title: 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.
abstract: Dengue 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.
authors.affiliation: Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhunan Town, Miaoli, Taiwan, Republic of China.
authors.forename: Chi-Chen
authors.initials: CC
authors.lastname: Yang
authors.forename: Han-Shu
authors.initials: HS
authors.lastname: Hu
authors.forename: Ren-Huang
authors.initials: RH
authors.lastname: Wu
authors.forename: Szu-Huei
authors.initials: SH
authors.lastname: Wu
authors.forename: Shiow-Ju
authors.initials: SJ
authors.lastname: Lee
authors.forename: Weir-Torn
authors.initials: WT
authors.lastname: Jiaang
authors.forename: Jyh-Haur
authors.initials: JH
authors.lastname: Chern
authors.forename: Zhi-Shun
authors.initials: ZS
authors.lastname: Huang
authors.forename: Huey-Nan
authors.initials: HN
authors.lastname: Wu
authors.forename: Chung-Ming
authors.initials: CM
authors.lastname: Chang
authors.forename: Andrew
authors.initials: A
authors.lastname: Yueh
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D006224:Cricetinae; D003716:Dengue Virus / Q000187:drug effects* / Q000201:enzymology; D024882:Drug Resistance, Viral; D012093:Replicon / Q000187:drug effects*; D012697:Serine Endopeptidases / Q000378:metabolism; D054852:Small Molecule Libraries; D014779:Virus Replication / Q000187:drug effects*; D000085242:Viral Proteases
chemical_list: D000998:Antiviral Agents; D012697:Serine Endopeptidases; D054852:Small Molecule Libraries; C504449:NS3 protease, dengue virus; D000085242:Viral Proteases