Eric M. Merten and John D. Sears and Tina M. Leisner and P. Brian Hardy and Anirban Ghoshal and Mohammad Anwar Hossain and Kesatebrhan Haile Asressu and Peter J. Brown and Edwin G. Tse and Michael A. Stashko and Kelin Li and Jacqueline L. Norris-Drouin and Laura E. Herring and Angie L. Mordant and Thomas S. Webb and Christine A. Mills and Natalie K. Barker and Zachary J. Streblow and Sumera Perveen and Cheryl H. Arrowsmith and Rafael Miguez Couñago and Jamie J. Arnold and Craig E. Cameron and Daniel N. Streblow and Nathaniel J. Moorman and Mark T. Heise and Timothy M. Willson and Konstantin I. Popov and Kenneth H. Pearce
Proc. Natl. Acad. Sci. U.S.A. 2024 121 (42) e2409166121
https://doi.org/10.1073/pnas.2409166121
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 kinact /KI of 6.4 × 103 M−1s−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.
