Identification of a cell- active chikungunya virus nsP2 protease inhibitor using a covalent fragment- based screening approach

• 成果类型: Article
• 署名作者: Merten, Eric M.; Sears, John D.; Leisner, Tina M.; Hardy, P. Brian; Ghoshal, Anirban; Hossain, Mohammad Anwar; Asressu, Kesatebrhan Haile; Brown, Peter J.; Tse, Edwin G.; Stashko, Michael A.; Li, Kelin; Drouin, Jacqueline L. Norris -; Herring, Laura E.; Mordant, Angie L.; Webb, Thomas S.; Mills, Christine A.; Barker, Natalie K.; Streblow, Zachary J.; Perveen, Sumera; Arrowsmith, Cheryl H.; Counago, Rafael Miguez; Arnold, Jamie J.; Cameron, Craig E.; Streblow, Daniel N.; Moorman, Nathaniel J.; Heise, Mark T.; Willson, Timothy M.; Popov, Konstantin I.; Pearce, Kenneth H.
• 署名单位: University of North Carolina; University of North Carolina Chapel Hill; University of North Carolina School of Medicine; University of North Carolina; University of North Carolina Chapel Hill; University of North Carolina School of Medicine; University of North Carolina; University of North Carolina Chapel Hill; University of North Carolina School of Medicine; University of North Carolina; University of North Carolina Chapel Hill; University of North Carolina School of Medicine; Oregon Health & Science University; University of Toronto; Structural Genomics Consortium; Universidade Estadual de Campinas; University of North Carolina; University of North Carolina Chapel Hill; University of North Carolina School of Medicine; University of North Carolina; University of North Carolina Chapel Hill; University of North Carolina School of Medicine
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-15343
• DOI: 10.1073/pnas.2409166121
• 发表日期: 2024-10-15
• 关键词: equine encephalitis-virus; replication; DESIGN

摘要

Chikungunya virus (CHIKV) is a mosquito- borne alphavirus that has been responsible for numerous large- scale outbreaks in the last twenty years. Currently, there are no FDA- approved therapeutics for any alphavirus infection. CHIKV nonstructural protein 2 (nsP2), which contains a cysteine protease domain, is essential for viral replication, making it an attractive target for a drug discovery campaign. Here, we optimized a CHIKV nsP2 protease (nsP2pro) biochemical assay for the screening of a 6,120- compound cysteine- directed covalent fragment library. Using a 50% inhibition threshold, we identified 153 hits (2.5% hit rate). In dose-response follow- up, RA- 0002034, a covalent fragment that contains a vinyl sulfone warhead, inhibited CHIKV nsP2pro with an IC50 of 58 +/- 17 nM, and further analysis with time- dependent inhibition studies yielded a k inact /K I of 6.4 x 103 M -1 s -1 . LC-MS/MS analysis determined that RA- 0002034 covalently modified the catalytic cysteine in a site- specific manner. Additionally, RA- 0002034 showed no significant off- target reactivity in proteomic experiments or against a panel of cysteine proteases. In addition to the potent biochemical inhibition of CHIKV nsP2pro activity and exceptional selectivity, RA- 0002034 was tested in cellular models of alphavirus infection and effectively inhibited viral replication of both CHIKV and related alphaviruses. This study highlights the identification and characterization of the chemical probe RA- 0002034 as a promising hit compound from covalent fragment- based screening for development toward a CHIKV or pan- alphavirus therapeutic.