A covalent chemical probe for Chikungunya nsP2 cysteine protease with antialphaviral activity and proteome-wide selectivity

Anirban Ghoshal; Edwin G Tse; Mohammad Anwar Hossain; Kesatebrhan Haile Asressu; Eric M Merten; John D Sears; Stefanie Howell; Sumera Perveen; Jane Burdick; Noah L Morales; Sabian A Martinez; Isabella Law; Bennett J Davenport; Thomas E Morrison; Zachary J Streblow; Daniel N Streblow; Angie L Mordant; Thomas S Webb; Aurora Cabrera; Laura E Herring; Cheryl H Arrowsmith; Kenneth H Pearce; Nathaniel J Moorman; Mark T Heise; Rafael M Couñago; Peter J Brown; Timothy M Willson

doi:10.1038/s41598-025-91673-x

A covalent chemical probe for Chikungunya nsP2 cysteine protease with antialphaviral activity and proteome-wide selectivity

Sci Rep. 2025 Mar 1;15(1):7264. doi: 10.1038/s41598-025-91673-x.

Authors

Anirban Ghoshal^{1

2}, Edwin G Tse^{1

2}, Mohammad Anwar Hossain^{1

2}, Kesatebrhan Haile Asressu^{1

2}, Eric M Merten^{2

3}, John D Sears^{2

4}, Stefanie Howell^{1

2}, Sumera Perveen^{2

5}, Jane Burdick^{2

6}, Noah L Morales^{2

6}, Sabian A Martinez^{2

6}, Isabella Law^{2

6}, Bennett J Davenport^{2

7}, Thomas E Morrison^{2

7}, Zachary J Streblow^{2

8}, Daniel N Streblow^{2

8}, Angie L Mordant⁹, Thomas S Webb⁹, Aurora Cabrera⁹, Laura E Herring⁹, Cheryl H Arrowsmith^{2

5}, Kenneth H Pearce^{2

3}, Nathaniel J Moorman^{2

4}, Mark T Heise^{2

4

6}, Rafael M Couñago^{1

2

10}, Peter J Brown^{1

2}, Timothy M Willson^{11

12}

Affiliations

¹ Structural Genomics Consortium, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.
² READDI Avidd Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.
³ Center for Integrative Chemical Biology and Drug Discovery, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.
⁴ Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.
⁵ Structural Genomics Consortium, University of Toronto, Toronto, ON, M5G 1L7, Canada.
⁶ Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.
⁷ Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO, 80045, USA.
⁸ Vaccine & Gene Therapy Institute, Oregon Health & Science University, Beaverton, OR, 97006, USA.
⁹ UNC Metabolomics and Proteomics Core, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.
¹⁰ Center of Medicinal Chemistry, Center for Molecular Biology and Genetic Engineering, University of Campinas, Campinas, SP, 13083-886, Brazil.
¹¹ Structural Genomics Consortium, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA. tim.willson@unc.edu.
¹² READDI Avidd Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA. tim.willson@unc.edu.

Abstract

Chikungunya is a mosquito-borne viral disease that causes fever and severe joint pain for which there is no direct acting drug treatments. Vinyl sulfone SGC-NSP2PRO-1 (3) was identified as a potent inhibitor of the nsP2 cysteine protease (nsP2pro) that reduced viral titer against infectious isolates of Chikungunya and other alphaviruses. The covalent warhead in 3 captured the active site C478 and inactivated nsP2pro with a k_inact/K_i ratio of 5950 M^-1 s^-1. The vinyl sulfone 3 was inactive across a panel of 23 other cysteine proteases and demonstrated remarkable proteome-wide selectivity by two chemoproteomic methods. A negative control analog SGC-NSP2PRO-1N (4) retained the isoxazole core and covalent warhead but demonstrated > 100-fold decrease in enzyme inhibition. Both 3 and 4 were stable across a wide range of pH in solution and upon prolonged storage as solids. Vinyl sulfone 3 and its negative control 4 will find utility as high-quality chemical probes to study the role of the nsP2pro in cellular studies of alphaviral replication and virulence.

Keywords: Alphavirus; Antiviral; Chemical probe; Covalent inhibitor; Cysteine protease.

MeSH terms

Animals
Antiviral Agents* / chemistry
Antiviral Agents* / pharmacology
Chikungunya Fever / drug therapy
Chikungunya Fever / virology
Chikungunya virus* / drug effects
Chikungunya virus* / enzymology
Cysteine Endopeptidases
Cysteine Proteases* / chemistry
Cysteine Proteases* / metabolism
Cysteine Proteinase Inhibitors* / chemistry
Cysteine Proteinase Inhibitors* / pharmacology
Humans
Molecular Probes* / chemistry
Molecular Probes* / pharmacology
Proteome* / metabolism
Sulfones / chemistry
Sulfones / pharmacology

Substances

Sulfones
Antiviral Agents
Proteome
divinyl sulfone
nsP2 proteinase
Cysteine Proteases
Cysteine Proteinase Inhibitors
Molecular Probes
Cysteine Endopeptidases

Abstract

MeSH terms

Substances

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