Peptide inhibitors of dengue virus NS3 protease. Part 1: Warhead

Yin, Zheng; Patel, Sonika; Wang, Weiling; Wang, Gang; Chan, Wai-Ling; Rao, K. R. Ranga; Alam, Jenefer; Jeyaraj, Duraiswamy A.; Ngew, Xinyi; Patel, Viral; Beer, David; Lim, Siew Pheng; Vasudevan, Subhash G.; Keller, Thomas H.

doi:10.1016/j.bmcl.2005.09.062

Cited by 150 publications

(130 citation statements)

References 25 publications

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“…Peptide Aldehyde Inhibitor Synthesis and Purification-The peptide Benzoyl-Nle-Lys-Arg-H (Bz-nKRR-H) with a C-terminal aldehyde moiety was synthesized according to a previous report (16) and purified by reverse-phase HPLC (Shimadzu, Japan) using acetonitrile and water solvent.…”

Section: Methodsmentioning

confidence: 99%

NMR Analysis of a Novel Enzymatically Active Unlinked Dengue NS2B-NS3 Protease Complex

Kim

Gayen

Kang

et al. 2013

Journal of Biological Chemistry

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178

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Background: Dengue protease is a two-component protease that is important for viral replication. Results: An unlinked protease complex containing the NS2B regulatory region and the NS3 protease domain was obtained. Conclusion:The unlinked protease complex produces dispersed cross-peaks in NMR spectra and exists predominantly in a closed conformation in solution.Significance: This new construct will be a useful tool for drug discovery against the dengue virus.

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Section: Methodsmentioning

confidence: 99%

NMR Analysis of a Novel Enzymatically Active Unlinked Dengue NS2B-NS3 Protease Complex

Kim

Gayen

Kang

et al. 2013

Journal of Biological Chemistry

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178

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“…The protease domain is contained in nonstructural protein 3 (NS3) [9], and its activity is greatly enhanced by interactions with the NS2B protein, which acts as its cofactor [10,11]. Several efforts to find inhibitors for different flavivirus proteases (West Nile virus [WNV], dengue, and yellow fever virus [YFV]) have been reported, with several studies focusing on peptidic substrate-based inhibitors [12][13][14][15][16][17]. Nonpeptidic inhibitors have been identified by in vitro screening [18][19][20] or in silico throughput docking [21].…”

Section: B S T R a C Tmentioning

confidence: 99%

“…Nonpeptidic inhibitors have been identified by in vitro screening [18][19][20] or in silico throughput docking [21]. Most of these compounds do not possess the appropriate properties for drug development, either because the scaffold is too labile [15][16][17]19] or because the inhibitors bind too weakly [18,20].…”

Section: B S T R a C Tmentioning

confidence: 99%

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A fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease

Bodenreider

Beer

Keller

et al. 2009

Analytical Biochemistry

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In drug discovery, the occurrence of false positives is a major hurdle in the search for lead compounds that can be developed into drugs. A small-molecular-weight compound that inhibits dengue virus protease at low micromolar levels was identified in a screening campaign. Binding to the enzyme was confirmed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). However, a structure-activity relationship study that ensued did not yield more potent leads. To further characterize the parental compound and its analogues, we developed a high-speed, low-cost, quantitative fluorescence quenching assay. We observed that specific analogues quenched dengue protease fluorescence and showed variation in IC (50) values. In contrast, nonspecifically binding compounds did not quench its fluorescence and showed similar IC(50) values with steep dose-response curves. We validated the assay using single Trp-to-Ala protease mutants and the competitive protease inhibitor aprotinin. Specific compounds detected in the binding assay were further analyzed by competitive ITC, NMR, and surface plasmon resonance, and the assay's utility in comparison with these biophysical methods is discussed. The sensitivity of this assay makes it highly useful for hit finding and validation in drug discovery. Furthermore, the technique can be readily adapted for studying other protein-ligand interactions. b s t r a c tIn drug discovery, the occurrence of false positives is a major hurdle in the search for lead compounds that can be developed into drugs. A small-molecular-weight compound that inhibits dengue virus protease at low micromolar levels was identified in a screening campaign. Binding to the enzyme was confirmed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). However, a structure-activity relationship study that ensued did not yield more potent leads. To further characterize the parental compound and its analogues, we developed a high-speed, low-cost, quantitative fluorescence quenching assay. We observed that specific analogues quenched dengue protease fluorescence and showed variation in IC 50 values. In contrast, nonspecifically binding compounds did not quench its fluorescence and showed similar IC 50 values with steep dose-response curves. We validated the assay using single Trp-to-Ala protease mutants and the competitive protease inhibitor aprotinin. Specific compounds detected in the binding assay were further analyzed by competitive ITC, NMR, and surface plasmon resonance, and the assay's utility in comparison with these biophysical methods is discussed. The sensitivity of this assay makes it highly useful for hit finding and validation in drug discovery. Furthermore, the technique can be readily adapted for studying other proteinligand interactions. Ó 2009 Elsevier Inc. All rights reserved.High-throughput screening (HTS) 2 is a major method in drug discovery for new small lead molecules. In high-throughput enzyme inhibition assays, more than a million compounds are usua...

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“…The development of an efficient vaccine or antiviral drugs is thus extremely necessary. However, vaccine development against DENV is challenging because of the antibodydependent enhancement effect (ADE) (ie, infection by one serotype of DENV can enhance the probability of infection by any of the other three serotypes) [6] , and for more complicated barriers in anti-virus drug discovery [7][8][9] , there are not yet effective antiviral drugs in the market against DENV, although many drug lead compounds have been discovered [10,11] . The DENV genome, a positive-sense single-stranded RNA, encodes a single poly-protein precursor that consists of three structural proteins (C, M, and E) and seven nonstructural 1127 www.chinaphar.com Wu DW et al Acta Pharmacologica Sinica npg proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).…”

Section: Introductionmentioning

confidence: 99%

Policresulen, a novel NS2B/NS3 protease inhibitor, effectively inhibits the replication of DENV2 virus in BHK-21 cells

Mao

et al. 2015

Acta Pharmacol Sin

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Aim: Dengue is a severe epidemic disease caused by dengue virus (DENV) infection, for which no effective treatment is available. The protease complex, consisting of nonstructural protein 3 (NS3) and its cofactor NS2B, plays a pivotal role in the replication of DENV, thus may be a potential target for anti-DENV drugs. Here, we report a novel inhibitor of DENV2 NS2B/NS3 protease and its antiviral action. Methods: An enzymatic inhibition assay was used for screening DENV2 NS2B/NS3 inhibitors. Cytotoxicity to BHK-21 cells was assessed with MTT assay. Antiviral activity was evaluated in BHK-21 cells transfected with Rlu-DENV-Rep. The molecular mechanisms of the antiviral action was analyzed using surface plasmon resonance, ultraviolet-visible spectral analysis and differential scanning calorimetry assays, as well as molecular docking analysis combined with site-directed mutagenesis. Results: In our in-house library of old drugs (~1000 compounds), a topical hemostatic and antiseptic 2-hydroxy-3,5-bis[(4-hydroxy-2-methyl-5-sulfophenyl)methyl]-4-methyl-benzene-sulfonic acid (policresulen) was found to be a potent inhibitor of DENV2 NS2B/NS3 protease with IC 50 of 0.48 μg/mL. Furthermore, policresulen inhibited DENV2 replication in BHK-21 cells with IC 50 of 4.99 μg/mL, whereas its IC 50 for cytotoxicity to BHK-21 cells was 459.45 μg/mL. Policresulen acted as a competitive inhibitor of the protease, and slightly affected the protease stability. Using biophysical technology-based assays and molecular docking analysis combined with sitedirected mutagenesis, we demonstrated that the residues Gln106 and Arg133 of DENV2 NS2B/NS3 protease directly interacted with policresulen via hydrogen bonding. Conclusion: Policresulen is a potent inhibitor of DENV2 NS2B/NS3 protease that inhibits DENV2 replication in BHK-21 cells. The binding mode of the protease and policresulen provides useful hints for designing new type of inhibitors against the protease.

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Peptide inhibitors of dengue virus NS3 protease. Part 1: Warhead

Cited by 150 publications

References 25 publications

NMR Analysis of a Novel Enzymatically Active Unlinked Dengue NS2B-NS3 Protease Complex

NMR Analysis of a Novel Enzymatically Active Unlinked Dengue NS2B-NS3 Protease Complex

A fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease

Policresulen, a novel NS2B/NS3 protease inhibitor, effectively inhibits the replication of DENV2 virus in BHK-21 cells

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