Combining NMR and X-ray crystallography in fragment-based drug discovery: discovery of highly potent and selective BACE-1 inhibitors.

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Wyss, Daniel F; Wang, Yu-Sen; Eaton, Hugh L; Strickland, Corey; Voigt, Johannes H; Zhu, Zhaoning; Stamford, Andrew W

2012-01-01

Fragment-based drug discovery (FBDD) has become increasingly popular over the last decade. We review here how we have used highly structure-driven fragment-based approaches to complement more traditional lead discovery to tackle high priority targets and those struggling for leads. Combining biomolecular nuclear magnetic resonance (NMR), X-ray crystallography, and molecular modeling with structure-assisted chemistry and innovative biology as an integrated approach for FBDD can solve very difficult problems, as illustrated in this chapter. Here, a successful FBDD campaign is described that has allowed the development of a clinical candidate for BACE-1, a challenging CNS drug target. Crucial to this achievement were the initial identification of a ligand-efficient isothiourea fragment through target-based NMR screening and the determination of its X-ray crystal structure in complex with BACE-1, which revealed an extensive H-bond network with the two active site aspartate residues. This detailed 3D structural information then enabled the design and validation of novel, chemically stable and accessible heterocyclic acylguanidines as aspartic acid protease inhibitor cores. Structure-assisted fragment hit-to-lead optimization yielded iminoheterocyclic BACE-1 inhibitors that possess desirable molecular properties as potential therapeutic agents to test the amyloid hypothesis of Alzheimer's disease in a clinical setting.

From BACE1 Inhibitor to Multifunctionality of Tryptoline and Tryptamine Triazole Derivatives for Alzheimer’s Disease

OpenAIRE

Jiaranaikulwanitch, Jutamas; Govitrapong, Piyarat; Fokin, Valery V.; Vajragupta, Opa

2012-01-01

Efforts to discover new drugs for Alzheimer’s disease emphasizing multiple targets was conducted seeking to inhibit amyloid oligomer formation and to prevent radical formation. The tryptoline and tryptamine cores of BACE1 inhibitors previously identified by virtual screening were modified in silico for additional modes of action. These core structures were readily linked to different side chains using 1,2,3-triazole rings as bridges by copper catalyzed azide-alkyne cycloa...

Structure-Based Design of an Iminoheterocyclic β-Site Amyloid Precursor Protein Cleaving Enzyme (BACE) Inhibitor that Lowers Central Aβ in Nonhuman Primates

Energy Technology Data Exchange (ETDEWEB)

Mandal, Mihirbaran; Wu, Yusheng; Misiaszek, Jeffrey; Li, Guoqing; Buevich, Alexei; Caldwell, John P.; Liu, Xiaoxiang; Mazzola, Robert D.; Orth, Peter; Strickland, Corey; Voigt, Johannes; Wang, Hongwu; Zhu, Zhaoning; Chen, Xia; Grzelak, Michael; Hyde, Lynn A.; Kuvelkar, Reshma; Leach, Prescott T.; Terracina, Giuseppe; Zhang, Lili; Zhang, Qi; Michener, Maria S.; Smith, Brad; Cox, Kathleen; Grotz, Diane; Favreau, Leonard; Mitra, Kaushik; Kazakevich, Irina; McKittrick, Brian A.; Greenlee, William; Kennedy, Matthew E.; Parker, Eric M.; Cumming, Jared N.; Stamford, Andrew W. (Merck)

2016-04-14

We describe successful efforts to optimize the in vivo profile and address off-target liabilities of a series of BACE1 inhibitors represented by 6 that embodies the recently validated fused pyrrolidine iminopyrimidinone scaffold. Employing structure-based design, truncation of the cyanophenyl group of 6 that binds in the S3 pocket of BACE1 followed by modification of the thienyl group in S1 was pursued. Optimization of the pyrimidine substituent that binds in the S2'–S2'' pocket of BACE1 remediated time-dependent CYP3A4 inhibition of earlier analogues in this series and imparted high BACE1 affinity. These efforts resulted in the discovery of difluorophenyl analogue 9 (MBi-4), which robustly lowered CSF and cortex Aβ40 in both rats and cynomolgus monkeys following a single oral dose. Compound 9 represents a unique molecular shape among BACE inhibitors reported to potently lower central Aβ in nonrodent preclinical species.

Sebestenoids A-D, BACE1 inhibitors from Cordia sebestena.

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Dai, Jingqiu; Sorribas, Analia; Yoshida, Wesley Y; Williams, Philip G

2010-12-01

Bioassay-guided fractionation of an extract prepared from the fruits of Cordia sebestena led to the isolation of sebestenoids A-D (1-4). Their structures were elucidated on the basis of extensive NMR experiments and mass spectroscopic measurements. Compounds 1-4 exhibited moderate inhibition of the aspartic protease BACE1. Copyright © 2010 Elsevier Ltd. All rights reserved.

Quantum mechanics study of the hydroxyethylamines-BACE-1 active site interaction energies

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Gueto-Tettay, Carlos; Drosos, Juan Carlos; Vivas-Reyes, Ricardo

2011-06-01

The identification of BACE-1, a key enzyme in the production of Amyloid-β (Aβ) peptides, generated by the proteolytic processing of amyloid precursor protein, was a major advance in the field of Alzheimer's disease as this pathology is characterized by the presence of extracellular senile plaques, mainly comprised of Aβ peptides. Hydroxyethylamines have demonstrated a remarkable potential, like candidate drugs, for this disease using BACE-1 as target. Density Functional Theory calculations were employed to estimate interaction energies for the complexes formed between the hydroxyethylamine derivated inhibitors and 24 residues in the BACE-1 active site. The collected data offered not only a general but a particular quantitative description that gives a deep insight of the interactions in the active site, showing at the same time how ligand structural variations affect them. Polar interactions are the major energetic contributors for complex stabilization and those ones with charged aspartate residues are highlighted, as they contribute over 90% of the total attractive interaction energy. Ligand-ARG296 residue interaction reports the most repulsive value and decreasing of the magnitude of this repulsion can be a key feature for the design of novel and more potent BACE-1 inhibitors. Also it was explained why sultam derivated BACE-1 inhibitors are better ones than lactam based. Hydrophobic interactions concentrated at S1 zone and other relevant repulsions and attractions were also evaluated. The comparison of two different theory levels (X3LYP and M062X) allowed to confirm the relevance of the detected interactions as each theory level has its own strength to depict the forces involved, as is the case of M062X which is better describing the hydrophobic interactions. Those facts were also evaluated and confirmed by comparing the quantitative trend, of selected ligand-residue interactions, with MP2 theory level as reference standard method for electrostatic plus

Heparan sulfate regulates amyloid precursor protein processing by BACE1, the Alzheimer's β-secretase

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Scholefield, Zoe; Yates, Edwin A.; Wayne, Gareth; Amour, Augustin; McDowell, William; Turnbull, Jeremy E.

2003-01-01

Cleavage of amyloid precursor protein (APP) by the Alzheimer's β-secretase (BACE1) is a key step in generating amyloid β-peptide, the main component of amyloid plaques. Here we report evidence that heparan sulfate (HS) interacts with β-site APP-cleaving enzyme (BACE) 1 and regulates its cleavage of APP. We show that HS and heparin interact directly with BACE1 and inhibit in vitro processing of peptide and APP substrates. Inhibitory activity is dependent on saccharide size and specific structural characteristics, and the mechanism of action involves blocking access of substrate to the active site. In cellular assays, HS specifically inhibits BACE1 cleavage of APP but not alternative cleavage by α-secretase. Endogenous HS immunoprecipitates with BACE1 and colocalizes with BACE1 in the Golgi complex and at the cell surface, two of its putative sites of action. Furthermore, inhibition of cellular HS synthesis results in enhanced BACE1 activity. Our findings identify HS as a natural regulator of BACE1 and suggest a novel mechanism for control of APP processing. PMID:14530380

Reversing hypomyelination in BACE1-null mice with Akt-DD overexpression.

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Hu, Xiangyou; Schlanger, Rita; He, Wanxia; Macklin, Wendy B; Yan, Riqiang

2013-05-01

β-Site amyloid precursor protein convertase enzyme 1 (BACE1), a type I transmembrane aspartyl protease required to cleave amyloid precursor protein for releasing a toxic amyloid peptide, also cleaves type I and type III neuregulin-1 (Nrg-1). BACE1 deficiency in mice causes hypomyelination during development and impairs remyelination if injured. In BACE1-null mice, the abolished cleavage of neuregulin-1 by BACE1 is speculated to cause reduced myelin sheath thickness in both the central nervous system and peripheral nervous system because reduced cleavage of Nrg-1 correlates with reduced Akt phosphorylation, a downstream signaling molecule of the Nrg-1/ErbB pathway. Here we tested specifically whether increasing Akt activity alone in oligodendrocytes would be sufficient to reverse the hypomyelination phenotype in BACE1-null mice. BACE1-null mice were bred with transgenic mice expressing constitutively active Akt (Akt-DD; mutations with D(308)T and D(473)S) in oligodendrocytes. Relative to littermate BACE1-null controls, BACE1(-/-)/Akt-DD mice exhibited enhanced expression of myelin basic protein and promoter of proteolipid protein. The elevated expression of myelin proteins correlated with a thicker myelin sheath in optic nerves; comparison of quantified g ratios with statistic significance was used to confirm this reversion. However, it appeared that myelin sheath thickness in the sciatic nerves was not increased in BACE1(-/-)/Akt-DD mice, as the g ratio was not significantly different from the control. Hence, increased Akt activity in BACE1-null myelinating cells only compensates for the loss of BACE1 activity in the central nervous system, which is consistent with the observation that overexpression of Akt-DD in Schwann cells did not induce hypermyelination. Our results suggest that signaling activity other than Akt may also contribute to proper myelination in peripheral nerves.

Encompassing receptor flexibility in virtual screening using ensemble docking-based hybrid QSAR: discovery of novel phytochemicals for BACE1 inhibition.

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Chakraborty, Sandipan; Ramachandran, Balaji; Basu, Soumalee

2014-10-01

Mimicking receptor flexibility during receptor-ligand binding is a challenging task in computational drug design since it is associated with a large increase in the conformational search space. In the present study, we have devised an in silico design strategy incorporating receptor flexibility in virtual screening to identify potential lead compounds as inhibitors for flexible proteins. We have considered BACE1 (β-secretase), a key target protease from a therapeutic perspective for Alzheimer's disease, as the highly flexible receptor. The protein undergoes significant conformational transitions from open to closed form upon ligand binding, which makes it a difficult target for inhibitor design. We have designed a hybrid structure-activity model containing both ligand based descriptors and energetic descriptors obtained from molecular docking based on a dataset of structurally diverse BACE1 inhibitors. An ensemble of receptor conformations have been used in the docking study, further improving the prediction ability of the model. The designed model that shows significant prediction ability judged by several statistical parameters has been used to screen an in house developed 3-D structural library of 731 phytochemicals. 24 highly potent, novel BACE1 inhibitors with predicted activity (Ki) ≤ 50 nM have been identified. Detailed analysis reveals pharmacophoric features of these novel inhibitors required to inhibit BACE1.

The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation.

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Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

2016-07-22

The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation*

Science.gov (United States)

Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

2016-01-01

The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. PMID:27302062

Neuronal human BACE1 knockin induces systemic diabetes in mice.

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Plucińska, Kaja; Dekeryte, Ruta; Koss, David; Shearer, Kirsty; Mody, Nimesh; Whitfield, Phillip D; Doherty, Mary K; Mingarelli, Marco; Welch, Andy; Riedel, Gernot; Delibegovic, Mirela; Platt, Bettina

2016-07-01

β-Secretase 1 (BACE1) is a key enzyme in Alzheimer's disease pathogenesis that catalyses the amyloidogenic cleavage of amyloid precursor protein (APP). Recently, global Bace1 deletion was shown to protect against diet-induced obesity and diabetes, suggesting that BACE1 is a potential regulator of glucose homeostasis. Here, we investigated whether increased neuronal BACE1 is sufficient to alter systemic glucose metabolism, using a neuron-specific human BACE1 knockin mouse model (PLB4). Glucose homeostasis and adiposity were determined by glucose tolerance tests and EchoMRI, lipid species were measured by quantitative lipidomics, and biochemical and molecular alterations were assessed by western blotting, quantitative PCR and ELISAs. Glucose uptake in the brain and upper body was measured via (18)FDG-PET imaging. Physiological and molecular analyses demonstrated that centrally expressed human BACE1 induced systemic glucose intolerance in mice from 4 months of age onward, alongside a fatty liver phenotype and impaired hepatic glycogen storage. This diabetic phenotype was associated with hypothalamic pathology, i.e. deregulation of the melanocortin system, and advanced endoplasmic reticulum (ER) stress indicated by elevated central C/EBP homologous protein (CHOP) signalling and hyperphosphorylation of its regulator eukaryotic translation initiation factor 2α (eIF2α). In vivo (18)FDG-PET imaging further confirmed brain glucose hypometabolism in these mice; this corresponded with altered neuronal insulin-related signalling, enhanced protein tyrosine phosphatase 1B (PTP1B) and retinol-binding protein 4 (RBP4) levels, along with upregulation of the ribosomal protein and lipid translation machinery. Increased forebrain and plasma lipid accumulation (i.e. ceramides, triacylglycerols, phospholipids) was identified via lipidomics analysis. Our data reveal that neuronal BACE1 is a key regulator of metabolic homeostasis and provide a potential mechanism for the high

The Alzheimer's β-secretase enzyme BACE1 is required for accurate axon guidance of olfactory sensory neurons and normal glomerulus formation in the olfactory bulb

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Rajapaksha Tharinda W

2011-12-01

Full Text Available Abstract Background The β-secretase, β-site amyloid precursor protein cleaving enzyme 1 (BACE1, is a prime therapeutic target for lowering cerebral β-amyloid (Aβ levels in Alzheimer's disease (AD. Clinical development of BACE1 inhibitors is being intensely pursued. However, little is known about the physiological functions of BACE1, and the possibility exists that BACE1 inhibition may cause mechanism-based side effects. Indeed, BACE1-/- mice exhibit a complex neurological phenotype. Interestingly, BACE1 co-localizes with presynaptic neuronal markers, indicating a role in axons and/or terminals. Moreover, recent studies suggest axon guidance molecules are potential BACE1 substrates. Here, we used a genetic approach to investigate the function of BACE1 in axon guidance of olfactory sensory neurons (OSNs, a well-studied model of axon targeting in vivo. Results We bred BACE1-/- mice with gene-targeted mice in which GFP is expressed from the loci of two odorant-receptors (ORs, MOR23 and M72, and olfactory marker protein (OMP to produce offspring that were heterozygous for MOR23-GFP, M72-GFP, or OMP-GFP and were either BACE1+/+ or BACE1-/-. BACE1-/- mice had olfactory bulbs (OBs that were smaller and weighed less than OBs of BACE1+/+ mice. In wild-type mice, BACE1 was present in OSN axon terminals in OB glomeruli. In whole-mount preparations and tissue sections, many OB glomeruli from OMP-GFP; BACE1-/- mice were malformed compared to wild-type glomeruli. MOR23-GFP; BACE1-/- mice had an irregular MOR23 glomerulus that was innervated by randomly oriented, poorly fasciculated OSN axons compared to BACE1+/+ mice. Most importantly, M72-GFP; BACE1-/- mice exhibited M72 OSN axons that were mis-targeted to ectopic glomeruli, indicating impaired axon guidance in BACE1-/- mice. Conclusions Our results demonstrate that BACE1 is required for the accurate targeting of OSN axons and the proper formation of glomeruli in the OB, suggesting a role for BACE1 in

Oleic acid and linoleic acid from Tenebrio molitor larvae inhibit BACE1 activity in vitro: molecular docking studies.

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Youn, Kumju; Yun, Eun-Young; Lee, Jinhyuk; Kim, Ji-Young; Hwang, Jae-Sam; Jeong, Woo-Sik; Jun, Mira

2014-02-01

In our ongoing research to find therapeutic compounds for Alzheimer's disease (AD) from natural resources, the inhibitory activity of the BACE1 enzyme by Tenebrio molitor larvae and its major compounds were evaluated. The T. molitor larvae extract and its fractions exhibited strong BACE1 suppression. The major components of hexane fraction possessing both high yield and strong BACE1 inhibition were determined by thin layer chromatography, gas chromatography, and nuclear magnetic resonance analysis. A remarkable composition of unsaturated long chain fatty acids, including oleic acid and linoleic acid, were identified. Oleic acid, in particular, noncompetitively attenuated BACE1 activity with a half-maximal inhibitory concentration (IC₅₀) value of 61.31 μM and Ki value of 34.3 μM. Furthermore, the fatty acids were stably interacted with BACE1 at different allosteric sites of the enzyme bound with the OH of CYS319 and the NH₃ of TYR320 for oleic acid and with the C=O group of GLN304 for linoleic acid. Here, we first revealed novel pharmacophore features of oleic acids and linoleic acid to BACE1 by in silico docking studies. The present findings would clearly suggest potential guidelines for designing novel BACE1 selective inhibitors.

Ubiquitin regulates GGA3-mediated degradation of BACE1.

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Kang, Eugene L; Cameron, Andrew N; Piazza, Fabrizio; Walker, Kendall R; Tesco, Giuseppina

2010-07-30

BACE1 (beta-site amyloid precursor protein-cleaving enzyme 1) is a membrane-tethered member of the aspartyl proteases, essential for the production of beta-amyloid, a toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. The BACE1 C-terminal fragment contains a DXXLL motif that has been shown to bind the VHS (VPS27, Hrs, and STAM) domain of GGA1-3 (Golgi-localized gamma-ear-containing ARF-binding proteins). GGAs are trafficking molecules involved in the transport of proteins containing the DXXLL signal from the Golgi complex to endosomes. Moreover, GGAs bind ubiquitin and traffic synthetic and endosomal ubiquitinated cargoes to lysosomes. We have previously shown that depletion of GGA3 results in increased BACE1 levels and activity because of impaired lysosomal degradation. Here, we report that the accumulation of BACE1 is rescued by the ectopic expression of GGA3 in H4 neuroglioma cells depleted of GGA3. Accordingly, the overexpression of GGA3 reduces the levels of BACE1 and beta-amyloid. We then established that mutations in the GGA3 VPS27, Hrs, and STAM domain (N91A) or in BACE1 di-leucine motif (L499A/L500A), able to abrogate their binding, did not affect the ability of ectopically expressed GGA3 to rescue BACE1 accumulation in cells depleted of GGA3. Instead, we found that BACE1 is ubiquitinated at lysine 501 and is mainly monoubiquitinated and Lys-63-linked polyubiquitinated. Finally, a GGA3 mutant with reduced ability to bind ubiquitin (GGA3L276A) was unable to regulate BACE1 levels both in rescue and overexpression experiments. These findings indicate that levels of GGA3 tightly and inversely regulate BACE1 levels via interaction with ubiquitin sorting machinery.

Ubiquitin Regulates GGA3-mediated Degradation of BACE1*

Science.gov (United States)

Kang, Eugene L.; Cameron, Andrew N.; Piazza, Fabrizio; Walker, Kendall R.; Tesco, Giuseppina

2010-01-01

BACE1 (β-site amyloid precursor protein-cleaving enzyme 1) is a membrane-tethered member of the aspartyl proteases, essential for the production of β-amyloid, a toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. The BACE1 C-terminal fragment contains a DXXLL motif that has been shown to bind the VHS (VPS27, Hrs, and STAM) domain of GGA1–3 (Golgi-localized γ-ear-containing ARF-binding proteins). GGAs are trafficking molecules involved in the transport of proteins containing the DXXLL signal from the Golgi complex to endosomes. Moreover, GGAs bind ubiquitin and traffic synthetic and endosomal ubiquitinated cargoes to lysosomes. We have previously shown that depletion of GGA3 results in increased BACE1 levels and activity because of impaired lysosomal degradation. Here, we report that the accumulation of BACE1 is rescued by the ectopic expression of GGA3 in H4 neuroglioma cells depleted of GGA3. Accordingly, the overexpression of GGA3 reduces the levels of BACE1 and β-amyloid. We then established that mutations in the GGA3 VPS27, Hrs, and STAM domain (N91A) or in BACE1 di-leucine motif (L499A/L500A), able to abrogate their binding, did not affect the ability of ectopically expressed GGA3 to rescue BACE1 accumulation in cells depleted of GGA3. Instead, we found that BACE1 is ubiquitinated at lysine 501 and is mainly monoubiquitinated and Lys-63-linked polyubiquitinated. Finally, a GGA3 mutant with reduced ability to bind ubiquitin (GGA3L276A) was unable to regulate BACE1 levels both in rescue and overexpression experiments. These findings indicate that levels of GGA3 tightly and inversely regulate BACE1 levels via interaction with ubiquitin sorting machinery. PMID:20484053

BACE1-Dependent Neuregulin-1 Signaling: An Implication for Schizophrenia

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Zhengrong Zhang

2017-09-01

Full Text Available Schizophrenia is a chronic psychiatric disorder with a lifetime prevalence of about 1% in the general population. Recent studies have shown that Neuregulin-1 (Nrg1 is a candidate gene for schizophrenia. At least 15 alternative splicing of NRG1 isoforms all contain an extracellular epidermal growth factor (EGF-like domain, which is sufficient for Nrg1 biological activity including the formation of myelin sheaths and the regulation of synaptic plasticity. It is known that Nrg1 can be cleaved by β-secretase (BACE1 and the resulting N-terminal fragment (Nrg1-ntf binds to receptor tyrosine kinase ErbB4, which activates Nrg1/ErbB4 signaling. While changes in Nrg1 expression levels in schizophrenia still remain controversial, understanding the BACE1-cleaved Nrg1-ntf and Nrg1/ErbB4 signaling in schizophrenia neuropathogenesis is essential and important. In this review paper, we included three major parts: (1 Nrg1 structure and cleavage pattern by BACE1; (2 BACE1-dependent Nrg1 cleavage associated with schizophrenia in human studies; and (3 Animal studies of Nrg1 and BACE1 mutations with behavioral observations. Our review will provide a better understanding of Nrg1 in schizophrenia and a potential strategy for using BACE1 cleavage of Nrg1 as a unique biomarker for diagnosis, as well as a new therapeutic target, of schizophrenia.

Depletion of GGA1 and GGA3 mediates postinjury elevation of BACE1.

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Walker, Kendall R; Kang, Eugene L; Whalen, Michael J; Shen, Yong; Tesco, Giuseppina

2012-07-25

Traumatic brain injury (TBI) is one of the most robust environmental risk factors for Alzheimer's disease (AD). Compelling evidence is accumulating that a single event of TBI is associated with increased levels of Aβ. However, the underlying molecular mechanisms remain unknown. We report here that the BACE1 interacting protein, GGA3, is depleted while BACE1 levels increase in the acute phase after injury (48 h) in a mouse model of TBI. We further demonstrated the role of GGA3 in the regulation of BACE1 in vivo by showing that BACE1 levels are increased in the brain of GGA3-null mice. We next found that head trauma potentiates BACE1 elevation in GGA3-null mice in the acute phase after TBI, and discovered that GGA1, a GGA3 homolog, is a novel caspase-3 substrate depleted at 48 h after TBI. Moreover, GGA1 silencing potentiates BACE1 elevation induced by GGA3 deletion in neurons in vitro, indicating that GGA1 and GGA3 synergistically regulate BACE1. Accordingly, we found that levels of both GGA1 and GGA3 are depleted while BACE1 levels are increased in a series of postmortem AD brains. Finally, we show that GGA3 haploinsufficiency results in sustained elevation of BACE1 and Aβ levels while GGA1 levels are restored in the subacute phase (7 d) after injury. In conclusion, our data indicate that depletion of GGA1 and GGA3 engender a rapid and robust elevation of BACE1 in the acute phase after injury. However, the efficient disposal of the acutely accumulated BACE1 solely depends on GGA3 levels in the subacute phase of injury.

Gadd153 and NF-κB crosstalk regulates 27-hydroxycholesterol-induced increase in BACE1 and β-amyloid production in human neuroblastoma SH-SY5Y cells.

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Gurdeep Marwarha

Full Text Available β-amyloid (Aβ peptide, accumulation of which is a culprit for Alzheimer's disease (AD, is derived from the initial cleavage of amyloid precursor protein by the aspartyl protease BACE1. Identification of cellular mechanisms that regulate BACE1 production is of high relevance to the search for potential disease-modifying therapies that inhibit BACE1 to reduce Aβ accumulation and AD progression. In the present study, we show that the cholesterol oxidation product 27-hydroxycholesterol (27-OHC increases BACE1 and Aβ levels in human neuroblastoma SH-SY5Y cells. This increase in BACE1 involves a crosstalk between the two transcription factors NF-κB and the endoplasmic reticulum stress marker, the growth arrest and DNA damage induced gene-153 (gadd153, also called CHOP. We specifically show that 27-OHC induces a substantial increase in NF-κB binding to the BACE1 promoter and subsequent increase in BACE1 transcription and Aβ production. The NF-κB inhibitor, sc514, significantly attenuated the 27-OHC-induced increase in NF-κB-mediated BACE1 expression and Aβ genesis. We further show that the 27-OHC-induced NF-κB activation and increased NF-κB-mediated BACE1 expression is contingent on the increased activation of gadd153. Silencing gadd153 expression with siRNA alleviated the 27-OHC-induced increase in NF-κB activation, NF-κB binding to the BACE1 promoter, and subsequent increase in BACE1 transcription and Aβ production. We also show that increased levels of BACE1 in the triple transgenic mouse model for AD is preceded by gadd153 and NF-κB activation. In summary, our study demonstrates that gadd153 and NF-κB work in concert to regulate BACE1 expression. Agents that inhibit gadd153 activation and subsequent interaction with NF-κB might be promising targets to reduce BACE1 and Aβ overproduction and may ultimately serve as disease-modifying treatments for AD.

Population PKPD modeling of BACE1 inhibitor-induced reduction in Aβ levels in vivo and correlation to in vitro potency in primary cortical neurons from mouse and guinea pig.

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Janson, Juliette; Eketjäll, Susanna; Tunblad, Karin; Jeppsson, Fredrik; Von Berg, Stefan; Niva, Camilla; Radesäter, Ann-Cathrin; Fälting, Johanna; Visser, Sandra A G

2014-03-01

The aims were to quantify the in vivo time-course between the oral dose, the plasma and brain exposure and the inhibitory effect on Amyloid β (Aβ) in brain and cerebrospinal fluid, and to establish the correlation between in vitro and in vivo potency of novel β-secretase (BACE1) inhibitors. BACE1-mediated inhibition of Aβ was quantified in in vivo dose- and/or time-response studies and in vitro in SH-SY5Y cells, N2A cells, and primary cortical neurons (PCN). An indirect response model with inhibition on Aβ production rate was used to estimate unbound in vivo IC 50 in a population pharmacokinetic-pharmacodynamic modeling approach. Estimated in vivo inhibitory potencies varied between 1 and 1,000 nM. The turnover half-life of Aβ40 in brain was predicted to be 0.5 h in mouse and 1 h in guinea pig. An excellent correlation between PCN and in vivo potency was observed. Moreover, a strong correlation in potency was found between human SH-SY5Y cells and mouse PCN, being 4.5-fold larger in SH-SY5Y cells. The strong in vivo-in vitro correlation increased the confidence in using human cell lines for screening and optimization of BACE1 inhibitors. This can optimize the design and reduce the number of preclinical in vivo effect studies.

Depletion of GGA1 and GGA3 mediates post-injury elevation of BACE1

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Walker, Kendall R.; Kang, Eugene L.; Whalen, Michael J.; Shen, Yong; Tesco, Giuseppina

2012-01-01

Traumatic brain injury (TBI) is one of the most robust environmental risk factors for Alzheimer’s disease (AD). Compelling evidence is accumulating that a single event of TBI is associated with increased levels of Aβ. However, the underlying molecular mechanisms remain unknown. We report here that the BACE1 interacting protein, GGA3, is depleted while BACE1 levels increase in the acute phase post-injury (48hrs) in a mouse model of TBI. We further demonstrated the role of GGA3 in the regulation of BACE1 in vivo by showing that BACE1 levels are increased in the brain of GGA3 null mice. We next found that head trauma potentiates BACE1 elevation in GGA3 null mice in the acute phase post-TBI and discovered that GGA1, a GGA3 homologue, is a novel caspase-3 substrate depleted at 48 hrs post-TBI. Moreover, GGA1 silencing potentiates BACE1 elevation induced by GGA3 deletion in neurons in vitro indicating that GGA1 and GGA3 synergistically regulate BACE1. Accordingly, we found that levels of both GGA1 and GGA3 are depleted while BACE1 levels are increased in a series of post-mortem AD brains. Finally, we show that GGA3 haploinsufficiency results in sustained elevation of BACE1 and Aβ levels while GGA1 levels are restored in the subacute phase (7 days) post-injury. In conclusion, our data indicate that depletion of GGA1 and GGA3 engender a rapid and robust elevation of BACE1 in the acute phase post-injury. However, the efficient disposal of the acutely accumulated BACE1 solely depends on GGA3 levels in the sub-acute phase of injury. PMID:22836275

Linking vascular disorders and Alzheimer’s disease: Potential involvement of BACE1

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Cole, Sarah L.; Vassar, Robert

2012-01-01

The etiology of Alzheimer’s disease (AD) remains unknown. However, specific risk factors have been identified, and aging is the strongest AD risk factor. The majority of cardiovascular events occur in older people and a close relationship between vascular disorders and AD exists. Amyloid plaques, composed of the beta amyloid peptide (Aβ), are hallmark lesions in AD and evidence indicates that Aβ plays a central role in AD pathophysiology. The BACE1 enzyme is essential for Aβ generation, and BACE1 levels are elevated in AD brain. The cause(s) of this BACE1 elevation remains undetermined. Here we review the potential contribution of vascular disease to AD pathogenesis. We examine the putative vasoactive properties of Aβ and how the cellular changes associated with vascular disease may elevate BACE1 levels. Despite increasing evidence, the exact role(s) vascular disorders play in AD remains to be determined. However, given that vascular diseases can be addressed by lifestyle and pharmacologic interventions, the potential benefits of these therapies in delaying the clinical appearance and progression of AD may warrant investigation. PMID:18289733

Differential regulation of BACE1 expression by oxidative and nitrosative signals

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Xu Huaxi

2011-03-01

Full Text Available Abstract Background It is well established that both cerebral hypoperfusion/stroke and type 2 diabetes are risk factors for Alzheimer's disease (AD. Recently, the molecular link between ischemia/hypoxia and amyloid precursor protein (APP processing has begun to be established. However, the role of the key common denominator, namely nitric oxide (NO, in AD is largely unknown. In this study, we investigated redox regulation of BACE1, the rate-limiting enzyme responsible for the β-cleavage of APP to Aβ peptides. Results Herein, we studied events such as S-nitrosylation, a covalent modification of cysteine residues by NO, and H2O2-mediated oxidation. We found that NO and H2O2 differentially modulate BACE1 expression and enzymatic activity: NO at low concentrations (2O2 (1-10 μM induces BACE1 expression via transcriptional activation, resulting in increased enzymatic activity. The differential effects of NO and H2O2 on BACE1 expression and activity are also reflected in their opposing effects on Aβ generation in cultured neurons in a dose-dependent manner. Furthermore, we found that BACE1 is highly S-nitrosylated in normal aging brains while S-nitrosylation is markedly reduced in AD brains. Conclusion This study demonstrates for the first time that BACE1 is highly modified by NO via multiple mechanisms: low and high levels of NO suppress BACE1 via transcriptional and post translational regulation, in contrast with the upregulation of BACE1 by H2O2-mediated oxidation. These novel NO-mediated regulatory mechanisms likely protect BACE1 from being further oxidized by excessive oxidative stress, as from H2O2 and peroxynitrite which are known to upregulate BACE1 and activate the enzyme, resulting in excessive cleavage of APP and Aβ generation; they likely represent the crucial house-keeping mechanism for BACE1 expression/activation under physiological conditions.

Thermodynamic and kinetic characterization of hydroxyethylamine β-secretase-1 inhibitors

Energy Technology Data Exchange (ETDEWEB)

Mondal, Kalyani; Regnstrom, Karin; Morishige, Winse; Barbour, Robin; Probst, Gary; Xu, Ying-Zi; Artis, Dean R.; Yao, Nanhua; Beroza, Paul; Bova, Michael P., E-mail: mpbova2001@yahoo.com

2013-11-15

Highlights: •Kinetic and thermodynamic characterization of 10 hydroxyethylamine BACE-1 inhibitors. •Equilibrium binding of inhibitors was enthalpy driven for BACE-1. •Negative entropy of binding was observed towards BACE-1, but not Cathepsin-D. •Structural analysis demonstrates ligand binding induces a major conformational change. •Structural analysis and SPR analysis corroborate induced fit and negative entropy of binding. -- Abstract: Alzheimer’s disease (AD) is a devastating neurodegenerative disease affecting millions of people. β-Secretase-1 (BACE-1), an enzyme involved in the processing of the amyloid precursor protein (APP) to form Aβ, is a well validated target for AD. Herein, the authors characterize 10 randomly selected hydroxyethylamine (HEA) BACE-1 inhibitors in terms of their association and dissociation rate constants and thermodynamics of binding using surface plasmon resonance (SPR). Rate constants of association (k{sub a}) measured at 25 °C ranged from a low of 2.42 × 10{sup 4} M{sup −1} s{sup −1} to the highest value of 8.3 × 10{sup 5} M{sup −1} s{sup −1}. Rate constants of dissociation (k{sub d}) ranged from 1.09 × 10{sup −4} s{sup −1} (corresponding to a residence time of close to three hours), to the fastest of 0.028 s{sup −1}. Three compounds were selected for further thermodynamic analysis where it was shown that equilibrium binding was enthalpy driven while unfavorable entropy of binding was observed. Structural analysis revealed that upon ligand binding, the BACE-1flap folds down over the bound ligand causing an induced fit. The maximal difference between alpha carbon positions in the open and closed conformations of the flap was over 5 Å. Thus the negative entropy of binding determined using SPR analysis was consistent with an induced fit observed by structural analysis.

BACE1 Deficiency Causes Abnormal Neuronal Clustering in the Dentate Gyrus

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Hailong Hou

2017-07-01

Full Text Available BACE1 is validated as Alzheimer's β-secretase and a therapeutic target for Alzheimer's disease. In examining BACE1-null mice, we discovered that BACE1 deficiency develops abnormal clusters of immature neurons, forming doublecortin-positive neuroblasts, in the developing dentate gyrus, mainly in the subpial zone (SPZ. Such clusters were rarely observed in wild-type SPZ and not reported in other mouse models. To understand their origins and fates, we examined how neuroblasts in BACE1-null SPZ mature and migrate during early postnatal development. We show that such neuroblasts are destined to form Prox1-positive granule cells in the dentate granule cell layer, and mainly mature to form excitatory neurons, but not inhibitory neurons. Mechanistically, higher levels of reelin potentially contribute to abnormal neurogenesis and timely migration in BACE1-null SPZ. Altogether, we demonstrate that BACE1 is a critical regulator in forming the dentate granule cell layer through timely maturation and migration of SPZ neuroblasts.

Increased CSF-BACE1 activity associated with decreased hippocampus volume in Alzheimer's disease.

LENUS (Irish Health Repository)

Ewers, Michael

2012-02-01

The enzyme beta-secretase (BACE1) is essentially involved in the production of cerebral amyloidogenic pathology in Alzheimer\\'s disease (AD). The measurement of BACE1 activity in cerebrospinal fluid (CSF) has been reported, which may render CSF measurement of BACE1 a potential biomarker candidate of AD. In order to investigate whether BACE1 protein activity is correlated with regional brain atrophy in AD, we investigated the association between CSF levels of BACE1 and MRI-assessed hippocampus volume in patients with AD (n = 30). An increase in CSF-BACE1 activity was associated with decreased left and right hippocampus volume corrected for global head volume in the AD patients. Boot-strapped regression analysis showed that increased CSF levels of BACE1 activity were associated with increased CSF concentration of total tau but not amyloid-beta1-42 in AD. White matter hyperintensities did not influence the results. BACE1 activity and protein levels were significantly increased in AD compared to 19 elderly healthy controls. Thus, the CSF biomarker candidate of BACE1 activity was associated with hippocampus atrophy in AD in a robust manner and may reflect neurotoxic amyloid-beta-related processes.

GGA1 regulates signal-dependent sorting of BACE1 to recycling endosomes, which moderates Aβ production

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Toh, Wei Hong; Chia, Pei Zhi Cheryl; Hossain, Mohammed Iqbal; Gleeson, Paul A.

2018-01-01

The diversion of the membrane-bound β-site amyloid precursor protein–(APP) cleaving enzyme (BACE1) from the endolysosomal pathway to recycling endosomes represents an important transport step in the regulation of amyloid beta (Aβ) production. However, the mechanisms that regulate endosome sorting of BACE1 are poorly understood. Here we assessed the transport of BACE1 from early to recycling endosomes and have identified essential roles for the sorting nexin 4 (SNX4)-mediated, signal-independent pathway and for a novel signal-mediated pathway. The signal-mediated pathway is regulated by the phosphorylation of the DXXLL-motif sequence DISLL in the cytoplasmic tail of BACE1. The phosphomimetic S498D BACE1 mutant was trafficked to recycling endosomes at a faster rate compared with wild-type BACE1 or the nonphosphorylatable S498A mutant. The rapid transit of BACE1 S498D from early endosomes was coupled with reduced levels of amyloid precursor protein processing and Aβ production, compared with the S498A mutant. We show that the adaptor, GGA1, and retromer are essential to mediate rapid trafficking of phosphorylated BACE1 to recycling endosomes. In addition, the BACE1 DISLL motif is phosphorylated and regulates endosomal trafficking, in primary neurons. Therefore, post-translational phosphorylation of DISLL enhances the exit of BACE1 from early endosomes, a pathway mediated by GGA1 and retromer, which is important in regulating Aβ production. PMID:29142073

BACE is degraded via the lysosomal pathway.

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Koh, Young Ho; von Arnim, Christine A F; Hyman, Bradley T; Tanzi, Rudolph E; Tesco, Giuseppina

2005-09-16

Amyloid plaques are formed by aggregates of amyloid-beta-peptide, a 37-43-amino acid fragment (primarily Abeta(40) and Abeta(42)) generated by proteolytic processing of the amyloid precursor protein (APP) by beta- and gamma-secretases. A type I transmembrane aspartyl protease, BACE (beta-site APP cleaving enzyme), has been identified to be the beta-secretase. BACE is targeted through the secretory pathway to the plasma membrane where it can be internalized to endosomes. The carboxyl terminus of BACE contains a di-leucine-based signal for sorting of transmembrane proteins to endosomes and lysosomes. In this study, we set out to determine whether BACE is degraded by the lysosomal pathway and whether the di-leucine motif is necessary for targeting BACE to the lysosomes. Here we show that lysosomal inhibitors, chloroquine and NH(4)Cl, lead to accumulation of endogenous and ectopically expressed BACE in a variety of cell types, including primary neurons. Furthermore, the inhibition of lysosomal hydrolases results in the redistribution and accumulation of BACE in the late endosomal/lysosomal compartments (lysosome-associated membrane protein 2 (LAMP2)-positive). In contrast, the BACE-LL/AA mutant, in which Leu(499) and Leu(500) in the COOH-terminal sequence (DDISLLK) were replaced by alanines, only partially co-localized with LAMP2-positive compartments following inhibition of lysosomal hydrolases. Collectively, our data indicate that BACE is transported to the late endosomal/lysosomal compartments where it is degraded via the lysosomal pathway and that the di-leucine motif plays a role in sorting BACE to lysosomes.

β-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses.

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Hartmann, Stephanie; Zheng, Fang; Kyncl, Michele C; Karch, Sandra; Voelkl, Kerstin; Zott, Benedikt; D'Avanzo, Carla; Lomoio, Selene; Tesco, Giuseppina; Kim, Doo Y; Alzheimer, Christian; Huth, Tobias

2018-04-04

The β-secretase β-site APP-cleaving enzyme 1 (BACE1) is deemed a major culprit in Alzheimer's disease, but accumulating evidence indicates that there is more to the enzyme than driving the amyloidogenic processing of the amyloid precursor protein. For example, BACE1 has emerged as an important regulator of neuronal activity through proteolytic and, most unexpectedly, also through nonproteolytic interactions with several ion channels. Here, we identify and characterize the voltage-gated K + channel 3.4 (Kv3.4) as a new and functionally relevant interaction partner of BACE1. Kv3.4 gives rise to A-type current with fast activating and inactivating kinetics and serves to repolarize the presynaptic action potential. We found that BACE1 and Kv3.4 are highly enriched and remarkably colocalized in hippocampal mossy fibers (MFs). In BACE1 -/- mice of either sex, Kv3.4 surface expression was significantly reduced in the hippocampus and, in synaptic fractions thereof, Kv3.4 was specifically diminished, whereas protein levels of other presynaptic K + channels such as K Ca 1.1 and K Ca 2.3 remained unchanged. The apparent loss of presynaptic Kv3.4 affected the strength of excitatory transmission at the MF-CA3 synapse in hippocampal slices of BACE1 -/- mice when probed with the Kv3 channel blocker BDS-I. The effect of BACE1 on Kv3.4 expression and function should be bidirectional, as predicted from a heterologous expression system, in which BACE1 cotransfection produced a concomitant upregulation of Kv3.4 surface level and current based on a physical interaction between the two proteins. Our data show that, by targeting Kv3.4 to presynaptic sites, BACE1 endows the terminal with a powerful means to regulate the strength of transmitter release. SIGNIFICANCE STATEMENT The β-secretase β-site APP-cleaving enzyme 1 (BACE1) is infamous for its crucial role in the pathogenesis of Alzheimer's disease, but its physiological functions in the intact nervous system are only gradually

BACE inhibition-dependent repair of Alzheimer's pathophysiology.

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Keskin, Aylin D; Kekuš, Maja; Adelsberger, Helmuth; Neumann, Ulf; Shimshek, Derya R; Song, Beomjong; Zott, Benedikt; Peng, Tingying; Förstl, Hans; Staufenbiel, Matthias; Nelken, Israel; Sakmann, Bert; Konnerth, Arthur; Busche, Marc Aurel

2017-08-08

Amyloid-β (Aβ) is thought to play an essential pathogenic role in Alzheimer´s disease (AD). A key enzyme involved in the generation of Aβ is the β-secretase BACE, for which powerful inhibitors have been developed and are currently in use in human clinical trials. However, although BACE inhibition can reduce cerebral Aβ levels, whether it also can ameliorate neural circuit and memory impairments remains unclear. Using histochemistry, in vivo Ca 2+ imaging, and behavioral analyses in a mouse model of AD, we demonstrate that along with reducing prefibrillary Aβ surrounding plaques, the inhibition of BACE activity can rescue neuronal hyperactivity, impaired long-range circuit function, and memory defects. The functional neuronal impairments reappeared after infusion of soluble Aβ, mechanistically linking Aβ pathology to neuronal and cognitive dysfunction. These data highlight the potential benefits of BACE inhibition for the effective treatment of a wide range of AD-like pathophysiological and cognitive impairments.

Astrocytic expression of the Alzheimer's disease beta-secretase (BACE1) is stimulus-dependent

DEFF Research Database (Denmark)

Hartlage-Rübsamen, Maike; Zeitschel, Ulrike; Apelt, Jenny

2003-01-01

The beta-site APP-cleaving enzyme (BACE1) is a prerequisite for the generation of beta-amyloid peptides, which give rise to cerebrovascular and parenchymal beta-amyloid deposits in the brain of Alzheimer's disease patients. BACE1 is neuronally expressed in the brains of humans and experimental...... paradigms studied. In contrast, BACE1 expression by reactive astrocytes was evident in chronic but not in acute models of gliosis. Additionally, we observed BACE1-immunoreactive astrocytes in proximity to beta-amyloid plaques in the brains of aged Tg2576 mice and Alzheimer's disease patients....

BACE1 protein endocytosis and trafficking are differentially regulated by ubiquitination at lysine 501 and the Di-leucine motif in the carboxyl terminus.

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Kang, Eugene L; Biscaro, Barbara; Piazza, Fabrizio; Tesco, Giuseppina

2012-12-14

β-Site amyloid precursor protein-cleaving enzyme (BACE1) is a membrane-tethered member of the aspartyl proteases that has been identified as β-secretase. BACE1 is targeted through the secretory pathway to the plasma membrane and then is internalized to endosomes. Sorting of membrane proteins to the endosomes and lysosomes is regulated by the interaction of signals present in their carboxyl-terminal fragment with specific trafficking molecules. The BACE1 carboxyl-terminal fragment contains a di-leucine sorting signal ((495)DDISLL(500)) and a ubiquitination site at Lys-501. Here, we report that lack of ubiquitination at Lys-501 (BACE1K501R) does not affect the rate of endocytosis but produces BACE1 stabilization and accumulation of BACE1 in early and late endosomes/lysosomes as well as at the cell membrane. In contrast, the disruption of the di-leucine motif (BACE1LLAA) greatly impairs BACE1 endocytosis and produces a delayed retrograde transport of BACE1 to the trans-Golgi network (TGN) and a delayed delivery of BACE1 to the lysosomes, thus decreasing its degradation. Moreover, the combination of the lack of ubiquitination at Lys-501 and the disruption of the di-leucine motif (BACE1LLAA/KR) produces additive effects on BACE1 stabilization and defective internalization. Finally, BACE1LLAA/KR accumulates in the TGN, while its levels are decreased in EEA1-positive compartments indicating that both ubiquitination at Lys-501 and the di-leucine motif are necessary for the trafficking of BACE1 from the TGN to early endosomes. Our studies have elucidated a differential role for the di-leucine motif and ubiquitination at Lys-501 in BACE1 endocytosis, trafficking, and degradation and suggest the involvement of multiple adaptor molecules.

BACE1 Protein Endocytosis and Trafficking Are Differentially Regulated by Ubiquitination at Lysine 501 and the Di-leucine Motif in the Carboxyl Terminus*

Science.gov (United States)

Kang, Eugene L.; Biscaro, Barbara; Piazza, Fabrizio; Tesco, Giuseppina

2012-01-01

β-Site amyloid precursor protein-cleaving enzyme (BACE1) is a membrane-tethered member of the aspartyl proteases that has been identified as β-secretase. BACE1 is targeted through the secretory pathway to the plasma membrane and then is internalized to endosomes. Sorting of membrane proteins to the endosomes and lysosomes is regulated by the interaction of signals present in their carboxyl-terminal fragment with specific trafficking molecules. The BACE1 carboxyl-terminal fragment contains a di-leucine sorting signal (495DDISLL500) and a ubiquitination site at Lys-501. Here, we report that lack of ubiquitination at Lys-501 (BACE1K501R) does not affect the rate of endocytosis but produces BACE1 stabilization and accumulation of BACE1 in early and late endosomes/lysosomes as well as at the cell membrane. In contrast, the disruption of the di-leucine motif (BACE1LLAA) greatly impairs BACE1 endocytosis and produces a delayed retrograde transport of BACE1 to the trans-Golgi network (TGN) and a delayed delivery of BACE1 to the lysosomes, thus decreasing its degradation. Moreover, the combination of the lack of ubiquitination at Lys-501 and the disruption of the di-leucine motif (BACE1LLAA/KR) produces additive effects on BACE1 stabilization and defective internalization. Finally, BACE1LLAA/KR accumulates in the TGN, while its levels are decreased in EEA1-positive compartments indicating that both ubiquitination at Lys-501 and the di-leucine motif are necessary for the trafficking of BACE1 from the TGN to early endosomes. Our studies have elucidated a differential role for the di-leucine motif and ubiquitination at Lys-501 in BACE1 endocytosis, trafficking, and degradation and suggest the involvement of multiple adaptor molecules. PMID:23109336

Mechanistic insights into mode of action of potent natural antagonists of BACE-1 for checking Alzheimer’s plaque pathology

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Dhanjal, Jaspreet Kaur [School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067 (India); Goyal, Sukriti [Apaji Institute of Mathematics and Applied Computer Technology, Banasthali University, Tonk 304022, Rajasthan (India); Sharma, Sudhanshu [Department of Biotechnology, Delhi Technological University, New Delhi 110042 (India); Hamid, Rabia [Department of Biochemistry, University of Kashmir, Srinagar 190006 (India); Grover, Abhinav, E-mail: abhinavgr@gmail.com [School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067 (India)

2014-01-17

Highlights: •Accumulation of Aβ plaques is one of the major pathology associated with Alzheimer’s disease. •Inhibition of β-Secretase or BACE-1 offers a viable prospect to check the growth of these plaques. •A large virtual dataset of natural compounds was screened against BACE-1. •Top two hits were analyzed for thermodynamic and structural stability using MD simulations. •Their detailed binding mode of actions were elucidated. -- Abstract: Alzheimer’s is a neurodegenerative disorder resulting in memory loss and decline in cognitive abilities. Accumulation of extracellular beta amyloidal plaques is one of the major pathology associated with this disease. β-Secretase or BACE-1 performs the initial and rate limiting step of amyloidic pathway in which 37–43 amino acid long peptides are generated which aggregate to form plaques. Inhibition of this enzyme offers a viable prospect to check the growth of these plaques. Numerous efforts have been made in recent years for the generation of BACE-1 inhibitors but many of them failed during the preclinical or clinical trials due to drug related or drug induced toxicity. In the present work, we have used computational methods to screen a large dataset of natural compounds to search for small molecules having BACE-1 inhibitory activity with low toxicity to normal cells. Molecular dynamics simulations were performed to analyze molecular interactions between the screened compounds and the active residues of the enzyme. Herein, we report two natural compounds of inhibitory nature active against β-secretase enzyme of amyloidic pathway and are potent lead molecules against Alzheimer’s disease.

Mechanistic insights into mode of action of potent natural antagonists of BACE-1 for checking Alzheimer’s plaque pathology

International Nuclear Information System (INIS)

Dhanjal, Jaspreet Kaur; Goyal, Sukriti; Sharma, Sudhanshu; Hamid, Rabia; Grover, Abhinav

2014-01-01

Highlights: •Accumulation of Aβ plaques is one of the major pathology associated with Alzheimer’s disease. •Inhibition of β-Secretase or BACE-1 offers a viable prospect to check the growth of these plaques. •A large virtual dataset of natural compounds was screened against BACE-1. •Top two hits were analyzed for thermodynamic and structural stability using MD simulations. •Their detailed binding mode of actions were elucidated. -- Abstract: Alzheimer’s is a neurodegenerative disorder resulting in memory loss and decline in cognitive abilities. Accumulation of extracellular beta amyloidal plaques is one of the major pathology associated with this disease. β-Secretase or BACE-1 performs the initial and rate limiting step of amyloidic pathway in which 37–43 amino acid long peptides are generated which aggregate to form plaques. Inhibition of this enzyme offers a viable prospect to check the growth of these plaques. Numerous efforts have been made in recent years for the generation of BACE-1 inhibitors but many of them failed during the preclinical or clinical trials due to drug related or drug induced toxicity. In the present work, we have used computational methods to screen a large dataset of natural compounds to search for small molecules having BACE-1 inhibitory activity with low toxicity to normal cells. Molecular dynamics simulations were performed to analyze molecular interactions between the screened compounds and the active residues of the enzyme. Herein, we report two natural compounds of inhibitory nature active against β-secretase enzyme of amyloidic pathway and are potent lead molecules against Alzheimer’s disease

Plasma long non-coding RNA BACE1 as a novel biomarker for diagnosis of Alzheimer disease.

Science.gov (United States)

Feng, Liang; Liao, Yu-Ting; He, Jin-Cai; Xie, Cheng-Long; Chen, Si-Yan; Fan, Hui-Hui; Su, Zhi-Peng; Wang, Zhen

2018-01-09

Long non-coding RNA (LncRNA) have been reported to be involved in the pathogenesis of neurodegenerative diseases, but whether it can serve as a biomarker for Alzheimer disease (AD) is not yet known. The present study selected four specific LncRNA (17A, 51A, BACE1 and BC200) as possible AD biomarker. RT-qPCR was performed to validate the LncRNA. Receiver operating characteristic curve (ROC) and area under the ROC curve (AUC) were applied to study the potential of LncRNA as a biomarker in a population of 88 AD patients and 72 control individuals. We found that the plasma LncRNA BACE1 level of AD patients was significantly higher than that of healthy controls (p = 0.006). Plasma level of LncRNA 17A, 51A and BC200 did not show a significant difference between two groups (p = 0.098, p = 0.204 and p = 0.232, respectively). ROC curve analysis showed that LncRNA BACE1 was the best candidate of these LncRNA (95% CI: 0.553-0.781, p = 0.003). In addition, no correlation was found for expression of these LncRNA in both control and AD groups with age or MMSE scale (p > 0.05). Our present study compared the plasma level of four LncRNA between AD and non-AD patients, and found that the level of the BACE1 is increased in the plasma of AD patients and have a high specificity (88%) for AD, indicating BACE1 may be a potential candidate biomarker to predict AD.

β-secretase inhibitor; a promising novel therapeutic drug in AD

Directory of Open Access Journals (Sweden)

Kelly Willemijn Menting

2014-07-01

Full Text Available Alzheimer’s disease (AD and vascular dementia are responsible for up to 90% of dementia cases. According to the World Health Organization (WHO, a staggering number of 35.6 million people are currently diagnosed with dementia. Blocking disease progression or preventing AD altogether is desirable for both social and economic reasons and recently focus has shifted to a new and promising drug: the β-secretase inhibitor. Much of AD research has investigated the amyloid cascade hypothesis, which postulates that AD is caused by changes in amyloid beta (Aβ stability and aggregation. Blocking Aβ production by inhibiting the first protease required for its generation, β-secretase/BACE1, may be the next step in blocking AD progression. In April 2012, promising phase I data on inhibitor MK-8931 was presented. This drug reduced Aβ CSF levels up to 92% and was well tolerated by patients. In March 2013 data was added from a one week trial in 32 mild to moderate AD patients, showing CSF Aβ levels decreased up to 84%. However, BACE1 inhibitors require further research. First, greatly reducing Aβ levels through BACE1 inhibition may have harmful side effects. Second, BACE1 inhibitors have yet to pass clinical trial phase II/III and no data on possible side effects on AD patients are available. And third, there remains doubt about the clinical efficacy of BACE1 inhibitors. In moderate AD patients, Aβ plaques have already been formed. BACE1 inhibitors prevent production of new Aβ plaques, but hypothetically do not influence already existing Aβ peptides. Therefore, BACE1 inhibitors are potentially better at preventing AD instead of having therapeutic use.

Molecular Docking and Dynamic Simulation of AZD3293 and Solanezumab Effects Against BACE1 to Treat Alzheimer's Disease

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Mubashir Hassan

2018-06-01

Full Text Available The design of novel inhibitors to target BACE1 with reduced cytotoxicity effects is a promising approach to treat Alzheimer's disease (AD. Multiple clinical drugs and antibodies such as AZD3293 and Solanezumab are being tested to investigate their therapeutical potential against AD. The current study explores the binding pattern of AZD3293 and Solanezumab against their target proteins such as β-secretase (BACE1 and mid-region amyloid-beta (Aβ (PDBIDs: 2ZHV & 4XXD, respectively using molecular docking and dynamic simulation (MD approaches. The molecular docking results show that AZD3293 binds within the active region of BACE1 by forming hydrogen bonds against Asp32 and Lys107 with distances 2.95 and 2.68 Å, respectively. However, the heavy chain of Solanezumab interacts with Lys16 and Asp23 of amyloid beta having bond length 2.82, 2.78, and 3.00 Å, respectively. The dynamic cross correlations and normal mode analyses show that BACE1 depicted good residual correlated motions and fluctuations, as compared to Solanezumab. Using MD, the Root Mean Square Deviation and Fluctuation (RMSD/F graphs show that AZD3293 residual fluctuations and RMSD value (0.2 nm was much better compared to Solanezumab (0.7 nm. Moreover, the radius of gyration (Rg results also depicts the significance of AZD3293 docked complex compared to Solanezumab through residual compactness. Our comparative results show that AZD3293 is a better therapeutic agent for treating AD than Solanezumab.

β-Secretase BACE1 regulates hippocampal and reconstituted M-currents in a β-subunit-like fashion.

Science.gov (United States)

Hessler, Sabine; Zheng, Fang; Hartmann, Stephanie; Rittger, Andrea; Lehnert, Sandra; Völkel, Meike; Nissen, Matthias; Edelmann, Elke; Saftig, Paul; Schwake, Michael; Huth, Tobias; Alzheimer, Christian

2015-02-25

The β-secretase BACE1 is widely known for its pivotal role in the amyloidogenic pathway leading to Alzheimer's disease, but how its action on transmembrane proteins other than the amyloid precursor protein affects the nervous system is only beginning to be understood. We report here that BACE1 regulates neuronal excitability through an unorthodox, nonenzymatic interaction with members of the KCNQ (Kv7) family that give rise to the M-current, a noninactivating potassium current with slow kinetics. In hippocampal neurons from BACE1(-/-) mice, loss of M-current enhanced neuronal excitability. We relate the diminished M-current to the previously reported epileptic phenotype of BACE1-deficient mice. In HEK293T cells, BACE1 amplified reconstituted M-currents, altered their voltage dependence, accelerated activation, and slowed deactivation. Biochemical evidence strongly suggested that BACE1 physically associates with channel proteins in a β-subunit-like fashion. Our results establish BACE1 as a physiologically essential constituent of regular M-current function and elucidate a striking new feature of how BACE1 impacts on neuronal activity in the intact and diseased brain. Copyright © 2015 the authors 0270-6474/15/353298-14$15.00/0.

Fluorescence Microspectroscopy for Testing the Dimerization Hypothesis of BACE1 Protein in Cultured HEK293 Cells

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Gardeen, Spencer; Johnson, Joseph L.; Heikal, Ahmed A.

2016-06-01

Alzheimer's Disease (AD) is a neurodegenerative disorder that results from the formation of beta-amyloid plaques in the brain that trigger the known symptoms of memory loss in AD patients. The beta-amyloid plaques are formed by the proteolytic cleavage of the amyloid precursor protein (APP) by the proteases BACE1 and gamma-secretase. These enzyme-facilitated cleavages lead to the production of beta-amyloid fragments that aggregate to form plaques, which ultimately lead to neuronal cell death. Recent detergent protein extraction studies suggest that BACE1 protein forms a dimer that has significantly higher catalytic activity than its monomeric counterpart. In this contribution, we examine the dimerization hypothesis of BACE1 in cultured HEK293 cells using complementary fluorescence spectroscopy and microscopy methods. Cells were transfected with a BACE1-EGFP fusion protein construct and imaged using confocal, and differential interference contrast to monitor the localization and distribution of intracellular BACE1. Complementary fluorescence lifetime and anisotropy measurements enabled us to examine the conformational and environmental changes of BACE1 as a function of substrate binding. Using fluorescence correlation spectroscopy, we also quantified the diffusion coefficient of BACE1-EGFP on the plasma membrane as a means to test the dimerization hypothesis as a fucntion of substrate-analog inhibitition. Our results represent an important first towards examining the substrate-mediated dimerization hypothesis of BACE1 in live cells.

Seamless integration of dose-response screening and flow chemistry: efficient generation of structure-activity relationship data of β-secretase (BACE1) inhibitors.

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Werner, Michael; Kuratli, Christoph; Martin, Rainer E; Hochstrasser, Remo; Wechsler, David; Enderle, Thilo; Alanine, Alexander I; Vogel, Horst

2014-02-03

Drug discovery is a multifaceted endeavor encompassing as its core element the generation of structure-activity relationship (SAR) data by repeated chemical synthesis and biological testing of tailored molecules. Herein, we report on the development of a flow-based biochemical assay and its seamless integration into a fully automated system comprising flow chemical synthesis, purification and in-line quantification of compound concentration. This novel synthesis-screening platform enables to obtain SAR data on b-secretase (BACE1) inhibitors at an unprecedented cycle time of only 1 h instead of several days. Full integration and automation of industrial processes have always led to productivity gains and cost reductions, and this work demonstrates how applying these concepts to SAR generation may lead to a more efficient drug discovery process. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Kinetics and molecular docking studies of loganin, morroniside and 7-O-galloyl-D-sedoheptulose derived from Corni fructus as cholinesterase and β-secretase 1 inhibitors.

Science.gov (United States)

Bhakta, Himanshu Kumar; Park, Chan Hum; Yokozawa, Takako; Min, Byung-Sun; Jung, Hyun Ah; Choi, Jae Sue

2016-06-01

We evaluated the major active components isolated from Corni Fructus: loganin, morroniside, and 7-O-galloyl-D-sedoheptulose as inhibitors of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) for use in Alzheimer's disease treatment. These compounds exhibited predominant cholinesterase (ChEs) inhibitory effects with IC50 values of 0.33, 3.95, and 10.50 ± 1.16 µM, respectively, for AChE, and 33.02, 37.78, and 87.94 ± 4.66 µM, respectively, for BChE. Kinetics studies revealed that loganin and 7-O-galloyl-D-sedoheptulose inhibited AChE with characteristics typical of mixed inhibitors, while morroniside was found to be a noncompetitive inhibitor against AChE and also exerted mixed BChE inhibitory activities. For BACE1, loganin showed noncompetitive type inhibitory effects, while morroniside and 7-O-galloyl-D-sedoheptulose were found to be mixed inhibitors. Furthermore, these compounds exhibited dose-dependent inhibitory activity with ONOO(-)-mediated protein tyrosine nitration. Molecular docking simulation of these compounds demonstrated negative binding energies for ChEs, and BACE1, indicating high affinity and tighter binding capacity for the active site of the enzyme. Loganin was the most potent inhibitor against both ChEs and BACE1. The data suggest that these compounds together can act as a triple inhibitor of AChE, BChE, and BACE1, providing a preventive and therapeutic strategy for Alzheimer's disease treatment.

A PM7 dynamic residue-ligand interactions energy landscape of the BACE1 inhibitory pathway by hydroxyethylamine compounds. Part I: The flap closure process.

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Gueto-Tettay, Carlos; Martinez-Consuegra, Alejandro; Zuchniarz, Joshua; Gueto-Tettay, Luis Roberto; Drosos-Ramírez, Juan Carlos

2017-09-01

BACE1 is an enzyme of scientific interest because it participates in the progression of Alzheimer's disease. Hydroxyethylamines (HEAs) are a family of compounds which exhibit inhibitory activity toward BACE1 at a nanomolar level, favorable pharmacokinetic properties and oral bioavailability. The first step in the inhibition of BACE1 by HEAs consists of their entrance into the protease active site and the resultant conformational change in the protein, from Apo to closed form. These two conformations differ in the position of an antiparallel loop (called the flap) which covers the entrance to the catalytic site. For BACE1, closure of this flap is vital to its catalytic activity and to inhibition of the enzyme due to the new interactions thereby formed with the ligand. In the present study a dynamic energy landscape of residue-ligand interaction energies (ReLIE) measured for 112 amino acids in the BACE1 active site and its immediate vicinity during the closure of the flap induced by 8 HEAs of different inhibitory power is presented. A total of 6.272 million ReLIE calculations, based on the PM7 semiempirical method, provided a deep and quantitative view of the first step in the inhibition of the aspartyl protease. The information suggests that residues Asp93, Asp289, Thr292, Thr293, Asn294 and Arg296 are anchor points for the ligand, accounting for approximately 45% of the total protein-ligand interaction. Additionally, flap closure improved the BACE1-HEA interaction by around 25%. Furthermore, the inhibitory activity of HEAs could be related to the capacity of these ligands to form said anchor point interactions and maintain them over time: the lack of some of these anchor interactions delayed flap closure or impeded it completely, or even caused the flap to reopen. The methodology employed here could be used as a tool to evaluate future structural modifications which lead to improvements in the favorability and stability of BACE1-HEA ReLIEs, aiding in the design of

Fetzima (levomilnacipran), a drug for major depressive disorder as a dual inhibitor for human serotonin transporters and beta-site amyloid precursor protein cleaving enzyme-1.

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Rizvi, Syed Mohd Danish; Shaikh, Sibhghatulla; Khan, Mahiuddin; Biswas, Deboshree; Hameed, Nida; Shakil, Shazi

2014-01-01

Pharmacological management of Major Depressive Disorder includes the use of serotonin reuptake inhibitors which targets serotonin transporters (SERT) to increase the synaptic concentrations of serotonin. Beta-site amyloid precursor protein cleaving enzyme-1 (BACE-1) is responsible for amyloid β plaque formation. Hence it is an interesting target for Alzheimer's disease (AD) therapy. This study describes molecular interactions of a new Food and Drug Administration approved antidepressant drug named 'Fetzima' with BACE-1 and SERT. Fetzima is chemically known as levomilnacipran. The study has explored a possible link between the treatment of Depression and AD. 'Autodock 4.2' was used for docking study. The free energy of binding (ΔG) values for 'levomilnacipran-SERT' interaction and 'levomilnacipran-BACE1' interaction were found to be -7.47 and -8.25 kcal/mol, respectively. Levomilnacipran was found to interact with S438, known to be the most important amino acid residue of serotonin binding site of SERT during 'levomilnacipran-SERT' interaction. In the case of 'levomilnacipran-BACE1' interaction, levomilnacipran interacted with two very crucial aspartic acid residues of BACE-1, namely, D32 and D228. These residues are accountable for the cleavage of amyloid precursor protein and the subsequent formation of amyloid β plaques in AD brain. Hence, Fetzima (levomilnacipran) might act as a potent dual inhibitor of SERT and BACE-1 and expected to form the basis of a future dual therapy against depression and AD. It is an established fact that development of AD is associated with Major Depressive Disorder. Therefore, the design of new BACE-1 inhibitors based on antidepressant drug scaffolds would be particularly beneficial.

Triazolyl tryptoline derivatives as β-secretase inhibitors

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Jiaranaikulwanitch, Jutamas; Boonyarat, Chantana; Fokin, Valery V.; Vajragupta, Opa

2010-01-01

Tryptoline, a core structure of ochrolifuanine E, which is a hit compound from virtual screening of the Thai herbal database against BACE1 was used as a scaffold for the design of BACE1 inhibitors. The tryptoline was linked with different side chains by 1,2,3-triazole ring readily synthesized by catalytic azide-alkyne cycloaddition reactions. Twenty two triazolyl tryptoline derivatives were synthesized and screened for the inhibitory action against BACE1. JJCA-140 was the most potent inhibito...

Dose-dependent inhibition of BACE-1 by the monoterpenoid 2,3,4,4-tetramethyl-5-methylenecyclopent-2-enone in cellular and mouse models of Alzheimer's disease.

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Videira, Rita; Castanheira, Pedro; Grãos, Mário; Resende, Rosa; Salgueiro, Lígia; Faro, Carlos; Cavaleiro, Carlos

2014-06-27

BACE-1 is an aspartic protease involved in the conversion of amyloid precursor protein (APP) to amyloid-β (Aβ) in vivo, which is one of the key steps in the development and progression of Alzheimer's disease. In a previous screening procedure for inhibitors of BACE-1 activity, the oil of Lavandula luisieri was identified as the most potent among several essential oils. The inhibitory effect of this essential oil on Aβ production was also demonstrated in a cellular assay. The composition of the volatile oil and the isolation of the compound responsible for the inhibitory activity were also reported. The present work focused on the characterization of the inhibition of BACE-1 by this active compound, a monoterpene necrodane ketone, 2,3,4,4-tetramethyl-5-methylenecyclopent-2-enone (1), with assessment of its Ki value and the type of inhibition. The dose-related effects of the compound were also evaluated using two different cell lines, with determinations of the respective EC50 values. The entire oil and the 2,3,4,4-tetramethyl-5-methylenecyclopent-2-enone (1) were tested on a triple transgenic mouse model of Alzheimer's disease. The overall results showed that compound 1 displayed a dose-dependent inhibition of BACE-1 in cellular and mouse models of Alzheimer's disease and is therefore capable of passing through cellular membranes and the blood-brain barrier.

BACE1 elevation engendered by GGA3 deletion increases β-amyloid pathology in association with APP elevation and decreased CHL1 processing in 5XFAD mice.

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Kim, WonHee; Ma, Liang; Lomoio, Selene; Willen, Rachel; Lombardo, Sylvia; Dong, Jinghui; Haydon, Philip G; Tesco, Giuseppina

2018-02-02

β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the rate-limiting enzyme in the production of amyloid beta (Aβ), the toxic peptide that accumulates in the brains of Alzheimer's disease (AD) patients. Our previous studies have shown that the clathrin adaptor Golgi-localized γ-ear-containing ARF binding protein 3 (GGA3) plays a key role in the trafficking of BACE1 to lysosomes, where it is normally degraded. GGA3 depletion results in BACE1 stabilization both in vitro and in vivo. Moreover, levels of GGA3 are reduced and inversely related to BACE1 levels in post-mortem brains of AD patients. In order to assess the effect of GGA3 deletion on AD-like phenotypes, we crossed GGA3 -/- mice with 5XFAD mice. BACE1-mediated processing of APP and the cell adhesion molecule L1 like protein (CHL1) was measured as well as levels of Aβ42 and amyloid burden. In 5XFAD mice, we found that hippocampal and cortical levels of GGA3 decreased while BACE1 levels increased with age, similar to what is observed in human AD brains. GGA3 deletion prevented age-dependent elevation of BACE1 in GGA3KO;5XFAD mice. We also found that GGA3 deletion resulted in increased hippocampal levels of Aβ42 and amyloid burden in 5XFAD mice at 12 months of age. While levels of BACE1 did not change with age and gender in GGAKO;5XFAD mice, amyloid precursor protein (APP) levels increased with age and were higher in female mice. Moreover, elevation of APP was associated with a decreased BACE1-mediated processing of CHL1 not only in 12 months old 5XFAD mice but also in human brains from subjects affected by Down syndrome, most likely due to substrate competition. This study demonstrates that GGA3 depletion is a leading candidate mechanism underlying elevation of BACE1 in AD. Furthermore, our findings suggest that BACE1 inhibition could exacerbate mechanism-based side effects in conditions associated with APP elevation (e.g. Down syndrome) owing to impairment of BACE1-mediated processing of CHL1

Discovery and X-ray crystallographic analysis of a spiropiperidine iminohydantoin inhibitor of beta-secretase.

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Barrow, James C; Stauffer, Shaun R; Rittle, Kenneth E; Ngo, Phung L; Yang, ZhiQiang; Selnick, Harold G; Graham, Samuel L; Munshi, Sanjeev; McGaughey, Georgia B; Holloway, M Katharine; Simon, Adam J; Price, Eric A; Sankaranarayanan, Sethu; Colussi, Dennis; Tugusheva, Katherine; Lai, Ming-Tain; Espeseth, Amy S; Xu, Min; Huang, Qian; Wolfe, Abigail; Pietrak, Beth; Zuck, Paul; Levorse, Dorothy A; Hazuda, Daria; Vacca, Joseph P

2008-10-23

A high-throughput screen at 100 microM inhibitor concentration for the BACE-1 enzyme revealed a novel spiropiperidine iminohydantoin aspartyl protease inhibitor template. An X-ray cocrystal structure with BACE-1 revealed a novel mode of binding whereby the inhibitor interacts with the catalytic aspartates via bridging water molecules. Using the crystal structure as a guide, potent compounds with good brain penetration were designed.

VPS35 regulates developing mouse hippocampal neuronal morphogenesis by promoting retrograde trafficking of BACE1

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Chun-Lei Wang

2012-10-01

VPS35, a major component of the retromer, plays an important role in the selective endosome-to-Golgi retrieval of membrane proteins. Dysfunction of retromer is a risk factor for neurodegenerative disorders, but its function in developing mouse brain remains poorly understood. Here we provide evidence for VPS35 promoting dendritic growth and maturation, and axonal protein transport in developing mouse hippocampal neurons. Embryonic hippocampal CA1 neurons suppressing Vps35 expression by in utero electroporation of its micro RNAs displayed shortened apical dendrites, reduced dendritic spines, and swollen commissural axons in the neonatal stage, those deficits reflecting a defective protein transport/trafficking in developing mouse neurons. Further mechanistic studies showed that Vps35 depletion in neurons resulted in an impaired retrograde trafficking of BACE1 (β1-secretase and altered BACE1 distribution. Suppression of BACE1 expression in CA1 neurons partially rescued both dendritic and axonal deficits induced by Vps35-deficiency. These results thus demonstrate that BACE1 acts as a critical cargo of retromer in vitro and in vivo, and suggest that VPS35 plays an essential role in regulating apical dendritic maturation and in preventing axonal spheroid formation in developing hippocampal neurons.

Mild oxidative stress induces redistribution of BACE1 in non-apoptotic conditions and promotes the amyloidogenic processing of Alzheimer's disease amyloid precursor protein.

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Jiang-Li Tan

Full Text Available BACE1 is responsible for β-secretase cleavage of the amyloid precursor protein (APP, which represents the first step in the production of amyloid β (Aβ peptides. Previous reports, by us and others, have indicated that the levels of BACE1 protein and activity are increased in the brain cortex of patients with Alzheimer's disease (AD. The association between oxidative stress (OS and AD has prompted investigations that support the potentiation of BACE1 expression and enzymatic activity by OS. Here, we have established conditions to analyse the effects of mild, non-lethal OS on BACE1 in primary neuronal cultures, independently from apoptotic mechanisms that were shown to impair BACE1 turnover. Six-hour treatment of mouse primary cortical cells with 10-40 µM hydrogen peroxide did not significantly compromise cell viability but it did produce mild oxidative stress (mOS, as shown by the increased levels of reactive radical species and activation of p38 stress kinase. The endogenous levels of BACE1 mRNA and protein were not significantly altered in these conditions, whereas a toxic H2O2 concentration (100 µM caused an increase in BACE1 protein levels. Notably, mOS conditions resulted in increased levels of the BACE1 C-terminal cleavage product of APP, β-CTF. Subcellular fractionation techniques showed that mOS caused a major rearrangement of BACE1 localization from light to denser fractions, resulting in an increased distribution of BACE1 in fractions containing APP and markers for trans-Golgi network and early endosomes. Collectively, these data demonstrate that mOS does not modify BACE1 expression but alters BACE1 subcellular compartmentalization to favour the amyloidogenic processing of APP, and thus offer new insight in the early molecular events of AD pathogenesis.

Discovery of the 3-Imino-1,2,4-thiadiazinane 1,1-Dioxide Derivative Verubecestat (MK-8931)–A β-Site Amyloid Precursor Protein Cleaving Enzyme 1 Inhibitor for the Treatment of Alzheimer’s Disease

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Scott, Jack D.; Li, Sarah W.; Brunskill, Andrew P.J.; Chen, Xia; Cox, Kathleen; Cumming, Jared N.; Forman, Mark; Gilbert, Eric J.; Hodgson, Robert A.; Hyde, Lynn A.; Jiang, Qin; Iserloh, Ulrich; Kazakevich, Irina; Kuvelkar, Reshma; Mei, Hong; Meredith, John; Misiaszek, Jeffrey; Orth, Peter; Rossiter, Lana M.; Slater, Meagan; Stone, Julie; Strickland, Corey O.; Voigt, Johannes H.; Wang, Ganfeng; Wang, Hongwu; Wu, Yusheng; Greenlee, William J.; Parker, Eric M.; Kennedy, Matthew E.; Stamford, Andrew W. (Merck)

2016-12-08

Verubecestat 3 (MK-8931), a diaryl amide-substituted 3-imino-1,2,4-thiadiazinane 1,1-dioxide derivative, is a high-affinity β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor currently undergoing Phase 3 clinical evaluation for the treatment of mild to moderate and prodromal Alzheimer’s disease. Although not selective over the closely related aspartyl protease BACE2, verubecestat has high selectivity for BACE1 over other key aspartyl proteases, notably cathepsin D, and profoundly lowers CSF and brain Aβ levels in rats and nonhuman primates and CSF Aβ levels in humans. In this annotation, we describe the discovery of 3, including design, validation, and selected SAR around the novel iminothiadiazinane dioxide core as well as aspects of its preclinical and Phase 1 clinical characterization.

Identification of myo-inositol hexakisphosphate (IP6) as a β-secretase 1 (BACE1) inhibitory molecule in rice grain extract and digest.

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Abe, Takako K; Taniguchi, Masayuki

2014-01-01

Alzheimer's disease (AD) is widely considered to be caused by amyloid-β peptide (Aβ) accumulation in the brain. Aβ is excised from amyloid-β precursor protein through sequential cleavage by β-secretase 1 (BACE1) and γ-secretase. Thus, BACE1 inhibition could prevent Aβ accumulation. Here, we identified myo-inositol hexakisphosphate (IP6) as a BACE1 inhibitory molecule in rice grain extract and digest. The rice digest and IP6 significantly inhibited Aβ production in neuroblastoma cells without cytotoxicity. These results suggested that rice components, including IP6, may be promising starting materials for the development of potent and safe drugs and/or food to prevent AD.

Glimepiride attenuates Aβ production via suppressing BACE1 activity in cortical neurons.

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Liu, Feiyang; Wang, Yijin; Yan, Ming; Zhang, Luyong; Pang, Tao; Liao, Hong

2013-12-17

Numerous lines of evidence suggest a strong link between diabetes mellitus and Alzheimer's disease (AD). Impaired insulin signaling and insulin resistance occur not only in diabetes but also in the brain of AD. Recent evidence has indicated that peroxisome proliferator-activated receptor γ (PPARγ) agonists thiazolidinediones (TZDs) can decrease β-amyloid peptide (Aβ) deposition, which is the core component of senile plaques in AD, but the underlying mechanisms still remain unclear. In this study, we investigated whether glimepiride with PPARγ-stimulating activity, an oral anti-diabetic drug, has similar effects on Aβ production in primary cortical neurons. We demonstrated that glimepiride decreased extracellular Aβ40 and Aβ42 levels. The effect of glimepiride on reduction of Aβ40 generation was mediated by downregulation of β-site APP-cleaving enzyme 1 (BACE1) mRNA and protein expression, and by suppression of BACE1 activity. In addition, we found that high glucose condition enhanced Aβ40 production and glimepiride significantly decreased high glucose-induced Aβ40 production. Finally, a specific PPARγ antagonist GW9662 reversed glimepiride inhibitory effect on Aβ40 generation, suggesting a PPARγ-dependent mechanism may be involved. Our data indicated that glimepiride may serve as a promising drug for the treatment of AD associated with diabetes. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

First demonstration of cerebrospinal fluid and plasma A beta lowering with oral administration of a beta-site amyloid precursor protein-cleaving enzyme 1 inhibitor in nonhuman primates.

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Sankaranarayanan, Sethu; Holahan, Marie A; Colussi, Dennis; Crouthamel, Ming-Chih; Devanarayan, Viswanath; Ellis, Joan; Espeseth, Amy; Gates, Adam T; Graham, Samuel L; Gregro, Allison R; Hazuda, Daria; Hochman, Jerome H; Holloway, Katharine; Jin, Lixia; Kahana, Jason; Lai, Ming-tain; Lineberger, Janet; McGaughey, Georgia; Moore, Keith P; Nantermet, Philippe; Pietrak, Beth; Price, Eric A; Rajapakse, Hemaka; Stauffer, Shaun; Steinbeiser, Melissa A; Seabrook, Guy; Selnick, Harold G; Shi, Xiao-Ping; Stanton, Matthew G; Swestock, John; Tugusheva, Katherine; Tyler, Keala X; Vacca, Joseph P; Wong, Jacky; Wu, Guoxin; Xu, Min; Cook, Jacquelynn J; Simon, Adam J

2009-01-01

beta-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) 1 cleavage of amyloid precursor protein is an essential step in the generation of the potentially neurotoxic and amyloidogenic A beta 42 peptides in Alzheimer's disease. Although previous mouse studies have shown brain A beta lowering after BACE1 inhibition, extension of such studies to nonhuman primates or man was precluded by poor potency, brain penetration, and pharmacokinetics of available inhibitors. In this study, a novel tertiary carbinamine BACE1 inhibitor, tertiary carbinamine (TC)-1, was assessed in a unique cisterna magna ported rhesus monkey model, where the temporal dynamics of A beta in cerebrospinal fluid (CSF) and plasma could be evaluated. TC-1, a potent inhibitor (IC(50) approximately 0.4 nM), has excellent passive membrane permeability, low susceptibility to P-glycoprotein transport, and lowered brain A beta levels in a mouse model. Intravenous infusion of TC-1 led to a significant but transient lowering of CSF and plasma A beta levels in conscious rhesus monkeys because it underwent CYP3A4-mediated metabolism. Oral codosing of TC-1 with ritonavir, a potent CYP3A4 inhibitor, twice daily over 3.5 days in rhesus monkeys led to sustained plasma TC-1 exposure and a significant and sustained reduction in CSF sAPP beta, A beta 40, A beta 42, and plasma A beta 40 levels. CSF A beta 42 lowering showed an EC(50) of approximately 20 nM with respect to the CSF [TC-1] levels, demonstrating excellent concordance with its potency in a cell-based assay. These results demonstrate the first in vivo proof of concept of CSF A beta lowering after oral administration of a BACE1 inhibitor in a nonhuman primate.

Effects of curcuminoids identified in rhizomes of Curcuma longa on BACE-1 inhibitory and behavioral activity and lifespan of Alzheimer’s disease Drosophila models

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2014-01-01

Background Alzheimer’s disease (AD) is the most common type of presenile and senile dementia. The human β-amyloid precursor cleavage enzyme (BACE-1) is a key enzyme responsible for amyloid plaque production, which implicates the progress and symptoms of AD. Here we assessed the anti-BACE-1 and behavioral activities of curcuminoids from rhizomes of Curcuma longa (Zingiberaceae), diarylalkyls curcumin (CCN), demethoxycurcumin (DMCCN), and bisdemethoxycurcumin (BDMCCN) against AD Drosophila melanogaster models. Methods Neuro-protective ability of the curcuminoids was assessed using Drosophila melanogaster model system overexpressing BACE-1 and its substrate APP in compound eyes and entire neurons. Feeding and climbing activity, lifespan, and morphostructural changes in fly eyes also were evaluated. Results BDMCCN has the strongest inhibitory activity toward BACE-1 with 17 μM IC50, which was 20 and 13 times lower than those of CCN and DMCCN respectively. Overexpression of APP/BACE-1 resulted in the progressive and measurable defects in morphology of eyes and locomotion. Remarkably, supplementing diet with either 1 mM BDMCCN or 1 mM CCN rescued APP/BACE1-expressing flies and kept them from developing both morphological and behavioral defects. Our results suggest that structural characteristics, such as degrees of saturation, types of carbon skeleton and functional group, and hydrophobicity appear to play a role in determining inhibitory potency of curcuminoids on BACE-1. Conclusion Further studies will warrant possible applications of curcuminoids as therapeutic BACE-1 blockers. PMID:24597901

Rationalizing fragment based drug discovery for BACE1: insights from FB-QSAR, FB-QSSR, multi objective (MO-QSPR) and MIF studies.

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Manoharan, Prabu; Vijayan, R S K; Ghoshal, Nanda

2010-10-01

The ability to identify fragments that interact with a biological target is a key step in FBDD. To date, the concept of fragment based drug design (FBDD) is increasingly driven by bio-physical methods. To expand the boundaries of QSAR paradigm, and to rationalize FBDD using In silico approach, we propose a fragment based QSAR methodology referred here in as FB-QSAR. The FB-QSAR methodology was validated on a dataset consisting of 52 Hydroxy ethylamine (HEA) inhibitors, disclosed by GlaxoSmithKline Pharmaceuticals as potential anti-Alzheimer agents. To address the issue of target selectivity, a major confounding factor in the development of selective BACE1 inhibitors, FB-QSSR models were developed using the reported off target activity values. A heat map constructed, based on the activity and selectivity profile of the individual R-group fragments, and was in turn used to identify superior R-group fragments. Further, simultaneous optimization of multiple properties, an issue encountered in real-world drug discovery scenario, and often overlooked in QSAR approaches, was addressed using a Multi Objective (MO-QSPR) method that balances properties, based on the defined objectives. MO-QSPR was implemented using Derringer and Suich desirability algorithm to identify the optimal level of independent variables (X) that could confer a trade-off between selectivity and activity. The results obtained from FB-QSAR were further substantiated using MIF (Molecular Interaction Fields) studies. To exemplify the potentials of FB-QSAR and MO-QSPR in a pragmatic fashion, the insights gleaned from the MO-QSPR study was reverse engineered using Inverse-QSAR in a combinatorial fashion to enumerate some prospective novel, potent and selective BACE1 inhibitors.

Rationalizing fragment based drug discovery for BACE1: insights from FB-QSAR, FB-QSSR, multi objective (MO-QSPR) and MIF studies

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Manoharan, Prabu; Vijayan, R. S. K.; Ghoshal, Nanda

2010-10-01

The ability to identify fragments that interact with a biological target is a key step in FBDD. To date, the concept of fragment based drug design (FBDD) is increasingly driven by bio-physical methods. To expand the boundaries of QSAR paradigm, and to rationalize FBDD using In silico approach, we propose a fragment based QSAR methodology referred here in as FB-QSAR. The FB-QSAR methodology was validated on a dataset consisting of 52 Hydroxy ethylamine (HEA) inhibitors, disclosed by GlaxoSmithKline Pharmaceuticals as potential anti-Alzheimer agents. To address the issue of target selectivity, a major confounding factor in the development of selective BACE1 inhibitors, FB-QSSR models were developed using the reported off target activity values. A heat map constructed, based on the activity and selectivity profile of the individual R-group fragments, and was in turn used to identify superior R-group fragments. Further, simultaneous optimization of multiple properties, an issue encountered in real-world drug discovery scenario, and often overlooked in QSAR approaches, was addressed using a Multi Objective (MO-QSPR) method that balances properties, based on the defined objectives. MO-QSPR was implemented using Derringer and Suich desirability algorithm to identify the optimal level of independent variables ( X) that could confer a trade-off between selectivity and activity. The results obtained from FB-QSAR were further substantiated using MIF (Molecular Interaction Fields) studies. To exemplify the potentials of FB-QSAR and MO-QSPR in a pragmatic fashion, the insights gleaned from the MO-QSPR study was reverse engineered using Inverse-QSAR in a combinatorial fashion to enumerate some prospective novel, potent and selective BACE1 inhibitors.

Evaluation of Small-Animal PET Outcome Measures to Detect Disease Modification Induced by BACE Inhibition in a Transgenic Mouse Model of Alzheimer Disease.

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Deleye, Steven; Waldron, Ann-Marie; Verhaeghe, Jeroen; Bottelbergs, Astrid; Wyffels, Leonie; Van Broeck, Bianca; Langlois, Xavier; Schmidt, Mark; Stroobants, Sigrid; Staelens, Steven

2017-12-01

In this study, we investigated the effects of chronic administration of an inhibitor of the β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) on Alzheimer-related pathology by multitracer PET imaging in transgenic APPPS1-21 (TG) mice. Methods: Wild-type (WT) and TG mice received vehicle or BACE inhibitor (60 mg/kg) starting at 7 wk of age. Outcome measures of brain metabolism, neuroinflammation, and amyloid-β pathology were obtained through small-animal PET imaging with 18 F-FDG, 18 F-peripheral benzodiazepine receptor ( 18 F-PBR), and 18 F-florbetapir ( 18 F-AV45), respectively. Baseline scans were acquired at 6-7 wk of age and follow-up scans at 4, 7, and 12 mo. 18 F-AV45 uptake was measured at 8 and 13 mo of age. After the final scans, histologic measures of amyloid-β (4G8), microglia (ionized calcium binding adaptor molecule 1), astrocytes (glial fibrillary acidic protein), and neuronal nuclei were performed. Results: TG mice demonstrated significant age-associated increases in 18 F-AV45 uptake. An effect of treatment was observed in the cortex ( P = 0.0014), hippocampus ( P = 0.0005), and thalamus ( P treatment, TG mice demonstrated significantly lower 18 F-FDG uptake than WT mice in the thalamus ( P = 0.0004) and hippocampus ( P = 0.0332). Neuronal nucleus staining was lower in both TG groups in the thalamus and cortex. 18 F-PBR111 detected a significant age-related increase in TG mice ( P treatment-induced reduction in activated microglia as demonstrated by histology. Conclusion: Although 18 F-FDG, 18 F-PBR111, and 18 F-AV45 all detected pathologic alterations between TG and WT mice, only 18 F-AV45 could detect an effect of BACE inhibitor treatment. However, changes in WT binding of 18 F-AV45 undermine the specificity of this effect. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Depletion of GGA3 stabilizes BACE and enhances β-secretase activity

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Tesco, Giuseppina; Koh, Young Ho; Kang, Eugene; Cameron, Andrew; Das, Shinjita; Sena-Esteves, Miguel; Hiltunen, Mikko; Yang, Shao-Hua; Zhong, Zhenyu; Shen, Yong; Simpkins, James; Tanzi, Rudolph E.

2007-01-01

Summary Beta-site APP-cleaving enzyme (BACE) is required for production of the Alzheimer's disease (AD)-associated Aβ protein. BACE levels are elevated in AD brain, and increasing evidence reveals BACE as a stress-related protease that is upregulated following cerebral ischemia. However, the molecular mechanism responsible is unknown. We show that increases in BACE and β-secretase activity are due to post-translational stabilization following caspase activation. We also found that during cerebral ischemia, levels of GGA3, an adaptor protein involved in BACE trafficking, are reduced, while BACE levels are increased. RNAi silencing of GGA3 also elevated levels of BACE and Aβ. Finally, in AD brain samples, GGA3 protein levels were significantly decreased and inversely correlated with increased levels of BACE. In summary, we have elucidated a novel GGA3-dependent mechanism regulating BACE levels and β-secretase activity. This mechanism may explain increased cerebral levels of BACE and Aβ following cerebral ischemia and in AD. PMID:17553422

Depletion of GGA3 stabilizes BACE and enhances beta-secretase activity.

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Tesco, Giuseppina; Koh, Young Ho; Kang, Eugene L; Cameron, Andrew N; Das, Shinjita; Sena-Esteves, Miguel; Hiltunen, Mikko; Yang, Shao-Hua; Zhong, Zhenyu; Shen, Yong; Simpkins, James W; Tanzi, Rudolph E

2007-06-07

Beta-site APP-cleaving enzyme (BACE) is required for production of the Alzheimer's disease (AD)-associated Abeta protein. BACE levels are elevated in AD brain, and increasing evidence reveals BACE as a stress-related protease that is upregulated following cerebral ischemia. However, the molecular mechanism responsible is unknown. We show that increases in BACE and beta-secretase activity are due to posttranslational stabilization following caspase activation. We also found that during cerebral ischemia, levels of GGA3, an adaptor protein involved in BACE trafficking, are reduced, while BACE levels are increased. RNAi silencing of GGA3 also elevated levels of BACE and Abeta. Finally, in AD brain samples, GGA3 protein levels were significantly decreased and inversely correlated with increased levels of BACE. In summary, we have elucidated a GGA3-dependent mechanism regulating BACE levels and beta-secretase activity. This mechanism may explain increased cerebral levels of BACE and Abeta following cerebral ischemia and existing in AD.

G Protein-Coupled Receptors (GPCRs in Alzheimer’s Disease: A Focus on BACE1 Related GPCRs

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Juan eZhao

2016-03-01

Full Text Available The G protein coupled receptors (GPCRs have been considered as one of the largest families of validated drug targets, which involve in almost overall physiological functions and pathological processes. Meanwhile, Alzheimer’s disease (AD, the most common type of dementia, affects thinking, learning, memory and behavior of elderly people, that has become the hotspot nowadays for its increasing risks and incurability. The above fields have been intensively studied, and the link between the two has been demonstrated, whereas the way how GPCRs perturb AD progress are yet to be further explored given their complexities. In this review, we summarized recent progress regarding the GPCRs interacted with β-site APP cleaving enzyme 1 (BACE1, a key secretase in AD pathogenesis. Then we discussed the current findings on the regulatory roles of GPCRs on BACE1, and the possibility for pharmaceutical treatment of AD patients by the allosteric modulators and biased ligands of GPCRs. We hope this review can provide new insights into the understanding of mechanistic link between GPCRs and BACE1, and highlight the potential of GPCRs as therapeutic target for AD.

Anti-BACE1 and Antimicrobial Activities of Steroidal Compounds Isolated from Marine Urechis unicinctus

Directory of Open Access Journals (Sweden)

Yong-Zhe Zhu

2018-03-01

Full Text Available The human β-site amyloid cleaving enzyme (BACE1 has been considered as an effective drug target for treatment of Alzheimer’s disease (AD. In this study, Urechis unicinctus (U. unicinctus, which is a Far East specialty food known as innkeeper worm, ethanol extract was studied by bioassay-directed fractionation and isolation to examine its potential β-site amyloid cleaving enzyme inhibitory and antimicrobial activity. The following compounds were characterized: hecogenin, cholest-4-en-3-one, cholesta-4,6-dien-3-ol, and hurgadacin. These compounds were identified by their mass spectrometry, 1H, and 13C NMR spectral data, comparing those data with NIST/EPA/NIH Mass spectral database (NIST11 and published values. Hecogenin and cholest-4-en-3-one showed significant inhibitory activity against BACE1 with EC50 values of 116.3 and 390.6 µM, respectively. Cholesta-4,6-dien-3-ol and hurgadacin showed broad spectrum antimicrobial activity, particularly strongly against Escherichia coli (E. coli, Salmonella enterica (S. enterica, Pasteurella multocida (P. multocida, and Physalospora piricola (P. piricola, with minimal inhibitory concentration (MIC ranging from 0.46 to 0.94 mg/mL. This is the first report regarding those four known compounds that were isolated from U. unicinctus and their anti-BACE1 and antimicrobial activity, highlighting the fact that known natural compounds may be a critical source of new medicine leads. These findings provide scientific evidence for potential application of those bioactive compounds for the development of AD drugs and antimicrobial agents.

Potential anti-cholinesterase and β-site amyloid precursor protein cleaving enzyme 1 inhibitory activities of cornuside and gallotannins from Cornus officinalis fruits.

Science.gov (United States)

Bhakta, Himanshu Kumar; Park, Chan Hum; Yokozawa, Takako; Tanaka, Takashi; Jung, Hyun Ah; Choi, Jae Sue

2017-07-01

Cholinesterase (ChE) and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors are promising agents for the treatment of Alzheimer's disease (AD). In the present study, we examined the inhibitory activity of seven compounds isolated from the fruits of Cornus officinalis, cornuside, polymeric proanthocyanidins, 1,2,3-tri-O-galloyl-β-D-glucose, 1,2,3,6-tetra-O-galloyl-β-D-glucose, tellimagrandin I, tellimagrandin II, and isoterchebin, against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and BACE1. All of the compounds displayed concentration-dependent in vitro inhibitory activity toward the ChEs and BACE1. Among them, tellimagrandin II exhibited the best inhibitory activity toward ChEs, whereas the best BACE1 inhibitor was 1,2,3,6-tetra-O-galloyl-β-D-glucose. Isoterchebin and polymeric proanthocyanidins were also significant ChE inhibitors. The kinetic and docking studies demonstrated that all compounds interacted with both the catalytic active sites and the peripheral anionic sites of the ChEs and BACE1. Tellimagrandin II, isoterchebin, and the polymeric proanthocyanidins exhibited concentration-dependent inhibition of peroxynitrite-mediated protein tyrosine nitration. In conclusion, we identified significant ChE and BACE1 inhibitors from Corni Fructus that could have value as new multi-targeted compounds for anti-AD agents.

Anti-BACE1 and Antimicrobial Activities of Steroidal Compounds Isolated from Marine Urechis unicinctus.

Science.gov (United States)

Zhu, Yong-Zhe; Liu, Jing-Wen; Wang, Xue; Jeong, In-Hong; Ahn, Young-Joon; Zhang, Chuan-Jie

2018-03-14

The human β-site amyloid cleaving enzyme (BACE1) has been considered as an effective drug target for treatment of Alzheimer's disease (AD). In this study, Urechis unicinctus (U. unicinctus) , which is a Far East specialty food known as innkeeper worm, ethanol extract was studied by bioassay-directed fractionation and isolation to examine its potential β-site amyloid cleaving enzyme inhibitory and antimicrobial activity. The following compounds were characterized: hecogenin, cholest-4- en -3-one, cholesta-4,6- dien -3-ol, and hurgadacin. These compounds were identified by their mass spectrometry, ¹H, and 13 C NMR spectral data, comparing those data with NIST/EPA/NIH Mass spectral database (NIST11) and published values. Hecogenin and cholest-4- en -3-one showed significant inhibitory activity against BACE1 with EC 50 values of 116.3 and 390.6 µM, respectively. Cholesta-4,6- dien -3-ol and hurgadacin showed broad spectrum antimicrobial activity, particularly strongly against Escherichia coli (E. coli) , Salmonella enterica (S. enterica) , Pasteurella multocida (P. multocida) , and Physalospora piricola (P. piricola) , with minimal inhibitory concentration (MIC) ranging from 0.46 to 0.94 mg/mL. This is the first report regarding those four known compounds that were isolated from U. unicinctus and their anti-BACE1 and antimicrobial activity, highlighting the fact that known natural compounds may be a critical source of new medicine leads. These findings provide scientific evidence for potential application of those bioactive compounds for the development of AD drugs and antimicrobial agents.

The Golgi-Localized γ-Ear-Containing ARF-Binding (GGA Proteins Alter Amyloid-β Precursor Protein (APP Processing through Interaction of Their GAE Domain with the Beta-Site APP Cleaving Enzyme 1 (BACE1.

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Bjoern von Einem

Full Text Available Proteolytic processing of amyloid-β precursor protein (APP by beta-site APP cleaving enzyme 1 (BACE1 is the initial step in the production of amyloid beta (Aβ, which accumulates in senile plaques in Alzheimer's disease (AD. Essential for this cleavage is the transport and sorting of both proteins through endosomal/Golgi compartments. Golgi-localized γ-ear-containing ARF-binding (GGA proteins have striking cargo-sorting functions in these pathways. Recently, GGA1 and GGA3 were shown to interact with BACE1, to be expressed in neurons, and to be decreased in AD brain, whereas little is known about GGA2. Since GGA1 impacts Aβ generation by confining APP to the Golgi and perinuclear compartments, we tested whether all GGAs modulate BACE1 and APP transport and processing. We observed decreased levels of secreted APP alpha (sAPPα, sAPPβ, and Aβ upon GGA overexpression, which could be reverted by knockdown. GGA-BACE1 co-immunoprecipitation was impaired upon GGA-GAE but not VHS domain deletion. Autoinhibition of the GGA1-VHS domain was irrelevant for BACE1 interaction. Our data suggest that all three GGAs affect APP processing via the GGA-GAE domain.

Iminopyrimidinones: A novel pharmacophore for the development of orally active renin inhibitors

Energy Technology Data Exchange (ETDEWEB)

McKittrick, Brian A.; Caldwell, John P.; Bara, Thomas; Boykow, George; Chintala, Madhu; Clader, John; Czarniecki, Michael; Courneya, Brandy; Duffy, Ruth; Fleming, Linda; Giessert, Rachel; Greenlee, William J.; Heap, Charles; Hong, Liwu; Huang, Ying; Iserloh, Ulrich; Josien, Hubert; Khan, Tanweer; Korfmacher, Walter; Liang, Xian; Mazzola, Robert; Mitra, Soumya; Moore, Kristina; Orth, Peter; Rajagopalan, Murali; Roy, Sudipta; Sakwa, Samuel; Strickland, Corey; Vaccaro, Henry; Voigt, Johannes; Wang, Hongwu; Wong, Jesse; Zhang, Rumin; Zych, Andrew (Merck); (Albany MR)

2015-04-01

The development of renin inhibitors with favorable oral pharmacokinetic profiles has been a longstanding challenge for the pharmaceutical industry. As part of our work to identify inhibitors of BACE1, we have previously developed iminopyrimidinones as a novel pharmacophore for aspartyl protease inhibition. In this letter we describe how we modified substitution around this pharmacophore to develop a potent, selective and orally active renin inhibitor.

Mapping the conformational space accessible to BACE2 using surface mutants and cocrystals with Fab fragments, Fynomers and Xaperones.

Science.gov (United States)

Banner, David W; Gsell, Bernard; Benz, Jörg; Bertschinger, Julian; Burger, Dominique; Brack, Simon; Cuppuleri, Simon; Debulpaep, Maja; Gast, Alain; Grabulovski, Dragan; Hennig, Michael; Hilpert, Hans; Huber, Walter; Kuglstatter, Andreas; Kusznir, Eric; Laeremans, Toon; Matile, Hugues; Miscenic, Christian; Rufer, Arne C; Schlatter, Daniel; Steyaert, Jan; Stihle, Martine; Thoma, Ralf; Weber, Martin; Ruf, Armin

2013-06-01

The aspartic protease BACE2 is responsible for the shedding of the transmembrane protein Tmem27 from the surface of pancreatic β-cells, which leads to inactivation of the β-cell proliferating activity of Tmem27. This role of BACE2 in the control of β-cell maintenance suggests BACE2 as a drug target for diabetes. Inhibition of BACE2 has recently been shown to lead to improved control of glucose homeostasis and to increased insulin levels in insulin-resistant mice. BACE2 has 52% sequence identity to the well studied Alzheimer's disease target enzyme β-secretase (BACE1). High-resolution BACE2 structures would contribute significantly to the investigation of this enzyme as either a drug target or anti-target. Surface mutagenesis, BACE2-binding antibody Fab fragments, single-domain camelid antibody VHH fragments (Xaperones) and Fyn-kinase-derived SH3 domains (Fynomers) were used as crystallization helpers to obtain the first high-resolution structures of BACE2. Eight crystal structures in six different packing environments define an ensemble of low-energy conformations available to the enzyme. Here, the different strategies used for raising and selecting BACE2 binders for cocrystallization are described and the crystallization success, crystal quality and the time and resources needed to obtain suitable crystals are compared.

Caspase-3 cleavage of GGA3 stabilizes BACE: implications for Alzheimer's disease.

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Vassar, Robert

2007-06-07

BACE initiates the production of beta-amyloid (Abeta), the likely cause of Alzheimer's disease (AD). In this issue of Neuron, Tesco et al. show that during apoptosis caspase-3 cleaves the adaptor protein GGA3, which is required for BACE lysosomal degradation, consequently stabilizing BACE and elevating Abeta generation.

BACE1 Is Necessary for Experience-Dependent Homeostatic Synaptic Plasticity in Visual Cortex

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Emily Petrus

2014-01-01

Full Text Available Alzheimer’s disease (AD is the most common form of age-related dementia, which is thought to result from overproduction and/or reduced clearance of amyloid-beta (Aβ peptides. Studies over the past few decades suggest that Aβ is produced in an activity-dependent manner and has physiological relevance to normal brain functions. Similarly, physiological functions for β- and γ-secretases, the two key enzymes that produce Aβ by sequentially processing the amyloid precursor protein (APP, have been discovered over recent years. In particular, activity-dependent production of Aβ has been suggested to play a role in homeostatic regulation of excitatory synaptic function. There is accumulating evidence that activity-dependent immediate early gene Arc is an activity “sensor,” which acts upstream of Aβ production and triggers AMPA receptor endocytosis to homeostatically downregulate the strength of excitatory synaptic transmission. We previously reported that Arc is critical for sensory experience-dependent homeostatic reduction of excitatory synaptic transmission in the superficial layers of visual cortex. Here we demonstrate that mice lacking the major neuronal β-secretase, BACE1, exhibit a similar phenotype: stronger basal excitatory synaptic transmission and failure to adapt to changes in visual experience. Our results indicate that BACE1 plays an essential role in sensory experience-dependent homeostatic synaptic plasticity in the neocortex.

Electrical conductivity and chemical stability of BaCe0·8−xAxGd0 ...

Indian Academy of Sciences (India)

−1 in air at 1073 K. BaCe0·7In0·1Gd0·2O3−δ and BaCe0·7Zr0·1Gd0·2O3−δ ceramics exhibit an ... cells convert chemical energy directly into electrical energy and their ... peratures in the presence of CO2 and water vapour (Haile. 2003 ...

Anti-BACE1 and Antimicrobial Activities of Steroidal Compounds Isolated from Marine Urechis unicinctus

OpenAIRE

Yong-Zhe Zhu; Jing-Wen Liu; Xue Wang; In-Hong Jeong; Young-Joon Ahn; Chuan-Jie Zhang

2018-01-01

The human β-site amyloid cleaving enzyme (BACE1) has been considered as an effective drug target for treatment of Alzheimer’s disease (AD). In this study, Urechis unicinctus (U. unicinctus), which is a Far East specialty food known as innkeeper worm, ethanol extract was studied by bioassay-directed fractionation and isolation to examine its potential β-site amyloid cleaving enzyme inhibitory and antimicrobial activity. The following compounds were characterized: hecogenin, cholest-4-en-3-one,...

Electrical conductivity and chemical stability of BaCe0· 8− xAxGd0 ...

Indian Academy of Sciences (India)

... K. BaCe0.7In0.1Gd0.2O3− and BaCe0.7Zr0.1Gd0.2O3− ceramics exhibit an excellent chemical stability against boiling water. Indium is a suitable doping element to promote the sintering densification and to enhance both electrical conductivity and chemical stability of Gd-doped BaCeO3 at operating temperatures.

Differential contributions of ApoE4 and female sex to BACE1 activity and expression mediate Aβ deposition and learning and memory in mouse models of Alzheimer’s disease

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Xu eHou

2015-10-01

Full Text Available Alzheimer’s disease (AD, the most common form of dementia, disproportionately affects women in both prevalence and severity. This increased vulnerability to AD in women is strongly associated with age-related ovarian hormone loss and apolipoprotein E 4 allele (ApoE4, the most important genetic risk factor for sporadic AD. Up to date, the mechanism involved in the interaction between ApoE4 and sex/gender in AD is still unclear. This study evaluated the sex-dependent ApoE4 effects on learning and memory, Aβ deposition and potential mechanisms, using mice bearing both sporadic (ApoE4 and familial (APPSwe, PS1M146V, tauP301L; 3xTg AD risk factors and compared with sex- and age-matched 3xTg or nonTg mice. Compared to nonTg mice, transgenic mice of both sexes showed spatial learning and memory deficits in the radial arm water maze and novel arm discrimination tests at 20 months of age. However, at 10 months, only ApoE4/3xTg mice showed significant learning and memory impairment. Moreover, molecular studies of hippocampal tissue revealed significantly higher protein levels of Aβ species, β-site APP cleavage enzyme (BACE1 and Sp1, a transcription factor of BACE1, in female ApoE4/3xTg when compared with female nonTg, female 3xTg and male ApoE4/3xTg mice. Significantly increased BACE1 enzymatic activities were observed in both male and female mice carrying ApoE4; however, only the females showed significant higher BACE1 expressions. Together, these data suggest that ApoE4 allele is associated with increased BACE1 enzymatic activity, while female sex plays an important role in increasing BACE1 expression. The combination of both provides a molecular basis for high Aβ pathology and the resultant hippocampus-dependent learning and memory deficits in female ApoE4 carriers.

Cholesterol-dependent energy transfer between fluorescent proteins-insights into protein proximity of APP and BACE1 in different membranes in Niemann-Pick type C disease cells.

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von Einem, Bjoern; Weber, Petra; Wagner, Michael; Malnar, Martina; Kosicek, Marko; Hecimovic, Silva; Arnim, Christine A F von; Schneckenburger, Herbert

2012-11-26

Förster resonance energy transfer (FRET) -based techniques have recently been applied to study the interactions between β-site APP-cleaving enzyme-GFP (BACE1-GFP) and amyloid precursor protein-mRFP (APP-mRFP) in U373 glioblastoma cells. In this context, the role of APP-BACE1 proximity in Alzheimer's disease (AD) pathogenesis has been discussed. FRET was found to depend on intracellular cholesterol levels and associated alterations in membrane stiffness. Here, NPC1 null cells (CHO-NPC1-/-), exhibiting increased cholesterol levels and disturbed cholesterol transport similar to that observed in Niemann-Pick type C disease (NPC), were used to analyze the influence of altered cholesterol levels on APP-BACE1 proximity. Fluorescence lifetime measurements of whole CHO-wild type (WT) and CHO-NPC1-/- cells (EPI-illumination microscopy), as well as their plasma membranes (total internal reflection fluorescence microscopy, TIRFM), were performed. Additionally, generalized polarization (GP) measurements of CHO-WT and CHO-NPC1-/- cells incubated with the fluorescence marker laurdan were performed to determine membrane stiffness of plasma- and intracellular-membranes. CHO-NPC1-/- cells showed higher membrane stiffness at intracellular- but not plasma-membranes, equivalent to cholesterol accumulation in late endosomes/lysosomes. Along with higher membrane stiffness, the FRET efficiency between BACE1-GFP and APP-mRFP was reduced at intracellular membranes, but not within the plasma membrane of CHO-NPC1-/-. Our data show that FRET combined with TIRF is a powerful technique to determine protein proximity and membrane fluidity in cellular models of neurodegenerative diseases.

Isozyme-specific fluorescent inhibitor of glutathione s-transferase omega 1.

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Son, Junghyun; Lee, Jae-Jung; Lee, Jun-Seok; Schüller, Andreas; Chang, Young-Tae

2010-05-21

Recently, the glutathione S-transferase omega 1 (GSTO1) is suspected to be involved in certain cancers and neurodegenerative diseases. However, profound investigation on the pathological roles of GSTO1 has been hampered by the lack of specific methods to determine or modulate its activity in biological systems containing other isoforms with similar catalytic function. Here, we report a fluorescent compound that is able to inhibit and monitor the activity of GSTO1. We screened 43 fluorescent chemicals and found a compound (6) that binds specifically to the active site of GSTO1. We observed that compound 6 inhibits GSTO1 by covalent modification but spares other isoforms in HEK293 cells and demonstrated that compound 6 could report the activity of GSTO1 in NIH/3T3 or HEK293 cells by measuring the fluorescence intensity of the labeled amount of GSTO1 in SDS-PAGE. Compound 6 is a useful tool to study GSTO1, applicable as a specific inhibitor and an activity reporter.

Identification of STAT1 and STAT3 specific inhibitors using comparative virtual screening and docking validation.

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Malgorzata Szelag

Full Text Available Signal transducers and activators of transcription (STATs facilitate action of cytokines, growth factors and pathogens. STAT activation is mediated by a highly conserved SH2 domain, which interacts with phosphotyrosine motifs for specific STAT-receptor contacts and STAT dimerization. The active dimers induce gene transcription in the nucleus by binding to a specific DNA-response element in the promoter of target genes. Abnormal activation of STAT signaling pathways is implicated in many human diseases, like cancer, inflammation and auto-immunity. Searches for STAT-targeting compounds, exploring the phosphotyrosine (pTyr-SH2 interaction site, yielded many small molecules for STAT3 but sparsely for other STATs. However, many of these inhibitors seem not STAT3-specific, thereby questioning the present modeling and selection strategies of SH2 domain-based STAT inhibitors. We generated new 3D structure models for all human (hSTATs and developed a comparative in silico docking strategy to obtain further insight into STAT-SH2 cross-binding specificity of a selection of previously identified STAT3 inhibitors. Indeed, by primarily targeting the highly conserved pTyr-SH2 binding pocket the majority of these compounds exhibited similar binding affinity and tendency scores for all STATs. By comparative screening of a natural product library we provided initial proof for the possibility to identify STAT1 as well as STAT3-specific inhibitors, introducing the 'STAT-comparative binding affinity value' and 'ligand binding pose variation' as selection criteria. In silico screening of a multi-million clean leads (CL compound library for binding of all STATs, likewise identified potential specific inhibitors for STAT1 and STAT3 after docking validation. Based on comparative virtual screening and docking validation, we developed a novel STAT inhibitor screening tool that allows identification of specific STAT1 and STAT3 inhibitory compounds. This could increase our

Synthesis, Sintering, and Electrical Properties of BaCe0.9−xZrxY0.1O3−δ

DEFF Research Database (Denmark)

Ricote, S.; Caboche, G.; Estournes, C.

2008-01-01

BaCe0.9-xZrxY0.1O3-delta powders were synthesized by a solid-state reaction. Different contents of cerium and zirconium were studied. Pellets were sintered using either conventional sintering in air at 1700 degrees C or the Spark Plasma Sintering (SPS) technique. The density of the samples sintered...

Increased CSF-BACE 1 Activity Is Associated with ApoE-[Epsilon]4 Genotype in Subjects with Mild Cognitive Impairment and Alzheimer's Disease

Science.gov (United States)

Ewers, Michael; Zhong, Zhenyu; Burger, Katharina; Wallin, Anders; Blennow, Kaj; Teipel, Stefan J.; Shen, Yong; Hampel, Harald

2008-01-01

The Apolipoprotein (ApoE) [epsilon]4 allele is a major genetic risk factor of Alzheimer's disease, and may affect the production of amyloid beta (A[beta][subscript 1-42]). Recently, we have shown that [beta]-secretase (BACE 1) activity can be reliably detected within the brain and human CSF. Here, we have examined an association between the ApoE…

Three Dimensional Human Neuro-Spheroid Model of Alzheimer’s Disease Based on Differentiated Induced Pluripotent Stem Cells

Science.gov (United States)

Lee, Han-Kyu; Velazquez Sanchez, Clara; Chen, Mei; Morin, Peter J.; Wells, John M.; Hanlon, Eugene B.

2016-01-01

The testing of candidate drugs to slow progression of Alzheimer’s disease (AD) requires clinical trials that are lengthy and expensive. Efforts to model the biochemical milieu of the AD brain may be greatly facilitated by combining two cutting edge technologies to generate three-dimensional (3D) human neuro-spheroid from induced pluripotent stem cells (iPSC) derived from AD subjects. We created iPSC from blood cells of five AD patients and differentiated them into 3D human neuronal culture. We characterized neuronal markers of our 3D neurons by immunocytochemical staining to validate the differentiation status. To block the generation of pathologic amyloid β peptides (Aβ), the 3D-differentiated AD neurons were treated with inhibitors targeting β-secretase (BACE1) and γ-secretases. As predicted, both BACE1 and γ-secretase inhibitors dramatically decreased Aβ generation in iPSC-derived neural cells derived from all five AD patients, under standard two-dimensional (2D) differentiation conditions. However, BACE1 and γ-secretase inhibitors showed less potency in decreasing Aβ levels in neural cells differentiated under 3D culture conditions. Interestingly, in a single subject AD1, we found that BACE1 inhibitor treatment was not able to significantly reduce Aβ42 levels. To investigate underlying molecular mechanisms, we performed proteomic analysis of 3D AD human neuronal cultures including AD1. Proteomic analysis revealed specific reduction of several proteins that might contribute to a poor inhibition of BACE1 in subject AD1. To our knowledge, this is the first iPSC-differentiated 3D neuro-spheroid model derived from AD patients’ blood. Our results demonstrate that our 3D human neuro-spheroid model can be a physiologically relevant and valid model for testing efficacy of AD drug. PMID:27684569

Covalent cross-linking of insulin-like growth factor-1 to a specific inhibitor from human serum

International Nuclear Information System (INIS)

Ooi, G.T.; Herington, A.C.

1986-01-01

Previous studies have shown that a specific inhibitor of insulin-like growth factor (IGF) action in vitro can be isolated from normal human serum and subsequently partially purified on an IGF-affinity column. The ability of the inhibitor to bind the IGFs has now been confirmed directly using covalent cross-linking techniques. When 125 I-IGF-1 was cross-linked to inhibitor using disuccinimidyl suberate, five specifically labelled bands were seen on SDS-PAGE and autoradiography. Two bands (MW 21.5 K and 25.5 K) were intensely labelled, while the remaining three (MW 37 K, 34 K and 18 K) appeared as minor bands only. Inhibitor bioactivity, following further analysis by hydrophobic interaction chromatography or Con A-Sepharose affinity chromatography, was always associated with the presence of the 21.5 K and/or 25.5 K bands

Membrane-Associated Effects of Glucocorticoid on BACE1 Upregulation and Aβ Generation: Involvement of Lipid Raft-Mediated CREB Activation.

Science.gov (United States)

Choi, Gee Euhn; Lee, Sei-Jung; Lee, Hyun Jik; Ko, So Hee; Chae, Chang Woo; Han, Ho Jae

2017-08-30

Glucocorticoid has been widely accepted to induce Alzheimer's disease, but the nongenomic effect of glucocorticoid on amyloid β (Aβ) generation has yet to be studied. Here, we investigated the effect of the nongenomic pathway induced by glucocorticoid on amyloid precursor protein processing enzymes as well as Aβ production using male ICR mice and human neuroblastoma SK-N-MC cells. Mice groups exposed to restraint stress or intracerebroventricular injection of Aβ showed impaired cognition, decreased intracellular glucocorticoid receptor (GR) level, but elevated level of membrane GR (mGR). In this respect, we identified the mGR-dependent pathway evoked by glucocorticoid using impermeable cortisol conjugated to BSA (cortisol-BSA) on SK-N-MC cells. Cortisol-BSA augmented the expression of β-site amyloid precursor protein cleaving enzyme 1 (BACE1), the level of C-terminal fragment β of amyloid precursor protein (C99) and Aβ production, which were maintained even after blocking intracellular GR. We also found that cortisol-BSA enhanced the interaction between mGR and Gαs, which colocalized in the lipid raft. The subsequently activated CREB by cortisol-BSA bound to the CRE site of the BACE1 promoter increasing its expression, which was downregulated by inhibiting CBP. Consistently, blocking CBP attenuated cognitive impairment and Aβ production induced by corticosterone treatment or intracerebroventricular injection of Aβ more efficiently than inhibiting intracellular GR in mice. In conclusion, glucocorticoid couples mGR with Gαs and triggers cAMP-PKA-CREB axis dependent on the lipid raft to stimulate BACE1 upregulation and Aβ generation. SIGNIFICANCE STATEMENT Patients with Alzheimer's disease (AD) have been growing sharply and stress is considered as the major environment factor of AD. Glucocorticoid is the primarily responsive factor to stress and is widely known to induce AD. However, most AD patients usually have impaired genomic pathway of glucocorticoid

Profound Understanding of Effect of Transition Metal Dopant, Sintering Temperature, and pO2 on the Electrical and Optical Properties of Proton Conducting BaCe0.9Sm0.1O3-δ.

Science.gov (United States)

Handal, Hala T; Hassan, Azfar; Leeson, Ryan; Eloui, Sherif M; Fitzpatrick, Martin; Thangadurai, Venkataraman

2016-01-19

This study reports the effect of transition metal (TM) substitution on the electrical and optical properties of BaCe0.9Sm0.1O3-δ (BCS). Concentrations of 5-10 mol % of each of Fe and Co have been doped for the B-site of BCS by citric acid autocombustion method. Powder X-ray diffraction has revealed the formation of an orthorhombic perovskite-type structure. FTIR confirmed a distortion in the lattice upon TM-doping in BCS. Scanning electron microscopy (SEM) images of 1400 °C sintered samples have manifested a higher densification in BaCe0.8Sm0.1Co0.1O3-δ (BCSC10) with a grain size ∼11 μm compared to the parent compound BCS (∼2 μm). Thermogravimetric (TG) analysis showed a water uptake in case of BaCe0.85Sm0.1Co0.05O3-δ (BCSC5), while BaCe0.85Sm0.1Fe0.05O3-δ (BCSF5) did not show a noteworthy uptake of water. TG has also proved that the incorporation of Fe and Co in BCS did not improve the chemical stability in CO2 at elevated temperature. The band gap estimated using Kubelka-Munk model based on the diffuse reflectance data was found to be the lowest for BCSC5 (2.47 eV). However, it increases upon lowering oxygen partial pressure (pO2), which was interpreted by a band structure modifications. Among the samples investigated, BCSC10 sintered at 1400 °C showed the highest electrical conductivity of 0.02 S cm(-1) in air at 600 °C, while its proton mobility appears to be negligible under the investigated humidity atmosphere.

In silico simulations of STAT1 and STAT3 inhibitors predict SH2 domain cross-binding specificity.

Science.gov (United States)

Szelag, Malgorzata; Sikorski, Krzysztof; Czerwoniec, Anna; Szatkowska, Katarzyna; Wesoly, Joanna; Bluyssen, Hans A R

2013-11-15

Signal transducers and activators of transcription (STATs) comprise a family of transcription factors that are structurally related and which participate in signaling pathways activated by cytokines, growth factors and pathogens. Activation of STAT proteins is mediated by the highly conserved Src homology 2 (SH2) domain, which interacts with phosphotyrosine motifs for specific contacts between STATs and receptors and for STAT dimerization. By generating new models for human (h)STAT1, hSTAT2 and hSTAT3 we applied comparative in silico docking to determine SH2-binding specificity of the STAT3 inhibitor stattic, and of fludarabine (STAT1 inhibitor). Thus, we provide evidence that by primarily targeting the highly conserved phosphotyrosine (pY+0) SH2 binding pocket stattic is not a specific hSTAT3 inhibitor, but is equally effective towards hSTAT1 and hSTAT2. This was confirmed in Human Micro-vascular Endothelial Cells (HMECs) in vitro, in which stattic inhibited interferon-α-induced phosphorylation of all three STATs. Likewise, fludarabine inhibits both hSTAT1 and hSTAT3 phosphorylation, but not hSTAT2, by competing with the highly conserved pY+0 and pY-X binding sites, which are less well-preserved in hSTAT2. Moreover we observed that in HMECs in vitro fludarabine inhibits cytokine and lipopolysaccharide-induced phosphorylation of hSTAT1 and hSTAT3 but does not affect hSTAT2. Finally, multiple sequence alignment of STAT-SH2 domain sequences confirmed high conservation between hSTAT1 and hSTAT3, but not hSTAT2, with respect to stattic and fludarabine binding sites. Together our data offer a molecular basis that explains STAT cross-binding specificity of stattic and fludarabine, thereby questioning the present selection strategies of SH2 domain-based competitive small inhibitors. © 2013 Elsevier B.V. All rights reserved.

Water vapour solubility and conductivity study of the proton conductor BaCe(0.9 − x)ZrxY0.1O(3 − δ)

DEFF Research Database (Denmark)

Ricote, Sandrine; Bonanos, Nikolaos; Caboche, G:

2009-01-01

The perovskite BaCe(0.9 − x)ZrxY0.1O(3 − δ) has been prepared by solid state reaction at 1400 °C and conventional sintering at 1700 °C. Water uptake experiments performed between 400 and 600 °C, at a water vapour pressure of 0.02 atm, provide data on the concentration of protons incorporated in t...

Structural and conductivity study of the proton conductor BaCe(0.9−x)ZrxY0.1O(3−δ) at intermediate temperatures

DEFF Research Database (Denmark)

Ricote, Sandrine; Bonanos, Nikolaos; Marco de Lucas, M.C.

2009-01-01

The perovskite BaCe(0.9−x)ZrxY0.1O(3−δ) is prepared by solid-state reaction at 1400 °C and sintering at 1700 °C. It is characterised using X-ray diffraction, Raman spectroscopy and electrical measurements. A distortion from the cubic structure at room temperature is noticeable in the Raman spectr...

Novel β-amyloid aggregation inhibitors possessing a turn mimic.

Science.gov (United States)

Hamada, Yoshio; Miyamoto, Naoko; Kiso, Yoshiaki

2015-04-01

Amyloid β peptide, the main component of senile plaques found in the brain of Alzheimer disease (AD) patients, is a molecular target for AD therapeutic intervention. A number of potential AD therapeutics have been reported, including inhibitors of β-secretase, γ-secretase, and Aβ aggregation, and anti-amyloid agents, such as neprilysin, insulin degrading enzyme (IDE), and Aβ antibodies. Recently, we reported potent small-sized β-secretase (BACE1) inhibitors, which could serve as anti-AD drugs. However AD is a progressive disorder, where dementia symptoms gradually worsen over several decades, and therefore may require many years to get cured. One possible way to achieve a greater therapeutic effect is through simultaneous administration of multiple drugs, similar to those used in Highly Active Anti-Retroviral Therapy (HAART) used to treat AIDS. In order to overcome AD, we took a drug discovery approach to evaluate, novel β-amyloid aggregation inhibitors. Previously, we reported that a tong-type compound possessing a turn mimic as the inhibitor of HIV-1 protease dimerization. Oligomerized amyloid β peptides contain a turn structure within the molecule. Here, we designed and synthesized novel β-amyloid aggregation inhibitors with a turn-mimic template, based on the turn conformer of the oligomerized amyloid β peptides. Copyright © 2015 Elsevier Ltd. All rights reserved.

Evaluation of the specificity of antigen assays for plasminogen activator inhibitor 1 : Comparison of two new commercial kits

NARCIS (Netherlands)

Huisman, L.G.M.; Meijer, P.; Griensven, J. van; Kluft, C.

1992-01-01

t-PA depleted citrated plasma was used to prepare standards of different molecular forms of plasminogen activator inhibitor 1 (PAI-1). These standards were used to evaluate the specificity of two new PAI-1 antigen assays: the TintElize PAI-1 antigen assay (cat. no. 210221) and the Innotest PAI-1.

Cyclic peptide inhibitors of lysine-specific demethylase 1 with improved potency identified by alanine scanning mutagenesis.

Science.gov (United States)

Kumarasinghe, Isuru R; Woster, Patrick M

2018-03-25

Lysine-specific demethylase 1 (LSD1) is a chromatin-remodeling enzyme that plays an important role in cancer. Over-expression of LSD1 decreases methylation at histone 3 lysine 4, and aberrantly silences tumor suppressor genes. Inhibitors of LSD1 have been designed as chemical probes and potential antitumor agents. We recently reported the cyclic peptide 9, which potently and reversibly inhibits LSD1 (IC 50 2.1 μM; K i 385 nM). Systematic alanine mutagenesis of 9 revealed residues that are critical for LSD1 inhibition, and these mutated peptides were evaluated as LSD1 inhibitors. Alanine substitution at positions 2, 3, 4, 6 and 11-17 preserved inhibition, while substitution of alanine at positions 8 and 9 resulted in complete loss of activity. Cyclic mutant peptides 11 and 16 produced the greatest LSD1 inhibition, and 11, 16, 27 and 28 increased global H3K4me2 in K562 cells. In addition, 16, 27 and 28 promoted significant increases in H3K4me2 levels at the promoter sites of the genes IGFBP2 and FEZ1. Data from these LSD1 inhibitors will aid in the design of peptidomimetics with improved stability and pharmacokinetics. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

BACE1 inhibition by microdose lithium formulation NP03 rescues memory loss and early stage amyloid neuropathology.

Science.gov (United States)

Wilson, E N; Do Carmo, S; Iulita, M F; Hall, H; Ducatenzeiler, A; Marks, A R; Allard, S; Jia, D T; Windheim, J; Cuello, A C

2017-08-01

Lithium is first-line therapy for bipolar affective disorder and has recently been shown to have protective effects in populations at risk for Alzheimer's disease (AD). However, the mechanism underlying this protection is poorly understood and consequently limits its possible therapeutic application in AD. Moreover, conventional lithium formulations have a narrow therapeutic window and are associated with a severe side effect profile. Here we evaluated a novel microdose formulation of lithium, coded NP03, in a well-characterized rat model of progressive AD-like amyloid pathology. This formulation allows microdose lithium delivery to the brain in the absence of negative side effects. We found that NP03 rescued key initiating components of AD pathology, including inactivating GSK-3β, reducing BACE1 expression and activity, and reducing amyloid levels. Notably, NP03 rescued memory loss, impaired CRTC1 promoter binding of synaptic plasticity genes and hippocampal neurogenesis. These results raise the possibility that NP03 be of therapeutic value in the early or preclinical stages of AD.

Composite Fe - BaCe0.2Zr0.6Y0.2O2.9 Anodes for Proton Conductor Fuel Cells

DEFF Research Database (Denmark)

Lapina, Alberto; Chatzichristodoulou, Christodoulos; Holtappels, Peter

2014-01-01

Symmetrical cells with Fe - BaCe0.2Zr0.6Y0.2O2.9 composite electrodes are produced by screen printing and infiltration, using BaCe0.2Zr0.6Y0.2O2.9 as electrolyte. The electrochemical performance of the composite electrode is studied by impedance spectroscopy at 250–500◦C in dry and wet hydrogen/n...

LaCoO3: Promising cathode material for protonic ceramic fuel cells based on a BaCe0.2Zr0.7Y0.1O3−δ electrolyte

DEFF Research Database (Denmark)

Ricote, Sandrine; Bonanos, Nikolaos; Lenrick, Filip

2012-01-01

Symmetric cells (cathode/electrolyte/cathode) were prepared using BaCe0.2Zr0.7Y0.1O3−δ (BCZY27) as proton conducting electrolyte and LaCoO3 (LC) infiltrated into a porous BCZY27 backbone as cathode. Single phased LC was formed after annealing in air at 600 °C for 2 h. Scanning electron micrograph...... that the presence of oxide ion conduction in the cathode material is not necessary for good performance.......Symmetric cells (cathode/electrolyte/cathode) were prepared using BaCe0.2Zr0.7Y0.1O3−δ (BCZY27) as proton conducting electrolyte and LaCoO3 (LC) infiltrated into a porous BCZY27 backbone as cathode. Single phased LC was formed after annealing in air at 600 °C for 2 h. Scanning electron micrographs...... showed the presence of the infiltrated LC in the full cathode depth. Transmission electron micrographs revealed LC grains (60–80 nm) covering partly the BCZY27 grains (200 nm–1 μm). Impedance spectra were recorded at 500 °C and 600 °C, varying the oxygen partial pressure and the water vapour pressure...

Cleavage of ST6Gal I by Radiation-Induced BACE1 Inhibits Golgi-Anchored ST6Gal I-Mediated Sialylation of Integrin β1 and Migration in Colon Cancer Cells

International Nuclear Information System (INIS)

Lee, Minyoung; Park, Jung-Jin; Ko, Young-Gyu; Lee, Yun-Sil

2012-01-01

Previously, we found that β-galactoside α2,6-sialyltransferase (ST6Gal I), an enzyme that adds sialic acids to N-linked oligosaccharides of glycoproteins and is frequently overexpressed in cancer cells, is up-regulated by ionizing radiation (IR) and cleaved to a form possessing catalytic activity comparable to that of the Golgi-localized enzyme. Moreover, this soluble form is secreted into the culture media. Induction of ST6Gal I significantly increased the migration of colon cancer cells via sialylation of integrin β1. Here, we further investigated the mechanisms underlying ST6Gal I cleavage, solubilization and release from cells, and addressed its functions, focusing primarily on cancer cell migration. We performed immunoblotting and lectin affinity assay to analyze the expression of ST6 Gal I and level of sialylated integrin β1. After ionizing radiation, migration of cells was measured by in vitro migration assay. α2, 6 sialylation level of cell surface was analyzed by flow cytometry. Cell culture media were concentrated and then analyzed for soluble ST6Gal I levels using an α2, 6 sialyltransferase sandwich ELISA. We found that ST6Gal I was cleaved by BACE1 (β-site amyloid precursor protein-cleaving enzyme), which was specifically overexpressed in response to IR. The soluble form of ST6Gal I, which also has sialyltransferase enzymatic activity, was cleaved from the Golgi membrane and then released into the culture media. Both non-cleaved and cleaved forms of ST6Gal I significantly increased colon cancer cell migration in a sialylation-dependent manner. The pro-migratory effect of the non-cleaved form of ST6Gal I was dependent on integrin β1 sialylation, whereas that of the cleaved form of ST6Gal I was not, suggesting that other intracellular sialylated molecules apart from cell surface molecules such as integrin β1 might be involved in mediating the pro-migratory effects of the soluble form of ST6Gal I. Moreover, production of soluble form ST6Gal I by

Inhibition of β-Secretase Activity by Monoterpenes, Sesquiterpenes, and C13 Norisoprenoids.

Science.gov (United States)

Marumoto, Shinsuke; Okuno, Yoshiharu; Miyazawa, Mitsuo

2017-08-01

Inhibition of β-secretase (BACE1) is currently regarded as the leading treatment strategy for Alzheimer's disease. In the present study, we aimed to screen the in vitro inhibitory activity of 80 types of aroma compounds (monoterpenes, sesquiterpenes, and C 13 norisoprenoids), including plant-based types, at a 200-μM concentration against a recombinant human BACE1. The results showed that the most potent inhibitor of BACE1 was geranyl acetone followed by (+)-camphor, (-)-fenchone, (+)-fenchone, and (-)-camphor with the half-maximal inhibitory concentration (IC 50 ) values of 51.9 ± 3.9, 95.9 ± 11.0, 106.3 ± 14.9, 117.0 ± 18.6, and 134.1 ± 16.4 μM, respectively. Furthermore, the mechanism of inhibition of BACE1 by geranyl acetone was analyzed using Dixon kinetics plus Cornish-Bowden plots, which revealed mixed-type mode. Therefore aroma compounds may be used as potential lead molecules for designing anti-BACE1 agents.

Discovery of core-structurally novel PTP1B inhibitors with specific selectivity containing oxindole-fused spirotetrahydrofurochroman by one-pot reaction.

Science.gov (United States)

Dong, Suzhen; Lei, Yubing; Jia, Shikun; Gao, Lixin; Li, Jia; Zhu, Tong; Liu, Shunying; Hu, Wenhao

2017-02-15

Protein tyrosine phosphatase 1B (PTP1B) has been proposed to be an ideal target for treatment of type II diabetes and obesity. However, no druggable PTP1B inhibitor has been established and there is still an urgent demand for the development of structurally novel PTPIB inhibitor. Herein, we reported core-structurally novel PTP1B inhibitors with low micromole-ranged inhibitory activity by one-pot reaction from simple starting materials. Further studies demonstrated some of these active compounds had a specific selectivity over other PTPs. The structure and activity relationship was also described. The best active and selective compound 5e inhibited PTP1B activity with an IC 50 of 4.53μM. Molecular docking analysis further demonstrated that compound 5e bound to the active pocket of PTP1B. The results might provide some insights for further development of new drugs for type II diabetes and obesity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Prion protein is decreased in Alzheimer's brain and inversely correlates with BACE1 activity, amyloid-β levels and Braak stage.

Directory of Open Access Journals (Sweden)

Isobel J Whitehouse

Full Text Available The cellular prion protein (PrP(C has been implicated in the development of Alzheimer's disease (AD. PrP(C decreases amyloid-β (Aβ production, which is involved in AD pathogenesis, by inhibiting β-secretase (BACE1 activity. Contactin 5 (CNTN5 has also been implicated in the development of AD by a genome-wide association study. Here we measured PrP(C and CNTN5 in frontal cortex samples from 24 sporadic AD and 24 age-matched control brains and correlated the expression of these proteins with markers of AD. PrP(C was decreased in sporadic AD compared to controls (by 49%, p = 0.014 but there was no difference in CNTN5 between sporadic AD and controls (p = 0.217. PrP(C significantly inversely correlated with BACE1 activity (rs = -0.358, p = 0.006, Aβ load (rs = -0.456, p = 0.001, soluble Aβ (rs = -0.283, p = 0.026 and insoluble Aβ (rs = -0.353, p = 0.007 and PrP(C also significantly inversely correlated with the stage of disease, as indicated by Braak tangle stage (rs = -0.377, p = 0.007. CNTN5 did not correlate with Aβ load (rs = 0.040, p = 0.393, soluble Aβ (rs = 0.113, p = 0.223 or insoluble Aβ (rs = 0.169, p = 0.125. PrP(C was also measured in frontal cortex samples from 9 Down's syndrome (DS and 8 age-matched control brains. In contrast to sporadic AD, there was no difference in PrP(C in the DS brains compared to controls (p = 0.625. These data are consistent with a role for PrP(C in regulating Aβ production and indicate that brain PrP(C level may be important in influencing the onset and progression of sporadic AD.

A specific pharmacophore model of sodium-dependent glucose co-transporter 2 (SGLT2) inhibitors.

Science.gov (United States)

Tang, Chunlei; Zhu, Xiaoyun; Huang, Dandan; Zan, Xin; Yang, Baowei; Li, Ying; Du, Xiaoyong; Qian, Hai; Huang, Wenlong

2012-06-01

Sodium-dependent glucose co-transporter 2 (SGLT2) plays a pivotal role in maintaining glucose equilibrium in the human body, emerging as one of the most promising targets for the treatment of diabetes mellitus type 2. Pharmacophore models of SGLT2 inhibitors have been generated with a training set of 25 SGLT2 inhibitors using Discovery Studio V2.1. The best hypothesis (Hypo1(SGLT2)) contains one hydrogen bond donor, five excluded volumes, one ring aromatic and three hydrophobic features, and has a correlation coefficient of 0.955, cost difference of 68.76, RMSD of 0.85. This model was validated by test set, Fischer randomization test and decoy set methods. The specificity of Hypo1(SGLT2) was evaluated. The pharmacophore features of Hypo1(SGLT2) were different from the best pharmacophore model (Hypo1(SGLT1)) of SGLT1 inhibitors we developed. Moreover, Hypo1(SGLT2) could effectively distinguish selective inhibitors of SGLT2 from those of SGLT1. These results indicate that a highly predictive and specific pharmacophore model of SGLT2 inhibitors has been successfully obtained. Then Hypo1(SGLT2) was used as a 3D query to screen databases including NCI and Maybridge for identifying new inhibitors of SGLT2. The hit compounds were subsequently subjected to filtering by Lipinski's rule of five. And several compounds selected from the top ranked hits have been suggested for further experimental assay studies.

The design strategy of selective PTP1B inhibitors over TCPTP.

Science.gov (United States)

Li, XiangQian; Wang, LiJun; Shi, DaYong

2016-08-15

Protein tyrosine phosphatase 1B (PTP1B) has already been well studied as a highly validated therapeutic target for diabetes and obesity. However, the lack of selectivity limited further studies and clinical applications of PTP1B inhibitors, especially over T-cell protein tyrosine phosphatase (TCPTP). In this review, we enumerate the published specific inhibitors of PTP1B, discuss the structure-activity relationships by analysis of their X-ray structures or docking results, and summarize the characteristic of selectivity related residues and groups. Furthermore, the design strategy of selective PTP1B inhibitors over TCPTP is also proposed. We hope our work could provide an effective way to gain specific PTP1B inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

Profile of yttrium segregation in BaCe0,9Y0,1O3-δ as function of sintering temperature

International Nuclear Information System (INIS)

Hosken, C.M.; Souza, D.P.F. de

2010-01-01

Researches on solid oxide fuel cells indicate barium cerate perovskite as a very attractive material for using as electrolyte due to its high protonic conductivity. The objective of this work is investigate the yttrium segregation during sintering of BaCe 0,9 Y 0,1 O 3-δ doped with Zn O as a sintering aid. The powders were prepared by citrate process. Powders were isostatic pressed into pellets and sintered in air at 1200, 1275, 1325 and 1400 deg C. The samples were characterized by scanning electron microscopy, X-ray diffraction and impedance spectroscopy. Secondary phase containing Yttrium and Cerium was detected as sintering temperature increased. Increase of the lattice parameter and activation energy for electrical conductivity were also detected on samples sintered at 1400 deg C. (author)

Seed-Specific Stable Expression of the ?-AI1 Inhibitor in Coffee Grains and the In Vivo Implications for the Development of the Coffee Berry Borer

OpenAIRE

Albuquerque, ?rika V. S.; Bezerra, Caroline A.; Romero, Juan V.; Valencia, Jorge W. A.; Valencia-Jim?nez, Arnubio; Pimenta, Lucas M.; Barbosa, Aulus E. A. D.; Silva, Maria C. M.; Meneguim, Ana M.; S?, Maria Eug?nia L.; Engler, Gilbert; de Almeida-Engler, Janice; Fernandez, Diana; Grossi-de-S?, Maria F.

2015-01-01

Genetic transformation of coffee (Coffea spp.), the second most traded commodity worldwide, is an alternative approach to introducing features that cannot be introgressed by traditional crossings. The transgenic stability, heritability and quantitative and spatial expression patterns of the seed-specific promoter phytohemagglutinin (PHA-L) from Phaseolus vulgaris were characterized in genetically modified C. arabica expressing the ?-amylase inhibitor-1 (?-AI1) gene. The ?-AI1 inhibitor shows ...

IMD-4690, a novel specific inhibitor for plasminogen activator inhibitor-1, reduces allergic airway remodeling in a mouse model of chronic asthma via regulating angiogenesis and remodeling-related mediators.

Directory of Open Access Journals (Sweden)

Toshifumi Tezuka

Full Text Available Plasminogen activator inhibitor (PAI-1 is the principal inhibitor of plasminogen activators, and is responsible for the degradation of fibrin and extracellular matrix. IMD-4690 is a newly synthesized inhibitor for PAI-1, whereas the effect on allergic airway inflammation and remodeling is still unclear. We examined the in vivo effects by using a chronic allergen exposure model of bronchial asthma in mice. The model was generated by an immune challenge for 8 weeks with house dust mite antigen, Dermatophagoides pteronyssinus (Dp. IMD-4690 was intraperitoneally administered during the challenge. Lung histopathology, hyperresponsiveness and the concentrations of mediators in lung homogenates were analyzed. The amount of active PAI-1 in the lungs was increased in mice treated with Dp. Administration with IMD-4690 reduced an active/total PAI-1 ratio. IMD-4690 also reduced the number of bronchial eosinophils in accordance with the decreased expressions of Th2 cytokines in the lung homogenates. Airway remodeling was inhibited by reducing subepithelial collagen deposition, smooth muscle hypertrophy, and angiogenesis. The effects of IMD-4690 were partly mediated by the regulation of TGF-β, HGF and matrix metalloproteinase. These results suggest that PAI-1 plays crucial roles in airway inflammation and remodeling, and IMD-4690, a specific PAI-1 inhibitor, may have therapeutic potential for patients with refractory asthma due to airway remodeling.

IMD-4690, a novel specific inhibitor for plasminogen activator inhibitor-1, reduces allergic airway remodeling in a mouse model of chronic asthma via regulating angiogenesis and remodeling-related mediators.

Science.gov (United States)

Tezuka, Toshifumi; Ogawa, Hirohisa; Azuma, Masahiko; Goto, Hisatsugu; Uehara, Hisanori; Aono, Yoshinori; Hanibuchi, Masaki; Yamaguchi, Yoichi; Fujikawa, Tomoyuki; Itai, Akiko; Nishioka, Yasuhiko

2015-01-01

Plasminogen activator inhibitor (PAI)-1 is the principal inhibitor of plasminogen activators, and is responsible for the degradation of fibrin and extracellular matrix. IMD-4690 is a newly synthesized inhibitor for PAI-1, whereas the effect on allergic airway inflammation and remodeling is still unclear. We examined the in vivo effects by using a chronic allergen exposure model of bronchial asthma in mice. The model was generated by an immune challenge for 8 weeks with house dust mite antigen, Dermatophagoides pteronyssinus (Dp). IMD-4690 was intraperitoneally administered during the challenge. Lung histopathology, hyperresponsiveness and the concentrations of mediators in lung homogenates were analyzed. The amount of active PAI-1 in the lungs was increased in mice treated with Dp. Administration with IMD-4690 reduced an active/total PAI-1 ratio. IMD-4690 also reduced the number of bronchial eosinophils in accordance with the decreased expressions of Th2 cytokines in the lung homogenates. Airway remodeling was inhibited by reducing subepithelial collagen deposition, smooth muscle hypertrophy, and angiogenesis. The effects of IMD-4690 were partly mediated by the regulation of TGF-β, HGF and matrix metalloproteinase. These results suggest that PAI-1 plays crucial roles in airway inflammation and remodeling, and IMD-4690, a specific PAI-1 inhibitor, may have therapeutic potential for patients with refractory asthma due to airway remodeling.

Acetylation site specificities of lysine deacetylase inhibitors in human cells

DEFF Research Database (Denmark)

Schölz, Christian; Weinert, Brian Tate; Wagner, Sebastian A

2015-01-01

Lysine deacetylases inhibitors (KDACIs) are used in basic research, and many are being investigated in clinical trials for treatment of cancer and other diseases. However, their specificities in cells are incompletely characterized. Here we used quantitative mass spectrometry (MS) to obtain acety......1-α, providing a possible mechanistic explanation of its adverse, pro-inflammatory effects. Our results offer a systems view of KDACI specificities, providing a framework for studying function of acetylation and deacetylases....

Structure based design of 11β-HSD1 inhibitors.

Science.gov (United States)

Singh, Suresh; Tice, Colin

2010-11-01

Controlling elevated tissue-specific levels of cortisol may provide a novel therapeutic approach for treating metabolic syndrome. This concept has spurred large scale medicinal chemistry efforts in the pharmaceutical industry for the design of 11β-HSD1 inhibitors. High resolution X-ray crystal structures of inhibitors in complex with the enzyme have facilitated the structure-based design of diverse classes of molecules. A summary of binding modes, trends in structure-activity relationships, and the pharmacodynamic data of inhibitors from each class is presented.

Design, Synthesis, and Biological Evaluation of 2-(Benzylamino-2-HydroxyalkylIsoindoline-1,3-Diones Derivatives as Potential Disease-Modifying Multifunctional Anti-Alzheimer Agents

Directory of Open Access Journals (Sweden)

Dawid Panek

2018-02-01

Full Text Available The complex nature of Alzheimer’s disease calls for multidirectional treatment. Consequently, the search for multi-target-directed ligands may lead to potential drug candidates. The aim of the present study is to seek multifunctional compounds with expected activity against disease-modifying and symptomatic targets. A series of 15 drug-like various substituted derivatives of 2-(benzylamino-2-hydroxyalkylisoindoline-1,3-diones was designed by modification of cholinesterase inhibitors toward β-secretase inhibition. All target compounds have been synthesized and tested against eel acetylcholinesterase (eeAChE, equine serum butyrylcholinesterase (eqBuChE, human β-secretase (hBACE-1, and β-amyloid (Aβ-aggregation. The most promising compound, 12 (2-(5-(benzylamino-4-hydroxypentylisoindoline-1,3-dione, displayed inhibitory potency against eeAChE (IC50 = 3.33 μM, hBACE-1 (43.7% at 50 μM, and Aβ-aggregation (24.9% at 10 μM. Molecular modeling studies have revealed possible interaction of compound 12 with the active sites of both enzymes—acetylcholinesterase and β-secretase. In conclusion: modifications of acetylcholinesterase inhibitors led to the discovery of a multipotent anti-Alzheimer’s agent, with moderate and balanced potency, capable of inhibiting acetylcholinesterase, a symptomatic target, and disease-modifying targets: β-secretase and Aβ-aggregation.

Discovery of novel selenium derivatives as Pin1 inhibitors by high-throughput screening

International Nuclear Information System (INIS)

Subedi, Amit; Shimizu, Takeshi; Ryo, Akihide; Sanada, Emiko; Watanabe, Nobumoto; Osada, Hiroyuki

2016-01-01

Peptidyl prolyl cis/trans isomerization by Pin1 regulates various oncogenic signals during cancer progression, and its inhibition through multiple approaches has established Pin1 as a therapeutic target. However, lack of simplified screening systems has limited the discovery of potent Pin1 inhibitors. We utilized phosphorylation-dependent binding of Pin1 to its specific substrate to develop a screening system for Pin1 inhibitors. Using this system, we screened a chemical library, and identified a novel selenium derivative as Pin1 inhibitor. Based on structure-activity guided chemical synthesis, we developed more potent Pin1 inhibitors that inhibited cancer cell proliferation. -- Highlights: •Novel screening for Pin1 inhibitors based on Pin1 binding is developed. •A novel selenium compound is discovered as Pin1 inhibitor. •Activity guided chemical synthesis of selenium derivatives resulted potent Pin1 inhibitors.

Bioassay-Guided Isolated Compounds from Morinda officinalis Inhibit Alzheimer’s Disease Pathologies

Directory of Open Access Journals (Sweden)

Yoon Kyoung Lee

2017-09-01

Full Text Available Due to the side effects of synthetic drugs, the therapeutic potential of natural products for Alzheimer’s disease (AD has gained interest. Morinda officinalis has demonstrated inhibitory effects on geriatric diseases, such as bone loss and osteoporosis. However, although AD is a geriatric disease, M. officinalis has not been evaluated in an AD bioassay. Therefore, M. officinalis extracts and fractions were tested for AD-related activity, including inhibition of acetylcholinesterase (AChE, butyrylcholinesterase (BChE, β-site amyloid precursor protein cleaving enzyme 1 (BACE1, and advanced glycation end-product (AGE formation. A bioassay-guided approach led to isolation of 10 active compounds, eight anthraquinones (1–8, one coumarin (9, and one phytosterol (10, from n-hexane and ethyl acetate fractions of M. officinalis. The five anthraquinones (4–8 were stronger inhibitors of AChE than were other compounds. Compounds 3 and 9 were good inhibitors of BChE, and compounds 3 and 8 were good inhibitors of BACE1. Compounds 1–5 and 7–9 were more active than the positive control in inhibiting AGE formation. In addition, we first suggested a structure-activity relationship by which anthraquinones inhibit AChE and BACE1. Our findings demonstrate the preventive and therapeutic efficacy of M. officinalis for AD and its potential use as a natural alternative medicine.

Saururus cernuus lignans-Potent small molecule inhibitors of hypoxia-inducible factor-1

International Nuclear Information System (INIS)

Hossain, Chowdhury Faiz; Kim, Yong-Pil; Baerson, Scott R.; Zhang Lei; Bruick, Richard K.; Mohammed, Kaleem A.; Agarwal, Ameeta K.; Nagle, Dale G.; Zhou Yudong

2005-01-01

Hypoxia-inducible factor-1 (HIF-1) represents an important tumor-selective therapeutic target for solid tumors. In search of novel small molecule HIF-1 inhibitors, 5400 natural product-rich extracts from plants, marine organisms, and microbes were examined for HIF-1 inhibitory activities using a cell-based reporter assay. Bioassay-guided fractionation and isolation, followed by structure elucidation, yielded three potent natural product-derived HIF-1 inhibitors and two structurally related inactive compounds. In a T47D cell-based reporter assay, manassantin B 1 , manassantin A, and 4-O-methylsaucerneol inhibited hypoxia-induced HIF-1 activation with IC 50 values of 3, 3, and 20 nM, respectively. All three compounds are relatively hypoxia-specific inhibitors of HIF-1 activation, in comparison to other stimuli. The hypoxic induction of HIF-1 target genes CDKN1A, VEGF, and GLUT-1 were also inhibited. These compounds inhibit HIF-1 by blocking hypoxia-induced nuclear HIF-1α protein accumulation without affecting HIF-1α mRNA levels. In addition, preliminary structure-activity studies suggest specific structural requirements for this class of HIF-1 inhibitors

PF-4708671, a specific inhibitor of p70 ribosomal S6 kinase 1, activates Nrf2 by promoting p62-dependent autophagic degradation of Keap1

Energy Technology Data Exchange (ETDEWEB)

Park, Jeong Su [Severance Biomedical Science Institute (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Kang, Dong Hoon [Department of Life Science and Ewha Research Center for Systems Biology (Korea, Republic of); The Research Center for Cell Homeostasis, Ewha Womans University, Seoul 127-750 (Korea, Republic of); Lee, Da Hyun [Severance Biomedical Science Institute (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Bae, Soo Han, E-mail: soohanbae@yuhs.ac [Severance Biomedical Science Institute (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of)

2015-10-23

p70 ribosomal S6 kinase 1 (S6K1) is an important serine/threonine kinase and downstream target of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. PF-4708671 is a specific inhibitor of S6K1, and prevents S6K1-mediated phosphorylation of the S6 protein. PF-4708671 treatment often leads to apoptotic cell death. However, the protective mechanism against PF-4708671-induced cell death has not been elucidated. The nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway is essential for protecting cells against oxidative stress. p62, an adaptor protein in the autophagic process, enhances Nrf2 activation through the impairment of Keap1 activity. In this study, we showed that PF-4708671 induces autophagic Keap1 degradation-mediated Nrf2 activation in p62-dependent manner. Furthermore, p62-dependent Nrf2 activation plays a crucial role in protecting cells from PF-4708671-mediated apoptosis. - Highlights: • PF-4708671, a S6K1-specific inhibitor, prevents S6K1-mediated S6 phosphorylation. • However, PF-4708671 treatment often leads to apoptotic cell death. • Protective mechanism against PF-4708671-induced cell death remains to be elucidated. • PF-4708671 induced p62-dependent, autophagic Keap1 degradation-mediated Nrf2 activation. • p62-dependent Nrf2 activation protects cells from PF-4708671-mediated apoptosis.

Discovery of natural mouse serum derived HIV-1 entry inhibitor(s).

Science.gov (United States)

Wei, M; Chen, Y; Xi, J; Ru, S; Ji, M; Zhang, D; Fang, Q; Tang, B

Among rationally designed human immunodeficiency virus 1 (HIV-1) inhibitors, diverse natural factors have showed as potent anti-HIV activity in human blood. We have discovered that the boiled supernatant of healthy mouse serum could suppress HIV-1 entry, and exhibited reduced inhibitory activity after trypsin digestion. Further analysis demonstrated that only the fraction containing 10-25 K proteins could inhibit HIV-1 mediated cell-cell fusion. These results suggest that the 10-25 K protein(s) is novel natural HIV-1 entry inhibitor(s). Our findings provide important information about novel natural HIV entry inhibitors in mouse serum.

Role of synthetic route on the transport properties of BaCe1-xYxO3 proton conductor

International Nuclear Information System (INIS)

Chiodelli, Gaetano; Malavasi, Lorenzo; Tealdi, Cristina; Barison, Simona; Battagliarin, Marino; Doubova, Lioudmila; Fabrizio, Monica; Mortalo, Cecilia; Gerbasi, Rosalba

2009-01-01

In this paper the role of the preparation route on the transport properties of the BaCe 1-x Y x O 3-δ solid solution with x = 0, 0.1, 0.15, and 0.2 has been studied. In particular, the samples were synthesized by means of the solid-state reaction and by a modified Pechini method. The effect of grain size on the sintering behaviour of the two samples batches was investigated by means of impedance spectroscopy and electron microscopy. It was found that a good sintering of the pellets can be achieved at 1250 deg. C for the samples prepared through the Pechini method. The ceramic route, even at higher temperatures, does not lead to good density values. The extensive conductivity measurements as a function of doping and gas environment (pure oxygen and argon + 10% H 2 + water) we carried out showed that the optimal Y-doping is around 15% and that a significant proton conductivity can be achieved for T lower than 500 deg. C. Above this temperature, the role of oxygen defects starts becoming relevant. Finally, the presence of a slope change in all the Arrhenius plots, irrespective to the gas environment, at about 450-500 deg. C was observed and qualitatively interpreted as due to a change in the nature of the main charge carriers involved

FoxM1 is a general target for proteasome inhibitors.

Directory of Open Access Journals (Sweden)

Uppoor G Bhat

2009-08-01

Full Text Available Proteasome inhibitors are currently in the clinic or in clinical trials, but the mechanism of their anticancer activity is not completely understood. The oncogenic transcription factor FoxM1 is one of the most overexpressed genes in human tumors, while its expression is usually halted in normal non-proliferating cells. Previously, we established that thiazole antibiotics Siomycin A and thiostrepton inhibit FoxM1 and induce apoptosis in human cancer cells. Here, we report that Siomycin A and thiostrepton stabilize the expression of a variety of proteins, such as p21, Mcl-1, p53 and hdm-2 and also act as proteasome inhibitors in vitro. More importantly, we also found that well-known proteasome inhibitors such as MG115, MG132 and bortezomib inhibit FoxM1 transcriptional activity and FoxM1 expression. In addition, overexpression of FoxM1 specifically protects against bortezomib-, but not doxorubicin-induced apoptosis. These data suggest that negative regulation of FoxM1 by proteasome inhibitors is a general feature of these drugs and it may contribute to their anticancer properties.

The effects of residual platelets in plasma on plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays.

Directory of Open Access Journals (Sweden)

Marlien Pieters

Full Text Available Due to controversial evidence in the literature pertaining to the activity of plasminogen activator inhibitor-1 in platelets, we examined the effects of residual platelets present in plasma (a potential pre-analytical variable on various plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays. Blood samples were collected from 151 individuals and centrifuged at 352 and 1500 g to obtain plasma with varying numbers of platelet. In a follow-up study, blood samples were collected from an additional 23 individuals, from whom platelet-poor (2000 g, platelet-containing (352 g and platelet-rich plasma (200 g were prepared and analysed as fresh-frozen and after five defrost-refreeze cycles (to determine the contribution of in vitro platelet degradation. Plasminogen activator inhibitor-1 activity, plasminogen activator inhibitor-1 antigen, tissue plasminogen activator/plasminogen activator inhibitor-1 complex, plasma clot lysis time, β-thromboglobulin and plasma platelet count were analysed. Platelet α-granule release (plasma β-thromboglobulin showed a significant association with plasminogen activator inhibitor-1 antigen levels but weak associations with plasminogen activator inhibitor-1 activity and a functional marker of fibrinolysis, clot lysis time. Upon dividing the study population into quartiles based on β-thromboglobulin levels, plasminogen activator inhibitor-1 antigen increased significantly across the quartiles while plasminogen activator inhibitor-1 activity and clot lysis time tended to increase in the 4th quartile only. In the follow-up study, plasma plasminogen activator inhibitor-1 antigen was also significantly influenced by platelet count in a concentration-dependent manner. Plasma plasminogen activator inhibitor-1 antigen levels increased further after complete platelet degradation. Residual platelets in plasma significantly influence plasma plasminogen activator inhibitor-1 antigen levels mainly

Anti-adipogenic effects of KD025 (SLx-2119), a ROCK2-specific inhibitor, in 3T3-L1 cells.

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Diep, Duy Trong Vien; Hong, Kyungki; Khun, Triyeng; Zheng, Mei; Ul-Haq, Asad; Jun, Hee-Sook; Kim, Young-Bum; Chun, Kwang-Hoon

2018-02-06

Adipose tissue is a specialized organ that synthesizes and stores fat. During adipogenesis, Rho and Rho-associated kinase (ROCK) 2 are inactivated, which enhances the expression of pro-adipogenic genes and induces the loss of actin stress fibers. Furthermore, pan ROCK inhibitors enhance adipogenesis in 3T3-L1 cells. Here, we show that KD025 (formerly known as SLx-2119), a ROCK2-specific inhibitor, suppresses adipogenesis in 3T3-L1 cells partially through a ROCK2-independent mechanism. KD025 downregulated the expression of key adipogenic transcription factors PPARγ and C/EBPα during adipogenesis in addition to lipogenic factors FABP4 and Glut4. Interestingly, adipogenesis was blocked by KD025 during days 1~3 of differentiation; after differentiation terminated, lipid accumulation was unaffected. Clonal expansion occurred normally in KD025-treated cells. These results suggest that KD025 could function during the intermediate stage after clonal expansion. Data from depletion of ROCKs showed that KD025 suppressed cell differentiation partially independent of ROCK's activity. Furthermore, no further loss of actin stress fibers emerged in KD025-treated cells during and after differentiation compared to control cells. These results indicate that in contrast to the pro-adipogenic effect of pan-inhibitors, KD025 suppresses adipogenesis in 3T3-L1 cells by regulating key pro-adipogenic factors. This outcome further implies that KD025 could be a potential anti-adipogenic/obesity agent.

Enzyme immunoassay for measurement of murine plasminogen activator inhibitor-1, employing a specific antibody produced by the DNA vaccine method.

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Yamada, Takayuki; Takagi, Akira; Takeshita, Kyosuke; Yamamoto, Koji; Ito, Masafumi; Matsushita, Tadashi; Murate, Takashi; Saito, Hidehiko; Kojima, Tetsuhito

2003-01-01

We developed a sensitive immunoassay to determine the concentration of mouse plasminogen activator inhibitor-1. The assay was a non-competitive sandwich enzyme-linked immunosorbent assay (ELISA) based on the production of a specific polyclonal antibody against mouse plasminogen activator inhibitor type-1 (PAI-1) used both as a trapping and detecting antibody. This antibody was raised in a rabbit by direct introduction of the expression vector plasmid DNA encoding mouse PAI-1, instead of conventional immunization with the purified protein. The standard curve was constructed with a recombinant glutathione S-transferase (GST)-mouse PAI-1 fusion protein (GST-mPAI-1) and dose-response of the assay was linear for GST-mPAI-1 between 6.25 and 100 pM. In order to assess the consistency of the assay, we measured PAI-1 antigen in normal mouse pooled plasma several times. We found that the intra-assay and inter-assay coefficients of variation (CV) were 4.8% and 9.2%, respectively, indicating that the ELISA would be sufficiently repeatable and reproducible. In this assay, lipopolysaccharide (LPS)-injected mice showed substantially higher levels (22-fold) of plasma PAI-1 antigen than did control mice (12.5+/-2.4 vs. 0.58+/-0.16 nM), similar to results reported elsewhere. Taken together, the DNA vaccine method is extremely useful for preparing specific antibodies against mouse PAI-1, which can be utilized to establish the ELISA and analyze the profile of PAI-1 distributions in mice under various conditions. This approach might also be useful for immunological investigation of other coagulation factors and related proteins.

Inhibitin: a specific inhibitor of sodium/sodium exchange in erythrocytes.

OpenAIRE

Morgan, K; Brown, R C; Spurlock, G; Southgate, K; Mir, M A

1986-01-01

An inhibitor of ouabain-insensitive sodium/sodium exchange in erythrocytes has been isolated from leukemic promyelocytes. To explore the specific effects of this inhibitor, named inhibitin, sodium transport experiments were carried out in human erythrocytes. Inhibitin reduced ouabain-insensitive bidirectional sodium transport. It did not change net sodium fluxes, had no significant effect on rubidium influx, and did not inhibit sodium-potassium-ATPase activity. The inhibitory effect of inhibi...

Specific CLK inhibitors from a novel chemotype for regulation of alternative splicing

DEFF Research Database (Denmark)

Fedorov, Oleg; Huber, Kilian; Eisenreich, Andreas

2011-01-01

There is a growing recognition of the importance of protein kinases in the control of alternative splicing. To define the underlying regulatory mechanisms, highly selective inhibitors are needed. Here, we report the discovery and characterization of the dichloroindolyl enaminonitrile KH-CB19......, a potent and highly specific inhibitor of the CDC2-like kinase isoforms 1 and 4 (CLK1/CLK4). Cocrystal structures of KH-CB19 with CLK1 and CLK3 revealed a non-ATP mimetic binding mode, conformational changes in helix aC and the phosphate binding loop and halogen bonding to the kinase hinge region. KH-CB19...... effectively suppressed phosphorylation of SR (serine/arginine) proteins in cells, consistent with its expected mechanism of action. Chemical inhibition of CLK1/CLK4 generated a unique pattern of splicing factor dephosphorylation and had at low nM concentration a profound effect on splicing of the two tissue...

A screen for kinase inhibitors identifies antimicrobial imidazopyridine aminofurazans as specific inhibitors of the Listeria monocytogenes PASTA kinase PrkA.

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Schaenzer, Adam J; Wlodarchak, Nathan; Drewry, David H; Zuercher, William J; Rose, Warren E; Striker, Rob; Sauer, John-Demian

2017-10-13

Bacterial signaling systems such as protein kinases and quorum sensing have become increasingly attractive targets for the development of novel antimicrobial agents in a time of rising antibiotic resistance. The family of bacterial P enicillin-binding-protein A nd S erine/ T hreonine kinase- A ssociated (PASTA) kinases is of particular interest due to the role of these kinases in regulating resistance to β-lactam antibiotics. As such, small-molecule kinase inhibitors that target PASTA kinases may prove beneficial as treatments adjunctive to β-lactam therapy. Despite this interest, only limited progress has been made in identifying functional inhibitors of the PASTA kinases that have both activity against the intact microbe and high kinase specificity. Here, we report the results of a small-molecule screen that identified GSK690693, an imidazopyridine aminofurazan-type kinase inhibitor that increases the sensitivity of the intracellular pathogen Listeria monocytogenes to various β-lactams by inhibiting the PASTA kinase PrkA. GSK690693 potently inhibited PrkA kinase activity biochemically and exhibited significant selectivity for PrkA relative to the Staphylococcus aureus PASTA kinase Stk1. Furthermore, other imidazopyridine aminofurazans could effectively inhibit PrkA and potentiate β-lactam antibiotic activity to varying degrees. The presence of the 2-methyl-3-butyn-2-ol (alkynol) moiety was important for both biochemical and antimicrobial activity. Finally, mutagenesis studies demonstrated residues in the back pocket of the active site are important for GSK690693 selectivity. These data suggest that targeted screens can successfully identify PASTA kinase inhibitors with both biochemical and antimicrobial specificity. Moreover, the imidazopyridine aminofurazans represent a family of PASTA kinase inhibitors that have the potential to be optimized for selective PASTA kinase inhibition.

Identification of tyrosinase specific inhibitors from Xanthium strumarium fruit extract using ultrafiltration-high performance liquid chromatography.

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Wang, Zhiqiang; Hwang, Seung Hwan; Huang, Bo; Lim, Soon Sung

2015-10-01

In this study, a strategy based on ultrafiltration-high performance liquid chromatography coupled with diode array detection (UF-HPLC-DAD) was proposed for screening tyrosinase specific inhibitors in Xanthii fructus. The false negatives were distinguished by optimizing the UF-HPLC-DAD parameters to reduce the background noise; the false positives were distinguished by introducing a blocked tyrosinase in the control group for comparison. To obtain the best blocker, the competitive experiments were performed using various known ligands. Using this strategy, three competitive inhibitors (protocatechuic acid; 3,5-di-O-caffeoylquinic acid; and 1,5-di-O-caffeoylquinic acid) and one mixed-type inhibitor (chlorogenic acid) were identified. These results were verified using tyrosinase inhibition assay, kinetic analysis, and structural simulation of the complex. Our experimental results suggest that the proposed strategy could be useful for high-throughput identification of tyrosinase specific inhibitors in natural products. Copyright © 2015 Elsevier B.V. All rights reserved.

Changing the inhibitory specificity and function of Cucurbita maxima trypsin inhibitor-V by site-directed mutagenesis.

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Wen, L; Lee, I; Chen, G; Huang, J K; Gong, Y; Krishnamoorthi, R

1995-02-27

Cucurbita maxima trypsin inhibitor-V (CMTI-V) is also a specific inhibitor of human blood coagulation factor beta-factor XIIa. A recombinant version of CMTI-V has allowed probing of roles of individual amino acid residues including the reactive site residue, lysine (P1), by site-directed mutagenesis. The K44R showed at least a 5-fold increase in inhibitory activity toward human beta-factor XIIa, while there was no change toward bovine trypsin. This result demonstrates that beta-factor-XIIa prefers an arginine residue over lysine residue, while trypsin is non-specific to lysine or arginine in its binding pocket. On the other hand, the specificity of CMTI-V could be changed from trypsin to chymotrypsin inhibition by mutation of the P1 residue to either leucine or methionine (K44L or K44M).

Tissue-specific expression and regulation by 1,25(OH)2D3 of chick protein kinase inhibitor (PKI) mRNA.

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Marchetto, G S; Henry, H L

1997-02-01

The heat-stable protein kinase inhibitor (PKI) protein is a specific and potent competitive inhibitor of the catalytic subunit of cAMP-dependent protein kinase (PKA). Previously, it has been shown that vitamin D status affects chick kidney PKI activity: a 5- to 10-fold increase in PKI activity was observed in kidneys of chronically vitamin D-deficient chicks and treatment with 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) in cultured kidney cells resulted in a 95% decrease in PKI activity. The authors have recently cloned the cDNA for chick kidney PKI and have used the coding sequence to study the regulation of PKI mRNA. Northern analysis showed the expression of two PKI messages, which are 2.7 and 3.3 kb in size. These mRNAs are expressed in brain, muscle, testis, and kidney, but not in pancreas, liver, or intestine. PKI mRNA steady-state levels are downregulated by 47% in kidneys from vitamin D-replete chicks as compared to vitamin D-deficient chicks. PKI mRNA levels in brain, muscle, and testis are not affected by vitamin D status. Treatment of primary chick kidney cultures treated with 10(-7) M 1,25(OH)2D3 for 24h resulted in a 20-30% decrease in PKI mRNA. 1,25(OH)2D3 treatment does not affect the stability of PKI mRNA as determined by treatment of cell cultures with actinomycin D. This study shows that 1,25(OH)2D3 directly and tissue-specifically downregulates PKI mRNA in the chick kidney.

A dual-specificity isoform of the protein kinase inhibitor PKI produced by alternate gene splicing.

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Kumar, Priyadarsini; Walsh, Donal A

2002-03-15

We have previously shown that the protein kinase inhibitor beta (PKIbeta) form of the cAMP-dependent protein kinase inhibitor exists in multiple isoforms, some of which are specific inhibitors of the cAMP-dependent protein kinase, whereas others also inhibit the cGMP-dependent enzyme [Kumar, Van Patten and Walsh (1997), J. Biol. Chem. 272, 20011-20020]. We have now demonstrated that the switch from a cAMP-dependent protein kinase (PKA)-specific inhibitor to one with dual specificity arises as a consequence of alternate gene splicing. We have confirmed using bacterially produced pure protein that a single inhibitor species has dual specificity for both PKA and cGMP-dependent protein kinase (PKG), inhibiting each with very high and closely similar inhibitory potencies. The gene splicing converted a protein with 70 amino acids into one of 109 amino acids, and did not change the inhibitory potency to PKA, but changed it from a protein that had no detectable PKG inhibitory activity to one that now inhibited PKG in the nanomolar range.

Novel Tacrine-Benzofuran Hybrids as Potent Multitarget-Directed Ligands for the Treatment of Alzheimer's Disease: Design, Synthesis, Biological Evaluation, and X-ray Crystallography.

Science.gov (United States)

Zha, Xiaoming; Lamba, Doriano; Zhang, Lili; Lou, Yinghan; Xu, Changxu; Kang, Di; Chen, Li; Xu, Yungen; Zhang, Luyong; De Simone, Angela; Samez, Sarah; Pesaresi, Alessandro; Stojan, Jure; Lopez, Manuela G; Egea, Javier; Andrisano, Vincenza; Bartolini, Manuela

2016-01-14

Twenty-six new tacrine-benzofuran hybrids were designed, synthesized, and evaluated in vitro on key molecular targets for Alzheimer's disease. Most hybrids exhibited good inhibitory activities on cholinesterases and β-amyloid self-aggregation. Selected compounds displayed significant inhibition of human β-secretase-1 (hBACE-1). Among the 26 hybrids, 2e showed the most interesting profile as a subnanomolar selective inhibitor of human acetylcholinesterase (hAChE) (IC50 = 0.86 nM) and a good inhibitor of both β-amyloid aggregation (hAChE- and self-induced, 61.3% and 58.4%, respectively) and hBACE-1 activity (IC50 = 1.35 μM). Kinetic studies showed that 2e acted as a slow, tight-binding, mixed-type inhibitor, while X-ray crystallographic studies highlighted the ability of 2e to induce large-scale structural changes in the active-site gorge of Torpedo californica AChE (TcAChE), with significant implications for structure-based drug design. In vivo studies confirmed that 2e significantly ameliorates performances of scopolamine-treated ICR mice. Finally, 2e administration did not exhibit significant hepatotoxicity.

Design of novel HIV-1 protease inhibitors incorporating isophthalamide-derived P2-P3 ligands: Synthesis, biological evaluation and X-ray structural studies of inhibitor-HIV-1 protease complex

Energy Technology Data Exchange (ETDEWEB)

Ghosh, Arun K.; Brindisi, Margherita; Nyalapatla, Prasanth R.; Takayama, Jun; Ella-Menye, Jean-Rene; Yashchuk, Sofiya; Agniswamy, Johnson; Wang, Yuan-Fang; Aoki, Manabu; Amano, Masayuki; Weber, Irene T.; Mitsuya, Hiroaki

2017-10-01

Based upon molecular insights from the X-ray structures of inhibitor-bound HIV-1 protease complexes, we have designed a series of isophthalamide-derived inhibitors incorporating substituted pyrrolidines, piperidines and thiazolidines as P2-P3 ligands for specific interactions in the S2-S3 extended site. Compound 4b has shown an enzyme Ki of 0.025 nM and antiviral IC50 of 69 nM. An X-ray crystal structure of inhibitor 4b-HIV-1 protease complex was determined at 1.33 Å resolution. We have also determined X-ray structure of 3b-bound HIV-1 protease at 1.27 Å resolution. These structures revealed important molecular insight into the inhibitor–HIV-1 protease interactions in the active site.

Potent and Selective Peptidyl Boronic Acid Inhibitors of the Serine Protease Prostate-Specific Antigen

Science.gov (United States)

LeBeau, Aaron M.; Singh, Pratap; Isaacs, John T.; Denmeade, Samuel R.

2012-01-01

SUMMARY Prostate cancer cells produce high (microgram to milligram/milliliter) levels of the serine protease Prostate-Specific Antigen (PSA). PSA is enzymatically active in the extracellular fluid surrounding prostate cancers but is found at 1,000- to 10,000-fold lower concentrations in the circulation, where it is inactivated due to binding to abundant serum protease inhibitors. The exclusive presence of high levels of active PSA within prostate cancer sites makes PSA an attractive candidate for targeted imaging and therapeutics. A synthetic approach based on a peptide substrate identified first peptide aldehyde and then boronic acid inhibitors of PSA. The best of these had the sequence Cbz-Ser-Ser-Lys-Leu-(boro)Leu, with a Ki for PSA of 65 nM. The inhibitor had a 60-fold higher Ki for chymotrypsin. A validated model of PSA’s catalytic site confirmed the critical interactions between the inhibitor and residues within the PSA enzyme. PMID:18635003

Docking Studies and Biological Evaluation of a Potential β-Secretase Inhibitor of 3-Hydroxyhericenone F from Hericium erinaceus.

Science.gov (United States)

Diling, Chen; Tianqiao, Yong; Jian, Yang; Chaoqun, Zheng; Ou, Shuai; Yizhen, Xie

2017-01-01

Alzheimer's disease (AD) is the most common neurodegenerative disorder, affecting approximately more than 5% of the population worldwide over the age 65, annually. The incidence of AD is expected to be higher in the next 10 years. AD patients experience poor prognosis and as a consequence new drugs and therapeutic strategies are required in order to improve the clinical responses and outcomes of AD. The purpose of the present study was to screen a certain number of potential compounds from herbal sources and investigate their corresponding mode of action. In the present study, the learning and memory effects of ethanol:water (8:2) extracts from Hericium erinaceus were evaluated on a dementia rat model. The model was established by intraperitoneal injection of 100 mg/kg/d D-galactose in rats. The results indicated that the extracts can significantly ameliorate the learning and memory abilities. Specific active ingredients were screened in vivo assays and the results were combined with molecular docking studies. Potential receptor-ligand interactions on the BACE1-inhibitor namely, 3-Hydroxyhericenone F (3HF) were investigated. The isolation of a limited amount of 3HF from the fruit body of H. erinaceus by chemical separation was conducted, and the mode of action of this compound was verified in NaN 3 -induced PC12 cells. The cell-based assays demonstrated that 3HF can significantly down-regulate the expression of BACE1 ( p < 0.01), while additional AD intracellular markers namely, p-Tau and Aβ 1-42 were further down-regulated ( p < 0.05). The data further indicate that 3HF can ameliorate certain mitochondrial dysfunction conditions by the reversal of the decreasing level of mitochondrial respiratory chain complexes, the calcium ion levels ([Ca 2+ ]), the inhibiton in the production of ROS, the increase in the mitochondrial membrane potential and ATP levels, and the regulation of the expression levels of the genes encoding for the p21, COX I, COX II, PARP1, and NF

Synthesis of ceramic BaCe_0_._7Zr_0_,_1Y_0_._2O_3_-_δ electrolyte containing ZnO through the method hidrogel

International Nuclear Information System (INIS)

Vieira, J.H.A.; Fagury Neto, E.; Rabelo, A.A.

2014-01-01

The solid oxide fuel cells are promising sources of sustainable energy, once they produce electricity from an electrochemical reaction only with steam water as the product of this reaction. In this paper the synthesis of ceramic electrolyte BaCe_0_._7Zr_0_._1Y_0_._2O_3_-_δ using the hydrogel method which involves using an organic gelatin inexpensive and nontoxic modifying the conventional route of polymeric precursor was performed. It was used different amounts of the sintering additive ZnO to decrease the sintering temperature, typically around 1600 °C, towards a minimum relative density of 95%. The polymeric resin was pyrolyzed at 300 °C, subjected to calcination at 600 °C and sintering at temperatures of 1350, 1400 and 1450 °C for 2 hours. Characterizations were carried out by determination of relative density and apparent porosity and using the techniques of differential thermal analysis and simultaneous thermogravimetric, X-ray diffraction and scanning electron microscopy.(author)

Bilingual Academic and Career Education Services for Hispanic High School Students (Project BACES). Final Evaluation Report, 1992-93. OER Report.

Science.gov (United States)

Clarke, Candice

This report presents an evaluation of the Bilingual Academic and Career Education Services for Hispanic High School Students (Project BACES), an Elementary and Secondary Education Act Title VII-funded project in its third year of operation at DeWitt Clinton High School in the Bronx and George Washington High School in Manhattan. The project served…

Discovery and study of novel protein tyrosine phosphatase 1B inhibitors

Science.gov (United States)

Zhang, Qian; Chen, Xi; Feng, Changgen

2017-10-01

Protein tyrosine phosphatase 1B (PTP1B) is considered to be a target for therapy of type II diabetes and obesity. So it is of great significance to take advantage of a computer aided drug design protocol involving the structured-based virtual screening with docking simulations for fast searching small molecule PTP1B inhibitors. Based on optimized complex structure of PTP1B bound with specific inhibitor of IX1, structured-based virtual screening against a library of natural products containing 35308 molecules, which was constructed based on Traditional Chinese Medicine database@ Taiwan (TCM database@ Taiwan), was conducted to determine the occurrence of PTP1B inhibitors using the Lubbock module and CDOCKER module from Discovery Studio 3.1 software package. The results were further filtered by predictive ADME simulation and predictive toxic simulation. As a result, 2 good drug-like molecules, namely para-benzoquinone compound 1 and Clavepictine analogue 2 were identified ultimately with the dock score of original inhibitor (IX1) and the receptor as a threshold. Binding model analyses revealed that these two candidate compounds have good interactions with PTP1B. The PTP1B inhibitory activity of compound 2 hasn't been reported before. The optimized compound 2 has higher scores and deserves further study.

PEGylated DX-1000: Pharmacokinetics and Antineoplastic Activity of a Specific Plasmin Inhibitor

Directory of Open Access Journals (Sweden)

Laetitia Devy

2007-11-01

Full Text Available Novel inhibitors of the urokinase-mediated plasminogen (plg activation system are potentially of great clinical benefit as anticancer treatments. Using phage display, we identified DX-1000 a tissue factor pathway inhibitor-derived Kunitz domain protein which is a specific high-affinity inhibitor of plasmin (pin (Ki = 99 pM. When tested in vitro, DX-1000 blocks plasminmediated pro-matrix metal loproteinase-9 (proMMP-9 activation on cells and dose-dependently inhibits tube formation, while not significantly affecting hemostasis and coagulation. However, this low-molecular weight protein inhibitor (~ 7 kDa exhibits rapid plasma clearance in mice and rabbits, limiting its potential clinical use in chronic diseases. After site-specific PEGylation, DX-1000 retains its activity and exhibits a decreased plasma clearance. This PEGylated derivative is effective in vitro, as well as potent in inhibiting tumor growth of green fluorescent protein (GFP-labeled MDA-MB-231 cells. 4PEG-DX-1000 treatment causes a significant reduction of urokinase-type plasminogen activator (uPA and plasminogen expressions, a reduction of tumor proliferation, and vascularization. 4PEG-DX-1000 treatment significantly decreases the level of active mitogenactivated protein kinase (MAPK in the primary tumors and reduces metastasis incidence. Together, our results demonstrate the potential value of plasmin inhibitors as therapeutic agents for blocking breast cancer growth and metastasis.

Crystal structures of HIV-1 nonnucleoside reverse transcriptase inhibitors: N-benzyl-4-methyl-benzimidazoles

Science.gov (United States)

Ziółkowska, Natasza E.; Michejda, Christopher J.; Bujacz, Grzegorz D.

2009-07-01

HIV-1 nonnucleoside reverse transcriptase inhibitors are potentially specific and effective drugs in AIDS therapy. The presence of two aromatic systems with an angled orientation in the molecule of the inhibitor is crucial for interactions with HIV-1 RT. The inhibitor drives like a wedge into the cluster of aromatic residues of RT HIV-1 and restrains the enzyme in a conformation that blocks the chemical step of nucleotide incorporation. Structural studies provide useful information for designing new, more active inhibitors. The crystal structures of four NNRTIs are presented here. The investigated compounds are derivatives of N-benzyl-4-methyl-benzimidazole with various aliphatic and aromatic substituents at carbon 2 positions and a 2,6-dihalogeno-substituted N-benzyl moiety. Structural data reported here show that the conformation of the investigated compounds is relatively rigid. Such feature is important for the nonnucleoside inhibitor binding to HIV-1 reverse transcriptase.

Structure-Based Design of Novel HIV-1 Protease Inhibitors to Combat Drug Resistance

Energy Technology Data Exchange (ETDEWEB)

Ghosh,A.; Sridhar, P.; Leshchenko, S.; Hussain, A.; Li, J.; Kovalevsky, A.; Walters, D.; Wedelind, J.; Grum-Tokars, V.; et al.

2006-01-01

Structure-based design and synthesis of novel HIV protease inhibitors are described. The inhibitors are designed specifically to interact with the backbone of HIV protease active site to combat drug resistance. Inhibitor 3 has exhibited exceedingly potent enzyme inhibitory and antiviral potency. Furthermore, this inhibitor maintains impressive potency against a wide spectrum of HIV including a variety of multi-PI-resistant clinical strains. The inhibitors incorporated a stereochemically defined 5-hexahydrocyclopenta[b]furanyl urethane as the P2-ligand into the (R)-(hydroxyethylamino)sulfonamide isostere. Optically active (3aS,5R,6aR)-5-hydroxy-hexahydrocyclopenta[b]furan was prepared by an enzymatic asymmetrization of meso-diacetate with acetyl cholinesterase, radical cyclization, and Lewis acid-catalyzed anomeric reduction as the key steps. A protein-ligand X-ray crystal structure of inhibitor 3-bound HIV-1 protease (1.35 Angstroms resolution) revealed extensive interactions in the HIV protease active site including strong hydrogen bonding interactions with the backbone. This design strategy may lead to novel inhibitors that can combat drug resistance.

Ribonuclease inhibitor 1 regulates erythropoiesis by controlling GATA1 translation.

Science.gov (United States)

Chennupati, Vijaykumar; Veiga, Diogo Ft; Maslowski, Kendle M; Andina, Nicola; Tardivel, Aubry; Yu, Eric Chi-Wang; Stilinovic, Martina; Simillion, Cedric; Duchosal, Michel A; Quadroni, Manfredo; Roberts, Irene; Sankaran, Vijay G; MacDonald, H Robson; Fasel, Nicolas; Angelillo-Scherrer, Anne; Schneider, Pascal; Hoang, Trang; Allam, Ramanjaneyulu

2018-04-02

Ribosomal proteins (RP) regulate specific gene expression by selectively translating subsets of mRNAs. Indeed, in Diamond-Blackfan anemia and 5q- syndrome, mutations in RP genes lead to a specific defect in erythroid gene translation and cause anemia. Little is known about the molecular mechanisms of selective mRNA translation and involvement of ribosomal-associated factors in this process. Ribonuclease inhibitor 1 (RNH1) is a ubiquitously expressed protein that binds to and inhibits pancreatic-type ribonucleases. Here, we report that RNH1 binds to ribosomes and regulates erythropoiesis by controlling translation of the erythroid transcription factor GATA1. Rnh1-deficient mice die between embryonic days E8.5 and E10 due to impaired production of mature erythroid cells from progenitor cells. In Rnh1-deficient embryos, mRNA levels of Gata1 are normal, but GATA1 protein levels are decreased. At the molecular level, we found that RNH1 binds to the 40S subunit of ribosomes and facilitates polysome formation on Gata1 mRNA to confer transcript-specific translation. Further, RNH1 knockdown in human CD34+ progenitor cells decreased erythroid differentiation without affecting myelopoiesis. Our results reveal an unsuspected role for RNH1 in the control of GATA1 mRNA translation and erythropoiesis.

Interaction of HIV-1 reverse transcriptase ribonuclease H with an acylhydrazone inhibitor.

Science.gov (United States)

Gong, Qingguo; Menon, Lakshmi; Ilina, Tatiana; Miller, Lena G; Ahn, Jinwoo; Parniak, Michael A; Ishima, Rieko

2011-01-01

HIV-1 reverse transcriptase is a bifunctional enzyme, having both DNA polymerase (RNA- and DNA-dependent) and ribonuclease H activities. HIV-1 reverse transcriptase has been an exceptionally important target for antiretroviral therapeutic development, and nearly half of the current clinically used antiretrovirals target reverse transcriptase DNA polymerase. However, no inhibitors of reverse transcriptase ribonuclease H are on the market or in preclinical development. Several drug-like small molecule inhibitors of reverse transcriptase ribonuclease H have been described, but little structural information is available about the interactions between reverse transcriptase ribonuclease H and inhibitors that exhibit antiviral activity. In this report, we describe NMR studies of the interaction of a new ribonuclease H inhibitor, BHMP07, with a catalytically active HIV-1 reverse transcriptase ribonuclease H domain fragment. We carried out solution NMR experiments to identify the interaction interface of BHMP07 with the ribonuclease H domain fragment. Chemical shift changes of backbone amide signals at different BHMP07 concentrations clearly demonstrate that BHMP07 mainly recognizes the substrate handle region in the ribonuclease H fragment. Using ribonuclease H inhibition assays and reverse transcriptase mutants, the binding specificity of BHMP07 was compared with another inhibitor, dihydroxy benzoyl naphthyl hydrazone. Our results provide a structural characterization of the ribonuclease H inhibitor interaction and are likely to be useful for further improvements of the inhibitors. © 2010 John Wiley & Sons A/S.

High resolution crystal structure of rat long chain hydroxy acid oxidase in complex with the inhibitor 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1, 2, 3-thiadiazole. Implications for inhibitor specificity and drug design

Energy Technology Data Exchange (ETDEWEB)

Chen, Zhi-wei; Vignaud, Caroline; Jaafar, Adil; Lévy, Bernard; Guéritte, Françoise; Guénard, Daniel; Lederer, Florence; Mathews, F. Scott (CNRS-UMR); (WU-MED)

2012-05-24

Long chain hydroxy acid oxidase (LCHAO) is responsible for the formation of methylguanidine, a toxic compound with elevated serum levels in patients with chronic renal failure. Its isozyme glycolate oxidase (GOX), has a role in the formation of oxalate, which can lead to pathological deposits of calcium oxalate, in particular in the disease primary hyperoxaluria. Inhibitors of these two enzymes may have therapeutic value. These enzymes are the only human members of the family of FMN-dependent L-2-hydroxy acid-oxidizing enzymes, with yeast flavocytochrome b{sub 2} (Fcb2) among its well studied members. We screened a chemical library for inhibitors, using in parallel rat LCHAO, human GOX and the Fcb2 flavodehydrogenase domain (FDH). Among the hits was an inhibitor, CCPST, with an IC{sub 50} in the micromolar range for all three enzymes. We report here the crystal structure of a complex between this compound and LCHAO at 1.3 {angstrom} resolution. In comparison with a lower resolution structure of this enzyme, binding of the inhibitor induces a conformational change in part of the TIM barrel loop 4, as well as protonation of the active site histidine. The CCPST interactions are compared with those it forms with human GOX and those formed by two other inhibitors with human GOX and spinach GOX. These compounds differ from CCPST in having the sulfur replaced with a nitrogen in the five-membered ring as well as different hydrophobic substituents. The possible reason for the {approx}100-fold difference in affinity between these two series of inhibitors is discussed. The present results indicate that specificity is an issue in the quest for therapeutic inhibitors of either LCHAO or GOX, but they may give leads for this quest.

HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors

DEFF Research Database (Denmark)

Vanangamudi, Murugesan; Poongavanam, Vasanthanathan; Namasivayam, Vigneshwaran

2017-01-01

BACKGROUND: Design of inhibitors for HIV-1 reverse transcriptase inhibition (HIV-1 RT) is one of the successful chemotherapies for the treatment of HIV infection. Among the inhibitors available for HIV-1 RT, non-nucleoside reverse transcriptase inhibitors (NNRTIs) have shown to be very promising......: The conformation dependent-alignment based (CoMFA and CoMSIA) methods have been proven very successful ligand based strategy in the drug design. Here, CoMFA and CoMSIA studies reported for structurally distinct NNRTIs including thiazolobenzimidazole, dipyridodiazepinone, 1,1,3-trioxo [1,2,4]-thiadiazine...

Seed-specific stable expression of the alpha-AI1 inhibitor in coffee grains and the in vivo implications for the development of the coffee berry borer

OpenAIRE

Albuquerque, E. V. S.; Bezerra, C. A.; Romero, J. V.; Valencia, J. W. A.; Valencia-Jimenez, A.; Pimenta, L. M.; Barbosa, Aead; Silva, M. C. M.; Meneguim, A. M.; Sa, M. E. L.; Engler, G.; de Almeida-Engler, J.; Fernandez, Diana; Grossi-de-Sa, M. F.

2015-01-01

Genetic transformation of coffee (Coffea spp.), the second most traded commodity worldwide, is an alternative approach to introducing features that cannot be introgressed by traditional crossings. The transgenic stability, heritability and quantitative and spatial expression patterns of the seed-specific promoter phytohemagglutinin (PHA-L) from Phaseolus vulgaris were characterized in genetically modified C. arabica expressing the alpha-amylase inhibitor-1 (alpha-AI1) gene. The alpha-AI1 inhi...

Propiconazole is a specific and accessible brassinosteroid (BR) biosynthesis inhibitor for Arabidopsis and maize.

Science.gov (United States)

Hartwig, Thomas; Corvalan, Claudia; Best, Norman B; Budka, Joshua S; Zhu, Jia-Ying; Choe, Sunghwa; Schulz, Burkhard

2012-01-01

Brassinosteroids (BRs) are steroidal hormones that play pivotal roles during plant development. In addition to the characterization of BR deficient mutants, specific BR biosynthesis inhibitors played an essential role in the elucidation of BR function in plants. However, high costs and limited availability of common BR biosynthetic inhibitors constrain their key advantage as a species-independent tool to investigate BR function. We studied propiconazole (Pcz) as an alternative to the BR inhibitor brassinazole (Brz). Arabidopsis seedlings treated with Pcz phenocopied BR biosynthetic mutants. The steady state mRNA levels of BR, but not gibberellic acid (GA), regulated genes increased proportional to the concentrations of Pcz. Moreover, root inhibition and Pcz-induced expression of BR biosynthetic genes were rescued by 24epi-brassinolide, but not by GA(3) co-applications. Maize seedlings treated with Pcz showed impaired mesocotyl, coleoptile, and true leaf elongation. Interestingly, the genetic background strongly impacted the tissue specific sensitivity towards Pcz. Based on these findings we conclude that Pcz is a potent and specific inhibitor of BR biosynthesis and an alternative to Brz. The reduced cost and increased availability of Pcz, compared to Brz, opens new possibilities to study BR function in larger crop species.

Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours

Energy Technology Data Exchange (ETDEWEB)

Lasko, Loren M.; Jakob, Clarissa G.; Edalji, Rohinton P.; Qiu, Wei; Montgomery, Debra; Digiammarino, Enrico L.; Hansen, T. Matt; Risi, Roberto M.; Frey, Robin; Manaves, Vlasios; Shaw, Bailin; Algire, Mikkel; Hessler, Paul; Lam, Lloyd T.; Uziel, Tamar; Faivre, Emily; Ferguson, Debra; Buchanan, Fritz G.; Martin, Ruth L.; Torrent, Maricel; Chiang, Gary G.; Karukurichi, Kannan; Langston, J. William; Weinert, Brian T.; Choudhary, Chunaram; de Vries, Peter; Van Drie, John H.; McElligott, David; Kesicki, Ed; Marmorstein, Ronen; Sun, Chaohong; Cole, Philip A.; Rosenberg, Saul H.; Michaelides, Michael R.; Lai, Albert; Bromberg, Kenneth D. (AbbVie); (UCopenhagen); (Petra Pharma); (UPENN); (JHU); (Van Drie); (Faraday)

2017-09-27

The dynamic and reversible acetylation of proteins, catalysed by histone acetyltransferases (HATs) and histone deacetylases (HDACs), is a major epigenetic regulatory mechanism of gene transcription1 and is associated with multiple diseases. Histone deacetylase inhibitors are currently approved to treat certain cancers, but progress on the development of drug-like histone actyltransferase inhibitors has lagged behind2. The histone acetyltransferase paralogues p300 and CREB-binding protein (CBP) are key transcriptional co-activators that are essential for a multitude of cellular processes, and have also been implicated in human pathological conditions (including cancer3). Current inhibitors of the p300 and CBP histone acetyltransferase domains, including natural products4, bi-substrate analogues5 and the widely used small molecule C6466,7, lack potency or selectivity. Here, we describe A-485, a potent, selective and drug-like catalytic inhibitor of p300 and CBP. We present a high resolution (1.95 Å) co-crystal structure of a small molecule bound to the catalytic active site of p300 and demonstrate that A-485 competes with acetyl coenzyme A (acetyl-CoA). A-485 selectively inhibited proliferation in lineage-specific tumour types, including several haematological malignancies and androgen receptor-positive prostate cancer. A-485 inhibited the androgen receptor transcriptional program in both androgen-sensitive and castration-resistant prostate cancer and inhibited tumour growth in a castration-resistant xenograft model. These results demonstrate the feasibility of using small molecule inhibitors to selectively target the catalytic activity of histone acetyltransferases, which may provide effective treatments for transcriptional activator-driven malignancies and diseases.

Does plasminogen activator inhibitor-1 drive lymphangiogenesis?

DEFF Research Database (Denmark)

Bruyère, Françoise; Melen-Lamalle, Laurence; Blacher, Silvia

2010-01-01

The purpose of this study is to explore the function of plasminogen activator inhibitor-1 (PAI-1) during pathological lymphangiogenesis. PAI-1, the main physiological inhibitor of plasminogen activators is involved in pathological angiogenesis at least by controlling extracellular proteolysis and...

Optimizing fluorescently tethered Hsp90 inhibitor dose for maximal specific uptake by breast tumors

Science.gov (United States)

Crouch, Brian T.; Duer, Joy; Wang, Roujia; Gallagher, Jennifer; Hall, Allison; Soo, Mary Scott; Hughes, Philip; Haystead, Timothy A. J.; Ramanujam, Nirmala

2018-03-01

Despite improvements in surgical resection, 20-40% of patients undergoing breast conserving surgery require at least one additional re-excision. Leveraging the unique surface expression of heat shock protein 90 (Hsp90), a chaperone protein involved in several key hallmarks of cancer, in breast cancer provides an exciting opportunity to identify residual disease during surgery. We developed a completely non-destructive strategy using HS-27, a fluorescently-tethered Hsp90 inhibitor, to assay surface Hsp90 expression on intact tissue specimens using a fluorescence microendoscope with a field of view of 750 μm and subcellular resolution of 4 μm. HS-27 consists of an FDA approved Hsp90 inhibitor tethered to fluorescein isothiocyanate (EX 488nm, EM 525nm). Here, we optimized ex vivo HS-27 administration in pre-clinical breast cancer models and validated our approach on 21 patients undergoing standard of care ultrasound guided core needle biopsy. HS-27 administration time was fixed at 1- minute to minimize imaging impact on clinical workflow. HS-27 and HS-217 (non-specific control) doses were modulated from 1 μM up to 100 μM to identify the dose maximizing the ratio of specific uptake (HS-27 fluorescence) to non-specific uptake (HS-217 fluorescence). The specificity ratio was maximized at 100 μM and was significantly greater than all other doses (pcancer makes this technology attractive for assessing tumor margins, as one agent can be used for all subtypes.

Seed-Specific Stable Expression of the α-AI1 Inhibitor in Coffee Grains and the In Vivo Implications for the Development of the Coffee Berry Borer.

Science.gov (United States)

Albuquerque, Érika V S; Bezerra, Caroline A; Romero, Juan V; Valencia, Jorge W A; Valencia-Jiménez, Arnubio; Pimenta, Lucas M; Barbosa, Aulus E A D; Silva, Maria C M; Meneguim, Ana M; Sá, Maria Eugênia L; Engler, Gilbert; de Almeida-Engler, Janice; Fernandez, Diana; Grossi-de-Sá, Maria F

Genetic transformation of coffee ( Coffea spp.), the second most traded commodity worldwide, is an alternative approach to introducing features that cannot be introgressed by traditional crossings. The transgenic stability, heritability and quantitative and spatial expression patterns of the seed-specific promoter phytohemagglutinin (PHA-L) from Phaseolus vulgaris were characterized in genetically modified C. arabica expressing the α-amylase inhibitor-1 ( α-AI1 ) gene. The α-AI1 inhibitor shows considerable activity toward digestive enzymes of the coffee berry borer (CBB) Hypothenemus hampei . This insect pest expends its life cycle almost entirely in coffee berries. Transgene containment in the fruit is important to meeting food and environmental safety requirements for releasing genetically modified (GM) crops. PCR analysis of T2 coffee plants showed a Mendelian single-copy segregation pattern. Ectopic transgene expression was only detected in coffee grains, as demonstrated by reverse transcription-PCR analysis of different plant tissues. An intense immunocytochemical signal associated with α-AI1 protein expression was localized to endospermic cells. In addition, a delay in the larval development of CBB was observed after challenging transgenic coffee seeds with the insect. These results indicate that the PHA-L promoter might be a useful tool in coffee for the seed-specific expression of genes related to coffee bean productivity, quality and pest protection. The biotechnological applicability of the α-AI1 gene for controlling CBB is also discussed. This work is the first report showing a seed-specific transgene expression in coffee plants.

JAK inhibitors for the treatment of myeloproliferative neoplasms and other disorders [version 1; referees: 2 approved

Directory of Open Access Journals (Sweden)

William Vainchenker

2018-01-01

Full Text Available JAK inhibitors have been developed following the discovery of the JAK2V617F in 2005 as the driver mutation of the majority of non-BCR-ABL1 myeloproliferative neoplasms (MPNs. Subsequently, the search for JAK2 inhibitors continued with the discovery that the other driver mutations (CALR and MPL also exhibited persistent JAK2 activation. Several type I ATP-competitive JAK inhibitors with different specificities were assessed in clinical trials and exhibited minimal hematologic toxicity. Interestingly, these JAK inhibitors display potent anti-inflammatory activity. Thus, JAK inhibitors targeting preferentially JAK1 and JAK3 have been developed to treat inflammation, autoimmune diseases, and graft-versus-host disease. Ten years after the beginning of clinical trials, only two drugs have been approved by the US Food and Drug Administration: one JAK2/JAK1 inhibitor (ruxolitinib in intermediate-2 and high-risk myelofibrosis and hydroxyurea-resistant or -intolerant polycythemia vera and one JAK1/JAK3 inhibitor (tofacitinib in methotrexate-resistant rheumatoid arthritis. The non-approved compounds exhibited many off-target effects leading to neurological and gastrointestinal toxicities, as seen in clinical trials for MPNs. Ruxolitinib is a well-tolerated drug with mostly anti-inflammatory properties. Despite a weak effect on the cause of the disease itself in MPNs, it improves the clinical state of patients and increases survival in myelofibrosis. This limited effect is related to the fact that ruxolitinib, like the other type I JAK2 inhibitors, inhibits equally mutated and wild-type JAK2 (JAK2WT and also the JAK2 oncogenic activation. Thus, other approaches need to be developed and could be based on either (1 the development of new inhibitors specifically targeting JAK2V617F or (2 the combination of the actual JAK2 inhibitors with other therapies, in particular with molecules targeting pathways downstream of JAK2 activation or the stability of JAK2

Binding of the Inhibitor Protein IF1 to Bovine F1-ATPase

Science.gov (United States)

Bason, John V.; Runswick, Michael J.; Fearnley, Ian M.; Walker, John E.

2011-01-01

In the structure of bovine F1-ATPase inhibited with residues 1–60 of the bovine inhibitor protein IF1, the α-helical inhibitor interacts with five of the nine subunits of F1-ATPase. In order to understand the contributions of individual amino acid residues to this complex binding mode, N-terminal deletions and point mutations have been introduced, and the binding properties of each mutant inhibitor protein have been examined. The N-terminal region of IF1 destabilizes the interaction of the inhibitor with F1-ATPase and may assist in removing the inhibitor from its binding site when F1Fo-ATPase is making ATP. Binding energy is provided by hydrophobic interactions between residues in the long α-helix of IF1 and the C-terminal domains of the βDP-subunit and βTP-subunit and a salt bridge between residue E30 in the inhibitor and residue R408 in the C-terminal domain of the βDP-subunit. Several conserved charged amino acids in the long α-helix of IF1 are also required for establishing inhibitory activity, but in the final inhibited state, they are not in contact with F1-ATPase and occupy aqueous cavities in F1-ATPase. They probably participate in the pathway from the initial interaction of the inhibitor and the enzyme to the final inhibited complex observed in the structure, in which two molecules of ATP are hydrolysed and the rotor of the enzyme turns through two 120° steps. These findings contribute to the fundamental understanding of how the inhibitor functions and to the design of new inhibitors for the systematic analysis of the catalytic cycle of the enzyme. PMID:21192948

Tofacitinib and analogs as inhibitors of the histone kinase PRK1 (PKN1).

Science.gov (United States)

Ostrovskyi, Dmytro; Rumpf, Tobias; Eib, Julia; Lumbroso, Alexandre; Slynko, Inna; Klaeger, Susan; Heinzlmeir, Stephanie; Forster, Michael; Gehringer, Matthias; Pfaffenrot, Ellen; Bauer, Silke Mona; Schmidtkunz, Karin; Wenzler, Sandra; Metzger, Eric; Kuster, Bernhard; Laufer, Stefan; Schüle, Roland; Sippl, Wolfgang; Breit, Bernhard; Jung, Manfred

2016-09-01

The histone kinase PRK1 has been identified as a potential target to combat prostate cancer but selective PRK1 inhibitors are lacking. The US FDA -approved JAK1-3 inhibitor tofacitinib also potently inhibits PRK1 in vitro. We show that tofacitinib also inhibits PRK1 in a cellular setting. Using tofacitinib as a starting point for structure-activity relationship studies, we identified a more potent and another more selective PRK1 inhibitor compared with tofacitinib. Furthermore, we found two potential PRK1/JAK3-selectivity hotspots. The identified inhibitors and the selectivity hotspots lay the basis for the development of selective PRK1 inhibitors. The identification of PRK1, but also of other cellular tofacitinib targets, has implications on its clinical use and on future development of tofacitinib-like JAK inhibitors. [Formula: see text].

Dipicolinic Acid Derivatives as Inhibitors of New Delhi Metallo-β-lactamase-1.

Science.gov (United States)

Chen, Allie Y; Thomas, Pei W; Stewart, Alesha C; Bergstrom, Alexander; Cheng, Zishuo; Miller, Callie; Bethel, Christopher R; Marshall, Steven H; Credille, Cy V; Riley, Christopher L; Page, Richard C; Bonomo, Robert A; Crowder, Michael W; Tierney, David L; Fast, Walter; Cohen, Seth M

2017-09-14

The efficacy of β-lactam antibiotics is threatened by the emergence and global spread of metallo-β-lactamase (MBL) mediated resistance, specifically New Delhi metallo-β-lactamase-1 (NDM-1). By utilization of fragment-based drug discovery (FBDD), a new class of inhibitors for NDM-1 and two related β-lactamases, IMP-1 and VIM-2, was identified. On the basis of 2,6-dipicolinic acid (DPA), several libraries were synthesized for structure-activity relationship (SAR) analysis. Inhibitor 36 (IC 50 = 80 nM) was identified to be highly selective for MBLs when compared to other Zn(II) metalloenzymes. While DPA displayed a propensity to chelate metal ions from NDM-1, 36 formed a stable NDM-1:Zn(II):inhibitor ternary complex, as demonstrated by 1 H NMR, electron paramagnetic resonance (EPR) spectroscopy, equilibrium dialysis, intrinsic tryptophan fluorescence emission, and UV-vis spectroscopy. When coadministered with 36 (at concentrations nontoxic to mammalian cells), the minimum inhibitory concentrations (MICs) of imipenem against clinical isolates of Eschericia coli and Klebsiella pneumoniae harboring NDM-1 were reduced to susceptible levels.

Discovery of Specific Inhibitors for Intestinal E. coli  β-Glucuronidase through In Silico Virtual Screening

Directory of Open Access Journals (Sweden)

Ta-Chun Cheng

2015-01-01

Full Text Available Glucuronidation is a major metabolism process of detoxification for carcinogens, 4-(methylnitrosamino-1-(3-pyridy-1-butanone (NNK and 1,2-dimethylhydrazine (DMH, of reactive oxygen species (ROS. However, intestinal E. coli   β-glucuronidase (eβG has been considered pivotal to colorectal carcinogenesis. Specific inhibition of eβG may prevent reactivating the glucuronide-carcinogen and protect the intestine from ROS-mediated carcinogenesis. In order to develop specific eβG inhibitors, we found that 59 candidate compounds obtained from the initial virtual screening had high inhibition specificity against eβG but not human βG. In particular, we found that compounds 7145 and 4041 with naphthalenylidene-benzenesulfonamide (NYBS are highly effective and selective to inhibit eβG activity. Compound 4041  (IC50=2.8 μM shows a higher inhibiting ability than compound 7145  (IC50=31.6 μM against eβG. Furthermore, the molecular docking analysis indicates that compound 4041 has two hydrophobic contacts to residues L361 and I363 in the bacterial loop, but 7145 has one contact to L361. Only compound 4041 can bind to key residue (E413 at active site of eβG via hydrogen-bonding interactions. These novel NYBS-based eβG specific inhibitors may provide as novel candidate compounds, which specifically inhibit eβG to reduce eβG-based carcinogenesis and intestinal injury.

Substrate and inhibitor specificity of kynurenine monooxygenase from Cytophaga hutchinsonii.

Science.gov (United States)

Phillips, Robert S; Anderson, Andrew D; Gentry, Harvey G; Güner, Osman F; Bowen, J Phillip

2017-04-15

Kynurenine monooxygenase (KMO) is a potential drug target for treatment of neurodegenerative disorders such as Huntington's and Alzheimer's diseases. We have evaluated substituted kynurenines as substrates or inhibitors of KMO from Cytophaga hutchinsonii. Kynurenines substituted with a halogen at the 5-position are excellent substrates, with values of k cat and k cat /K m comparable to or higher than kynurenine. However, kynurenines substituted in the 3-position are competitive inhibitors, with K I values lower than the K m for kynurenine. Bromination also enhances inhibition, and 3,5-dibromokynurenine is a potent competitive inhibitor with a K I value of 1.5μM. A pharmacophore model of KMO was developed, and predicted that 3,4-dichlorohippuric acid would be an inhibitor. The K I for this compound was found to be 34μM, thus validating the pharmacophore model. We are using these results and our model to design more potent inhibitors of KMO. Copyright © 2017 Elsevier Ltd. All rights reserved.

Docking Studies and Biological Evaluation of a Potential β-Secretase Inhibitor of 3-Hydroxyhericenone F from Hericium erinaceus

Directory of Open Access Journals (Sweden)

Chen Diling

2017-05-01

Full Text Available Alzheimer’s disease (AD is the most common neurodegenerative disorder, affecting approximately more than 5% of the population worldwide over the age 65, annually. The incidence of AD is expected to be higher in the next 10 years. AD patients experience poor prognosis and as a consequence new drugs and therapeutic strategies are required in order to improve the clinical responses and outcomes of AD. The purpose of the present study was to screen a certain number of potential compounds from herbal sources and investigate their corresponding mode of action. In the present study, the learning and memory effects of ethanol:water (8:2 extracts from Hericium erinaceus were evaluated on a dementia rat model. The model was established by intraperitoneal injection of 100 mg/kg/d D-galactose in rats. The results indicated that the extracts can significantly ameliorate the learning and memory abilities. Specific active ingredients were screened in vivo assays and the results were combined with molecular docking studies. Potential receptor–ligand interactions on the BACE1-inhibitor namely, 3-Hydroxyhericenone F (3HF were investigated. The isolation of a limited amount of 3HF from the fruit body of H. erinaceus by chemical separation was conducted, and the mode of action of this compound was verified in NaN3-induced PC12 cells. The cell-based assays demonstrated that 3HF can significantly down-regulate the expression of BACE1 (p < 0.01, while additional AD intracellular markers namely, p-Tau and Aβ1-42 were further down-regulated (p < 0.05. The data further indicate that 3HF can ameliorate certain mitochondrial dysfunction conditions by the reversal of the decreasing level of mitochondrial respiratory chain complexes, the calcium ion levels ([Ca2+], the inhibiton in the production of ROS, the increase in the mitochondrial membrane potential and ATP levels, and the regulation of the expression levels of the genes encoding for the p21, COX I, COX II, PARP1

A new class of HIV-1 protease inhibitor: the crystallographic structure, inhibition and chemical synthesis of an aminimide peptide isostere.

Science.gov (United States)

Rutenber, E E; McPhee, F; Kaplan, A P; Gallion, S L; Hogan, J C; Craik, C S; Stroud, R M

1996-09-01

The essential role of HIV-1 protease (HIV-1 PR) in the viral life cycle makes it an attractive target for the development of substrate-based inhibitors that may find efficacy as anti-AIDS drugs. However, resistance has arisen to potent peptidomimetic drugs necessitating the further development of novel chemical backbones for diversity based chemistry focused on probing the active site for inhibitor interactions and binding modes that evade protease resistance. AQ148 is a potent inhibitor of HIV-1 PR and represents a new class of transition state analogues incorporating an aminimide peptide isostere. A 3-D crystallographic structure of AQ148, a tetrapeptide isostere, has been determined in complex with its target HIV-1 PR to a resolution of 2.5 A and used to evaluate the specific structural determinants of AQ148 potency and to correlate structure-activity relationships within the class of related compounds. AQ148 is a competitive inhibitor of HIV-1 PR with a Ki value of 137 nM. Twenty-nine derivatives have been synthesized and chemical modifications have been made at the P1, P2, P1', and P2' sites. The atomic resolution structure of AQ148 bound to HIV-1 PR reveals both an inhibitor binding mode that closely resembles that of other peptidomimetic inhibitors and specific protein/inhibitor interactions that correlate with structure-activity relationships. The structure provides the basis for the design, synthesis and evaluation of the next generation of hydroxyethyl aminimide inhibitors. The aminimide peptide isostere is a scaffold with favorable biological properties well suited to both the combinatorial methods of peptidomimesis and the rational design of potent and specific substrate-based analogues.

The effects of residual platelets in plasma on plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays

NARCIS (Netherlands)

M. Pieters (Marlien); S.A. Barnard (Sunelle A.); D.T. Loots (Du Toit); D.C. Rijken (Dingeman)

2017-01-01

textabstractDue to controversial evidence in the literature pertaining to the activity of plasminogen activator inhibitor-1 in platelets, we examined the effects of residual platelets present in plasma (a potential pre-analytical variable) on various plasminogen activator inhibitor-1 and plasminogen

The common inhalation anesthetic isoflurane induces caspase activation and increases amyloid beta-protein level in vivo.

Science.gov (United States)

Xie, Zhongcong; Culley, Deborah J; Dong, Yuanlin; Zhang, Guohua; Zhang, Bin; Moir, Robert D; Frosch, Matthew P; Crosby, Gregory; Tanzi, Rudolph E

2008-12-01

An estimated 200 million patients worldwide have surgery each year. Anesthesia and surgery have been reported to facilitate emergence of Alzheimer's disease. The commonly used inhalation anesthetic isoflurane has previously been reported to induce apoptosis, and to increase levels and aggregation of Alzheimer's disease-associated amyloid beta-protein (Abeta) in cultured cells. However, the in vivo relevance has not been addressed. We therefore set out to determine effects of isoflurane on caspase activation and levels of beta-site amyloid precursor protein-cleaving enzyme (BACE) and Abeta in naive mice, using Western blot, immunohistochemistry, and reverse transcriptase polymerase chain reaction. Here we show for the first time that a clinically relevant isoflurane anesthesia (1.4% isoflurane for 2 hours) leads to caspase activation and modest increases in levels of BACE 6 hours after anesthesia in mouse brain. Isoflurane anesthesia induces caspase activation, and increases levels of BACE and Abeta up to 24 hours after anesthesia. Isoflurane may increase BACE levels by reducing BACE degradation. Moreover, the Abeta aggregation inhibitor, clioquinol, was able to attenuate isoflurane-induced caspase-3 activation in vivo. Given that transient insults to brain may lead to long-term brain damage, these findings suggest that isoflurane may promote Alzheimer's disease neuropathogenesis and, as such, have implications for use of isoflurane in humans, pending human study confirmation.

Total levels of tissue inhibitor of metalloproteinases 1 in plasma yield high diagnostic sensitivity and specificity in patients with colon cancer

DEFF Research Database (Denmark)

Holten-Andersen, Mads N; Christensen, Ib Jarle; Nielsen, Hans Jørgen

2002-01-01

: Total TIMP-1 plasma levels were measured by ELISA in blood samples from two different blood donor populations from IBD patients, and preoperative samples from patients with primary colon cancer (CC), rectal cancer (RC), or breast cancer. RESULTS: There were no significant differences in plasma TIMP-1......PURPOSE: The purpose of this study was to measure total levels of tissue inhibitor of metalloproteinases (TIMP-1) by ELISA in plasma from blood donors, patients with inflammatory bowel disease (IBD), and patients with cancer and to correlate the results to patient diagnosis. EXPERIMENTAL DESIGN...... levels between healthy donors and IBD or breast cancer patients, whereas patients with CC or RC had significantly elevated TIMP-1 levels. Total TIMP-1 levels identified patients with CC with a sensitivity of 63% at 98% specificity, patients with early CC (Dukes' A+B) with a sensitivity of 56% at 98...

Tissue- and agonist-specific regulation of human and murine plasminogen activator inhibitor-1 promoters in transgenic mice.

Science.gov (United States)

Eren, M; Painter, C A; Gleaves, L A; Schoenhard, J A; Atkinson, J B; Brown, N J; Vaughan, D E

2003-11-01

Numerous studies have described regulatory factors and sequences that control transcriptional responses in vitro. However, there is a paucity of information on the qualitative and quantitative regulation of heterologous promoters using transgenic strategies. In order to investigate the physiological regulation of human plasminogen activator inhibitor type-1 (hPAI-1) expression in vivo compared to murine PAI-1 (mPAI-1) and to test the physiological relevance of regulatory mechanisms described in vitro, we generated transgenic mice expressing enhanced green fluorescent protein (EGFP) driven by the proximal -2.9 kb of the hPAI-1 promoter. Transgenic animals were treated with Ang II, TGF-beta1 and lipopolysaccharide (LPS) to compare the relative activation of the human and murine PAI-1 promoters. Ang II increased EGFP expression most effectively in brain, kidney and spleen, while mPAI-1 expression was quantitatively enhanced most prominently in heart and spleen. TGF-beta1 failed to induce activation of the hPAI-1 promoter but potently stimulated mPAI-1 in kidney and spleen. LPS administration triggered robust expression of mPAI-1 in liver, kidney, pancreas, spleen and lung, while EGFP was induced only modestly in heart and kidney. These results indicate that the transcriptional response of the endogenous mPAI-1 promoter varies widely in terms of location and magnitude of response to specific stimuli. Moreover, the physiological regulation of PAI-1 expression likely involves a complex interaction of transcription factors and DNA sequences that are not adequately replicated by in vitro functional studies focused on the proximal -2.9 kb promoter.

Hydrogen separation through tailored dual phase membranes with nominal composition BaCe0.8Eu0.2O3-δ:Ce0.8Y0.2O2-δ at intermediate temperatures

Science.gov (United States)

Ivanova, Mariya E.; Escolástico, Sonia; Balaguer, Maria; Palisaitis, Justinas; Sohn, Yoo Jung; Meulenberg, Wilhelm A.; Guillon, Olivier; Mayer, Joachim; Serra, Jose M.

2016-11-01

Hydrogen permeation membranes are a key element in improving the energy conversion efficiency and decreasing the greenhouse gas emissions from energy generation. The scientific community faces the challenge of identifying and optimizing stable and effective ceramic materials for H2 separation membranes at elevated temperature (400-800 °C) for industrial separations and intensified catalytic reactors. As such, composite materials with nominal composition BaCe0.8Eu0.2O3-δ:Ce0.8Y0.2O2-δ revealed unprecedented H2 permeation levels of 0.4 to 0.61 mL·min-1·cm-2 at 700 °C measured on 500 μm-thick-specimen. A detailed structural and phase study revealed single phase perovskite and fluorite starting materials synthesized via the conventional ceramic route. Strong tendency of Eu to migrate from the perovskite to the fluorite phase was observed at sintering temperature, leading to significant Eu depletion of the proton conducing BaCe0.8Eu0.2O3-δ phase. Composite microstructure was examined prior and after a variety of functional tests, including electrical conductivity, H2-permeation and stability in CO2 containing atmospheres at elevated temperatures, revealing stable material without morphological and structural changes, with segregation-free interfaces and no further diffusive effects between the constituting phases. In this context, dual phase material based on BaCe0.8Eu0.2O3-δ:Ce0.8Y0.2O2-δ represents a very promising candidate for H2 separating membrane in energy- and environmentally-related applications.

PD-1/PD-L1 Inhibitors for Immuno-oncology: From Antibodies to Small Molecules.

Science.gov (United States)

Geng, Qiaohong; Jiao, Peifu; Jin, Peng; Su, Gaoxing; Dong, Jinlong; Yan, Bing

2018-02-12

The recent regulatory approvals of immune checkpoint protein inhibitors, such as ipilimumab, pembrolizumab, nivolumab, atezolizumab, durvalumab, and avelumab ushered a new era in cancer therapy. These inhibitors do not attack tumor cells directly but instead mobilize the immune system to re-recognize and eradicate tumors, which endows them with unique advantages including durable clinical responses and substantial clinical benefits. PD-1/PD-L1 inhibitors, a pillar of immune checkpoint protein inhibitors, have demonstrated unprecedented clinical efficacy in more than 20 cancer types. Besides monoclonal antibodies, diverse PD- 1/PD-L1 inhibiting candidates, such as peptides, small molecules have formed a powerful collection of weapons to fight cancer. The goal of this review is to summarize and discuss the current PD-1/PD-L1 inhibitors including candidates under clinical development, their molecular interactions with PD-1 or PD-L1, the disclosed structureactivity relationships of peptides and small molecules as inhibitors. Current PD-1/PD-L1 inhibitors under clinical development are exclusively dominated by antibodies. The molecular interactions of therapeutic antibodies with PD-1 or PD-L1 have been gradually elucidated for the design of novel inhibitors. Various peptides and traditional small molecules have been investigated in preclinical model to discover novel PD-1/PD-L1 inhibitors. Peptides and small molecules may play an important role in immuno-oncology because they may bind to multiple immune checkpoint proteins via rational design, opening opportunity for a new generation of novel PD-1/PD-L1 inhibitors. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Structural studies of series HIV-1 nonnucleoside reverse transcriptase inhibitors 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)-benzimidazoles with different 4-substituents

Science.gov (United States)

Ziółkowska, Natasza E.; Michejda, Christopher J.; Bujacz, Grzegorz D.

2010-03-01

Over the past 10 years, several anti-viral drugs have become available to fight the HIV infection. Antiretroviral treatment reduces the mortality of AIDS. Nonnucleoside inhibitors of HIV-1 reverse transcriptase are specific and potentially nontoxic drugs against AIDS. The crystal structures of five nonnucleoside inhibitors of HIV-1 reverse transcriptase are presented here. The structural parameters, especially those describing the angular orientation of the π-electron systems and influencing biological activity, were determined for all of the investigated inhibitors. The chemical character and orientation of the substituent at C4 position of the benzimidazole moiety substantially influences the anti-viral activity. The structural data of the investigated inhibitors is a good basis for modeling enzyme-inhibitor interactions for structure-assisted drug design.

Fasxiator, a novel factor XIa inhibitor from snake venom, and its site-specific mutagenesis to improve potency and selectivity.

Science.gov (United States)

Chen, W; Carvalho, L P D; Chan, M Y; Kini, R M; Kang, T S

2015-02-01

Bleeding remains a major limitation of standard anticoagulant drugs that target the extrinsic and common coagulation pathways. Recently, intrinsic coagulation factors are increasingly being investigated as alternative targets for developing anticoagulant drugs with lower bleeding risk. Goals were to (i) identify novel anticoagulants selectively targeting intrinsic coagulation pathway and (ii) characterize and further improve the properties of the identified anticoagulants. We have isolated and sequenced a specific factor XIa (FXIa) inhibitor, henceforth named Fasxiator, from the venom of the banded krait snake, Bungarus fasciatus. It is a Kunitz-type protease inhibitor that prolonged activated partial thromboplastin time without significant effects on prothrombin time. Fasxiator was recombinantly expressed (rFasxiator), purified, and characterized to be a slow-type inhibitor of FXIa that exerts its anticoagulant activities (doubled activated partial thromboplastin time at ~ 3 μmol L(-1) ) by selectively inhibiting human FXIa in in vitro assays. A series of mutants were subsequently generated to improve the potency and selectivity of recombinant rFasxiator. rFasxiatorN17R,L19E showed the best balance between potency (IC50 ~ 1 nmol L(-1) ) and selectivity (> 100 times). rFasxiatorN17R,L19E is a competitive slow-type inhibitor of FXIa (Ki  = 0.86 nmol L(-1) ), possesses anticoagulant activity that is ~ 10 times stronger in human plasma than in murine plasma, and prolonged the occlusion time of mice carotid artery in FeCl3 -induced thrombosis models. We have isolated an exogenous FXIa specific inhibitor, engineered it to improve its potency by ~ 1000 times and demonstrated its in vitro and in vivo efficacy. These proof-of-principle data supported the further development of Fasxiator as a novel anticoagulant candidate. © 2014 International Society on Thrombosis and Haemostasis.

99mTc-labeling and evaluation of a HYNIC modified small-molecular inhibitor of prostate-specific membrane antigen

International Nuclear Information System (INIS)

Xu, Xiaoping; Zhang, Jianping; Hu, Silong; He, Simin; Bao, Xiao; Ma, Guang; Luo, Jianmin; Cheng, Jingyi; Zhang, Yingjian

2017-01-01

Introduction: Prostate-specific membrane antigen (PSMA) is a well-established target in the development of radiopharmaceuticals for the diagnosis and therapy of prostate cancer (PCa). In this study, we evaluated a novel 99m Tc-labeled small molecular inhibitor of PSMA. Methods: This new small-molecular inhibitor of PSMA, 6-hydrazinonicotinate-Aminocaproic acid-Lysine-Urea-Glutamate (HYNIC-ALUG) was radiolabeled by 99m Tc and was evaluated both in vitro and in vivo using PCa models (PC-3 and LNCaP). Radiation dosimetry was assessed in mice. Results: 99m Tc-HYNIC-ALUG showed excellent stability in different media. A cell assay preliminarily displayed its specificity for PSMA. The inhibitor showed good pharmacokinetics making it suitable for in vivo imaging. PC-3-derived tumors showed no obvious radioactive uptake; however, the LNCaP-derived tumors showed very high radioactive uptake which was significantly decreased by the selective PSMA inhibitor 2-PMPA. Biodistribution in LNCaP xenografts showed an optimum tumor-to-blood ratio of 24.23 ± 3.54 at 2 h. Tumor uptake was also decreased in the inhibition experiment with 2-PMPA (19.45 ± 2.14%ID/g versus 1.42 ± 0.15%ID/g at 2 h). The effective dose of the 99m Tc-HYNIC-ALUG was 8.4E-04 mSv/MBq. Conclusions: A new 99m Tc-labeled PSMA inhibitor with specific accumulation in PSMA-positive tumors and low background in other organs was synthesized. The radiopharmaceutical also showed very low radiation dosimetry. This agent may significantly improve the diagnosis, staging, and subsequent monitoring of therapeutic effects in PCa patients.

Checkpoint inhibitors in cancer immunotherapy: Cross reactivity of a CTLA-4 antibody and IDO-inhibitor L-1MT in pigs

DEFF Research Database (Denmark)

Al-Shatrawi, Zina Adil; Frøsig, Thomas Mørch; Jungersen, Gregers

a non-specific activation of porcine T cells. This will be further investigated to provide the basis for in vivo studies investigating checkpoint inhibitor blockade in combination with other cancer immunotherapies. Eventually our goal is to establish pigs as an alternative large animal model......Blockade of checkpoint inhibitors has recently shown very convincing results in the treatment of cancer. One key target is CTLA-4, which has been demonstrated to be a potent negative regulator of lymphocyte activation. The treatment with the FDA-approved fully human CTLA-4 monoclonal antibody...... Ipilimumab increases anticancer T-cell reactivity and overall survival of metastatic cancer patients. Indole-amine 2,3-dioxygenase (IDO) is another checkpoint inhibitor which suppresses T-cell immunity by the depletion of tryptophan in the T-cell microenvironment, and also inhibition of IDO by L-1...

RAGE-Specific Inhibitor FPS-ZM1 Attenuates AGEs-Induced Neuroinflammation and Oxidative Stress in Rat Primary Microglia.

Science.gov (United States)

Shen, Chao; Ma, Yingjuan; Zeng, Ziling; Yin, Qingqing; Hong, Yan; Hou, Xunyao; Liu, Xueping

2017-10-01

Advanced glycation end products (AGEs) enhance microglial activation and intensify the inflammatory response and oxidative stress in the brain. This process may occur due to direct cytotoxicity or interacting with AGEs receptors (RAGE), which are expressed on the surface of microglia. FPS-ZM1 is a high-affinity but nontoxic RAGE-specific inhibitor that has been recently shown to attenuate the Aβ-induced inflammatory response by blocking the ligation of Aβ to RAGE. In this study, we further investigated the effect of FPS-ZM1 on the AGEs/RAGE interaction and downstream elevation of neuroinflammation and oxidative stress in primary microglia cells. The results suggested that FPS-ZM1 significantly suppressed AGEs-induced RAGE overexpression, RAGE-dependent microglial activation, nuclear translocation of nuclear factor kappaB p65 (NF-κB p65), and the expression of downstream inflammatory mediators such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), cyclooxygenase 2 (COX-2)/prostaglandin E2 (PGE2) and inducible nitric oxide synthase (iNOS)/nitric oxide (NO). Furthermore, FPS-ZM1 attenuated AGEs-stimulated NADPH oxidase (NOX) activation and reactive oxygen species (ROS) expression. Finally, FPS-ZM1 elevated the levels of transcription factors nuclear-factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 (HO-1), as well as decreased antioxidant capacity and increased production of oxidative species. Our results suggest that FPS-ZM1 may be neuroprotective through attenuating microglial activation, oxidative stress and inflammation by blocking RAGE.

Engineering the substrate and inhibitor specificities of human coagulation Factor VIIa

DEFF Research Database (Denmark)

Larsen, Katrine S; Østergaard, Henrik; Bjelke, Jais R

2007-01-01

The remarkably high specificity of the coagulation proteases towards macromolecular substrates is provided by numerous interactions involving the catalytic groove and remote exosites. For FVIIa [activated FVII (Factor VII)], the principal initiator of coagulation via the extrinsic pathway, several...... for FVIIa by marked changes in primary substrate specificity and decreased rates of antithrombin III inhibition. Interestingly, these changes do not necessarily coincide with an altered ability to activate Factor X, demonstrating that inhibitor and macromolecular substrate selectivity may be engineered...

Pre-equilibrium competitive library screening for tuning inhibitor association rate and specificity toward serine proteases.

Science.gov (United States)

Cohen, Itay; Naftaly, Si; Ben-Zeev, Efrat; Hockla, Alexandra; Radisky, Evette S; Papo, Niv

2018-04-16

High structural and sequence similarity within protein families can pose significant challenges to the development of selective inhibitors, especially toward proteolytic enzymes. Such enzymes usually belong to large families of closely similar proteases and may also hydrolyze, with different rates, protein- or peptide-based inhibitors. To address this challenge, we employed a combinatorial yeast surface display library approach complemented with a novel pre-equilibrium, competitive screening strategy for facile assessment of the effects of multiple mutations on inhibitor association rates and binding specificity. As a proof of principle for this combined approach, we utilized this strategy to alter inhibitor/protease association rates and to tailor the selectivity of the amyloid β-protein precursor Kunitz protease inhibitor domain (APPI) for inhibition of the oncogenic protease mesotrypsin, in the presence of three competing serine proteases, anionic trypsin, cationic trypsin and kallikrein-6. We generated a variant, designated APPI P13W/M17G/I18F/F34V , with up to 30-fold greater specificity relative to the parental APPI M17G/I18F/F34V protein, and 6500- to 230 000-fold improved specificity relative to the wild-type APPI protein in the presence of the other proteases tested. A series of molecular docking simulations suggested a mechanism of interaction that supported the biochemical results. These simulations predicted that the selectivity and specificity are affected by the interaction of the mutated APPI residues with nonconserved enzyme residues located in or near the binding site. Our strategy will facilitate a better understanding of the binding landscape of multispecific proteins and will pave the way for design of new drugs and diagnostic tools targeting proteases and other proteins. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Development of a disease-specific quality of life questionnaire for adult patients with hereditary angioedema due to C1 inhibitor deficiency (HAE-QoL): Spanish multi-centre research project.

Science.gov (United States)

Prior, Nieves; Remor, Eduardo; Gómez-Traseira, Carmen; López-Serrano, Concepción; Cabañas, Rosario; Contreras, Javier; Campos, Ángel; Cardona, Victoria; Cimbollek, Stefan; González-Quevedo, Teresa; Guilarte, Mar; de Rojas, Dolores Hernández Fernández; Marcos, Carmen; Rubio, María; Tejedor-Alonso, Miguel Ángel; Caballero, Teresa

2012-07-20

There is a need for a disease-specific instrument for assessing health-related quality of life in adults with hereditary angioedema due to C1 inhibitor deficiency, a rare, disabling and life-threatening disease. In this paper we report the protocol for the development and validation of a specific questionnaire, with details on the results of the process of item generation, domain selection, and the expert and patient rating phase. Semi-structured interviews were completed by 45 patients with hereditary angioedema and 8 experts from 8 regions in Spain. A qualitative content analysis of the responses was carried out. Issues raised by respondents were grouped into categories. Content analysis identified 240 different responses, which were grouped into 10 conceptual domains. Sixty- four items were generated. A total of 8 experts and 16 patients assessed the items for clarity, relevance to the disease, and correct dimension assignment. The preliminary version of the specific health-related quality of life questionnaire for hereditary angioedema (HAE-QoL v 1.1) contained 44 items grouped into 9 domains. To the best of our knowledge, this is the first multi-centre research project that aims to develop a specific health-related quality of life questionnaire for adult patients with hereditary angioedema due to C1 inhibitor deficiency. A preliminary version of the specific HAE-QoL questionnaire was obtained. The qualitative analysis of interviews together with the expert and patient rating phase helped to ensure content validity. A pilot study will be performed to assess the psychometric properties of the questionnaire and to decide on the final version.

Structure of ‘linkerless’ hydroxamic acid inhibitor-HDAC8 complex confirms the formation of an isoform-specific subpocket

Energy Technology Data Exchange (ETDEWEB)

Tabackman, Alexa A.; Frankson, Rochelle; Marsan, Eric S.; Perry, Kay; Cole, Kathryn E. (Ithaca); (Cornell); (Christopher Newport U)

2016-11-04

Histone deacetylases (HDACs) catalyze the hydrolysis of acetylated lysine side chains in histone and non-histone proteins, and play a critical role in the regulation of many biological processes, including cell differentiation, proliferation, senescence, and apoptosis. Aberrant HDAC activity is associated with cancer, making these enzymes important targets for drug design. In general, HDAC inhibitors (HDACi) block the proliferation of tumor cells by inducing cell differentiation, cell cycle arrest, and/or apoptosis, and comprise some of the leading therapies in cancer treatments. To date, four HDACi have been FDA approved for the treatment of cancers: suberoylanilide hydroxamic acid (SAHA, Vorinostat, Zolinza®), romidepsin (FK228, Istodax®), belinostat (Beleodaq®), and panobinostat (Farydak®). Most current inhibitors are pan-HDACi, and non-selectively target a number of HDAC isoforms. Six previously reported HDACi were rationally designed, however, to target a unique sub-pocket found only in HDAC8. While these inhibitors were indeed potent against HDAC8, and even demonstrated specificity for HDAC8 over HDACs 1 and 6, there were no structural data to confirm the mode of binding. Here we report the X-ray crystal structure of Compound 6 complexed with HDAC8 to 1.98 Å resolution. We also describe the use of molecular docking studies to explore the binding interactions of the other 5 related HDACi. Our studies confirm that the HDACi induce the formation of and bind in the HDAC8-specific subpocket, offering insights into isoform-specific inhibition.

A potent, selective, and orally bioavailable inhibitor of the protein-tyrosine phosphatase PTP1B improves insulin and leptin signaling in animal models.

Science.gov (United States)

Krishnan, Navasona; Konidaris, Konstantis F; Gasser, Gilles; Tonks, Nicholas K

2018-02-02

The protein-tyrosine phosphatase PTP1B is a negative regulator of insulin and leptin signaling and a highly validated therapeutic target for diabetes and obesity. Conventional approaches to drug development have produced potent and specific PTP1B inhibitors, but these inhibitors lack oral bioavailability, which limits their potential for drug development. Here, we report that DPM-1001, an analog of the specific PTP1B inhibitor trodusquemine (MSI-1436), is a potent, specific, and orally bioavailable inhibitor of PTP1B. DPM-1001 also chelates copper, which enhanced its potency as a PTP1B inhibitor. DPM-1001 displayed anti-diabetic properties that were associated with enhanced signaling through insulin and leptin receptors in animal models of diet-induced obesity. Therefore, DPM-1001 represents a proof of concept for a new approach to therapeutic intervention in diabetes and obesity. Although the PTPs have been considered undruggable, the findings of this study suggest that allosteric PTP inhibitors may help reinvigorate drug development efforts that focus on this important family of signal-transducing enzymes. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Fragment-based discovery of potent inhibitors of the anti-apoptotic MCL-1 protein.

Science.gov (United States)

Petros, Andrew M; Swann, Steven L; Song, Danying; Swinger, Kerren; Park, Chang; Zhang, Haichao; Wendt, Michael D; Kunzer, Aaron R; Souers, Andrew J; Sun, Chaohong

2014-03-15

Apoptosis is regulated by the BCL-2 family of proteins, which is comprised of both pro-death and pro-survival members. Evasion of apoptosis is a hallmark of malignant cells. One way in which cancer cells achieve this evasion is thru overexpression of the pro-survival members of the BCL-2 family. Overexpression of MCL-1, a pro-survival protein, has been shown to be a resistance factor for Navitoclax, a potent inhibitor of BCL-2 and BCL-XL. Here we describe the use of fragment screening methods and structural biology to drive the discovery of novel MCL-1 inhibitors from two distinct structural classes. Specifically, cores derived from a biphenyl sulfonamide and salicylic acid were uncovered in an NMR-based fragment screen and elaborated using high throughput analog synthesis. This culminated in the discovery of selective and potent inhibitors of MCL-1 that may serve as promising leads for medicinal chemistry optimization efforts. Copyright © 2014 Elsevier Ltd. All rights reserved.

Molecular insights on analogs of HIV PR inhibitors toward HTLV-1 PR through QM/MM interactions and molecular dynamics studies: comparative structure analysis of wild and mutant HTLV-1 PR.

Science.gov (United States)

Selvaraj, Chandrabose; Singh, Poonam; Singh, Sanjeev Kumar

2014-12-01

Retroviruses HTLV-1 and HIV-1 are the primary causative agents of fatal adult T-cell leukemia and acquired immune deficiency syndrome (AIDS) disease. Both retroviruses are similar in characteristics mechanism, and it encodes for protease that mainly involved in the viral replication process. On the basis of the therapeutic success of HIV-1 PR inhibitors, the protease of HTLV-1 is mainly considered as a potential target for chemotherapy. At the same time, structural similarities in both enzymes that originate HIV PR inhibitors can also be an HTLV-1 PR inhibitor. But the expectations failed because of rejection of HIV PR inhibitors from the HTLV-1 PR binding pocket. In this present study, the reason for the HIV PR inhibitor rejection from the HTLV-1 binding site was identified through sequence analysis and molecular dynamics simulation method. Functional analysis of M37A mutation in HTLV PR clearly shows that the MET37 specificity and screening of potential inhibitors targeting MET37 is performed by using approved 90% similar HIV PR inhibitor compounds. From this approach, we report few compounds with a tendency to accept/donate electron specifically to an important site residue MET37 in HTLV-1 PR binding pocket. Copyright © 2014 John Wiley & Sons, Ltd.

Development of a High-Throughput Screen for Inhibitors of Epstein-Barr Virus EBNA1

Science.gov (United States)

Thompson, Scott; Messick, Troy; Schultz, David C.; Reichman, Melvin; Lieberman, Paul M.

2012-01-01

Latent infection with Epstein-Barr Virus (EBV) is a carcinogenic cofactor in several lymphoid and epithelial cell malignancies. At present, there are no small molecule inhibitors that specifically target EBV latent infection or latency-associated oncoproteins. EBNA1 is an EBV-encoded sequence-specific DNA-binding protein that is consistently expressed in EBV-associated tumors and required for stable maintenance of the viral genome in proliferating cells. EBNA1 is also thought to provide cell survival function in latently infected cells. In this work we describe the development of a biochemical high-throughput screening (HTS) method using a homogenous fluorescence polarization (FP) assay monitoring EBNA1 binding to its cognate DNA binding site. An FP-based counterscreen was developed using another EBV-encoded DNA binding protein, Zta, and its cognate DNA binding site. We demonstrate that EBNA1 binding to a fluorescent labeled DNA probe provides a robust assay with a Z-factor consistently greater than 0.6. A pilot screen of a small molecule library of ~14,000 compounds identified 3 structurally related molecules that selectively inhibit EBNA1, but not Zta. All three compounds had activity in a cell-based assay specific for the disruption of EBNA1 transcription repression function. One of the compounds was effective in reducing EBV genome copy number in Raji Burkitt lymphoma cells. These experiments provide a proof-of-concept that small molecule inhibitors of EBNA1 can be identified by biochemical high-throughput screening of compound libraries. Further screening in conjunction with medicinal chemistry optimization may provide a selective inhibitor of EBNA1 and EBV latent infection. PMID:20930215

CINPA1 Is an Inhibitor of Constitutive Androstane Receptor That Does Not Activate Pregnane X Receptor

Science.gov (United States)

Cherian, Milu T; Lin, Wenwei; Wu, Jing

2015-01-01

Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic sensors that enhance the detoxification and elimination of xenobiotics and endobiotics by modulating the expression of genes encoding drug-metabolizing enzymes and transporters. Elevated levels of drug-metabolizing enzymes and efflux transporters, resulting from CAR activation in various cancers, promote the elimination of chemotherapeutic agents, leading to reduced therapeutic effectiveness and acquired drug resistance. CAR inhibitors, in combination with existing chemotherapeutics, could therefore be used to attenuate multidrug resistance in cancers. Interestingly, all previously reported CAR inverse-agonists are also activators of PXR, rendering them mechanistically counterproductive in tissues where both these xenobiotic receptors are present and active. We used a directed high-throughput screening approach, followed by subsequent mechanistic studies, to identify novel, potent, and specific small-molecule CAR inhibitors that do not activate PXR. We describe here one such inhibitor, CINPA1 (CAR inhibitor not PXR activator 1), capable of reducing CAR-mediated transcription with an IC50 of ∼70 nM. CINPA1 1) is a specific xenobiotic receptor inhibitor and has no cytotoxic effects up to 30 µM; 2) inhibits CAR-mediated gene expression in primary human hepatocytes, where CAR is endogenously expressed; 3) does not alter the protein levels or subcellular localization of CAR; 4) increases corepressor and reduces coactivator interaction with the CAR ligand-binding domain in mammalian two-hybrid assays; and 5) disrupts CAR binding to the promoter regions of target genes in chromatin immunoprecipitation assays. CINPA1 could be used as a novel molecular tool for understanding CAR function. PMID:25762023

Effect of specific enzyme inhibitors on replication, total genome DNA repair and on gene-specific DNA repair after UV irradiation in CHO cells

Energy Technology Data Exchange (ETDEWEB)

Jones, J.C.; Stevsner, Tinna; Bohr, Vilhelm A. (National Cancer Institute, NIH, Bethesda, MD (USA). Division of Cancer Treatment, Laboratory of Molecular Pharmacology); Mattern, M.R. (Smith Kline Beecham Pharmaceuticals, King of Prussia, PA (USA). Department of Biomolecular Discovery)

1991-09-01

The effects were studied of some specific enzyme inhibitors on DNA repair and replication after UV damage in Chinese hamster ovary cells. The DNA repair was studied at the level of the average, overall genome and also in the active dihydrofolate reductase gene. Replication was measured in the overall genome. The inhibitors were tested of DNA poly-merase {alpha} and {delta} (aphidicolin), of poly(ADPr) polymerase (3-aminobenzamide), of ribonucleotide reductase (hydroxyurea), of topo-isomerase I (camptothecin), and of topoisomerase II (merbarone, VP-16). In addition, the effects were tested of the potential topoisomerase I activator, {beta}-lapachone. All of these compounds inhibited genome replication and all topoisomerase inhibitors affected the overall genome repair; {beta}-lapachone stimulated it. None of these compounds had any effect on the gene-specific repair. (author). 36 refs.; 3 figs.; 2 tabs.

Accessibility of receptor-bound urokinase to type-1 plasminogen activator inhibitor

Energy Technology Data Exchange (ETDEWEB)

Cubellis, M.V.; Andreasen, P.; Ragno, P.; Mayer, M.; Dano, K.; Blasi, F. (Univ. of Copenhagen (Denmark))

1989-07-01

Urokinase plasminogen activator (uPA) interacts with a surface receptor and with specific inhibitors, such as plasminogen activator inhibitor type 1 (PAI-1). These interactions are mediated by two functionally independent domains of the molecule: the catalytic domain (at the carboxyl terminus) and the growth factor domain (at the amino terminus). The authors have now investigated whether PAI-1 can bind and inhibit receptor-bound uPA. Binding of {sup 125}I-labeled ATF (amino-terminal fragment of uPA) to human U937 monocyte-like cells can be competed for by uPA-PAI-1 complexes, but not by PAI-1 alone. Preformed {sup 125}I-labeled uPA-PAI-1 complexes can bind to uPA receptor with the same binding specificity as uPA. PAI-1 also binds to, and inhibits the activity of, receptor-bound uPA in U937 cells, as shown in U937 cells by a caseinolytic plaque assay. Plasminogen activator activity of these cells is dependent on exogenous uPA, is competed for by receptor-binding diisopropyl fluorophosphate-treated uPA, and is inhibited by the addition of PAI-1. In conclusion, in U937 cells the binding to the receptor does not shield uPA from the action of PAI-1. The possibility that in adherent cells a different localization of PAI-1 and uPA leads to protection of uPA from PAI-1 is to be considered.

Indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors activate the aryl hydrocarbon receptor

Energy Technology Data Exchange (ETDEWEB)

Moyer, Benjamin J. [Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth, Dartmouth Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756 (United States); Rojas, Itzel Y. [Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Dartmouth Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756 (United States); Murray, Iain A. [Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, University Park, PA 16802 (United States); Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, PA 16802 (United States); Lee, Seokwon; Hazlett, Haley F. [Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Dartmouth Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756 (United States); Perdew, Gary H. [Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, University Park, PA 16802 (United States); Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, PA 16802 (United States); Tomlinson, Craig R., E-mail: Craig.R.Tomlinson@Dartmouth.edu [Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth, Dartmouth Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756 (United States); Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Dartmouth Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756 (United States)

2017-05-15

Indoleamine 2,3-dioxygenase 1 (IDO1) plays a key role in the immune system by regulating tryptophan levels and T cell differentiation. Several tumor types overexpress IDO1 to avoid immune surveillance making IDO1 of interest as a target for therapeutic intervention. As a result, several IDO1 inhibitors are currently being tested in clinical trials for cancer treatment as well as several other diseases. Many of the IDO1 inhibitors in clinical trials naturally bear structural similarities to the IDO1 substrate tryptophan, as such, they fulfill many of the structural and functional criteria as potential AHR ligands. Using mouse and human cell-based luciferase gene reporter assays, qPCR confirmation experiments, and CYP1A1 enzyme activity assays, we report that some of the promising clinical IDO1 inhibitors also act as agonists for the aryl hydrocarbon receptor (AHR), best known for its roles in xenobiotic metabolism and as another key regulator of the immune response. The dual role as IDO antagonist and AHR agonist for many of these IDO target drugs should be considered for full interrogation of their biological mechanisms and clinical outcomes. - Highlights: • Indoleamine-2,3-dioxygenase 1 (IDO1) inhibitors are in cancer clinical trials. • Some IDO1 inhibitors also potently activate AHR signaling. • The dual role of the IDO1 inhibitors may explain some past paradoxical findings. • AHR induction studies must be included in assessing clinical suitability.

Indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors activate the aryl hydrocarbon receptor

International Nuclear Information System (INIS)

Moyer, Benjamin J.; Rojas, Itzel Y.; Murray, Iain A.; Lee, Seokwon; Hazlett, Haley F.; Perdew, Gary H.; Tomlinson, Craig R.

2017-01-01

Indoleamine 2,3-dioxygenase 1 (IDO1) plays a key role in the immune system by regulating tryptophan levels and T cell differentiation. Several tumor types overexpress IDO1 to avoid immune surveillance making IDO1 of interest as a target for therapeutic intervention. As a result, several IDO1 inhibitors are currently being tested in clinical trials for cancer treatment as well as several other diseases. Many of the IDO1 inhibitors in clinical trials naturally bear structural similarities to the IDO1 substrate tryptophan, as such, they fulfill many of the structural and functional criteria as potential AHR ligands. Using mouse and human cell-based luciferase gene reporter assays, qPCR confirmation experiments, and CYP1A1 enzyme activity assays, we report that some of the promising clinical IDO1 inhibitors also act as agonists for the aryl hydrocarbon receptor (AHR), best known for its roles in xenobiotic metabolism and as another key regulator of the immune response. The dual role as IDO antagonist and AHR agonist for many of these IDO target drugs should be considered for full interrogation of their biological mechanisms and clinical outcomes. - Highlights: • Indoleamine-2,3-dioxygenase 1 (IDO1) inhibitors are in cancer clinical trials. • Some IDO1 inhibitors also potently activate AHR signaling. • The dual role of the IDO1 inhibitors may explain some past paradoxical findings. • AHR induction studies must be included in assessing clinical suitability.

1-Methoxy-agroclavine from Penicillium sp. WC75209, a novel inhibitor of the Lck tyrosine kinase.

Science.gov (United States)

Padmanabha, R; Shu, Y Z; Cook, L S; Veitch, J A; Donovan, M; Lowe, S; Huang, S; Pirnik, D; Manly, S P

1998-03-17

A high-throughput screen was developed and implemented to identify inhibitors of the Lck tyrosine kinase. This report describes the identification of a specific inhibitor of this enzyme from the solid fermentation culture of the Penicillium sp., WC75209. The active compound was isolated and structurally characterized as 1-methoxy-5R, 10S-agroclavine, a new member of the ergot alkaloid family.

In silico design and biological evaluation of a dual specificity kinase inhibitor targeting cell cycle progression and angiogenesis.

Science.gov (United States)

Latham, Antony M; Kankanala, Jayakanth; Fearnley, Gareth W; Gage, Matthew C; Kearney, Mark T; Homer-Vanniasinkam, Shervanthi; Wheatcroft, Stephen B; Fishwick, Colin W G; Ponnambalam, Sreenivasan

2014-01-01

Protein kinases play a central role in tumor progression, regulating fundamental processes such as angiogenesis, proliferation and metastasis. Such enzymes are an increasingly important class of drug target with small molecule kinase inhibitors being a major focus in drug development. However, balancing drug specificity and efficacy is problematic with off-target effects and toxicity issues. We have utilized a rational in silico-based approach to demonstrate the design and study of a novel compound that acts as a dual inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2) and cyclin-dependent kinase 1 (CDK1). This compound acts by simultaneously inhibiting pro-angiogenic signal transduction and cell cycle progression in primary endothelial cells. JK-31 displays potent in vitro activity against recombinant VEGFR2 and CDK1/cyclin B proteins comparable to previously characterized inhibitors. Dual inhibition of the vascular endothelial growth factor A (VEGF-A)-mediated signaling response and CDK1-mediated mitotic entry elicits anti-angiogenic activity both in an endothelial-fibroblast co-culture model and a murine ex vivo model of angiogenesis. We deduce that JK-31 reduces the growth of both human endothelial cells and human breast cancer cells in vitro. This novel synthetic molecule has broad implications for development of similar multi-kinase inhibitors with anti-angiogenic and anti-cancer properties. In silico design is an attractive and innovative method to aid such drug discovery.

Inhibitor specificity of recombinant and endogenous caspase-9.

Science.gov (United States)

Ryan, Ciara A; Stennicke, Henning R; Nava, Victor E; Burch, Jennifer B; Hardwick, J Marie; Salvesen, Guy S

2002-01-01

Apoptosis triggered through the intrinsic pathway by radiation and anti-neoplastic drugs is initiated by the activation of caspase-9. To elucidate control mechanisms in this pathway we used a range of synthetic and natural reagents. The inhibitory potency of acetyl-Asp-Glu-Val-Asp-aldehyde ('Ac-DEVD-CHO'), benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone ('Z-VAD-FMK') and the endogenous caspase inhibitor X-chromosome-linked inhibitor of apoptosis protein ('XIAP') against recombinant caspase-9 were predictive of the efficacy of these compounds in a cell-free system. However, the viral proteins CrmA and p35, although potent inhibitors of recombinant caspase-9, had almost no ability to block caspase-9 in this system. These findings were also mirrored in cell expression studies. We hypothesize that the viral inhibitors CrmA and p35 are excluded from reacting productively with the natural form of active caspase-9 in vivo, making the potency of inhibitors highly context-dependent. This is supported by survival data from a mouse model of apoptosis driven by Sindbis virus expressing either p35 or a catalytic mutant of caspase-9. These results consolidate previous findings that CrmA is a potent inhibitor of caspase-9 in vitro, yet fails to block caspase-9-mediated cell death. PMID:12067274

Tackling Cancer Resistance by Immunotherapy: Updated Clinical Impact and Safety of PD-1/PD-L1 Inhibitors

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Shifaa M. Abdin

2018-01-01

Full Text Available Cancer therapy has been constantly evolving with the hope of finding the most effective agents with the least toxic effects to eradicate tumors. Cancer immunotherapy is currently among the most promising options, fulfilling this hope in a wide range of tumors. Immunotherapy aims to activate immunity to fight cancer in a very specific and targeted manner; however, some abnormal immune reactions known as immune-related adverse events (IRAEs might occur. Therefore, many researchers are aiming to define the most proper protocols for managing these complications without interfering with the anticancer effect. One of these targeted approaches is the inhibition of the interaction between the checkpoint protein, programmed death-receptor 1 (PD-1, and its ligand, programmed death-ligand 1 (PD-L1, via a class of antibodies known as PD-1/PD-L1 inhibitors. These antibodies achieved prodigious success in a wide range of malignancies, including those where optimal treatment is not yet fully identified. In this review, we have critically explored and discussed the outcome of the latest PD-1 and PD-L1 inhibitor studies in different malignancies compared to standard chemotherapeutic alternatives with a special focus on the clinical efficacy and safety. The approval of the clinical applications of nivolumab, pembrolizumab, atezolizumab, avelumab, and durvalumab in the last few years clearly highlights the hopeful future of PD-1/PD-L1 inhibitors for cancer patients. These promising results of PD-1/PD-L1 inhibitors have encouraged many ongoing preclinical and clinical trials to explore the extent of antitumor activity, clinical efficacy and safety as well as to extend their applications.

Tackling Cancer Resistance by Immunotherapy: Updated Clinical Impact and Safety of PD-1/PD-L1 Inhibitors.

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Abdin, Shifaa M; Zaher, Dana M; Arafa, El-Shaimaa A; Omar, Hany A

2018-01-25

Cancer therapy has been constantly evolving with the hope of finding the most effective agents with the least toxic effects to eradicate tumors. Cancer immunotherapy is currently among the most promising options, fulfilling this hope in a wide range of tumors. Immunotherapy aims to activate immunity to fight cancer in a very specific and targeted manner; however, some abnormal immune reactions known as immune-related adverse events (IRAEs) might occur. Therefore, many researchers are aiming to define the most proper protocols for managing these complications without interfering with the anticancer effect. One of these targeted approaches is the inhibition of the interaction between the checkpoint protein, programmed death-receptor 1 (PD-1), and its ligand, programmed death-ligand 1 (PD-L1), via a class of antibodies known as PD-1/PD-L1 inhibitors. These antibodies achieved prodigious success in a wide range of malignancies, including those where optimal treatment is not yet fully identified. In this review, we have critically explored and discussed the outcome of the latest PD-1 and PD-L1 inhibitor studies in different malignancies compared to standard chemotherapeutic alternatives with a special focus on the clinical efficacy and safety. The approval of the clinical applications of nivolumab, pembrolizumab, atezolizumab, avelumab, and durvalumab in the last few years clearly highlights the hopeful future of PD-1/PD-L1 inhibitors for cancer patients. These promising results of PD-1/PD-L1 inhibitors have encouraged many ongoing preclinical and clinical trials to explore the extent of antitumor activity, clinical efficacy and safety as well as to extend their applications.

Synergistic apoptosis induction in leukemic cells by the phosphatase inhibitor salubrinal and proteasome inhibitors.

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Hannes C A Drexler

Full Text Available Cells adapt to endoplasmic reticulum (ER-stress by arresting global protein synthesis while simultaneously activating specific transcription factors and their downstream targets. These processes are mediated in part by the phosphorylation-dependent inactivation of the translation initiation factor eIF2alpha. Following restoration of homeostasis protein synthesis is resumed when the serine/threonine-protein phosphatase PP1 dephosphorylates and reactivates eIF2alpha. Proteasome inhibitors, used to treat multiple myeloma patients evoke ER-stress and apoptosis by blocking the ER-associated degradation of misfolded proteins (ERAD, however, the role of eIF2alpha phosphorylation in leukemic cells under conditions of proteasome inhibitor-mediated ER stress is currently unclear.Bcr-Abl-positive and negative leukemic cell lines were used to investigate the functional implications of PP1-related phosphatase activities on eIF2alpha phosphorylation in proteasome inhibitor-mediated ER stress and apoptosis. Rather unexpectedly, salubrinal, a recently identified PP1 inhibitor capable to protect against ER stress in various model systems, strongly synergized with proteasome inhibitors to augment apoptotic death of different leukemic cell lines. Salubrinal treatment did not affect the phosphorlyation status of eIF2alpha. Furthermore, the proapoptotic effect of salubrinal occurred independently from the chemical nature of the proteasome inhibitor, was recapitulated by a second unrelated phosphatase inhibitor and was unaffected by overexpression of a dominant negative eIF2alpha S51A variant that can not be phosphorylated. Salubrinal further aggravated ER-stress and proteotoxicity inflicted by the proteasome inhibitors on the leukemic cells since characteristic ER stress responses, such as ATF4 and CHOP synthesis, XBP1 splicing, activation of MAP kinases and eventually apoptosis were efficiently abrogated by the translational inhibitor cycloheximide.Although PP1

Inhibition of herpes simplex virus type 1 entry by chloride channel inhibitors tamoxifen and NPPB

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Zheng, Kai [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); College of Life Science and Technology, Jinan University, Guangzhou (China); Chen, Maoyun [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); College of pharmacy, Jinan University, Guangzhou (China); Xiang, Yangfei; Ma, Kaiqi [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); Jin, Fujun [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); College of pharmacy, Jinan University, Guangzhou (China); Wang, Xiao [School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006 (China); Wang, Xiaoyan; Wang, Shaoxiang [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); Wang, Yifei, E-mail: twang-yf@163.com [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China)

2014-04-18

Highlights: • We analyze the anti-HSV potential of chloride channel inhibitors. • Tamoxifen and NPPB show anti-HSV-1 and anti-ACV-resistant HSV-1 activities. • HSV-1 infection induces intracellular chloride concentration increasing. • Tamoxifen and NPPB inhibit HSV-1 early infection. • Tamoxifen and NPPB prevent the fusion process of HSV-1. - Abstract: Herpes simplex virus type 1 (HSV-1) infection is very common worldwide and can cause significant health problems from periodic skin and corneal lesions to encephalitis. Appearance of drug-resistant viruses in clinical therapy has made exploring novel antiviral agents emergent. Here we show that chloride channel inhibitors, including tamoxifen and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB), exhibited extensive antiviral activities toward HSV-1 and ACV-resistant HSV viruses. HSV-1 infection induced chloride ion influx while treatment with inhibitors reduced the increase of intracellular chloride ion concentration. Pretreatment or treatment of inhibitors at different time points during HSV-1 infection all suppressed viral RNA synthesis, protein expression and virus production. More detailed studies demonstrated that tamoxifen and NPPB acted as potent inhibitors of HSV-1 early entry step by preventing viral binding, penetration and nuclear translocation. Specifically the compounds appeared to affect viral fusion process by inhibiting virus binding to lipid rafts and interrupting calcium homeostasis. Taken together, the observation that tamoxifen and NPPB can block viral entry suggests a stronger potential for these compounds as well as other ion channel inhibitors in antiviral therapy against HSV-1, especially the compound tamoxifen is an immediately actionable drug that can be reused for treatment of HSV-1 infections.

Inhibition of herpes simplex virus type 1 entry by chloride channel inhibitors tamoxifen and NPPB

International Nuclear Information System (INIS)

Zheng, Kai; Chen, Maoyun; Xiang, Yangfei; Ma, Kaiqi; Jin, Fujun; Wang, Xiao; Wang, Xiaoyan; Wang, Shaoxiang; Wang, Yifei

2014-01-01

Highlights: • We analyze the anti-HSV potential of chloride channel inhibitors. • Tamoxifen and NPPB show anti-HSV-1 and anti-ACV-resistant HSV-1 activities. • HSV-1 infection induces intracellular chloride concentration increasing. • Tamoxifen and NPPB inhibit HSV-1 early infection. • Tamoxifen and NPPB prevent the fusion process of HSV-1. - Abstract: Herpes simplex virus type 1 (HSV-1) infection is very common worldwide and can cause significant health problems from periodic skin and corneal lesions to encephalitis. Appearance of drug-resistant viruses in clinical therapy has made exploring novel antiviral agents emergent. Here we show that chloride channel inhibitors, including tamoxifen and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB), exhibited extensive antiviral activities toward HSV-1 and ACV-resistant HSV viruses. HSV-1 infection induced chloride ion influx while treatment with inhibitors reduced the increase of intracellular chloride ion concentration. Pretreatment or treatment of inhibitors at different time points during HSV-1 infection all suppressed viral RNA synthesis, protein expression and virus production. More detailed studies demonstrated that tamoxifen and NPPB acted as potent inhibitors of HSV-1 early entry step by preventing viral binding, penetration and nuclear translocation. Specifically the compounds appeared to affect viral fusion process by inhibiting virus binding to lipid rafts and interrupting calcium homeostasis. Taken together, the observation that tamoxifen and NPPB can block viral entry suggests a stronger potential for these compounds as well as other ion channel inhibitors in antiviral therapy against HSV-1, especially the compound tamoxifen is an immediately actionable drug that can be reused for treatment of HSV-1 infections

Effect of catalase-specific inhibitor 3-amino-1,2,4-triazole on yeast peroxisomal catalase in vivo.

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Ueda, Mitsuyoshi; Kinoshita, Hiroshi; Yoshida, Tomoko; Kamasawa, Naomi; Osumi, Masako; Tanaka, Atsuo

2003-02-14

3-Amino-1,2,4-triazole (3-AT) is known as an inhibitor of catalase to whose active center it specifically and covalently binds. Subcellular fractionation and immunoelectronmicroscopic observation of the yeast Candida tropicalis revealed that, in 3-AT-treated cells in which the 3-AT was added to the n-alkane medium from the beginning of cultivation, catalase transported into peroxisomes was inactivated and was present as insoluble aggregated forms in the organelle. The aggregation of catalase in peroxisomes occurred only in these 3-AT-treated cells and not in cells in which 3-AT was added at the late exponential growth phase. Furthermore, 3-AT did not affect the transportation of catalase into peroxisomes. The appearance of aggregation only in cells to which 3-AT was added from the beginning of cultivation suggests that, in the process of catalase transportation into yeast peroxisomes, some conformational change may take place and that correct folding may be inhibited by the binding of 3-AT to the active center of catalase. Accordingly, 3-AT will be an interesting compound for investigation of the transport machinery of the peroxisomal tetrameric catalase.

Glycyrrhetinic acid and its derivatives as inhibitors of poly(ADP-ribosepolymerases 1 and 2, apurinic/apyrimidinic endonuclease 1 and DNA polymerase β

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Salakhutdinov N. F.

2012-06-01

Full Text Available Aim. For strengthening the efficiency of monofunctional alkylating antineoplastic drugs it is important to lower the capacity of base excision repair (BER system which corrects the majority of DNA damages caused by these reagents. The objective was to create inhibitors of the key BER enzymes (PARP1, PARP2, DNA polymerase β, and APE1 by the directed modification of glycyrrhetinic acid (GA. Methods. Amides of GA were produced from the GA acetate by formation of the corresponding acyl chloride, amidation with the appropriate amine and subsequent deacylation. Small library of 2-cyano substituted derivatives of GA methyl esters was obtained by the structural modification of GA framework and carboxylic acid group. The inhibitory capacity of the compounds was estimated by comparison of the enzyme activities in specific tests in the presence of compounds versus their absence. Results. None of tested compounds inhibits PARP1 significantly. Unmodified GA and its morpholinic derivative were shown to be weak inhibitors of PARP2. The derivatives of GA containing keto-group in 11 triterpene framework were shown to be moderate inhibitors of pol β. Compound 3, containing 12-oxo-9(11-en moiety in the ring C, was shown to be a single inhibitor of APE1 among all compounds studied. Conclusions. The class of GA derivatives, selective pol β inhibitors, was found out. The selective inhibitor of APE1 and weak selective inhibitor of PARP2 were also revealed.

Subnanomolar Inhibitor of Cytochrome bc1 Complex Designed via Optimizing Interaction with Conformationally Flexible Residues

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Zhao, Pei-Liang; Wang, Le; Zhu, Xiao-Lei; Huang, Xiaoqin; Zhan, Chang-Guo; Wu, Jia-Wei; Yang, Guang-Fu

2009-01-01

Cytochrome bc1 complex (EC 1.10.2.2, bc1), an essential component of the cellular respiratory chain and the photosynthetic apparatus in photosynthetic bacteria, has been identified as a promising target for new drugs and agricultural fungicides. X-ray diffraction structures of the free bc1 complex and its complexes with various inhibitors revealed that the phenyl group of Phe274 in the binding pocket exhibited significant conformational flexibility upon different inhibitors binding to optimize respective π-π interactions, whereas the side chains of other hydrophobic residues showed conformational stability. Therefore, in the present study, a strategy of optimizing the π-π interaction with conformationally flexible residues was proposed to design and discover new bc1 inhibitors with a higher potency. Eight new compounds were designed and synthesized, among which compound 5c with a Ki value of 570 pM was identified as the most promising drug or fungicide candidate, significantly more potent than the commercially available bc1 inhibitors including azoxystrobin (AZ), kresoxim-methyl (KM), and pyraclostrobin (PY). To our knowledge, this is the first bc1 inhibitor discovered from structure-based design with a potency of subnanomolar Ki value. For all of the compounds synthesized and assayed, the calculated binding free energies correlated reasonably well with the binding free energies derived from the experimental Ki values with a correlation coefficient of r2 = 0.89. The further inhibitory kinetics studies revealed that compound 5c is a non-competitive inhibitor with respect to substrate cytochrome c, but is a competitive inhibitor with respect to substrate ubiquinol. Due to its subnanomolar Ki potency and slow dissociation rate constant (k−0 = 0.00358 s−1), compound 5c could be used as a specific probe for further elucidation of the mechanism of bc1 function and as a new lead compound for future drug discovery. PMID:19928849

Long-term electromagnetic pulse exposure induces Abeta deposition and cognitive dysfunction through oxidative stress and overexpression of APP and BACE1.

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Jiang, Da-Peng; Li, Jin-Hui; Zhang, Jie; Xu, Sheng-Long; Kuang, Fang; Lang, Hai-Yang; Wang, Ya-Feng; An, Guang-Zhou; Li, Jing; Guo, Guo-Zhen

2016-07-01

A progressively expanded literature has been devoted in the past years to the noxious or beneficial effects of electromagnetic field (EMF) to Alzheimer׳s disease (AD). This study concerns the relationship between electromagnetic pulse (EMP) exposure and the occurrence of AD in rats and the underlying mechanisms, focusing on the role of oxidative stress (OS). 55 healthy male Sprague Dawley (SD) rats were used and received continuous exposure for 8 months. Morris water maze (MWM) test was conducted to test the ability of cognitive and memory. The level of OS was detected by superoxide dismutase (SOD) activity and glutathione (GSH) content. We found that long-term EMP exposure induced cognitive damage in rats. The content of β-amyloid (Aβ) protein in hippocampus was increased after long-term EMP exposure. OS of hippocampal neuron was detected. Western blotting and immunohistochemistry (IHC) assay showed that the content of Aβ protein and its oligomers in EMP-exposed rats were higher than that of sham-exposed rats. The content of Beta Site App Cleaving Enzyme (BACE1) and microtubule-associated protein 1 light chain 3-II (LC3-II) in EMP-exposed rats hippocampus were also higher than that of sham-exposed rats. SOD activity and GSH content in EMP-exposed rats were lower than sham-exposed rats (p<0.05). Several mechanisms were proposed based on EMP exposure-induced OS, including increased amyloid precursor protein (APP) aberrant cleavage. Although further study is needed, the present results suggest that long-term EMP exposure is harmful to cognitive ability in rats and could induce AD-like pathological manifestation. Copyright © 2016 Elsevier B.V. All rights reserved.

Consensus QSAR model for identifying novel H5N1 inhibitors.

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Sharma, Nitin; Yap, Chun Wei

2012-08-01

Due to the importance of neuraminidase in the pathogenesis of influenza virus infection, it has been regarded as the most important drug target for the treatment of influenza. Resistance to currently available drugs and new findings related to structure of the protein requires novel neuraminidase 1 (N1) inhibitors. In this study, a consensus QSAR model with defined applicability domain (AD) was developed using published N1 inhibitors. The consensus model was validated using an external validation set. The model achieved high sensitivity, specificity, and overall accuracy along with low false positive rate (FPR) and false discovery rate (FDR). The performance of model on the external validation set and training set were comparable, thus it was unlikely to be overfitted. The low FPR and low FDR will increase its accuracy in screening large chemical libraries. Screening of ZINC library resulted in 64,772 compounds as probable N1 inhibitors, while 173,674 compounds were defined to be outside the AD of the consensus model. The advantage of the current model is that it was developed using a large and diverse dataset and has a defined AD which prevents its use on compounds that it is not capable of predicting. The consensus model developed in this study is made available via the free software, PaDEL-DDPredictor.

CINPA1 is an inhibitor of constitutive androstane receptor that does not activate pregnane X receptor.

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Cherian, Milu T; Lin, Wenwei; Wu, Jing; Chen, Taosheng

2015-05-01

Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic sensors that enhance the detoxification and elimination of xenobiotics and endobiotics by modulating the expression of genes encoding drug-metabolizing enzymes and transporters. Elevated levels of drug-metabolizing enzymes and efflux transporters, resulting from CAR activation in various cancers, promote the elimination of chemotherapeutic agents, leading to reduced therapeutic effectiveness and acquired drug resistance. CAR inhibitors, in combination with existing chemotherapeutics, could therefore be used to attenuate multidrug resistance in cancers. Interestingly, all previously reported CAR inverse-agonists are also activators of PXR, rendering them mechanistically counterproductive in tissues where both these xenobiotic receptors are present and active. We used a directed high-throughput screening approach, followed by subsequent mechanistic studies, to identify novel, potent, and specific small-molecule CAR inhibitors that do not activate PXR. We describe here one such inhibitor, CINPA1 (CAR inhibitor not PXR activator 1), capable of reducing CAR-mediated transcription with an IC50 of ∼70 nM. CINPA1 1) is a specific xenobiotic receptor inhibitor and has no cytotoxic effects up to 30 µM; 2) inhibits CAR-mediated gene expression in primary human hepatocytes, where CAR is endogenously expressed; 3) does not alter the protein levels or subcellular localization of CAR; 4) increases corepressor and reduces coactivator interaction with the CAR ligand-binding domain in mammalian two-hybrid assays; and 5) disrupts CAR binding to the promoter regions of target genes in chromatin immunoprecipitation assays. CINPA1 could be used as a novel molecular tool for understanding CAR function. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

The prototype HIV-1 maturation inhibitor, bevirimat, binds to the CA-SP1 cleavage site in immature Gag particles

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Nguyen Albert T

2011-12-01

Full Text Available Abstract Background Bevirimat, the prototype Human Immunodeficiency Virus type 1 (HIV-1 maturation inhibitor, is highly potent in cell culture and efficacious in HIV-1 infected patients. In contrast to inhibitors that target the active site of the viral protease, bevirimat specifically inhibits a single cleavage event, the final processing step for the Gag precursor where p25 (CA-SP1 is cleaved to p24 (CA and SP1. Results In this study, photoaffinity analogs of bevirimat and mass spectrometry were employed to map the binding site of bevirimat to Gag within immature virus-like particles. Bevirimat analogs were found to crosslink to sequences overlapping, or proximal to, the CA-SP1 cleavage site, consistent with previous biochemical data on the effect of bevirimat on Gag processing and with genetic data from resistance mutations, in a region predicted by NMR and mutational studies to have α-helical character. Unexpectedly, a second region of interaction was found within the Major Homology Region (MHR. Extensive prior genetic evidence suggests that the MHR is critical for virus assembly. Conclusions This is the first demonstration of a direct interaction between the maturation inhibitor, bevirimat, and its target, Gag. Information gained from this study sheds light on the mechanisms by which the virus develops resistance to this class of drug and may aid in the design of next-generation maturation inhibitors.

Novel orally bioavailable EZH1/2 dual inhibitors with greater antitumor efficacy than an EZH2 selective inhibitor.

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Honma, Daisuke; Kanno, Osamu; Watanabe, Jun; Kinoshita, Junzo; Hirasawa, Makoto; Nosaka, Emi; Shiroishi, Machiko; Takizawa, Takeshi; Yasumatsu, Isao; Horiuchi, Takao; Nakao, Akira; Suzuki, Keisuke; Yamasaki, Tomonori; Nakajima, Katsuyoshi; Hayakawa, Miho; Yamazaki, Takanori; Yadav, Ajay Singh; Adachi, Nobuaki

2017-10-01

Polycomb repressive complex 2 (PRC2) methylates histone H3 lysine 27 and represses gene expression to regulate cell proliferation and differentiation. Enhancer of zeste homolog 2 (EZH2) or its close homolog EZH1 functions as a catalytic subunit of PRC2, so there are two PRC2 complexes containing either EZH2 or EZH1. Tumorigenic functions of EZH2 and its synthetic lethality with some subunits of SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complexes have been observed. However, little is known about the function of EZH1 in tumorigenesis. Herein, we developed novel, orally bioavailable EZH1/2 dual inhibitors that strongly and selectively inhibited methyltransferase activity of both EZH2 and EZH1. EZH1/2 dual inhibitors suppressed trimethylation of histone H3 lysine 27 in cells more than EZH2 selective inhibitors. They also showed greater antitumor efficacy than EZH2 selective inhibitor in vitro and in vivo against diffuse large B-cell lymphoma cells harboring gain-of-function mutation in EZH2. A hematological cancer panel assay indicated that EZH1/2 dual inhibitor has efficacy against some lymphomas, multiple myeloma, and leukemia with fusion genes such as MLL-AF9, MLL-AF4, and AML1-ETO. A solid cancer panel assay demonstrated that some cancer cell lines are sensitive to EZH1/2 dual inhibitor in vitro and in vivo. No clear correlation was detected between sensitivity to EZH1/2 dual inhibitor and SWI/SNF mutations, with a few exceptions. Severe toxicity was not seen in rats treated with EZH1/2 dual inhibitor for 14 days at drug levels higher than those used in the antitumor study. Our results indicate the possibility of EZH1/2 dual inhibitors for clinical applications. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Molecular dynamics of conformation-specific dopamine transporter-inhibitor complexes.

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Jean, Bernandie; Surratt, Christopher K; Madura, Jeffry D

2017-09-01

The recreational psychostimulant cocaine inhibits dopamine reuptake from the synapse, resulting in excessive stimulation of postsynaptic dopamine receptors in brain areas associated with reward and addiction. Cocaine binds to and stabilizes the outward- (extracellular-) facing conformation of the dopamine transporter (DAT) protein, while the low abuse potential DAT inhibitor benztropine prefers the inward- (cytoplasmic-) facing conformation. A correlation has been previously postulated between psychostimulant abuse potential and preference for the outward-facing DAT conformation. The 3β-aryltropane cocaine analogs LX10 and LX11, however, differ only in stereochemistry and share a preference for the outward-facing DAT, yet are reported to vary widely in abuse potential in an animal model. In search of the molecular basis for DAT conformation preference, complexes of cocaine, benztropine, LX10 or LX11 bound to each DAT conformation were subjected to 100ns of all-atom molecular dynamics simulation. Results were consistent with previous findings from cysteine accessibility assays used to assess an inhibitor's DAT conformation preference. The respective 2β- and 2α-substituted phenyltropanes of LX10 and LX11 interacted with hydrophobic regions of the DAT S1 binding site that were inaccessible to cocaine. Solvent accessibility measurements also revealed subtle differences in inhibitor positioning within a given DAT conformation. This work serves to advance our understanding of the conformational selectivity of DAT inhibitors and suggests that MD may be useful in antipsychostimulant therapeutic design. Copyright © 2017 Elsevier Inc. All rights reserved.

Inhibitors of serotonin reuptake and specific imipramine binding in human blood plasma

International Nuclear Information System (INIS)

Brusov, O.S.; Fomenko, A.M.; Katasonov, A.B.; Lidemann, R.R.

1985-01-01

This paper describes a method of extraction of endogenous inhibitors of specific IMI binding and of 5-HT reuptake, from human blood plasma and the heterogeneity of these compounds is demonstrated. Specific binding was determined as the difference between binding of 3 H-IMI in the absence and in the presence of 50 microM IMI. Under these conditions, specific binding amounted to 70-80% of total binding of 3 H-IMI. It is shown that extract obtained from human blood contains a material which inhibits dose-dependently both 5-HT reuptake and specific binding of 3 H-IMI. Gel-chromatography of extracts of human blood plasma on Biogel P-2 is also shown

In silico design and biological evaluation of a dual specificity kinase inhibitor targeting cell cycle progression and angiogenesis.

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Antony M Latham

Full Text Available Protein kinases play a central role in tumor progression, regulating fundamental processes such as angiogenesis, proliferation and metastasis. Such enzymes are an increasingly important class of drug target with small molecule kinase inhibitors being a major focus in drug development. However, balancing drug specificity and efficacy is problematic with off-target effects and toxicity issues.We have utilized a rational in silico-based approach to demonstrate the design and study of a novel compound that acts as a dual inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2 and cyclin-dependent kinase 1 (CDK1. This compound acts by simultaneously inhibiting pro-angiogenic signal transduction and cell cycle progression in primary endothelial cells. JK-31 displays potent in vitro activity against recombinant VEGFR2 and CDK1/cyclin B proteins comparable to previously characterized inhibitors. Dual inhibition of the vascular endothelial growth factor A (VEGF-A-mediated signaling response and CDK1-mediated mitotic entry elicits anti-angiogenic activity both in an endothelial-fibroblast co-culture model and a murine ex vivo model of angiogenesis.We deduce that JK-31 reduces the growth of both human endothelial cells and human breast cancer cells in vitro. This novel synthetic molecule has broad implications for development of similar multi-kinase inhibitors with anti-angiogenic and anti-cancer properties. In silico design is an attractive and innovative method to aid such drug discovery.

Plasminogen Activator Inhibitor-1 Controls Vascular Integrity by Regulating VE-Cadherin Trafficking.

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Anna E Daniel

Full Text Available Plasminogen activator inhibitor-1 (PAI-1, a serine protease inhibitor, is expressed and secreted by endothelial cells. Patients with PAI-1 deficiency show a mild to moderate bleeding diathesis, which has been exclusively ascribed to the function of PAI-1 in down-regulating fibrinolysis. We tested the hypothesis that PAI-1 function plays a direct role in controlling vascular integrity and permeability by keeping endothelial cell-cell junctions intact.We utilized PAI-039, a specific small molecule inhibitor of PAI-1, to investigate the role of PAI-1 in protecting endothelial integrity. In vivo inhibition of PAI-1 resulted in vascular leakage from intersegmental vessels and in the hindbrain of zebrafish embryos. In addition PAI-1 inhibition in human umbilical vein endothelial cell (HUVEC monolayers leads to a marked decrease of transendothelial resistance and disrupted endothelial junctions. The total level of the endothelial junction regulator VE-cadherin was reduced, whereas surface VE-cadherin expression was unaltered. Moreover, PAI-1 inhibition reduced the shedding of VE-cadherin. Finally, we detected an accumulation of VE-cadherin at the Golgi apparatus.Our findings indicate that PAI-1 function is important for the maintenance of endothelial monolayer and vascular integrity by controlling VE-cadherin trafficking to and from the plasma membrane. Our data further suggest that therapies using PAI-1 antagonists like PAI-039 ought to be used with caution to avoid disruption of the vessel wall.

Inhibitors of Deubiquitinating Enzymes Block HIV-1 Replication and Augment the Presentation of Gag-Derived MHC-I Epitopes.

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Setz, Christian; Friedrich, Melanie; Rauch, Pia; Fraedrich, Kirsten; Matthaei, Alina; Traxdorf, Maximilian; Schubert, Ulrich

2017-08-12

In recent years it has been well established that two major constituent parts of the ubiquitin proteasome system (UPS)-the proteasome holoenzymes and a number of ubiquitin ligases-play a crucial role, not only in virus replication but also in the regulation of the immunogenicity of human immunodeficiency virus type 1 (HIV-1). However, the role in HIV-1 replication of the third major component, the deubiquitinating enzymes (DUBs), has remained largely unknown. In this study, we show that the DUB-inhibitors (DIs) P22077 and PR-619, specific for the DUBs USP7 and USP47, impair Gag processing and thereby reduce the infectivity of released virions without affecting viral protease activity. Furthermore, the replication capacity of X4- and R5-tropic HIV-1 NL4-3 in human lymphatic tissue is decreased upon treatment with these inhibitors without affecting cell viability. Most strikingly, combinatory treatment with DIs and proteasome inhibitors synergistically blocks virus replication at concentrations where mono-treatment was ineffective, indicating that DIs can boost the therapeutic effect of proteasome inhibitors. In addition, P22077 and PR-619 increase the polyubiquitination of Gag and thus its entry into the UPS and the major histocompatibility complex (MHC)-I pathway. In summary, our data point towards a model in which specific inhibitors of DUBs not only interfere with virus spread but also increase the immune recognition of HIV-1 expressing cells.

Molecular phylogeny of C1 inhibitor depicts two immunoglobulin-like domains fusion in fishes and ray-finned fishes specific intron insertion after separation from zebrafish

International Nuclear Information System (INIS)

Kumar, Abhishek; Bhandari, Anita; Sarde, Sandeep J.; Goswami, Chandan

2014-01-01

Highlights: • C1 inhibitors of fishes have two Ig domains fused in the N-terminal end. • Spliceosomal introns gain in two Ig domains of selected ray-finned fishes. • C1 inhibitors gene is maintained from 450 MY on the same locus. • C1 inhibitors gene is missing in frog and lampreys. • C1 inhibitors of tetrapod and fishes differ in the RCL region. - Abstract: C1 inhibitor (C1IN) is a multi-facet serine protease inhibitor in the plasma cascades, inhibiting several proteases, notably, regulates both complement and contact system activation. Despite huge advancements in the understanding of C1IN based on biochemical properties and its roles in the plasma cascades, the phylogenetic history of C1IN remains uncharacterized. To date, there is no comprehensive study illustrating the phylogenetic history of C1IN. Herein, we explored phylogenetic history of C1IN gene in vertebrates. Fishes have C1IN with two immunoglobulin like domains attached in the N-terminal region. The RCL regions of CIIN from fishes and tetrapod genomes have variations at the positions P2 and P1′. Gene structures of C1IN gene from selected ray-finned fishes varied in the Ig domain region with creation of novel intron splitting exon Im2 into Im2a and Im2b. This intron is limited to ray-finned fishes with genome size reduced below 1 Gb. Hence, we suggest that genome compaction and associated double-strand break repairs are behind this intron gain. This study reveals the evolutionary history of C1IN and confirmed that this gene remains the same locus for ∼450 MY in 52 vertebrates analysed, but it is not found in frogs and lampreys

Molecular phylogeny of C1 inhibitor depicts two immunoglobulin-like domains fusion in fishes and ray-finned fishes specific intron insertion after separation from zebrafish

Energy Technology Data Exchange (ETDEWEB)

Kumar, Abhishek, E-mail: akumar@bot.uni-kiel.de [Department of Genetics and Molecular Biology in Botany, Institute of Botany, Christian-Albrechts-University at Kiel, Kiel (Germany); Bhandari, Anita [Molecular Physiology, Zoological Institute, Christian-Albrechts-University at Kiel, Kiel (Germany); Sarde, Sandeep J. [Department of Genetics and Molecular Biology in Botany, Institute of Botany, Christian-Albrechts-University at Kiel, Kiel (Germany); Goswami, Chandan [National Institute of Science Education and Research, Bhubaneswar, Orissa (India)

2014-07-18

Highlights: • C1 inhibitors of fishes have two Ig domains fused in the N-terminal end. • Spliceosomal introns gain in two Ig domains of selected ray-finned fishes. • C1 inhibitors gene is maintained from 450 MY on the same locus. • C1 inhibitors gene is missing in frog and lampreys. • C1 inhibitors of tetrapod and fishes differ in the RCL region. - Abstract: C1 inhibitor (C1IN) is a multi-facet serine protease inhibitor in the plasma cascades, inhibiting several proteases, notably, regulates both complement and contact system activation. Despite huge advancements in the understanding of C1IN based on biochemical properties and its roles in the plasma cascades, the phylogenetic history of C1IN remains uncharacterized. To date, there is no comprehensive study illustrating the phylogenetic history of C1IN. Herein, we explored phylogenetic history of C1IN gene in vertebrates. Fishes have C1IN with two immunoglobulin like domains attached in the N-terminal region. The RCL regions of CIIN from fishes and tetrapod genomes have variations at the positions P2 and P1′. Gene structures of C1IN gene from selected ray-finned fishes varied in the Ig domain region with creation of novel intron splitting exon Im2 into Im2a and Im2b. This intron is limited to ray-finned fishes with genome size reduced below 1 Gb. Hence, we suggest that genome compaction and associated double-strand break repairs are behind this intron gain. This study reveals the evolutionary history of C1IN and confirmed that this gene remains the same locus for ∼450 MY in 52 vertebrates analysed, but it is not found in frogs and lampreys.

Chemical probing of the human sirtuin 5 active site reveals its substrate acyl specificity and peptide-based inhibitors.

Science.gov (United States)

Roessler, Claudia; Nowak, Theresa; Pannek, Martin; Gertz, Melanie; Nguyen, Giang T T; Scharfe, Michael; Born, Ilona; Sippl, Wolfgang; Steegborn, Clemens; Schutkowski, Mike

2014-09-26

Sirtuins are NAD(+)-dependent deacetylases acting as sensors in metabolic pathways and stress response. In mammals there are seven isoforms. The mitochondrial sirtuin 5 is a weak deacetylase but a very efficient demalonylase and desuccinylase; however, its substrate acyl specificity has not been systematically analyzed. Herein, we investigated a carbamoyl phosphate synthetase 1 derived peptide substrate and modified the lysine side chain systematically to determine the acyl specificity of Sirt5. From that point we designed six potent peptide-based inhibitors that interact with the NAD(+) binding pocket. To characterize the interaction details causing the different substrate and inhibition properties we report several X-ray crystal structures of Sirt5 complexed with these peptides. Our results reveal the Sirt5 acyl selectivity and its molecular basis and enable the design of inhibitors for Sirt5. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Interdependence of Inhibitor Recognition in HIV-1 Protease.

Science.gov (United States)

Paulsen, Janet L; Leidner, Florian; Ragland, Debra A; Kurt Yilmaz, Nese; Schiffer, Celia A

2017-05-09

Molecular recognition is a highly interdependent process. Subsite couplings within the active site of proteases are most often revealed through conditional amino acid preferences in substrate recognition. However, the potential effect of these couplings on inhibition and thus inhibitor design is largely unexplored. The present study examines the interdependency of subsites in HIV-1 protease using a focused library of protease inhibitors, to aid in future inhibitor design. Previously a series of darunavir (DRV) analogs was designed to systematically probe the S1' and S2' subsites. Co-crystal structures of these analogs with HIV-1 protease provide the ideal opportunity to probe subsite interdependency. All-atom molecular dynamics simulations starting from these structures were performed and systematically analyzed in terms of atomic fluctuations, intermolecular interactions, and water structure. These analyses reveal that the S1' subsite highly influences other subsites: the extension of the hydrophobic P1' moiety results in 1) reduced van der Waals contacts in the P2' subsite, 2) more variability in the hydrogen bond frequencies with catalytic residues and the flap water, and 3) changes in the occupancy of conserved water sites both proximal and distal to the active site. In addition, one of the monomers in this homodimeric enzyme has atomic fluctuations more highly correlated with DRV than the other monomer. These relationships intricately link the HIV-1 protease subsites and are critical to understanding molecular recognition and inhibitor binding. More broadly, the interdependency of subsite recognition within an active site requires consideration in the selection of chemical moieties in drug design; this strategy is in contrast to what is traditionally done with independent optimization of chemical moieties of an inhibitor.

Evaluation of spiropiperidine hydantoins as a novel class of antimalarial agents.

Science.gov (United States)

Meyers, Marvin J; Anderson, Elizabeth J; McNitt, Sarah A; Krenning, Thomas M; Singh, Megh; Xu, Jing; Zeng, Wentian; Qin, Limei; Xu, Wanwan; Zhao, Siting; Qin, Li; Eickhoff, Christopher S; Oliva, Jonathan; Campbell, Mary A; Arnett, Stacy D; Prinsen, Michael J; Griggs, David W; Ruminski, Peter G; Goldberg, Daniel E; Ding, Ke; Liu, Xiaorong; Tu, Zhengchao; Tortorella, Micky D; Sverdrup, Francis M; Chen, Xiaoping

2015-08-15

Given the rise of parasite resistance to all currently used antimalarial drugs, the identification of novel chemotypes with unique mechanisms of action is of paramount importance. Since Plasmodium expresses a number of aspartic proteases necessary for its survival, we have mined antimalarial datasets for drug-like aspartic protease inhibitors. This effort led to the identification of spiropiperidine hydantoins, bearing similarity to known inhibitors of the human aspartic protease β-secretase (BACE), as new leads for antimalarial drug discovery. Spiropiperidine hydantoins have a dynamic structure-activity relationship profile with positions identified as being tolerant of a variety of substitution patterns as well as a key piperidine N-benzyl phenol pharmacophore. Lead compounds 4e (CWHM-123) and 12k (CWHM-505) are potent antimalarials with IC50 values against Plasmodium falciparum 3D7 of 0.310 μM and 0.099 μM, respectively, and the former features equivalent potency on the chloroquine-resistant Dd2 strain. Remarkably, these compounds do not inhibit human aspartic proteases BACE, cathepsins D and E, or Plasmodium plasmepsins II and IV despite their similarity to known BACE inhibitors. Although the current leads suffer from poor metabolic stability, they do fit into a drug-like chemical property space and provide a new class of potent antimalarial agents for further study. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effects of specific inhibitors on anammox and denitrification in marine sediments.

Science.gov (United States)

Jensen, Marlene Mark; Thamdrup, Bo; Dalsgaard, Tage

2007-05-01

The effects of three metabolic inhibitors (acetylene, methanol, and allylthiourea [ATU]) on the pathways of N2 production were investigated by using short anoxic incubations of marine sediment with a 15N isotope technique. Acetylene inhibited ammonium oxidation through the anammox pathway as the oxidation rate decreased exponentially with increasing acetylene concentration; the rate decay constant was 0.10+/-0.02 microM-1, and there was 95% inhibition at approximately 30 microM. Nitrous oxide reduction, the final step of denitrification, was not sensitive to acetylene concentrations below 10 microM. However, nitrous oxide reduction was inhibited by higher concentrations, and the sensitivity was approximately one-half the sensitivity of anammox (decay constant, 0.049+/-0.004 microM-1; 95% inhibition at approximately 70 microM). Methanol specifically inhibited anammox with a decay constant of 0.79+/-0.12 mM-1, and thus 3 to 4 mM methanol was required for nearly complete inhibition. This level of methanol stimulated denitrification by approximately 50%. ATU did not have marked effects on the rates of anammox and denitrification. The profile of inhibitor effects on anammox agreed with the results of studies of the process in wastewater bioreactors, which confirmed the similarity between the anammox bacteria in bioreactors and natural environments. Acetylene and methanol can be used to separate anammox and denitrification, but the effects of these compounds on nitrification limits their use in studies of these processes in systems where nitrification is an important source of nitrate. The observed differential effects of acetylene and methanol on anammox and denitrification support our current understanding of the two main pathways of N2 production in marine sediments and the use of 15N isotope methods for their quantification.

Screening and identification of potential PTP1B allosteric inhibitors using in silico and in vitro approaches.

Science.gov (United States)

Shinde, Ranajit Nivrutti; Kumar, G Siva; Eqbal, Shahbaz; Sobhia, M Elizabeth

2018-01-01

Protein tyrosine phosphatase 1B (PTP1B) is a validated therapeutic target for Type 2 diabetes due to its specific role as a negative regulator of insulin signaling pathways. Discovery of active site directed PTP1B inhibitors is very challenging due to highly conserved nature of the active site and multiple charge requirements of the ligands, which makes them non-selective and non-permeable. Identification of the PTP1B allosteric site has opened up new avenues for discovering potent and selective ligands for therapeutic intervention. Interactions made by potent allosteric inhibitor in the presence of PTP1B were studied using Molecular Dynamics (MD). Computationally optimized models were used to build separate pharmacophore models of PTP1B and TCPTP, respectively. Based on the nature of interactions the target residues offered, a receptor based pharmacophore was developed. The pharmacophore considering conformational flexibility of the residues was used for the development of pharmacophore hypothesis to identify potentially active inhibitors by screening large compound databases. Two pharmacophore were successively used in the virtual screening protocol to identify potential selective and permeable inhibitors of PTP1B. Allosteric inhibition mechanism of these molecules was established using molecular docking and MD methods. The geometrical criteria values confirmed their ability to stabilize PTP1B in an open conformation. 23 molecules that were identified as potential inhibitors were screened for PTP1B inhibitory activity. After screening, 10 molecules which have good permeability values were identified as potential inhibitors of PTP1B. This study confirms that selective and permeable inhibitors can be identified by targeting allosteric site of PTP1B.

Two-dimensional NMR studies of squash family inhibitors. Sequence-specific proton assignments and secondary structure of reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor III

Energy Technology Data Exchange (ETDEWEB)

Krisnamoorthi, R.; Yuxi Gong; Chanlan Sun Lin (Kansas State Univ., Manhattan (United States)); VanderVelde, D. (Univ. of Kansas, Lawrence (United States))

1992-01-28

The solution structure of reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor III (CMTI-III*) was investigated by two-dimensional proton nuclear magnetic resonance (2D NMR) spectroscopy. CMTI-III*, prepared by reacting CMTI-III with trypsin which cleaved the Arg5-Ile6 peptide bond, had the two fragments held together by a disulfide linkage. Sequence-specific {sup 1}H NMR resonance assignments were made for all the 29 amino acid residues of the protein. The secondary structure of CMTI-III*, as deduced from NOESY cross peaks and identification of slowly exchanging hydrogens, contains two turns, a 3{sub 10}-helix, and a triple-stranded {beta}-sheet. Sequential proton assignments were also made for the virgin inhibitor, CMTI-III, at pH 4.71, 30C. Comparison of backbone hydrogen chemical shifts of CMTI-III and CMTI-III* revealed significant changes for residues located far away from the reactive-site region as well as for those located near it, indicating tertiary structural changes that are transmitted through most of the 29 residues of the inhibitor protein. These chemical shift changes were relatively small compared to changes that occurred upon hydrolysis of the reactive-site peptide bond between Arg 5 and Ile6 in CMTI-III.

Identification of a novel polyprenylated acylphloroglucinol‑derived SIRT1 inhibitor with cancer‑specific anti-proliferative and invasion-suppressing activities.

Science.gov (United States)

Zhu, Lijia; Qi, Ji; Chiao, Christine Ya-Chi; Zhang, Qiang; Porco, John A; Faller, Douglas V; Dai, Yan

2014-11-01

SIRT1, a class III histone deacetylase, plays a critical role in regulating cancer cell growth, migration and invasion, which makes it a potential target for cancer therapeutics. In this study, we screened derivatives of several groups of natural products and identified a novel SIRT1 inhibitor JQ-101, a synthetic derivative of the polyprenylated acylphloroglucinol (PPAP) natural products, with an IC(50) for SIRT1 of 30 µM in vitro, with 5-fold higher activity for SIRT1 vs. SIRT2. Exposure of tumor cells to JQ-101 significantly enhanced acetylation of p53 and histone H4K16 at known sites of SIRT1 deacetylation, validating SIRT1 as its cellular target. JQ-101 suppressed cancer cell growth and survival by targeting SIRT1, and also exhibited selective cytotoxicity towards a panel of human tumor cell lines, while producing no toxicity in two normal human cell types at comparable concentrations. JQ-101 induced both apoptosis and cell senescence, and suppressed cancer cell invasion in vitro. In summary, we have identified JQ-101 as a new SIRT1 inhibitor which may have potential application in cancer treatment through its ability to induce tumor cell apoptosis and senescence and suppress cancer cell invasion.

Identification of a novel polyprenylated acylphloroglucinol-derived SIRT1 inhibitor with cancer-specific anti-proliferative and invasion-suppressing activities

Science.gov (United States)

ZHU, LIJIA; QI, JI; CHIAO, CHRISTINE YA-CHI; ZHANG, QIANG; PORCO, JOHN A.; FALLER, DOUGLAS V.; DAI, YAN

2014-01-01

SIRT1, a class III histone deacetylase, plays a critical role in regulating cancer cell growth, migration and invasion, which makes it a potential target for cancer therapeutics. In this study, we screened derivatives of several groups of natural products and identified a novel SIRT1 inhibitor JQ-101, a synthetic derivative of the polyprenylated acylphloroglucinol (PPAP) natural products, with an IC50 for SIRT1 of 30 μM in vitro, with 5-fold higher activity for SIRT1 vs. SIRT2. Exposure of tumor cells to JQ-101 significantly enhanced acetylation of p53 and histone H4K16 at known sites of SIRT1 deacetylation, validating SIRT1 as its cellular target. JQ-101 suppressed cancer cell growth and survival by targeting SIRT1, and also exhibited selective cytotoxicity towards a panel of human tumor cell lines, while producing no toxicity in two normal human cell types at comparable concentrations. JQ-101 induced both apoptosis and cell senescence, and suppressed cancer cell invasion in vitro. In summary, we have identified JQ-101 as a new SIRT1 inhibitor which may have potential application in cancer treatment through its ability to induce tumor cell apoptosis and senescence and suppress cancer cell invasion. PMID:25189993

C1-esterase inhibitor blocks T lymphocyte proliferation and cytotoxic T lymphocyte generation in vitro

DEFF Research Database (Denmark)

Nissen, Mogens Holst; Bregenholt, S; Nording, J A

1998-01-01

We have previously shown that activated C1s complement and activated T cells cleave beta2-microglobulin (beta2m) in vitro leading to the formation of desLys58 beta2m. This process can specifically be inhibited by C1-esterase inhibitor (C1-inh). Furthermore we showed that exogenously added desLys58...

Isolation and characterization of a specific endogenous Na+, K+-ATPase inhibitor from bovine adrenal

International Nuclear Information System (INIS)

Tamura, M.; Lam, T.T.; Inagami, T.

1988-01-01

In order to identify a specific endogenous Na + ,K + -ATPase inhibitor which could possibly be related to salt-dependent hypertension, the authors looked for substances in the methanol extract of bovine whole adrenal which show all of the following properties: (i) inhibitory activity for Na + ,K + -ATPase; (ii) competitive displacing activity against [ 3 H]ouabain binding to the enzyme; (iii) inhibitory activity for 86 Rb uptake into intact human erythrocytes; and (iv) cross-reactivity with sheep anti-digoxin-specific antibody. After stepwise fractionation of the methanol extract of bovine adrenal glands by chromatography on a C 18 open column, a 0-15% acetonitrile fraction was fractionated by high-performance liquid chromatography on a Zorbax octadecylsilane column. One of the most active fractions in 0-15% acetonitrile was found to exhibit all of the four types of the activities. It was soluble in water and was distinct from various substances which have been known to inhibit Na + ,K + -ATPase. These results strongly suggest that this water-soluble nonpeptidic Na + ,K + -ATPase inhibitor may be a specific endogenous regulator for the ATPase

High-throughput screening reveals alsterpaullone, 2-cyanoethyl as a potent p27Kip1 transcriptional inhibitor.

Directory of Open Access Journals (Sweden)

Brandon J Walters

Full Text Available p27Kip1 is a cell cycle inhibitor that prevents cyclin dependent kinase (CDK/cyclin complexes from phosphorylating their targets. p27Kip1 is a known tumor suppressor, as the germline loss of p27Kip1 results in sporadic pituitary formation in aged rodents, and its presence in human cancers is indicative of a poor prognosis. In addition to its role in cancer, loss of p27Kip1 results in regenerative phenotypes in some tissues and maintenance of stem cell pluripotency, suggesting that p27Kip1 inhibitors could be beneficial for tissue regeneration. Because p27Kip1 is an intrinsically disordered protein, identifying direct inhibitors of the p27Kip1 protein is difficult. Therefore, we pursued a high-throughput screening strategy to identify novel p27Kip1 transcriptional inhibitors. We utilized a luciferase reporter plasmid driven by the p27Kip1 promoter to transiently transfect HeLa cells and used cyclohexamide as a positive control for non-specific inhibition. We screened a "bioactive" library consisting of 8,904 (4,359 unique compounds, of which 830 are Food and Drug Administration (FDA approved. From this screen, we successfully identified 111 primary hits with inhibitory effect against the promoter of p27Kip1. These hits were further refined using a battery of secondary screens. Here we report four novel p27Kip1 transcriptional inhibitors, and further demonstrate that our most potent hit compound (IC50 = 200 nM Alsterpaullone 2-cyanoethyl, inhibits p27Kip1 transcription by preventing FoxO3a from binding to the p27Kip1 promoter. This screen represents one of the first attempts to identify inhibitors of p27Kip1 and may prove useful for future tissue regeneration studies.

Overview of hereditary angioedema caused by C1-inhibitor deficiency: assessment and clinical management.

Science.gov (United States)

Bork, K; Davis-Lorton, M

2013-02-01

Hereditary angioedema due to C1-inhibitor deficiency (HAE-C1-INH) is a rare, autosomal-dominant disease. HAE-C1-INH is characterized by recurrent attacks of marked, diffuse, nonpitting and nonpruritic skin swellings, painful abdominal attacks, and laryngeal edema. The extremities and the gastrointestinal tract are most commonly affected. Swelling of the upper respiratory mucosa poses the greatest risk because death from asphyxiation can result from laryngealedema. HAE-C1-INH attacks are variable, unpredictable, and may be induced by a variety of stimuli, including stress or physical trauma. Because the clinical presentation of HAE-C1-INH is similar to other types of angioedema, the condition may be a challenge to diagnose. Accurate identification of HAE-C1-INH is critical in order to avoid asphyxiation by laryngeal edema and to improve the burden of disease. Based on an understanding of the underlying pathophysiology of IHAE-C1-INH, drugs targeted specifically to the disease, such as C1-inhibitor therapy, bradykinin B2-receptor antagonists, and kallikrein-inhibitors, have become available for both treatment and prevention of angioedema attacks. This article reviews the clinical features, differential diagnosis, and current approaches to management of HAE-C1-INH.

Ticlopidine in Its Prodrug Form Is a Selective Inhibitor of Human NTPDase1

Directory of Open Access Journals (Sweden)

Joanna Lecka

2014-01-01

Full Text Available Nucleoside triphosphate diphosphohydrolase-1 (NTPDase1, like other ectonucleotidases, controls extracellular nucleotide levels and consequently their (pathophysiological responses such as in thrombosis, inflammation, and cancer. Selective NTPDase1 inhibitors would therefore be very useful. We previously observed that ticlopidine in its prodrug form, which does not affect P2 receptor activity, inhibited the recombinant form of human NTPDase1 (Ki=14 μM. Here we tested whether ticlopidine can be used as a selective inhibitor of NTPDase1. We confirmed that ticlopidine inhibits NTPDase1 in different forms and in different assays. The ADPase activity of intact HUVEC as well as of COS-7 cells transfected with human NTPDase1 was strongly inhibited by 100 µM ticlopidine, 99 and 86%, respectively. Ticlopidine (100 µM completely inhibited the ATPase activity of NTPDase1 in situ as shown by enzyme histochemistry with human liver and pancreas sections. Ticlopidine also inhibited the activity of rat and mouse NTPDase1 and of potato apyrase. At 100 µM ticlopidine did not affect the activity of human NTPDase2, NTPDase3, and NTPDase8, nor of NPP1 and NPP3. Weak inhibition (10–20% of NTPDase3 and -8 was observed at 1 mM ticlopidine. These results show that ticlopidine is a specific inhibitor of NTPDase1 that can be used in enzymatic and histochemistry assays.

Structural and functional analysis of cyclin D1 reveals p27 and substrate inhibitor binding requirements.

Science.gov (United States)

Liu, Shu; Bolger, Joshua K; Kirkland, Lindsay O; Premnath, Padmavathy N; McInnes, Campbell

2010-12-17

An alternative strategy for inhibition of the cyclin dependent kinases (CDKs) in antitumor drug discovery is afforded through the substrate recruitment site on the cyclin positive regulatory subunit. Critical CDK substrates such as the Rb and E2F families must undergo cyclin groove binding before phosphorylation, and hence inhibitors of this interaction also block substrate specific kinase activity. This approach offers the potential to generate highly selective and cell cycle specific CDK inhibitors and to reduce the inhibition of transcription mediated through CDK7 and 9, commonly observed with ATP competitive compounds. While highly potent peptide and small molecule inhibitors of CDK2/cyclin A, E substrate recruitment have been reported, little information has been generated on the determinants of inhibitor binding to the cyclin groove of the CDK4/cyclin D1 complex. CDK4/cyclin D is a validated anticancer drug target and continues to be widely pursued in the development of new therapeutics based on cell cycle blockade. We have therefore investigated the structural basis for peptide binding to its cyclin groove and have examined the features contributing to potency and selectivity of inhibitors. Peptidic inhibitors of CDK4/cyclin D of pRb phosphorylation have been synthesized, and their complexes with CDK4/cyclin D1 crystal structures have been generated. Based on available structural information, comparisons of the cyclin grooves of cyclin A2 and D1 are presented and provide insights into the determinants for peptide binding and the basis for differential binding and inhibition. In addition, a complex structure has been generated in order to model the interactions of the CDKI, p27(KIP)¹, with cyclin D1. This information has been used to shed light onto the endogenous inhibition of CDK4 and also to identify unique aspects of cyclin D1 that can be exploited in the design of cyclin groove based CDK inhibitors. Peptidic and nonpeptidic compounds have been

Allosteric Mutant IDH1 Inhibitors Reveal Mechanisms for IDH1 Mutant and Isoform Selectivity

Energy Technology Data Exchange (ETDEWEB)

Xie, Xiaoling; Baird, Daniel; Bowen, Kimberly; Capka, Vladimir; Chen, Jinyun; Chenail, Gregg; Cho, YoungShin; Dooley, Julia; Farsidjani, Ali; Fortin, Pascal; Kohls, Darcy; Kulathila, Raviraj; Lin, Fallon; McKay, Daniel; Rodrigues, Lindsey; Sage, David; Touré, B. Barry; van der Plas, Simon; Wright, Kirk; Xu, Ming; Yin, Hong; Levell, Julian; Pagliarini, Raymond A. (Novartis)

2017-03-01

Oncogenic IDH1 and IDH2 mutations contribute to cancer via production of R-2-hydroxyglutarate (2-HG). Here, we characterize two structurally distinct mutant- and isoform-selective IDH1 inhibitors that inhibit 2-HG production. Both bind to an allosteric pocket on IDH1, yet shape it differently, highlighting the plasticity of this site. Oncogenic IDH1R132H mutation destabilizes an IDH1 “regulatory segment,” which otherwise restricts compound access to the allosteric pocket. Regulatory segment destabilization in wild-type IDH1 promotes inhibitor binding, suggesting that destabilization is critical for mutant selectivity. We also report crystal structures of oncogenic IDH2 mutant isoforms, highlighting the fact that the analogous segment of IDH2 is not similarly destabilized. This intrinsic stability of IDH2 may contribute to observed inhibitor IDH1 isoform selectivity. Moreover, discrete residues in the IDH1 allosteric pocket that differ from IDH2 may also guide IDH1 isoform selectivity. These data provide a deeper understanding of how IDH1 inhibitors achieve mutant and isoform selectivity.

Ligand-based modeling of Akt3 lead to potent dual Akt1/Akt3 inhibitor.

Science.gov (United States)

Al-Sha'er, Mahmoud A; Taha, Mutasem O

2018-02-13

Akt1 and Akt3 are important serine/threonine-specific protein kinases involved in G2 phase required by cancer cells to maintain cell cycle and to prevent cell death. Accordingly, inhibitors of these kinases should have potent anti-cancer properties. This prompted us to use pharmacophore/QSAR modeling to identify optimal binding models and physicochemical descriptors that explain bioactivity variation within a set of 74 diverse Akt3 inhibitors. Two successful orthogonal pharmacophores were identified and further validated using receiver operating characteristic (ROC) curve analyses. The pharmacophoric models and associated QSAR equation were applied to screen the national cancer institute (NCI) list of compounds for new Akt3 inhibitors. Six hits showed significant experimental anti-Akt3 IC 50 values, out of which one compound exhibited dual low micromolar anti-Akt1 and anti-Akt3 inhibitory profiles. Copyright © 2018 Elsevier Inc. All rights reserved.

Caspase-1 inhibitor regulates humoral responses in experimental autoimmune myasthenia gravis via IL-6- dependent inhibiton of STAT3.

Science.gov (United States)

Wang, Cong-Cong; Zhang, Min; Li, Heng; Li, Xiao-Li; Yue, Long-Tao; Zhang, Peng; Liu, Ru-Tao; Chen, Hui; Li, Yan-Bin; Duan, Rui-Sheng

2017-08-24

We have previously demonstrated that Cysteinyl aspartate-specific proteinase-1 (caspase-1) inhibitor ameliorates experimental autoimmune myasthenia gravis (EAMG) by inhibited cellular immune response, via suppressing DC IL-1 β, CD4 + T and γdT cells IL-17 pathways. In this study, we investigated the effect of caspase-1 inhibitor on humoral immune response of EAMG and further explore the underlying mechanisms. An animal model of MG was induced by region 97-116 of the rat AChR α subunit (R97-116 peptide) in Lewis rats. Rats were treated with caspase-1 inhibitor Ac-YVAD-cmk intraperitoneally (i.p.) every second day from day 13 after the first immunization. Flow cytometry, western blot, immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) were performed to evaluate the neuroprotective effect of caspase-1 inhibitor on humoral immune response of EAMG. The results showed that caspase-1 inhibitor reduced the relative affinity of anti-R97-116 IgG, suppressed germinal center response, decreased follicular helper T cells, and increased follicular regulatory T cells and regulatory B cells. In addition, we found that caspase-1 inhibitor inhibited humoral immunity response in EAMG rats via suppressing IL-6-STAT3-Bcl-6 pathways. These results suggest that caspase-1 inhibitor ameliorates EAMG by regulating humoral immune response, thus providing new insights into the development of myasthenia gravis and other autoimmune diseases therapies. Copyright © 2017 Elsevier B.V. All rights reserved.

PTP1B inhibitor promotes endothelial cell motility by activating the DOCK180/Rac1 pathway.

Science.gov (United States)

Wang, Yuan; Yan, Feng; Ye, Qing; Wu, Xiao; Jiang, Fan

2016-04-07

Promoting endothelial cell (EC) migration is important not only for therapeutic angiogenesis, but also for accelerating re-endothelialization after vessel injury. Several recent studies have shown that inhibition of protein tyrosine phosphatase 1B (PTP1B) may promote EC migration and angiogenesis by enhancing the vascular endothelial growth factor receptor-2 (VEGFR2) signalling. In the present study, we demonstrated that PTP1B inhibitor could promote EC adhesion, spreading and migration, which were abolished by the inhibitor of Rac1 but not RhoA GTPase. PTP1B inhibitor significantly increased phosphorylation of p130Cas, and the interactions among p130Cas, Crk and DOCK180; whereas the phosphorylation levels of focal adhesion kinase, Src, paxillin, or Vav2 were unchanged. Gene silencing of DOCK180, but not Vav2, abrogated the effects of PTP1B inhibitor on EC motility. The effects of PTP1B inhibitor on EC motility and p130Cas/DOCK180 activation persisted in the presence of the VEGFR2 antagonist. In conclusion, we suggest that stimulation of the DOCK180 pathway represents an alternative mechanism of PTP1B inhibitor-stimulated EC motility, which does not require concomitant VEGFR2 activation as a prerequisite. Therefore, PTP1B inhibitor may be a useful therapeutic strategy for promoting EC migration in cardiovascular patients in which the VEGF/VEGFR functions are compromised.

HAEdb: a novel interactive, locus-specific mutation database for the C1 inhibitor gene.

Science.gov (United States)

Kalmár, Lajos; Hegedüs, Tamás; Farkas, Henriette; Nagy, Melinda; Tordai, Attila

2005-01-01

Hereditary angioneurotic edema (HAE) is an autosomal dominant disorder characterized by episodic local subcutaneous and submucosal edema and is caused by the deficiency of the activated C1 esterase inhibitor protein (C1-INH or C1INH; approved gene symbol SERPING1). Published C1-INH mutations are represented in large universal databases (e.g., OMIM, HGMD), but these databases update their data rather infrequently, they are not interactive, and they do not allow searches according to different criteria. The HAEdb, a C1-INH gene mutation database (http://hae.biomembrane.hu) was created to contribute to the following expectations: 1) help the comprehensive collection of information on genetic alterations of the C1-INH gene; 2) create a database in which data can be searched and compared according to several flexible criteria; and 3) provide additional help in new mutation identification. The website uses MySQL, an open-source, multithreaded, relational database management system. The user-friendly graphical interface was written in the PHP web programming language. The website consists of two main parts, the freely browsable search function, and the password-protected data deposition function. Mutations of the C1-INH gene are divided in two parts: gross mutations involving DNA fragments >1 kb, and micro mutations encompassing all non-gross mutations. Several attributes (e.g., affected exon, molecular consequence, family history) are collected for each mutation in a standardized form. This database may facilitate future comprehensive analyses of C1-INH mutations and also provide regular help for molecular diagnostic testing of HAE patients in different centers.

HuD Regulates Coding and Noncoding RNA to Induce APP→Aβ Processing

Directory of Open Access Journals (Sweden)

Min-Ju Kang

2014-06-01

Full Text Available The primarily neuronal RNA-binding protein HuD is implicated in learning and memory. Here, we report the identification of several HuD target transcripts linked to Alzheimer’s disease (AD pathogenesis. HuD interacted with the 3′ UTRs of APP mRNA (encoding amyloid precursor protein and BACE1 mRNA (encoding β-site APP-cleaving enzyme 1 and increased the half-lives of these mRNAs. HuD also associated with and stabilized the long noncoding (lncRNA BACE1AS, which partly complements BACE1 mRNA and enhances BACE1 expression. Consistent with HuD promoting production of APP and APP-cleaving enzyme, the levels of APP, BACE1, BACE1AS, and Aβ were higher in the brain of HuD-overexpressing mice. Importantly, cortex (superior temporal gyrus from patients with AD displayed significantly higher levels of HuD and, accordingly, elevated APP, BACE1, BACE1AS, and Aβ than did cortical tissue from healthy age-matched individuals. We propose that HuD jointly promotes the production of APP and the cleavage of its amyloidogenic fragment, Aβ.

Adapted J6/JFH1-based Hepatitis C virus recombinants with genotype-specific NS4A show similar efficacies against lead protease inhibitors, alpha interferon, and a putative NS4A inhibitor

DEFF Research Database (Denmark)

Gottwein, Judith M; Jensen, Sanne B; Serre, Stéphanie B N

2013-01-01

To facilitate studies of hepatitis C virus (HCV) NS4A, we aimed at developing J6/JFH1-based recombinants with genotype 1- to 7-specific NS4A proteins. We developed efficient culture systems expressing NS4A proteins of genotypes (isolates) 1a (H77 and TN), 1b (J4), 2a (J6), 4a (ED43), 5a (SA13), 6a...... (HK6a), and 7a (QC69), with peak infectivity titers of ∼3.5 to 4.5 log10 focus-forming units per ml. Except for genotype 2a (J6), growth depended on adaptive mutations identified in long-term culture. Genotype 1a, 1b, and 4a recombinants were adapted by amino acid substitutions F772S (p7) and V1663A...... (NS4A), while 5a, 6a, and 7a recombinants required additional substitutions in the NS3 protease and/or NS4A. We demonstrated applicability of the developed recombinants for study of antivirals. Genotype 1 to 7 NS4A recombinants showed similar responses to the protease inhibitors telaprevir (VX-950...

Classification of Cytochrome P450 1A2 Inhibitors and Non-Inhibitors by Machine Learning Techniques

DEFF Research Database (Denmark)

Vasanthanathan, Poongavanam; Taboureau, Olivier; Oostenbrink, Chris

2009-01-01

of CYP1A2 inhibitors and non-inhibitors. Training and test sets consisted of about 400 and 7000 compounds, respectively. Various machine learning techniques, like binary QSAR, support vector machine (SVM), random forest, kappa nearest neighbors (kNN), and decision tree methods were used to develop...

Identification and characterization of naturally occurring inhibitors against UDP-glucuronosyltransferase 1A1 in Fructus Psoraleae (Bu-gu-zhi)

International Nuclear Information System (INIS)

Wang, Xin-Xin; Lv, Xia; Li, Shi-Yang; Hou, Jie; Ning, Jing; Wang, Jia-Yue; Cao, Yun-Feng; Ge, Guang-Bo; Guo, Bin; Yang, Ling

2015-01-01

As an edible traditional Chinese herb, Fructus psoraleae (FP) has been widely used in Asia for the treatment of vitiligo, bone fracture and osteoporosis. Several cases on markedly elevated bilirubin and acute liver injury following administration of FP and its related proprietary medicine have been reported, but the mechanism in FP-associated toxicity has not been well investigated yet. This study aimed to investigate the inhibitory effects of FP extract and its major constituents against human UDP-glucuronosyltransferase 1A1 (UGT1A1), the key enzyme responsible for metabolic elimination of bilirubin. To this end, N-(3-carboxy propyl)-4-hydroxy-1,8-naphthalimide (NCHN), a newly developed specific fluorescent probe for UGT1A1, was used to evaluate the inhibitory effects of FP extract or its fractions in human liver microsomes (HLM), while LC-UV fingerprint and UGT1A1 inhibition profile were combined to identity and characterize the naturally occurring inhibitors of UGT1A1 in FP. Our results demonstrated that both the extract of FP and five major components of FP displayed evident inhibitory effects on UGT1A1 in HLM. Among these five identified naturally occurring inhibitors, bavachin and corylifol A were found to be strong inhibitors of UGT1A1 with the inhibition kinetic parameters (K i ) values lower than 1 μM, while neobavaisoflavone, isobavachalcone, and bavachinin displayed moderate inhibitory effects against UGT1A1 in HLM, with the K i values ranging from 1.61 to 9.86 μM. These findings suggested that FP contains natural compounds with potent inhibitory effects against human UGT1A1, which may be one of the important reasons for triggering FP-associated toxicity, including elevated bilirubin levels and liver injury. - Graphical abstract: LC-UV fingerprint and UGT1A1 inhibition profiles were combined to identity and characterize the natural inhibitors of UGT1A1 in F. psoraleae for the first time. Five major components in F. psoraleae were identified as strong

Identification and characterization of naturally occurring inhibitors against UDP-glucuronosyltransferase 1A1 in Fructus Psoraleae (Bu-gu-zhi)

Energy Technology Data Exchange (ETDEWEB)

Wang, Xin-Xin [Liaoning Medical University, Jinzhou, Liaoning (China); Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023 (China); Lv, Xia; Li, Shi-Yang [Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023 (China); Hou, Jie [Dalian Medical University, Dalian 116044 (China); Ning, Jing [Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023 (China); Dalian Medical University, Dalian 116044 (China); Wang, Jia-Yue [Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023 (China); Cao, Yun-Feng [Liaoning Medical University, Jinzhou, Liaoning (China); Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023 (China); Ge, Guang-Bo, E-mail: geguangbo@dicp.ac.cn [Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023 (China); Guo, Bin, E-mail: jyguobin@126.com [Liaoning Medical University, Jinzhou, Liaoning (China); Yang, Ling [Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023 (China); Jiangxi University of Traditional Chinese Medicine, Nanchang 330006 (China)

2015-11-15

As an edible traditional Chinese herb, Fructus psoraleae (FP) has been widely used in Asia for the treatment of vitiligo, bone fracture and osteoporosis. Several cases on markedly elevated bilirubin and acute liver injury following administration of FP and its related proprietary medicine have been reported, but the mechanism in FP-associated toxicity has not been well investigated yet. This study aimed to investigate the inhibitory effects of FP extract and its major constituents against human UDP-glucuronosyltransferase 1A1 (UGT1A1), the key enzyme responsible for metabolic elimination of bilirubin. To this end, N-(3-carboxy propyl)-4-hydroxy-1,8-naphthalimide (NCHN), a newly developed specific fluorescent probe for UGT1A1, was used to evaluate the inhibitory effects of FP extract or its fractions in human liver microsomes (HLM), while LC-UV fingerprint and UGT1A1 inhibition profile were combined to identity and characterize the naturally occurring inhibitors of UGT1A1 in FP. Our results demonstrated that both the extract of FP and five major components of FP displayed evident inhibitory effects on UGT1A1 in HLM. Among these five identified naturally occurring inhibitors, bavachin and corylifol A were found to be strong inhibitors of UGT1A1 with the inhibition kinetic parameters (K{sub i}) values lower than 1 μM, while neobavaisoflavone, isobavachalcone, and bavachinin displayed moderate inhibitory effects against UGT1A1 in HLM, with the K{sub i} values ranging from 1.61 to 9.86 μM. These findings suggested that FP contains natural compounds with potent inhibitory effects against human UGT1A1, which may be one of the important reasons for triggering FP-associated toxicity, including elevated bilirubin levels and liver injury. - Graphical abstract: LC-UV fingerprint and UGT1A1 inhibition profiles were combined to identity and characterize the natural inhibitors of UGT1A1 in F. psoraleae for the first time. Five major components in F. psoraleae were identified as

Type 1 plaminogen activator inhibitor gene: Functional analysis and glucocorticoid regulation of its promoter

International Nuclear Information System (INIS)

Van Zonneveld, A.J.; Curriden, S.A.; Loskutoff, D.J.

1988-01-01

Plasminogen activator inhibitor type 1 is an important component of the fibrinolytic system and its biosynthesis is subject to complex regulation. To study this regulation at the level of transcription, the authors have identified and sequenced the promoter of the human plasminogen activator inhibitor type 1 gene. Nuclease protection experiments were performed by using endothelial cell mRNA and the transcription initiation (cap) site was established. Sequence analysis of the 5' flanking region of the gene revealed a perfect TATA box at position -28 to position -23, the conserved distance from the cap site. Comparative functional studies with the firefly luciferase gene as a reporter gene showed that fragments derived from this 5' flanking region exhibited high promoter activity when transfected into bovine aortic endothelial cells and mouse Ltk - fibroblasts but were inactive when introduced into HeLa cells. These studies indicate that the fragments contain the plasminogen activator inhibitor type 1 promoter and that it is expressed in a tissue-specific manner. Although the fragments were also silent in rat FTO2B hepatoma cells, their promoter activity could be induced up to 40-fold with the synthetic glucocorticoid dexamethasone. Promoter deletion mapping experiments and studies involving the fusion of promoter fragments to a heterologous gene indicated that dexamethasone induction is mediated by a glucocorticoid responsive element with enhancer-like properties located within the region between nucleotides -305 and +75 of the plasminogen activator inhibitor type 1 gene

Two-dimensional NMR studies of squash family inhibitors. Sequence-specific proton assignments and secondary structure of reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor III.

Science.gov (United States)

Krishnamoorthi, R; Gong, Y X; Lin, C L; VanderVelde, D

1992-01-28

The solution structure of reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor III (CMTI-III*) was investigated by two-dimensional proton nuclear magnetic resonance (2D NMR) spectroscopy. CMTI-III*, prepared by reacting CMTI-III with trypsin which cleaved the Arg5-Ile6 peptide bond, had the two fragments held together by a disulfide linkage. Sequence-specific 1H NMR resonance assignments were made for all the 29 amino acid residues of the protein. The secondary structure of CMTI-III*, as deduced from NOESY cross peaks and identification of slowly exchanging hydrogens, contains two turns (residues 8-12 and 24-27), a 3(10)-helix (residues 13-16), and a triple-stranded beta-sheet (residues 8-10, 29-27, and 21-25). This secondary structure is similar to that of CMTI-I [Holak, T. A., Gondol, D., Otlewski, J., & Wilusz, T. (1989) J. Mol. Biol. 210, 635-648], which has a Glu instead of a Lys at position 9. Sequential proton assignments were also made for the virgin inhibitor, CMTI-III, at pH 4.71, 30 degrees C. Comparison of backbone hydrogen chemical shifts of CMTI-III and CMTI-III* revealed significant changes for residues located far away from the reactive-site region as well as for those located near it, indicating tertiary structural changes that are transmitted through most of the 29 residues of the inhibitor protein. Many of these residues are functionally important in that they make contact with atoms of the enzyme in the trypsin-inhibitor complex, as revealed by X-ray crystallography [Bode, W., Greyling, H. J., Huber, R., Otlewski, J., & Wilusz, T. (1989) FEBS Lett. 242, 285-292].(ABSTRACT TRUNCATED AT 250 WORDS)

A Rhodium(III)-Based Inhibitor of Lysine-Specific Histone Demethylase 1 as an Epigenetic Modulator in Prostate Cancer Cells.

Science.gov (United States)

Yang, Chao; Wang, Wanhe; Liang, Jia-Xin; Li, Guodong; Vellaisamy, Kasipandi; Wong, Chun-Yuen; Ma, Dik-Lung; Leung, Chung-Hang

2017-03-23

We report herein a novel rhodium(III) complex 1 as a new LSD1 targeting agent and epigenetic modulator. Complex 1 disrupted the interaction of LSD1-H3K4me2 in human prostate carcinoma cells and enhanced the amplification of p21, FOXA2, and BMP2 gene promoters. Complex 1 was selective for LSD1 over other histone demethylases, such as KDM2b, KDM7, and MAO activities, and also showed antiproliferative activity toward human cancer cells. To date, complex 1 is the first metal-based inhibitor of LSD1 activity.

Structure-Based Design of Potent and Selective 3-Phosphoinositide-Dependent Kinase-1 (PDK1) Inhibitors

Energy Technology Data Exchange (ETDEWEB)

Medina, Jesus R.; Becker, Christopher J.; Blackledge, Charles W.; Duquenne, Celine; Feng, Yanhong; Grant, Seth W.; Heerding, Dirk; Li, William H.; Miller, William H.; Romeril, Stuart P.; Scherzer, Daryl; Shu, Arthur; Bobko, Mark A.; Chadderton, Antony R.; Dumble, Melissa; Gardiner, Christine M.; Gilbert, Seth; Liu, Qi; Rabindran, Sridhar K.; Sudakin, Valery; Xiang, Hong; Brady, Pat G.; Campobasso, Nino; Ward, Paris; Axten, Jeffrey M. (GSKPA)

2014-10-02

Phosphoinositide-dependent protein kinase-1(PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway. As this pathway is among the most commonly deregulated across all cancers, a selective inhibitor of PDK1 might have utility as an anticancer agent. Herein we describe our lead optimization of compound 1 toward highly potent and selective PDK1 inhibitors via a structure-based design strategy. The most potent and selective inhibitors demonstrated submicromolar activity as measured by inhibition of phosphorylation of PDK1 substrates as well as antiproliferative activity against a subset of AML cell lines. In addition, reduction of phosphorylation of PDK1 substrates was demonstrated in vivo in mice bearing OCl-AML2 xenografts. These observations demonstrate the utility of these molecules as tools to further delineate the biology of PDK1 and the potential pharmacological uses of a PDK1 inhibitor.

Synthesis and in vitro Evaluation of 2-heteroarylidene-1-tetralone Derivatives as Monoamine Oxidase Inhibitors.

Science.gov (United States)

Amakali, Klaudia T; Legoabe, Lesetja J; Petzer, Anél; Petzer, Jacobus P

2018-05-14

The present study investigates the human monoamine oxidase (MAO) inhibition properties of a series of twelve 2-heteroarylidene-1-tetralone derivatives. Also included are related cyclohexylmethylidene, cyclopentylmethylidene and benzylidene substituted 1-tetralones. These compounds are related to the 2-benzylidene-1-indanone class of compounds which has previously been shown to inhibit the MAOs, with specificity for the MAO-B isoform. The target compounds were synthesised by the Claisen-Schmidt condensation between 7-methoxy-1-tetralone or 1-tetralone, and various aldehydes, under acid (hydrochloric acid) or base (potassium hydroxide) catalysis. The results of the MAO inhibition studies showed that the 2-heteroarylidene-1-tetralone and related derivatives are in most instances more selective inhibitors of the MAO-B isoform compared to MAO-A. (2E)-2-Benzylidene-7-methoxy-3,4-dihydronaphthalen-1(2 H)-one (IC 50 =0.707 μM) was found to be the most potent MAO-B inhibitor, while the most potent MAO-A inhibitor was (2E)-2-[(2-chloropyridin-3-yl)methylidene]-7-methoxy-3,4-dihydronaphthalen-1(2 H)-one (IC 50 =1.37 μM). The effect of the heteroaromatic substituent on MAO-B inhibition activity, in decreasing order was found to be: cyclohexyl, phenyl>thiophene>pyridine, furane, pyrrole, cyclopentyl. This study concludes that, although some 2-heteroarylidene-1-tetralone derivatives are good potency MAO inhibitors, in general their inhibition potencies, particularly for MAO-B, are lower than structurally related chalcones and 1-indanone derivatives that were previously studied. © Georg Thieme Verlag KG Stuttgart · New York.

Opportunities to Target Specific Contractile Abnormalities with Smooth Muscle Protein Kinase Inhibitors

Directory of Open Access Journals (Sweden)

Annegret Ulke-Lemée

2010-05-01

Full Text Available Smooth muscle is a major component of most hollow organ systems (e.g., airways, vasculature, bladder and gut/gastrointestine; therefore, the coordinated regulation of contraction is a key property of smooth muscle. When smooth muscle functions normally, it contributes to general health and wellness, but its dysfunction is associated with morbidity and mortality. Rho-associated protein kinase (ROCK is central to calcium-independent, actomyosin-mediated contractile force generation in the vasculature, thereby playing a role in smooth muscle contraction, cell motility and adhesion. Recent evidence supports an important role for ROCK in the increased vasoconstriction and remodeling observed in various models of hypertension. This review will provide a commentary on the development of specific ROCK inhibitors and their clinical application. Fasudil will be discussed as an example of bench-to-bedside development of a clinical therapeutic that is used to treat conditions of vascular hypercontractility. Due to the wide spectrum of biological processes regulated by ROCK, many additional clinical indications might also benefit from ROCK inhibition. Apart from the importance of ROCK in smooth muscle contraction, a variety of other protein kinases are known to play similar roles in regulating contractile force. The zipper-interacting protein kinase (ZIPK and integrin-linked kinase (ILK are two well-described regulators of contraction. The relative contribution of each kinase to contraction depends on the muscle bed as well as hormonal and neuronal stimulation. Unfortunately, specific inhibitors for ZIPK and ILK are still in the development phase, but the success of fasudil suggests that inhibitors for these other kinases may also have valuable clinical applications. Notably, the directed inhibition of ZIPK with a pseudosubstrate molecule shows unexpected effects on the contractility of gastrointestinal smooth muscle.

Structural characterization of human heme oxygenase-1 in complex with azole-based inhibitors.

Science.gov (United States)

Rahman, Mona N; Vlahakis, Jason Z; Roman, Gheorghe; Vukomanovic, Dragic; Szarek, Walter A; Nakatsu, Kanji; Jia, Zongchao

2010-03-01

The development of inhibitors specific for heme oxygenases (HO) aims to provide powerful tools in understanding the HO system. Based on the lead structure (2S, 4S)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-4-[((4-aminophenyl)thio)methyl]-1,3-dioxolane (azalanstat, QC-1) we have synthesized structural modifications to develop novel and selective HO inhibitors. The structural study of human HO-1 (hHO-1) in complex with a select group of the inhibitors was initiated using X-ray crystallographic techniques. Comparison of the structures of four such compounds each in complex with hHO-1 revealed a common binding mode, despite having different structural fragments. The compounds bind to the distal side of heme through an azole "anchor" which coordinates with the heme iron. An expansion of the distal pocket, mainly due to distal helix flexibility, allows accommodation of the compounds without displacing heme or the critical Asp140 residue. Rather, binding displaces a catalytically critical water molecule and disrupts an ordered hydrogen-bond network involving Asp140. The presence of a triazole "anchor" may provide further stability via a hydrogen bond with the protein. A hydrophobic pocket acts to stabilize the region occupied by the phenyl or adamantanyl moieties of these compounds. Further, a secondary hydrophobic pocket is formed via "induced fit" to accommodate bulky substituents at the 4-position of the dioxolane ring. Copyright 2009 Elsevier Inc. All rights reserved.

Identification of Leishmania donovani Topoisomerase 1 inhibitors via intuitive scaffold hopping and bioisosteric modification of known Top 1 inhibitors

Science.gov (United States)

Mamidala, Rajinikanth; Majumdar, Papiya; Jha, Kunal Kumar; Bathula, Chandramohan; Agarwal, Rahul; Chary, M. Thirumala; Mazumdar, H. K.; Munshi, Parthapratim; Sen, Subhabrata

2016-05-01

A library of arylidenefuropyridinediones was discovered as potent inhibitors of Leishmania donovani Topoisomerase 1 (LdTop1) where the active molecules displayed considerable inhibition with single digit micromolar EC50 values. This molecular library was designed via intuitive scaffold hopping and bioisosteric modification of known topoisomerase 1 inhibitors such as camptothecin, edotecarin and etc. The design was rationalized by molecular docking analysis of the compound prototype with human topoisomerase 1 (HTop1) and Leishmania donovani topoisomerase 1(LdTop1). The most active compound 4 displayed no cytotoxicity against normal mammalian COS7 cell line (~100 fold less inhibition at the EC50). Similar to camptothecin, 4 interacted with free LdTop1 as observed in the preincubation DNA relaxation inhibition experiment. It also displayed anti-protozoal activity against Leishmania donovani promastigote. Crystal structure investigation of 4 and its molecular modelling with LdTop1 revealed putative binding sites in the enzyme that could be harnessed to generate molecules with better potency.

Optimal Classes of Chemotherapeutic Agents Sensitized by Specific Small-Molecule Inhibitors of Akt In Vitro and In Vivo

Directory of Open Access Journals (Sweden)

Yan Shi

2005-11-01

Full Text Available Akt is a serine/threonine kinase that transduces survival signals from survival/growth factors. Deregulation and signal imbalance in cancer cells make them prone to apoptosis. Upregulation or activation of Akt to aid the survival of cancer cells is a common theme in human malignancies. We have developed small-molecule Akt inhibitors that are potent and specific. These Akt inhibitors can inhibit Akt activity and block phosphorylation by Akt on multiple downstream targets in cells. Synergy in apoptosis induction was observed when Akt inhibitors were combined with doxorubicin or camptothecin. Akt inhibitor-induced enhancement of topoisomerase inhibitor cytotoxicity was also evident in long-term cell survival assay. Synergy with paclitaxel in apoptosis induction was evident in cells pretreated with paclitaxel, and enhancement of tumor delay by paclitaxel was demonstrated through cotreatment with Akt inhibitor Compound A (A-443654. Combination with other classes of chemotherapeutic agents did not yield any enhancement of cytotoxicity. These findings provide important guidance in selecting appropriate classes of chemotherapeutic agents for combination with Akt inhibitors in cancer treatment.

Oncogenic PKC-ι activates Vimentin during epithelial-mesenchymal transition in melanoma; a study based on PKC-ι and PKC-ζ specific inhibitors.

Science.gov (United States)

Ratnayake, Wishrawana S; Apostolatos, Christopher A; Apostolatos, André H; Schutte, Ryan J; Huynh, Monica A; Ostrov, David A; Acevedo-Duncan, Mildred

2018-05-21

Melanoma is one of the fastest growing cancers in the United States and is accompanied with a poor prognosis owing to tumors being resistant to most therapies. Atypical protein kinase Cs (aPKC) are involved in malignancy in many cancers. We previously reported that aPKCs play a key role in melanoma's cell motility by regulating cell signaling pathways which induce epithelial-mesenchymal Transition (EMT). We tested three novel inhibitors; [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1T) along with its nucleoside analog 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) which are specific to protein kinase C-iota (PKC-ι) and 8-hydroxy-1,3,6-naphthalenetrisulfonic acid (ζ-Stat) which is specific to PKC-zeta (PKC-ζ) on cell proliferation, apoptosis, migration and invasion of two malignant melanoma cell lines compared to normal melanocytes. Molecular modeling was used to identify potential binding sites for the inhibitors and to predict selectivity. Kinase assay showed >50% inhibition for specified targets beyond 5 μM for all inhibitors. Both ICA-1 and ζ-Stat significantly reduced cell proliferation and induced apoptosis, while ICA-1 also significantly reduced migration and melanoma cell invasion. PKC-ι stimulated EMT via TGFβ/Par6/RhoA pathway and activated Vimentin by phosphorylation at S39. Both ICA-1 and ζ-Stat downregulate TNF-α induced NF-κB translocation to the nucleus there by inducing apoptosis. Results suggest that PKC-ι is involved in melanoma malignancy than PKC-ζ. Inhibitors proved to be effective under in-vitro conditions and need to be tested in-vivo for the validity as effective therapeutics. Overall, results show that aPKCs are essential for melanoma progression and metastasis and that they could be used as effective therapeutic targets for malignant melanoma.

Identification of the hot spot residues for pyridine derivative inhibitor CCT251455 and ATP substrate binding on monopolar spindle 1 (MPS1) kinase by molecular dynamic simulation.

Science.gov (United States)

Chen, Kai; Duan, Wenxiu; Han, Qianqian; Sun, Xuan; Li, Wenqian; Hu, Shuangyun; Wan, Jiajia; Wu, Jiang; Ge, Yushu; Liu, Dan

2018-03-08

Protein kinase monopolar spindle 1 plays an important role in spindle assembly checkpoint at the onset of mitosis. Over expression of MPS1 correlated with a wide range of human tumors makes it an attractive target for finding an effective and specific inhibitor. In this work, we performed molecular dynamics simulations of protein MPS1 itself as well as protein bound systems with the inhibitor and natural substrate based on crystal structures. The reported orally bioavailable 1 h-pyrrolo [3,2-c] pyridine inhibitors of MPS1 maintained stable binding in the catalytic site, while natural substrate ATP could not stay. Comparative study of stability and flexibility of three systems reveals position shifting of β-sheet region within the catalytic site, which indicates inhibition mechanism was through stabilizing the β-sheet region. Binding free energies calculated with MM-GB/PBSA method shows different binding affinity for inhibitor and ATP. Finally, interactions between protein and inhibitor during molecular dynamic simulations were measured and counted. Residue Gly605 and Leu654 were suggested as important hot spots for stable binding of inhibitor by molecular dynamic simulation. Our results reveal an important position shifting within catalytic site for non-inhibited proteins. Together with hot spots found by molecular dynamic simulation, the results provide important information of inhibition mechanism and will be referenced for designing novel inhibitors.

Structure-Activity Relationships of the Human Immunodeficiency Virus Type 1 Maturation Inhibitor PF-46396.

Science.gov (United States)

Murgatroyd, Christopher; Pirrie, Lisa; Tran, Fanny; Smith, Terry K; Westwood, Nicholas J; Adamson, Catherine S

2016-09-15

target the Gag protein, specifically by inhibiting CA-SP1 proteolytic cleavage. The lack of high-resolution structural information of the CA-SP1 target in Gag has hindered our understanding of the inhibitor-binding pocket and maturation inhibitor mode of action. Therefore, we utilized analogues of the maturation inhibitor PF-46396 as chemical tools to determine the chemical components of PF-46396 that contribute to antiviral activity and Gag binding and the relationship between these essential properties. This is the first study to report structure-activity relationships of the maturation inhibitor PF-46396. PF-46396 is chemically distinct from betulinic acid-derived maturation inhibitors; therefore, our data provide a foundation of knowledge that will aid our understanding of how structurally distinct maturation inhibitors act by similar modes of action. Copyright © 2016 Murgatroyd et al.

A novel anti-tumor inhibitor identified by virtual screen with PLK1 structure and zebrafish assay.

Directory of Open Access Journals (Sweden)

Jing Lu

Full Text Available Polo-like kinase 1 (PLK1, one of the key regulators of mitosis, is a target for cancer therapy due to its abnormally high activity in several tumors. Plk1 is highly conserved and shares a nearly identical 3-D structure between zebrafish and humans. The initial 10 mitoses of zebrafish embryonic cleavages occur every∼30 minutes, and therefore provide a rapid assay to evaluate mitosis inhibitors including those targeting Plk1. To increase efficiency and specificity, we first performed a computational virtual screen of∼60000 compounds against the human Plk1 3-D structure docked to both its kinase and Polo box domain. 370 candidates with the top free-energy scores were subjected to zebrafish assay and 3 were shown to inhibit cell division. Compared to general screen for compounds inhibiting zebrafish embryonic cleavage, computation increased the efficiency by 11 folds. One of the 3 compounds, named I2, was further demonstrated to effectively inhibit multiple tumor cell proliferation in vitro and PC3 prostate cancer growth in Xenograft mouse model in vivo. Furthermore, I2 inhibited Plk1 enzyme activity in a dose dependent manner. The IC50 values of I2 in these assays are compatible to those of ON-01910, a Plk1 inhibitor currently in Phase III clinic trials. Our studies demonstrate that zebrafish assays coupled with computational screening significantly improves the efficiency of identifying specific regulators of biological targets. The PLK1 inhibitor I2, and its analogs, may have potential in cancer therapeutics.

PD-1 Checkpoint Inhibitor Associated Autoimmune Encephalitis

Directory of Open Access Journals (Sweden)

Stephanie Schneider

2017-05-01

Full Text Available Objective: To report first-hand narrative experience of autoimmune encephalitis and to briefly review currently available evidence of autoimmune encephalitis in cancer patients treated with immune checkpoint inhibitors. Setting: A case study is presented on the management of a patient who developed autoimmune encephalitis during nivolumab monotherapy occurring after 28 weeks on anti-PD-1 monotherapy (nivolumab 3 mg/kg every 2 weeks for non-small cell lung cancer. Results: No substantial improvement was observed by antiepileptic treatment. After administration of 80 mg methylprednisolone, neurologic symptoms disappeared within 24 h and the patient fully recovered. Conclusions: Immune checkpoint inhibitor treatment can lead to autoimmune encephalitis. Clinical trial data indicate a frequency of autoimmune encephalitis of ≥0.1 to <1% with a higher probability during combined or sequential anti-CTLA-4/anti-PD-1 therapy than during anti-PD-1 or anti-PD-L1 monotherapy. Further collection of evidence and translational research is warranted.

Anti-tumor effects of dehydroaltenusin, a specific inhibitor of mammalian DNA polymerase α

International Nuclear Information System (INIS)

Maeda, Naoki; Kokai, Yasuo; Ohtani, Seiji; Sahara, Hiroeki; Kuriyama, Isoko; Kamisuki, Shinji; Takahashi, Shunya; Sakaguchi, Kengo; Sugawara, Fumio; Yoshida, Hiromi; Sato, Noriyuki; Mizushina, Yoshiyuki

2007-01-01

In the screening of selective inhibitors of eukaryotic DNA polymerases (pols), dehydroaltenusin was found to be an inhibitor of pol α from a fungus (Alternaria tennuis). We succeeded in chemically synthesizing dehydroaltenusin, and the compound inhibited only mammalian pol α with IC 50 value of 0.5 μM, and did not influence the activities of other replicative pols such as pols δ and ε, but also showed no effect on pol α activity from another vertebrate, fish, or from a plant species. Dehydroaltenusin also had no influence on the other pols and DNA metabolic enzymes tested. The compound also inhibited the proliferation of human cancer cells with LD 50 values of 38.0-44.4 μM. In an in vivo anti-tumor assay on nude mice bearing solid tumors of HeLa cells, dehydroaltenusin was shown to be a promising suppressor of solid tumors. Histopathological examination revealed that increased tumor necrosis and decreased mitotic index were apparently detected by the compound in vivo. Therefore, dehydroaltenusin could be of interest as not only a mammalian pol α-specific inhibitor, but also as a candidate drug for anti-cancer treatment

Structural Basis for Specific Inhibition of tRNA Synthetase by an ATP Competitive Inhibitor

OpenAIRE

Fang, Pengfei; Han, Hongyan; Wang, Jing; Chen, Kaige; Chen, Xin; Guo, Min

2015-01-01

Pharmaceutical inhibitors of aminoacyl-tRNA synthetases demand high species and family specificity. The antimalarial ATP-mimetic cladosporin selectively inhibits P. falciparum LysRS (PfLysRS). How the binding to a universal ATP site achieves the specificity is unknown. Here we report 3 crystal structures of cladosporin with human LysRS, PfLysRS, and a Pf-like human LysRS mutant. In all 3 structures, cladosporin occupies the class defining ATP-binding pocket, replacing the adenosine portion of...

Antimalarial activity of HIV-1 protease inhibitor in chromone series.

Science.gov (United States)

Lerdsirisuk, Pradith; Maicheen, Chirattikan; Ungwitayatorn, Jiraporn

2014-12-01

Increasing parasite resistance to nearly all available antimalarial drugs becomes a serious problem to human health and necessitates the need to continue the search for new effective drugs. Recent studies have shown that clinically utilized HIV-1 protease (HIV-1 PR) inhibitors can inhibit the in vitro and in vivo growth of Plasmodium falciparum. In this study, a series of chromone derivatives possessing HIV-1 PR inhibitory activity has been tested for antimalarial activity against P. falciparum (K1 multi-drug resistant strain). Chromone 15, the potent HIV-1 PR inhibitor (IC50=0.65μM), was found to be the most potent antimalarial compound with IC50=0.95μM while primaquine and tafenoquine showed IC50=2.41 and 1.95μM, respectively. Molecular docking study of chromone compounds against plasmepsin II, an aspartic protease enzyme important in hemoglobin degradation, revealed that chromone 15 exhibited the higher binding affinity (binding energy=-13.24kcal/mol) than the known PM II inhibitors. Thus, HIV-1 PR inhibitor in chromone series has the potential to be a new class of antimalarial agent. Copyright © 2014 Elsevier Inc. All rights reserved.

Inhibitors of nuclease and redox activity of apurinic/apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1).

Science.gov (United States)

Laev, Sergey S; Salakhutdinov, Nariman F; Lavrik, Olga I

2017-05-01

Human apurinic/apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1) is a multifunctional protein which is essential in the base excision repair (BER) pathway of DNA lesions caused by oxidation and alkylation. This protein hydrolyzes DNA adjacent to the 5'-end of an apurinic/apyrimidinic (AP) site to produce a nick with a 3'-hydroxyl group and a 5'-deoxyribose phosphate moiety or activates the DNA-binding activity of certain transcription factors through its redox function. Studies have indicated a role for APE1/Ref-1 in the pathogenesis of cancer and in resistance to DNA-interactive drugs. Thus, this protein has potential as a target in cancer treatment. As a result, major efforts have been directed to identify small molecule inhibitors against APE1/Ref-1 activities. These agents have the potential to become anticancer drugs. The aim of this review is to present recent progress in studies of all published small molecule APE1/Ref-1 inhibitors. The structures and activities of APE1/Ref-1 inhibitors, that target both DNA repair and redox activities, are presented and discussed. To date, there is an urgent need for further development of the design and synthesis of APE1/Ref-1 inhibitors due to high importance of this protein target. Copyright © 2017 Elsevier Ltd. All rights reserved.

Synthesis and biological evaluation of novel thiadiazole amides as potent Cdc25B and PTP1B inhibitors.

Science.gov (United States)

Li, Yingjun; Yu, Yang; Jin, Kun; Gao, Lixin; Luo, Tongchuan; Sheng, Li; Shao, Xin; Li, Jia

2014-09-01

A series of novel thiadiazole amide derivatives have been synthesized and evaluated for inhibitory activities against Cdc25B and PTP1B. Most of them showed inhibitory activities against Cdc25B (IC50=1.18-8.01 μg/mL) and PTP1B (IC50=0.85-8.75 μg/mL), respectively. Moreover, compounds 5b and 4l were most potent with IC50 values of 1.18 and 0.85 μg/mL for Cdc25B and PTP1B, respectively, compared with reference drugs Na3VO4 (IC50=0.93 μg/mL) and oleanolic acid (IC50=0.85 μg/mL). The results of selectivity experiments showed that the target compounds were selective inhibitors against PTP1B and Cdc25B. Enzyme kinetic experiments demonstrated that compound 5k was a specific inhibitor with the typical characteristics of a mixed inhibitor. Copyright © 2014 Elsevier Ltd. All rights reserved.

Medicinal Chemistry Projects Requiring Imaginative Structure-Based Drug Design Methods.

Science.gov (United States)

Moitessier, Nicolas; Pottel, Joshua; Therrien, Eric; Englebienne, Pablo; Liu, Zhaomin; Tomberg, Anna; Corbeil, Christopher R

2016-09-20

cytochrome P450 enzymes (CYPs)-for toxicology studies-the program Impacts was derived from Fitted and helped us to reveal a complex metabolism with unforeseen stereocenter isomerizations. These efforts, combined with those of other docking software developers, have strengthened our understanding of the complex drug-protein binding process while providing the medicinal chemistry community with useful tools that have led to drug discoveries. In this Account, we describe our contributions over the past 15 years-within their historical context-to the design of drug candidates, including BACE-1 inhibitors, POP covalent inhibitors, G-quadruplex binders, and aminoglycosides binding to nucleic acids. We also remark the necessary developments of docking programs, specifically Fitted, that enabled structure-based design to flourish and yielded multiple fruitful, rational medicinal chemistry campaigns.

MKP1 phosphatase mediates G1-specific dephosphorylation of H3Serine10P in response to DNA damage

Energy Technology Data Exchange (ETDEWEB)

Sharma, Ajit K.; Khan, Shafqat A.; Sharda, Asmita; Reddy, Divya V; Gupta, Sanjay, E-mail: sgupta@actrec.gov.in

2015-08-15

Highlights: • Reversible reduction of H3S10 phosphorylation after DNA damage is G1 phase specific. • Dynamic balance between MAP kinases, MKP1 and MSK1 regulate H3S10P during DDR. • MKP1 associates with chromatin bearing γH2AX in response to DNA damage. • Inhibition of MKP1 activity with specific inhibitor promotes radiation-induced cell death. - Abstract: Histone mark, H3S10 phosphorylation plays a dual role in a cell by maintaining relaxed chromatin for active transcription in interphase and condensed chromatin state in mitosis. The level of H3S10P has also been shown to alter on DNA damage; however, its cell cycle specific behavior and regulation during DNA damage response is largely unexplored. In the present study, we demonstrate G1 cell cycle phase specific reversible loss of H3S10P in response to IR-induced DNA damage is mediated by opposing activities of phosphatase, MKP1 and kinase, MSK1 of the MAP kinase pathway. We also show that the MKP1 recruits to the chromatin in response to DNA damage and correlates with the decrease of H3S10P, whereas MKP1 is released from chromatin during recovery phase of DDR. Furthermore, blocking of H3S10 dephosphorylation by MKP1 inhibition impairs DNA repair process and results in poor survival of WRL68 cells. Collectively, our data proposes a pathway regulating G1 cell cycle phase specific reversible reduction of H3S10P on IR induced DNA damage and also raises the possibility of combinatorial modulation of H3S10P with specific inhibitors to target the cancer cells in G1-phase of cell cycle.

Developing HIV-1 Protease Inhibitors through Stereospecific Reactions in Protein Crystals.

Science.gov (United States)

Olajuyigbe, Folasade M; Demitri, Nicola; De Zorzi, Rita; Geremia, Silvano

2016-10-31

Protease inhibitors are key components in the chemotherapy of HIV infection. However, the appearance of viral mutants routinely compromises their clinical efficacy, creating a constant need for new and more potent inhibitors. Recently, a new class of epoxide-based inhibitors of HIV-1 protease was investigated and the configuration of the epoxide carbons was demonstrated to play a crucial role in determining the binding affinity. Here we report the comparison between three crystal structures at near-atomic resolution of HIV-1 protease in complex with the epoxide-based inhibitor, revealing an in-situ epoxide ring opening triggered by a pH change in the mother solution of the crystal. Increased pH in the crystal allows a stereospecific nucleophile attack of an ammonia molecule onto an epoxide carbon, with formation of a new inhibitor containing amino-alcohol functions. The described experiments open a pathway for the development of new stereospecific protease inhibitors from a reactive lead compound.

Developing HIV-1 Protease Inhibitors through Stereospecific Reactions in Protein Crystals

Directory of Open Access Journals (Sweden)

Folasade M. Olajuyigbe

2016-10-01

Full Text Available Protease inhibitors are key components in the chemotherapy of HIV infection. However, the appearance of viral mutants routinely compromises their clinical efficacy, creating a constant need for new and more potent inhibitors. Recently, a new class of epoxide-based inhibitors of HIV-1 protease was investigated and the configuration of the epoxide carbons was demonstrated to play a crucial role in determining the binding affinity. Here we report the comparison between three crystal structures at near-atomic resolution of HIV-1 protease in complex with the epoxide-based inhibitor, revealing an in-situ epoxide ring opening triggered by a pH change in the mother solution of the crystal. Increased pH in the crystal allows a stereospecific nucleophile attack of an ammonia molecule onto an epoxide carbon, with formation of a new inhibitor containing amino-alcohol functions. The described experiments open a pathway for the development of new stereospecific protease inhibitors from a reactive lead compound.

Histone deacetylase inhibitors (HDACIs: multitargeted anticancer agents

Directory of Open Access Journals (Sweden)

Ververis K

2013-02-01

Full Text Available Katherine Ververis,1 Alison Hiong,1 Tom C Karagiannis,1,* Paul V Licciardi2,*1Epigenomic Medicine, Alfred Medical Research and Education Precinct, 2Allergy and Immune Disorders, Murdoch Childrens Research Institute, Melbourne, VIC, Australia*These authors contributed equally to this workAbstract: Histone deacetylase (HDAC inhibitors are an emerging class of therapeutics with potential as anticancer drugs. The rationale for developing HDAC inhibitors (and other chromatin-modifying agents as anticancer therapies arose from the understanding that in addition to genetic mutations, epigenetic changes such as dysregulation of HDAC enzymes can alter phenotype and gene expression, disturb homeostasis, and contribute to neoplastic growth. The family of HDAC inhibitors is large and diverse. It includes a range of naturally occurring and synthetic compounds that differ in terms of structure, function, and specificity. HDAC inhibitors have multiple cell type-specific effects in vitro and in vivo, such as growth arrest, cell differentiation, and apoptosis in malignant cells. HDAC inhibitors have the potential to be used as monotherapies or in combination with other anticancer therapies. Currently, there are two HDAC inhibitors that have received approval from the US FDA for the treatment of cutaneous T-cell lymphoma: vorinostat (suberoylanilide hydroxamic acid, Zolinza and depsipeptide (romidepsin, Istodax. More recently, depsipeptide has also gained FDA approval for the treatment of peripheral T-cell lymphoma. Many more clinical trials assessing the effects of various HDAC inhibitors on hematological and solid malignancies are currently being conducted. Despite the proven anticancer effects of particular HDAC inhibitors against certain cancers, many aspects of HDAC enzymes and HDAC inhibitors are still not fully understood. Increasing our understanding of the effects of HDAC inhibitors, their targets and mechanisms of action will be critical for the

The binding mechanism of a peptidic cyclic serine protease inhibitor

DEFF Research Database (Denmark)

Jiang, Longguang; Svane, Anna Sigrid P.; Sørensen, Hans Peter

2011-01-01

Serine proteases are classical objects for studies of catalytic and inhibitory mechanisms as well as interesting as therapeutic targets. Since small-molecule serine protease inhibitors generally suffer from specificity problems, peptidic inhibitors, isolated from phage-displayed peptide libraries......, have attracted considerable attention. Here, we have investigated the mechanism of binding of peptidic inhibitors to serine protease targets. Our model is upain-1 (CSWRGLENHRMC), a disulfide-bond-constrained competitive inhibitor of human urokinase-type plasminogen activator with a noncanonical...... inhibitory mechanism and an unusually high specificity. Using a number of modified variants of upain-1, we characterised the upain-1-urokinase-type plasminogen activator complex using X-ray crystal structure analysis, determined a model of the peptide in solution by NMR spectroscopy, and analysed binding...

Task-specific ionic liquid as a new green inhibitor of mild steel corrosion

Energy Technology Data Exchange (ETDEWEB)

Kowsari, E., E-mail: kowsarie@aut.ac.ir [Department of Chemistry, Amirkabir University of Technology, No. 424, Hafez Avenue, 1591634311 Tehran (Iran, Islamic Republic of); Payami, M. [Department of Chemistry, Amirkabir University of Technology, No. 424, Hafez Avenue, 1591634311 Tehran (Iran, Islamic Republic of); Amini, R.; Ramezanzadeh, B. [Department of Surface Coating and Corrosion, Institute for Color Science and Technology (ICST), PO 16765-654 Tehran (Iran, Islamic Republic of); Javanbakht, M. [Department of Chemistry, Amirkabir University of Technology, No. 424, Hafez Avenue, 1591634311 Tehran (Iran, Islamic Republic of)

2014-01-15

The corrosion inhibition effects of an imidazolium-based task specific ionic liquid (TSIL) were investigated on a low carbon steel in 1 M HCl solution. Samples were exposed to 1 M HCl solution without and with different concentrations of TSIL. Weight loss measurements, potentiodynamic polarization test, electrochemical impedance spectroscopy (EIS), scanning electron microscope (SEM), atomic force microscope (AFM) and contact angle measurements were utilized to investigate the inhibition effects of TSIL. The results obtained from the polarization studies revealed that both the anodic and cathodic branches slopes affected in the presence of TSIL. It was shown that TSIL behaved as a mixed type inhibitor with a dominant effect on the anodic reaction rate depression. It was shown that the increase in polarization resistance and the decrease in corrosion current density were more pronounced using 100 mg/L of TSIL after 2 h immersion time. It was also shown that the adsorption of TSIL followed a Langmuir adsorption isotherm.

Inhibitors of Histone Deacetylases Are Weak Activators of the FMR1 Gene in Fragile X Syndrome Cell Lines

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Alexander A. Dolskiy

2017-01-01

Full Text Available Fragile X syndrome is the most common cause of inherited intellectual disability in humans. It is a result of CGG repeat expansion in the 5′ untranslated region (5′ UTR of the FMR1 gene. This gene encodes the FMRP protein that is involved in neuronal development. Repeat expansion leads to heterochromatinization of the promoter, gene silencing, and the subsequent absence of FMRP. To date, there is no specific therapy for the syndrome. All treatments in clinic practice provide symptomatic therapy. The development of drug therapy for Fragile X syndrome treatment is connected with the search for inhibitors of enzymes that are responsible for heterochromatinization. Here, we report a weak transcriptional activity of the FMR1 gene and the absence of FMRP protein after Fragile X syndrome cell lines treatment with two FDA approved inhibitors of histone deacetylases, romidepsin and vorinostat. We demonstrate that romidepsin, an inhibitor of class I histone deacetylases, does not activate FMR1 expression in patient cell cultures, whereas vorinostat, an inhibitor of classes I and II histone deacetylases, activates a low level of FMR1 expression in some patient cell lines.

1-Methyl-beta-carboline (harmane), a potent endogenous inhibitor of benzodiazepine receptor binding.

Science.gov (United States)

Rommelspacher, H; Nanz, C; Borbe, H O; Fehske, K J; Müller, W E; Wollert, U

1980-10-01

The interaction of several beta-carbolines with specific [3H]-flunitrazepam binding to benzodiazepine receptors in rat brain membranes was investigated. Out of the investigated compounds, harmane and norharmane were the most potent inhibitors of specific [3H]-flunitrazepam binding, with IC50-values in the micromolar range. All other derivatives, including harmine, harmaline, and several tetrahydroderivatives were at least ten times less potent. Harmane has been previously found in rat brain and human urine, so it is the most potent endogenous inhibitor of specific [3H]-flunitrazepam binding known so far, with a several fold higher affinity for the benzodiazepine receptor than inosine and hypoxanthine. Thus, we suggest that harmane or other related beta-carbolines could be potential candidates as endogenous ligands of the benzodiazepine receptor.

Curcumin derivatives as HIV-1 protease inhibitors

Energy Technology Data Exchange (ETDEWEB)

Sui, Z.; Li, J.; Craik, C.S.; Ortiz de Montellano, P.R. [Univ. of California, San Francisco, CA (United States)

1993-12-31

Curcumin, a non-toxic natural compound from Curcuma longa, has been found to be an HIV-1 protease inhibitor. Some of its derivatives were synthesized and their inhibitory activity against the HIV-1 protease was tested. Curcumin analogues containing boron enhanced the inhibitory activity. At least of the the synthesized compounds irreversibly inhibits the HIV-1 protease.

Interaction of small molecule inhibitors of HIV-1 entry with CCR5

International Nuclear Information System (INIS)

Seibert, Christoph; Ying Weiwen; Gavrilov, Svetlana; Tsamis, Fotini; Kuhmann, Shawn E.; Palani, Anandan; Tagat, Jayaram R.; Clader, John W.; McCombie, Stuart W.; Baroudy, Bahige M.; Smith, Steven O.; Dragic, Tatjana; Moore, John P.; Sakmar, Thomas P.

2006-01-01

The CC-chemokine receptor 5 (CCR5) is the major coreceptor for macrophage-tropic (R5) HIV-1 strains. Several small molecule inhibitors of CCR5 that block chemokine binding and HIV-1 entry are being evaluated as drug candidates. Here we define how CCR5 antagonists TAK-779, AD101 (SCH-350581) and SCH-C (SCH-351125), which inhibit HIV-1 entry, interact with CCR5. Using a mutagenesis approach in combination with a viral entry assay to provide a direct functional read out, we tested predictions based on a homology model of CCR5 and analyzed the functions of more than 30 amino acid residues. We find that a key set of aromatic and aliphatic residues serves as a hydrophobic core for the ligand binding pocket, while E283 is critical for high affinity interaction, most likely by acting as the counterion for a positively charged nitrogen atom common to all three inhibitors. These results provide a structural basis for understanding how specific antagonists interact with CCR5, and may be useful for the rational design of new, improved CCR5 ligands

Sibiriline, a new small chemical inhibitor of receptor-interacting protein kinase 1, prevents immune-dependent hepatitis.

Science.gov (United States)

Le Cann, Fabienne; Delehouzé, Claire; Leverrier-Penna, Sabrina; Filliol, Aveline; Comte, Arnaud; Delalande, Olivier; Desban, Nathalie; Baratte, Blandine; Gallais, Isabelle; Piquet-Pellorce, Claire; Faurez, Florence; Bonnet, Marion; Mettey, Yvette; Goekjian, Peter; Samson, Michel; Vandenabeele, Peter; Bach, Stéphane; Dimanche-Boitrel, Marie-Thérèse

2017-09-01

Necroptosis is a regulated form of cell death involved in several disease models including in particular liver diseases. Receptor-interacting protein kinases, RIPK1 and RIPK3, are the main serine/threonine kinases driving this cell death pathway. We screened a noncommercial, kinase-focused chemical library which allowed us to identify Sibiriline as a new inhibitor of necroptosis induced by tumor necrosis factor (TNF) in Fas-associated protein with death domain (FADD)-deficient Jurkat cells. Moreover, Sib inhibits necroptotic cell death induced by various death ligands in human or mouse cells while not protecting from caspase-dependent apoptosis. By using competition binding assay and recombinant kinase assays, we demonstrated that Sib is a rather specific competitive RIPK1 inhibitor. Molecular docking analysis shows that Sib is trapped closed to human RIPK1 adenosine triphosphate-binding site in a relatively hydrophobic pocket locking RIPK1 in an inactive conformation. In agreement with its RIPK1 inhibitory property, Sib inhibits both TNF-induced RIPK1-dependent necroptosis and RIPK1-dependent apoptosis. Finally, Sib protects mice from concanavalin A-induced hepatitis. These results reveal the small-molecule Sib as a new RIPK1 inhibitor potentially of interest for the treatment of immune-dependent hepatitis. © 2017 Federation of European Biochemical Societies.

Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in diagnosis of pleural effusion of malignant origin.

Science.gov (United States)

Fiorelli, Alfonso; Ricci, Serena; Feola, Antonia; Mazzella, Antonio; D'Angelo, Luigi; Santini, Mario; Di Domenico, Marina; Di Carlo, Angelina

2016-04-01

The aim of the present study was to evaluate the diagnostic accuracy of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in differentiating benign from malignant exudative pleural effusions. This is a unicentre observational study including 97 consecutive patients with exudative pleural effusions. Metalloproteinase-9, tissue inhibitor of metalloproteinase-1, lactate dehydrogenase, ferritin, carcinoembryonic antigen and carbohydrate antigen 15-3 were measured in pleural effusion and serum by enzyme-linked immunosorbent assay. The activity of metalloproteinase-9 was also evaluated by substrate zymography. The data were correlated with final diagnosis of pleural effusions to evaluate the diagnostic accuracy. Of the 97 eligible patients, 6 were excluded. Of the 91 patients included in the study, 70 had malignant pleural effusions and 21 had benign pleural effusions. Both in sera and pleural effusions, matrix metalloproteinase-9 (P effusion (P effusion metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 levels showed higher value of sensitivity (97 and 91%, respectively) and specificity (90 and 95%, respectively) compared with other standard markers. Serum metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 levels showed similar results. Among 70 neoplastic patients, 29 had negative pleural cytology. Of these, 25 presented elevated levels of metalloproteinase-9 and tissue inhibitor of metalloproteinase-1, whereas 4 patients had elevated levels of one of the two markers. Our results showed that metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 might be valuable markers in differentiating benign from malignant pleural effusions. Their levels are neither influenced by the histology and tumour origin nor by the presence of tumour cells in pleural effusions. Thus, their use in clinical practice could help in the selection of patients needing more invasive procedures, such as thoracoscopic biopsy. © The Author 2016

Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours

DEFF Research Database (Denmark)

Lasko, Loren M; Jakob, Clarissa G; Edalji, Rohinton P

2017-01-01

-specific tumour types, including several haematological malignancies and androgen receptor-positive prostate cancer. A-485 inhibited the androgen receptor transcriptional program in both androgen-sensitive and castration-resistant prostate cancer and inhibited tumour growth in a castration-resistant xenograft...... to treat certain cancers, but progress on the development of drug-like histone actyltransferase inhibitors has lagged behind. The histone acetyltransferase paralogues p300 and CREB-binding protein (CBP) are key transcriptional co-activators that are essential for a multitude of cellular processes, and have...... also been implicated in human pathological conditions (including cancer). Current inhibitors of the p300 and CBP histone acetyltransferase domains, including natural products, bi-substrate analogues and the widely used small molecule C646, lack potency or selectivity. Here, we describe A-485, a potent...

Medicinal chemistry insights in the discovery of novel LSD1 inhibitors.

Science.gov (United States)

Wang, Xueshun; Huang, Boshi; Suzuki, Takayoshi; Liu, Xinyong; Zhan, Peng

2015-01-01

LSD1 is an epigenetic modulator associated with transcriptional regulation of genes involved in a broad spectrum of key cellular processes, and its activity is often altered under pathological conditions. LSD1 inhibitors are considered to be candidates for therapy of cancer, viral diseases and neurodegeneration. Many LSD1 inhibitors with various scaffolds have been disclosed, and a few potent molecules are in different stages of clinical development. In this review, we summarize recent biological findings on the roles of LSD1 and the current understanding of the clinical significance of LSD1, and focus on the medicinal chemistry strategies used in the design and development of LSD1 inhibitors as drug-like epigenetic modulators since 2012, including a brief consideration of structure-activity relationships.

Selective serotonin reuptake inhibitor antidepressant use in first trimester pregnancy and risk of specific congenital anomalies

DEFF Research Database (Denmark)

Wemakor, Anthony; Casson, Karen; Garne, Ester

2015-01-01

Evidence of an association between early pregnancy exposure to selective serotonin reuptake inhibitors (SSRI) and congenital heart defects (CHD) has contributed to recommendations to weigh benefits and risks carefully. The objective of this study was to determine the specificity of association be...

Identification of brain-specific angiogenesis inhibitor 2 as an interaction partner of glutaminase interacting protein

International Nuclear Information System (INIS)

Zencir, Sevil; Ovee, Mohiuddin; Dobson, Melanie J.; Banerjee, Monimoy; Topcu, Zeki; Mohanty, Smita

2011-01-01

Highlights: → Brain-specific angiogenesis inhibitor 2 (BAI2) is a new partner protein for GIP. → BAI2 interaction with GIP was revealed by yeast two-hybrid assay. → Binding of BAI2 to GIP was characterized by NMR, CD and fluorescence. → BAI2 and GIP binding was mediated through the C-terminus of BAI2. -- Abstract: The vast majority of physiological processes in living cells are mediated by protein-protein interactions often specified by particular protein sequence motifs. PDZ domains, composed of 80-100 amino acid residues, are an important class of interaction motif. Among the PDZ-containing proteins, glutaminase interacting protein (GIP), also known as Tax Interacting Protein TIP-1, is unique in being composed almost exclusively of a single PDZ domain. GIP has important roles in cellular signaling, protein scaffolding and modulation of tumor growth and interacts with a number of physiological partner proteins, including Glutaminase L, β-Catenin, FAS, HTLV-1 Tax, HPV16 E6, Rhotekin and Kir 2.3. To identify the network of proteins that interact with GIP, a human fetal brain cDNA library was screened using a yeast two-hybrid assay with GIP as bait. We identified brain-specific angiogenesis inhibitor 2 (BAI2), a member of the adhesion-G protein-coupled receptors (GPCRs), as a new partner of GIP. BAI2 is expressed primarily in neurons, further expanding GIP cellular functions. The interaction between GIP and the carboxy-terminus of BAI2 was characterized using fluorescence, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy assays. These biophysical analyses support the interaction identified in the yeast two-hybrid assay. This is the first study reporting BAI2 as an interaction partner of GIP.

The significance of fibrin binding by plasminogen activator inhibitor 1 for the mechanism of tissue-type plasminogen activator-mediated fibrinolysis

NARCIS (Netherlands)

Stringer, H. A.; Pannekoek, H.

1995-01-01

The specific, reversible interaction between plasminogen activator inhibitor 1 (PAI-1) and intact fibrin polymers was studied using both purified components and isolated activated platelets as a source of PAI-1. A key reagent in these experiments is a PAI-1 mutant, having its P1 reactive center

Structure-Activity Relationships of Pentacyclic Triterpenoids as Potent and Selective Inhibitors against Human Carboxylesterase 1

Directory of Open Access Journals (Sweden)

Li-Wei Zou

2017-06-01

Full Text Available Human carboxylesterase 1 (hCE1, one of the most important serine hydrolases distributed in liver and adipocytes, plays key roles in endobiotic homeostasis and xenobiotic metabolism. This study aimed to find potent and selective inhibitors against hCE1 from phytochemicals and their derivatives. To this end, a series of natural triterpenoids were collected and their inhibitory effects against human carboxylesterases (hCEs were assayed using D-Luciferin methyl ester (DME and 6,8-dichloro-9,9-dimethyl-7-oxo-7,9-dihydroacridin-2-yl benzoate (DDAB as specific optical substrate for hCE1, and hCE2, respectively. Following screening of a series of natural triterpenoids, oleanolic acid (OA, and ursolic acid (UA were found with strong inhibitory effects on hCE1 and relative high selectivity over hCE2. In order to get the highly selective and potent inhibitors of hCE1, a series of OA and UA derivatives were synthesized from OA and UA by chemical modifications including oxidation, reduction, esterification, and amidation. The inhibitory effects of these derivatives on hCEs were assayed and the structure-activity relationships of tested triterpenoids as hCE1 inhibitors were carefully investigated. The results demonstrated that the carbonyl group at the C-28 site is essential for hCE1 inhibition, the modifications of OA or UA at this site including esters, amides and alcohols are unbeneficial for hCE1 inhibition. In contrast, the structural modifications on OA and UA at other sites, such as converting the C-3 hydroxy group to 3-O-β-carboxypropionyl (compounds 20 and 22, led to a dramatically increase of the inhibitory effects against hCE1 and very high selectivity over hCE2. 3D-QSAR analysis of all tested triterpenoids including OA and UA derivatives provide new insights into the fine relationships linking between the inhibitory effects on hCE1 and the steric-electrostatic properties of triterpenoids. Furthermore, both inhibition kinetic analyses and docking

PTP1B Inhibitors from the Entomogenous Fungi Isaria fumosorosea

OpenAIRE

Jun Zhang; Lin-Lin Meng; Jing-Jing Wei; Peng Fan; Sha-Sha Liu; Wei-Yu Yuan; You-Xing Zhao; Du-Qiang Luo

2017-01-01

Protein tyrosine phosphatase 1B (PTP1B) is implicated as a negative regulator of insulin receptor (IR) signaling and a potential drug target for the treatment of type II diabetes and other associated metabolic syndromes. Thus, small molecule inhibitors of PTP1B can be considered as an attractive approach for the design of new therapeutic agents of type II diabetes and cancer diseases. In a continuing search for new PTP1B inhibitors, a new tetramic acid possessing a rare pyrrolidinedione skele...

Progress of PD-1/PD-L1 Inhibitors in Non-small Cell Lung Cancer

Directory of Open Access Journals (Sweden)

Zhansheng JIANG

2017-02-01

Full Text Available Pembrolizumab, an inhibitor target programmed death 1 (PD-1, was approved into the first line therapy in advanced non-small cell lung cancer (NSCLC. It was a milestone that immune checkpoints drugs have played an important role in the treatment system of NSCLC. The results of clinical trials revealed the superiority of PD-1/programmed death ligand 1 (PD-L1 inhibitors compared with chemotherapy in first-line, second-line and multidrug resistance phase therapy. Objective response rate (ORR was up to 80% with pembrolizumab plus chemotherapy, and progression-free survival (PFS with single pembrolizumab in first line was nearly 1 year (10.3 months, the hazard ratio for death fell by 40%. Overall survival (OS was more or less 1 year with single drug pembrolizumab, nivolumab and atezolizumab for second line therapy. PD-L1 expression was a predictor of PD-1/PD-L1 inhibitors. The positive rate of PD-L1 (more than 1% in advanced NSCLC was about 60% with little difference between the tissue types. However, there was no gold standard test of PD-L1 expression.

Identification of danthron as an isoform-specific inhibitor of HEME OXYGENASE-1/cytochrome P450 reductase interaction with anti-tumor activity.

Science.gov (United States)

Chou, Yi-Tai; Hsu, Fu-Fei; Hu, Dun-Yao; Chen, Ying-Chih; Hsu, Yuan-Hao; Hsu, John T-A; Chau, Lee-Young

2018-01-23

Heme oxygenase (HO) catalyzes NADPH-dependent degradation of heme to liberate iron, carbon monoxide and biliverdin. The interaction between HO and cytochrome P450 reductase (CPR), an electron donor, is essential for HO activity. HO-1 is a stress-inducible isoform whereas HO-2 is constitutively expressed. HO-1 induction is commonly seen in cancers and impacts disease progression, supporting the possibility of targeting HO-1 for cancer therapy. We employed a cell-based bioluminescence resonance energy transfer assay to screen compounds with ability to inhibit HO-1/CPR interaction. The effect of the identified compound on HO-1/CPR interaction was confirmed by pull down assay. Moreover, the anti-tumorigenic activity of the identified compound on HO-1-enhanced tumor growth and migration was assessed by trypan blue exclusion method and wound healing assay. Danthron was identified as an effective small molecule able to interfere with the interaction between HO-1 and CPR but not HO-2 and CPR. Additional experiments with structural analogues of danthron revealed that the positions of hydroxyl moieties significantly affected the potency of inhibition on HO-1/CPR interaction. Pull-down assay confirmed that danthron inhibited the interaction of CPR with HO-1 but not HO-2. Danthron suppressed growth and migration of HeLa cells with stable HO-1 overexpression but not mock cells. In contrast, anthrarufin, a structural analog with no ability to interfere HO-1/CPR interaction, exhibited no significant effect on HO-1-overexpressing HeLa cells. These findings demonstrate that danthron is an isoform-specific inhibitor for HO-1/CPR interaction and may serve as a lead compound for novel anticancer drug.

The cardiovascular safety trials of DPP-4 inhibitors, GLP-1 agonists, and SGLT2 inhibitors.

Science.gov (United States)

Secrest, Matthew H; Udell, Jacob A; Filion, Kristian B

2017-04-01

In this paper, we review the results of large, double-blind, placebo-controlled randomized trials mandated by the US Food and Drug Administration to examine the cardiovascular safety of newly-approved antihyperglycemic agents in patients with type 2 diabetes. The cardiovascular effects of dipeptidyl peptidase-4 (DPP-4) inhibitors remain controversial: while these drugs did not reduce or increase the risk of primary, pre-specified composite cardiovascular outcomes, one DPP-4 inhibitor (saxagliptin) increased the risk of hospitalization for heart failure in the overall population; another (alogliptin) demonstrated inconsistent effects on heart failure hospitalization across subgroups of patients, and a third (sitagliptin) demonstrated no effect on heart failure. Evidence for cardiovascular benefits of glucagon-like peptide-1 (GLP-1) agonists has been similarly heterogeneous, with liraglutide and semaglutide reducing the risk of composite cardiovascular outcomes, but lixisenatide having no reduction or increase in cardiovascular risk. The effect of GLP-1 agonists on retinopathy remains a potential concern. In the only completed trial to date to assess a sodium-glucose cotransporter-2 (SGLT2) inhibitor, empagliflozin reduced the risk of composite cardiovascular endpoints, predominantly through its impact on cardiovascular mortality and heart failure hospitalization. Copyright © 2017 Elsevier Inc. All rights reserved.

Generation and Characterization of Inhibitory Antibodies Specific to Guinea Pig CXCR1 and CXCR2.

Science.gov (United States)

Tanaka, Kento; Yoshimura, Chigusa; Shiina, Tetsuo; Terauchi, Tomoko; Yoshitomi, Tomomi; Hirahara, Kazuki

2017-04-01

CXCR1 and CXCR2 are chemokine receptors that have different selectivity of chemokine ligands, but the distinct role of each receptor is not clearly understood. This is due to the absence of specific inhibitors in guinea pigs, which are the appropriate species for investigation of CXCR1 and CXCR2 because of their functional similarity to humans. In this study, we generated and evaluated monoclonal antibodies that specifically bound to guinea pig CXCR1 (gpCXCR1) and guinea pig CXCR2 (gpCXCR2) for acquisition of specific inhibitors. To assess the activity of antibodies, we established CHO-K1 cells stably expressing either gpCXCR1 or gpCXCR2 (CHO/gpCXCR1 or CHO/gpCXCR2). CHO/gpCXCR1 showed migration in response to guinea pig interleukin (IL)-8, and CHO/gpCXCR2 showed migration in response to both guinea pig IL-8 and guinea pig growth-regulated oncogene α. The receptor selectivities of the chemokines of guinea pigs were the same as the human orthologs. The inhibitory activities of the anti-gpCXCR1 and anti-gpCXCR2 monoclonal antibodies on cell migration were observed in a concentration-dependent manner. In conclusion, we successfully obtained inhibitory antibodies specific to gpCXCR1 and gpCXCR2. These inhibitory antibodies will be useful to clarify the physiological roles of CXCR1 and CXCR2 in guinea pigs.

Biochemical Importance of Glycosylation of Plasminogen Activator Inhibitor-1

DEFF Research Database (Denmark)

Gils, Ann; Pedersen, Katrine Egelund; Skottrup, Peter

2003-01-01

The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous glycosyla......The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous...... with the glycosylation sites could be excluded as explanation for the differential reactivity. The latency transition of non-glycosylated, but not of glycosylated PAI-1, was strongly accelerated by a non-ionic detergent. The different biochemical properties of glycosylated and non-glycosylated PAI-1 depended...

Largazole, a class I histone deacetylase inhibitor, enhances TNF-α-induced ICAM-1 and VCAM-1 expression in rheumatoid arthritis synovial fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Ahmed, Salahuddin, E-mail: Salah.Ahmed@utoledo.edu [Department of Pharmacology, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, OH (United States); Riegsecker, Sharayah; Beamer, Maria; Rahman, Ayesha; Bellini, Joseph V. [Department of Pharmacology, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, OH (United States); Bhansali, Pravin; Tillekeratne, L.M. Viranga [Department of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, OH (United States)

2013-07-15

In the present study, we evaluated the effect of largazole (LAR), a marine-derived class I HDAC inhibitor, on tumor necrosis factor-α (TNF-α)-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and matrix metalloproteinase-2 (MMP-2) activity. LAR (1–5 μM) had no adverse effect on the viability of RA synovial fibroblasts. Among the different class I HDACs screened, LAR (0.5–5 μM) inhibited the constitutive expression of HDAC1 (0–30%). Surprisingly, LAR increased class II HDAC [HDAC6] by ∼ 220% with a concomitant decrease in HDAC5 [30–58%] expression in RA synovial fibroblasts. SAHA (5 μM), a pan-HDAC inhibitor, also induced HDAC6 expression in RA synovial fibroblasts. Pretreatment of RA synovial fibroblasts with LAR further enhanced TNF-α-induced ICAM-1 and VCAM-1 expression. However, LAR inhibited TNF-α-induced MMP-2 activity in RA synovial fibroblasts by 35% when compared to the TNF-α-treated group. Further, the addition of HDAC6 specific inhibitor Tubastatin A with LAR suppressed TNF-α + LAR-induced ICAM-1 and VCAM-1 expression and completely blocked MMP-2 activity, suggesting a role of HDAC6 in LAR-induced ICAM-1 and VCAM-1 expression. LAR also enhanced TNF-α-induced phospho-p38 and phospho-AKT expression, but inhibited the expression of phospho-JNK and nuclear translocation of NF-κBp65 in RA synovial fibroblasts. These results suggest that LAR activates p38 and Akt pathways and influences class II HDACs, in particular HDAC6, to enhance some of the detrimental effects of TNF-α in RA synovial fibroblasts. Understanding the exact role of different HDAC isoenzymes in RA pathogenesis is extremely important in order to develop highly effective HDAC inhibitors for the treatment of RA. - Highlights: • Largazole enhances TNF-α-induced ICAM-1 and VCAM-1. • Largazole upregulates class II HDAC (HDAC6) in RA synovial fibroblasts. • Largazole also induces the expression of phospho-p38

Effects of kinase inhibitors and potassium phosphate (KPi) on site-specific phosphorylation of branched chain α-ketoacid dehydrogenase (BCKDH)

International Nuclear Information System (INIS)

Kuntz, M.J.; Shimomura, Y.; Ozawa, T.; Harris, R.A.

1987-01-01

BCKDH is phosphorylated by a copurifying kinase at two serine residues on the Elα subunit. Phosphorylation of both sites occurs at about the same rate initially, but inactivation is believed associated only with site 1 phosphorylation. The effects of KPi and known inhibitors of BCKDH kinase, α-chloroisocaproate (CIC) and branched chain α-ketoacids (BCKA), on the phosphorylation of purified rat liver BCKDH were studied. Site-specific phosphorylation was quantitated by thin-layer electrophoresis of tryptic peptides followed by densitometric scanning of autoradiograms. Addition of 5 mM KPi was found necessary to stabilize the BCKDH activity at 37 0 C. Increasing the KPi to 50 mM dramatically increased the CIC and BCKA inhibition of site 1 and site 2 phosphorylation. The finding of enhanced sensitivity of inhibitors with 50 mM KPi may facilitate identification of physiologically important kinase effectors. Regardless of the KPi concentration, CIC and the BCKA showed much more effective inhibition of site 2 than site 1 phosphorylation. Although site 1 is the primary inactivating site, predominant inhibition of site 2 phosphorylation may provide a means of modulating kinase/phosphatase control of BCKDH activity under steady state conditions

Isolation and characterization of a specific endogenous Na/sup +/, K/sup +/-ATPase inhibitor from bovine adrenal

Energy Technology Data Exchange (ETDEWEB)

Tamura, M.; Lam, T.T.; Inagami, T.

1988-06-14

In order to identify a specific endogenous Na/sup +/,K/sup +/-ATPase inhibitor which could possibly be related to salt-dependent hypertension, the authors looked for substances in the methanol extract of bovine whole adrenal which show all of the following properties: (i) inhibitory activity for Na/sup +/,K/sup +/-ATPase; (ii) competitive displacing activity against (/sup 3/H)ouabain binding to the enzyme; (iii) inhibitory activity for /sup 86/Rb uptake into intact human erythrocytes; and (iv) cross-reactivity with sheep anti-digoxin-specific antibody. After stepwise fractionation of the methanol extract of bovine adrenal glands by chromatography on a C/sub 18/ open column, a 0-15% acetonitrile fraction was fractionated by high-performance liquid chromatography on a Zorbax octadecylsilane column. One of the most active fractions in 0-15% acetonitrile was found to exhibit all of the four types of the activities. It was soluble in water and was distinct from various substances which have been known to inhibit Na/sup +/,K/sup +/-ATPase. These results strongly suggest that this water-soluble nonpeptidic Na/sup +/,K/sup +/-ATPase inhibitor may be a specific endogenous regulator for the ATPase.

Functional C1-inhibitor diagnostics in hereditary angioedema: assay evaluation and recommendations

DEFF Research Database (Denmark)

Wagenaar-Bos, Ineke G A; Drouet, Christian; Aygören-Pursun, Emel

2008-01-01

Hereditary angioedema (HAE) is an autosomal dominant disease characterized by recurrent episodes of potentially life-threatening angioedema. The most widespread underlying genetic deficiency is a heterozygous deficiency of the serine protease inhibitor C1 esterase inhibitor (C1-Inh). In addition ...

Escape from Human Immunodeficiency Virus Type 1 (HIV-1 Entry Inhibitors

Directory of Open Access Journals (Sweden)

Carol D. Weiss

2012-12-01

Full Text Available The human immunodeficiency virus (HIV enters cells through a series of molecular interactions between the HIV envelope protein and cellular receptors, thus providing many opportunities to block infection. Entry inhibitors are currently being used in the clinic, and many more are under development. Unfortunately, as is the case for other classes of antiretroviral drugs that target later steps in the viral life cycle, HIV can become resistant to entry inhibitors. In contrast to inhibitors that block viral enzymes in intracellular compartments, entry inhibitors interfere with the function of the highly variable envelope glycoprotein as it continuously adapts to changing immune pressure and available target cells in the extracellular environment. Consequently, pathways and mechanisms of resistance for entry inhibitors are varied and often involve mutations across the envelope gene. This review provides a broad overview of entry inhibitor resistance mechanisms that inform our understanding of HIV entry and the design of new inhibitors and vaccines.

Adventures in Scaffold Morphing: Discovery of Fused Ring Heterocyclic Checkpoint Kinase 1 (CHK1) Inhibitors.

Science.gov (United States)

Yang, Bin; Vasbinder, Melissa M; Hird, Alexander W; Su, Qibin; Wang, Haixia; Yu, Yan; Toader, Dorin; Lyne, Paul D; Read, Jon A; Breed, Jason; Ioannidis, Stephanos; Deng, Chun; Grondine, Michael; DeGrace, Nancy; Whitston, David; Brassil, Patrick; Janetka, James W

2018-02-08

Checkpoint kinase 1 (CHK1) inhibitors are potential cancer therapeutics that can be utilized for enhancing the efficacy of DNA damaging agents. Multiple small molecule CHK1 inhibitors from different chemical scaffolds have been developed and evaluated in clinical trials in combination with chemotherapeutics and radiation treatment. Scaffold morphing of thiophene carboxamide ureas (TCUs), such as AZD7762 (1) and a related series of triazoloquinolines (TZQs), led to the identification of fused-ring bicyclic CHK1 inhibitors, 7-carboxamide thienopyridines (7-CTPs), and 7-carboxamide indoles. X-ray crystal structures reveal a key intramolecular noncovalent sulfur-oxygen interaction in aligning the hinge-binding carboxamide group to the thienopyridine core in a coplanar fashion. An intramolecular hydrogen bond to an indole NH was also effective in locking the carboxamide in the preferred bound conformation to CHK1. Optimization on the 7-CTP series resulted in the identification of lead compound 44, which displayed respectable drug-like properties and good in vitro and in vivo potency.

Discovery of pyrazolo[1,5-a]pyrimidine-based CHK1 inhibitors: A template-based approach-Part 2

Energy Technology Data Exchange (ETDEWEB)

Labroli, Marc; Paruch, Kamil; Dwyer, Michael P.; Alvarez, Carmen; Keertikar, Kartik; Poker, Cory; Rossman, Randall; Duca, Jose S.; Fischmann, Thierry O.; Madison, Vincent; Parry, David; Davis, Nicole; Seghezzi, Wolfgang; Wiswell, Derek; Guzi, Timothy J. [Merck

2013-11-20

Previous efforts by our group have established pyrazolo[1,5-a]pyrimidine as a viable core for the development of potent and selective CDK inhibitors. As part of an effort to utilize the pyrazolo[1,5-a]pyrimidine core as a template for the design and synthesis of potent and selective kinase inhibitors, we focused on a key regulator in the cell cycle progression, CHK1. Continued SAR development of the pyrazolo[1,5-a]pyrimidine core at the C5 and C6 positions, in conjunction with previously disclosed SAR at the C3 and C7 positions, led to the discovery of potent and selective CHK1 inhibitors.

Lipid raft-like liposomes used for targeted delivery of a chimeric entry-inhibitor peptide with anti-HIV-1 activity.

Science.gov (United States)

Gómara, María José; Pérez-Pomeda, Ignacio; Gatell, José María; Sánchez-Merino, Victor; Yuste, Eloisa; Haro, Isabel

2017-02-01

The work reports the design and synthesis of a chimeric peptide that is composed of the peptide sequences of two entry inhibitors which target different sites of HIV-1 gp41. The chimeric peptide offers the advantage of targeting two gp41 regions simultaneously: the fusion peptide and the loop both of which are membrane active and participate in the membrane fusion process. We therefore use lipid raft-like liposomes as a tool to specifically direct the chimeric inhibitor peptide to the membrane domains where the HIV-1 envelope protein is located. Moreover, the liposomes that mimic the viral membrane composition protect the chimeric peptide against proteolytic digestion thereby increasing the stability of the peptide. The described liposome preparations are suitable nanosystems for managing hydrophobic entry-inhibitor peptides as putative therapeutics. Copyright © 2016 Elsevier Inc. All rights reserved.

CD8+ T Cells Specific to Apoptosis-Associated Antigens Predict the Response to Tumor Necrosis Factor Inhibitor Therapy in Rheumatoid Arthritis.

Directory of Open Access Journals (Sweden)

Alessandra Citro

Full Text Available CD8+ T cells specific to caspase-cleaved antigens derived from apoptotic T cells (apoptotic epitopes represent a principal player in chronic immune activation, which is known to amplify immunopathology in various inflammatory diseases. The purpose of the present study was to investigate the relationship involving these autoreactive T cells, the rheumatoid arthritis immunopathology, and the response to tumor necrosis factor-α inhibitor therapy. The frequency of autoreactive CD8+ T cells specific to various apoptotic epitopes, as detected by both enzyme-linked immunospot assay and dextramers of major histocompatibility complex class I molecules complexed with relevant apoptotic epitopes, was longitudinally analyzed in the peripheral blood of rheumatoid arthritis patients who were submitted to etanercept treatment (or other tumor necrosis factor inhibitors as a control. The percentage of apoptotic epitope-specific CD8+ T cells was significantly higher in rheumatoid arthritis patients than in healthy donors, and correlated with the disease activity. More important, it was significantly more elevated in responders to tumor necrosis factor-α inhibitor therapy than in non-responders before the start of therapy; it significantly dropped only in the former following therapy. These data indicate that apoptotic epitope-specific CD8+ T cells may be involved in rheumatoid arthritis immunopathology through the production of inflammatory cytokines and that they may potentially represent a predictive biomarker of response to tumor necrosis factor-α inhibitor therapy to validate in a larger cohort of patients.

Structural investigation of HIV-1 nonnucleoside reverse transcriptase inhibitors: 2-Aryl-substituted benzimidazoles

Science.gov (United States)

Ziółkowska, Natasza E.; Michejda, Christopher J.; Bujacz, Grzegorz D.

2009-11-01

Acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV) is one of the most destructive epidemics in history. Inhibitors of HIV enzymes are the main targets to develop drugs against that disease. Nonnucleoside reverse transcriptase inhibitors of HIV-1 (NNRTIs) are potentially effective and nontoxic. Structural studies provide information necessary to design more active compounds. The crystal structures of four NNRTI derivatives of 2-aryl-substituted N-benzyl-benzimidazole are presented here. Analysis of the geometrical parameters shows that the structures of the investigated inhibitors are rigid. The important geometrical parameter is the dihedral angle between the planes of the π-electron systems of the benzymidazole and benzyl moieties. The values of these dihedral angles are in a narrow range for all investigated inhibitors. There is no significant difference between the structure of the free inhibitor and the inhibitor in the complex with RT HIV-1. X-ray structures of the investigated inhibitors are a good basis for modeling enzyme-inhibitor interactions in rational drug design.

[Specific inhibitors of cyclooxygenase-2 (COX-2): current knowledge and perspectives].

Science.gov (United States)

Rioda, W T; Nervetti, A

2001-01-01

The Authors summarize the current knowledge on a new class of nonsteroidal anti-inflammatory drugs (NSAIDs), the coxib (celecoxib and rofecoxib), in the treatment of rheumatic diseases. Celecoxib and rofecoxib are selective cyclooxygenase-2 (COX-2) inhibitors which possess the same anti-inflammatory and analgesic activities, but a better gastric tolerability compared to the non-selective COX-1 and COX-2 inhibitors. The Authors also report other possible therapeutic effects of these NSADIs as evidenced by the more recent data of the literature. Celecoxib seems to reduce the incidence of new polyps in patients with familial adenomatous polyposis. It has been suggested the use of celecoxib as a protective drug against the development of colorectal cancer. Other (neoplastic) or pre-neoplastic conditions, such as bladder dysplasia, Barret esophagus, attinic keratosis and Alzheimer's disease seem to have benefit from this class of drugs.

Full-length cellular β-secretase has a trimeric subunit stoichiometry, and its sulfur-rich transmembrane interaction site modulates cytosolic copper compartmentalization.

Science.gov (United States)

Liebsch, Filip; Aurousseau, Mark R P; Bethge, Tobias; McGuire, Hugo; Scolari, Silvia; Herrmann, Andreas; Blunck, Rikard; Bowie, Derek; Multhaup, Gerd

2017-08-11

The β-secretase (BACE1) initiates processing of the amyloid precursor protein (APP) into Aβ peptides, which have been implicated as central players in the pathology of Alzheimer disease. BACE1 has been described as a copper-binding protein and its oligomeric state as being monomeric, dimeric, and/or multimeric, but the native cellular stoichiometry has remained elusive. Here, by using single-molecule fluorescence and in vitro cross-linking experiments with photo-activatable unnatural amino acids, we show that full-length BACE1, independently of its subcellular localization, exists as trimers in human cells. We found that trimerization requires the BACE1 transmembrane sequences (TMSs) and cytoplasmic domains, with residues Ala 463 and Cys 466 buried within the trimer interface of the sulfur-rich core of the TMSs. Our 3D model predicts that the sulfur-rich core of the trimeric BACE1 TMS is accessible to metal ions, but copper ions did not trigger trimerization. The results of functional assays of endogenous BACE1 suggest that it has a role in intracellular copper compartmentalization by transferring cytosolic copper to intracellular compartments, while leaving the overall cellular copper concentration unaltered. Adding to existing physiological models, our results provide novel insight into the atypical interactions between copper and BACE1 and into its non-enzymatic activities. In conclusion, therapeutic Alzheimer disease prevention strategies aimed at decreasing BACE1 protein levels should be regarded with caution, because adverse effects in copper homeostasis may occur. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Fluorescence Resonance Energy Transfer Assay for High-Throughput Screening of ADAMTS1 Inhibitors

Directory of Open Access Journals (Sweden)

Guanhua Du

2011-12-01

Full Text Available A disintegrin and metalloprotease with thrombospondin type I motifs-1 (ADAMTS1 plays a crucial role in inflammatory joint diseases and its inhibitors are potential candidates for anti-arthritis drugs. For the purposes of drug discovery, we reported the development and validation of fluorescence resonance energy transfer (FRET assay for high-throughput screening (HTS of the ADAMTS1 inhibitors. A FRET substrate was designed for a quantitative assay of ADAMTS1 activity and enzyme kinetics studies. The assay was developed into a 50-µL, 384-well assay format for high throughput screening of ADAMTS1 inhibitors with an overall Z’ factor of 0.89. ADAMTS1 inhibitors were screened against a diverse library of 40,960 total compounds with the established HTS system. Four structurally related hits, naturally occurring compounds, kuwanon P, kuwanon X, albafuran C and mulberrofuran J, extracted from the Chinese herb Morus alba L., were identified for further investigation. The results suggest that this FRET assay is an excellent tool, not only for measurement of ADAMTS1 activity but also for discovery of novel ADAMTS1 inhibitors with HTS.

Structural Principles in the Development of Cyclic Peptidic Enzyme Inhibitors

Science.gov (United States)

Xu, Peng; Andreasen, Peter A.; Huang, Mingdong

2017-01-01

This review summarizes our studies in the development of small cyclic peptides for specifically modulating enzyme activity. Serine proteases share highly similar active sites but perform diverse physiological and pathological functions. From a phage-display peptide library, we isolated two mono-cyclic peptides, upain-1 (CSWRGLENHRMC) and mupain-1 (CPAYSRYLDC), which inhibit the activity of human and murine urokinase-type plasminogen activators (huPA and muPA) with Ki values in the micromolar or sub-micromolar range, respectively. The following affinity maturations significantly enhanced the potencies of the two peptides, 10-fold and >250-fold for upain-1 and mupain-1, respectively. The most potent muPA inhibitor has a potency (Ki = 2 nM) and specificity comparable to mono-clonal antibodies. Furthermore, we also found an unusual feature of mupain-1 that its inhibitory potency can be enhanced by increasing the flexibility, which challenges the traditional viewpoint that higher rigidity leading to higher affinity. Moreover, by changing a few key residues, we converted mupain-1 from a uPA inhibitor to inhibitors of other serine proteases, including plasma kallikrein (PK) and coagulation factor XIa (fXIa). PK and fXIa inhibitors showed Ki values in the low nanomolar range and high specificity. Our studies demonstrate the versatility of small cyclic peptides to engineer inhibitory potency against serine proteases and to provide a new strategy for generating peptide inhibitors of serine proteases. PMID:29104489

Cortical Astrocytes Acutely Exposed to the Monomethylarsonous Acid (MMAIII) Show Increased Pro-inflammatory Cytokines Gene Expression that is Consistent with APP and BACE-1: Over-expression.

Science.gov (United States)

Escudero-Lourdes, C; Uresti-Rivera, E E; Oliva-González, C; Torres-Ramos, M A; Aguirre-Bañuelos, P; Gandolfi, A J

2016-10-01

Long-term exposure to inorganic arsenic (iAs) through drinking water has been associated with cognitive impairment in children and adults; however, the related pathogenic mechanisms have not been completely described. Increased or chronic inflammation in the brain is linked to impaired cognition and neurodegeneration; iAs induces strong inflammatory responses in several cells, but this effect has been poorly evaluated in central nervous system (CNS) cells. Because astrocytes are the most abundant cells in the CNS and play a critical role in brain homeostasis, including regulation of the inflammatory response, any functional impairment in them can be deleterious for the brain. We propose that iAs could induce cognitive impairment through inflammatory response activation in astrocytes. In the present work, rat cortical astrocytes were acutely exposed in vitro to the monomethylated metabolite of iAs (MMA III ), which accumulates in glial cells without compromising cell viability. MMA III LD 50 in astrocytes was 10.52 μM, however, exposure to sub-toxic MMA III concentrations (50-1000 nM) significantly increased IL-1β, IL-6, TNF-α, COX-2, and MIF-1 gene expression. These effects were consistent with amyloid precursor protein (APP) and β-secretase (BACE-1) increased gene expression, mainly for those MMA III concentrations that also induced TNF-α over-expression. Other effects of MMA III on cortical astrocytes included increased proliferative and metabolic activity. All tested MMA III concentrations led to an inhibition of intracellular lactate dehydrogenase (LDH) activity. Results suggest that MMA III induces important metabolic and functional changes in astrocytes that may affect brain homeostasis and that inflammation may play a major role in cognitive impairment-related pathogenicity in As-exposed populations.

Squash inhibitor family of serine proteinases

International Nuclear Information System (INIS)

Otlewski, J.; Krowarsch, D.

1996-01-01

Squash inhibitors of serine proteinases form an uniform family of small proteins. They are built of 27-33 amino-acid residues and cross-linked with three disulfide bridges. The reactive site peptide bond (P1-P1') is between residue 5 (Lys, Arg or Leu) and 6 (always Ile). High resolution X-ray structures are available for two squash inhibitors complexed with trypsin. NMR solution structures have also been determined for free inhibitors. The major structural motif is a distorted, triple-stranded antiparallel beta-sheet. A similar folding motif has been recently found in a number of proteins, including: conotoxins from fish-hunting snails, carboxypeptidase inhibitor from potato, kalata B1 polypeptide, and in some growth factors (e.g. nerve growth factor, transforming growth factor β2, platelet-derived growth factor). Squash inhibitors are highly stable and rigid proteins. They inhibit a number of serine proteinases: trypsin, plasmin, kallikrein, blood clotting factors: X a and XII a , cathepsin G. The inhibition spectrum can be much broadened if specific amino-acid substitutions are introduced, especially at residues which contact proteinase. Squash inhibitors inhibit proteinases via the standard mechanism. According to the mechanism, inhibitors are substrates which exhibit at neutral pH a high k cat /K m index for hydrolysis and resynthesis of the reactive site, and a low value of the hydrolysis constant. (author)

DISTRIBUTION AND ELIMINATION OF THE GLYCOSIDASE INHIBITORS 1-DEOXYMANNOJIRIMYCIN AND N-METHYL-1-DEOXYNOJIRIMYCIN IN THE RAT INVIVO

NARCIS (Netherlands)

FABER, ED; NEEFJES, JJ; PLOEGH, HL; MEIJER, DKF

1992-01-01

We studied the pharmacokinetics of two synthetic derivatives of 1-deoxynojirimycin in the rat after intravenous administration. The mannosidase IA/B inhibitor 1-deoxymannojirimycin and the glucosidase inhibitor N-methyl-1-deoxynojirimycin exhibited minimal plasma protein binding and showed a rapid

Acidic tumor microenvironment abrogates the efficacy of mTORC1 inhibitors.

Science.gov (United States)

Faes, Seraina; Duval, Adrian P; Planche, Anne; Uldry, Emilie; Santoro, Tania; Pythoud, Catherine; Stehle, Jean-Christophe; Horlbeck, Janine; Letovanec, Igor; Riggi, Nicolo; Demartines, Nicolas; Dormond, Olivier

2016-12-05

Blocking the mechanistic target of rapamycin complex-1 (mTORC1) with chemical inhibitors such as rapamycin has shown limited clinical efficacy in cancer. The tumor microenvironment is characterized by an acidic pH which interferes with cancer therapies. The consequences of acidity on the anti-cancer efficacy of mTORC1 inhibitors have not been characterized and are thus the focus of our study. Cancer cell lines were treated with rapamycin in acidic or physiological conditions and cell proliferation was investigated. The effect of acidity on mTORC1 activity was determined by Western blot. The anticancer efficacy of rapamycin in combination with sodium bicarbonate to increase the intratumoral pH was tested in two different mouse models and compared to rapamycin treatment alone. Histological analysis was performed on tumor samples to evaluate proliferation, apoptosis and necrosis. Exposing cancer cells to acidic pH in vitro significantly reduced the anti-proliferative effect of rapamycin. At the molecular level, acidity significantly decreased mTORC1 activity, suggesting that cancer cell proliferation is independent of mTORC1 in acidic conditions. In contrast, the activation of mitogen-activated protein kinase (MAPK) or AKT were not affected by acidity, and blocking MAPK or AKT with a chemical inhibitor maintained an anti-proliferative effect at low pH. In tumor mouse models, the use of sodium bicarbonate increased mTORC1 activity in cancer cells and potentiated the anti-cancer efficacy of rapamycin. Combining sodium bicarbonate with rapamycin resulted in increased tumor necrosis, increased cancer cell apoptosis and decreased cancer cell proliferation as compared to single treatment. Taken together, these results emphasize the inefficacy of mTORC1 inhibitors in acidic conditions. They further highlight the potential of combining sodium bicarbonate with mTORC1 inhibitors to improve their anti-tumoral efficacy.

Structures of human Golgi-resident glutaminyl cyclase and its complexes with inhibitors reveal a large loop movement upon inhibitor binding.

Science.gov (United States)

Huang, Kai-Fa; Liaw, Su-Sen; Huang, Wei-Lin; Chia, Cho-Yun; Lo, Yan-Chung; Chen, Yi-Ling; Wang, Andrew H-J

2011-04-08

Aberrant pyroglutamate formation at the N terminus of certain peptides and proteins, catalyzed by glutaminyl cyclases (QCs), is linked to some pathological conditions, such as Alzheimer disease. Recently, a glutaminyl cyclase (QC) inhibitor, PBD150, was shown to be able to reduce the deposition of pyroglutamate-modified amyloid-β peptides in brain of transgenic mouse models of Alzheimer disease, leading to a significant improvement of learning and memory in those transgenic animals. Here, we report the 1.05-1.40 Å resolution structures, solved by the sulfur single-wavelength anomalous dispersion phasing method, of the Golgi-luminal catalytic domain of the recently identified Golgi-resident QC (gQC) and its complex with PBD150. We also describe the high-resolution structures of secretory QC (sQC)-PBD150 complex and two other gQC-inhibitor complexes. gQC structure has a scaffold similar to that of sQC but with a relatively wider and negatively charged active site, suggesting a distinct substrate specificity from sQC. Upon binding to PBD150, a large loop movement in gQC allows the inhibitor to be tightly held in its active site primarily by hydrophobic interactions. Further comparisons of the inhibitor-bound structures revealed distinct interactions of the inhibitors with gQC and sQC, which are consistent with the results from our inhibitor assays reported here. Because gQC and sQC may play different biological roles in vivo, the different inhibitor binding modes allow the design of specific inhibitors toward gQC and sQC.

Multimerized CHR-derived peptides as HIV-1 fusion inhibitors.

Science.gov (United States)

Nomura, Wataru; Hashimoto, Chie; Suzuki, Takaharu; Ohashi, Nami; Fujino, Masayuki; Murakami, Tsutomu; Yamamoto, Naoki; Tamamura, Hirokazu

2013-08-01

To date, several HIV-1 fusion inhibitors based on the carboxy-terminal leucine/isoleucine heptad repeat (CHR) region of an HIV-1 envelope protein gp41 have been discovered. We have shown that a synthetic peptide mimetic of a trimer form of the CHR-derived peptide C34 has potent inhibitory activity against the HIV-1 fusion mechanism, compared to a monomer C34 peptide. The present study revealed that a dimeric form of C34 is evidently structurally critical for fusion inhibitors, and that the activity of multimerized CHR-derived peptides in fusion inhibition is affected by the properties of the unit peptides C34, SC34EK, and T20. The fluorescence-based study suggested that the N36-interactive sites of the C34 trimer, including hydrophobic residues, are exposed outside the trimer and that trimerization of C34 caused a remarkable increase in fusion inhibitory activity. The present results could be useful in the design of fusion inhibitors against viral infections which proceed via membrane fusion with host cells. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

Cell-Specific Establishment of Poliovirus Resistance to an Inhibitor Targeting a Cellular Protein

Science.gov (United States)

Viktorova, Ekaterina G.; Nchoutmboube, Jules; Ford-Siltz, Lauren A.

2015-01-01

ABSTRACT It is hypothesized that targeting stable cellular factors involved in viral replication instead of virus-specific proteins may raise the barrier for development of resistant mutants, which is especially important for highly adaptable small (+)RNA viruses. However, contrary to this assumption, the accumulated evidence shows that these viruses easily generate mutants resistant to the inhibitors of cellular proteins at least in some systems. We investigated here the development of poliovirus resistance to brefeldin A (BFA), an inhibitor of the cellular protein GBF1, a guanine nucleotide exchange factor for the small cellular GTPase Arf1. We found that while resistant viruses can be easily selected in HeLa cells, they do not emerge in Vero cells, in spite that in the absence of the drug both cultures support robust virus replication. Our data show that the viral replication is much more resilient to BFA than functioning of the cellular secretory pathway, suggesting that the role of GBF1 in the viral replication is independent of its Arf activating function. We demonstrate that the level of recruitment of GBF1 to the replication complexes limits the establishment and expression of a BFA resistance phenotype in both HeLa and Vero cells. Moreover, the BFA resistance phenotype of poliovirus mutants is also cell type dependent in different cells of human origin and results in a fitness loss in the form of reduced efficiency of RNA replication in the absence of the drug. Thus, a rational approach to the development of host-targeting antivirals may overcome the superior adaptability of (+)RNA viruses. IMPORTANCE Compared to the number of viral diseases, the number of available vaccines is miniscule. For some viruses vaccine development has not been successful after multiple attempts, and for many others vaccination is not a viable option. Antiviral drugs are needed for clinical practice and public health emergencies. However, viruses are highly adaptable and can

Human recombinant beta-secretase immobilized enzyme reactor for fast hits' selection and characterization from a virtual screening library.

Science.gov (United States)

De Simone, Angela; Mancini, Francesca; Cosconati, Sandro; Marinelli, Luciana; La Pietra, Valeria; Novellino, Ettore; Andrisano, Vincenza

2013-01-25

In the present work, a human recombinant BACE1 immobilized enzyme reactor (hrBACE1-IMER) has been applied for the sensitive fast screening of 38 compounds selected through a virtual screening approach. HrBACE1-IMER was inserted into a liquid chromatograph coupled with a fluorescent detector. A fluorogenic peptide substrate (M-2420), containing the β-secretase site of the Swedish mutation of APP, was injected and cleaved in the on-line HPLC-hrBACE1-IMER system, giving rise to the fluorescent product. The compounds of the library were tested for their ability to inhibit BACE1 in the immobilized format and to reduce the area related to the chromatographic peak of the fluorescent enzymatic product. The results were validated in solution by using two different FRET methods. Due to the efficient virtual screening methodology, more than fifty percent of the selected compounds showed a measurable inhibitory activity. One of the most active compound (a bis-indanone derivative) was characterized in terms of IC(50) and K(i) determination on the hrBACE1-IMER. Thus, the hrBACE1-IMER has been confirmed as a valid tool for the throughput screening of different chemical entities with potency lower than 30μM for the fast hits' selection and for mode of action determination. Copyright © 2012 Elsevier B.V. All rights reserved.

Tissue inhibitor of metalloproteinase-2 (TIMP-2) regulates myogenesis and β1 integrin expression in vitro

International Nuclear Information System (INIS)

Lluri, Gentian; Langlois, Garret D.; Soloway, Paul D.; Jaworski, Diane M.

2008-01-01

Myogenesis in vitro involves myoblast cell cycle arrest, migration, and fusion to form multinucleated myotubes. Extracellular matrix (ECM) integrity during these processes is maintained by the opposing actions of matrix metalloproteinase (MMP) proteases and their inhibitors, the tissue inhibitor of metalloproteinases (TIMPs). Here, we report that TIMP-2, MMP-2, and MT1-MMP are differentially expressed during mouse myoblast differentiation in vitro. A specific role for TIMP-2 in myogenesis is demonstrated by altered TIMP-2 -/- myotube formation. When differentiated in horse serum-containing medium, TIMP-2 -/- myotubes are larger than wild-type myotubes. In contrast, when serum-free medium is used, TIMP-2 -/- myotubes are smaller than wild-type myotubes. Regardless of culture condition, myotube size is directly correlated with MMP activity and inversely correlated with β1 integrin expression. Treatment with recombinant TIMP-2 rescues reduced TIMP-2 -/- myotube size and induces increased MMP-9 activation and decreased β1 integrin expression. Treatment with either MMP-2 or MMP-9 similarly rescues reduced myotube size, but has no effect on β1 integrin expression. These data suggest a specific regulatory relationship between TIMP-2 and β1 integrin during myogenesis. Elucidating the role of TIMP-2 in myogenesis in vitro may lead to new therapeutic options for the use of TIMP-2 in myopathies and muscular dystrophies in vivo

Biochemical Importance of Glycosylation of Plasminogen Activator Inhibitor-1

DEFF Research Database (Denmark)

Gils, Ann; Pedersen, Katrine Egelund; Skottrup, Peter Durand

2003-01-01

The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous glycosyla...

Inhibitor discovery of full-length New Delhi metallo-β-lactamase-1 (NDM-1.

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Bingzheng Shen

Full Text Available New Delhi metallo-β-lactmase-1 (NDM-1 has recently attracted extensive attention for its biological activities to catalyze the hydrolysis of almost all of β-lactam antibiotics. To study the catalytic property of NDM-1, the steady-kinetic parameters of NDM-1 toward several kinds of β-lactam antibiotics have been detected. It could effectively hydrolyze most β-lactams (k cat/K m ratios between 0.03 to 1.28 µmol⁻¹.s⁻¹, except aztreonam. We also found that thiophene-carboxylic acid derivatives could inhibit NDM-1 and have shown synergistic antibacterial activity in combination with meropenem. Flexible docking and quantum mechanics (QM study revealed electrostatic interactions between the sulfur atom of thiophene-carboxylic acid derivatives and the zinc ion of NDM-1, along with hydrogen bond between inhibitor and His189 of NDM-1. The interaction models proposed here can be used in rational design of NDM-1 inhibitors.

Plasminogen activator inhibitor-1 is an independent prognostic factor of ovarian cancer and IMD-4482, a novel plasminogen activator inhibitor-1 inhibitor, inhibits ovarian cancer peritoneal dissemination.

Science.gov (United States)

Nakatsuka, Erika; Sawada, Kenjiro; Nakamura, Koji; Yoshimura, Akihito; Kinose, Yasuto; Kodama, Michiko; Hashimoto, Kae; Mabuchi, Seiji; Makino, Hiroshi; Morii, Eiichi; Yamaguchi, Yoichi; Yanase, Takeshi; Itai, Akiko; Morishige, Ken-Ichirou; Kimura, Tadashi

2017-10-27

In the present study, the therapeutic potential of targeting plasminogen activator inhibitor-1 (PAI-1) in ovarian cancer was tested. Tissues samples from 154 cases of ovarian carcinoma were immunostained with anti-PAI-1 antibody, and the prognostic value was analyzed. Among the samples, 67% (104/154) showed strong PAI-1 expression; this was significantly associated with poor prognosis (progression-free survival: 20 vs. 31 months, P = 0.0033). In particular, among patients with stage II-IV serous adenocarcinoma, PAI-1 expression was an independent prognostic factor. The effect of a novel PAI-1 inhibitor, IMD-4482, on ovarian cancer cell lines was assessed and its therapeutic potential was examined using a xenograft mouse model of ovarian cancer. IMD-4482 inhibited in vitro cell adhesion to vitronectin in PAI-1-positive ovarian cancer cells, followed by the inhibition of extracellular signal-regulated kinase and focal adhesion kinase phosphorylation through dissociation of the PAI-urokinase receptor complex from integrin αVβ3. IMD-4482 caused G0/G1 cell arrest and inhibited the proliferation of PAI-1-positive ovarian cancer cells. In the xenograft model, IMD-4482 significantly inhibited peritoneal dissemination with the reduction of PAI-1 expression and the inhibition of focal adhesion kinase phosphorylation. Collectively, the functional inhibition of PAI-1 significantly inhibited ovarian cancer progression, and targeting PAI-1 may be a potential therapeutic strategy in ovarian cancer.

Aldehyde Dehydrogenase 1 and Raf Kinase Inhibitor Protein ...

African Journals Online (AJOL)

Aldehyde Dehydrogenase 1 and Raf Kinase Inhibitor Protein Expression Defines the Proliferative Nature of Cervical Cancer Stem Cells. ... of cervical cancer stem cells and also to validate them in initial and advanced stages of cervical cancer. Keywords: Cervical cancer, ALDH1, BALB/c-nu/nu, HeLa cells, RKIP, Sox2 ...

Mycobacterium tuberculosis, but not vaccine BCG, specifically upregulates matrix metalloproteinase-1.

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Elkington, Paul T G; Nuttall, Robert K; Boyle, Joseph J; O'Kane, Cecilia M; Horncastle, Donna E; Edwards, Dylan R; Friedland, Jon S

2005-12-15

Pulmonary cavitation is fundamental to the global success of Mycobacterium tuberculosis. However, the mechanisms of this lung destruction are poorly understood. The biochemistry of lung matrix predicts matrix metalloproteinase (MMP) involvement in immunopathology. We investigated gene expression of all MMPs, proteins with a disintegrin and metalloproteinase domain, and tissue inhibitors of metalloproteinases in M. tuberculosis-infected human macrophages by real-time polymerase chain reaction. MMP secretion was measured by zymography and Western analysis, and expression in patients with pulmonary tuberculosis was localized by immunohistochemistry. MMP-1 and MMP-7 gene expression and secretion are potently upregulated by M. tuberculosis, and no increase in tissue inhibitor of metalloproteinase expression occurs to oppose their activity. Dexamethasone completely suppresses MMP-1 but not MMP-7 gene expression and secretion. In patients with active tuberculosis, macrophages express MMP-1 and MMP-7 adjacent to areas of tissue destruction. MMP-1 but not MMP-7 expression and secretion are relatively M. tuberculosis specific, are not upregulated by tuberculosis-associated cytokines, and are prostaglandin dependent. In contrast, the vaccine M. bovis bacillus Calmette-Guérin (BCG) does not stimulate MMP-1 secretion from human macrophages, although M. tuberculosis and BCG do upregulate MMP-7 equally. BCG-infected macrophages secrete reduced prostaglandin E2 concentrations compared with M. tuberculosis-infected macrophages, and prostaglandin pathway supplementation augments MMP-1 secretion from BCG-infected cells. M. tuberculosis specifically upregulates MMP-1 in a cellular model of human infection and in patients with tuberculosis. In contrast, vaccine BCG, which does not cause lung cavitation, does not upregulate prostaglandin E2-dependent MMP-1 secretion.

Comparison of Clot-based, Chromogenic, and Fluorescence Assays for Measurement of Factor VIII Inhibitors in the U.S. Hemophilia Inhibitor Research Study

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Miller, Connie H.; Rice, Anne S.; Boylan, Brian; Shapiro, Amy D.; Lentz, Steven R.; Wicklund, Brian M.; Kelly, Fiona M.; Soucie, J. Michael

2015-01-01

Summary Background Detection and validation of inhibitors (antibodies) to hemophilia treatment products are important for clinical care, evaluation of product safety, and assessment of population trends. Methods Centralized monitoring for factor VIII (FVIII) inhibitors was conducted for patients in the Hemophilia Inhibitor Research Study using a previously reported modified Nijmegen-Bethesda clotting assay (NBA), a chromogenic Bethesda assay (CBA), and a novel fluorescence immunoassay (FLI). Results NBA and CBA were performed on 1005 specimens and FLI on 272 specimens. CBA was negative on 880/883 specimens (99.7%) with Nijmegen-Bethesda units (NBU)NBA and negative CBA, 58.1% were FLI-negative, 12.9% had evidence of lupus anticoagulant, and 35.5% had non-time-dependent inhibition. CBA and FLI were positive on 72.4% and 100% of 1.0–1.9 NBU specimens and 43.1% and 50.0% of 0.5–0.9 NBU specimens. FLI detected antibodies in 98.0% of CBA-positive and 81.6% of NBA-positive specimens (P=0.004). Among 21 new inhibitors detected by NBA, 5 (23.8%) with 0.7–1.3 NBU did not react in CBA or FLI. Among previously positive patients with 0.5–1.9 NBU, 7/25 (28%) were not CBA or FLI positive. FLI was positive on 36/169 NBU-negative specimens (21.3%). Conclusions FVIII specificity could not be demonstrated by CBA or FLI for 26% of inhibitors of 0.5–1.9 NBU; such results must be interpreted with caution. Low titer inhibitors detected in clot-based assays should always be repeated, with consideration given to evaluating their reactivity with FVIII using more specific assays. PMID:23601690

Genetics Home Reference: complete plasminogen activator inhibitor 1 deficiency

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... well studied in a large family belonging to the Old Order Amish population of eastern and southern Indiana. Additional cases in North ... Human plasminogen activator inhibitor-1 (PAI-1) deficiency: characterization of a large kindred with a null mutation in the PAI-1 gene. Blood. 1997 Jul 1;90( ...

Molecular dynamics simulation study of PTP1B with allosteric inhibitor and its application in receptor based pharmacophore modeling

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Bharatham, Kavitha; Bharatham, Nagakumar; Kwon, Yong Jung; Lee, Keun Woo

2008-12-01

Allosteric inhibition of protein tyrosine phosphatase 1B (PTP1B), has paved a new path to design specific inhibitors for PTP1B, which is an important drug target for the treatment of type II diabetes and obesity. The PTP1B1-282-allosteric inhibitor complex crystal structure lacks α7 (287-298) and moreover there is no available 3D structure of PTP1B1-298 in open form. As the interaction between α7 and α6-α3 helices plays a crucial role in allosteric inhibition, α7 was modeled to the PTP1B1-282 in open form complexed with an allosteric inhibitor (compound-2) and a 5 ns MD simulation was performed to investigate the relative orientation of the α7-α6-α3 helices. The simulation conformational space was statistically sampled by clustering analyses. This approach was helpful to reveal certain clues on PTP1B allosteric inhibition. The simulation was also utilized in the generation of receptor based pharmacophore models to include the conformational flexibility of the protein-inhibitor complex. Three cluster representative structures of the highly populated clusters were selected for pharmacophore model generation. The three pharmacophore models were subsequently utilized for screening databases to retrieve molecules containing the features that complement the allosteric site. The retrieved hits were filtered based on certain drug-like properties and molecular docking simulations were performed in two different conformations of protein. Thus, performing MD simulation with α7 to investigate the changes at the allosteric site, then developing receptor based pharmacophore models and finally docking the retrieved hits into two distinct conformations will be a reliable methodology in identifying PTP1B allosteric inhibitors.

Wheat Subtilisin/Chymotrypsin Inhibitor (WSCI) as a scaffold for novel serine protease inhibitors with a given specificity.

Science.gov (United States)

Tedeschi, Francesca; Di Maro, Antimo; Facchiano, Angelo; Costantini, Susan; Chambery, Angela; Bruni, Natalia; Capuzzi, Valeria; Ficca, Anna Grazia; Poerio, Elia

2012-10-30

WSCI (Wheat Subtilisin/Chymotrypsin Inhibitor) is a small protein belonging to the Potato inhibitor I family exhibiting a high content of essential amino acid. In addition to bacterial subtilisins and mammalian chymotrypsins, WSCI inhibits chymotrypsin-like activities isolated from digestive traits of a number of insect larvae. In vivo, as suggested for many plant proteinase inhibitors, WSCI seems to play a role of natural defence against attacks of pests and pathogens. The functional region of WSCI, containing the inhibitor reactive site (Met48-Glu49), corresponds to an extended flexible loop (Val42-Asp53) whose architecture is somehow stabilized by a number of secondary interactions established with a small β-sheet located underneath. The aim of this study was to employ a WSCI molecule as a stable scaffold to obtain recombinant inhibitors with new acquired anti-proteinase activity or, alternatively, inactive WSCI variants. A gene sequence coding for the native WSCI, along with genes coding for muteins with different specficities, could be exploited to obtain transformed non-food use plants with improved insect resistance. On the other hand, the genetic transformation of cereal plants over-expressing inactive WSCI muteins could represent a possible strategy to improve the nutritional quality of cereal-based foods, without risk of interference with human or animal digestive enzymes. Here, we described the characterization of four muteins containing single/multiple amino acid substitutions at the WSCI reactive site and/or at its proximity. Modalities of interaction of these muteins with proteinases (subtilisin, trypsin and chymotrypsin) were investigated by time course hydrolysis and molecular simulations studies.

A cyclic peptidic serine protease inhibitor

DEFF Research Database (Denmark)

Zhao, Baoyu; Xu, Peng; Jiang, Longguang

2014-01-01

Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase...... pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending......, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity...

Changes in glucose-induced plasma active glucagon-like peptide-1 levels by co-administration of sodium–glucose cotransporter inhibitors with dipeptidyl peptidase-4 inhibitors in rodents

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Takahiro Oguma

2016-12-01

Full Text Available We investigated whether structurally different sodium–glucose cotransporter (SGLT 2 inhibitors, when co-administered with dipeptidyl peptidase-4 (DPP4 inhibitors, could enhance glucagon-like peptide-1 (GLP-1 secretion during oral glucose tolerance tests (OGTTs in rodents. Three different SGLT inhibitors—1-(β-d-Glucopyranosyl-4-chloro-3-[5-(6-fluoro-2-pyridyl-2-thienylmethyl]benzene (GTB, TA-1887, and canagliflozin—were examined to assess the effect of chemical structure. Oral treatment with GTB plus a DPP4 inhibitor enhanced glucose-induced plasma active GLP-1 (aGLP-1 elevation and suppressed glucose excursions in both normal and diabetic rodents. In DPP4-deficient rats, GTB enhanced glucose-induced aGLP-1 elevation without affecting the basal level, whereas metformin, previously reported to enhance GLP-1 secretion, increased both the basal level and glucose-induced elevation. Oral treatment with canagliflozin and TA-1887 also enhanced glucose-induced aGLP-1 elevation when co-administered with either teneligliptin or sitagliptin. These data suggest that structurally different SGLT2 inhibitors enhance plasma aGLP-1 elevation and suppress glucose excursions during OGTT when co-administered with DPP4 inhibitors, regardless of the difference in chemical structure. Combination treatment with DPP4 inhibitors and SGLT2 inhibitors having moderate SGLT1 inhibitory activity may be a promising therapeutic option for improving glycemic control in patients with type 2 diabetes mellitus.

Imidazopyridine- and purine-thioacetamide derivatives: potent inhibitors of nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1).

Science.gov (United States)

Chang, Lei; Lee, Sang-Yong; Leonczak, Piotr; Rozenski, Jef; De Jonghe, Steven; Hanck, Theodor; Müller, Christa E; Herdewijn, Piet

2014-12-11

Nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) belongs to the family of ecto-nucleotidases, which control extracellular nucleotide, nucleoside, and (di)phosphate levels. To study the (patho)physiological roles of NPP1 potent and selective inhibitors with drug-like properties are required. Therefore, a compound library was screened for NPP1 inhibitors using a colorimetric assay with p-nitrophenyl 5'-thymidine monophosphate (p-Nph-5'-TMP) as an artificial substrate. This led to the discovery of 2-(3H-imidazo[4,5-b]pyridin-2-ylthio)-N-(3,4-dimethoxyphenyl)acetamide (5a) as a hit compound with a Ki value of 217 nM. Subsequent structure-activity relationship studies led to the development of purine and imidazo[4,5-b]pyridine analogues with high inhibitory potency (Ki values of 5.00 nM and 29.6 nM, respectively) when assayed with p-Nph-5'-TMP as a substrate. Surprisingly, the compounds were significantly less potent when tested versus ATP as a substrate, with Ki values in the low micromolar range. A prototypic inhibitor was investigated for its mechanism of inhibition and found to be competitive versus both substrates.

High-throughput screening identifies Ceefourin 1 and Ceefourin 2 as highly selective inhibitors of multidrug resistance protein 4 (MRP4).

Science.gov (United States)

Cheung, Leanna; Flemming, Claudia L; Watt, Fujiko; Masada, Nanako; Yu, Denise M T; Huynh, Tony; Conseil, Gwenaëlle; Tivnan, Amanda; Polinsky, Alexander; Gudkov, Andrei V; Munoz, Marcia A; Vishvanath, Anasuya; Cooper, Dermot M F; Henderson, Michelle J; Cole, Susan P C; Fletcher, Jamie I; Haber, Michelle; Norris, Murray D

2014-09-01

Multidrug resistance protein 4 (MRP4/ABCC4), a member of the ATP-binding cassette (ABC) transporter superfamily, is an organic anion transporter capable of effluxing a wide range of physiologically important signalling molecules and drugs. MRP4 has been proposed to contribute to numerous functions in both health and disease; however, in most cases these links remain to be unequivocally established. A major limitation to understanding the physiological and pharmacological roles of MRP4 has been the absence of specific small molecule inhibitors, with the majority of established inhibitors also targeting other ABC transporter family members, or inhibiting the production, function or degradation of important MRP4 substrates. We therefore set out to identify more selective and well tolerated inhibitors of MRP4 that might be used to study the many proposed functions of this transporter. Using high-throughput screening, we identified two chemically distinct small molecules, Ceefourin 1 and Ceefourin 2, that inhibit transport of a broad range of MRP4 substrates, yet are highly selective for MRP4 over other ABC transporters, including P-glycoprotein (P-gp), ABCG2 (Breast Cancer Resistance Protein; BCRP) and MRP1 (multidrug resistance protein 1; ABCC1). Both compounds are more potent MRP4 inhibitors in cellular assays than the most widely used inhibitor, MK-571, requiring lower concentrations to effect a comparable level of inhibition. Furthermore, Ceefourin 1 and Ceefourin 2 have low cellular toxicity, and high microsomal and acid stability. These newly identified inhibitors should be of great value for efforts to better understand the biological roles of MRP4, and may represent classes of compounds with therapeutic application. Copyright © 2014 Elsevier Inc. All rights reserved.

Discovery of Selective Phosphodiesterase 1 Inhibitors with Memory Enhancing Properties.

Science.gov (United States)

Dyck, Brian; Branstetter, Bryan; Gharbaoui, Tawfik; Hudson, Andrew R; Breitenbucher, J Guy; Gomez, Laurent; Botrous, Iriny; Marrone, Tami; Barido, Richard; Allerston, Charles K; Cedervall, E Peder; Xu, Rui; Sridhar, Vandana; Barker, Ryan; Aertgeerts, Kathleen; Schmelzer, Kara; Neul, David; Lee, Dong; Massari, Mark Eben; Andersen, Carsten B; Sebring, Kristen; Zhou, Xianbo; Petroski, Robert; Limberis, James; Augustin, Martin; Chun, Lawrence E; Edwards, Thomas E; Peters, Marco; Tabatabaei, Ali

2017-04-27

A series of potent thienotriazolopyrimidinone-based PDE1 inhibitors was discovered. X-ray crystal structures of example compounds from this series in complex with the catalytic domain of PDE1B and PDE10A were determined, allowing optimization of PDE1B potency and PDE selectivity. Reduction of hERG affinity led to greater than a 3000-fold selectivity for PDE1B over hERG. 6-(4-Methoxybenzyl)-9-((tetrahydro-2H-pyran-4-yl)methyl)-8,9,10,11-tetrahydropyrido[4',3':4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-5(6H)-one was identified as an orally bioavailable and brain penetrating PDE1B enzyme inhibitor with potent memory-enhancing effects in a rat model of object recognition memory.

Elucidating the Mechanism of Gain of Toxic Function From Mutant C1 Inhibitor Proteins in Hereditary Angioedema

Science.gov (United States)

2017-10-01

antibodies to 5 specifically blot wild-type C1INH in the pathologic polymers.. A FLAG tag was placed into the wild-type C1INH cDNA located immediately...resulted in decreased secretion of the 3x-FLAG-WT-C1INH when cotransfected with the mutant cDNA . This was an important confirmation of our...C1INH plus mutant C1INH cDNA in the presence or absence of a lactacystin, a proteasome inhibitor. As shown in figure 2, blocking degradation of

Effects and Mechanisms of Checkpoint Inhibitors (CTLA-4, PD-1 and PD-L1 Inhibitors as New Immunotherapeutic Agents for Bladder Cancer

Directory of Open Access Journals (Sweden)

Serdar Çelik

2018-04-01

Full Text Available Since intravesical Bacillus Calmette-Guerin (BCG began to be used for bladder cancer, our understanding of the importance of immune mechanisms in bladder cancer has steadily grown. With developments in immunotherapy in recent years, the use of new immunotherapeutic agents for bladder cancer, especially chemotherapy-resistant invasive and metastatic cancers, has opened the way for research in this area. Of these new therapeutic agents, this article reviews studies published on PubMed or listed on the ClinicalTrials.gov website as of December 2017 regarding the effects and mechanisms of action of checkpoint inhibitors [cytotoxic t-lymphocyte associated protein-4, programmed cell death 1 receptor (PD-1 and PD-1 ligand inhibitors] on bladder cancer. Because checkpoint inhibitors were first used for chemotherapy-resistant bladder cancer after identification of positive expression in tumor cells and especially in tumor-infiltrating mononuclear cells, significant objective response rates and survival advantages have been reported. Research continues regarding the use of these agents as first- and second-line treatment for metastatic disease in combination with chemotherapy; their efficacy in neoadjuvant, adjuvant, and bladder-preserving approaches to muscle-invasive bladder cancer (MIBC disease, and their use in non-muscle-invasize bladder cancer (NMIBC, especially BCG-refractory disease. Depending on the results of these ongoing studies, immunotherapy may direct the treatment of bladder cancer in the future.

Multicenter Evaluation of the Tolerability of Combined Treatment With PD-1 and CTLA-4 Immune Checkpoint Inhibitors and Palliative Radiation Therapy

Energy Technology Data Exchange (ETDEWEB)

Bang, Andrew [Department of Radiation Oncology, Brigham and Women' s Hospital/Dana-Farber Cancer Institute, Boston, Massachusetts (United States); Division of Radiation Oncology, University of Ottawa, Ottawa, Ontario (Canada); Wilhite, Tyler J. [Harvard Medical School, Boston, Massachusetts (United States); Pike, Luke R.G. [Harvard Radiation Oncology Program, Brigham and Women' s Hospital/Dana-Farber Cancer Institute, Boston, Massachusetts (United States); Cagney, Daniel N.; Aizer, Ayal A.; Taylor, Allison; Spektor, Alexander; Krishnan, Monica [Department of Radiation Oncology, Brigham and Women' s Hospital/Dana-Farber Cancer Institute, Boston, Massachusetts (United States); Ott, Patrick A. [Department of Medical Oncology and Center for Immuno-Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts (United States); Balboni, Tracy A. [Department of Radiation Oncology, Brigham and Women' s Hospital/Dana-Farber Cancer Institute, Boston, Massachusetts (United States); Hodi, F. Stephen [Department of Medical Oncology and Center for Immuno-Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts (United States); Schoenfeld, Jonathan D., E-mail: jdschoenfeld@partners.org [Department of Radiation Oncology, Brigham and Women' s Hospital/Dana-Farber Cancer Institute, Boston, Massachusetts (United States)

2017-06-01