Identification and characterization of nonsubstrate based inhibitors of the essential dengue and West Nile virus proteases

Ganesh, Vannakambadi K.; Muller, Nik; Judge, K.; Luan, Chi Hao; Padmanabhan, Radhakrishnan; Murthy, Krishna H. M.

doi:10.1016/j.bmc.2004.09.036

Cited by 76 publications

(59 citation statements)

References 41 publications

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“…By analogy with the success of the HIV and HCV protease inhibitors, a number of approaches have been employed to search for DENV NS3 protease inhibitors (reviewed in references 8 and 34-37). One strategy used for this purpose involves a specific target-based approach, e.g., an enzyme-based biochemical assay or structure-based virtual screen (38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49). An alternative strategy is to use a replication-based approach, e.g., screening through virus infection assays or replicon cell lines (50)(51)(52).…”

Section: Engue Virus (Denv) (Serotypes 1 To 4) Belongs To the Familymentioning

confidence: 99%

A Novel Dengue Virus Inhibitor, BP13944, Discovered by High-Throughput Screening with Dengue Virus Replicon Cells Selects for Resistance in the Viral NS2B/NS3 Protease

Yang

et al. 2014

Antimicrob Agents Chemother

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bDengue virus (DENV) causes disease globally, resulting in an estimated 25 to 100 million new infections per year. No effective DENV vaccine is available, and the current treatment is only supportive. Thus, there is an urgent need to develop therapeutic agents to cure this epidemic disease. In the present study, we identified a potential small-molecule inhibitor, BP13944, via highthroughput screening (HTS) of 60,000 compounds using a stable cell line harboring an efficient luciferase replicon of DENV serotype 2 (DENV-2). BP13944 reduced the expression of the DENV replicon reporter in cells, showing a 50% effective concentration (EC 50 ) of 1.03 ؎ 0.09 M. Without detectable cytotoxicity, the compound inhibited replication or viral RNA synthesis in all four serotypes of DENV but not in Japanese encephalitis virus (JEV). Sequencing analyses of several individual clones derived from BP13944-resistant RNAs purified from cells harboring the DENV-2 replicon revealed a consensus amino acid substitution (E66G) in the region of the NS3 protease domain. Introduction of E66G into the DENV replicon, an infectious DENV cDNA clone, and recombinant NS2B/NS3 protease constructs conferred 15.2-, 17.2-, and 3.1-fold resistance to BP13944, respectively. Our results identify an effective small-molecule inhibitor, BP13944, which likely targets the DENV NS3 protease. BP13944 could be considered part of a more effective treatment regime for inhibiting DENV in the future. D engue virus (DENV) (serotypes 1 to 4) belongs to the familyFlaviviridae, a group of enveloped RNA viruses that includes the genera Hepacivirus, Flavivirus, and Pestivirus. The genus Flavivirus consists of arthropod-borne disease agents such as yellow fever virus (YFV), Japanese encephalitis virus (JEV), West Nile virus (WNV), tick-borne encephalitis virus (TBEV), and DENV (1). More than 70 members of the Flavivirus genus are important human pathogens that cause significant morbidity and mortality (2). DENV is a public health threat to an estimated 2.5 billion people living in areas where dengue is epidemic, leading to 50 to 100 million human infections each year (3, 4). DENV infection frequently leads to dengue fever, life-threatening dengue hemorrhagic fever (DHF), or dengue shock syndrome (DSS) (5-7). Approximately 500,000 cases of DHF and DSS have been reported among more than 100 countries, causing approximately 12,500 deaths per year (3). Despite the tremendous efforts invested in anti-DENV research, no clinically approved vaccine or antiviral therapeutic agents are available for humans, and disease treatment is limited to supportive care (4,8,9). Considering the spread of this epidemic and the severity of DENV, an effective anti-DENV drug is urgently needed.DENV is an enveloped RNA virus consisting of a 10.7-kb single-stranded, positive-polarity RNA genome associated with multiple copies of capsid proteins. DENV RNA is translated as a single polyprotein upon entering the host cell and is cleaved by host proteases and the virus-encoded two-component protease (NS...

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Section: Engue Virus (Denv) (Serotypes 1 To 4) Belongs To the Familymentioning

confidence: 99%

A Novel Dengue Virus Inhibitor, BP13944, Discovered by High-Throughput Screening with Dengue Virus Replicon Cells Selects for Resistance in the Viral NS2B/NS3 Protease

Yang

et al. 2014

Antimicrob Agents Chemother

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“…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]. 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%

“…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%

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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“…It is also difficult to design potent inhibitors against dengue virus NS2B/NS3 protease because the active site of dengue virus NS2B/NS3 protease is relatively flat (10) and charged (36). Some progress has been realized in the developing HTS and peptidomimetic approaches (7,12,24,33,48,49), although to date there are no dengue virus protease inhibitors in clinical trials.…”

mentioning

confidence: 99%

Novel Dengue Virus-Specific NS2B/NS3 Protease Inhibitor, BP2109, Discovered by a High-Throughput Screening Assay

Yang

Hsieh

Lee

et al. 2011

Antimicrob Agents Chemother

100

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Dengue virus (DENV) causes disease globally, with an estimated 25 to 100 million new infections per year. At present, no effective vaccine is available, and treatment is supportive. In this study, we identified BP2109, a potent and selective small-molecule inhibitor of the DENV NS2B/NS3 protease, by a high-throughput screening assay using a recombinant protease complex consisting of the central hydrophilic portion of NS2B and the N terminus of the protease domain. BP2109 inhibited DENV (serotypes 1 to 4), but not Japanese encephalitis virus (JEV), replication and viral RNA synthesis without detectable cytotoxicity. The compound inhibited recombinant DENV-2 NS2B/NS3 protease with a 50% inhibitory concentration (IC 50 ) of 15.43 ؎ 2.12 M and reduced the reporter expression of the DENV-2 replicon with a 50% effective concentration (EC 50 ) of 0.17 ؎ 0.01 M. Sequencing analyses of several individual clones derived from BP2109-resistant DENV-2 RNAs revealed that two amino acid substitutions (R55K and E80K) are found in the region of NS2B, a cofactor of the NS2B/NS3 protease complex. The introduction of R55K and E80K double mutations into the dengue virus NS2B/NS3 protease and a dengue virus replicon construct conferred 10.3-and 73.8-fold resistance to BP2109, respectively. The E80K mutation was further determined to be the key mutation conferring dengue virus replicon resistance (61.3-fold) to BP2109, whereas the R55K mutation alone did not affect resistance to BP2109. Both the R55K and E80K mutations are located in the central hydrophilic portion of the NS2B cofactor, where extensive interactions with the NS3pro domain exist. Thus, our data provide evidence that BP2109 likely inhibits DENV by a novel mechanism.Dengue virus serotypes 1 to 4 (DENV-1 to -4) belongs to the family Flaviviridae, a group of enveloped RNA viruses that includes the genera Hepacivirus, Flavivirus, and Pestivirus. The genus Flavivirus consists of arthropod-borne disease agents, such as the yellow fever virus (YFV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and DENV (14). Many members of the genus Flavivirus are important human pathogens and cause significant morbidity and mortality. DENV alone poses a public health threat to an estimated 2.5 billion people living in areas where dengue is epidemic and leads to 50 to 100 million human infections each year (17,18,38). DENV infection often leads to dengue fever, life-threatening dengue hemorrhagic fever (DHF), or dengue shock syndrome (DSS) (15,19,37). Approximately 500,000 cases of DHF and DSS have been reported in over 100 countries and have caused approximately 25,000 deaths per year (16). Despite the tremendous efforts invested in anti-DENV research, no clinically approved vaccine or antiviral therapy for humans is available for DENV (25,32,36). Considering the spread of this epidemic and the severity of DENV, the discovery of an effective anti-DENV drug is now an urgent need.DENV is an enveloped RNA virus that consists of a 10.7-kb single-stranded, positive-polarity genomic RN...

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Identification and characterization of nonsubstrate based inhibitors of the essential dengue and West Nile virus proteases

Cited by 76 publications

References 41 publications

A Novel Dengue Virus Inhibitor, BP13944, Discovered by High-Throughput Screening with Dengue Virus Replicon Cells Selects for Resistance in the Viral NS2B/NS3 Protease

A Novel Dengue Virus Inhibitor, BP13944, Discovered by High-Throughput Screening with Dengue Virus Replicon Cells Selects for Resistance in the Viral NS2B/NS3 Protease

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

Novel Dengue Virus-Specific NS2B/NS3 Protease Inhibitor, BP2109, Discovered by a High-Throughput Screening Assay

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