Naturally Occurring Mutations of SARS-CoV-2 Main Protease Confer Drug Resistance to Nirmatrelvir

Yanmei Hu; Eric M Lewandowski; Haozhou Tan; Xiaoming Zhang; Ryan T Morgan; Xiujun Zhang; Lian M C Jacobs; Shane G Butler; Maura V Gongora; John Choy; Xufang Deng; Yu Chen; Jun Wang

doi:10.1021/acscentsci.3c00538

Naturally Occurring Mutations of SARS-CoV-2 Main Protease Confer Drug Resistance to Nirmatrelvir

ACS Cent Sci. 2023 Jul 24;9(8):1658-1669. doi: 10.1021/acscentsci.3c00538. eCollection 2023 Aug 23.

Authors

Yanmei Hu¹, Eric M Lewandowski², Haozhou Tan¹, Xiaoming Zhang^{3

4}, Ryan T Morgan², Xiujun Zhang², Lian M C Jacobs², Shane G Butler², Maura V Gongora², John Choy⁵, Xufang Deng^{3

4}, Yu Chen², Jun Wang¹

Affiliations

¹ Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, the State University of New Jersey, New Brunswick, New Jersey 08854, United States.
² Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, Florida 33612, United States.
³ Department Physiological Sciences, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma 74078, United States.
⁴ Oklahoma Center for Respiratory and Infectious Diseases, Oklahoma State University, Stillwater, Oklahoma 74078, United States.
⁵ Department Biology, School of Arts and Sciences, the Catholic University of America, Washington, DC 20064, United States.

Abstract

The SARS-CoV-2 main protease (M^pro) is the drug target of Pfizer's oral drug nirmatrelvir. The emergence of SARS-CoV-2 variants with mutations in M^pro raised the alarm of potential drug resistance. To identify potential clinically relevant drug-resistant mutants, we systematically characterized 102 naturally occurring M^pro mutants located at 12 residues at the nirmatrelvir-binding site, among which 22 mutations in 5 residues, including S144M/F/A/G/Y, M165T, E166 V/G/A, H172Q/F, and Q192T/S/L/A/I/P/H/V/W/C/F, showed comparable enzymatic activity to the wild-type (k_cat/K_m < 10-fold change) while being resistant to nirmatrelvir (K_i > 10-fold increase). X-ray crystal structures were determined for six representative mutants with and/or without GC-376/nirmatrelvir. Using recombinant SARS-CoV-2 viruses generated from reverse genetics, we confirmed the drug resistance in the antiviral assay and showed that M^pro mutants with reduced enzymatic activity had attenuated viral replication. Overall, our study identified several drug-resistant hotspots in M^pro that warrant close monitoring for possible clinical evidence of nirmatrelvir resistance, some of which have already emerged in independent viral passage assays conducted by others.