In Vitro and In Vivo Inhibition of the 2 Active Sites of ACE by Omapatrilat, a Vasopeptidase Inhibitor

Background: ,2,3-Triazoles and imidazoles are important five-membered heterocyclic scaffolds due to their extensive biological activities. These products have been an area of growing interest to many researchers around the world because of their enormous pharmaceutical scope. Methods: The in vivo and in vitro enzyme inhibition of some thioglycosides encompassing 1,2,4- triazole N1, N2, and N3 and/or imidazole moieties N4, N5, and N6. The effect on the antioxidant enzymes (superoxide dismutase, glutathione S-transferase, glutathione peroxidase and catalase) was investigated as well as their effect on α-glucosidase and β-glucuronidase. Molecular docking studies were carried out to investigate the mode of the binding interaction of the compounds with α- glucosidase and β -glucuronidase. In addition, quantitative structure-activity relationship (QSAR) investigation was applied to find out the correlation between toxicity and physicochemical properties. Results: The decrease of the antioxidant status was revealed by the in vivo effect of the tested compounds. Furthermore, the in vivo and in vitro inhibitory effects of the tested compounds were clearly pronounced on α-glucosidase, but not β-glucuronidase. The IC50 and Ki values revealed that the thioglycoside - based 1,2,4-triazole N3 possesses a high inhibitory action. In addition, the in vitro studies demonstrated that the whole tested 1,2,4-triazole are potent inhibitors with a Ki magnitude of 10-6 and exhibited a competitive type inhibition. On the other hand, the thioglycosides - based imidazole ring showed an antioxidant activity and exerted a slight in vivo stimulation of α-glucosidase and β- glucuronidase. Molecular docking proved that the compounds exhibited binding affinity with the active sites of α -glucosidase and β-glucuronidase (docking score ranged from -2.320 to -4.370 kcal/mol). Furthermore, QSAR study revealed that the HBD and RB were found to have an overall significant correlation with the toxicity. Conclusion: These data suggest that the inhibition of α-glucosidase is accompanied by an oxidative stress action.

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TrioGEF1 controls Rac- and Cdc42-dependent cell structures through the direct activation of rhoG

Journal of Cell Science ◽

10.1242/jcs.113.4.729 ◽

2000 ◽

Vol 113 (4) ◽

pp. 729-739 ◽

Cited By ~ 15

Author(s):

A. Blangy ◽

E. Vignal ◽

S. Schmidt ◽

A. Debant ◽

C. Gauthier-Rouviere ◽

...

Keyword(s):

Rho Gtpases ◽

Active Sites ◽

Dominant Negative ◽

Signaling Cascade ◽

Microtubule Network ◽

Cell Structures ◽

Dependent Cell

Rho GTPases regulate the morphology of cells stimulated by extracellular ligands. Their activation is controlled by guanine exchange factors (GEF) that catalyze their binding to GTP. The multidomain Trio protein represents an emerging class of Ρ regulators that contain two GEF domains of distinct specificities. We report here the characterization of Rho signaling pathways activated by the N-terminal GEF domain of Trio (TrioD1). In fibroblasts, TrioD1 triggers the formation of particular cell structures, similar to those elicited by RhoG, a GTPase known to activate both Rac1 and Cdc42Hs. In addition, the activity of TrioD1 requires the microtubule network and relocalizes RhoG at the active sites of the plasma membrane. Using a classical in vitro exchange assay, TrioD1 displays a higher GEF activity on RhoG than on Rac1. In fibroblasts, expression of dominant negative RhoG mutants totally abolished TrioD1 signaling, whereas dominant negative Rac1 and Cdc42Hs only led to partial and complementary inhibitions. Finally, expression of a Rho Binding Domain that specifically binds RhoG(GTP) led to the complete abolition of TrioD1 signaling, which strongly supports Rac1 not being activated by TrioD1 in vivo. These data demonstrate that Trio controls a signaling cascade that activates RhoG, which in turn activates Rac1 and Cdc42Hs.

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Discovery of a dual inhibitor of NQO1 and GSTP1 for treating glioblastoma

Journal of Hematology & Oncology ◽

10.1186/s13045-020-00979-y ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Kecheng Lei ◽

Xiaoxia Gu ◽

Alvaro G. Alvarado ◽

Yuhong Du ◽

Shilin Luo ◽

...

Keyword(s):

Oxidative Stress ◽

Crystal Structures ◽

Active Sites ◽

Redox Homeostasis ◽

Growth Factor Receptor ◽

Quinone Oxidoreductase ◽

Cancer Proliferation ◽

Dual Inhibitor

Abstract Background Glioblastoma (GBM) is a universally lethal tumor with frequently overexpressed or mutated epidermal growth factor receptor (EGFR). NADPH quinone oxidoreductase 1 (NQO1) and glutathione-S-transferase Pi 1 (GSTP1) are commonly upregulated in GBM. NQO1 and GSTP1 decrease the formation of reactive oxygen species (ROS), which mediates the oxidative stress and promotes GBM cell proliferation. Methods High-throughput screen was used for agents selectively active against GBM cells with EGFRvIII mutations. Co-crystal structures were revealed molecular details of target recognition. Pharmacological and gene knockdown/overexpression approaches were used to investigate the oxidative stress in vitro and in vivo. Results We identified a small molecular inhibitor, “MNPC,” that binds to both NQO1 and GSTP1 with high affinity and selectivity. MNPC inhibits NQO1 and GSTP1 enzymes and induces apoptosis in GBM, specifically inhibiting the growth of cell lines and primary GBM bearing the EGFRvIII mutation. Co-crystal structures between MNPC and NQO1, and molecular docking of MNPC with GSTP1 reveal that it binds the active sites and acts as a potent dual inhibitor. Inactivation of both NQO1 and GSTP1 with siRNA or MNPC results in imbalanced redox homeostasis, leading to apoptosis and mitigated cancer proliferation in vitro and in vivo. Conclusions Thus, MNPC, a dual inhibitor for both NQO1 and GSTP1, provides a novel lead compound for treating GBM via the exploitation of specific vulnerabilities created by mutant EGFR.

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Functional properties of the two redox-active sites in yeast protein disulphide isomerase in Vitro and in Vivo 1 1Edited by G. Von Heijne

Journal of Molecular Biology ◽

10.1006/jmbi.1999.2560 ◽

1999 ◽

Vol 286 (4) ◽

pp. 1229-1239 ◽

Cited By ~ 25

Author(s):

Vibeke Westphal ◽

Nigel J Darby ◽

Jakob R Winther

Keyword(s):

Functional Properties ◽

Active Sites ◽

Yeast Protein ◽

Protein Disulphide Isomerase ◽

Redox Active

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A Proteasome Specific Binding Assay for Quantitation of Constitutive and Immunoproteasome Active Sites.

Blood ◽

10.1182/blood.v106.11.2472.2472 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2472-2472

Author(s):

Mark K. Bennett ◽

Monette A. Aujay ◽

Tonia J. Buchholz ◽

Susan D. Demo ◽

Guy J. Laidig ◽

...

Keyword(s):

Active Sites ◽

Antigen Processing ◽

Specific Binding ◽

Peripheral Blood Mononuclear ◽

Peptide Substrates ◽

Enzyme Preparations ◽

Proteolytic Activities ◽

Fluorogenic Peptide

Abstract The ubiquitin-proteasome pathway constitutes a major intracellular system for protein degradation. Substrates for this pathway include misfolded or unassembled proteins as well as short-lived regulatory proteins that play key roles in signaling and proliferative pathways. The majority of cell types express the standard, or “constitutive”, form of the proteasome, while cells of the immune system also express the immunoproteasome, a form of the proteasome that contributes to class I major histocompatibility complex restricted antigen processing. Non-immune cells can also express immunoproteasome in response to interferon gamma exposure. The immunoproteasome retains the same structural subunits as the constitutive proteasome but has three different catalytic subunits. The catalytic activities of both forms of the proteasome have been traditionally characterized with purified enzyme preparations and fluorogenic peptide substrates. Such fluorogenic peptide substrates suffer from two characteristics that limit their utility in measuring proteasome activities in complex cell or tissue lysates: 1) they cannot distinguish proteasome activities from other proteolytic activities within the lysate; and 2) they can not distinguish between constitutive and immunoproteasome activities. We have developed an ELISA-based proteasome-specific binding (PSB) assay that can detect and quantify the chymotryptic-like proteasome active sites of the beta-5 constitutive proteasome subunit and the LMP7 immunoproteasome subunit. The assay utilizes a biotin-modified peptide epoxyketone probe that covalently and irreversibly interacts with the active site threonine present in catalytic proteasome subunits. Once bound to the probe, the labeled subunits are recovered on streptavidin-conjugated beads and detected with subunit-specific antibodies. The PSB assay is both quantitative and sensitive. We have demonstrated that the assay is capable of measuring constitutive proteasome and immunoproteasome binding activity in human whole blood and peripheral blood mononuclear cell preparations, respectively. In experiments with the epoxyketone-based proteasome inhibitor PR-171, the dose response for inhibition of the PSB assay is equivalent to that measured with a conventional fluorogenic peptide proteasome substrate. In addition, the PSB assay can effectively measure the level of PR-171 mediated inhibition of both the constitutive and immunoproteasome in the RPMI-8226 multiple myeloma cell line that co-expresses both proteasome types. Thus, the PSB assay overcomes the limitations of conventional fluorogenic substrate-based proteasome activity assays when applied to cell or tissue lysates that contain multiple proteolytic activities or mixtures of constitutive and immunoproteasomes. Potential applications of the PSB assay include the measurement of the pharmacodynamic response to proteasome inhibitors and the evaluation of constitutive vs. immunoproteasome selectivity of inhibitors both in vitro and in vivo.

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Anti-Hyperglycaemic Evaluation of Buddleia indica Leaves Using In Vitro, In Vivo and In Silico Studies and Its Correlation with the Major Phytoconstituents

Plants ◽

10.3390/plants10112351 ◽

2021 ◽

Vol 10 (11) ◽

pp. 2351

Author(s):

Sameh S. Elhady ◽

Fadia S. Youssef ◽

Abdulrahman M. Alahdal ◽

Diena M. Almasri ◽

Mohamed L. Ashour

Keyword(s):

Active Sites ◽

Biological Activities ◽

Serum Insulin ◽

Ethyl Acetate Fraction ◽

In Vivo Studies ◽

Stress Markers ◽

In Silico Studies ◽

Streptozotocin Induced Diabetes

Buddleia indica Lam. is an ornamental evergreen shrub with few reports concerning its phytoconstituents and biological activities. Herein, the antihyperglycaemic activity of B. indica leaves methanol extract (BIT) was evaluated for the first time using in vitro and in vivo studies. Molecular modelling was performed for its major phytoconstituents that were further subjected to ADME/TOPAKT (absorption, distribution, metabolism, excretion and toxicity) prediction. BIT revealed considerable reduction in glucose concentration by 9.93% at 50 μg/mL using 3T3-L1 adipocyte culture. It displayed substantial inhibition versus α-glucosidase and α-amylase with IC50 205.2 and 385.06 μg/mL, respectively. In vivo antihyperglycaemic activity of BIT and the ethyl acetate fraction (BIE) was performed using streptozotocin-induced diabetes in rat model. BIT and BIE effectively ameliorate oxidative stress markers in addition to reducing serum blood glucose by 56.08 and 54.00%, respectively, and are associated with a substantial increase in serum insulin by 4.1 and 12.7%, respectively. This can be attributed to its richness with polyphenolic compounds comprising flavonoids, phenolic acids, phenyl propanoids and iridoids. Molecular docking showed that verbascoside and kaempferol displayed the highest fitting within human α-amylase (HA) and human α-glucosidase (HG) active sites, respectively. They showed reasonable pharmacokinetic, pharmacodynamic and toxicity properties, as revealed by ADME/TOPKAT study.

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Structure-based analysis of a natural GOT1-inhibitor Aspulvinone H arrests pancreatic ductal adenocarcinoma cells growth

10.22541/au.161881470.02929068/v1 ◽

2021 ◽

Author(s):

Zhang Yonghui ◽

Shan Yan ◽

Wei Song ◽

Qianqian Xu ◽

Changxing Qi ◽

...

Keyword(s):

Pancreatic Ductal Adenocarcinoma ◽

Anticancer Agents ◽

Active Sites ◽

Redox Homeostasis ◽

Xenograft Model ◽

Glutamine Metabolism ◽

Ductal Adenocarcinoma ◽

Biological Study

Pancreatic ductal adenocarcinoma (PDAC) cells are Gln-metabolism dependence, which can preferentially utilize glutamic oxaloacetate transaminase 1 (GOT1) to maintain the redox homeostasis of cancer cells. Therefore, small molecule inhibitors targeting GOT1 can be used as a new strategy for developing cancer therapies. Here, we identified a cyclobutyrolactone lignan, Aspulvinone H (AH), showing significant GOT1 inhibitory activity in vitro. The complex crystal structure of GOT1-AH elucidated the molecular mechanism, which AH and the cofactor pyrido-aldehyde 5-phosphate (PLP) competitively bound to the active sites of GOT1. Structure-activity relationship (SAR) analysis exhibited that the π-π stacking and isopentenyl side chain of aspulvinone were related to the inhibition of GOT1 activity. Further biological study indicated that AH could suppress glutamine metabolism, which made PDAC cells sensitive to oxidative stress and inhibited cell proliferation. Besides, AH exhibited potent in vivo antitumor activity in the SW1990 cell-induced xenograft model. These findings suggest that AH could be considered as a promising lead molecule for the development of PDAC anticancer agents.

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Molecular Docking and Preclinical Study of Five-MemberedS,S-Palladaheterocycle as Hepatoprotective Agent

Advanced Pharmaceutical Bulletin ◽

10.15171/apb.2019.079 ◽

2019 ◽

Vol 9 (4) ◽

pp. 674-684 ◽

Cited By ~ 1

Author(s):

Nail Salavatovich Akhmadiev ◽

Albina Midkhatovna Galimova ◽

Vnira Rakhimovna Akhmetova ◽

Veronika Radievna Khairullina ◽

Rozaliia Akramovna Galimova ◽

...

Keyword(s):

Molecular Docking ◽

Survival Data ◽

Active Sites ◽

Cytochromes P450 ◽

Hepatoprotective Activity ◽

Preclinical Study ◽

Therapeutic Effects ◽

Preclinical Trials

Purpose: In order to investigate mechanisms underlying the hepatoprotective action of S,Spalladaheterocycle,inhibition of cytochromes P450 has been modeled by molecular dockingof four palladaheterocycle stereoisomers to the active sites of an enzymatic oxidase system. Toobtain a deeper insight into biochemical aspects providing a basis for the therapeutic effects offive-membered palladacycles (as mixture of stereoisomers), a number of preclinical trials hasbeen conductedMethods: 2D and 3D structures of palladaheterocycle stereoisomers were obtained viaconverting into SDF files by means of software MarvinSketch. Binding of palladaheterocycle atthe active sites of cytochromes P450 2E1 and P450 2C9 has been studied by molecular dockingusing LeadIT 2.3.2. Hepatoprotective activity of palladaheterocycle at 2.5, 25 and 250 mg/kgdoses has been studied based on a model of acute intoxication by CCl4 using in vivo methods.Results: By molecular docking it was identify amino acid fragments responsible for bindingwith palladacyclic isomers. The tested compound is comparable, in terms of its activity tothe hepatoprotective drug SAM according to the in vivo and in vitro experiments such asanimal survival data, the efficiency of correction of the cytolytic syndrome, the liver excretoryfunction, carbohydrate, protein and lipid metabolism, and the correction efficiency of the liverantitoxic function (the latter has been determined based on the results of a hexobarbital controlexperiment).Conclusion: Taking into account results obtained in vivo, in vitro and in silico, it can be concludedthat the five-membered S,S-palladaheterocycle effectively protect the liver against acute damagecaused by CCl4, via activation of catalase and glucuronyltransferase, as well as via inhibition ofthe oxidative stress enzymes.<br />

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A Novel, Nonpeptidic, Orally Active Bivalent Inhibitor of Human β-Tryptase

Pharmacology ◽

10.1159/000492078 ◽

2018 ◽

Vol 102 (5-6) ◽

pp. 233-243 ◽

Cited By ~ 2

Author(s):

Sarah F. Giardina ◽

Douglas S. Werner ◽

Maneesh Pingle ◽

Donald E. Bergstrom ◽

Lee D. Arnold ◽

...

Keyword(s):

Active Sites ◽

Rational Design ◽

Therapeutic Potential ◽

X Ray Crystallography ◽

Inflammatory Stimuli ◽

Invaluable Tool ◽

Xenograft Models ◽

Orally Bioavailable

β-Tryptase is released from mast cells upon degranulation in response to allergic and inflammatory stimuli. Human tryptase is a homotetrameric serine protease with 4 identical active sites directed toward a central pore. These active sites present an optimized scenario for the rational design of bivalent inhibitors, which bridge 2 adjacent active sites. Using (3-[1-acylpiperidin-4-yl]phenyl)methanamine as the pharmacophoric core and a disiloxane linker to span 2 active sites we have successfully produced a novel bivalent tryptase inhibitor, compound 1a, with a comparable profile to previously described inhibitors. Pharmacological properties of compound 1a were studied in a range of in vitro enzymic and cellular screening assays, and in vivo xenograft models. This non-peptide inhibitor of tryptase demonstrated superior activity (IC50 at 100 pmol/L tryptase = 1.82 nmol/L) compared to monomeric modes of inhibition. X-ray crystallography validated the dimeric mechanism of inhibition, and 1a demonstrated good oral bioavailability and efficacy in HMC-1 xenograft models. Furthermore, compound 1a demonstrated extremely slow off rates and high selectivity against-related proteases. This highly potent, orally bioavailable and selective inhibitor of human tryptase will be an invaluable tool in future studies to explore the therapeutic potential of attenuating the activity of this elusive target.

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RISC-interacting clearing 3’- 5’ exoribonucleases (RICEs) degrade uridylated cleavage fragments to maintain functional RISC in Arabidopsis thaliana

eLife ◽

10.7554/elife.24466 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 16

Author(s):

Zhonghui Zhang ◽

Fuqu Hu ◽

Min Woo Sung ◽

Chang Shu ◽

Claudia Castillo-González ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Active Sites ◽

Ectopic Expression ◽

Single Strand ◽

Target Mrnas ◽

Rna Fragments

RNA-induced silencing complex (RISC) is composed of miRNAs and AGO proteins. AGOs use miRNAs as guides to slice target mRNAs to produce truncated 5' and 3' RNA fragments. The 5' cleaved RNA fragments are marked with uridylation for degradation. Here, we identified novel cofactors of Arabidopsis AGOs, named RICE1 and RICE2. RICE proteins specifically degraded single-strand (ss) RNAs in vitro; but neither miRNAs nor miRNA*s in vivo. RICE1 exhibited a DnaQ-like exonuclease fold and formed a homohexamer with the active sites located at the interfaces between RICE1 subunits. Notably, ectopic expression of catalytically-inactive RICE1 not only significantly reduced miRNA levels; but also increased 5' cleavage RISC fragments with extended uridine tails. We conclude that RICEs act to degrade uridylated 5’ products of AGO cleavage to maintain functional RISC. Our study also suggests a possible link between decay of cleaved target mRNAs and miRNA stability in RISC.

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