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

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 IC<sub>50</sub> of 58 ± 17 nM, and further analysis with time-dependent inhibition studies yielded a k<sub>inact</sub> /K<sub>I</sub> of 6.4 × 10<sup>3</sup> M<sup>-1</sup>s<sup>-1</sup>. 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.

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Publication Details

Journal
Proceedings of the National Academy of Sciences
Published
2024-10-10
DOI
https://doi.org/10.1073/pnas.2409166121
Citations
25
Primary Topic
Mosquito-borne diseases and control
Type
article
Field-Weighted Citation Impact
13.57

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article

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

Kesatebrhan Haile Asressu, Thomas S. Webb, C.H. Arrowsmith, Daniel N. Streblow et al.
25 citations
Proceedings of the National Academy of Sciences
Mosquito-borne diseases and control
13.57
article

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

Kesatebrhan Haile Asressu, Thomas S. Webb, C.H. Arrowsmith, Daniel N. Streblow, Sumera Perveen, John D. Sears, Zachary J. Streblow, Mark T. Heise, Craig E. Cameron, Kenneth H. Pearce, Anirban Ghoshal, Christine A. Mills, Natalie K. Barker, Eric M. Merten, Angie L. Mordant, Jamie J. Arnold, Nathaniel J. Moorman, Michael A. Stashko, Laura E. Herring, Rafael M. Couñago, Konstantin Popov, P. Brian Hardy, Kelin Li, Timothy M. Willson, Mohammad Anwar Hossain, Peter J. Brown, Edwin G. Tse, Tina M. Leisner, Jacqueline L. Norris‐Drouin
article en
25 citations

Abstract

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 IC<sub>50</sub> of 58 ± 17 nM, and further analysis with time-dependent inhibition studies yielded a k<sub>inact</sub> /K<sub>I</sub> of 6.4 × 10<sup>3</sup> M<sup>-1</sup>s<sup>-1</sup>. 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.

Proceedings of the National Academy of SciencesVol. 121(42)
University of North Carolina at Chapel Hill (US), Oregon Health & Science University (US), Universidade Estadual de Campinas (UNICAMP) (BR), University of Toronto (CA), Structural Genomics Consortium (CA), Children's Cancer Therapy Development Institute (US)
Bristol-Myers Squibb, Pfizer, Bayer, North Carolina Biotechnology Center, McGill University, Genome Canada, Merck KGaA, Ontario Genomics, European Federation of Pharmaceutical Industries and Associations, Ontario Genomics Institute, National Institutes of Health, Genentech, National Cancer Institute, National Institute of Allergy and Infectious Diseases, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill
Openalex Percentile: Top 1%
Mosquito-borne diseases and control
13.57
DOIOpen Access PDFOpenAlex
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