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 Miguez Couñago; Peter J Brown; Timothy M Willson

doi:10.21203/rs.3.rs-5363451/v1

A Covalent Chemical Probe for Chikungunya nsP2 Cysteine Protease with Antialphaviral Activity and Proteome-wide Selectivity

Res Sq [Preprint]. 2024 Nov 12:rs.3.rs-5363451. doi: 10.21203/rs.3.rs-5363451/v1.

Authors

Anirban Ghoshal^{1

2}, Edwin G Tse^{1

2}, Mohammad Anwar Hossain^{1

2}, Kesatebrhan Haile Asressu^{1

2}, Eric M Merten^{3

2}, John D Sears^{4

2}, Stefanie Howell^{1

2}, Sumera Perveen^{5

2}, Jane Burdick^{6

2}, Noah L Morales^{6

2}, Sabian A Martinez^{6

2}, Isabella Law^{6

2}, Bennett J Davenport^{7

2}, Thomas E Morrison^{7

2}, Zachary J Streblow^{8

2}, Daniel N Streblow^{8

2}, Angie L Mordant⁹, Thomas S Webb⁹, Aurora Cabrera⁹, Laura E Herring⁹, Cheryl H Arrowsmith^{5

2}, Kenneth H Pearce^{3

2}, Nathaniel J Moorman^{4

2}, Mark T Heise^{6

4

2}, Rafael Miguez Couñago^{1

10

2}, Peter J Brown^{1

2}, Timothy M Willson^{1

2}

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 UNC Eshelman School of Pharmacy, Center for Integrative Chemical Biology and Drug Discovery, 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, Ontario, 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, 13083-886-Campinas, SP, Brazil.

Abstract

RA-0003022 (3) was identified as a high-quality covalent chemical probe for Chikungunya nsP2 cysteine protease (nsP2pro). Isoxazole 3 covalently captured the active site C478 and inactivated the enzyme with a k _inact/K _i ratio of 6000 M^-1s^-1. A negative control analog RA-0025453 (4) retained the covalent warhead but demonstrated >100-fold decrease in enzyme inhibition. Isoxazoles 3 and 4 were stable across a wide range of pH in solution and upon prolonged storage as solids. The covalent chemical probe 3 was inactive across a panel of 23 human and viral cysteine proteases and demonstrated remarkable proteome-wide selectivity by two chemoproteomic methods. Isoxazole 3 reduced viral titer against infectious isolates of Chikungunya, Mayaro, and Venezuelan Equine Encephalitis viruses demonstrating its activity on both New and Old World alphaviruses. Isoxazole 3 and its negative control 4 will find utility as covalent chemical probes to study the role of the nsP2pro in alphaviral replication and virulence.

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Preprint

Abstract

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