2. Academic Validation
  3. Identification of a cell-active chikungunya virus nsP2 protease inhibitor using a covalent fragment-based screening approach

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

  • Proc Natl Acad Sci U S A. 2024 Oct 15;121(42):e2409166121. doi: 10.1073/pnas.2409166121.
Eric M Merten 1,  John D Sears 2,  Tina M Leisner 1,  P Brian Hardy 1,  Anirban Ghoshal 3,  Mohammad Anwar Hossain 3,  Kesatebrhan Haile Asressu 3,  Peter J Brown 3,  Edwin G Tse 3,  Michael A Stashko 1,  Kelin Li 1,  Jacqueline L Norris-Drouin 1,  Laura E Herring 4,  Angie L Mordant 4,  Thomas S Webb 4,  Christine A Mills 4,  Natalie K Barker 4,  Zachary J Streblow 5,  Sumera Perveen 6,  Cheryl H Arrowsmith 6,  Rafael Miguez Couñago 3 7,  Jamie J Arnold 2,  Craig E Cameron 2,  Daniel N Streblow 5,  Nathaniel J Moorman 2,  Mark T Heise 2 8,  Timothy M Willson 3,  Konstantin I Popov 1,  Kenneth H Pearce 1 9
Affiliations

Affiliations

  • 1 Eshelman School of Pharmacy, Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.
  • 2 Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.
  • 3 Structural Genomics Consortium, Division of Chemical Biology and Medicinal Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.
  • 4 Proteomics Core Facility, Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.
  • 5 Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR 97006.
  • 6 Structural Genomics Consortium, University of Toronto, Toronto, ON M5G 1L7, Canada.
  • 7 Center of Medicinal Chemistry, Center for Molecular Biology and Genetic Engineering, University of Campinas, Campinas, SP 13083-886, Brazil.
  • 8 Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.
  • 9 Lineberger Comprehensive Cancer Center, Molecular Therapeutics Research Program, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.
PMID: 39388272 DOI: 10.1073/pnas.2409166121
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 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.

Keywords

chikungunya virus; covalent inhibitor; drug discovery; fragment; nsP2 protease.

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