States)

Morin, Mario A.; Ficarazzo, Francesco

2006-04-01

Rock fragmentation is considered the most important aspect of production blasting because of its direct effects on the costs of drilling and blasting and on the economics of the subsequent operations of loading, hauling and crushing. Over the past three decades, significant progress has been made in the development of new technologies for blasting applications. These technologies include increasingly sophisticated computer models for blast design and blast performance prediction. Rock fragmentation depends on many variables such as rock mass properties, site geology, in situ fracturing and blasting parameters and as such has no complete theoretical solution for its prediction. However, empirical models for the estimation of size distribution of rock fragments have been developed. In this study, a blast fragmentation Monte Carlo-based simulator, based on the Kuz-Ram fragmentation model, has been developed to predict the entire fragmentation size distribution, taking into account intact and joints rock properties, the type and properties of explosives and the drilling pattern. Results produced by this simulator were quite favorable when compared with real fragmentation data obtained from a blast quarry. It is anticipated that the use of Monte Carlo simulation will increase our understanding of the effects of rock mass and explosive properties on the rock fragmentation by blasting, as well as increase our confidence in these empirical models. This understanding will translate into improvements in blasting operations, its corresponding costs and the overall economics of open pit mines and rock quarries.

Screening Mixtures of Small Molecules for Binding to Multiple Sites on the Surface Tetanus Toxin C Fragment by Bioaffinity NMR

International Nuclear Information System (INIS)

Cosman, M; Zeller, L; Lightstone, F C; Krishnan, V V; Balhorn, R

2002-01-01

The clostridial neurotoxins include the closely related tetanus (TeNT) and botulinum (BoNT) toxins. Botulinum toxin is used to treat severe muscle disorders and as a cosmetic wrinkle reducer. Large quantities of botulinum toxin have also been produced by terrorists for use as a biological weapon. Because there are no known antidotes for these toxins, they thus pose a potential threat to human health whether by an accidental overdose or by a hostile deployment. Thus, the discovery of high specificity and affinity compounds that can inhibit their binding to neural cells can be used as antidotes or in the design of chemical detectors. Using the crystal structure of the C fragment of the tetanus toxin (TetC), which is the cell recognition and cell surface binding domain, and the computational program DOCK, sets of small molecules have been predicted to bind to two different sites located on the surface of this protein. While Site-1 is common to the TeNT and BoNTs, Site-2 is unique to TeNT. Pairs of these molecules from each site can then be linked together synthetically to thereby increase the specificity and affinity for this toxin. Electrospray ionization mass spectroscopy was used to experimentally screen each compound for binding. Mixtures containing binders were further screened for activity under biologically relevant conditions using nuclear magnetic resonance (NMR) methods. The screening of mixtures of compounds offers increased efficiency and throughput as compared to testing single compounds and can also evaluate how possible structural changes induced by the binding of one ligand can influence the binding of the second ligand. In addition, competitive binding experiments with mixtures containing ligands predicted to bind the same site could identify the best binder for that site. NMR transfer nuclear Overhauser effect (trNOE) confirm that TetC binds doxorubicin but that this molecule is displaced by N-acetylneuraminic acid (sialic acid) in a mixture that

Use of computational modeling in preparation and evaluation of surface imprinted xerogels for binding tetracycline

International Nuclear Information System (INIS)

Pace, Samantha J.; Nguyen, Eric; Baria, Maximillian P.; Mojica, Elmer-Rico E.

2015-01-01

Linear alkyl alkoxysilanes (methoxy and ethoxy-based) of varying length were used in preparing tetracycline surface imprinted silica xerogels by the sol–gel process. The resulting xerogels were characterized in terms of binding tetracycline (TC) by using tritium-labeled TC. Results showed preferential binding in the ethoxysilane based xerogels in comparison to methoxysilane based xerogels. A computational approach using the interaction energy (IE) between TC and each alkyl alkoxysilane was deduced as a rational way of predicting the formulation that would provide the best analytical performance for a given molecularly imprinted xerogel (MIX). Hartree-Fock calculations revealed an increase in IE as the length of the carbon chain increases until an optimum value at C6 in both alkoxysilanes. This is consistent with the experimental results wherein the C6 xerogel formulation has the highest imprinting factor. These results show the potential of using computational modeling as a rational way of preparing surface imprinted materials. (author)

Application of the fragment molecular orbital method analysis to fragment-based drug discovery of BET (bromodomain and extra-terminal proteins) inhibitors.

Science.gov (United States)

Ozawa, Motoyasu; Ozawa, Tomonaga; Ueda, Kazuyoshi

2017-06-01

The molecular interactions of inhibitors of bromodomains (BRDs) were investigated. BRDs are protein interaction modules that recognizing ε-N-acetyl-lysine (εAc-Lys) motifs found in histone tails and are promising protein-protein interaction (PPI) targets. First, we analyzed a peptide ligand containing εAc-Lys to evaluate native PPIs. We then analyzed tetrahydroquinazoline-6-yl-benzensulfonamide derivatives found by fragment-based drug design (FBDD) and examined their interactions with the protein compared with the peptide ligand in terms of the inter-fragment interaction energy. In addition, we analyzed benzodiazepine derivatives that are high-affinity ligands for BRDs and examined differences in the CH/π interactions of the amino acid residues. We further surveyed changes in the charges of the amino acid residues among individual ligands, performed pair interaction energy decomposition analysis and estimated the water profile within the ligand binding site. Thus, useful insights for drug design were provided. Through these analyses and considerations, we show that the FMO method is a useful drug design tool to evaluate the process of FBDD and to explore PPI inhibitors. Copyright © 2017 Elsevier Inc. All rights reserved.

Zonula occludens toxin structure-function analysis. Identification of the fragment biologically active on tight junctions and of the zonulin receptor binding domain.

Science.gov (United States)

Di Pierro, M; Lu, R; Uzzau, S; Wang, W; Margaretten, K; Pazzani, C; Maimone, F; Fasano, A

2001-06-01

Zonula occludens toxin (Zot) is an enterotoxin elaborated by Vibrio cholerae that increases intestinal permeability by interacting with a mammalian cell receptor with subsequent activation of intracellular signaling leading to the disassembly of the intercellular tight junctions. Zot localizes in the bacterial outer membrane of V. cholerae with subsequent cleavage and secretion of a carboxyl-terminal fragment in the host intestinal milieu. To identify the Zot domain(s) directly involved in the protein permeating effect, several zot gene deletion mutants were constructed and tested for their biological activity in the Ussing chamber assay and their ability to bind to the target receptor on intestinal epithelial cell cultures. The Zot biologically active domain was localized toward the carboxyl terminus of the protein and coincided with the predicted cleavage product generated by V. cholerae. This domain shared a putative receptor-binding motif with zonulin, the Zot mammalian analogue involved in tight junction modulation. Amino acid comparison between the Zot active fragment and zonulin, combined with site-directed mutagenesis experiments, confirmed the presence of an octapeptide receptor-binding domain toward the amino terminus of the processed Zot.

Establish an automated flow injection ESI-MS method for the screening of fragment based libraries: Application to Hsp90.

Science.gov (United States)

Riccardi Sirtori, Federico; Caronni, Dannica; Colombo, Maristella; Dalvit, Claudio; Paolucci, Mauro; Regazzoni, Luca; Visco, Carlo; Fogliatto, Gianpaolo

2015-08-30

ESI-MS is a well established technique for the study of biopolymers (nucleic acids, proteins) and their non covalent adducts, due to its capacity to detect ligand-target complexes in the gas phase and allows inference of ligand-target binding in solution. In this article we used this approach to investigate the interaction of ligands to the Heat Shock Protein 90 (Hsp90). This enzyme is a molecular chaperone involved in the folding and maturation of several proteins which has been subjected in the last years to intensive drug discovery efforts due to its key role in cancer. In particular, reference compounds, with a broad range of dissociation constants from 40pM to 100μM, were tested to assess the reliability of ESI-MS for the study of protein-ligand complexes. A good agreement was found between the values measured with a fluorescence polarization displacement assay and those determined by mass spectrometry. After this validation step we describe the setup of a medium throughput screening method, based on ESI-MS, suitable to explore interactions of therapeutic relevance biopolymers with chemical libraries. Our approach is based on an automated flow injection ESI-MS method (AFI-MS) and has been applied to screen the Nerviano Medical Sciences proprietary fragment library of about 2000 fragments against Hsp90. In order to discard false positive hits and to discriminate those of them interacting with the N-terminal ATP binding site, competition experiments were performed using a reference inhibitor. Gratifyingly, this group of hits matches with the ligands previously identified by NMR FAXS techniques and confirmed by X-ray co-crystallization experiments. These results support the use of AFI-MS for the screening of medium size libraries, including libraries of small molecules with low affinity typically used in fragment based drug discovery. AFI-MS is a valid alternative to other techniques with the additional opportunities to identify compounds interacting with

Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGFβ1 antibody

Science.gov (United States)

Honey, Denise M.; Best, Annie; Qiu, Huawei

2018-01-01

ABSTRACT Metelimumab (CAT192) is a human IgG4 monoclonal antibody developed as a TGFβ1-specific antagonist. It was tested in clinical trials for the treatment of scleroderma but later terminated due to lack of efficacy. Subsequent characterization of CAT192 indicated that its TGFβ1 binding affinity was reduced by ∼50-fold upon conversion from the parental single-chain variable fragment (scFv) to IgG4. We hypothesized this result was due to decreased conformational flexibility of the IgG that could be altered via engineering. Therefore, we designed insertion mutants in the elbow region and screened for binding and potency. Our results indicated that increasing the elbow region linker length in each chain successfully restored the isoform-specific and high affinity binding of CAT192 to TGFβ1. The crystal structure of the high binding affinity mutant displays large conformational rearrangements of the variable domains compared to the wild-type antigen-binding fragment (Fab) and the low binding affinity mutants. Insertion of two glycines in both the heavy and light chain elbow regions provided sufficient flexibility for the variable domains to extend further apart than the wild-type Fab, and allow the CDR3s to make additional interactions not seen in the wild-type Fab structure. These interactions coupled with the dramatic conformational changes provide a possible explanation of how the scFv and elbow-engineered Fabs bind TGFβ1 with high affinity. This study demonstrates the benefits of re-examining both structure and function when converting scFv to IgG molecules, and highlights the potential of structure-based engineering to produce fully functional antibodies. PMID:29333938

Metabolite identification through multiple kernel learning on fragmentation trees.

Science.gov (United States)

Shen, Huibin; Dührkop, Kai; Böcker, Sebastian; Rousu, Juho

2014-06-15

Metabolite identification from tandem mass spectrometric data is a key task in metabolomics. Various computational methods have been proposed for the identification of metabolites from tandem mass spectra. Fragmentation tree methods explore the space of possible ways in which the metabolite can fragment, and base the metabolite identification on scoring of these fragmentation trees. Machine learning methods have been used to map mass spectra to molecular fingerprints; predicted fingerprints, in turn, can be used to score candidate molecular structures. Here, we combine fragmentation tree computations with kernel-based machine learning to predict molecular fingerprints and identify molecular structures. We introduce a family of kernels capturing the similarity of fragmentation trees, and combine these kernels using recently proposed multiple kernel learning approaches. Experiments on two large reference datasets show that the new methods significantly improve molecular fingerprint prediction accuracy. These improvements result in better metabolite identification, doubling the number of metabolites ranked at the top position of the candidates list. © The Author 2014. Published by Oxford University Press.

Identification of N-ethylmethylamine as a novel scaffold for inhibitors of soluble epoxide hydrolase by crystallographic fragment screening.

Science.gov (United States)

Amano, Yasushi; Tanabe, Eiki; Yamaguchi, Tomohiko

2015-05-15

Soluble epoxide hydrolase (sEH) is a potential target for the treatment of inflammation and hypertension. X-ray crystallographic fragment screening was used to identify fragment hits and their binding modes. Eight fragment hits were identified via soaking of sEH crystals with fragment cocktails, and the co-crystal structures of these hits were determined via individual soaking. Based on the binding mode, N-ethylmethylamine was identified as a promising scaffold that forms hydrogen bonds with the catalytic residues of sEH, Asp335, Tyr383, and Tyr466. Compounds containing this scaffold were selected from an in-house chemical library and assayed. Although the starting fragment had a weak inhibitory activity (IC50: 800μM), we identified potent inhibitors including 2-({[2-(adamantan-1-yl)ethyl]amino}methyl)phenol exhibiting the highest inhibitory activity (IC50: 0.51μM). This corresponded to a more than 1500-fold increase in inhibitory activity compared to the starting fragment. Co-crystal structures of the hit compounds demonstrate that the binding of N-ethylmethylamine to catalytic residues is similar to that of the starting fragment. We therefore consider crystallographic fragment screening to be appropriate for the identification of weak but promising fragment hits. Copyright © 2015 Elsevier Ltd. All rights reserved.

Modified cytokeratins expressed on the surface of carcinoma cells undergo endocytosis upon binding of human monoclonal antibody and its recombinant Fab fragment

DEFF Research Database (Denmark)

Ditzel, H J; Garrigues, U; Andersen, C B

1997-01-01

display selection and the human Fab fragment was expressed in bacteria. Analysis by confocal laser scanning microscopy demonstrated that COU-1 bound in a uniform punctate pattern to the surface of viable carcinoma cells stained at 4 degrees C, and binding increased significantly when cells were cultured...... was significantly reduced. Similar results were obtained using intact IgM COU-1 and the recombinant Fab fragment. Immunohistological studies indicated that COU-1, in contrast to murine monoclonal antibodies against normal cytokeratin 8 and 18, could differentiate between malignant and normal colon epithelia...

Leveraging structure determination with fragment screening for infectious disease drug targets: MECP synthase from Burkholderia pseudomallei

Energy Technology Data Exchange (ETDEWEB)

Begley, Darren W.; Hartley, Robert C.; Davies, Douglas R.; Edwards, Thomas E.; Leonard, Jess T.; Abendroth, Jan; Burris, Courtney A.; Bhandari, Janhavi; Myler, Peter J.; Staker, Bart L.; Stewart, Lance J. (UWASH); (Emerald)

2011-09-28

As part of the Seattle Structural Genomics Center for Infectious Disease, we seek to enhance structural genomics with ligand-bound structure data which can serve as a blueprint for structure-based drug design. We have adapted fragment-based screening methods to our structural genomics pipeline to generate multiple ligand-bound structures of high priority drug targets from pathogenic organisms. In this study, we report fragment screening methods and structure determination results for 2C-methyl-D-erythritol-2,4-cyclo-diphosphate (MECP) synthase from Burkholderia pseudomallei, the gram-negative bacterium which causes melioidosis. Screening by nuclear magnetic resonance spectroscopy as well as crystal soaking followed by X-ray diffraction led to the identification of several small molecules which bind this enzyme in a critical metabolic pathway. A series of complex structures obtained with screening hits reveal distinct binding pockets and a range of small molecules which form complexes with the target. Additional soaks with these compounds further demonstrate a subset of fragments to only bind the protein when present in specific combinations. This ensemble of fragment-bound complexes illuminates several characteristics of MECP synthase, including a previously unknown binding surface external to the catalytic active site. These ligand-bound structures now serve to guide medicinal chemists and structural biologists in rational design of novel inhibitors for this enzyme.

Estimation of kinetic and thermodynamic ligand-binding parameters using computational strategies.

Science.gov (United States)

Deganutti, Giuseppe; Moro, Stefano

2017-04-01

Kinetic and thermodynamic ligand-protein binding parameters are gaining growing importance as key information to consider in drug discovery. The determination of the molecular structures, using particularly x-ray and NMR techniques, is crucial for understanding how a ligand recognizes its target in the final binding complex. However, for a better understanding of the recognition processes, experimental studies of ligand-protein interactions are needed. Even though several techniques can be used to investigate both thermodynamic and kinetic profiles for a ligand-protein complex, these procedures are very often laborious, time consuming and expensive. In the last 10 years, computational approaches have enormous potential in providing insights into each of the above effects and in parsing their contributions to the changes in both kinetic and thermodynamic binding parameters. The main purpose of this review is to summarize the state of the art of computational strategies for estimating the kinetic and thermodynamic parameters of a ligand-protein binding.

Conformational changes in fragments D and double-D from human fibrin(ogen) upon binding the peptide ligand Gly-His-Arg-Pro-amide.

Science.gov (United States)

Everse, S J; Spraggon, G; Veerapandian, L; Doolittle, R F

1999-03-09

The structure of fragment double-D from human fibrin has been solved in the presence and absence of the peptide ligands that simulate the two knobs exposed by the removal of fibrinopeptides A and B, respectively. All told, six crystal structures have been determined, three of which are reported here for the first time: namely, fragments D and double-D with the peptide GHRPam alone and double-D in the absence of any peptide ligand. Comparison of the structures has revealed a series of conformational changes that are brought about by the various knob-hole interactions. Of greatest interest is a moveable "flap" of two negatively charged amino acids (Glubeta397 and Aspbeta398) whose side chains are pinned back to the coiled coil with a calcium atom bridge until GHRPam occupies the beta-chain pocket. Additionally, in the absence of the peptide ligand GPRPam, GHRPam binds to the gamma-chain pocket, a new calcium-binding site being formed concomitantly.

A computational method for identification of vaccine targets from protein regions of conserved human leukocyte antigen binding

DEFF Research Database (Denmark)

Olsen, Lars Rønn; Simon, Christian; Kudahl, Ulrich J.

2015-01-01

Background: Computational methods for T cell-based vaccine target discovery focus on selection of highly conserved peptides identified across pathogen variants, followed by prediction of their binding of human leukocyte antigen molecules. However, experimental studies have shown that T cells ofte...... or proteome using human leukocyte antigen binding predictions and made a web-accessible software implementation freely available at http://met-hilab.cbs.dtu.dk/blockcons/....

An Immunosensor Based on Antibody Binding Fragments Attached to Gold Nanoparticles for the Detection of Peptides Derived from Avian Influenza Hemagglutinin H5

Directory of Open Access Journals (Sweden)

Urszula Jarocka

2014-08-01

Full Text Available This paper concerns the development of an immunosensor for detection of peptides derived from avian influenza hemagglutinin H5. Its preparation consists of successive gold electrode modification steps: (i modification with 1,6-hexanedithiol and gold colloidal nanoparticles; (ii immobilization of antibody-binding fragments (Fab’ of anti-hemagglutinin H5 monoclonal antibodies Mab 6-9-1 via S-Au covalent bonds; and (iii covering the remaining free space on the electrode surfaces with bovine serum albumin. The interactions between Fab’ fragments and hemagglutinin (HA variants have been explored with electrochemical impedance spectroscopy (EIS in the presence of [Fe(CN6]3−/4− as an electroactive marker. The immunosensor was able to recognize three different His-tagged variants of recombinant hemagglutinin from H5N1 viruses: H1 subunit (17–340 residues of A/swan/Poland/305-135V08/2006, the long HA (17–530 residues A/Bar-headed Goose/Qinghai/12/2005 and H1 subunit (1–345 residues of A/Vietnam/1194/2004. The strongest response has been observed for the long variant with detection limit of 2.2 pg/mL and dynamic range from 4.0 to 20.0 pg/mL.

Fragment Based Optimization of Metabotropic Glutamate Receptor 2 (mGluR2) Positive Allosteric Modulators in the Absence of Structural Information.

Science.gov (United States)

Szabó, György; Túrós, György I; Kolok, Sándor; Vastag, Mónika; Sánta, Zsuzsanna; Dékány, Miklós; Lévay, György I; Greiner, István; Natsumi, Minami; Tatsuya, Watanabe; Keserű, György M

2018-03-14

Metabotropic glutamate receptor 2 (mGluR2) positive allosteric modulators (PAMs) have been implicated as potential pharmacotherapy for psychiatric conditions. Screening our corporate compound deck, we identified a benzotriazole fragment (4) that was rapidly optimized to a potent and metabolically stable early lead (16). The highly lipophilic character of 16, together with its limited solubility, permeability, and high protein binding, however, did not allow reaching of the proof of concept in vivo. Since further attempts on the optimization of druglike properties were unsuccessful, the original hit 4 has been revisited and was optimized following the principles of fragment based drug discovery (FBDD). Lacking structural information on the receptor-ligand complex, we implemented a group efficiency (GE) based strategy and identified a new fragment like lead (60) with more balanced profile. Significant improvement achieved on the druglike properties nominated the compound for in vivo proof of concept studies that revealed the chemotype being a promising PAM lead targeting mGluR2 receptors.

Binding Mode and Induced Fit Predictions for Prospective Computational Drug Design.

Science.gov (United States)

Grebner, Christoph; Iegre, Jessica; Ulander, Johan; Edman, Karl; Hogner, Anders; Tyrchan, Christian

2016-04-25

Computer-aided drug design plays an important role in medicinal chemistry to obtain insights into molecular mechanisms and to prioritize design strategies. Although significant improvement has been made in structure based design, it still remains a key challenge to accurately model and predict induced fit mechanisms. Most of the current available techniques either do not provide sufficient protein conformational sampling or are too computationally demanding to fit an industrial setting. The current study presents a systematic and exhaustive investigation of predicting binding modes for a range of systems using PELE (Protein Energy Landscape Exploration), an efficient and fast protein-ligand sampling algorithm. The systems analyzed (cytochrome P, kinase, protease, and nuclear hormone receptor) exhibit different complexities of ligand induced fit mechanisms and protein dynamics. The results are compared with results from classical molecular dynamics simulations and (induced fit) docking. This study shows that ligand induced side chain rearrangements and smaller to medium backbone movements are captured well in PELE. Large secondary structure rearrangements, however, remain challenging for all employed techniques. Relevant binding modes (ligand heavy atom RMSD PELE method within a few hours of simulation, positioning PELE as a tool applicable for rapid drug design cycles.

Development of tight-binding, chemical-reaction-dynamics simulator for combinatorial computational chemistry

International Nuclear Information System (INIS)

Kubo, Momoji; Ando, Minako; Sakahara, Satoshi; Jung, Changho; Seki, Kotaro; Kusagaya, Tomonori; Endou, Akira; Takami, Seiichi; Imamura, Akira; Miyamoto, Akira

2004-01-01

Recently, we have proposed a new concept called 'combinatorial computational chemistry' to realize a theoretical, high-throughput screening of catalysts and materials. We have already applied our combinatorial, computational-chemistry approach, mainly based on static first-principles calculations, to various catalysts and materials systems and its applicability to the catalysts and materials design was strongly confirmed. In order to realize more effective and efficient combinatorial, computational-chemistry screening, a high-speed, chemical-reaction-dynamics simulator based on quantum-chemical, molecular-dynamics method is essential. However, to the best of our knowledge, there is no chemical-reaction-dynamics simulator, which has an enough high-speed ability to perform a high-throughput screening. In the present study, we have succeeded in the development of a chemical-reaction-dynamics simulator based on our original, tight-binding, quantum-chemical, molecular-dynamics method, which is more than 5000 times faster than the regular first-principles, molecular-dynamics method. Moreover, its applicability and effectiveness to the atomistic clarification of the methanol-synthesis dynamics at reaction temperature were demonstrated

The use of many-body expansions and geometry optimizations in fragment-based methods.

Science.gov (United States)

Fedorov, Dmitri G; Asada, Naoya; Nakanishi, Isao; Kitaura, Kazuo

2014-09-16

Conspectus Chemists routinely work with complex molecular systems: solutions, biochemical molecules, and amorphous and composite materials provide some typical examples. The questions one often asks are what are the driving forces for a chemical phenomenon? How reasonable are our views of chemical systems in terms of subunits, such as functional groups and individual molecules? How can one quantify the difference in physicochemical properties of functional units found in a different chemical environment? Are various effects on functional units in molecular systems additive? Can they be represented by pairwise potentials? Are there effects that cannot be represented in a simple picture of pairwise interactions? How can we obtain quantitative values for these effects? Many of these questions can be formulated in the language of many-body effects. They quantify the properties of subunits (fragments), referred to as one-body properties, pairwise interactions (two-body properties), couplings of two-body interactions described by three-body properties, and so on. By introducing the notion of fragments in the framework of quantum chemistry, one obtains two immense benefits: (a) chemists can finally relate to quantum chemistry, which now speaks their language, by discussing chemically interesting subunits and their interactions and (b) calculations become much faster due to a reduced computational scaling. For instance, the somewhat academic sounding question of the importance of three-body effects in water clusters is actually another way of asking how two hydrogen bonds affect each other, when they involve three water molecules. One aspect of this is the many-body charge transfer (CT), because the charge transfers in the two hydrogen bonds are coupled to each other (not independent). In this work, we provide a generalized view on the use of many-body expansions in fragment-based methods, focusing on the general aspects of the property expansion and a contraction of a

Computational analysis of protein-protein interfaces involving an alpha helix: insights for terphenyl-like molecules binding.

Science.gov (United States)

Isvoran, Adriana; Craciun, Dana; Martiny, Virginie; Sperandio, Olivier; Miteva, Maria A

2013-06-14

Protein-Protein Interactions (PPIs) are key for many cellular processes. The characterization of PPI interfaces and the prediction of putative ligand binding sites and hot spot residues are essential to design efficient small-molecule modulators of PPI. Terphenyl and its derivatives are small organic molecules known to mimic one face of protein-binding alpha-helical peptides. In this work we focus on several PPIs mediated by alpha-helical peptides. We performed computational sequence- and structure-based analyses in order to evaluate several key physicochemical and surface properties of proteins known to interact with alpha-helical peptides and/or terphenyl and its derivatives. Sequence-based analysis revealed low sequence identity between some of the analyzed proteins binding alpha-helical peptides. Structure-based analysis was performed to calculate the volume, the fractal dimension roughness and the hydrophobicity of the binding regions. Besides the overall hydrophobic character of the binding pockets, some specificities were detected. We showed that the hydrophobicity is not uniformly distributed in different alpha-helix binding pockets that can help to identify key hydrophobic hot spots. The presence of hydrophobic cavities at the protein surface with a more complex shape than the entire protein surface seems to be an important property related to the ability of proteins to bind alpha-helical peptides and low molecular weight mimetics. Characterization of similarities and specificities of PPI binding sites can be helpful for further development of small molecules targeting alpha-helix binding proteins.

DNA fragments assembly based on nicking enzyme system.

Directory of Open Access Journals (Sweden)

Rui-Yan Wang

Full Text Available A couple of DNA ligation-independent cloning (LIC methods have been reported to meet various requirements in metabolic engineering and synthetic biology. The principle of LIC is the assembly of multiple overlapping DNA fragments by single-stranded (ss DNA overlaps annealing. Here we present a method to generate single-stranded DNA overlaps based on Nicking Endonucleases (NEases for LIC, the method was termed NE-LIC. Factors related to cloning efficiency were optimized in this study. This NE-LIC allows generating 3'-end or 5'-end ss DNA overlaps of various lengths for fragments assembly. We demonstrated that the 10 bp/15 bp overlaps had the highest DNA fragments assembling efficiency, while 5 bp/10 bp overlaps showed the highest efficiency when T4 DNA ligase was added. Its advantage over Sequence and Ligation Independent Cloning (SLIC and Uracil-Specific Excision Reagent (USER was obvious. The mechanism can be applied to many other LIC strategies. Finally, the NEases based LIC (NE-LIC was successfully applied to assemble a pathway of six gene fragments responsible for synthesizing microbial poly-3-hydroxybutyrate (PHB.

Process Fragment Libraries for Easier and Faster Development of Process-based Applications

Directory of Open Access Journals (Sweden)

David Schumm

2011-01-01

Full Text Available The term “process fragment” is recently gaining momentum in business process management research. We understand a process fragment as a connected and reusable process structure, which has relaxed completeness and consistency criteria compared to executable processes. We claim that process fragments allow for an easier and faster development of process-based applications. As evidence to this claim we present a process fragment concept and show a sample collection of concrete, real-world process fragments. We present advanced application scenarios for using such fragments in development of process-based applications. Process fragments are typically managed in a repository, forming a process fragment library. On top of a process fragment library from previous work, we discuss the potential impact of using process fragment libraries in cross-enterprise collaboration and application integration.

Computational scheme for pH-dependent binding free energy calculation with explicit solvent.

Science.gov (United States)

Lee, Juyong; Miller, Benjamin T; Brooks, Bernard R

2016-01-01

We present a computational scheme to compute the pH-dependence of binding free energy with explicit solvent. Despite the importance of pH, the effect of pH has been generally neglected in binding free energy calculations because of a lack of accurate methods to model it. To address this limitation, we use a constant-pH methodology to obtain a true ensemble of multiple protonation states of a titratable system at a given pH and analyze the ensemble using the Bennett acceptance ratio (BAR) method. The constant pH method is based on the combination of enveloping distribution sampling (EDS) with the Hamiltonian replica exchange method (HREM), which yields an accurate semi-grand canonical ensemble of a titratable system. By considering the free energy change of constraining multiple protonation states to a single state or releasing a single protonation state to multiple states, the pH dependent binding free energy profile can be obtained. We perform benchmark simulations of a host-guest system: cucurbit[7]uril (CB[7]) and benzimidazole (BZ). BZ experiences a large pKa shift upon complex formation. The pH-dependent binding free energy profiles of the benchmark system are obtained with three different long-range interaction calculation schemes: a cutoff, the particle mesh Ewald (PME), and the isotropic periodic sum (IPS) method. Our scheme captures the pH-dependent behavior of binding free energy successfully. Absolute binding free energy values obtained with the PME and IPS methods are consistent, while cutoff method results are off by 2 kcal mol(-1) . We also discuss the characteristics of three long-range interaction calculation methods for constant-pH simulations. © 2015 The Protein Society.

HETC-3STEP included fragmentation process

Energy Technology Data Exchange (ETDEWEB)

Shigyo, Nobuhiro; Iga, Kiminori; Ishibashi, Kenji [Kyushu Univ., Fukuoka (Japan). Faculty of Engineering

1997-03-01

High Energy Transport Code (HETC) based on the cascade-evaporation model is modified to calculate the fragmentation cross section. For the cascade process, nucleon-nucleon cross sections are used for collision computation; effective in-medium-corrected cross sections are adopted instead of the original free-nucleon collision. The exciton model is adopted for improvement of backward nucleon-emission cross section for low-energy nucleon-incident events. The fragmentation reaction is incorporated into the original HETC as a subroutine set by the use of the systematics of the reaction. The modified HETC (HETC-3STEP/FRG) reproduces experimental fragment yields to a reasonable degree. (author)

Activation and binding of opsonic fragments of C3 on encapsulated Cryptococcus neoformans by using an alternative complement pathway reconstituted from six isolated proteins.

Science.gov (United States)

Kozel, T R; Wilson, M A; Pfrommer, G S; Schlageter, A M

1989-07-01

Encapsulated Cryptococcus neoformans yeast cells are potent activators of the complement system. We examined the interaction of the yeast cells with an alternative complement pathway reconstituted from isolated factor D, factor B, factor H, factor I, C3, and properdin. Incubation of encapsulated cryptococci with the reconstituted pathway led to activation and binding of C3 fragments to the yeast cells that was quantitatively and qualitatively identical to that observed with normal human serum. Incubation with either normal serum or a mixture of isolated proteins led to binding of 4 x 10(7) to 5 x 10(7) C3 molecules to the yeast cells. The kinetics for activation and binding of C3 were identical, with maximum binding observed after a 20-min incubation. Immunoglobulin G was not needed for optimal activation kinetics. C3 fragments eluted from the yeast cells by treatment with hydroxylamine and subsequent analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the presence primarily of iC3b on yeast cells incubated with either normal serum or the reconstituted pathway. Ultrastructural examination of the opsonized yeast cells showed that the cryptococcal capsule was the site for binding of C3 activated from normal serum or the reconstituted pathway, with a dense accumulation of C3 at the periphery of the capsule. Thus, incubation of encapsulated cryptococci in the reconstituted pathway led to deposition of opsonic complement fragments at a site that was appropriate for interaction with phagocyte receptors. Cryptococci opsonized with the reconstituted pathway showed a markedly enhanced interaction with cultured human monocytes compared with unopsonized yeast cells, indicating that the alternative pathway alone is opsonic for yeast cells. However, the results indicate that additional serum factors are needed for optimal opsonization of yeast cells because a 35% reduction in the number of cryptococci bound to macrophages was observed with

Computational assessment of the cooperativity between RNA binding proteins and MicroRNAs in Transcript Decay.

Science.gov (United States)

Jiang, Peng; Singh, Mona; Coller, Hilary A

2013-01-01

Transcript degradation is a widespread and important mechanism for regulating protein abundance. Two major regulators of transcript degradation are RNA Binding Proteins (RBPs) and microRNAs (miRNAs). We computationally explored whether RBPs and miRNAs cooperate to promote transcript decay. We defined five RBP motifs based on the evolutionary conservation of their recognition sites in 3'UTRs as the binding motifs for Pumilio (PUM), U1A, Fox-1, Nova, and UAUUUAU. Recognition sites for some of these RBPs tended to localize at the end of long 3'UTRs. A specific group of miRNA recognition sites were enriched within 50 nts from the RBP recognition sites for PUM and UAUUUAU. The presence of both a PUM recognition site and a recognition site for preferentially co-occurring miRNAs was associated with faster decay of the associated transcripts. For PUM and its co-occurring miRNAs, binding of the RBP to its recognition sites was predicted to release nearby miRNA recognition sites from RNA secondary structures. The mammalian miRNAs that preferentially co-occur with PUM binding sites have recognition seeds that are reverse complements to the PUM recognition motif. Their binding sites have the potential to form hairpin secondary structures with proximal PUM binding sites that would normally limit RISC accessibility, but would be more accessible to miRNAs in response to the binding of PUM. In sum, our computational analyses suggest that a specific set of RBPs and miRNAs work together to affect transcript decay, with the rescue of miRNA recognition sites via RBP binding as one possible mechanism of cooperativity.

AutoDrug: fully automated macromolecular crystallography workflows for fragment-based drug discovery

International Nuclear Information System (INIS)

Tsai, Yingssu; McPhillips, Scott E.; González, Ana; McPhillips, Timothy M.; Zinn, Daniel; Cohen, Aina E.; Feese, Michael D.; Bushnell, David; Tiefenbrunn, Theresa; Stout, C. David; Ludaescher, Bertram; Hedman, Britt; Hodgson, Keith O.; Soltis, S. Michael

2013-01-01

New software has been developed for automating the experimental and data-processing stages of fragment-based drug discovery at a macromolecular crystallography beamline. A new workflow-automation framework orchestrates beamline-control and data-analysis software while organizing results from multiple samples. AutoDrug is software based upon the scientific workflow paradigm that integrates the Stanford Synchrotron Radiation Lightsource macromolecular crystallography beamlines and third-party processing software to automate the crystallography steps of the fragment-based drug-discovery process. AutoDrug screens a cassette of fragment-soaked crystals, selects crystals for data collection based on screening results and user-specified criteria and determines optimal data-collection strategies. It then collects and processes diffraction data, performs molecular replacement using provided models and detects electron density that is likely to arise from bound fragments. All processes are fully automated, i.e. are performed without user interaction or supervision. Samples can be screened in groups corresponding to particular proteins, crystal forms and/or soaking conditions. A single AutoDrug run is only limited by the capacity of the sample-storage dewar at the beamline: currently 288 samples. AutoDrug was developed in conjunction with RestFlow, a new scientific workflow-automation framework. RestFlow simplifies the design of AutoDrug by managing the flow of data and the organization of results and by orchestrating the execution of computational pipeline steps. It also simplifies the execution and interaction of third-party programs and the beamline-control system. Modeling AutoDrug as a scientific workflow enables multiple variants that meet the requirements of different user groups to be developed and supported. A workflow tailored to mimic the crystallography stages comprising the drug-discovery pipeline of CoCrystal Discovery Inc. has been deployed and successfully

Isolation of Panels of Llama Single-Domain Antibody Fragments Binding All Nine Neuraminidase Subtypes of Influenza A Virus

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Guus Koch

2013-04-01

Full Text Available Avian influenza A virus comprises sixteen hemagglutinin (HA and nine neuraminidase (NA subtypes (N1–N9. To isolate llama single-domain antibody fragments (VHHs against all N subtypes, four llamas were immunized with mixtures of influenza viruses. Selections using influenza virus yielded predominantly VHHs binding to the highly immunogenic HA and nucleoprotein. However, selection using enzymatically active recombinant NA (rNA protein enabled us to isolate NA binding VHHs. Some isolated VHHs cross-reacted to other N subtypes. These were subsequently used for the capture of N subtypes that could not be produced as recombinant protein (rN6 or were enzymatically inactive (rN1, rN5 in phage display selection, yielding novel VHHs. In total we isolated 188 NA binding VHHs, 64 of which were expressed in yeast. Most VHHs specifically recognize a single N subtype, but some VHHs cross-react with other N-subtypes. At least one VHH bound to all N subtypes, except N4, identifying a conserved antigenic site. Thus, this work (1 describes methods for isolating NA binding VHHs, (2 illustrates the suitability of llama immunization with multiple antigens for retrieving many binders against different antigens and (3 describes 64 novel NA binding VHHs, including a broadly reactive VHH, which can be used in various assays for influenza virus subtyping, detection or serology.

Process of Fragment-Based Lead Discovery—A Perspective from NMR

Directory of Open Access Journals (Sweden)

Rongsheng Ma

2016-07-01

Full Text Available Fragment-based lead discovery (FBLD has proven fruitful during the past two decades for a variety of targets, even challenging protein–protein interaction (PPI systems. Nuclear magnetic resonance (NMR spectroscopy plays a vital role, from initial fragment-based screening to lead generation, because of its power to probe the intrinsically weak interactions between targets and low-molecular-weight fragments. Here, we review the NMR FBLD process from initial library construction to lead generation. We describe technical aspects regarding fragment library design, ligand- and protein-observed screening, and protein–ligand structure model generation. For weak binders, the initial hit-to-lead evolution can be guided by structural information retrieved from NMR spectroscopy, including chemical shift perturbation, transferred pseudocontact shifts, and paramagnetic relaxation enhancement. This perspective examines structure-guided optimization from weak fragment screening hits to potent leads for challenging PPI targets.

Computational design of trimeric influenza-neutralizing proteins targeting the hemagglutinin receptor binding site

Energy Technology Data Exchange (ETDEWEB)

Strauch, Eva-Maria; Bernard, Steffen M.; La, David; Bohn, Alan J.; Lee, Peter S.; Anderson, Caitlin E.; Nieusma, Travis; Holstein, Carly A.; Garcia, Natalie K.; Hooper, Kathryn A.; Ravichandran, Rashmi; Nelson, Jorgen W.; Sheffler, William; Bloom, Jesse D.; Lee, Kelly K.; Ward, Andrew B.; Yager, Paul; Fuller, Deborah H.; Wilson, Ian A.; Baker , David (UWASH); (Scripps); (FHCRC)

2017-06-12

Many viral surface glycoproteins and cell surface receptors are homo-oligomers1, 2, 3, 4, and thus can potentially be targeted by geometrically matched homo-oligomers that engage all subunits simultaneously to attain high avidity and/or lock subunits together. The adaptive immune system cannot generally employ this strategy since the individual antibody binding sites are not arranged with appropriate geometry to simultaneously engage multiple sites in a single target homo-oligomer. We describe a general strategy for the computational design of homo-oligomeric protein assemblies with binding functionality precisely matched to homo-oligomeric target sites5, 6, 7, 8. In the first step, a small protein is designed that binds a single site on the target. In the second step, the designed protein is assembled into a homo-oligomer such that the designed binding sites are aligned with the target sites. We use this approach to design high-avidity trimeric proteins that bind influenza A hemagglutinin (HA) at its conserved receptor binding site. The designed trimers can both capture and detect HA in a paper-based diagnostic format, neutralizes influenza in cell culture, and completely protects mice when given as a single dose 24 h before or after challenge with influenza.

Designing an orally available nontoxic p38 inhibitor with a fragment-based strategy.

Science.gov (United States)

Guarnieri, Frank

2015-01-01

The MAPK p38 became a focal point of inflammatory research when it was recognized that it played a key role in the production of the pro-inflammatory molecules TNF-alpha, IL-beta, and cyclooxygenase-2 (COX-2). The pharmaceutical industry devoted enormous efforts to creating p38 inhibitors, because blocking p38 had the potential of downregulating a group of pro-inflammatory mediators, and thus, one drug could have a cocktail effect. The market potential seemed to be clearly established (Bonafede et al., Clinicoecon Outcomes Res 6:381-388, 2014) with a multiplicity of TNF-alpha antibodies and a soluble receptor (Mewar and Wilson, Br J Pharmacol 162:785-791, 2011) already on the market, although the relationship between TNF-alpha production and p38 activation is a complicated two-way (Sabio and Davis, Semin Immunol 26:237-245, 2014) signal transduction process. With the discovery that activated p38 stabilizes (Mancini and Di Battista, Inflamm Res 60:1083-1092, 2011) COX-2 mRNA and upregulates expression of IL-beta (Bachstetter and Van Eldik, Aging Dis 1:199-211, 2010) probably in a similar manner, inhibiting p38 appeared to be a way of blocking TNF-alpha, COX-2, and IL-beta simultaneously. At Locus Pharmaceuticals we jumped on this opportunity, because we believed that our fragment-based drug discovery approach was ideally suited for making a potent small molecule p38 inhibitor that did not bind in the ATP site, but also had the solubility, lack of planarity, and low molecular weight required of a clinical candidate. Just to be clear, in our experience highly planar compounds often result in poor pharmacokinetics, because they tend to bind strongly to plasma proteins. At Locus we typically repeated assays by adding increasing amounts of plasma to check for potency degradation in the presence of blood. We found this to be a useful early indicator of pharmacokinetics and in vivo behavior. It became clear from our work and the work of others that binding to the ATP site

Fragmentation and structure of silicon microclusters

International Nuclear Information System (INIS)

Feuston, B.P.; Kalia, R.K.; Vashishta, P.

1987-01-01

It may be possible to determine the magic numbers and fragmentation spectra from the ground-state binding energies and structure, but the relationship between the lowest-energy zero-temperature configurations and the energetics of finite-temperature microclusters is not obvious. Recall fragmentation of Si clusters occurs at temperatures the order of the melting temperature (T∼2000 K). What is needed, a first-principles finite-temperature calculation, allowing the determination of all possible structures, their corresponding binding energies, and fragmentation spectra, is not presently possible. However, a molecular dynamics calculation does allow one to study the nature of fragmentation in addition to determination of the global ground-state structure and all mechanically stable configurations underlying the finite-temperature cluster, once given an interaction potential. The authors present results for such a calculation for Si/sub 2-14/ using the Stillinger-Weber 3-body potential. Their results indicate that the existence of magic numbers is determined by the topology and energetics of high-energy bound structures rather than the structure and ground-state energies at zero temperature

Characterization of Tumor-Avid Antibody Fragments Genetically Engineered for Mono-Specific Radionuclide Chelation

International Nuclear Information System (INIS)

Quinn, T.P.

2003-01-01

The successful clinical application of targeted-radiopharmaceuticals depends on the development of molecules that optimize tumor specific radionuclide deposition and minimize non-specific organ irradiation. To this end, this proposal outlines a research effort to identify and evaluate novel antibodies and antibody fragments that bind breast tumors. The tumor-avid antibodies will be investigated for as imaging and therapeutic agents and to gain a better understanding of the pharmacokinetics and metabolism of radiolabeled tumor-avid antibody fragments through the use of site-specifically labeled molecules. Antibodies or antibody fragments, that bind breast carcinoma carbohydrate antigens, will be obtained from hybridoma or bacteriophage library screening. More specifically, antibody fragments that bind the carcinoma-associated Thomsen-Friedenreich (T) antigen will be radiolabeled with 99m Tc and 188 Re at a natural amino acid chelation site and will be investigated in vivo for their abilities to target human breast tumors. In addition, site-specific radiolabeled antibody fragments will be biosynthesized using misacylated suppressor tRNAs. Homogeneously radiolabeled populations of antibody fragments will be used to investigate the effects of radionuclide location and chelation chemistries on their biodistribution and metabolism. It is hypothesized that site-specifically radiolabeled antibody fragments will possess enhanced tumor imaging and therapeutic properties due to optimal label location and conjugation chemistries. New insights into the factors that govern antibody metabolism in vivo are also expected from this work. Results from these studies should enhance our ability to design and synthesize radiolabeled antibody fragments that have improved pharmacokinetic properties. The studies in this proposal involve basic research into the development of antibody-based radiopharmaceuticals, with the ultimate goal of application in humans. This type of basic nuclear

Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin : Fragment-Based Drug Design Facilitated by Dynamic Combinatorial Chemistry

NARCIS (Netherlands)

Mondal, Milon; Radeva, Nedyalka; Fanlo-Virgos, Hugo; Otto, Sijbren; Klebe, Gerhard; Hirsch, Anna K. H.

2016-01-01

Fragment-based drug design (FBDD) affords active compounds for biological targets. While there are numerous reports on FBDD by fragment growing/optimization, fragment linking has rarely been reported. Dynamic combinatorial chemistry (DCC) has become a powerful hit-identification strategy for

A recombinant estrogen receptor fragment-based homogeneous fluorescent assay for rapid detection of estrogens.

Science.gov (United States)

Wang, Dan; Xie, Jiangbi; Zhu, Xiaocui; Li, Jinqiu; Zhao, Dongqin; Zhao, Meiping

2014-05-15

In this work, we demonstrate a novel estrogenic receptor fragment-based homogeneous fluorescent assay which enables rapid and sensitive detection of 17β-estradiol (E2) and other highly potent estrogens. A modified human estrogenic receptor fragment (N-His × 6-hER270-595-C-Strep tag II) has been constructed that contains amino acids 270-595 of wild-type human estrogenic receptor α (hER270-595) and two specific tags (6 × His and Strep tag II) fused to the N and C terminus, respectively. The designed receptor protein fragment could be easily produced by prokaryotic expression with high yield and high purity. The obtained protein exhibits high binding affinity to E2 and the two tags greatly facilitate the application of the recombinant protein. Taking advantage of the unique spectroscopic properties of coumestrol (CS), a fluorescent phytoestrogen, a CS/hER270-595-based fluorescent assay has been developed which can sensitively respond to E2 within 1.0 min with a linear working range from 0.1 to 20 ng/mL and a limit of detection of 0.1 ng/mL. The assay was successfully applied for rapid detection of E2 in the culture medium of rat hippocampal neurons. The method also holds great potential for high-throughput monitoring the variation of estrogen levels in complex biological fluids, which is crucial for investigation of the molecular basis of various estrogen-involved processes. Copyright © 2013 Elsevier B.V. All rights reserved.

The binding parameters of radiolabelled monoclonal F (ab')2 and Fab' fragments relative to immunoglobulin G in reactions with surface-bound antigens

International Nuclear Information System (INIS)

Fjeld, J.G.; Nustad, K.; Michaelsen, T.E.

1992-01-01

The binding parameters of iodine-125-labelled intact monoclonal immunoglobulin G (IgG), F(ab') 2 and Fab' fragments were compared. The study was carried out with the two monoclonal antibodies (MoAbs) K13 and K16 specific for human Ig light chains κ and λ, respectively. When testing the 125 I-MoAbs against monodisperse polymer particles coated with the specific antigens, the K a for the F(ab') 2 fragments were similar to that for IgG, while the K a for the Fab' fragments were reduced to 10%-20% of that for IgG. The number N of effective target sites revealed with Fab' was higher than with F(ab') and IgG, presumably because less surface area is occupied by the small Fab' molecules. The immunoreactive fraction F ranged according to IgG>F(ab') 2 >Fab'. The explanation of the moderate difference between the K a of the monoclonal Fab' and the divalent IgG and F(ab') 2 was that the divalent molecules were not divalently attached to the particles. When testing the same antibody preparations against humanlymphoma cells producing Ig with light chains κ or λ, the binding results were less reliable than when particles were utilised, presumably due to antigen shedding. Different MoAbs vary in their loss of immunoreactivity due to enzymatic degradation and the radiolabelling procedure. The preparation of the radiolabelled fragments should therefore be optimized for each MoAb, and evaluation is necessary before injection. Artificial targets with a low leakage of antigen, like the monodisperse polymer particles here applied, are recommended for the in vitro evaluation of the immunoreactivity of labelled MoAb preparations. (orig.)

Transforming fragments into candidates: small becomes big in medicinal chemistry.

Science.gov (United States)

de Kloe, Gerdien E; Bailey, David; Leurs, Rob; de Esch, Iwan J P

2009-07-01

Fragment-based drug discovery (FBDD) represents a logical and efficient approach to lead discovery and optimisation. It can draw on structural, biophysical and biochemical data, incorporating a wide range of inputs, from precise mode-of-binding information on specific fragments to wider ranging pharmacophoric screening surveys using traditional HTS approaches. It is truly an enabling technology for the imaginative medicinal chemist. In this review, we analyse a representative set of 23 published FBDD studies that describe how low molecular weight fragments are being identified and efficiently transformed into higher molecular weight drug candidates. FBDD is now becoming warmly endorsed by industry as well as academia and the focus on small interacting molecules is making a big scientific impact.

Binding of indomethacin methyl ester to cyclooxygenase-2. A computational study.

Science.gov (United States)

Sárosi, Menyhárt-Botond

2018-06-05

Inhibitors selective towards the second isoform of prostaglandin synthase (cyclooxygenase, COX-2) are promising nonsteroidal anti-inflammatory drugs and antitumor medications. Methylation of the carboxylate group in the relatively nonselective COX inhibitor indomethacin confers significant COX-2 selectivity. Several other modifications converting indomethacin into a COX-2 selective inhibitor have been reported. Earlier experimental and computational studies on neutral indomethacin derivatives suggest that the methyl ester derivative likely binds to COX-2 with a similar binding mode as that observed for the parent indomethacin. However, docking studies followed by molecular dynamics simulations revealed two possible binding modes in COX-2 for indomethacin methyl ester, which differs from the experimental binding mode found for indomethacin. Both alternative binding modes might explain the observed COX-2 selectivity of indomethacin methyl ester. Graphical abstract Binding of indomethacin methyl ester to cyclooxygenase-2.

DNABP: Identification of DNA-Binding Proteins Based on Feature Selection Using a Random Forest and Predicting Binding Residues.

Science.gov (United States)

Ma, Xin; Guo, Jing; Sun, Xiao

2016-01-01

DNA-binding proteins are fundamentally important in cellular processes. Several computational-based methods have been developed to improve the prediction of DNA-binding proteins in previous years. However, insufficient work has been done on the prediction of DNA-binding proteins from protein sequence information. In this paper, a novel predictor, DNABP (DNA-binding proteins), was designed to predict DNA-binding proteins using the random forest (RF) classifier with a hybrid feature. The hybrid feature contains two types of novel sequence features, which reflect information about the conservation of physicochemical properties of the amino acids, and the binding propensity of DNA-binding residues and non-binding propensities of non-binding residues. The comparisons with each feature demonstrated that these two novel features contributed most to the improvement in predictive ability. Furthermore, to improve the prediction performance of the DNABP model, feature selection using the minimum redundancy maximum relevance (mRMR) method combined with incremental feature selection (IFS) was carried out during the model construction. The results showed that the DNABP model could achieve 86.90% accuracy, 83.76% sensitivity, 90.03% specificity and a Matthews correlation coefficient of 0.727. High prediction accuracy and performance comparisons with previous research suggested that DNABP could be a useful approach to identify DNA-binding proteins from sequence information. The DNABP web server system is freely available at http://www.cbi.seu.edu.cn/DNABP/.

Evolutions in fragment-based drug design: the deconstruction–reconstruction approach

Science.gov (United States)

Chen, Haijun; Zhou, Xiaobin; Wang, Ailan; Zheng, Yunquan; Gao, Yu; Zhou, Jia

2014-01-01

Recent advances in the understanding of molecular recognition and protein–ligand interactions have facilitated rapid development of potent and selective ligands for therapeutically relevant targets. Over the past two decades, a variety of useful approaches and emerging techniques have been developed to promote the identification and optimization of leads that have high potential for generating new therapeutic agents. Intriguingly, the innovation of a fragment-based drug design (FBDD) approach has enabled rapid and efficient progress in drug discovery. In this critical review, we focus on the construction of fragment libraries and the advantages and disadvantages of various fragment-based screening (FBS) for constructing such libraries. We also highlight the deconstruction–reconstruction strategy by utilizing privileged fragments of reported ligands. PMID:25263697

Alignment-independent comparison of binding sites based on DrugScore potential fields encoded by 3D Zernike descriptors.

Science.gov (United States)

Nisius, Britta; Gohlke, Holger

2012-09-24

Analyzing protein binding sites provides detailed insights into the biological processes proteins are involved in, e.g., into drug-target interactions, and so is of crucial importance in drug discovery. Herein, we present novel alignment-independent binding site descriptors based on DrugScore potential fields. The potential fields are transformed to a set of information-rich descriptors using a series expansion in 3D Zernike polynomials. The resulting Zernike descriptors show a promising performance in detecting similarities among proteins with low pairwise sequence identities that bind identical ligands, as well as within subfamilies of one target class. Furthermore, the Zernike descriptors are robust against structural variations among protein binding sites. Finally, the Zernike descriptors show a high data compression power, and computing similarities between binding sites based on these descriptors is highly efficient. Consequently, the Zernike descriptors are a useful tool for computational binding site analysis, e.g., to predict the function of novel proteins, off-targets for drug candidates, or novel targets for known drugs.

Fragment-based {sup 13}C nuclear magnetic resonance chemical shift predictions in molecular crystals: An alternative to planewave methods

Energy Technology Data Exchange (ETDEWEB)

Hartman, Joshua D.; Beran, Gregory J. O., E-mail: gregory.beran@ucr.edu [Department of Chemistry, University of California, Riverside, California 92521 (United States); Monaco, Stephen; Schatschneider, Bohdan [The Pennsylvania State University, The Eberly Campus, 2201 University Dr, Lemont Furnace, Pennsylvania 15456 (United States)

2015-09-14

We assess the quality of fragment-based ab initio isotropic {sup 13}C chemical shift predictions for a collection of 25 molecular crystals with eight different density functionals. We explore the relative performance of cluster, two-body fragment, combined cluster/fragment, and the planewave gauge-including projector augmented wave (GIPAW) models relative to experiment. When electrostatic embedding is employed to capture many-body polarization effects, the simple and computationally inexpensive two-body fragment model predicts both isotropic {sup 13}C chemical shifts and the chemical shielding tensors as well as both cluster models and the GIPAW approach. Unlike the GIPAW approach, hybrid density functionals can be used readily in a fragment model, and all four hybrid functionals tested here (PBE0, B3LYP, B3PW91, and B97-2) predict chemical shifts in noticeably better agreement with experiment than the four generalized gradient approximation (GGA) functionals considered (PBE, OPBE, BLYP, and BP86). A set of recommended linear regression parameters for mapping between calculated chemical shieldings and observed chemical shifts are provided based on these benchmark calculations. Statistical cross-validation procedures are used to demonstrate the robustness of these fits.

Fragment screening by SPR and advanced application to GPCRs.

Science.gov (United States)

Shepherd, Claire A; Hopkins, Andrew L; Navratilova, Iva

2014-01-01

Surface plasmon resonance (SPR) is one of the primary biophysical methods for the screening of low molecular weight 'fragment' libraries, due to its low protein consumption and 'label-free' methodology. SPR biosensor interaction analysis is employed to both screen and confirm the binding of compounds in fragment screening experiments, as it provides accurate information on the affinity and kinetics of molecular interactions. The most advanced application of the use of SPR for fragment screening is against membrane protein drug targets, such G-protein coupled receptors (GPCRs). Biophysical GPCR assays using SPR have been validated with pharmacological measurements approximate to cell-based methods, yet provide the advantage of biophysical methods in their ability to measure the weak affinities of low molecular weight fragments. A number of SPR fragment screens against GPCRs have now been disclosed in the literature. SPR fragment screening is proving versatile to screen both thermostabilised GPCRs and solubilised wild type receptors. In this chapter, we discuss the state-of-the-art in GPCR fragment screening by SPR and the technical considerations in performing such experiments. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Transcription factor binding sites prediction based on modified nucleosomes.

Directory of Open Access Journals (Sweden)

Mohammad Talebzadeh

Full Text Available In computational methods, position weight matrices (PWMs are commonly applied for transcription factor binding site (TFBS prediction. Although these matrices are more accurate than simple consensus sequences to predict actual binding sites, they usually produce a large number of false positive (FP predictions and so are impoverished sources of information. Several studies have employed additional sources of information such as sequence conservation or the vicinity to transcription start sites to distinguish true binding regions from random ones. Recently, the spatial distribution of modified nucleosomes has been shown to be associated with different promoter architectures. These aligned patterns can facilitate DNA accessibility for transcription factors. We hypothesize that using data from these aligned and periodic patterns can improve the performance of binding region prediction. In this study, we propose two effective features, "modified nucleosomes neighboring" and "modified nucleosomes occupancy", to decrease FP in binding site discovery. Based on these features, we designed a logistic regression classifier which estimates the probability of a region as a TFBS. Our model learned each feature based on Sp1 binding sites on Chromosome 1 and was tested on the other chromosomes in human CD4+T cells. In this work, we investigated 21 histone modifications and found that only 8 out of 21 marks are strongly correlated with transcription factor binding regions. To prove that these features are not specific to Sp1, we combined the logistic regression classifier with the PWM, and created a new model to search TFBSs on the genome. We tested the model using transcription factors MAZ, PU.1 and ELF1 and compared the results to those using only the PWM. The results show that our model can predict Transcription factor binding regions more successfully. The relative simplicity of the model and capability of integrating other features make it a superior method

Fragment based drug discovery: practical implementation based on ¹⁹F NMR spectroscopy.

Science.gov (United States)

Jordan, John B; Poppe, Leszek; Xia, Xiaoyang; Cheng, Alan C; Sun, Yax; Michelsen, Klaus; Eastwood, Heather; Schnier, Paul D; Nixey, Thomas; Zhong, Wenge

2012-01-26

Fragment based drug discovery (FBDD) is a widely used tool for discovering novel therapeutics. NMR is a powerful means for implementing FBDD, and several approaches have been proposed utilizing (1)H-(15)N heteronuclear single quantum coherence (HSQC) as well as one-dimensional (1)H and (19)F NMR to screen compound mixtures against a target of interest. While proton-based NMR methods of fragment screening (FBS) have been well documented and are widely used, the use of (19)F detection in FBS has been only recently introduced (Vulpetti et al. J. Am. Chem. Soc.2009, 131 (36), 12949-12959) with the aim of targeting "fluorophilic" sites in proteins. Here, we demonstrate a more general use of (19)F NMR-based fragment screening in several areas: as a key tool for rapid and sensitive detection of fragment hits, as a method for the rapid development of structure-activity relationship (SAR) on the hit-to-lead path using in-house libraries and/or commercially available compounds, and as a quick and efficient means of assessing target druggability.

Benchmark fragment-based 1H, 13C, 15N and 17O chemical shift predictions in molecular crystals†

Science.gov (United States)

Hartman, Joshua D.; Kudla, Ryan A.; Day, Graeme M.; Mueller, Leonard J.; Beran, Gregory J. O.

2016-01-01

The performance of fragment-based ab initio 1H, 13C, 15N and 17O chemical shift predictions is assessed against experimental NMR chemical shift data in four benchmark sets of molecular crystals. Employing a variety of commonly used density functionals (PBE0, B3LYP, TPSSh, OPBE, PBE, TPSS), we explore the relative performance of cluster, two-body fragment, and combined cluster/fragment models. The hybrid density functionals (PBE0, B3LYP and TPSSh) generally out-perform their generalized gradient approximation (GGA)-based counterparts. 1H, 13C, 15N, and 17O isotropic chemical shifts can be predicted with root-mean-square errors of 0.3, 1.5, 4.2, and 9.8 ppm, respectively, using a computationally inexpensive electrostatically embedded two-body PBE0 fragment model. Oxygen chemical shieldings prove particularly sensitive to local many-body effects, and using a combined cluster/fragment model instead of the simple two-body fragment model decreases the root-mean-square errors to 7.6 ppm. These fragment-based model errors compare favorably with GIPAW PBE ones of 0.4, 2.2, 5.4, and 7.2 ppm for the same 1H, 13C, 15N, and 17O test sets. Using these benchmark calculations, a set of recommended linear regression parameters for mapping between calculated chemical shieldings and observed chemical shifts are provided and their robustness assessed using statistical cross-validation. We demonstrate the utility of these approaches and the reported scaling parameters on applications to 9-tertbutyl anthracene, several histidine co-crystals, benzoic acid and the C-nitrosoarene SnCl2(CH3)2(NODMA)2. PMID:27431490

Benchmark fragment-based (1)H, (13)C, (15)N and (17)O chemical shift predictions in molecular crystals.

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Hartman, Joshua D; Kudla, Ryan A; Day, Graeme M; Mueller, Leonard J; Beran, Gregory J O

2016-08-21

The performance of fragment-based ab initio(1)H, (13)C, (15)N and (17)O chemical shift predictions is assessed against experimental NMR chemical shift data in four benchmark sets of molecular crystals. Employing a variety of commonly used density functionals (PBE0, B3LYP, TPSSh, OPBE, PBE, TPSS), we explore the relative performance of cluster, two-body fragment, and combined cluster/fragment models. The hybrid density functionals (PBE0, B3LYP and TPSSh) generally out-perform their generalized gradient approximation (GGA)-based counterparts. (1)H, (13)C, (15)N, and (17)O isotropic chemical shifts can be predicted with root-mean-square errors of 0.3, 1.5, 4.2, and 9.8 ppm, respectively, using a computationally inexpensive electrostatically embedded two-body PBE0 fragment model. Oxygen chemical shieldings prove particularly sensitive to local many-body effects, and using a combined cluster/fragment model instead of the simple two-body fragment model decreases the root-mean-square errors to 7.6 ppm. These fragment-based model errors compare favorably with GIPAW PBE ones of 0.4, 2.2, 5.4, and 7.2 ppm for the same (1)H, (13)C, (15)N, and (17)O test sets. Using these benchmark calculations, a set of recommended linear regression parameters for mapping between calculated chemical shieldings and observed chemical shifts are provided and their robustness assessed using statistical cross-validation. We demonstrate the utility of these approaches and the reported scaling parameters on applications to 9-tert-butyl anthracene, several histidine co-crystals, benzoic acid and the C-nitrosoarene SnCl2(CH3)2(NODMA)2.

Production of a phage-displayed single chain variable fragment ...

African Journals Online (AJOL)

Abstract. Purpose: To develop specific single chain variable fragments (scFv) against ... libraries. The binding ability of the selected scFv antibody fragments against the IBDV particles was ..... Hermelink H, Koscielniak E. A human recombinant.

LETTER TO THE EDITOR: Phase transition in a random fragmentation problem with applications to computer science

Science.gov (United States)

Dean, David S.; Majumdar, Satya N.

2002-08-01

We study a fragmentation problem where an initial object of size x is broken into m random pieces provided x > x0 where x0 is an atomic cut-off. Subsequently, the fragmentation process continues for each of those daughter pieces whose sizes are bigger than x0. The process stops when all the fragments have sizes smaller than x0. We show that the fluctuation of the total number of splitting events, characterized by the variance, generically undergoes a nontrivial phase transition as one tunes the branching number m through a critical value m = mc. For m mc they are anomalously large and non-Gaussian. We apply this general result to analyse two different search algorithms in computer science.

The consequences of translational and rotational entropy lost by small molecules on binding to proteins

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Murray, Christopher W.; Verdonk, Marcel L.

2002-10-01

When a small molecule binds to a protein, it loses a significant amount of rigid body translational and rotational entropy. Estimates of the associated energy barrier vary widely in the literature yet accurate estimates are important in the interpretation of results from fragment-based drug discovery techniques. This paper describes an analysis that allows the estimation of the rigid body entropy barrier from the increase in binding affinities that results when two fragments of known affinity and known binding mode are joined together. The paper reviews the relatively rare number of examples where good quality data is available. From the analysis of this data, we estimate that the barrier to binding, due to the loss of rigid-body entropy, is 15-20 kJ/mol, i.e. around 3 orders of magnitude in affinity at 298 K. This large barrier explains why it is comparatively rare to observe multiple fragments binding to non-overlapping adjacent sites in enzymes. The barrier is also consistent with medicinal chemistry experience where small changes in the critical binding regions of ligands are often poorly tolerated by enzymes.

Gluon fragmentation into 3 PJ quarkonium

International Nuclear Information System (INIS)

Ma, J.P.

1995-01-01

The functions of the gluon fragmentation into 3 P j quarkonium are calculated to order α 2 s . With the recent progress in analysing quarkonium systems in QCD it is possible show how the so called divergence in the limit of the zero-binding energy, which is related to P-wave quarkonia, is treated correctly in the case of fragmentation functions. The obtained fragmentation functions satisfy explicitly at the order of α 2 s the Altarelli-Parisi equation and when z → 0 they behave as z -1 as expected. 19 refs., 7 figs

The effect of catastrophic collisional fragmentation and diffuse medium accretion on a computational interstellar dust system

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Liffman, Kurt

1990-01-01

The effects of catastrophic collisional fragmentation and diffuse medium accretion on a the interstellar dust system are computed using a Monte Carlo computer model. The Monte Carlo code has as its basis an analytic solution of the bulk chemical evolution of a two-phase interstellar medium, described by Liffman and Clayton (1989). The model is subjected to numerous different interstellar processes as it transfers from one interstellar phase to another. Collisional fragmentation was found to be the dominant physical process that shapes the size spectrum of interstellar dust. It was found that, in the diffuse cloud phase, 90 percent of the refractory material is locked up in the dust grains, primarily due to accretion in the molecular medium. This result is consistent with the observed depletions of silicon. Depletions were found to be affected only slightly by diffuse cloud accretion.

NMR characterization of weak interactions between RhoGDI2 and fragment screening hits.

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Liu, Jiuyang; Gao, Jia; Li, Fudong; Ma, Rongsheng; Wei, Qingtao; Wang, Aidong; Wu, Jihui; Ruan, Ke

2017-01-01

The delineation of intrinsically weak interactions between novel targets and fragment screening hits has long limited the pace of hit-to-lead evolution. Rho guanine-nucleotide dissociation inhibitor 2 (RhoGDI2) is a novel target that lacks any chemical probes for the treatment of tumor metastasis. Protein-observed and ligand-observed NMR spectroscopy was used to characterize the weak interactions between RhoGDI2 and fragment screening hits. We identified three hits of RhoGDI2 using streamlined NMR fragment-based screening. The binding site residues were assigned using non-uniformly sampled C α - and H α -based three dimensional NMR spectra. The molecular docking to the proposed geranylgeranyl binding pocket of RhoGDI2 was guided by NMR restraints of chemical shift perturbations and ligand-observed transferred paramagnetic relaxation enhancement. We further validated the weak RhoGDI2-hit interactions using mutagenesis and structure-affinity analysis. Weak interactions between RhoGDI2 and fragment screening hits were delineated using an integrated NMR approach. Binders to RhoGDI2 as a potential anti-cancer target have been first reported, and their weak interactions were depicted using NMR spectroscopy. Our work highlights the powerfulness and the versatility of the integrative NMR techniques to provide valuable structural insight into the intrinsically weak interactions between RhoGDI2 and the fragment screening hits, which could hardly be conceived using other biochemical techniques. Copyright © 2016 Elsevier B.V. All rights reserved.

Ab initio protein structure assembly using continuous structure fragments and optimized knowledge-based force field.

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Xu, Dong; Zhang, Yang

2012-07-01

Ab initio protein folding is one of the major unsolved problems in computational biology owing to the difficulties in force field design and conformational search. We developed a novel program, QUARK, for template-free protein structure prediction. Query sequences are first broken into fragments of 1-20 residues where multiple fragment structures are retrieved at each position from unrelated experimental structures. Full-length structure models are then assembled from fragments using replica-exchange Monte Carlo simulations, which are guided by a composite knowledge-based force field. A number of novel energy terms and Monte Carlo movements are introduced and the particular contributions to enhancing the efficiency of both force field and search engine are analyzed in detail. QUARK prediction procedure is depicted and tested on the structure modeling of 145 nonhomologous proteins. Although no global templates are used and all fragments from experimental structures with template modeling score >0.5 are excluded, QUARK can successfully construct 3D models of correct folds in one-third cases of short proteins up to 100 residues. In the ninth community-wide Critical Assessment of protein Structure Prediction experiment, QUARK server outperformed the second and third best servers by 18 and 47% based on the cumulative Z-score of global distance test-total scores in the FM category. Although ab initio protein folding remains a significant challenge, these data demonstrate new progress toward the solution of the most important problem in the field. Copyright © 2012 Wiley Periodicals, Inc.

Immunogenic properties of Streptococcus agalactiae FbsA fragments.

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Salvatore Papasergi

Full Text Available Several species of Gram-positive bacteria can avidly bind soluble and surface-associated fibrinogen (Fng, a property that is considered important in the pathogenesis of human infections. To gain insights into the mechanism by which group B Streptococcus (GBS, a frequent neonatal pathogen, interacts with Fng, we have screened two phage displayed genomic GBS libraries. All of the Fng-binding phage clones contained inserts encoding fragments of FbsA, a protein displaying multiple repeats. Since the functional role of this protein is only partially understood, representative fragments were recombinantly expressed and analyzed for Fng binding affinity and ability to induce immune protection against GBS infection. Maternal immunization with 6pGST, a fragment containing five repeats, significantly protected mouse pups against lethal GBS challenge and these protective effects could be recapitulated by administration of anti-6pGST serum from adult animals. Notably, a monoclonal antibody that was capable of neutralizing Fng binding by 6pGST, but not a non-neutralizing antibody, could significantly protect pups against lethal GBS challenge. These data suggest that FbsA-Fng interaction promotes GBS pathogenesis and that blocking such interaction is a viable strategy to prevent or treat GBS infections.

Straightforward hit identification approach in fragment-based discovery of bromodomain-containing protein 4 (BRD4) inhibitors.

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Borysko, Petro; Moroz, Yurii S; Vasylchenko, Oleksandr V; Hurmach, Vasyl V; Starodubtseva, Anastasia; Stefanishena, Natalia; Nesteruk, Kateryna; Zozulya, Sergey; Kondratov, Ivan S; Grygorenko, Oleksandr O

2018-05-09

A combination approach of a fragment screening and "SAR by catalog" was used for the discovery of bromodomain-containing protein 4 (BRD4) inhibitors. Initial screening of 3695-fragment library against bromodomain 1 of BRD4 using thermal shift assay (TSA), followed by initial hit validation, resulted in 73 fragment hits, which were used to construct a follow-up library selected from available screening collection. Additionally, analogs of inactive fragments, as well as a set of randomly selected compounds were also prepared (3 × 3200 compounds in total). Screening of the resulting sets using TSA, followed by re-testing at several concentrations, counter-screen, and TR-FRET assay resulted in 18 confirmed hits. Compounds derived from the initial fragment set showed better hit rate as compared to the other two sets. Finally, building dose-response curves revealed three compounds with IC 50  = 1.9-7.4 μM. For these compounds, binding sites and conformations in the BRD4 (4UYD) have been determined by docking. Copyright © 2018 Elsevier Ltd. All rights reserved.

Interaction Entropy: A New Paradigm for Highly Efficient and Reliable Computation of Protein-Ligand Binding Free Energy.

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Duan, Lili; Liu, Xiao; Zhang, John Z H

2016-05-04

Efficient and reliable calculation of protein-ligand binding free energy is a grand challenge in computational biology and is of critical importance in drug design and many other molecular recognition problems. The main challenge lies in the calculation of entropic contribution to protein-ligand binding or interaction systems. In this report, we present a new interaction entropy method which is theoretically rigorous, computationally efficient, and numerically reliable for calculating entropic contribution to free energy in protein-ligand binding and other interaction processes. Drastically different from the widely employed but extremely expensive normal mode method for calculating entropy change in protein-ligand binding, the new method calculates the entropic component (interaction entropy or -TΔS) of the binding free energy directly from molecular dynamics simulation without any extra computational cost. Extensive study of over a dozen randomly selected protein-ligand binding systems demonstrated that this interaction entropy method is both computationally efficient and numerically reliable and is vastly superior to the standard normal mode approach. This interaction entropy paradigm introduces a novel and intuitive conceptual understanding of the entropic effect in protein-ligand binding and other general interaction systems as well as a practical method for highly efficient calculation of this effect.

Fragment-based screening by protein crystallography: successes and pitfalls.

Science.gov (United States)

Chilingaryan, Zorik; Yin, Zhou; Oakley, Aaron J

2012-10-08

Fragment-based drug discovery (FBDD) concerns the screening of low-molecular weight compounds against macromolecular targets of clinical relevance. These compounds act as starting points for the development of drugs. FBDD has evolved and grown in popularity over the past 15 years. In this paper, the rationale and technology behind the use of X-ray crystallography in fragment based screening (FBS) will be described, including fragment library design and use of synchrotron radiation and robotics for high-throughput X-ray data collection. Some recent uses of crystallography in FBS will be described in detail, including interrogation of the drug targets β-secretase, phenylethanolamine N-methyltransferase, phosphodiesterase 4A and Hsp90. These examples provide illustrations of projects where crystallography is straightforward or difficult, and where other screening methods can help overcome the limitations of crystallography necessitated by diffraction quality.

Fragment-Based Screening by Protein Crystallography: Successes and Pitfalls

Directory of Open Access Journals (Sweden)

Aaron J. Oakley

2012-10-01

Full Text Available Fragment-based drug discovery (FBDD concerns the screening of low-molecular weight compounds against macromolecular targets of clinical relevance. These compounds act as starting points for the development of drugs. FBDD has evolved and grown in popularity over the past 15 years. In this paper, the rationale and technology behind the use of X-ray crystallography in fragment based screening (FBS will be described, including fragment library design and use of synchrotron radiation and robotics for high-throughput X-ray data collection. Some recent uses of crystallography in FBS will be described in detail, including interrogation of the drug targets β-secretase, phenylethanolamine N-methyltransferase, phosphodiesterase 4A and Hsp90. These examples provide illustrations of projects where crystallography is straightforward or difficult, and where other screening methods can help overcome the limitations of crystallography necessitated by diffraction quality.

Inhibition of ligand exchange kinetics via active-site trapping with an antibody fragment.

Science.gov (United States)

Oyen, David; Steyaert, Jan; Barlow, John N

2014-04-01

We describe the first example of an inhibitory antibody fragment (nanobody ca1697) that binds simultaneously to an enzyme (the enzyme dihydrofolate reductase from Escherichia coli) and its bound substrate (folate). Binding of the antibody to the substrate causes a 20-fold reduction in the rate of folate exchange kinetics. This work opens up the prospect of designing new types of antibody-based inhibitors of enzymes and receptors through suitable design of immunogens.

Monoclonal antibody to the rat glucocorticoid receptor. Relationship between the immunoreactive and DNA-binding domain

International Nuclear Information System (INIS)

Eisen, L.P.; Reichman, M.E.; Thompson, E.B.; Gametchu, B.; Harrison, R.W.; Eisen, H.J.

1985-01-01

The region of the glucocorticoid receptor that reacted with a monoclonal antibody (BUGR-1) was identified. In order to identify the immunoreactive region, the rat liver glucocorticoid receptor was subjected to limited proteolysis; immunoreactive fragments were identified by Western blotting. The monoclonal antibody reacted with both the undigested Mr approximately 97,000 receptor subunit and a Mr approximately 45,000 fragment containing the steroid-binding and DNA-binding domains. Digestion by trypsin also produced two steroid-binding fragments of Mr approximately 27,000 and 31,000 which did not react with the antibody and an immunoreactive Mr approximately 16,000 fragment. This Mr approximately 16,000 fragment was shown to bind to DNA-cellulose, indicating that it contained a DNA-binding domain of the receptor. The undigested receptor must have steroid associated with it to undergo activation to a DNA-binding form. However, the Mr approximately 16,000 immunoreactive fragment binds to DNA-cellulose even if it is obtained by digestion of the steroid-free holoreceptor which does not itself bind to DNA

Crius: A Novel Fragment-Based Algorithm of De Novo Substrate Prediction for Enzymes.

Science.gov (United States)

Yao, Zhiqiang; Jiang, Shuiqin; Zhang, Lujia; Gao, Bei; He, Xiao; Zhang, John Z H; Wei, Dongzhi

2018-05-03

The study of enzyme substrate specificity is vital for developing potential applications of enzymes. However, the routine experimental procedures require lot of resources in the discovery of novel substrates. This article reports an in silico structure-based algorithm called Crius, which predicts substrates for enzyme. The results of this fragment-based algorithm show good agreements between the simulated and experimental substrate specificities, using a lipase from Candida antarctica (CALB), a nitrilase from Cyanobacterium syechocystis sp. PCC6803 (Nit6803), and an aldo-keto reductase from Gluconobacter oxydans (Gox0644). This opens new prospects of developing computer algorithms that can effectively predict substrates for an enzyme. This article is protected by copyright. All rights reserved. © 2018 The Protein Society.

Validation of tautomeric and protomeric binding modes by free energy calculations. A case study for the structure based optimization of d-amino acid oxidase inhibitors

Science.gov (United States)

Orgován, Zoltán; Ferenczy, György G.; Steinbrecher, Thomas; Szilágyi, Bence; Bajusz, Dávid; Keserű, György M.

2018-02-01

Optimization of fragment size d-amino acid oxidase (DAAO) inhibitors was investigated using a combination of computational and experimental methods. Retrospective free energy perturbation (FEP) calculations were performed for benzo[d]isoxazole derivatives, a series of known inhibitors with two potential binding modes derived from X-ray structures of other DAAO inhibitors. The good agreement between experimental and computed binding free energies in only one of the hypothesized binding modes strongly support this bioactive conformation. Then, a series of 1-H-indazol-3-ol derivatives formerly not described as DAAO inhibitors was investigated. Binding geometries could be reliably identified by structural similarity to benzo[d]isoxazole and other well characterized series and FEP calculations were performed for several tautomers of the deprotonated and protonated compounds since all these forms are potentially present owing to the experimental pKa values of representative compounds in the series. Deprotonated compounds are proposed to be the most important bound species owing to the significantly better agreement between their calculated and measured affinities compared to the protonated forms. FEP calculations were also used for the prediction of the affinities of compounds not previously tested as DAAO inhibitors and for a comparative structure-activity relationship study of the benzo[d]isoxazole and indazole series. Selected indazole derivatives were synthesized and their measured binding affinity towards DAAO was in good agreement with FEP predictions.

Simulations of High Speed Fragment Trajectories

Science.gov (United States)

Yeh, Peter; Attaway, Stephen; Arunajatesan, Srinivasan; Fisher, Travis

2017-11-01

Flying shrapnel from an explosion are capable of traveling at supersonic speeds and distances much farther than expected due to aerodynamic interactions. Predicting the trajectories and stable tumbling modes of arbitrary shaped fragments is a fundamental problem applicable to range safety calculations, damage assessment, and military technology. Traditional approaches rely on characterizing fragment flight using a single drag coefficient, which may be inaccurate for fragments with large aspect ratios. In our work we develop a procedure to simulate trajectories of arbitrary shaped fragments with higher fidelity using high performance computing. We employ a two-step approach in which the force and moment coefficients are first computed as a function of orientation using compressible computational fluid dynamics. The force and moment data are then input into a six-degree-of-freedom rigid body dynamics solver to integrate trajectories in time. Results of these high fidelity simulations allow us to further understand the flight dynamics and tumbling modes of a single fragment. Furthermore, we use these results to determine the validity and uncertainty of inexpensive methods such as the single drag coefficient model.

Fragment-based drug discovery and molecular docking in drug design.

Science.gov (United States)

Wang, Tao; Wu, Mian-Bin; Chen, Zheng-Jie; Chen, Hua; Lin, Jian-Ping; Yang, Li-Rong

2015-01-01

Fragment-based drug discovery (FBDD) has caused a revolution in the process of drug discovery and design, with many FBDD leads being developed into clinical trials or approved in the past few years. Compared with traditional high-throughput screening, it displays obvious advantages such as efficiently covering chemical space, achieving higher hit rates, and so forth. In this review, we focus on the most recent developments of FBDD for improving drug discovery, illustrating the process and the importance of FBDD. In particular, the computational strategies applied in the process of FBDD and molecular-docking programs are highlighted elaborately. In most cases, docking is used for predicting the ligand-receptor interaction modes and hit identification by structurebased virtual screening. The successful cases of typical significance and the hits identified most recently are discussed.

Analytical and experimental evaluation of a proposed self-forging fragment munition

International Nuclear Information System (INIS)

Tuft, D.B.; Folsom, E.N.

1982-01-01

Analytical and experimental tools have been used to study the formation of a proposed self-forging fragment projectile. The primary objective of this study is the determination of the interior and exterior shape of the fully formed fragment, and to determine if the fragment tumbles in flight. In addition, it is of interest to compare computer predictions to experimental results. An experiment was performed using high speed photography and high-energy flash x-ray radiography to study liner and case motion and projectile formation. Fabrication and assembly tolerances were closely controlled in an effort to eliminate tolerances as a possible source of fragment instability. X-ray film-density contours were analyzed to determine the fully formed fragment interior and exterior shape. Down-range yaw screens showed fragment tumbling in flight. The computed fragment shape was compared to experimental results and it was found that a retaining ring in the computational model near the liner periphery had a significant effect on the final computed fragment shape. With the retaining ring in the computational model and full two-way sliding between all material interfaces, the final computed fragment showed very good agreement with the experiment on both exterior and interior shapes

Computational design of binding proteins to EGFR domain II.

Directory of Open Access Journals (Sweden)

Yoon Sup Choi

Full Text Available We developed a process to produce novel interactions between two previously unrelated proteins. This process selects protein scaffolds and designs protein interfaces that bind to a surface patch of interest on a target protein. Scaffolds with shapes complementary to the target surface patch were screened using an exhaustive computational search of the human proteome and optimized by directed evolution using phage display. This method was applied to successfully design scaffolds that bind to epidermal growth factor receptor (EGFR domain II, the interface of EGFR dimerization, with high reactivity toward the target surface patch of EGFR domain II. One potential application of these tailor-made protein interactions is the development of therapeutic agents against specific protein targets.

DNA Length Modulates the Affinity of Fragments of Genomic DNA for the Nuclear Matrix In Vitro.

Science.gov (United States)

García-Vilchis, David; Aranda-Anzaldo, Armando

2017-12-01

Classical observations have shown that during the interphase the chromosomal DNA of metazoans is organized in supercoiled loops attached to a compartment known as the nuclear matrix (NM). Fragments of chromosomal DNA able to bind the isolated NM in vitro are known as matrix associated/attachment/addressed regions or MARs. No specific consensus sequence or motif has been found that may constitute a universal, defining feature of MARs. On the other hand, high-salt resistant DNA-NM interactions in situ define true DNA loop anchorage regions or LARs, that might correspond to a subset of the potential MARs but are not necessarily identical to MARs characterized in vitro, since there are several examples of MARs able to bind the NM in vitro but which are not actually bound to the NM in situ. In the present work we assayed the capacity of two LARs, as well as of shorter fragments within such LARs, for binding to the NM in vitro. Paradoxically the isolated (≈2 kb) LARs cannot bind to the NM in vitro while their shorter (≈300 pb) sub-fragments and other non-related but equally short DNA fragments, bind to the NM in a high-salt resistant fashion. Our results suggest that the ability of a given DNA fragment for binding to the NM in vitro primarily depends on the length of the fragment, suggesting that binding to the NM is modulated by the local topology of the DNA fragment in suspension that it is known to depend on the DNA length. J. Cell. Biochem. 118: 4487-4497, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

A linear relationship between crystal size and fragment binding time observed crystallographically: implications for fragment library screening using acoustic droplet ejection.

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Krystal Cole

Full Text Available High throughput screening technologies such as acoustic droplet ejection (ADE greatly increase the rate at which X-ray diffraction data can be acquired from crystals. One promising high throughput screening application of ADE is to rapidly combine protein crystals with fragment libraries. In this approach, each fragment soaks into a protein crystal either directly on data collection media or on a moving conveyor belt which then delivers the crystals to the X-ray beam. By simultaneously handling multiple crystals combined with fragment specimens, these techniques relax the automounter duty-cycle bottleneck that currently prevents optimal exploitation of third generation synchrotrons. Two factors limit the speed and scope of projects that are suitable for fragment screening using techniques such as ADE. Firstly, in applications where the high throughput screening apparatus is located inside the X-ray station (such as the conveyor belt system described above, the speed of data acquisition is limited by the time required for each fragment to soak into its protein crystal. Secondly, in applications where crystals are combined with fragments directly on data acquisition media (including both of the ADE methods described above, the maximum time that fragments have to soak into crystals is limited by evaporative dehydration of the protein crystals during the fragment soak. Here we demonstrate that both of these problems can be minimized by using small crystals, because the soak time required for a fragment hit to attain high occupancy depends approximately linearly on crystal size.

When fragments link : a bibliometric perspective on the development of fragment-based drug discovery

NARCIS (Netherlands)

Romasanta, A.K.S.; van der Sijde, P.C.; Hellsten, I.; Hubbard, Roderick E.; Keseru, Gyorgy M.; van Muijlwijk-Koezen, Jacqueline E.; de Esch, I.J.P.

2018-01-01

Fragment-based drug discovery (FBDD) is a highly interdisciplinary field, rich in ideas integrated from pharmaceutical sciences, chemistry, biology, and physics, among others. To enrich our understanding of the development of the field, we used bibliometric techniques to analyze 3642 publications in

A focused fragment library targeting the antibiotic resistance enzyme - Oxacillinase-48: Synthesis, structural evaluation and inhibitor design.

Science.gov (United States)

Akhter, Sundus; Lund, Bjarte Aarmo; Ismael, Aya; Langer, Manuel; Isaksson, Johan; Christopeit, Tony; Leiros, Hanna-Kirsti S; Bayer, Annette

2018-02-10

β-Lactam antibiotics are of utmost importance when treating bacterial infections in the medical community. However, currently their utility is threatened by the emergence and spread of β-lactam resistance. The most prevalent resistance mechanism to β-lactam antibiotics is expression of β-lactamase enzymes. One way to overcome resistance caused by β-lactamases, is the development of β-lactamase inhibitors and today several β-lactamase inhibitors e.g. avibactam, are approved in the clinic. Our focus is the oxacillinase-48 (OXA-48), an enzyme reported to spread rapidly across the world and commonly identified in Escherichia coli and Klebsiella pneumoniae. To guide inhibitor design, we used diversely substituted 3-aryl and 3-heteroaryl benzoic acids to probe the active site of OXA-48 for useful enzyme-inhibitor interactions. In the presented study, a focused fragment library containing 49 3-substituted benzoic acid derivatives were synthesised and biochemically characterized. Based on crystallographic data from 33 fragment-enzyme complexes, the fragments could be classified into R 1 or R 2 binders by their overall binding conformation in relation to the binding of the R 1 and R 2 side groups of imipenem. Moreover, binding interactions attractive for future inhibitor design were found and their usefulness explored by the rational design and evaluation of merged inhibitors from orthogonally binding fragments. The best inhibitors among the resulting 3,5-disubstituted benzoic acids showed inhibitory potential in the low micromolar range (IC 50  = 2.9 μM). For these inhibitors, the complex X-ray structures revealed non-covalent binding to Arg250, Arg214 and Tyr211 in the active site and the interactions observed with the mono-substituted fragments were also identified in the merged structures. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Computational estimation of rainbow trout estrogen receptor binding affinities for environmental estrogens

International Nuclear Information System (INIS)

Shyu, Conrad; Cavileer, Timothy D.; Nagler, James J.; Ytreberg, F. Marty

2011-01-01

Environmental estrogens have been the subject of intense research due to their documented detrimental effects on the health of fish and wildlife and their potential to negatively impact humans. A complete understanding of how these compounds affect health is complicated because environmental estrogens are a structurally heterogeneous group of compounds. In this work, computational molecular dynamics simulations were utilized to predict the binding affinity of different compounds using rainbow trout (Oncorhynchus mykiss) estrogen receptors (ERs) as a model. Specifically, this study presents a comparison of the binding affinity of the natural ligand estradiol-17β to the four rainbow trout ER isoforms with that of three known environmental estrogens 17α-ethinylestradiol, bisphenol A, and raloxifene. Two additional compounds, atrazine and testosterone, that are known to be very weak or non-binders to ERs were tested. The binding affinity of these compounds to the human ERα subtype is also included for comparison. The results of this study suggest that, when compared to estradiol-17β, bisphenol A binds less strongly to all four receptors, 17α-ethinylestradiol binds more strongly, and raloxifene has a high affinity for the α subtype only. The results also show that atrazine and testosterone are weak or non-binders to the ERs. All of the results are in excellent qualitative agreement with the known in vivo estrogenicity of these compounds in the rainbow trout and other fishes. Computational estimation of binding affinities could be a valuable tool for predicting the impact of environmental estrogens in fish and other animals.

Residue preference mapping of ligand fragments in the Protein Data Bank.

Science.gov (United States)

Wang, Lirong; Xie, Zhaojun; Wipf, Peter; Xie, Xiang-Qun

2011-04-25

The interaction between small molecules and proteins is one of the major concerns for structure-based drug design because the principles of protein-ligand interactions and molecular recognition are not thoroughly understood. Fortunately, the analysis of protein-ligand complexes in the Protein Data Bank (PDB) enables unprecedented possibilities for new insights. Herein, we applied molecule-fragmentation algorithms to split the ligands extracted from PDB crystal structures into small fragments. Subsequently, we have developed a ligand fragment and residue preference mapping (LigFrag-RPM) algorithm to map the profiles of the interactions between these fragments and the 20 proteinogenic amino acid residues. A total of 4032 fragments were generated from 71 798 PDB ligands by a ring cleavage (RC) algorithm. Among these ligand fragments, 315 unique fragments were characterized with the corresponding fragment-residue interaction profiles by counting residues close to these fragments. The interaction profiles revealed that these fragments have specific preferences for certain types of residues. The applications of these interaction profiles were also explored and evaluated in case studies, showing great potential for the study of protein-ligand interactions and drug design. Our studies demonstrated that the fragment-residue interaction profiles generated from the PDB ligand fragments can be used to detect whether these fragments are in their favorable or unfavorable environments. The algorithm for a ligand fragment and residue preference mapping (LigFrag-RPM) developed here also has the potential to guide lead chemistry modifications as well as binding residues predictions.

Assessing the model transferability for prediction of transcription factor binding sites based on chromatin accessibility.

Science.gov (United States)

Liu, Sheng; Zibetti, Cristina; Wan, Jun; Wang, Guohua; Blackshaw, Seth; Qian, Jiang

2017-07-27

Computational prediction of transcription factor (TF) binding sites in different cell types is challenging. Recent technology development allows us to determine the genome-wide chromatin accessibility in various cellular and developmental contexts. The chromatin accessibility profiles provide useful information in prediction of TF binding events in various physiological conditions. Furthermore, ChIP-Seq analysis was used to determine genome-wide binding sites for a range of different TFs in multiple cell types. Integration of these two types of genomic information can improve the prediction of TF binding events. We assessed to what extent a model built upon on other TFs and/or other cell types could be used to predict the binding sites of TFs of interest. A random forest model was built using a set of cell type-independent features such as specific sequences recognized by the TFs and evolutionary conservation, as well as cell type-specific features derived from chromatin accessibility data. Our analysis suggested that the models learned from other TFs and/or cell lines performed almost as well as the model learned from the target TF in the cell type of interest. Interestingly, models based on multiple TFs performed better than single-TF models. Finally, we proposed a universal model, BPAC, which was generated using ChIP-Seq data from multiple TFs in various cell types. Integrating chromatin accessibility information with sequence information improves prediction of TF binding.The prediction of TF binding is transferable across TFs and/or cell lines suggesting there are a set of universal "rules". A computational tool was developed to predict TF binding sites based on the universal "rules".

Chromatin immunoprecipitation to analyze DNA binding sites of HMGA2.

Directory of Open Access Journals (Sweden)

Nina Winter

Full Text Available BACKGROUND: HMGA2 is an architectonic transcription factor abundantly expressed during embryonic and fetal development and it is associated with the progression of malignant tumors. The protein harbours three basically charged DNA binding domains and an acidic protein binding C-terminal domain. DNA binding induces changes of DNA conformation and hence results in global overall change of gene expression patterns. Recently, using a PCR-based SELEX (Systematic Evolution of Ligands by Exponential Enrichment procedure two consensus sequences for HMGA2 binding have been identified. METHODOLOGY/PRINCIPAL FINDINGS: In this investigation chromatin immunoprecipitation (ChIP experiments and bioinformatic methods were used to analyze if these binding sequences can be verified on chromatin of living cells as well. CONCLUSION: After quantification of HMGA2 protein in different cell lines the colon cancer derived cell line HCT116 was chosen for further ChIP experiments because of its 3.4-fold higher HMGA2 protein level. 49 DNA fragments were obtained by ChIP. These fragments containing HMGA2 binding sites have been analyzed for their AT-content, location in the human genome and similarities to sequences generated by a SELEX study. The sequences show a significantly higher AT-content than the average of the human genome. The artificially generated SELEX sequences and short BLAST alignments (11 and 12 bp of the ChIP fragments from living cells show similarities in their organization. The flanking regions are AT-rich, whereas a lower conservation is present in the center of the sequences.

Production of a phage-displayed single chain variable fragment ...

African Journals Online (AJOL)

Purpose: To develop specific single chain variable fragments (scFv) against infectious bursal disease virus (IBDV) via phage display technology. Methods: Purified viruses were initially applied for iterative panning rounds of scFv phage display libraries. The binding ability of the selected scFv antibody fragments against the ...

Highly efficient power system based on direct fission fragment energy conversion utilizing magnetic collimation

International Nuclear Information System (INIS)

Tsvetkov, Pavel V.; Hart, Ron R.; Parish, Theodore A.

2003-01-01

The present study was focused on developing a technologically feasible power system that is based on direct fission fragment energy conversion utilizing magnetic collimation. The new concept is an attempt to combine several advantageous design solutions, which have been proposed for application in both fission and fusion reactors, into one innovative system that can offer exceptional energy conversion efficiency. The analysis takes into consideration a wide range of operational aspects including fission fragment escape from the fuel, collimation, collection, criticality, long-term performance, energy conversion efficiency, heat removal, and safety characteristics. Specific characteristics of the individual system components and the entire system are evaluated. Consistent analysis and evaluation of the technological feasibility of the concept were achieved using state-of-the-art computer codes that allowed realistic and consistent modeling. The calculated energy conversion efficiencies for the presented designs without a thermodynamic cycle and with the heavy water cycle are 52% and 62%, respectively. The analysis indicates that efficiencies up to 90% are potentially achievable. (author)

Thermodynamic model of binding of flexible bivalent haptens to antibody

Energy Technology Data Exchange (ETDEWEB)

Dembo, M; Goldstein, B

1978-01-01

Studies by Wilder et al. of the binding of Fab' fragments to small haptens have shown that the cross-linking constant (the equilibrium constant for binding an additional Fab' fragment to a hapten-Fab' complex) is strongly dependent on the length of the hapten. We present a simple model for predicting the relationship between the intermolecular cross-linking constant and the monovalent hapten-antibody binding constant. In particular we used the model to obtain the dependence of the cross-linking constant on the length of th hapten, the depth to which the hapten fills th Fab' binding site, and the size of the Fab' fragment. To test the model, we devised expressions which allowed us to analyze the data of Wilder et al. From their data we determined the values of two parameters which we took to be unknown in the theory, the size of the Fab' fragment and the depth to which the hapten fills the Fab' binding site. The values arrived at in this way agreed well with published measurements of these parameters.

Fragment screening for drug leads by weak affinity chromatography (WAC-MS).

Science.gov (United States)

Ohlson, Sten; Duong-Thi, Minh-Dao

2018-02-23

Fragment-based drug discovery is an important tool for design of small molecule hit-to-lead compounds against various biological targets. Several approved drugs have been derived from an initial fragment screen and many such candidates are in various stages of clinical trials. Finding fragment hits, that are suitable for optimisation by medicinal chemists, is still a challenge as the binding between the small fragment and its target is weak in the range of mM to µM of K d and irrelevant non-specific interactions are abundant in this area of transient interactions. Fortunately, there are methods that can study weak interactions quite efficiently of which NMR, surface plasmon resonance (SPR) and X-ray crystallography are the most prominent. Now, a new technology based on zonal affinity chromatography, weak affinity chromatography (WAC), has been introduced which has remedied many of the problems with other technologies. By combining WAC with mass spectrometry (WAC-MS), it is a powerful tool to identify binders quantitatively in terms of affinity and kinetics either from fragment libraries or from complex mixtures of biological extracts. As WAC-MS can be multiplexed by analysing mixtures of fragments (20-100 fragments) in one sample, this approach yields high throughput, where a whole library of e.g. >2000 fragments can be analysed quantitatively within a day. WAC-MS is easy to perform, where the robustness and quality of HPLC is fully utilized. This review will highlight the rationale behind the application of WAC-MS for fragment screening in drug discovery. Copyright © 2018 Elsevier Inc. All rights reserved.

The antigen-binding fragment of human gamma immunoglobulin prevents amyloid β-peptide folding into β-sheet to form oligomers

Science.gov (United States)

Valls-Comamala, Victòria; Guivernau, Biuse; Bonet, Jaume; Puig, Marta; Perálvarez-Marín, Alex; Palomer, Ernest; Fernàndez-Busquets, Xavier; Altafaj, Xavier; Tajes, Marta; Puig-Pijoan, Albert; Vicente, Rubén; Oliva, Baldomero; Muñoz, Francisco J.

2017-01-01

The amyloid beta-peptide (Aβ) plays a leading role in Alzheimer's disease (AD) physiopathology. Even though monomeric forms of Aβ are harmless to cells, Aβ can aggregate into β-sheet oligomers and fibrils, which are both neurotoxic. Therefore, one of the main therapeutic approaches to cure or delay AD onset and progression is targeting Aβ aggregation. In the present study, we show that a pool of human gamma immunoglobulins (IgG) protected cortical neurons from the challenge with Aβ oligomers, as assayed by MTT reduction, caspase-3 activation and cytoskeleton integrity. In addition, we report the inhibitory effect of IgG on Aβ aggregation, as shown by Thioflavin T assay, size exclusion chromatography and atomic force microscopy. Similar results were obtained with Palivizumab, a human anti-sincitial virus antibody. In order to dissect the important domains, we cleaved the pool of human IgG with papain to obtain Fab and Fc fragments. Using these cleaved fragments, we functionally identified Fab as the immunoglobulin fragment inhibiting Aβ aggregation, a result that was further confirmed by an in silico structural model. Interestingly, bioinformatic tools show a highly conserved structure able to bind amyloid in the Fab region. Overall, our data strongly support the inhibitory effect of human IgG on Aβ aggregation and its neuroprotective role. PMID:28467807

Cold-inducible RNA-binding protein through TLR4 signaling induces mitochondrial DNA fragmentation and regulates macrophage cell death after trauma.

Science.gov (United States)

Li, Zhigang; Fan, Erica K; Liu, Jinghua; Scott, Melanie J; Li, Yuehua; Li, Song; Xie, Wen; Billiar, Timothy R; Wilson, Mark A; Jiang, Yong; Wang, Ping; Fan, Jie

2017-05-11

Trauma is a major cause of systemic inflammatory response syndrome and multiple organ dysfunction syndrome. Macrophages (Mφ) direct trauma-induced inflammation, and Mφ death critically influences the progression of the inflammatory response. In the current study, we explored an important role of trauma in inducing mitochondrial DNA (mtDNA) damage in Mφ and the subsequent regulation of Mφ death. Using an animal pseudo-fracture trauma model, we demonstrated that tissue damage induced NADPH oxidase activation and increased the release of reactive oxygen species via cold-inducible RNA-binding protein (CIRP)-TLR4-MyD88 signaling. This in turn, activates endonuclease G, which serves as an executor for the fragmentation of mtDNA in Mφ. We further showed that fragmented mtDNA triggered both p62-related autophagy and necroptosis in Mφ. However, autophagy activation also suppressed Mφ necroptosis and pro-inflammatory responses. This study demonstrates a previously unidentified intracellular regulation of Mφ homeostasis in response to trauma.

Discovery and validation of information theory-based transcription factor and cofactor binding site motifs.

Science.gov (United States)

Lu, Ruipeng; Mucaki, Eliseos J; Rogan, Peter K

2017-03-17

Data from ChIP-seq experiments can derive the genome-wide binding specificities of transcription factors (TFs) and other regulatory proteins. We analyzed 765 ENCODE ChIP-seq peak datasets of 207 human TFs with a novel motif discovery pipeline based on recursive, thresholded entropy minimization. This approach, while obviating the need to compensate for skewed nucleotide composition, distinguishes true binding motifs from noise, quantifies the strengths of individual binding sites based on computed affinity and detects adjacent cofactor binding sites that coordinate with the targets of primary, immunoprecipitated TFs. We obtained contiguous and bipartite information theory-based position weight matrices (iPWMs) for 93 sequence-specific TFs, discovered 23 cofactor motifs for 127 TFs and revealed six high-confidence novel motifs. The reliability and accuracy of these iPWMs were determined via four independent validation methods, including the detection of experimentally proven binding sites, explanation of effects of characterized SNPs, comparison with previously published motifs and statistical analyses. We also predict previously unreported TF coregulatory interactions (e.g. TF complexes). These iPWMs constitute a powerful tool for predicting the effects of sequence variants in known binding sites, performing mutation analysis on regulatory SNPs and predicting previously unrecognized binding sites and target genes. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Mapping of the antigenic and allergenic epitopes of Lol p VB using gene fragmentation.

Science.gov (United States)

Ong, E K; Knox, R B; Singh, M B

1995-03-01

The recombinant proteins of Lol p VA and Lol p VB expressed in E. coli reacted with IgE antibodies from sera of allergic patients and mAbs FMC A7 and PpV1. Cross-absorption analyses using these recombinant proteins showed that Lol p VA and Lol p VB possess both similar and unique IgE binding determinants. Gene fragmentation was utilized to localize the antigenic and allergenic determinants of Lol p VB. When full-length cDNA of Lol p VB was digested into three fragments and expressed as the fusions from the glutathione transferase of pGEX vectors, fragments Met1-Val196 and Asp197-Val339 bound IgE while fragment Met1-Pro96 did not. The data suggest that there are at least two IgE binding determinants in Lol p VB. In addition, only fragment Met1-Val196 reacted with mAb PpV1. The localization of these determinants was further resolved using random fragment expression libraries. The mAb PpV1 determinant was near the N-terminal region of Lol p VB molecule. The IgE binding determinants were distributed in the central region: region I (amino acids 111-195) and II (199-254). These IgE binding determinants are conserved in Lol p VA.

In silico fragmentation for computer assisted identification of metabolite mass spectra

Directory of Open Access Journals (Sweden)

Müller-Hannemann Matthias

2010-03-01

Full Text Available Abstract Background Mass spectrometry has become the analytical method of choice in metabolomics research. The identification of unknown compounds is the main bottleneck. In addition to the precursor mass, tandem MS spectra carry informative fragment peaks, but the coverage of spectral libraries of measured reference compounds are far from covering the complete chemical space. Compound libraries such as PubChem or KEGG describe a larger number of compounds, which can be used to compare their in silico fragmentation with spectra of unknown metabolites. Results We created the MetFrag suite to obtain a candidate list from compound libraries based on the precursor mass, subsequently ranked by the agreement between measured and in silico fragments. In the evaluation MetFrag was able to rank most of the correct compounds within the top 3 candidates returned by an exact mass query in KEGG. Compared to a previously published study, MetFrag obtained better results than the commercial MassFrontier software. Especially for large compound libraries, the candidates with a good score show a high structural similarity or just different stereochemistry, a subsequent clustering based on chemical distances reduces this redundancy. The in silico fragmentation requires less than a second to process a molecule, and MetFrag performs a search in KEGG or PubChem on average within 30 to 300 seconds, respectively, on an average desktop PC. Conclusions We presented a method that is able to identify small molecules from tandem MS measurements, even without spectral reference data or a large set of fragmentation rules. With today's massive general purpose compound libraries we obtain dozens of very similar candidates, which still allows a confident estimate of the correct compound class. Our tool MetFrag improves the identification of unknown substances from tandem MS spectra and delivers better results than comparable commercial software. MetFrag is available through a web

Target Immobilization as a Strategy for NMR-Based Fragment Screening: Comparison of TINS, STD, and SPR for Fragment Hit Identification

NARCIS (Netherlands)

Kobayashi, M.; Retra, K.; Figaroa, F.; Hollander, J.G.; Ab, E.; Heetebrij, R.J.; Irth, H.; Siegal, G.

2010-01-01

Fragment-based drug discovery (FBDD) has become a widely accepted tool that is complementary to high-throughput screening (HTS) in developing small-molecule inhibitors of pharmaceutical targets. Because a fragment campaign can only be as successful as the hit matter found, it is critical that the

incaRNAfbinv: a web server for the fragment-based design of RNA sequences

Science.gov (United States)

Drory Retwitzer, Matan; Reinharz, Vladimir; Ponty, Yann; Waldispühl, Jérôme; Barash, Danny

2016-01-01

Abstract In recent years, new methods for computational RNA design have been developed and applied to various problems in synthetic biology and nanotechnology. Lately, there is considerable interest in incorporating essential biological information when solving the inverse RNA folding problem. Correspondingly, RNAfbinv aims at including biologically meaningful constraints and is the only program to-date that performs a fragment-based design of RNA sequences. In doing so it allows the design of sequences that do not necessarily exactly fold into the target, as long as the overall coarse-grained tree graph shape is preserved. Augmented by the weighted sampling algorithm of incaRNAtion, our web server called incaRNAfbinv implements the method devised in RNAfbinv and offers an interactive environment for the inverse folding of RNA using a fragment-based design approach. It takes as input: a target RNA secondary structure; optional sequence and motif constraints; optional target minimum free energy, neutrality and GC content. In addition to the design of synthetic regulatory sequences, it can be used as a pre-processing step for the detection of novel natural occurring RNAs. The two complementary methodologies RNAfbinv and incaRNAtion are merged together and fully implemented in our web server incaRNAfbinv, available at http://www.cs.bgu.ac.il/incaRNAfbinv. PMID:27185893

A new in silico classification model for ready biodegradability, based on molecular fragments.

Science.gov (United States)

Lombardo, Anna; Pizzo, Fabiola; Benfenati, Emilio; Manganaro, Alberto; Ferrari, Thomas; Gini, Giuseppina

2014-08-01

Regulations such as the European REACH (Registration, Evaluation, Authorization and restriction of Chemicals) often require chemicals to be evaluated for ready biodegradability, to assess the potential risk for environmental and human health. Because not all chemicals can be tested, there is an increasing demand for tools for quick and inexpensive biodegradability screening, such as computer-based (in silico) theoretical models. We developed an in silico model starting from a dataset of 728 chemicals with ready biodegradability data (MITI-test Ministry of International Trade and Industry). We used the novel software SARpy to automatically extract, through a structural fragmentation process, a set of substructures statistically related to ready biodegradability. Then, we analysed these substructures in order to build some general rules. The model consists of a rule-set made up of the combination of the statistically relevant fragments and of the expert-based rules. The model gives good statistical performance with 92%, 82% and 76% accuracy on the training, test and external set respectively. These results are comparable with other in silico models like BIOWIN developed by the United States Environmental Protection Agency (EPA); moreover this new model includes an easily understandable explanation. Copyright © 2014 Elsevier Ltd. All rights reserved.

Ionization and fragmentation of DNA-RNA bases: a density functional theory study

International Nuclear Information System (INIS)

Sadr-Arani, Leila

2014-01-01

Ionizing radiation (IR) cross human tissue, deposit energy and dissipate fragmenting molecules. The resulting fragments may be highlighted by mass spectrometry. Despite the amount of information obtained experimentally by the interpretation of the mass spectrum, experience alone cannot answer all the questions of the mechanism of fragmentation of DNA/RNA bases and a theoretical study is a complement to this information. A theoretical study allows us to know the weakest bonds in the molecule during ionization and thus may help to provide mechanisms of dissociation and produced fragments. The purpose of this work, using the DFT with the PBE functional, is to study the ionization and fragmentation mechanisms of DNA/RNA bases (Uracil, Cytosine, Adenine and Guanine) and to identify the cations corresponding to each peak in mass spectra. For all RNA bases, the retro Diels-Alder reaction (elimination of HNCO or NCO*) is a major route for dissociating, with the exception of adenine for which there is no atom oxygen in its structure. Loss of NH 3 (NH 2 *) molecule is another common way to all bases that contain amine group. The possibility of the loss of hydrogen from the cations is also investigated, as well as the dissociation of dehydrogenated cations and protonated uracil. This work shows the interest of providing DFT calculation in the interpretation of mass spectra of DNA bases. (author)

Structural organization of the human glucocorticoid receptor determined by one- and two-dimensional gel electrophoresis of proteolytic receptor fragments

International Nuclear Information System (INIS)

Smith, A.C.; Harmon, J.M.

1987-01-01

The structural organization of the steroid-binding protein of the IM-9 cell glucocorticoid receptor was investigated by using one- and two-dimensional gel electrophoresis of proteolytic receptor fragments. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of receptor fragments isolated after trypsin digestion of immunopurified [ 3 H]dexamethasone 21-mesylate ([ 3 H]DM-) labeled receptor revealed the presence of a stable 26.5-kilodalton (kDa) steroid-containing non-DNA-binding fragment, derived from a larger, less stable, 29-kDa fragment. The 26.5-kDa tryptic fragment appeared to be completely contained within a 41-kDa, steroid-containing, DNA-binding species isolated after chymotrypsin digestion of the intact protein. Two-dimensional electrophoretic analysis of the [ 3 H]DM-labeled tryptic fragments resolved two 26.5-kDa and two 29-kDa components. This was the same number of isoforms seen in the intact protein, indicating that the charge heterogeneity of the steroid-binding protein is the result of modification within the steroid-containing, non-DNA-binding, 26.5-kDa tryptic fragment. Two-dimensional analysis of the 41-kDa [ 3 H]DM-labeled chymotryptic species revealed a pattern of isoforms more complex than that seen either in the intact protein or in the steroid-containing tryptic fragments. These results suggest that the 41-kDa [ 3 H]DM-labeled species resolved by one-dimensional SDS-PAGE after chymotrypsin digestion may be composed of several distinct proteolytic fragments

A computational method for identification of vaccine targets from protein regions of conserved human leukocyte antigen binding

DEFF Research Database (Denmark)

Olsen, Lars Rønn; Simon, Christian; Kudahl, Ulrich J.

2015-01-01

Background: Computational methods for T cell-based vaccine target discovery focus on selection of highly conserved peptides identified across pathogen variants, followed by prediction of their binding of human leukocyte antigen molecules. However, experimental studies have shown that T cells often...... target diverse regions in highly variable viral pathogens and this diversity may need to be addressed through redefinition of suitable peptide targets. Methods: We have developed a method for antigen assessment and target selection for polyvalent vaccines, with which we identified immune epitopes from...... variable regions, where all variants bind HLA. These regions, although variable, can thus be considered stable in terms of HLA binding and represent valuable vaccine targets. Results: We applied this method to predict CD8+ T-cell targets in influenza A H7N9 hemagglutinin and significantly increased...

Energy-weighted moments in the problems of fragmentation

International Nuclear Information System (INIS)

Kuz'min, V.A.

1986-01-01

The problem of fragmentation of simple nuclear states on the complex ones is reduced to real symmetrical matrix eigenvectors and eigenvalue problem. Based on spectral decomposition of this matrix the simple and economical from computing point of view algorithm to calculate energetically-weighted strength function moments is obtained. This permitted one to investigate the sensitivity of solving the fragmentation problem to reducing the basis of complex states. It is shown that the full width of strength function is determined only by the complex states connected directly with the simple ones

Peri/nuclear localization of intact insulin-like growth factor binding protein-2 and a distinct carboxyl-terminal IGFBP-2 fragment in vivo

International Nuclear Information System (INIS)

Hoeflich, A.; Reisinger, R.; Schuett, B.S.; Elmlinger, M.W.; Russo, V.C.; Vargas, G.A.; Jehle, P.M.; Lahm, H.; Renner-Mueller, I.; Wolf, E.

2004-01-01

Insulin-like growth factor binding protein-2 (IGFBP-2) as one of the most important IGFBPs has never been assessed in the intracellular compartment in vivo. Since there is evidence for novel intracellular functions of distinct IGFBPs, we investigated the presence of IGFBP-2 inside the cell. In peri/nuclear fractions of various tissues isolated from IGFBP-2 transgenic and non-transgenic mice we were able to show the presence of intact IGFBP-2. In addition, we demonstrate the presence of a highly conserved carboxyl-terminal IGFBP-2 fragment in the peri/nuclear fraction by using different peptide-induced antibodies. In pancreatic sections, confocal microscopy revealed the presence of IGFBP-2 on the nuclear surface but not within the nucleus. Our findings suggest novel functions of intact IGFBP-2 and IGFBP-2 fragments within the cell

Impact failure and fragmentation properties of metals

Energy Technology Data Exchange (ETDEWEB)

Grady, D.E. [Applied Research Associates, Albuquerque, NM (United States); Kipp, M.E. [Sandia National Labs., Albuquerque, NM (United States)

1998-03-01

In the present study we describe the development of an experimental fracture material property test method specific to dynamic fragmentation. Spherical test samples of the metals of interest are subjected to controlled impulsive stress loads by acceleration to high velocities with a light-gas launcher facility and subsequent normal impact on thin plates. Motion, deformation and fragmentation of the test samples are diagnosed with multiple flash radiography methods. The impact plate materials are selected to be transparent to the x-ray method so that only test metal material is imaged. Through a systematic series of such tests both strain-to-failure and fragmentation resistance properties are determined through this experimental method. Fragmentation property data for several steels, copper, aluminum, tantalum and titanium have been obtained to date. Aspects of the dynamic data have been analyzed with computational methods to achieve a better understanding of the processes leading to failure and fragmentation, and to test an existing computational fragmentation model.

Regulation of Cre recombinase by ligand-induced complementation of inactive fragments.

Science.gov (United States)

Jullien, Nicolas; Sampieri, François; Enjalbert, Alain; Herman, Jean-Paul

2003-11-01

Cre recombinase is extensively used to engineer the genome of experimental animals. However, its usefulness is still limited by the lack of an efficient temporal control over its activity. To overcome this, we have developed DiCre, a regulatable fragment complementation system for Cre. The enzyme was split into two moieties that were fused to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12-rapamycin-associated protein), respectively. These can be efficiently heterodimerized by rapamycin. Several variants, based on splitting Cre at different sites and using different linker peptides, were tested in an indicator cell line. The fusion proteins, taken separately, had no recombinase activity. Stable transformants, co-expressing complementing fragments based on splitting Cre between Asn59 and Asn60, displayed low background activity affecting 0.05-0.4% of the cells. Rapamycin induced a rapid recombination, reaching 100% by 48-72 h, with an EC50 of 0.02 nM. Thus, ligand-induced dimerization can efficiently regulate Cre, and should be useful to achieve a tight temporal control of its activity, such as in the case of the creation of conditional knock-out animals.

Experimental and numerical studies of high-velocity impact fragmentation

Energy Technology Data Exchange (ETDEWEB)

Kipp, M.E.; Grady, D.E.; Swegle, J.W.

1993-08-01

Developments are reported in both experimental and numerical capabilities for characterizing the debris spray produced in penetration events. We have performed a series of high-velocity experiments specifically designed to examine the fragmentation of the projectile during impact. High-strength, well-characterized steel spheres (6.35 mm diameter) were launched with a two-stage light-gas gun to velocities in the range of 3 to 5 km/s. Normal impact with PMMA plates, thicknesses of 0.6 to 11 mm, applied impulsive loads of various amplitudes and durations to the steel sphere. Multiple flash radiography diagnostics and recovery techniques were used to assess size, velocity, trajectory and statistics of the impact-induced fragment debris. Damage modes to the primary target plate (plastic) and to a secondary target plate (aluminum) were also evaluated. Dynamic fragmentation theories, based on energy-balance principles, were used to evaluate local material deformation and fracture state information from CTH, a three-dimensional Eulerian solid dynamics shock wave propagation code. The local fragment characterization of the material defines a weighted fragment size distribution, and the sum of these distributions provides a composite particle size distribution for the steel sphere. The calculated axial and radial velocity changes agree well with experimental data, and the calculated fragment sizes are in qualitative agreement with the radiographic data. A secondary effort involved the experimental and computational analyses of normal and oblique copper ball impacts on steel target plates. High-resolution radiography and witness plate diagnostics provided impact motion and statistical fragment size data. CTH simulations were performed to test computational models and numerical methods.

RCK: accurate and efficient inference of sequence- and structure-based protein-RNA binding models from RNAcompete data.

Science.gov (United States)

Orenstein, Yaron; Wang, Yuhao; Berger, Bonnie

2016-06-15

Protein-RNA interactions, which play vital roles in many processes, are mediated through both RNA sequence and structure. CLIP-based methods, which measure protein-RNA binding in vivo, suffer from experimental noise and systematic biases, whereas in vitro experiments capture a clearer signal of protein RNA-binding. Among them, RNAcompete provides binding affinities of a specific protein to more than 240 000 unstructured RNA probes in one experiment. The computational challenge is to infer RNA structure- and sequence-based binding models from these data. The state-of-the-art in sequence models, Deepbind, does not model structural preferences. RNAcontext models both sequence and structure preferences, but is outperformed by GraphProt. Unfortunately, GraphProt cannot detect structural preferences from RNAcompete data due to the unstructured nature of the data, as noted by its developers, nor can it be tractably run on the full RNACompete dataset. We develop RCK, an efficient, scalable algorithm that infers both sequence and structure preferences based on a new k-mer based model. Remarkably, even though RNAcompete data is designed to be unstructured, RCK can still learn structural preferences from it. RCK significantly outperforms both RNAcontext and Deepbind in in vitro binding prediction for 244 RNAcompete experiments. Moreover, RCK is also faster and uses less memory, which enables scalability. While currently on par with existing methods in in vivo binding prediction on a small scale test, we demonstrate that RCK will increasingly benefit from experimentally measured RNA structure profiles as compared to computationally predicted ones. By running RCK on the entire RNAcompete dataset, we generate and provide as a resource a set of protein-RNA structure-based models on an unprecedented scale. Software and models are freely available at http://rck.csail.mit.edu/ bab@mit.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by

Dynameomics: Data-driven methods and models for utilizing large-scale protein structure repositories for improving fragment-based loop prediction

Science.gov (United States)

Rysavy, Steven J; Beck, David AC; Daggett, Valerie

2014-01-01

Protein function is intimately linked to protein structure and dynamics yet experimentally determined structures frequently omit regions within a protein due to indeterminate data, which is often due protein dynamics. We propose that atomistic molecular dynamics simulations provide a diverse sampling of biologically relevant structures for these missing segments (and beyond) to improve structural modeling and structure prediction. Here we make use of the Dynameomics data warehouse, which contains simulations of representatives of essentially all known protein folds. We developed novel computational methods to efficiently identify, rank and retrieve small peptide structures, or fragments, from this database. We also created a novel data model to analyze and compare large repositories of structural data, such as contained within the Protein Data Bank and the Dynameomics data warehouse. Our evaluation compares these structural repositories for improving loop predictions and analyzes the utility of our methods and models. Using a standard set of loop structures, containing 510 loops, 30 for each loop length from 4 to 20 residues, we find that the inclusion of Dynameomics structures in fragment-based methods improves the quality of the loop predictions without being dependent on sequence homology. Depending on loop length, ∼25–75% of the best predictions came from the Dynameomics set, resulting in lower main chain root-mean-square deviations for all fragment lengths using the combined fragment library. We also provide specific cases where Dynameomics fragments provide better predictions for NMR loop structures than fragments from crystal structures. Online access to these fragment libraries is available at http://www.dynameomics.org/fragments. PMID:25142412

Dynameomics: data-driven methods and models for utilizing large-scale protein structure repositories for improving fragment-based loop prediction.

Science.gov (United States)

Rysavy, Steven J; Beck, David A C; Daggett, Valerie

2014-11-01

Protein function is intimately linked to protein structure and dynamics yet experimentally determined structures frequently omit regions within a protein due to indeterminate data, which is often due protein dynamics. We propose that atomistic molecular dynamics simulations provide a diverse sampling of biologically relevant structures for these missing segments (and beyond) to improve structural modeling and structure prediction. Here we make use of the Dynameomics data warehouse, which contains simulations of representatives of essentially all known protein folds. We developed novel computational methods to efficiently identify, rank and retrieve small peptide structures, or fragments, from this database. We also created a novel data model to analyze and compare large repositories of structural data, such as contained within the Protein Data Bank and the Dynameomics data warehouse. Our evaluation compares these structural repositories for improving loop predictions and analyzes the utility of our methods and models. Using a standard set of loop structures, containing 510 loops, 30 for each loop length from 4 to 20 residues, we find that the inclusion of Dynameomics structures in fragment-based methods improves the quality of the loop predictions without being dependent on sequence homology. Depending on loop length, ∼ 25-75% of the best predictions came from the Dynameomics set, resulting in lower main chain root-mean-square deviations for all fragment lengths using the combined fragment library. We also provide specific cases where Dynameomics fragments provide better predictions for NMR loop structures than fragments from crystal structures. Online access to these fragment libraries is available at http://www.dynameomics.org/fragments. © 2014 The Protein Society.

[Modification of retinal photoreceptor membranes and Ca ion binding].

Science.gov (United States)

Korchagin, V P; Berman, A L; Shukoliukov, S A; Rychkova, M P; Etingof, R N

1978-10-01

Calcium binding by modified photoreceptor membranes of cattle retina has been studied. Ca2+-binding the membranes significantly changes after C-phospholipase treatment, displaying the initial growth (less than 65% of lipid phosphorus removed) with subsequent decrease (more than 65% of phosphorus removed). Liposomes of the photoreceptor membranes lipids were found to bind more calcium than do the native photoreceptor membranes. Proteolytic enzymes (papaine, pronase) splitting some rhodopsin fragments do not affect the ability of the membrane to bind Ca2+. The increase of light-induced Ca-binding is observed only after the outer segments preincubation under conditions providing for rhodopsin phosphorylation. This effect was observed also after the splitting of the rhodopsin fragment by papaine. It is concluded that calcium binding in the photoreceptor membranes is mainly due to the phosphate groups of phospholipids.

Discovery of novel STAT3 small molecule inhibitors via in silico site-directed fragment-based drug design.

Science.gov (United States)

Yu, Wenying; Xiao, Hui; Lin, Jiayuh; Li, Chenglong

2013-06-13

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) has been validated as an attractive therapeutic target for cancer therapy. To stop both STAT3 activation and dimerization, a viable strategy is to design inhibitors blocking its SH2 domain phosphotyrosine binding site that is responsible for both actions. A new fragment-based drug design (FBDD) strategy, in silico site-directed FBDD, was applied in this study. A designed novel compound, 5,8-dioxo-6-(pyridin-3-ylamino)-5,8-dihydronaphthalene-1-sulfonamide (LY5), was confirmed to bind to STAT3 SH2 by fluorescence polarization assay. In addition, four out of the five chosen compounds have IC50 values lower than 5 μM for the U2OS cancer cells. 8 (LY5) has an IC50 range in 0.5-1.4 μM in various cancer cell lines. 8 also suppresses tumor growth in an in vivo mouse model. This study has demonstrated the utility of this approach and could be used to other drug targets in general.

Using physics-based pose predictions and free energy perturbation calculations to predict binding poses and relative binding affinities for FXR ligands in the D3R Grand Challenge 2

Science.gov (United States)

Athanasiou, Christina; Vasilakaki, Sofia; Dellis, Dimitris; Cournia, Zoe

2018-01-01

Computer-aided drug design has become an integral part of drug discovery and development in the pharmaceutical and biotechnology industry, and is nowadays extensively used in the lead identification and lead optimization phases. The drug design data resource (D3R) organizes challenges against blinded experimental data to prospectively test computational methodologies as an opportunity for improved methods and algorithms to emerge. We participated in Grand Challenge 2 to predict the crystallographic poses of 36 Farnesoid X Receptor (FXR)-bound ligands and the relative binding affinities for two designated subsets of 18 and 15 FXR-bound ligands. Here, we present our methodology for pose and affinity predictions and its evaluation after the release of the experimental data. For predicting the crystallographic poses, we used docking and physics-based pose prediction methods guided by the binding poses of native ligands. For FXR ligands with known chemotypes in the PDB, we accurately predicted their binding modes, while for those with unknown chemotypes the predictions were more challenging. Our group ranked #1st (based on the median RMSD) out of 46 groups, which submitted complete entries for the binding pose prediction challenge. For the relative binding affinity prediction challenge, we performed free energy perturbation (FEP) calculations coupled with molecular dynamics (MD) simulations. FEP/MD calculations displayed a high success rate in identifying compounds with better or worse binding affinity than the reference (parent) compound. Our studies suggest that when ligands with chemical precedent are available in the literature, binding pose predictions using docking and physics-based methods are reliable; however, predictions are challenging for ligands with completely unknown chemotypes. We also show that FEP/MD calculations hold predictive value and can nowadays be used in a high throughput mode in a lead optimization project provided that crystal structures of

Luciferase assay to study the activity of a cloned promoter DNA fragment.

Science.gov (United States)

Solberg, Nina; Krauss, Stefan

2013-01-01

Luciferase based assays have become an invaluable tool for the analysis of cloned promoter DNA fragments, both for verifying the ability of a potential promoter fragment to drive the expression of a luciferase reporter gene in various cellular contexts, and for dissecting binding elements in the promoter. Here, we describe the use of the Dual-Luciferase(®) Reporter Assay System created by Promega (Promega Corporation, Wisconsin, USA) to study the cloned 6.7 kilobases (kb) mouse (m) Tcf3 promoter DNA fragment in mouse embryonic derived neural stem cells (NSC). In this system, the expression of the firefly luciferase driven by the cloned mTcf3 promoter DNA fragment (including transcription initiation sites) is correlated with a co-transfected control reporter expressing Renilla luciferase from the herpes simplex virus (HSV) thymidine kinase promoter. Using an internal control reporter allows to normalize the activity of the experimental reporter to the internal control, which minimizes experimental variability.

Thermochemical Fragment Energy Method for Biomolecules: Application to a Collagen Model Peptide.

Science.gov (United States)

Suárez, Ernesto; Díaz, Natalia; Suárez, Dimas

2009-06-09

Herein, we first review different methodologies that have been proposed for computing the quantum mechanical (QM) energy and other molecular properties of large systems through a linear combination of subsystem (fragment) energies, which can be computed using conventional QM packages. Particularly, we emphasize the similarities among the different methods that can be considered as variants of the multibody expansion technique. Nevertheless, on the basis of thermochemical arguments, we propose yet another variant of the fragment energy methods, which could be useful for, and readily applicable to, biomolecules using either QM or hybrid quantum mechanical/molecular mechanics methods. The proposed computational scheme is applied to investigate the stability of a triple-helical collagen model peptide. To better address the actual applicability of the fragment QM method and to properly compare with experimental data, we compute average energies by carrying out single-point fragment QM calculations on structures generated by a classical molecular dynamics simulation. The QM calculations are done using a density functional level of theory combined with an implicit solvent model. Other free-energy terms such as attractive dispersion interactions or thermal contributions are included using molecular mechanics. The importance of correcting both the intermolecular and intramolecular basis set superposition error (BSSE) in the QM calculations is also discussed in detail. On the basis of the favorable comparison of our fragment-based energies with experimental data and former theoretical results, we conclude that the fragment QM energy strategy could be an interesting addition to the multimethod toolbox for biomolecular simulations in order to investigate those situations (e.g., interactions with metal clusters) that are beyond the range of applicability of common molecular mechanics methods.

Hypervelocity Impact Test Fragment Modeling: Modifications to the Fragment Rotation Analysis and Lightcurve Code

Science.gov (United States)

Gouge, Michael F.

2011-01-01

Hypervelocity impact tests on test satellites are performed by members of the orbital debris scientific community in order to understand and typify the on-orbit collision breakup process. By analysis of these test satellite fragments, the fragment size and mass distributions are derived and incorporated into various orbital debris models. These same fragments are currently being put to new use using emerging technologies. Digital models of these fragments are created using a laser scanner. A group of computer programs referred to as the Fragment Rotation Analysis and Lightcurve code uses these digital representations in a multitude of ways that describe, measure, and model on-orbit fragments and fragment behavior. The Dynamic Rotation subroutine generates all of the possible reflected intensities from a scanned fragment as if it were observed to rotate dynamically while in orbit about the Earth. This calls an additional subroutine that graphically displays the intensities and the resulting frequency of those intensities as a range of solar phase angles in a Probability Density Function plot. This document reports the additions and modifications to the subset of the Fragment Rotation Analysis and Lightcurve concerned with the Dynamic Rotation and Probability Density Function plotting subroutines.

Nonlinear regression analysis for evaluating tracer binding parameters using the programmable K1003 desk computer

International Nuclear Information System (INIS)

Sarrach, D.; Strohner, P.

1986-01-01

The Gauss-Newton algorithm has been used to evaluate tracer binding parameters of RIA by nonlinear regression analysis. The calculations were carried out on the K1003 desk computer. Equations for simple binding models and its derivatives are presented. The advantages of nonlinear regression analysis over linear regression are demonstrated

Solution and crystal structures of a C-terminal fragment of the neuronal isoform of the polypyrimidine tract binding protein (nPTB

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Amar Joshi

2014-03-01

Full Text Available The eukaryotic polypyrimidine tract binding protein (PTB serves primarily as a regulator of alternative splicing of messenger RNA, but is also co-opted to other roles such as RNA localisation and translation initiation from internal ribosome entry sites. The neuronal paralogue of PTB (nPTB is 75% identical in amino acid sequence with PTB. Although the two proteins have broadly similar RNA binding specificities and effects on RNA splicing, differential expression of PTB and nPTB can lead to the generation of alternatively spliced mRNAs. RNA binding by PTB and nPTB is mediated by four RNA recognition motifs (RRMs. We present here the crystal and solution structures of the C-terminal domain of nPTB (nPTB34 which contains RRMs 3 and 4. As expected the structures are similar to each other and to the solution structure of the equivalent fragment from PTB (PTB34. The result confirms that, as found for PTB, RRMs 3 and 4 of nPTB interact with one another to form a stable unit that presents the RNA-binding surfaces of the component RRMs on opposite sides that face away from each other. The major differences between PTB34 and nPTB34 arise from amino acid side chain substitutions on the exposed β-sheet surfaces and adjoining loops of each RRM, which are likely to modulate interactions with RNA.

Polymer fragmentation in extensional flow

Energy Technology Data Exchange (ETDEWEB)

Maroja, Armando M.; Oliveira, Fernando A.; Ciesla, Michal; Longa, Lech

2001-06-01

In this paper we present an analysis of fragmentation of dilute polymer solutions in extensional flow. The transition rate is investigated both from theoretical and computational approaches, where the existence of a Gaussian distribution for the breaking bonds has been controversial. We give as well an explanation for the low fragmentation frequency found in DNA experiments.

Graph Theory. 1. Fragmentation of Structural Graphs

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Lorentz JÄNTSCHI

2002-12-01

Full Text Available The investigation of structural graphs has many fields of applications in engineering, especially in applied sciences like as applied chemistry and physics, computer sciences and automation, electronics and telecommunication. The main subject of the paper is to express fragmentation criteria in graph using a new method of investigation: terminal paths. Using terminal paths are defined most of the fragmentation criteria that are in use in molecular topology, but the fields of applications are more generally than that, as I mentioned before. Graphical examples of fragmentation are given for every fragmentation criteria. Note that all fragmentation is made with a computer program that implements a routine for every criterion.[1] A web routine for tracing all terminal paths in graph can be found at the address: http://vl.academicdirect.ro/molecular_topology/tpaths/ [1] M. V. Diudea, I. Gutman, L. Jäntschi, Molecular Topology, Nova Science, Commack, New York, 2001, 2002.

Solution NMR Spectroscopy in Target-Based Drug Discovery.

Science.gov (United States)

Li, Yan; Kang, Congbao

2017-08-23

Solution NMR spectroscopy is a powerful tool to study protein structures and dynamics under physiological conditions. This technique is particularly useful in target-based drug discovery projects as it provides protein-ligand binding information in solution. Accumulated studies have shown that NMR will play more and more important roles in multiple steps of the drug discovery process. In a fragment-based drug discovery process, ligand-observed and protein-observed NMR spectroscopy can be applied to screen fragments with low binding affinities. The screened fragments can be further optimized into drug-like molecules. In combination with other biophysical techniques, NMR will guide structure-based drug discovery. In this review, we describe the possible roles of NMR spectroscopy in drug discovery. We also illustrate the challenges encountered in the drug discovery process. We include several examples demonstrating the roles of NMR in target-based drug discoveries such as hit identification, ranking ligand binding affinities, and mapping the ligand binding site. We also speculate the possible roles of NMR in target engagement based on recent processes in in-cell NMR spectroscopy.

Incorporation of rapid thermodynamic data in fragment-based drug discovery.

Science.gov (United States)

Kobe, Akihiro; Caaveiro, Jose M M; Tashiro, Shinya; Kajihara, Daisuke; Kikkawa, Masato; Mitani, Tomoya; Tsumoto, Kouhei

2013-03-14

Fragment-based drug discovery (FBDD) has enjoyed increasing popularity in recent years. We introduce SITE (single-injection thermal extinction), a novel thermodynamic methodology that selects high-quality hits early in FBDD. SITE is a fast calorimetric competitive assay suitable for automation that captures the essence of isothermal titration calorimetry but using significantly fewer resources. We describe the principles of SITE and identify a novel family of fragment inhibitors of the enzyme ketosteroid isomerase displaying high values of enthalpic efficiency.

Numerical simulation of fragmentation of hot metal and oxide melts with the computer code IVA3

International Nuclear Information System (INIS)

Mussa, S.; Tromm, W.

1994-01-01

The phenomena of fragmentation of melts caused by water-inlet from the bottom with the computer code IVA3/11,12,13/ are investigated. With the computer code IVA3 three-component-multiphase flows can be numerically simulated. Two geometrical models are used. Both consist of a cylindrical vessel for water lying beneath a cylindrical vessel for melt. The vessels are connected to each other through a hole. Steel and UO 2 melts are. The following parameters were varied: the type of the melt (steel,UO 2 ), the water supply pressure and the geometry of the hole in the bottom plate through which the water and melt vessels are connected. As results of the numerical simulations temperature and pressure versus time curves are plotted. Additionally the volume flow rates and the volume fractions of the various phases in the vessels and the increase in surface and enthalpy of the melt during the time of simulation are depicted. With steel melts the rate of fragmentation increases with increasing water pressure and melt temperature, whereby stable channels are formed in the melt layer showing a very low flow resistance for steam. With UO 2 the formations of channels are also observed. However, these channels are not so stable that they eventually break apart and lead to the fragmentation of the UO 2 melt in drops. The fragmentation of the steel melt in water vessel is less than that of UO 2 . No essential solidification of the melt is observed in the respective duration of the simulations. However, a small drop in the melt temperature is observed. With a slight or no water pressure the melt flows from the upper vessel into the water vessel via the connecting hole. The processes take place in a very slow manner and with such a low steam production so that despite the occuring pressure peaks no sign of steam explosions could be observed. (orig./HP) [de

Controlled isotropic fission fragment sources on the base of nuclear-physical facilities

International Nuclear Information System (INIS)

Sevast'yanov, V.D.; Maslov, G.N.

1995-01-01

Isotropic fission fragment sources (IFFS) are developed on the base of a neutron generator and pulse fast reactor. IFFS permit to calibrate fission fragment detectors. The IFFS consist of radiators with 235 U. The radiators are placed in a thermal neutron field of the neutron generator or in the reactor core center. The fragment activity is controlled by indications of an α-particle counter or by indications of a monitor of energy release in the core. 14 refs.; 1 fig.; 1 tab

Reducing the Flexibility of Type II Dehydroquinase for Inhibition: A Fragment-Based Approach and Molecular Dynamics Study.

Science.gov (United States)

Peón, Antonio; Robles, Adrián; Blanco, Beatriz; Convertino, Marino; Thompson, Paul; Hawkins, Alastair R; Caflisch, Amedeo; González-Bello, Concepción

2017-09-21

A multidisciplinary approach was used to identify and optimize a quinazolinedione-based ligand that would decrease the flexibility of the substrate-covering loop (catalytic loop) of the type II dehydroquinase from Helicobacter pylori. This enzyme, which is essential for the survival of this bacterium, is involved in the biosynthesis of aromatic amino acids. A computer-aided fragment-based protocol (ALTA) was first used to identify the aromatic fragments able to block the interface pocket that separates two neighboring enzyme subunits and is located at the active site entrance. Chemical modification of its non-aromatic moiety through an olefin cross-metathesis and Seebach's self-reproduction of chirality synthetic principle allowed the development of a quinazolinedione derivative that disables the catalytic loop plasticity, which is essential for the enzyme's catalytic cycle. Molecular dynamics simulations revealed that the ligand would force the catalytic loop into an inappropriate arrangement for catalysis by strong interactions with the catalytic tyrosine and by expelling the essential arginine out of the active site. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

In Silico Generation of Peptides by Replica Exchange Monte Carlo: Docking-Based Optimization of Maltose-Binding-Protein Ligands.

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Anna Russo

Full Text Available Short peptides can be designed in silico and synthesized through automated techniques, making them advantageous and versatile protein binders. A number of docking-based algorithms allow for a computational screening of peptides as binders. Here we developed ex-novo peptides targeting the maltose site of the Maltose Binding Protein, the prototypical system for the study of protein ligand recognition. We used a Monte Carlo based protocol, to computationally evolve a set of octapeptides starting from a polialanine sequence. We screened in silico the candidate peptides and characterized their binding abilities by surface plasmon resonance, fluorescence and electrospray ionization mass spectrometry assays. These experiments showed the designed binders to recognize their target with micromolar affinity. We finally discuss the obtained results in the light of further improvement in the ex-novo optimization of peptide based binders.

Fragment-based screening in tandem with phenotypic screening provides novel antiparasitic hits.

Science.gov (United States)

Blaazer, Antoni R; Orrling, Kristina M; Shanmugham, Anitha; Jansen, Chimed; Maes, Louis; Edink, Ewald; Sterk, Geert Jan; Siderius, Marco; England, Paul; Bailey, David; de Esch, Iwan J P; Leurs, Rob

2015-01-01

Methods to discover biologically active small molecules include target-based and phenotypic screening approaches. One of the main difficulties in drug discovery is elucidating and exploiting the relationship between drug activity at the protein target and disease modification, a phenotypic endpoint. Fragment-based drug discovery is a target-based approach that typically involves the screening of a relatively small number of fragment-like (molecular weight <300) molecules that efficiently cover chemical space. Here, we report a fragment screening on TbrPDEB1, an essential cyclic nucleotide phosphodiesterase (PDE) from Trypanosoma brucei, and human PDE4D, an off-target, in a workflow in which fragment hits and a series of close analogs are subsequently screened for antiparasitic activity in a phenotypic panel. The phenotypic panel contained T. brucei, Trypanosoma cruzi, Leishmania infantum, and Plasmodium falciparum, the causative agents of human African trypanosomiasis (sleeping sickness), Chagas disease, leishmaniasis, and malaria, respectively, as well as MRC-5 human lung cells. This hybrid screening workflow has resulted in the discovery of various benzhydryl ethers with antiprotozoal activity and low toxicity, representing interesting starting points for further antiparasitic optimization. © 2014 Society for Laboratory Automation and Screening.

Predicting fragmentation sizing profiles for different blasting patterns

International Nuclear Information System (INIS)

Sheikh, A.M.; Chung, S.H.

1987-01-01

This paper evaluates the efficiency of blasting in a large scale underground heap leaching operation. The prediction model is based on the dynamic tensile breaking strength of rock formation, the detonation characteristics of the explosives and the drill hole pattern. The modelling includes crack pattern development and fragmentation computation fitted by the Rosin-Rammler distribution equation

An Efficient Genome Fragment Assembling Using GA with Neighborhood Aware Fitness Function

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Satoko Kikuchi

2012-01-01

Full Text Available To decode a long genome sequence, shotgun sequencing is the state-of-the-art technique. It needs to properly sequence a very large number, sometimes as large as millions, of short partially readable strings (fragments. Arranging those fragments in correct sequence is known as fragment assembling, which is an NP-problem. Presently used methods require enormous computational cost. In this work, we have shown how our modified genetic algorithm (GA could solve this problem efficiently. In the proposed GA, the length of the chromosome, which represents the volume of the search space, is reduced with advancing generations, and thereby improves search efficiency. We also introduced a greedy mutation, by swapping nearby fragments using some heuristics, to improve the fitness of chromosomes. We compared results with Parsons’ algorithm which is based on GA too. We used fragments with partial reads on both sides, mimicking fragments in real genome assembling process. In Parsons’ work base-pair array of the whole fragment is known. Even then, we could obtain much better results, and we succeeded in restructuring contigs covering 100% of the genome sequences.

Restricted fragmentation of poliovirus type 1, 2, and 3 RNAs by ribonuclease III

Energy Technology Data Exchange (ETDEWEB)

Nomoto, A. (State Univ. of New York, Stony Brook); Lee, Y.F.; Babich, A.; Jacobson, A.; Dunn, J.J.; Wimmer, E.

1979-01-01

Cleavage of the genome RNAs of poliovirus type 1, 2, and 3 with the ribonuclease III of Escherichia coli has been investigated with the following results: (1) at or above physiological salt concentration, the RNAs are completely resistant to the action of the enzyme, an observation suggesting that the RNAs lack primary cleavage sites; (2) lowering the salt concentration to 0.1 M or below allows RNase III to cleave the RNAs at secondary sites. Both large and small fragments can be obtained in a reproducible manner depending on salt conditions chosen for cleavage. Fingerprints of three large fragments of poliovirus type 2 RNA show that they originate from unique segments and represent most if not all sequences of the genome. Based upon binding to poly(U) filters of poly(A)-linked fragments, a physical map of the large fragments of poliovirus type 2 RNA was constructed. The data suggest that RNase III cleavage of single-stranded RNA provides a useful method to fragment the RNA for further studies.

Universal elements of fragmentation

International Nuclear Information System (INIS)

Yanovsky, V. V.; Tur, A. V.; Kuklina, O. V.

2010-01-01

A fragmentation theory is proposed that explains the universal asymptotic behavior of the fragment-size distribution in the large-size range, based on simple physical principles. The basic principles of the theory are the total mass conservation in a fragmentation process and a balance condition for the energy expended in increasing the surface of fragments during their breakup. A flux-based approach is used that makes it possible to supplement the basic principles and develop a minimal theory of fragmentation. Such a supplementary principle is that of decreasing fragment-volume flux with increasing energy expended in fragmentation. It is shown that the behavior of the decreasing flux is directly related to the form of a power-law fragment-size distribution. The minimal theory is used to find universal asymptotic fragment-size distributions and to develop a natural physical classification of fragmentation models. A more general, nonlinear theory of strong fragmentation is also developed. It is demonstrated that solutions to a nonlinear kinetic equation consistent with both basic principles approach a universal asymptotic size distribution. Agreement between the predicted asymptotic fragment-size distributions and experimental observations is discussed.

Fragment-Based Drug Discovery of Potent Protein Kinase C Iota Inhibitors.

Science.gov (United States)

Kwiatkowski, Jacek; Liu, Boping; Tee, Doris Hui Ying; Chen, Guoying; Ahmad, Nur Huda Binte; Wong, Yun Xuan; Poh, Zhi Ying; Ang, Shi Hua; Tan, Eldwin Sum Wai; Ong, Esther Hq; Nurul Dinie; Poulsen, Anders; Pendharkar, Vishal; Sangthongpitag, Kanda; Lee, May Ann; Sepramaniam, Sugunavathi; Ho, Soo Yei; Cherian, Joseph; Hill, Jeffrey; Keller, Thomas H; Hung, Alvin W

2018-05-24

Protein kinase C iota (PKC-ι) is an atypical kinase implicated in the promotion of different cancer types. A biochemical screen of a fragment library has identified several hits from which an azaindole-based scaffold was chosen for optimization. Driven by a structure-activity relationship and supported by molecular modeling, a weakly bound fragment was systematically grown into a potent and selective inhibitor against PKC-ι.

Highly sensitive strain sensors based on fragmentized carbon nanotube/polydimethylsiloxane composites.

Science.gov (United States)

Gao, Yang; Fang, Xiaoliang; Tan, Jianping; Lu, Ting; Pan, Likun; Xuan, Fuzhen

2018-06-08

Wearable strain sensors based on nanomaterial/elastomer composites have potential applications in flexible electronic skin, human motion detection, human-machine interfaces, etc. In this research, a type of high performance strain sensors has been developed using fragmentized carbon nanotube/polydimethylsiloxane (CNT/PDMS) composites. The CNT/PDMS composites were ground into fragments, and a liquid-induced densification method was used to fabricate the strain sensors. The strain sensors showed high sensitivity with gauge factors (GFs) larger than 200 and a broad strain detection range up to 80%, much higher than those strain sensors based on unfragmentized CNT/PDMS composites (GF sensors is ascribed to the sliding of individual fragmentized-CNT/PDMS-composite particles during mechanical deformation, which causes significant resistance change in the strain sensors. The strain sensors can differentiate mechanical stimuli and monitor various human body motions, such as bending of the fingers, human breathing, and blood pulsing.

Bindings in working memory: The role of object-based attention.

Science.gov (United States)

Gao, Zaifeng; Wu, Fan; Qiu, Fangfang; He, Kaifeng; Yang, Yue; Shen, Mowei

2017-02-01

Over the past decade, it has been debated whether retaining bindings in working memory (WM) requires more attention than retaining constituent features, focusing on domain-general attention and space-based attention. Recently, we proposed that retaining bindings in WM needs more object-based attention than retaining constituent features (Shen, Huang, & Gao, 2015, Journal of Experimental Psychology: Human Perception and Performance, doi: 10.1037/xhp0000018 ). However, only unitized visual bindings were examined; to establish the role of object-based attention in retaining bindings in WM, more emperical evidence is required. We tested 4 new bindings that had been suggested requiring no more attention than the constituent features in the WM maintenance phase: The two constituent features of binding were stored in different WM modules (cross-module binding, Experiment 1), from auditory and visual modalities (cross-modal binding, Experiment 2), or temporally (cross-time binding, Experiments 3) or spatially (cross-space binding, Experiments 4-6) separated. In the critical condition, we added a secondary object feature-report task during the delay interval of the change-detection task, such that the secondary task competed for object-based attention with the to-be-memorized stimuli. If more object-based attention is required for retaining bindings than for retaining constituent features, the secondary task should impair the binding performance to a larger degree relative to the performance of constituent features. Indeed, Experiments 1-6 consistently revealed a significantly larger impairment for bindings than for the constituent features, suggesting that object-based attention plays a pivotal role in retaining bindings in WM.

Gamma-irradiation of homodeoxyoligonucleotides 32P-labelled at one end: computer simulation of the chain length distribution of the radioactive fragments

International Nuclear Information System (INIS)

Teoule, R.; Duplaa, A.M.

1987-01-01

Electrophoresis on polyacrylamide gels of the fragments resulting from γ-irradiation of single-stranded oligodeoxyribonucleotides labelled at their 5'- or 3'-end proved a potent tool for analysis of the radiation-induced chain breakage of DNA. Owing to the fact that the oligonucleotide may be ruptured at more than one site, counting of the electrophoresis bands must be corrected and it is necessary to assess the influence of the cleavage position on the band intensities. A complicating factor is the inhomogeneity of the system due to the presence of the four bases A, T, C and G. To circumvent this problem, the homooligodeoxyribonucleotides (dA) 15 , (dC) 15 , (dT) 15 were used as experimental probes. They were γ-irradiated in solution, heated in alkali and resulting fragments separated by gel electrophoresis. A computer simulation of band intensities was compiled based on the general assumption that the chain breakage is homogeneous. Experimental results obtained from the homooligodeoxyribonucleotides labelled at either the 5' or the 3'-end are in excellent agreement with theoretical calculations. Abacus giving the gel band intensities (percentage) against the nucleotide positions and the remaining intensity of the original oligonucleotide have been obtained. (author)

pH-tuneable binding of 2'-phospho-ADP-ribose to ketopantoate reductase: a structural and calorimetric study.

Science.gov (United States)

Ciulli, Alessio; Lobley, Carina M C; Tuck, Kellie L; Smith, Alison G; Blundell, Tom L; Abell, Chris

2007-02-01

The crystal structure of Escherichia coli ketopantoate reductase in complex with 2'-monophosphoadenosine 5'-diphosphoribose, a fragment of NADP+ that lacks the nicotinamide ring, is reported. The ligand is bound at the enzyme active site in the opposite orientation to that observed for NADP+, with the adenine ring occupying the lipophilic nicotinamide pocket. Isothermal titration calorimetry with R31A and N98A mutants of the enzyme is used to show that the unusual ;reversed binding mode' observed in the crystal is triggered by changes in the protonation of binding groups at low pH. This research has important implications for fragment-based approaches to drug design, namely that the crystallization conditions and the chemical modification of ligands can have unexpected effects on the binding modes.

Generation of single domain antibody fragments derived from camelids and generation of manifold constructs.

Science.gov (United States)

Vincke, Cécile; Gutiérrez, Carlos; Wernery, Ulrich; Devoogdt, Nick; Hassanzadeh-Ghassabeh, Gholamreza; Muyldermans, Serge

2012-01-01

Immunizing a camelid (camels and llamas) with soluble, properly folded proteins raises an affinity-matured immune response in the unique camelid heavy-chain only antibodies (HCAbs). The peripheral blood lymphocytes of the immunized animal are used to clone the antigen-binding antibody fragment from the HCAbs in a phage display vector. A representative aliquot of the library of these antigen-binding fragments is used to retrieve single domain antigen-specific binders by successive rounds of panning. These single domain antibody fragments are cloned in tandem to generate manifold constructs (bivalent, biparatopic or bispecific constructs) to increase their functional affinity, to increase specificity, or to connect two independent antigen molecules.

Fragmentation of atomic clusters: A theoretical study

International Nuclear Information System (INIS)

Lopez, M.J.; Jellinek, J.

1994-01-01

Collisionless fragmentation of nonrotating model n-atom metal clusters (n=12, 13, and 14) is studied using isoergic molecular-dynamics simulations. Minimum-energy paths for fragmentation are mapped out as functions of the distance between the centers of mass of the fragments. These paths provide information on the fragmentation energies for the different fragmentation channels. Fragmentation patterns (distributions of the fragmentation channel probabilities) and global and channel-specific fragmentation rate constants are computed and analyzed as functions of the internal energy and of the size of the clusters. The trends derived from the dynamics are compared with those obtained using the RRK and TST statistical approaches. The dynamics of the fragmentation process is analyzed in terms of characteristic quantities such as the distance between the centers of mass of the fragments, their relative translational energy, and their interaction energy, all considered as functions of time

Antibody or Antibody Fragments: Implications for Molecular Imaging and Targeted Therapy of Solid Tumors

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Katerina T. Xenaki

2017-10-01

Full Text Available The use of antibody-based therapeutics has proven very promising for clinical applications in cancer patients, with multiple examples of antibodies and antibody–drug conjugates successfully applied for the treatment of solid tumors and lymphomas. Given reported recurrence rates, improvements are clearly still necessary. A major factor limiting the efficacy of antibody-targeted cancer therapies may be the incomplete penetration of the antibody or antibody–drug conjugate into the tumor. Incomplete tumor penetration also affects the outcome of molecular imaging, when using such targeting agents. From the injection site until they arrive inside the tumor, targeting molecules are faced with several barriers that impact intratumoral distribution. The primary means of antibody transport inside tumors is based on diffusion. The diffusive penetration inside the tumor is influenced by both antibody properties, such as size and binding affinity, as well as tumor properties, such as microenvironment, vascularization, and targeted antigen availability. Engineering smaller antibody fragments has shown to improve the rate of tumor uptake and intratumoral distribution. However, it is often accompanied by more rapid clearance from the body and in several cases also by inherent destabilization and reduction of the binding affinity of the antibody. In this perspective, we discuss different cancer targeting approaches based on antibodies or their fragments. We carefully consider how their size and binding properties influence their intratumoral uptake and distribution, and how this may affect cancer imaging and therapy of solid tumors.

Mapping the Binding Interface of VEGF and a Monoclonal Antibody Fab-1 Fragment with Fast Photochemical Oxidation of Proteins (FPOP) and Mass Spectrometry

Science.gov (United States)

Zhang, Ying; Wecksler, Aaron T.; Molina, Patricia; Deperalta, Galahad; Gross, Michael L.

2017-05-01

We previously analyzed the Fab-1:VEGF (vascular endothelial growth factor) system described in this work, with both native top-down mass spectrometry and bottom-up mass spectrometry (carboxyl-group or GEE footprinting) techniques. This work continues bottom-up mass spectrometry analysis using a fast photochemical oxidation of proteins (FPOP) platform to map the solution binding interface of VEGF and a fragment antigen binding region of an antibody (Fab-1). In this study, we use FPOP to compare the changes in solvent accessibility by quantitating the extent of oxidative modification in the unbound versus bound states. Determining the changes in solvent accessibility enables the inference of the protein binding sites (epitope and paratopes) and a comparison to the previously published Fab-1:VEGF crystal structure, adding to the top-down and bottom-up data. Using this method, we investigated peptide-level and residue-level changes in solvent accessibility between the unbound proteins and bound complex. Mapping these data onto the Fab-1:VEGF crystal structure enabled successful characterization of both the binding region and regions of remote conformation changes. These data, coupled with our previous higher order structure (HOS) studies, demonstrate the value of a comprehensive toolbox of methods for identifying the putative epitopes and paratopes for biotherapeutic antibodies.

FRAGSION: ultra-fast protein fragment library generation by IOHMM sampling.

Science.gov (United States)

Bhattacharya, Debswapna; Adhikari, Badri; Li, Jilong; Cheng, Jianlin

2016-07-01

Speed, accuracy and robustness of building protein fragment library have important implications in de novo protein structure prediction since fragment-based methods are one of the most successful approaches in template-free modeling (FM). Majority of the existing fragment detection methods rely on database-driven search strategies to identify candidate fragments, which are inherently time-consuming and often hinder the possibility to locate longer fragments due to the limited sizes of databases. Also, it is difficult to alleviate the effect of noisy sequence-based predicted features such as secondary structures on the quality of fragment. Here, we present FRAGSION, a database-free method to efficiently generate protein fragment library by sampling from an Input-Output Hidden Markov Model. FRAGSION offers some unique features compared to existing approaches in that it (i) is lightning-fast, consuming only few seconds of CPU time to generate fragment library for a protein of typical length (300 residues); (ii) can generate dynamic-size fragments of any length (even for the whole protein sequence) and (iii) offers ways to handle noise in predicted secondary structure during fragment sampling. On a FM dataset from the most recent Critical Assessment of Structure Prediction, we demonstrate that FGRAGSION provides advantages over the state-of-the-art fragment picking protocol of ROSETTA suite by speeding up computation by several orders of magnitude while achieving comparable performance in fragment quality. Source code and executable versions of FRAGSION for Linux and MacOS is freely available to non-commercial users at http://sysbio.rnet.missouri.edu/FRAGSION/ It is bundled with a manual and example data. chengji@missouri.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Computational chemistry and metal-based radiopharmaceuticals

International Nuclear Information System (INIS)

Neves, M.; Fausto, R.

1998-01-01

Computer-assisted techniques have found extensive use in the design of organic pharmaceuticals but have not been widely applied on metal complexes, particularly on radiopharmaceuticals. Some examples of computer generated structures of complexes of In, Ga and Tc with N, S, O and P donor ligands are referred. Besides parameters directly related with molecular geometries, molecular properties of the predicted structures, as ionic charges or dipole moments, are considered to be related with biodistribution studies. The structure of a series of oxo neutral Tc-biguanide complexes are predicted by molecular mechanics calculations, and their interactions with water molecules or peptide chains correlated with experimental data of partition coefficients and percentage of human protein binding. The results stress the interest of using molecular modelling to predict molecular properties of metal-based radiopharmaceuticals, which can be successfully correlated with results of in vitro studies. (author)

Thioredoxin binding site of phosphoribulokinase overlaps the catalytic site

International Nuclear Information System (INIS)

Porter, M.A.; Hartman, F.C.

1986-01-01

The ATP-regulatory binding site of phosphoribulokinase was studied using bromoacetylethanolamine phosphate (BrAcNHEtOP). BrAcNHEtOP binds to the active-regulatory binding site of the protein. Following trypsin degradation of the labeled protein, fragments were separated by HPLC and sequenced. (DT)

Cost Function Network-based Design of Protein-Protein Interactions: predicting changes in binding affinity.

Science.gov (United States)

Viricel, Clément; de Givry, Simon; Schiex, Thomas; Barbe, Sophie

2018-02-20

Accurate and economic methods to predict change in protein binding free energy upon mutation are imperative to accelerate the design of proteins for a wide range of applications. Free energy is defined by enthalpic and entropic contributions. Following the recent progresses of Artificial Intelligence-based algorithms for guaranteed NP-hard energy optimization and partition function computation, it becomes possible to quickly compute minimum energy conformations and to reliably estimate the entropic contribution of side-chains in the change of free energy of large protein interfaces. Using guaranteed Cost Function Network algorithms, Rosetta energy functions and Dunbrack's rotamer library, we developed and assessed EasyE and JayZ, two methods for binding affinity estimation that ignore or include conformational entropic contributions on a large benchmark of binding affinity experimental measures. If both approaches outperform most established tools, we observe that side-chain conformational entropy brings little or no improvement on most systems but becomes crucial in some rare cases. as open-source Python/C ++ code at sourcesup.renater.fr/projects/easy-jayz. thomas.schiex@inra.fr and sophie.barbe@insa-toulouse.fr. Supplementary data are available at Bioinformatics online.

Rat embryo fibroblasts require both the cell-binding and the heparin-binding domains of fibronectin for survival

DEFF Research Database (Denmark)

Jeong, J; Han, I; Lim, Y

2001-01-01

of the cell-binding domain of FN with integrin is sufficient to rescue rat embryo fibroblasts (REFs) from detachment-induced apoptosis. REFs attached and spread normally after plating on substrates coated with either intact FN or a FN fragment, FN120, that contains the cell-binding domain but lacks the C...

Electrostatic field of the large fragment of Escherichia coli DNA polymerase I.

Science.gov (United States)

Warwicker, J; Ollis, D; Richards, F M; Steitz, T A

1985-12-05

The electrostatic field of the large fragment of Escherichia coli DNA polymerase I (Klenow fragment) has been calculated by the finite difference procedure on a 2 A grid. The potential field is substantially negative at physiological pH (reflecting the net negative charge at this pH). The largest regions of positive potential are in the deep crevice of the C-terminal domain, which is the proposed binding site for the DNA substrate. Within the crevice, the electrostatic potential has a partly helical form. If the DNA is positioned to fulfil stereochemical requirements, then the positive potential generally follows the major groove and (to a lesser extent) the negative potential is in the minor groove. Such an arrangement could stabilize DNA configurations related by screw symmetry. The histidine residues of the Klenow fragment give the positive field of the groove a sensitivity to relatively small pH changes around neutrality. We suggest that the histidine residues could change their ionization states in response to DNA binding, and that this effect could contribute to the protein-DNA binding energy.

Fragment-based drug discovery as alternative strategy to the drug development for neglected diseases.

Science.gov (United States)

Mello, Juliana da Fonseca Rezende E; Gomes, Renan Augusto; Vital-Fujii, Drielli Gomes; Ferreira, Glaucio Monteiro; Trossini, Gustavo Henrique Goulart

2017-12-01

Neglected diseases (NDs) affect large populations and almost whole continents, representing 12% of the global health burden. In contrast, the treatment available today is limited and sometimes ineffective. Under this scenery, the Fragment-Based Drug Discovery emerged as one of the most promising alternatives to the traditional methods of drug development. This method allows achieving new lead compounds with smaller size of fragment libraries. Even with the wide Fragment-Based Drug Discovery success resulting in new effective therapeutic agents against different diseases, until this moment few studies have been applied this approach for NDs area. In this article, we discuss the basic Fragment-Based Drug Discovery process, brief successful ideas of general applications and show a landscape of its use in NDs, encouraging the implementation of this strategy as an interesting way to optimize the development of new drugs to NDs. © 2017 John Wiley & Sons A/S.

Stochastic weighted particle methods for population balance equations with coagulation, fragmentation and spatial inhomogeneity

International Nuclear Information System (INIS)

Lee, Kok Foong; Patterson, Robert I.A.; Wagner, Wolfgang; Kraft, Markus

2015-01-01

Graphical abstract: -- Highlights: •Problems concerning multi-compartment population balance equations are studied. •A class of fragmentation weight transfer functions is presented. •Three stochastic weighted algorithms are compared against the direct simulation algorithm. •The numerical errors of the stochastic solutions are assessed as a function of fragmentation rate. •The algorithms are applied to a multi-dimensional granulation model. -- Abstract: This paper introduces stochastic weighted particle algorithms for the solution of multi-compartment population balance equations. In particular, it presents a class of fragmentation weight transfer functions which are constructed such that the number of computational particles stays constant during fragmentation events. The weight transfer functions are constructed based on systems of weighted computational particles and each of it leads to a stochastic particle algorithm for the numerical treatment of population balance equations. Besides fragmentation, the algorithms also consider physical processes such as coagulation and the exchange of mass with the surroundings. The numerical properties of the algorithms are compared to the direct simulation algorithm and an existing method for the fragmentation of weighted particles. It is found that the new algorithms show better numerical performance over the two existing methods especially for systems with significant amount of large particles and high fragmentation rates.

Stochastic weighted particle methods for population balance equations with coagulation, fragmentation and spatial inhomogeneity

Energy Technology Data Exchange (ETDEWEB)

Lee, Kok Foong [Department of Chemical Engineering and Biotechnology, University of Cambridge, New Museums Site, Pembroke Street, Cambridge CB2 3RA (United Kingdom); Patterson, Robert I.A.; Wagner, Wolfgang [Weierstrass Institute for Applied Analysis and Stochastics, Mohrenstraße 39, 10117 Berlin (Germany); Kraft, Markus, E-mail: mk306@cam.ac.uk [Department of Chemical Engineering and Biotechnology, University of Cambridge, New Museums Site, Pembroke Street, Cambridge CB2 3RA (United Kingdom); School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore, 637459 (Singapore)

2015-12-15

Graphical abstract: -- Highlights: •Problems concerning multi-compartment population balance equations are studied. •A class of fragmentation weight transfer functions is presented. •Three stochastic weighted algorithms are compared against the direct simulation algorithm. •The numerical errors of the stochastic solutions are assessed as a function of fragmentation rate. •The algorithms are applied to a multi-dimensional granulation model. -- Abstract: This paper introduces stochastic weighted particle algorithms for the solution of multi-compartment population balance equations. In particular, it presents a class of fragmentation weight transfer functions which are constructed such that the number of computational particles stays constant during fragmentation events. The weight transfer functions are constructed based on systems of weighted computational particles and each of it leads to a stochastic particle algorithm for the numerical treatment of population balance equations. Besides fragmentation, the algorithms also consider physical processes such as coagulation and the exchange of mass with the surroundings. The numerical properties of the algorithms are compared to the direct simulation algorithm and an existing method for the fragmentation of weighted particles. It is found that the new algorithms show better numerical performance over the two existing methods especially for systems with significant amount of large particles and high fragmentation rates.

Cloud computing approaches for prediction of ligand binding poses and pathways.

Science.gov (United States)

Lawrenz, Morgan; Shukla, Diwakar; Pande, Vijay S

2015-01-22

We describe an innovative protocol for ab initio prediction of ligand crystallographic binding poses and highly effective analysis of large datasets generated for protein-ligand dynamics. We include a procedure for setup and performance of distributed molecular dynamics simulations on cloud computing architectures, a model for efficient analysis of simulation data, and a metric for evaluation of model convergence. We give accurate binding pose predictions for five ligands ranging in affinity from 7 nM to > 200 μM for the immunophilin protein FKBP12, for expedited results in cases where experimental structures are difficult to produce. Our approach goes beyond single, low energy ligand poses to give quantitative kinetic information that can inform protein engineering and ligand design.

Complexity of Some Language Fragments of Fuzzy Logics

Czech Academy of Sciences Publication Activity Database

Haniková, Zuzana

2017-01-01

Roč. 21, č. 1 (2017), s. 69-77 ISSN 1432-7643 R&D Projects: GA ČR GAP202/11/1632 Institutional support: RVO:67985807 Keywords : fuzzy logic * propositional logic * language fragment * implicational fragment * commutative semigroup * equational theory * computational complexity Subject RIV: BA - General Mathematics OBOR OECD: Computer sciences, information science, bioinformathics (hardware development to be 2.2, social aspect to be 5.8) Impact factor: 2.472, year: 2016

A simple and robust approach to immobilization of antibody fragments.

Science.gov (United States)

Ikonomova, Svetlana P; He, Ziming; Karlsson, Amy J

2016-08-01

Antibody fragments, such as the single-chain variable fragment (scFv), have much potential in research and diagnostics because of their antigen-binding ability similar to a full-sized antibody and their ease of production in microorganisms. Some applications of antibody fragments require immobilization on a surface, and we have established a simple immobilization method that is based on the biotin-streptavidin interaction and does not require a separate purification step. We genetically fused two biotinylation tags-the biotin carboxyl carrier protein (BCCP) or the AviTag minimal sequence-to six different scFvs (scFv13R4, scFvD10, scFv26-10, scFv3, scFv5, and scFv12) for site-specific biotinylation in vivo by endogenous biotin ligases produced by Escherichia coli. The biotinylated scFvs were immobilized onto streptavidin-coated plates directly from cell lysates, and immobilization was detected through enzyme-linked immunosorbent assays. All scFvs fusions were successfully immobilized, and scFvs biotinylated via the BCCP tag tended to immobilize better than those biotinylated via the AviTag, even when biotinylation efficiency was improved with the biotin ligase BirA. The ability of immobilized scFvs to bind antigens was confirmed using scFv13R4 and scFvD10 with their respective targets β-galactosidase and bacteriophage lambda head protein D (gpD). The immobilized scFv13R4 bound to β-galactosidase at the same level for both biotinylation tags when the surface was saturated with the scFv, and immobilized scFvs retained their functionality for at least 100days after immobilization. The simplicity and robustness of our method make it a promising approach for future applications that require antibody fragment immobilization. Copyright © 2016 Elsevier B.V. All rights reserved.

Calcium binding promotes prion protein fragment 90-231 conformational change toward a membrane destabilizing and cytotoxic structure.

Directory of Open Access Journals (Sweden)

Sacha Sorrentino

Full Text Available The pathological form of prion protein (PrP(Sc, as other amyloidogenic proteins, causes a marked increase of membrane permeability. PrP(Sc extracted from infected Syrian hamster brains induces a considerable change in membrane ionic conductance, although the contribution of this interaction to the molecular mechanism of neurodegeneration process is still controversial. We previously showed that the human PrP fragment 90-231 (hPrP₉₀₋₂₃₁ increases ionic conductance across artificial lipid bilayer, in a calcium-dependent manner, producing an alteration similar to that observed for PrP(Sc. In the present study we demonstrate that hPrP₉₀₋₂₃₁, pre-incubated with 10 mM Ca⁺⁺ and then re-suspended in physiological external solution increases not only membrane conductance but neurotoxicity as well. Furthermore we show the existence of a direct link between these two effects as demonstrated by a highly statistically significant correlation in several experimental conditions. A similar correlation between increased membrane conductance and cell degeneration has been observed assaying hPrP₉₀₋₂₃₁ bearing pathogenic mutations (D202N and E200K. We also report that Ca⁺⁺ binding to hPrP₉₀₋₂₃₁ induces a conformational change based on an alteration of secondary structure characterized by loss of alpha-helix content causing hydrophobic amino acid exposure and proteinase K resistance. These features, either acquired after controlled thermal denaturation or induced by D202N and E200K mutations were previously identified as responsible for hPrP₉₀₋₂₃₁ cytotoxicity. Finally, by in silico structural analysis, we propose that Ca⁺⁺ binding to hPrP₉₀₋₂₃₁ modifies amino acid orientation, in the same way induced by E200K mutation, thus suggesting a pathway for the structural alterations responsible of PrP neurotoxicity.

Partial Gene Cloning and Enzyme Structure Modeling of Exolevanase Fragment from Bacillus subtilis

Science.gov (United States)

Azhar, M.; Natalia, D.; Syukur, S.; Andriani, N.; Jamsari, J.

2018-04-01

Inulin hydrolysis thermophilic and thermotolerant bacteria are potential sources of inulin hydrolysis enzymes. Partial gene that encodes inulin hydrolysis enzymes had been isolated from Bacillus subtilis using polymerase chain reaction (PCR) method with the DPE.slFandDPE.eR degenerative primers. The partial gene was cloned into pGEM-T Easy vector with E. coli as host cells and analyzed using BLASTx, CrustalW2, and Phyre2 programs. Size of thepartial gene had been found539 bp that encoded 179aminoacid residues of protein fragment. The sequences of protein fragment was more similar to exolevanase than exoinulinase. The protein fragment had conserved motif FSGS, and specific hits GH32 β-fructosidase. It had three residues of active site and five residues of substrate binding. The active site on the protein fragment were D (1-WLNDP-5), D (125-FRDPK-129) and E (177-WEC-179). Substrate binding on the protein fragment were ND (1-WLNDP-5), Q (18-FYQY-21), FS (60-FSGS-63) RD (125-FRDPK-129) and E (177-WEC-179).

Selectivity in ligand binding to uranyl compounds: A synthetic, structural, thermodynamic and computational study

Energy Technology Data Exchange (ETDEWEB)

Arnold, John [Univ. of California, Berkeley, CA (United States)

2015-01-21

The uranyl cation (UO₂²⁺) is the most abundant form of uranium on the planet. It is estimated that 4.5 billion tons of uranium in this form exist in sea water. The ability to bind and extract the uranyl cation from aqueous solution while separating it from other elements would provide a limitless source of nuclear fuel. A large body of research concerns the selective recognition and extraction of uranyl. A stable molecule, the cation has a linear O=U=O geometry. The short U-O bonds (1.78 Å) arise from the combination of uranium 5f/6d and oxygen 2p orbitals. Due to the oxygen moieties being multiply bonded, these sites were not thought to be basic enough for Lewis acidic coordination to be a viable approach to sequestration. The goal of this research is thus to broaden the coordination chemistry of the uranyl ion by studying new ligand systems via synthetic, structural, thermodynamic and computational methods. It is anticipated that this fundamental science will find use beyond actinide separation technologies in areas such as nuclear waste remediation and nuclear materials. The focus of this study is to synthesize uranyl complexes incorporating amidinate and guanidinate ligands. Both synthetic and computational methods are used to investigate novel equatorial ligand coordination and how this affects the basicity of the oxo ligands. Such an understanding will later apply to designing ligands incorporating functionalities that can bind uranyl both equatorially and axially for highly selective sequestration. Efficient and durable chromatography supports for lanthanide separation will be generated by (1) identifying robust peptoid-based ligands capable of binding different lanthanides with variable affinities, and (2) developing practical synthetic methods for the attachment of these ligands to Dowex ion exchange resins.

Selectivity in ligand binding to uranyl compounds: A synthetic, structural, thermodynamic and computational study

International Nuclear Information System (INIS)

Arnold, John

2015-01-01

The uranyl cation (UO 2 2+ ) is the most abundant form of uranium on the planet. It is estimated that 4.5 billion tons of uranium in this form exist in sea water. The ability to bind and extract the uranyl cation from aqueous solution while separating it from other elements would provide a limitless source of nuclear fuel. A large body of research concerns the selective recognition and extraction of uranyl. A stable molecule, the cation has a linear O=U=O geometry. The short U-O bonds (1.78 Å) arise from the combination of uranium 5f/6d and oxygen 2p orbitals. Due to the oxygen moieties being multiply bonded, these sites were not thought to be basic enough for Lewis acidic coordination to be a viable approach to sequestration. The goal of this research is thus to broaden the coordination chemistry of the uranyl ion by studying new ligand systems via synthetic, structural, thermodynamic and computational methods. It is anticipated that this fundamental science will find use beyond actinide separation technologies in areas such as nuclear waste remediation and nuclear materials. The focus of this study is to synthesize uranyl complexes incorporating amidinate and guanidinate ligands. Both synthetic and computational methods are used to investigate novel equatorial ligand coordination and how this affects the basicity of the oxo ligands. Such an understanding will later apply to designing ligands incorporating functionalities that can bind uranyl both equatorially and axially for highly selective sequestration. Efficient and durable chromatography supports for lanthanide separation will be generated by (1) identifying robust peptoid-based ligands capable of binding different lanthanides with variable affinities, and (2) developing practical synthetic methods for the attachment of these ligands to Dowex ion exchange resins.

Peptide synthesis, characterization and 68Ga-radiolabeling of NOTA-conjugated ubiquicidin fragments for prospective infection imaging with PET/CT

International Nuclear Information System (INIS)

Ebenhan, Thomas; Chadwick, Nicholas; Sathekge, Mike M.; Govender, Patrick; Govender, Thavendran; Kruger, Hendrik G.; Marjanovic-Painter, Biljana; Zeevaart, Jan Rijn

2014-01-01

Introduction: Human antimicrobial peptides are of interest for the development of positron emission tomography (PET) tracers as they exhibit desirable characteristics that make them good candidates for targeting vectors. Due to their natural role in the innate immune system they selectively bind to pathogenic bacteria and yeast, whilst remaining minimally immunogenic and cytotoxic to humans. Research into ubiquicidin (UBI)-based tracers has focused on 99m Tc as a radionuclide, however, the use of bi-functional chelators such as 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), in combination with 68 Ga as a radionuclide, allows for a simple radiolabeling procedure which is preferable in a clinical setting using PET/CT. Methods: The peptides fragments UBI29-41, UBI30-41 were synthesized by standard microwave Fmoc/tert-butyl (tBu)-solid phase synthetic protocols. Characterizations were performed using analytical HPLC and LC/MS. Both NOTA-conjugated peptides were exposed to nat Ga 3+ ; their complexed form was quantified by direct LC/MS injection. This complexation was utilized to testify bacterial and mammalian cell binding potential of fluorophore-linked NOTA-UBI29-41/30-41. 68 Ga labeled NOTA-UBI fragments were also tested for competitive interaction to Staphylococcus aureus to proof the binding target. 68 Ga was eluted from SnO 2 - and TiO 2 -based 68 Ge/ 68 Ga generators using fractionated elution and anion exchanged-based post-procession. NOTA-peptide radiolabeling was carried out including optimization of buffer molarity, NOTA-peptide concentration(s), incubation temperature and –duration as well as considering various SPE purification cartridges. Results: Pure UBI29-41, UBI30-41 and NOTA-UBI30-41 were successfully characterized. Both, NOTA-UBI fragments exhibited complexation rates to nat Ga 3+ ≥ 99%. The percentage binding was significantly higher to Staphylococcus aureus bacilli over Mt4 human leucocytes (P > 0.05) for NOTA-UBI29-41[Lys(Abz)] < NOTA

Current perspectives in fragment-based lead discovery (FBLD)

Science.gov (United States)

Lamoree, Bas; Hubbard, Roderick E.

2017-01-01

It is over 20 years since the first fragment-based discovery projects were disclosed. The methods are now mature for most ‘conventional’ targets in drug discovery such as enzymes (kinases and proteases) but there has also been growing success on more challenging targets, such as disruption of protein–protein interactions. The main application is to identify tractable chemical startpoints that non-covalently modulate the activity of a biological molecule. In this essay, we overview current practice in the methods and discuss how they have had an impact in lead discovery – generating a large number of fragment-derived compounds that are in clinical trials and two medicines treating patients. In addition, we discuss some of the more recent applications of the methods in chemical biology – providing chemical tools to investigate biological molecules, mechanisms and systems. PMID:29118093

Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment.

Science.gov (United States)

Kirley, Terence L; Norman, Andrew B

2015-01-01

Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region.

Computational identification of binding energy hot spots in protein-RNA complexes using an ensemble approach.

Science.gov (United States)

Pan, Yuliang; Wang, Zixiang; Zhan, Weihua; Deng, Lei

2018-05-01

Identifying RNA-binding residues, especially energetically favored hot spots, can provide valuable clues for understanding the mechanisms and functional importance of protein-RNA interactions. Yet, limited availability of experimentally recognized energy hot spots in protein-RNA crystal structures leads to the difficulties in developing empirical identification approaches. Computational prediction of RNA-binding hot spot residues is still in its infant stage. Here, we describe a computational method, PrabHot (Prediction of protein-RNA binding hot spots), that can effectively detect hot spot residues on protein-RNA binding interfaces using an ensemble of conceptually different machine learning classifiers. Residue interaction network features and new solvent exposure characteristics are combined together and selected for classification with the Boruta algorithm. In particular, two new reference datasets (benchmark and independent) have been generated containing 107 hot spots from 47 known protein-RNA complex structures. In 10-fold cross-validation on the training dataset, PrabHot achieves promising performances with an AUC score of 0.86 and a sensitivity of 0.78, which are significantly better than that of the pioneer RNA-binding hot spot prediction method HotSPRing. We also demonstrate the capability of our proposed method on the independent test dataset and gain a competitive advantage as a result. The PrabHot webserver is freely available at http://denglab.org/PrabHot/. leideng@csu.edu.cn. Supplementary data are available at Bioinformatics online.

Specific binding of a naturally occurring amyloidogenic fragment of Streptococcus mutans adhesin P1 to intact P1 on the cell surface characterized by solid state NMR spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Tang, Wenxing; Bhatt, Avni [University of Florida, Department of Biochemistry and Molecular Biology, College of Medicine (United States); Smith, Adam N. [University of Florida, Department of Chemistry, College of Liberal Arts and Sciences (United States); Crowley, Paula J.; Brady, L. Jeannine, E-mail: jbrady@dental.ufl.edu [University of Florida, Department of Oral Biology, College of Dentistry (United States); Long, Joanna R., E-mail: jrlong@ufl.edu [University of Florida, Department of Biochemistry and Molecular Biology, College of Medicine (United States)

2016-02-15

The P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 covalently attached to the cell wall and interacting with non-covalently associated proteolytic fragments thereof, particularly the ∼57-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we demonstrate that solid state NMR (ssNMR) spectroscopy can be used to (1) globally characterize cell walls isolated from a Gram-positive bacterium and (2) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent steps in biofilm formation via ssNMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin.

Specific binding of a naturally occurring amyloidogenic fragment of Streptococcus mutans adhesin P1 to intact P1 on the cell surface characterized by solid state NMR spectroscopy

International Nuclear Information System (INIS)

Tang, Wenxing; Bhatt, Avni; Smith, Adam N.; Crowley, Paula J.; Brady, L. Jeannine; Long, Joanna R.

2016-01-01

The P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 covalently attached to the cell wall and interacting with non-covalently associated proteolytic fragments thereof, particularly the ∼57-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we demonstrate that solid state NMR (ssNMR) spectroscopy can be used to (1) globally characterize cell walls isolated from a Gram-positive bacterium and (2) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent steps in biofilm formation via ssNMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin

Specific binding of a naturally occurring amyloidogenic fragment of Streptococcus mutans adhesin P1 to intact P1 on the cell surface characterized by solid state NMR spectroscopy.

Science.gov (United States)

Tang, Wenxing; Bhatt, Avni; Smith, Adam N; Crowley, Paula J; Brady, L Jeannine; Long, Joanna R

2016-02-01

The P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 covalently attached to the cell wall and interacting with non-covalently associated proteolytic fragments thereof, particularly the ~57-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we demonstrate that solid state NMR (ssNMR) spectroscopy can be used to (1) globally characterize cell walls isolated from a Gram-positive bacterium and (2) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent steps in biofilm formation via ssNMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin.

An overview of the prediction of protein DNA-binding sites.

Science.gov (United States)

Si, Jingna; Zhao, Rui; Wu, Rongling

2015-03-06

Interactions between proteins and DNA play an important role in many essential biological processes such as DNA replication, transcription, splicing, and repair. The identification of amino acid residues involved in DNA-binding sites is critical for understanding the mechanism of these biological activities. In the last decade, numerous computational approaches have been developed to predict protein DNA-binding sites based on protein sequence and/or structural information, which play an important role in complementing experimental strategies. At this time, approaches can be divided into three categories: sequence-based DNA-binding site prediction, structure-based DNA-binding site prediction, and homology modeling and threading. In this article, we review existing research on computational methods to predict protein DNA-binding sites, which includes data sets, various residue sequence/structural features, machine learning methods for comparison and selection, evaluation methods, performance comparison of different tools, and future directions in protein DNA-binding site prediction. In particular, we detail the meta-analysis of protein DNA-binding sites. We also propose specific implications that are likely to result in novel prediction methods, increased performance, or practical applications.

Development and validation of an antigen-binding capture ELISA for native and putrescine-modified anti-tetanus F(ab')2 fragments for the assessment of the cellular uptake and plasma kinetics of the antibodies.

OpenAIRE

Welfringer, Frédéric; D'Athis, Philippe; Scherrmann, Jean-Michel; Hervé, Françoise

2005-01-01

International audience; Cationization is a strategy to enhance the permeability of antibodies to physiological membranes for potential therapeutic and diagnostic applications of these proteins, with one of its crucial points being the retention of antigen binding activity. Here, we describe the cationization of horse polyclonal anti-tetanus F(ab')(2) fragments and the development and validation of an ELISA for quantitative measurements of the binding activity of the native and cationized F(ab...

Computational Study of the Binding Mechanism of Actin-Depolymerizing Factor 1 with Actin in Arabidopsis thaliana.

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Juan Du

Full Text Available Actin is a highly conserved protein. It plays important roles in cellular function and exists either in the monomeric (G-actin or polymeric form (F-actin. Members of the actin-depolymerizing factor (ADF/cofilin protein family bind to both G-actin and F-actin and play vital roles in actin dynamics by manipulating the rates of filament polymerization and depolymerization. It has been reported that the S6D and R98A/K100A mutants of actin-depolymerizing factor 1 (ADF1 in Arabidopsis thaliana decreased the binding affinity of ADF for the actin monomer. To investigate the binding mechanism and dynamic behavior of the ADF1-actin complex, we constructed a homology model of the AtADF1-actin complex based on the crystal structure of AtADF1 and the twinfilin C-terminal ADF-H domain in a complex with a mouse actin monomer. The model was then refined for subsequent molecular dynamics simulations. Increased binding energy of the mutated system was observed using the Molecular Mechanics Generalized Born Surface Area and Poisson-Boltzmann Surface Area (MM-GB/PBSA methods. To determine the residues that make decisive contributions to the ADF1 actin-binding affinity, per-residue decomposition and computational alanine scanning analyses were performed, which provided more detailed information on the binding mechanism. Root-mean-square fluctuation and principal component analyses confirmed that the S6D and R98A/K100A mutants induced an increased conformational flexibility. The comprehensive molecular insight gained from this study is of great importance for understanding the binding mechanism of ADF1 and G-actin.

Fragment-based discovery of novel pentacyclic triterpenoid derivatives as cholesteryl ester transfer protein inhibitors.

Science.gov (United States)

Chang, Yongzhi; Zhou, Shuxi; Li, Enqin; Zhao, Wenfeng; Ji, Yanpeng; Wen, Xiaoan; Sun, Hongbin; Yuan, Haoliang

2017-01-27

Cholesteryl Ester Transfer Protein (CETP) is an important therapeutic target for the treatment of atherosclerotic cardiovascular disease. Our molecular modeling study revealed that pentacyclic triterpenoid compounds could mimic the protein-ligand interactions of the endogenous ligand cholesteryl ester (CE) by occupying its binding site. Alignment of the docking conformations of oleanolic acid (OA), ursolic acid (UA) and the crystal conformations of known CETP inhibitor Torcetrapib in the active site proposed the applicability of fragment-based drug design (FBDD) approaches in this study. Accordingly, a series of pentacyclic triterpenoid derivatives have been designed and synthesized as novel CETP inhibitors. The most potent compound 12e (IC 50 :0.28 μM) validated our strategy for molecular design. Molecular dynamics simulations illustrated that the more stable hydrogen bond interaction of the UA derivative 12e with Ser191 and stronger hydrophobic interactions with Val198, Phe463 than those of OA derivative 12b mainly led to their significantly different CETP inhibitory activity. These novel potent CETP inhibitors based on ursane-type scaffold should deserve further investigation. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Ultrasensitive, Stretchable Strain Sensors Based on Fragmented Carbon Nanotube Papers

KAUST Repository

Zhou, Jian; Yu, Hu; Xu, Xuezhu; Han, Fei; Lubineau, Gilles

2017-01-01

The development of strain sensors featuring both ultra high sensitivity and high stretchability is still a challenge. We demonstrate that strain sensors based on fragmented single-walled carbon nanotube (SWCNT) paper embedded in poly

Calcium binding to an elastic portion of connectin/titin filaments.

Science.gov (United States)

Tatsumi, R; Maeda, K; Hattori, A; Takahashi, K

2001-01-01

Alpha-connectin/titin-1 exists as an elastic filament that links a thick filament with the Z-disk, keeping thick filaments centered within the sarcomere during force generation. We have shown that the connectin filament has an affinity for calcium ions and its binding site(s) is restricted to the beta-connectin/titin-2 portion. We now report the localization and the characterization of calcium-binding sites on beta-connectin. Purified beta-connectin was digested by trypsin into 1700- and 400-kDa fragments. which were then subjected to fluorescence calcium-binding assays. The 400-kDa fragment possesses calcium-binding activity; the binding constant was 1.0 x 10(7) M(-1) and the molar ratio of bound calcium ions to the 400-kDa fragment reached a maximum of 12 at a free calcium ion concentration of approximately 1.0 microM. Antibodies against the 400-kDa fragment formed a sharp dense stripe at the boundary of the A and the I bands, indicating that the calcium-binding domain constitutes the N-terminal region of beta-connectin, that is, the elastic portion of connectin filaments. Furthermore, we estimated the N-terminal location of beta-connectin of various origins (n = 26). Myofibrils were treated with a solution containing 0.1 mM CaCl2 and 70 microM leupeptin to split connectin filaments into beta-connectin and a subfragment, and chain weights of these polypeptides were estimated according to their mobility in 2% polyacrylamide slab gels. The subfragment exhibited a similar chain weight of 1200+/-33 kDa (mean+/-SD), while alpha- and beta-connectins were variable in size according to their origin. These results suggest that the apparent length of the 1200-kDa subfragment portion is almost constant in all instances, about 0.34 microm at the slack condition, therefore that the C-terminus of the 1200-kDa subfragment, that is, the N-terminus of the calcium-binding domain, is at the N2 line region of parent filaments in situ. Because the secondary structure of the 400-k

Ultrasensitive, Stretchable Strain Sensors Based on Fragmented Carbon Nanotube Papers

KAUST Repository

Zhou, Jian

2017-01-17

The development of strain sensors featuring both ultra high sensitivity and high stretchability is still a challenge. We demonstrate that strain sensors based on fragmented single-walled carbon nanotube (SWCNT) paper embedded in poly(dimethylsiloxane) (PDMS) can sustain their sensitivity even at very high strain levels (with a gauge factor of over 10(7) at 50% strain). This record sensitivity is ascribed to the low initial electrical resistance (5-28 Omega) of the SWCNT paper and the wide change in resistance (up to 10(6) Omega) governed by the percolated network of SWCNT in the cracked region. The sensor response remains nearly unchanged after 10 000 strain cycles at 20% proving the robustness of this technology. This fragmentation based sensing system brings opportunities to engineer highly sensitive stretchable sensors.

Predicting DNA-binding proteins and binding residues by complex structure prediction and application to human proteome.

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Huiying Zhao

Full Text Available As more and more protein sequences are uncovered from increasingly inexpensive sequencing techniques, an urgent task is to find their functions. This work presents a highly reliable computational technique for predicting DNA-binding function at the level of protein-DNA complex structures, rather than low-resolution two-state prediction of DNA-binding as most existing techniques do. The method first predicts protein-DNA complex structure by utilizing the template-based structure prediction technique HHblits, followed by binding affinity prediction based on a knowledge-based energy function (Distance-scaled finite ideal-gas reference state for protein-DNA interactions. A leave-one-out cross validation of the method based on 179 DNA-binding and 3797 non-binding protein domains achieves a Matthews correlation coefficient (MCC of 0.77 with high precision (94% and high sensitivity (65%. We further found 51% sensitivity for 82 newly determined structures of DNA-binding proteins and 56% sensitivity for the human proteome. In addition, the method provides a reasonably accurate prediction of DNA-binding residues in proteins based on predicted DNA-binding complex structures. Its application to human proteome leads to more than 300 novel DNA-binding proteins; some of these predicted structures were validated by known structures of homologous proteins in APO forms. The method [SPOT-Seq (DNA] is available as an on-line server at http://sparks-lab.org.

Direct fission fragment energy converter - Magnetic collimator option

International Nuclear Information System (INIS)

Tsvetkov, P. V.; Hart, R. R.

2006-01-01

The present study was focused on developing a technologically feasible power system that is based on direct fission fragment energy conversion utilizing magnetic collimation. The new concept is an attempt to combine several advantageous design solutions, which have been proposed for application in both fission and fusion reactors, into one innovative system that can offer exceptional energy conversion efficiency. The analysis takes into consideration a wide range of operational aspects including fission fragment escape from the fuel, collimation, collection, criticality, long-term performance, energy conversion efficiency, heat removal, and safety characteristics. Specific characteristics of the individual system components and the entire system are evaluated. Analysis and evaluation of the technological feasibility of the concept were achieved using state-of-the-art computer codes that allowed realistic and consistent modeling. In addition to the extensive computational effort, the scaled prototype experimental proof-of-principle program was conducted to validate basic physics of the concept. The program was focused on electromagnetic components and experimental demonstration of performance. This paper summarizes the final results of the 6-years research program including both computational and experimental efforts. Potential future research and development and anticipated applications are discussed. (authors)

The RNA-binding protein repertoire of Arabidopsis thaliana

KAUST Repository

Marondedze, Claudius; Thomas, Ludivine; Serano, Natalia Lorena Gorron; Lilley, Kathryn S.; Gehring, Christoph A

2016-01-01

RNA-binding proteins (RBPs) have essential roles in determining the fate of RNA from synthesis to decay and have been studied on a protein-by-protein basis, or computationally based on a number of well-characterised RNA-binding domains. Recently

Robust Object Tracking Using Valid Fragments Selection.

Science.gov (United States)

Zheng, Jin; Li, Bo; Tian, Peng; Luo, Gang

Local features are widely used in visual tracking to improve robustness in cases of partial occlusion, deformation and rotation. This paper proposes a local fragment-based object tracking algorithm. Unlike many existing fragment-based algorithms that allocate the weights to each fragment, this method firstly defines discrimination and uniqueness for local fragment, and builds an automatic pre-selection of useful fragments for tracking. Then, a Harris-SIFT filter is used to choose the current valid fragments, excluding occluded or highly deformed fragments. Based on those valid fragments, fragment-based color histogram provides a structured and effective description for the object. Finally, the object is tracked using a valid fragment template combining the displacement constraint and similarity of each valid fragment. The object template is updated by fusing feature similarity and valid fragments, which is scale-adaptive and robust to partial occlusion. The experimental results show that the proposed algorithm is accurate and robust in challenging scenarios.

Dhajala meteorite shower: atmospheric fragmentation and ablation based on cosmic ray track studies

Energy Technology Data Exchange (ETDEWEB)

Bagolia, C; Doshi, N; Gupta, S K; Kumar, S; Lal, D; Trivedi, J R [Physical Research Lab., Ahmedabad (India)

1977-06-01

Cosmic-ray track studies have been carried out in more than 250 fragments of Dhajala meteorite comprising greater than 70% of the recovered mass. In the case of larger fragments (namely, those with mass exceeding 250 g) several faces of each fragment have been analysed for track densities. Track densities are low, and fall generally in the range (10/sup 3/ to 10/sup 5/)cm/sup -2/, indicating appreciable ablation losses since the cosmic ray exposure age of Dhajala is about 7 m.y. (track measurements were confined to large olivine grains to minimize contributions to tracks due to the fission of uranium and extinct radionuclides). Attempts have been made to deduce information about fragmentation dynamics and the preatmospheric mass/radius of Dhajala, based on the present comprehensive study of track densities in the fragments. Correlations between the position of a fragment in the strewnfield and its track density have provided an approximate scenario for the fragmentation/ablation of the meteorite during its atmospheric flight. Observation of minimum track density in the fragments lead to a value of (38 +- 2)cm for the preatmospheric radius of the meteorite. It is estimated from these data that the collection of fragments was made with an overall efficiency of approximately 60% and that the ablation amounts to (86.7 +- 2.1)%. Estimated amounts of ablation for shells of different radii are also presented.

Automated NMR fragment based screening identified a novel interface blocker to the LARG/RhoA complex.

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Jia Gao

Full Text Available The small GTPase cycles between the inactive GDP form and the activated GTP form, catalyzed by the upstream guanine exchange factors. The modulation of such process by small molecules has been proven to be a fruitful route for therapeutic intervention to prevent the over-activation of the small GTPase. The fragment based approach emerging in the past decade has demonstrated its paramount potential in the discovery of inhibitors targeting such novel and challenging protein-protein interactions. The details regarding the procedure of NMR fragment screening from scratch have been rarely disclosed comprehensively, thus restricts its wider applications. To achieve a consistent screening applicable to a number of targets, we developed a highly automated protocol to cover every aspect of NMR fragment screening as possible, including the construction of small but diverse libray, determination of the aqueous solubility by NMR, grouping compounds with mutual dispersity to a cocktail, and the automated processing and visualization of the ligand based screening spectra. We exemplified our streamlined screening in RhoA alone and the complex of the small GTPase RhoA and its upstream guanine exchange factor LARG. Two hits were confirmed from the primary screening in cocktail and secondary screening over individual hits for LARG/RhoA complex, while one of them was also identified from the screening for RhoA alone. HSQC titration of the two hits over RhoA and LARG alone, respectively, identified one compound binding to RhoA.GDP at a 0.11 mM affinity, and perturbed the residues at the switch II region of RhoA. This hit blocked the formation of the LARG/RhoA complex, validated by the native gel electrophoresis, and the titration of RhoA to ¹⁵N labeled LARG in the absence and presence the compound, respectively. It therefore provides us a starting point toward a more potent inhibitor to RhoA activation catalyzed by LARG.

Structure and DNA-binding of meiosis-specific protein Hop2

Science.gov (United States)

Zhou, Donghua; Moktan, Hem; Pezza, Roberto

2014-03-01

Here we report structure elucidation of the DNA binding domain of homologous pairing protein 2 (Hop2), which is important to gene diversity when sperms and eggs are produced. Together with another protein Mnd1, Hop2 enhances the strand invasion activity of recombinase Dmc1 by over 30 times, facilitating proper synapsis of homologous chromosomes. However, the structural and biochemical bases for the function of Hop2 and Mnd1 have not been well understood. As a first step toward such understanding, we recently solved the structure for the N-terminus of Hop2 (1-84) using solution NMR. This fragment shows a typical winged-head conformation with recognized DNA binding activity. DNA interacting sites were then investigated by chemical shift perturbations in a titration experiment. Information of these sites was used to guide protein-DNA docking with MD simulation, revealing that helix 3 is stably lodged in the DNA major groove and that wing 1 (connecting strands 2 and 3) transiently comes in contact with the minor groove in nanosecond time scale. Mutagenesis analysis further confirmed the DNA binding sites in this fragment of the protein.

Protein-Templated Fragment Ligations-From Molecular Recognition to Drug Discovery.

Science.gov (United States)

Jaegle, Mike; Wong, Ee Lin; Tauber, Carolin; Nawrotzky, Eric; Arkona, Christoph; Rademann, Jörg

2017-06-19

Protein-templated fragment ligation is a novel concept to support drug discovery and can help to improve the efficacy of protein ligands. Protein-templated fragment ligations are chemical reactions between small molecules ("fragments") utilizing a protein's surface as a reaction vessel to catalyze the formation of a protein ligand with increased binding affinity. The approach exploits the molecular recognition of reactive small-molecule fragments by proteins both for ligand assembly and for the identification of bioactive fragment combinations. In this way, chemical synthesis and bioassay are integrated in one single step. This Review discusses the biophysical basis of reversible and irreversible fragment ligations and gives an overview of the available methods to detect protein-templated ligation products. The chemical scope and recent applications as well as future potential of the concept in drug discovery are reviewed. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modified stochastic fragmentation of an interval as an ageing process

Science.gov (United States)

Fortin, Jean-Yves

2018-02-01

We study a stochastic model based on modified fragmentation of a finite interval. The mechanism consists of cutting the interval at a random location and substituting a unique fragment on the right of the cut to regenerate and preserve the interval length. This leads to a set of segments of random sizes, with the accumulation of small fragments near the origin. This model is an example of record dynamics, with the presence of ‘quakes’ and slow dynamics. The fragment size distribution is a universal inverse power law with logarithmic corrections. The exact distribution for the fragment number as function of time is simply related to the unsigned Stirling numbers of the first kind. Two-time correlation functions are defined, and computed exactly. They satisfy scaling relations, and exhibit aging phenomena. In particular, the probability that the same number of fragments is found at two different times t>s is asymptotically equal to [4πlog(s)]-1/2 when s\\gg 1 and the ratio t/s is fixed, in agreement with the numerical simulations. The same process with a reset impedes the aging phenomenon-beyond a typical time scale defined by the reset parameter.

Identification and therapeutic potential of a vitronectin binding region of meningococcal msf.

Science.gov (United States)

Hill, Darryl J; Griffiths, Natalie J; Borodina, Elena; Andreae, Clio A; Sessions, Richard B; Virji, Mumtaz

2015-01-01

The human pathogen Neisseria meningitides (Nm) attains serum resistance via a number of mechanisms, one of which involves binding to the host complement regulator protein vitronectin. We have shown previously that the Meningococcal surface fibril (Msf), a trimeric autotransporter, binds to the activated form of vitronectin (aVn) to increase Nm survival in human serum. In this study, we aimed to identify the aVn-binding region of Msf to assess its potential as an antigen which can elicit antibodies that block aVn binding and/or possess bactericidal properties. Using several recombinant Msf fragments spanning its surface-exposed region, the smallest aVn-binding recombinants were found to span residues 1-86 and 39-124. The use of further deletion constructs and overlapping recombinant Msf fragments suggested that a region of Msf comprising residues 39-82 may be primarily important for aVn binding and that other regions may also be involved but to a lesser extent. Molecular modelling implicated K66 and K68, conserved in all available Msf sequences, to be involved in the interaction. Recombinant fragments which bound to aVn were able to reduce the survival advantage conveyed by aVn-interaction in serum bactericidal assays. Antibodies raised against one such fragment inhibited aVn binding to Msf. In addition, the antibodies enhanced specific killing of Msf-expressing Nm in a dose-dependent manner. Overall, this study identifies an aVn-binding region of Msf, an adhesin known to impart serum resistance properties to the pathogen; and shows that this region of Msf can elicit antibodies with dual properties which reduce pathogen survival within the host and thus has potential as a vaccine antigen.

Baculovirus display of functional antibody Fab fragments.

Science.gov (United States)

Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

2015-08-01

The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

Design of a multi-purpose fragment screening library using molecular complexity and orthogonal diversity metrics

Science.gov (United States)

Lau, Wan F.; Withka, Jane M.; Hepworth, David; Magee, Thomas V.; Du, Yuhua J.; Bakken, Gregory A.; Miller, Michael D.; Hendsch, Zachary S.; Thanabal, Venkataraman; Kolodziej, Steve A.; Xing, Li; Hu, Qiyue; Narasimhan, Lakshmi S.; Love, Robert; Charlton, Maura E.; Hughes, Samantha; van Hoorn, Willem P.; Mills, James E.

2011-07-01

Fragment Based Drug Discovery (FBDD) continues to advance as an efficient and alternative screening paradigm for the identification and optimization of novel chemical matter. To enable FBDD across a wide range of pharmaceutical targets, a fragment screening library is required to be chemically diverse and synthetically expandable to enable critical decision making for chemical follow-up and assessing new target druggability. In this manuscript, the Pfizer fragment library design strategy which utilized multiple and orthogonal metrics to incorporate structure, pharmacophore and pharmacological space diversity is described. Appropriate measures of molecular complexity were also employed to maximize the probability of detection of fragment hits using a variety of biophysical and biochemical screening methods. In addition, structural integrity, purity, solubility, fragment and analog availability as well as cost were important considerations in the selection process. Preliminary analysis of primary screening results for 13 targets using NMR Saturation Transfer Difference (STD) indicates the identification of uM-mM hits and the uniqueness of hits at weak binding affinities for these targets.

Fragment E1 labeled with I-123 in the detection of venous thrombosis

International Nuclear Information System (INIS)

Knight, L.C.; Maurer, A.H.; Robbins, P.S.; Malmud, L.S.; Budzynski, A.Z.

1985-01-01

Fragment E1, which has been shown to have specific binding affinity for thrombi in an animal model, was investigated in humans for its safety and ability to bind to venous thrombi. Human Fragment E1 was labeled with I-123 and administered intravenously to patients with proved or suspected deep vein thrombosis. The vascular distribution of radioactivity was documented by obtaining gamma camera images of the patients' legs for 30 minutes following administration of I-123-Fragment E1. All patients (n = 5) with documented venous thrombi had rapid localization of labeled Fragment E1 in the area of thrombus. Patients without evidence of thrombi (n = 5) showed no focal localization, although two of these patients showed diffuse uptake along the length of the veins, due to superficial phlebitis. Analysis of blood samples in four patients indicated that disappearance of Fragment E1 from the circulation was more rapid in individuals with thrombosis (t 1/2 = 20 min) than in individuals without thrombosis (t 1/2 = 90 min), and a radiolabeled species of high molecular weight was found in patients with thrombosis but was absent from patients without thrombosis. These early results suggest that radiolabeled Fragment E1 is a safe and potentially valuable agent for the rapid detection of venous thrombosis

pH-tuneable binding of 2′-phospho-ADP-ribose to ketopantoate reductase: a structural and calorimetric study

Science.gov (United States)

Ciulli, Alessio; Lobley, Carina M. C.; Tuck, Kellie L.; Smith, Alison G.; Blundell, Tom L.; Abell, Chris

2007-01-01

The crystal structure of Escherichia coli ketopantoate reductase in complex with 2′-monophosphoadenosine 5′-diphosphoribose, a fragment of NADP+ that lacks the nicotinamide ring, is reported. The ligand is bound at the enzyme active site in the opposite orientation to that observed for NADP+, with the adenine ring occupying the lipophilic nicotinamide pocket. Isothermal titration calorimetry with R31A and N98A mutants of the enzyme is used to show that the unusual ‘reversed binding mode’ observed in the crystal is triggered by changes in the protonation of binding groups at low pH. This research has important implications for fragment-based approaches to drug design, namely that the crystallization conditions and the chemical modification of ligands can have unexpected effects on the binding modes. PMID:17242510

Determination of the binding sites for oxaliplatin on insulin using mass spectrometry-based approaches

DEFF Research Database (Denmark)

Møller, Charlotte; Sprenger, Richard R.; Stürup, Stefan

2011-01-01

Using insulin as a model protein for binding of oxaliplatin to proteins, various mass spectrometric approaches and techniques were compared. Several different platinum adducts were observed, e.g. addition of one or two diaminocyclohexane platinum(II) (Pt(dach)) molecules. By top-down analysis...... and fragmentation of the intact insulin-oxaliplatin adduct using nano-electrospray ionisation quadrupole time-of-flight mass spectrometry (nESI-Q-ToF-MS), the major binding site was assigned to histidine5 on the insulin B chain. In order to simplify the interpretation of the mass spectrum, the disulphide bridges...... were reduced. This led to the additional identification of cysteine6 on the A chain as a binding site along with histidine5 on the B chain. Digestion of insulin-oxaliplatin with endoproteinase Glu-C (GluC) followed by reduction led to the formation of five peptides with Pt(dach) attached...

Lead identification for the K-Ras protein: virtual screening and combinatorial fragment-based approaches

Directory of Open Access Journals (Sweden)

Pathan AAK

2016-05-01

Full Text Available Akbar Ali Khan Pathan,1,2,* Bhavana Panthi,3,* Zahid Khan,1 Purushotham Reddy Koppula,4–6 Mohammed Saud Alanazi,1 Sachchidanand,3 Narasimha Reddy Parine,1 Mukesh Chourasia3,* 1Genome Research Chair (GRC, Department of Biochemistry, College of Science, King Saud University, 2Integrated Gulf Biosystems, Riyadh, Kingdom of Saudi Arabia; 3Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research, Hajipur, India; 4Department of Internal Medicine, School of Medicine, 5Harry S. Truman Memorial Veterans Affairs Hospital, 6Department of Radiology, School of Medicine, Columbia, MO, USA *These authors contributed equally to this work Objective: Kirsten rat sarcoma (K-Ras protein is a member of Ras family belonging to the small guanosine triphosphatases superfamily. The members of this family share a conserved structure and biochemical properties, acting as binary molecular switches. The guanosine triphosphate-bound active K-Ras interacts with a range of effectors, resulting in the stimulation of downstream signaling pathways regulating cell proliferation, differentiation, and apoptosis. Efforts to target K-Ras have been unsuccessful until now, placing it among high-value molecules against which developing a therapy would have an enormous impact. K-Ras transduces signals when it binds to guanosine triphosphate by directly binding to downstream effector proteins, but in case of guanosine diphosphate-bound conformation, these interactions get disrupted. Methods: In the present study, we targeted the nucleotide-binding site in the “on” and “off” state conformations of the K-Ras protein to find out suitable lead compounds. A structure-based virtual screening approach has been used to screen compounds from different databases, followed by a combinatorial fragment-based approach to design the apposite lead for the K-Ras protein. Results: Interestingly, the designed compounds exhibit a binding preference for the �

An Overview of the Prediction of Protein DNA-Binding Sites

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Jingna Si

2015-03-01

Full Text Available Interactions between proteins and DNA play an important role in many essential biological processes such as DNA replication, transcription, splicing, and repair. The identification of amino acid residues involved in DNA-binding sites is critical for understanding the mechanism of these biological activities. In the last decade, numerous computational approaches have been developed to predict protein DNA-binding sites based on protein sequence and/or structural information, which play an important role in complementing experimental strategies. At this time, approaches can be divided into three categories: sequence-based DNA-binding site prediction, structure-based DNA-binding site prediction, and homology modeling and threading. In this article, we review existing research on computational methods to predict protein DNA-binding sites, which includes data sets, various residue sequence/structural features, machine learning methods for comparison and selection, evaluation methods, performance comparison of different tools, and future directions in protein DNA-binding site prediction. In particular, we detail the meta-analysis of protein DNA-binding sites. We also propose specific implications that are likely to result in novel prediction methods, increased performance, or practical applications.

T cell receptor-like recognition of tumor in vivo by synthetic antibody fragment.

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Keith R Miller

Full Text Available A major difficulty in treating cancer is the inability to differentiate between normal and tumor cells. The immune system differentiates tumor from normal cells by T cell receptor (TCR binding of tumor-associated peptides bound to Major Histocompatibility Complex (pMHC molecules. The peptides, derived from the tumor-specific proteins, are presented by MHC proteins, which then serve as cancer markers. The TCR is a difficult protein to use as a recombinant protein because of production issues and has poor affinity for pMHC; therefore, it is not a good choice for use as a tumor identifier outside of the immune system. We constructed a synthetic antibody-fragment (Fab library in the phage-display format and isolated antibody-fragments that bind pMHC with high affinity and specificity. One Fab, fE75, recognizes our model cancer marker, the Human Epidermal growth factor Receptor 2 (HER2/neu peptide, E75, bound to the MHC called Human Leukocyte Antigen-A2 (HLA-A2, with nanomolar affinity. The fE75 bound selectively to E75/HLA-A2 positive cancer cell lines in vitro. The fE75 Fab conjugated with (64Cu selectively accumulated in E75/HLA-A2 positive tumors and not in E75/HLA-A2 negative tumors in an HLA-A2 transgenic mouse as probed using positron emission tomography/computed tomography (PET/CT imaging. Considering that hundreds to thousands of different peptides bound to HLA-A2 are present on the surface of each cell, the fact that fE75 arrives at the tumor at all shows extraordinary specificity. These antibody fragments have great potential for diagnosis and targeted drug delivery in cancer.

Proteolytic processing of the cilium adhesin MHJ_0194 (P123J ) in Mycoplasma hyopneumoniae generates a functionally diverse array of cleavage fragments that bind multiple host molecules.

Science.gov (United States)

Raymond, Benjamin B A; Jenkins, Cheryl; Seymour, Lisa M; Tacchi, Jessica L; Widjaja, Michael; Jarocki, Veronica M; Deutscher, Ania T; Turnbull, Lynne; Whitchurch, Cynthia B; Padula, Matthew P; Djordjevic, Steven P

2015-03-01

Mycoplasma hyopneumoniae, the aetiological agent of porcine enzootic pneumonia, regulates the presentation of proteins on its cell surface via endoproteolysis, including those of the cilial adhesin P123 (MHJ_0194). These proteolytic cleavage events create functional adhesins that bind to proteoglycans and glycoproteins on the surface of ciliated and non-ciliated epithelial cells and to the circulatory host molecule plasminogen. Two dominant cleavage events of the P123 preprotein have been previously characterized; however, immunoblotting studies suggest that more complex processing events occur. These extensive processing events are characterized here. The functional significance of the P97 cleavage fragments is also poorly understood. Affinity chromatography using heparin, fibronectin and plasminogen as bait and peptide arrays were used to expand our knowledge of the adhesive capabilities of P123 cleavage fragments and characterize a novel binding motif in the C-terminus of P123. Further, we use immunohistochemistry to examine in vivo, the biological significance of interactions between M. hyopneumoniae and fibronectin and show that M. hyopneumoniae induces fibronectin deposition at the site of infection on the ciliated epithelium. Our data supports the hypothesis that M. hyopneumoniae possesses the molecular machinery to influence key molecular communication pathways in host cells. © 2014 John Wiley & Sons Ltd.

QUEIMAP: a computer routine for punctual analysis of a nuclear fuel depletion with accumulation of fission fragments

International Nuclear Information System (INIS)

Couto, R.T.

1990-01-01

QUEIMAP is a computer routine for burnup calculation, composed of five FORTRAN-77 subroutines. Its objective is to solve depletion equation of four radionuclides conversion chain, U238, U235, Th232, as well as fission fragments equations. In this paper the burnup is considered punctual and evolutioned under cross section. It presents the solution algorithms employed by QUEIMAP, the validation of its results and the way of use it. (M.I.)

Development of an immunoassay for determination of 2,4-dichlorophenoxyacetic acid (2,4-D) based upon the recombinant Fab fragment of 2,4-D specific antibody

Science.gov (United States)

Nguyen, Van C.; Nguyen, Thi D. T.; Dau, Hung A.; Tham, Thu N.; Quyen, Dinh T.; Bachmman, Till; Schmid, Rolf D.

2001-09-01

To develop an immunoassay and further an immunosensor for 2,4-D based upon recombinant antibody, the Fab fragments of 2,4-D specific antibody were expressed in E. coli. Western blotting analysis of the periplasmic cell fractions shown that under the non-reducing condition only a single protein band at a molecular mass of 45-kDa, corresponding to the whole Fab fragment was detected. Antigen binding activity for 2,4-D was found only in the extract of cells bearing the 2,4-D plasmid. An immunoassay based on the competitive reaction of 2,4-D and enzyme tracer with 2,4-D Fab fragments immobilized on micro titer plates via rabbit anti-mouse IgC was developed. Using this assay, 2,4-D could be detected at concentration range of 0.5 (mu) g/1 to 10(mu) g/1. The center point of the 2,4-D test was found at a concentration of 5 (mu) g/l. The assay was applied for detection of 2,4-D in spiked orange samples, resulting in recovery rate of 90 percent. The immunoassay could be applied to monitor human exposure to 2,4-D from contamination in fruit samples.

Isolation of llama antibody fragments for prevention of dandruff by phage display in shampoo

NARCIS (Netherlands)

Dolk, E.; Vaart, M. van der; Lutje Hulsik, D.; Vriend, G.; Haard, H. de; Spinelli, S.; Cambillau, C.; Frenken, L.; Verrips, T.

As part of research exploring the feasibility of using antibody fragments to inhibit the growth of organisms implicated in dandruff, we isolated antibody fragments that bind to a cell surface protein of Malassezia furfur in the presence of shampoo. We found that phage display of llama

Intermetallic Competition in the Fragmentation of Trimetallic Au-Zn-Alkali Complexes.

Science.gov (United States)

Lang, Johannes; Cayir, Merve; Walg, Simon P; Di Martino-Fumo, Patrick; Thiel, Werner R; Niedner-Schatteburg, Gereon

2016-02-12

Cationization is a valuable tool to enable mass spectrometric studies on neutral transition-metal complexes (e.g., homogenous catalysts). However, knowledge of potential impacts on the molecular structure and catalytic reactivity induced by the cationization is indispensable to extract information about the neutral complex. In this study, we cationize a bimetallic complex [AuZnCl3 ] with alkali metal ions (M(+) ) and investigate the charged adducts [AuZnCl3 M](+) by electrospray ionization mass spectrometry (ESI-MS). Infrared multiple photon dissociation (IR-MPD) in combination with density functional theory (DFT) calculations reveal a μ(3) binding motif of all alkali ions to the three chlorido ligands. The cationization induces a reorientation of the organic backbone. Collision-induced dissociation (CID) studies reveal switches of fragmentation channels by the alkali ion and by the CID amplitude. The Li(+) and Na(+) adducts prefer the sole loss of ZnCl2 , whereas the K(+) , Rb(+) , and Cs(+) adducts preferably split off MCl2 ZnCl. Calculated energetics along the fragmentation coordinate profiles allow us to interpret the experimental findings to a level of subtle details. The Zn(2+) cation wins the competition for the nitrogen coordination sites against K(+) , Rb(+) , and Cs(+) , but it loses against Li(+) and Na(+) in a remarkable deviation from a naive hard and soft acids and bases (HSAB) concept. The computations indicate expulsion of MCl2 ZnCl rather than of MCl and ZnCl2 . © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Morphology of the posteromedial fragment in pertrochanteric fractures: A three-dimensional computed tomography analysis.

Science.gov (United States)

Sharma, Gaurav; Gn, Kiran Kumar; Khatri, Kavin; Singh, Ravijot; Gamanagatti, Shivanand; Sharma, Vijay

2017-02-01

In this study we describe the morphology of the posteromedial fragment in pertrochanteric fractures using 3D CT scans and answer two questions 1) Do differences exist between the 3D CT appearances of posteromedial fragments and the depictions made in the AO classification 2) Does the posteromedial fragment affect stability in pertrochanteric fractures, in terms of fracture collapse? Preoperative CT scans of eight 31-A1 and fifty 31-A2 fractures were analysed. The presence of PM fragment, its fragmentation, greater trochanter (GT) involvement, lesser trochanter (LT) fragment size (in terms of its posterior and medial extent as well as LT length), LT fragment displacement (in terms of medial displacement and rotation) were determined. All fractures were treated with a DHS. Fracture collapse was determined on postoperative radiographs. The relationship between fracture collapse and patient factors including age, gender, fracture type (A1 versus A2), characteristics of the posteromedial fragment, and the presence of a lateral wall fracture were determined. Three out of eight 31-A1 fractures demonstrated a separate GT fragment (three part fracture). Out of the 50 31-A2 fractures, 12 had a single PM fragment, which included the LT and GT in continuity. The more common four part fractures seem to form by further fragmentation of this basic form. In A2 fractures, the GT was almost always broken and the broken fragment comprised a mean 56% of normal GT. The LT fragment involved an average of 74% of the posterior wall, and an average of 36% of the medial wall of the proximal femur. Larger LT fragments were less displaced as compared to smaller fragments. Univariate regression analyses revealed that fracture collapse was significantly correlated with fracture type (A1 versus A2, p 0.036), GT size (p 0.002) and the presence of a lateral wall fracture (pfragmentation of the posteromedial fragment, nor the size of the lesser trochanter fragment was found to predict stability in

Paper-Based and Computer-Based Concept Mappings: The Effects on Computer Achievement, Computer Anxiety and Computer Attitude

Science.gov (United States)

Erdogan, Yavuz

2009-01-01

The purpose of this paper is to compare the effects of paper-based and computer-based concept mappings on computer hardware achievement, computer anxiety and computer attitude of the eight grade secondary school students. The students were randomly allocated to three groups and were given instruction on computer hardware. The teaching methods used…

pH-tuneable binding of 2′-phospho-ADP-ribose to ketopantoate reductase: a structural and calorimetric study

Energy Technology Data Exchange (ETDEWEB)

Ciulli, Alessio [University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW (United Kingdom); Lobley, Carina M. C. [Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA (United Kingdom); Tuck, Kellie L. [University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW (United Kingdom); Smith, Alison G. [Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA (United Kingdom); Blundell, Tom L. [Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA (United Kingdom); Abell, Chris, E-mail: ca26@cam.ac.uk [University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW (United Kingdom)

2007-02-01

A combined crystallographic, calorimetric and mutagenic study has been used to show how changes in pH give rise to two distinct binding modes of 2′-phospho-ADP-ribose to ketopantoate reductase. The crystal structure of Escherichia coli ketopantoate reductase in complex with 2′-monophosphoadenosine 5′-diphosphoribose, a fragment of NADP{sup +} that lacks the nicotinamide ring, is reported. The ligand is bound at the enzyme active site in the opposite orientation to that observed for NADP{sup +}, with the adenine ring occupying the lipophilic nicotinamide pocket. Isothermal titration calorimetry with R31A and N98A mutants of the enzyme is used to show that the unusual ‘reversed binding mode’ observed in the crystal is triggered by changes in the protonation of binding groups at low pH. This research has important implications for fragment-based approaches to drug design, namely that the crystallization conditions and the chemical modification of ligands can have unexpected effects on the binding modes.

pH-tuneable binding of 2′-phospho-ADP-ribose to ketopantoate reductase: a structural and calorimetric study

International Nuclear Information System (INIS)

Ciulli, Alessio; Lobley, Carina M. C.; Tuck, Kellie L.; Smith, Alison G.; Blundell, Tom L.; Abell, Chris

2007-01-01

A combined crystallographic, calorimetric and mutagenic study has been used to show how changes in pH give rise to two distinct binding modes of 2′-phospho-ADP-ribose to ketopantoate reductase. The crystal structure of Escherichia coli ketopantoate reductase in complex with 2′-monophosphoadenosine 5′-diphosphoribose, a fragment of NADP + that lacks the nicotinamide ring, is reported. The ligand is bound at the enzyme active site in the opposite orientation to that observed for NADP + , with the adenine ring occupying the lipophilic nicotinamide pocket. Isothermal titration calorimetry with R31A and N98A mutants of the enzyme is used to show that the unusual ‘reversed binding mode’ observed in the crystal is triggered by changes in the protonation of binding groups at low pH. This research has important implications for fragment-based approaches to drug design, namely that the crystallization conditions and the chemical modification of ligands can have unexpected effects on the binding modes

Screening of a Novel Fragment Library with Functional Complexity against Mycobacterium tuberculosis InhA.

Science.gov (United States)

Prati, Federica; Zuccotto, Fabio; Fletcher, Daniel; Convery, Maire A; Fernandez-Menendez, Raquel; Bates, Robert; Encinas, Lourdes; Zeng, Jingkun; Chung, Chun-Wa; De Dios Anton, Paco; Mendoza-Losana, Alfonso; Mackenzie, Claire; Green, Simon R; Huggett, Margaret; Barros, David; Wyatt, Paul G; Ray, Peter C

2018-04-06

Our findings reported herein provide support for the benefits of including functional group complexity (FGC) within fragments when screening against protein targets such as Mycobacterium tuberculosis InhA. We show that InhA fragment actives with FGC maintained their binding pose during elaboration. Furthermore, weak fragment hits with functional group handles also allowed for facile fragment elaboration to afford novel and potent InhA inhibitors with good ligand efficiency metrics for optimization. © 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Identification and therapeutic potential of a vitronectin binding region of meningococcal msf.

Directory of Open Access Journals (Sweden)

Darryl J Hill

Full Text Available The human pathogen Neisseria meningitides (Nm attains serum resistance via a number of mechanisms, one of which involves binding to the host complement regulator protein vitronectin. We have shown previously that the Meningococcal surface fibril (Msf, a trimeric autotransporter, binds to the activated form of vitronectin (aVn to increase Nm survival in human serum. In this study, we aimed to identify the aVn-binding region of Msf to assess its potential as an antigen which can elicit antibodies that block aVn binding and/or possess bactericidal properties. Using several recombinant Msf fragments spanning its surface-exposed region, the smallest aVn-binding recombinants were found to span residues 1-86 and 39-124. The use of further deletion constructs and overlapping recombinant Msf fragments suggested that a region of Msf comprising residues 39-82 may be primarily important for aVn binding and that other regions may also be involved but to a lesser extent. Molecular modelling implicated K66 and K68, conserved in all available Msf sequences, to be involved in the interaction. Recombinant fragments which bound to aVn were able to reduce the survival advantage conveyed by aVn-interaction in serum bactericidal assays. Antibodies raised against one such fragment inhibited aVn binding to Msf. In addition, the antibodies enhanced specific killing of Msf-expressing Nm in a dose-dependent manner. Overall, this study identifies an aVn-binding region of Msf, an adhesin known to impart serum resistance properties to the pathogen; and shows that this region of Msf can elicit antibodies with dual properties which reduce pathogen survival within the host and thus has potential as a vaccine antigen.

Complete photo-fragmentation of the deuterium molecule

International Nuclear Information System (INIS)

Weber, Thorsten; Czasch, Achim O.; Jagutzki, Ottmar; Muller, Alkis K.; Mergel, Volker; Kheifets, A.; Rotenberg, Eli; Meigs, George; Prior, Mike H.; Daveau, Sebastian; Landers, Allen; Cocke, C.L.; Osipov, Timur; Diez Muino, Ricardo; Schmidt-Bocking, Horst; Dorner, Reinhard

2004-01-01

All properties of molecules, from binding and excitation energies to their geometry, are determined by the highly correlated initial state wavefunction of the electrons and nuclei. Perhaps surprisingly, details of these correlations can be revealed by studying the break-up of these systems into their constituents. The fragmentation might be initiated by the absorption of a single photon [1, 2, 3, 4, 5, 6], collision with a charged particle [7, 8] or exposure to a strong laser pulse [9, 10]. If the exciting interaction is sufficiently understood, one can use the fragmentation process as a tool to learn about the bound initial state [11, 12]. However, often the interaction and the fragment motions pose formidable challenges to quantum theory [13, 14, 15]. Here we report the coincident measurement of the momenta of both nuclei and both electrons from the single photon induced fragmentation of the deuterium molecule. The results reveal that the correlated motion of the electrons is strongly dependent on the inter-nuclear separation in the molecular ground state at the instant of photon absorption

Tropical forest fragmentation affects floral visitors but not the structure of individual-based palm-pollinator networks.

Science.gov (United States)

Dáttilo, Wesley; Aguirre, Armando; Quesada, Mauricio; Dirzo, Rodolfo

2015-01-01

Despite increasing knowledge about the effects of habitat loss on pollinators in natural landscapes, information is very limited regarding the underlying mechanisms of forest fragmentation affecting plant-pollinator interactions in such landscapes. Here, we used a network approach to describe the effects of forest fragmentation on the patterns of interactions involving the understory dominant palm Astrocaryum mexicanum (Arecaceae) and its floral visitors (including both effective and non-effective pollinators) at the individual level in a Mexican tropical rainforest landscape. Specifically, we asked: (i) Does fragment size affect the structure of individual-based plant-pollinator networks? (ii) Does the core of highly interacting visitor species change along the fragmentation size gradient? (iii) Does forest fragment size influence the abundance of effective pollinators of A. mexicanum? We found that fragment size did not affect the topological structure of the individual-based palm-pollinator network. Furthermore, while the composition of peripheral non-effective pollinators changed depending on fragment size, effective core generalist species of pollinators remained stable. We also observed that both abundance and variance of effective pollinators of male and female flowers of A. mexicanum increased with forest fragment size. These findings indicate that the presence of effective pollinators in the core of all forest fragments could keep the network structure stable along the gradient of forest fragmentation. In addition, pollination of A. mexicanum could be more effective in larger fragments, since the greater abundance of pollinators in these fragments may increase the amount of pollen and diversity of pollen donors between flowers of individual plants. Given the prevalence of fragmentation in tropical ecosystems, our results indicate that the current patterns of land use will have consequences on the underlying mechanisms of pollination in remnant forests.

Tropical forest fragmentation affects floral visitors but not the structure of individual-based palm-pollinator networks.

Directory of Open Access Journals (Sweden)

Wesley Dáttilo

Full Text Available Despite increasing knowledge about the effects of habitat loss on pollinators in natural landscapes, information is very limited regarding the underlying mechanisms of forest fragmentation affecting plant-pollinator interactions in such landscapes. Here, we used a network approach to describe the effects of forest fragmentation on the patterns of interactions involving the understory dominant palm Astrocaryum mexicanum (Arecaceae and its floral visitors (including both effective and non-effective pollinators at the individual level in a Mexican tropical rainforest landscape. Specifically, we asked: (i Does fragment size affect the structure of individual-based plant-pollinator networks? (ii Does the core of highly interacting visitor species change along the fragmentation size gradient? (iii Does forest fragment size influence the abundance of effective pollinators of A. mexicanum? We found that fragment size did not affect the topological structure of the individual-based palm-pollinator network. Furthermore, while the composition of peripheral non-effective pollinators changed depending on fragment size, effective core generalist species of pollinators remained stable. We also observed that both abundance and variance of effective pollinators of male and female flowers of A. mexicanum increased with forest fragment size. These findings indicate that the presence of effective pollinators in the core of all forest fragments could keep the network structure stable along the gradient of forest fragmentation. In addition, pollination of A. mexicanum could be more effective in larger fragments, since the greater abundance of pollinators in these fragments may increase the amount of pollen and diversity of pollen donors between flowers of individual plants. Given the prevalence of fragmentation in tropical ecosystems, our results indicate that the current patterns of land use will have consequences on the underlying mechanisms of pollination in

The Office Software Learning and Examination System Design Based on Fragmented Learning Idea

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Xu Ling

2016-01-01

Full Text Available Fragmented learning is that through the segmentation of learning content or learning time, make learners can use the fragmented time for learning fragmentated content, have the characteristics of time flexibility, learning targeted and high learning efficiency. Based on the fragmented learning ideas, combined with the teaching idea of micro class and interactive teaching, comprehensive utilization of flash animation design software, .NET development platform, VSTO technology, multimedia development technology and so on, design and develop a system integrated with learning, practice and examination of the Office software, which is not only conducive to the effective and personalized learning of students, but also conducive to the understanding the students’ situation of teachers, and liberate teachers from the heavy labor of mechanical, focus on promoting the formation of students’ knowledge system.

Novel Small Molecule Inhibitors of Choline Kinase Identified by Fragment-Based Drug Discovery.

Science.gov (United States)

Zech, Stephan G; Kohlmann, Anna; Zhou, Tianjun; Li, Feng; Squillace, Rachel M; Parillon, Lois E; Greenfield, Matthew T; Miller, David P; Qi, Jiwei; Thomas, R Mathew; Wang, Yihan; Xu, Yongjin; Miret, Juan J; Shakespeare, William C; Zhu, Xiaotian; Dalgarno, David C

2016-01-28

Choline kinase α (ChoKα) is an enzyme involved in the synthesis of phospholipids and thereby plays key roles in regulation of cell proliferation, oncogenic transformation, and human carcinogenesis. Since several inhibitors of ChoKα display antiproliferative activity in both cellular and animal models, this novel oncogene has recently gained interest as a promising small molecule target for cancer therapy. Here we summarize our efforts to further validate ChoKα as an oncogenic target and explore the activity of novel small molecule inhibitors of ChoKα. Starting from weakly binding fragments, we describe a structure based lead discovery approach, which resulted in novel highly potent inhibitors of ChoKα. In cancer cell lines, our lead compounds exhibit a dose-dependent decrease of phosphocholine, inhibition of cell growth, and induction of apoptosis at low micromolar concentrations. The druglike lead series presented here is optimizable for improvements in cellular potency, drug target residence time, and pharmacokinetic parameters. These inhibitors may be utilized not only to further validate ChoKα as antioncogenic target but also as novel chemical matter that may lead to antitumor agents that specifically interfere with cancer cell metabolism.

28-mer Fragment Derived from Enterocin CRL35 Displays an Unexpected Bactericidal Effect on Listeria Cells.

Science.gov (United States)

Masias, Emilse; Sanches, Paulo R S; Dupuy, Fernando G; Acuna, Leonardo; Bellomio, Augusto; Cilli, Eduardo; Saavedra, Lucila; Minahk, Carlos

2015-01-01

Two shorter peptides derived from enterocin CRL35, a 43-mer bacteriocin, were synthesized i.e. the N-terminal fragment spanning from residues 1 to 15, and a 28-mer fragment that represents the C-terminal of enterocin CRL35, the residues 16 to 43. The separate peptides showed no activity when combined. On one hand, the 28-mer peptide displayed an unpredicted antimicrobial activity. On the other, 15- mer peptide had no consistent anti-Listeria effect. The dissociation constants calculated from experimental data indicated that all peptides could bind at similar extent to the sensitive cells. However, transmembrane electrical potential was not dissipated to the same level by the different peptides; whereas the full-length and the C-terminal 28-mer fragment induced almost full dissipation, 15-mer fragment produced only a slow and incomplete effect. Furthermore, a different interaction of each peptide with membranes was demonstrated based on studies carried out with liposomes, which led us to conclude that activity was related to structure rather than to net positive charges. These results open up the possibility of designing new peptides based on the 28-mer fragment with enhanced activity, which would represent a promising approach for combating Listeria and other pathogens.

Structure of calmodulin complexed with an olfactory CNG channel fragment and role of the central linker: Residual dipolar couplings to evaluate calmodulin binding modes outside the kinase family

International Nuclear Information System (INIS)

Contessa, Gian Marco; Orsale, Maria; Melino, Sonia; Torre, Vincent; Paci, Maurizio; Desideri, Alessandro; Cicero, Daniel O.

2005-01-01

The NMR high-resolution structure of calmodulin complexed with a fragment of the olfactory cyclic-nucleotide gated channel is described. This structure shows features that are unique for this complex, including an active role of the linker connecting the N- and C-lobes of calmodulin upon binding of the peptide. Such linker is not only involved in the formation of an hydrophobic pocket to accommodate a bulky peptide residue, but it also provides a positively charged region complementary to a negative charge of the target. This complex of calmodulin with a target not belonging to the kinase family was used to test the residual dipolar coupling (RDC) approach for the determination of calmodulin binding modes to peptides. Although the complex here characterized belongs to the (1--14) family, high Q values were obtained with all the 1:1 complexes for which crystalline structures are available. Reduction of the RDC data set used for the correlation analysis to structured regions of the complex allowed a clear identification of the binding mode. Excluded regions comprise calcium binding loops and loops connecting the EF-hand motifs

Locations of the three primary binding sites for long-chain fatty acids on bovine serum albumin

International Nuclear Information System (INIS)

Hamilton, J.A.; Era, S.; Bhamidipati, S.P.; Reed, R.G.

1991-01-01

Binding of 13 C-enriched oleic acid to bovine serum albumin and to three large proteolytic fragments of albumin - two complementary fragments corresponding to the two halved of albumin and one fragment corresponding to the carboxyl-terminal domain - yielded unique patterns of NMR resonances (chemical shifts and relative intensities) that were used to identify the locations of binding of the first 5 mol of oleic acid to the multidomain albumin molecule. The first 3 mol of oleic acid added to intact albumin generated three distinct NMR resonances as a result of simultaneous binding of oleic acid to three heterogeneous sites (primary sites). This distribution suggests albumin to be a less symmetrical binding molecule than theoretical models predict. This work also demonstrates the power of NMR for the study of microenvironments of individual fatty acid binding sites in specific domain

Challenges in Optimizing a Prostate Carcinoma Binding Peptide, Identified through the Phage Display Technology

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Jürgen Debus

2011-02-01

Full Text Available The transfer of peptides identified through the phage display technology to clinical applications is difficult. Major drawbacks are the metabolic degradation and label instability. The aim of our work is the optimization of DUP-1, a peptide which was identified by phage display to specifically target human prostate carcinoma. To investigate the influence of chelate conjugation, DOTA was coupled to DUP-1 and labeling was performed with 111In. To improve serum stability cyclization of DUP-1 and targeted D-amino acid substitution were carried out. Alanine scanning was performed for identification of the binding site and based on the results peptide fragments were chemically synthesized. The properties of modified ligands were investigated in in vitro binding and competition assays. In vivo biodistribution studies were carried out in mice, carrying human prostate tumors subcutaneously. DOTA conjugation resulted in different cellular binding kinetics, rapid in vivo renal clearance and increased tumor-to-organ ratios. Cyclization and D-amino acid substitution increased the metabolic stability but led to binding affinity decrease. Fragment investigation indicated that the sequence NRAQDY might be significant for target-binding. Our results demonstrate challenges in optimizing peptides, identified through phage display libraries, and show that careful investigation of modified derivatives is necessary in order to improve their characteristics.

Computing the binding affinity of a ligand buried deep inside a protein with the hybrid steered molecular dynamics

International Nuclear Information System (INIS)

Villarreal, Oscar D.; Yu, Lili; Rodriguez, Roberto A.; Chen, Liao Y.

2017-01-01

Computing the ligand-protein binding affinity (or the Gibbs free energy) with chemical accuracy has long been a challenge for which many methods/approaches have been developed and refined with various successful applications. False positives and, even more harmful, false negatives have been and still are a common occurrence in practical applications. Inevitable in all approaches are the errors in the force field parameters we obtain from quantum mechanical computation and/or empirical fittings for the intra- and inter-molecular interactions. These errors propagate to the final results of the computed binding affinities even if we were able to perfectly implement the statistical mechanics of all the processes relevant to a given problem. And they are actually amplified to various degrees even in the mature, sophisticated computational approaches. In particular, the free energy perturbation (alchemical) approaches amplify the errors in the force field parameters because they rely on extracting the small differences between similarly large numbers. In this paper, we develop a hybrid steered molecular dynamics (hSMD) approach to the difficult binding problems of a ligand buried deep inside a protein. Sampling the transition along a physical (not alchemical) dissociation path of opening up the binding cavity- -pulling out the ligand- -closing back the cavity, we can avoid the problem of error amplifications by not relying on small differences between similar numbers. We tested this new form of hSMD on retinol inside cellular retinol-binding protein 1 and three cases of a ligand (a benzylacetate, a 2-nitrothiophene, and a benzene) inside a T4 lysozyme L99A/M102Q(H) double mutant. In all cases, we obtained binding free energies in close agreement with the experimentally measured values. This indicates that the force field parameters we employed are accurate and that hSMD (a brute force, unsophisticated approach) is free from the problem of error amplification suffered by

Development of the Fragment Molecular Orbital Method for Calculating Nonlocal Excitations in Large Molecular Systems.

Science.gov (United States)

Fujita, Takatoshi; Mochizuki, Yuji

2018-04-19

We developed the fragment-based method for calculating nonlocal excitations in large molecular systems. This method is based on the multilayer fragment molecular orbital method and the configuration interaction single (CIS) wave function using localized molecular orbitals. The excited-state wave function for the whole system is described as a superposition of configuration state functions (CSFs) for intrafragment excitations and for interfragment charge-transfer excitations. The formulation and calculations of singlet excited-state Hamiltonian matrix elements in the fragment CSFs are presented in detail. The efficient approximation schemes for calculating the matrix elements are also presented. The computational efficiency and the accuracy were evaluated using the molecular dimers and molecular aggregates. We confirmed that absolute errors of 50 meV (relative to the conventional calculations) are achievable for the molecular systems in their equilibrium geometries. The perturbative electron correlation correction to the CIS excitation energies is also demonstrated. The present theory can compute a large number of excited states in large molecular systems; in addition, it allows for the systematic derivation of a model exciton Hamiltonian. These features are useful for studying excited-state dynamics in condensed molecular systems based on the ab initio electronic structure theory.

Assessing the lipophilicity of fragments and early hits

Science.gov (United States)

Mortenson, Paul N.; Murray, Christopher W.

2011-07-01

A key challenge in many drug discovery programs is to accurately assess the potential value of screening hits. This is particularly true in fragment-based drug design (FBDD), where the hits often bind relatively weakly, but are correspondingly small. Ligand efficiency (LE) considers both the potency and the size of the molecule, and enables us to estimate whether or not an initial hit is likely to be optimisable to a potent, druglike lead. While size is a key property that needs to be controlled in a small molecule drug, there are a number of additional properties that should also be considered. Lipophilicity is amongst the most important of these additional properties, and here we present a new efficiency index (LLEAT) that combines lipophilicity, size and potency. The index is intuitively defined, and has been designed to have the same target value and dynamic range as LE, making it easily interpretable by medicinal chemists. Monitoring both LE and LLEAT should help both in the selection of more promising fragment hits, and controlling molecular weight and lipophilicity during optimisation.

Chimeric recombinant antibody fragments in cardiac troponin I immunoassay.

Science.gov (United States)

Hyytiä, Heidi; Heikkilä, Taina; Brockmann, Eeva-Christine; Kekki, Henna; Hedberg, Pirjo; Puolakanaho, Tarja; Lövgren, Timo; Pettersson, Kim

2015-03-01

To introduce a novel nanoparticle-based immunoassay for cardiac troponin I (cTnI) utilizing chimeric antibody fragments and to demonstrate that removal of antibody Fc-part and antibody chimerization decrease matrix related interferences. A sandwich-type immunoassay for cTnI based on recombinant chimeric (mouse variable/human constant) antigen binding (cFab) antibodies and intrinsically fluorescent nanoparticles was developed. To test whether using chimeric antibody fragments helps to avoid matrix related interferences, samples (n=39) with known amounts of triglycerides, bilirubin, rheumatoid factor (RF) or human anti-mouse antibodies (HAMAs) were measured with the novel assay, along with a previously published nanoparticle-based research assay with the same antibody epitopes. The limit of detection (LoD) was 3.30ng/L. Within-laboratory precision for 29ng/L and 2819ng/L cTnI were 13.7% and 15.9%, respectively. Regression analysis with Siemens ADVIA Centaur® yielded a slope (95% confidence intervals) of 0.18 (0.17-1.19) and a y-intercept of 1.94 (-1.28-3.91) ng/L. When compared to a previously published nanoparticle-based assay, the novel assay showed substantially reduced interference in the tested interference prone samples, 15.4 vs. 51.3%. A rheumatoid factor containing sample was decreased from 241ng/L to fragments enabled the development of a sensitive (LoD=3.3ng/L) immunoassay for the detection of cTnI and decreased matrix related interferences, thus resulting in a lower number of falsely elevated cTnI-values. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Recent progress on perturbative QCD fragmentation functions

International Nuclear Information System (INIS)

Cheung, K.

1995-05-01

The recent development of perturbative QCD (PQCD) fragmentation functions has strong impact on quarkonium production. I shall summarize B c meson production based on these PQCD fragmentation functions, as well as, the highlights of some recent activities on applying these PQCD fragmentation functions to explain anomalous J/ψ and ψ' production at the Tevatron. Finally, I discuss a fragmentation model based on the PQCD fragmentation functions for heavy quarks fragmenting into heavy-light mesons

Computational Strategy for Quantifying Human Pesticide Exposure based upon a Saliva Measurement

Directory of Open Access Journals (Sweden)

Charles eTimchalk

2015-05-01

Full Text Available Quantitative exposure data is important for evaluating toxicity risk and biomonitoring is a critical tool for evaluating human exposure. Direct personal monitoring provides the most accurate estimation of a subject’s true dose, and non-invasive methods are advocated for quantifying exposure to xenobiotics. In this regard, there is a need to identify chemicals that are cleared in saliva at concentrations that can be quantified to support the implementation of this approach. This manuscript reviews the computational modeling approaches that are coupled to in vivo and in vitro experiments to predict salivary uptake and clearance of xenobiotics and provides additional insight on species-dependent differences in partitioning that are of key importance for extrapolation. The primary mechanism by which xenobiotics leave the blood and enter saliva involves paracellular transport, passive transcellular diffusion, or trancellular active transport with the majority of xenobiotics transferred by passive diffusion. The transcellular or paracellular diffusion of unbound chemicals in plasma to saliva has been computationally modeled using compartmental and physiologically based approaches. Of key importance for determining the plasma:saliva partitioning was the utilization of the Schmitt algorithm that calculates partitioning based upon the tissue composition, pH, chemical pKa and plasma protein-binding. Sensitivity analysis identified that both protein-binding and pKa (for weak acids and bases have significant impact on determining partitioning and species dependent differences based upon physiological variance. Future strategies are focused on an in vitro salivary acinar cell based system to experimentally determine and computationally predict salivary gland uptake and clearance for xenobiotics. It is envisioned that a combination of salivary biomonitoring and computational modeling will enable the non-invasive measurement of chemical exposures in human

Affinity of hemoglobin for the cytoplasmic fragment of human erythrocyte membrane band 3. Equilibrium measurements at physiological pH using matrix-bound proteins: the effects of ionic strength, deoxygenation and of 2,3-diphosphoglycerate.

Science.gov (United States)

Chétrite, G; Cassoly, R

1985-10-05

The cytoplasmic fragment of band 3 protein isolated from the human erythrocyte membrane was linked to a CNBr-activated Sepharose matrix in an attempt to measure, in batch experiments, its equilibrium binding constant with oxy- and deoxyhemoglobin at physiological pH and ionic strength values and in the presence or the absence of 2,3-diphosphoglycerate. All the experiments were done at pH 7.2, and equilibrium constants were computed on the basis of one hemoglobin tetramer bound per monomer of fragment. In 10 mM-phosphate buffer, a dissociation constant KD = 2 X 10(-4)M was measured for oxyhemoglobin and was shown to increase to 8 X 10(-4)M in the presence of 50 mM-NaCl. Association could not be demonstrated at higher salt concentrations. Diphosphoglycerate-stripped deoxyhemoglobin was shown to associate more strongly with the cytoplasmic fragment of band 3. In 10 mM-bis-Tris (pH 7.2) and in the presence of 120 mM-NaCl, a dissociation constant KD = 4 X 10(-4)M was measured. Upon addition of increasing amounts of 2,3-diphosphoglycerate, the complex formed between deoxyhemoglobin and the cytoplasmic fragment of band 3 was dissociated. On the reasonable assumption that the hemoglobin binding site present on band 3 fragment was not modified upon linking the protein to the Sepharose matrix, the results indicated that diphosphoglycerate-stripped deoxyhemoglobin or partially liganded hemoglobin tetramers in the T state could bind band 3 inside the intact human red blood cell.

Binding of MCM-interacting proteins to ATP-binding site in MCM6

Directory of Open Access Journals (Sweden)

Hosoi A

2016-03-01

Full Text Available Atsutoshi Hosoi, Taku Sakairi, Yukio Ishimi Graduate School of Science and Engineering, Ibaraki University, Mito, Ibaraki, Japan Abstract: The function of MCM2–7 complex that is a DNA helicase in DNA replication may be regulated by various MCM-interacting proteins, including CDC45, RPA, TIM, TIPIN, Claspin, MCM10, and MCM-BP. It has been shown by immunoprecipitation that human MCM6 interacts with all these proteins in coexpressed insect cells. To determine the region in MCM6 to interact with these proteins, we prepared various truncated forms of MCM6 and examined the interaction of these MCM6 fragments with the MCM-interacting proteins. All these proteins bound to C-terminal half of MCM6, and CDC45, RPA2, TIM, TIPIN, MCM-BP, and MCM10 bound to the fragments containing ATP-binding motifs. CDC45 and RPA2 bound to the smallest fragment containing Walker motif A. Only MCM-BP is bound to the N-terminal half of MCM6. Site-directed mutagenesis study suggests that hydrophobic interaction is involved in the interaction of MCM6 with CDC45 and TIM. These results suggest a possibility that MCM-interacting proteins regulate MCM2–7 function by modulating the ATP-binding ability of the MCM2–7. Keywords: DNA helicase, DNA replication, checkpoint, MCM2–7 proteins

Fragger: a protein fragment picker for structural queries.

Science.gov (United States)

Berenger, Francois; Simoncini, David; Voet, Arnout; Shrestha, Rojan; Zhang, Kam Y J

2017-01-01

Protein modeling and design activities often require querying the Protein Data Bank (PDB) with a structural fragment, possibly containing gaps. For some applications, it is preferable to work on a specific subset of the PDB or with unpublished structures. These requirements, along with specific user needs, motivated the creation of a new software to manage and query 3D protein fragments. Fragger is a protein fragment picker that allows protein fragment databases to be created and queried. All fragment lengths are supported and any set of PDB files can be used to create a database. Fragger can efficiently search a fragment database with a query fragment and a distance threshold. Matching fragments are ranked by distance to the query. The query fragment can have structural gaps and the allowed amino acid sequences matching a query can be constrained via a regular expression of one-letter amino acid codes. Fragger also incorporates a tool to compute the backbone RMSD of one versus many fragments in high throughput. Fragger should be useful for protein design, loop grafting and related structural bioinformatics tasks.

The split comets: gravitational interaction between the fragments

International Nuclear Information System (INIS)

Sekanina, Z.

1979-01-01

The n-body computer program by Schubart and Stumpff (1966) has been slightly modified to study the gravitational interaction between two fragments of a split comet nucleus in the sun's gravitational field. All calculations refer to the orbit of Comet West (1976 VI), the velocity of separation of the fragments is assumed to be equal in magnitude to the velocity of escape from the parent nucleus, and the numerical integration of the relative motion of one fragment (called the companion) with respect to the other (principal fragment) is carried over the period of 200 days from separation. (Auth.)

A Dynamic Object Behavior Model and Implementation Based on Computational Reflection

Institute of Scientific and Technical Information of China (English)

HE Cheng-wan; HE Fei; HE Ke-qing

2005-01-01

A dynamic object behavior model based on computational reflection is proposed. This model consists of function level and meta level, the meta objects in meta level manage the base objects and behaviors in function level, including dynamic binding and unbinding of base object and behavior.We implement this model with RoleJava Language, which is our self linguistic extension of the Java Language. Meta Objects are generated automatically at compile-time, this makes the reflecton mechanism transparent to programmers. Finally an example applying this model to a banking system is presented.

Heavy-residue isoscaling as a probe of the symmetry energy of hot fragments

International Nuclear Information System (INIS)

Souliotis, G.A.; Shetty, D.V.; Keksis, A.; Bell, E.; Jandel, M.; Veselsky, M.; Yennello, S.J.

2006-01-01

The isoscaling properties of isotopically resolved projectile residues from peripheral collisions of 86 Kr (25 MeV/nucleon) 64 Ni (25 MeV/nucleon), and 136 Xe (20 MeV/nucleon) beams on various target pairs are employed to probe the symmetry energy coefficient of the nuclear binding energy. The present study focuses on heavy projectile fragments produced in peripheral and semiperipheral collisions near the onset of multifragment emission (E * /A=2-3 MeV). For these fragments, the measured average velocities are used to extract excitation energies. The excitation energies, in turn, are used to estimate the temperatures of the fragmenting quasiprojectiles in the framework the Fermi gas model. The isoscaling analysis of the fragment yields provided the isoscaling parameters α that, in combination with temperatures and isospin asymmetries provided the symmetry energy coefficient of the nuclear binding energy of the hot fragmenting quasiprojectiles. The extracted values of the symmetry energy coefficient at this excitation energy range (2-3 MeV/nucleon) are lower than the typical liquid-drop model value ∼25 MeV corresponding to ground-state nuclei and show a monotonic decrease with increasing excitation energy. This result is of importance in the formation of hot nuclei in heavy-ion reactions and in hot stellar environments such as supernova

Target based drug design - a reality in virtual sphere.

Science.gov (United States)

Verma, Saroj; Prabhakar, Yenamandra S

2015-01-01

The target based drug design approaches are a series of computational procedures, including visualization tools, to support the decision systems of drug design/discovery process. In the essence of biological targets shaping the potential lead/drug molecules, this review presents a comprehensive position of different components of target based drug design which include target identification, protein modeling, molecular dynamics simulations, binding/catalytic sites identification, docking, virtual screening, fragment based strategies, substructure treatment of targets in tackling drug resistance, in silico ADMET, structural vaccinology, etc along with the key issues involved therein and some well investigated case studies. The concepts and working of these procedures are critically discussed to arouse interest and to advance the drug research.

Fragmenting networks by targeting collective influencers at a mesoscopic level

Science.gov (United States)

Kobayashi, Teruyoshi; Masuda, Naoki

2016-11-01

A practical approach to protecting networks against epidemic processes such as spreading of infectious diseases, malware, and harmful viral information is to remove some influential nodes beforehand to fragment the network into small components. Because determining the optimal order to remove nodes is a computationally hard problem, various approximate algorithms have been proposed to efficiently fragment networks by sequential node removal. Morone and Makse proposed an algorithm employing the non-backtracking matrix of given networks, which outperforms various existing algorithms. In fact, many empirical networks have community structure, compromising the assumption of local tree-like structure on which the original algorithm is based. We develop an immunization algorithm by synergistically combining the Morone-Makse algorithm and coarse graining of the network in which we regard a community as a supernode. In this way, we aim to identify nodes that connect different communities at a reasonable computational cost. The proposed algorithm works more efficiently than the Morone-Makse and other algorithms on networks with community structure.

Translation of Polioviral mRNA Is Inhibited by Cleavage of Polypyrimidine Tract-Binding Proteins Executed by Polioviral 3Cpro

Science.gov (United States)

Back, Sung Hoon; Kim, Yoon Ki; Kim, Woo Jae; Cho, Sungchan; Oh, Hoe Rang; Kim, Jung-Eun; Jang, Sung Key

2002-01-01

The translation of polioviral mRNA occurs through an internal ribosomal entry site (IRES). Several RNA-binding proteins, such as polypyrimidine tract-binding protein (PTB) and poly(rC)-binding protein (PCBP), are required for the poliovirus IRES-dependent translation. Here we report that a poliovirus protein, 3Cpro (and/or 3CDpro), cleaves PTB isoforms (PTB1, PTB2, and PTB4). Three 3Cpro target sites (one major target site and two minor target sites) exist in PTBs. PTB fragments generated by poliovirus infection are redistributed to the cytoplasm from the nucleus, where most of the intact PTBs are localized. Moreover, these PTB fragments inhibit polioviral IRES-dependent translation in a cell-based assay system. We speculate that the proteolytic cleavage of PTBs may contribute to the molecular switching from translation to replication of polioviral RNA. PMID:11836431