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

ABSTRACTDengue 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 high-throughput 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 (EC50) of 1.03 ± 0.09 μM. 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.

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Small Molecule Inhibitor Discovery for Dengue Virus Protease Using High-Throughput Screening

Dengue - Methods in Molecular Biology ◽

10.1007/978-1-4939-0348-1_20 ◽

2014 ◽

pp. 331-344 ◽

Cited By ~ 3

Author(s):

Mark Manzano ◽

Janak Padia ◽

Radhakrishnan Padmanabhan

Keyword(s):

Dengue Virus ◽

Small Molecule ◽

High Throughput ◽

High Throughput Screening ◽

Small Molecule Inhibitor ◽

Molecule Inhibitor ◽

Inhibitor Discovery

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High-Throughput Screening Reveals a Small-Molecule Inhibitor of the Renal Outer Medullary Potassium Channel and Kir7.1

Molecular Pharmacology ◽

10.1124/mol.109.059840 ◽

2009 ◽

Vol 76 (5) ◽

pp. 1094-1103 ◽

Cited By ~ 68

Author(s):

L. Michelle Lewis ◽

Gautam Bhave ◽

Brian A. Chauder ◽

Sreedatta Banerjee ◽

Katharina A. Lornsen ◽

...

Keyword(s):

Potassium Channel ◽

Small Molecule ◽

High Throughput ◽

High Throughput Screening ◽

Small Molecule Inhibitor ◽

Molecule Inhibitor

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Faculty Opinions recommendation of Application of high-throughput screening to identify a novel alphaIIb-specific small- molecule inhibitor of alphaIIbbeta3-mediated platelet interaction with fibrinogen.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1098248.554267 ◽

2007 ◽

Author(s):

Xiaoping Du

Keyword(s):

Small Molecule ◽

High Throughput ◽

High Throughput Screening ◽

Small Molecule Inhibitor ◽

Molecule Inhibitor

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Necrosulfonamide inhibits necroptosis by selectively targeting the mixed lineage kinase domain-like protein

MedChemComm ◽

10.1039/c3md00278k ◽

2014 ◽

Vol 5 (3) ◽

pp. 333-337 ◽

Cited By ~ 19

Author(s):

Daohong Liao ◽

Liming Sun ◽

Weilong Liu ◽

Sudan He ◽

Xiaodong Wang ◽

...

Keyword(s):

Small Molecule ◽

High Throughput ◽

High Throughput Screening ◽

Kinase Domain ◽

Small Molecule Inhibitor ◽

Mixed Lineage Kinase ◽

Sar Studies ◽

Structure Activity ◽

Molecule Inhibitor ◽

Smac Mimetic

Through high-throughput screening of 200 000 compounds and subsequent structure–activity relationship (SAR) studies we identified necrosulfonamide (NSA) as a potent small molecule inhibitor for necroptosis, induced by a combination of TNF-a, Smac mimetic, and z-VAD-fmk (T/S/Z).

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00855-10 ◽

2010 ◽

Vol 55 (1) ◽

pp. 229-238 ◽

Cited By ~ 80

Author(s):

Chi-Chen Yang ◽

Yi-Chen Hsieh ◽

Shiow-Ju Lee ◽

Szu-Huei Wu ◽

Ching-Len Liao ◽

...

Keyword(s):

Dengue Virus ◽

High Throughput ◽

High Throughput Screening ◽

Effective Vaccine ◽

Protease Domain ◽

Screening Assay ◽

Denv Serotypes ◽

Molecule Inhibitor ◽

High Throughput Screening Assay ◽

Ns3 Protease

ABSTRACTDengue 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 (IC50) of 15.43 ± 2.12 μM and reduced the reporter expression of the DENV-2 replicon with a 50% effective concentration (EC50) of 0.17 ± 0.01 μM. 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.

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