Identification of naturally occurring drug-resistant mutations of SARS-CoV-2 papain-like protease

  • Nat Commun. 2025 May 16;16(1):4548. doi: 10.1038/s41467-025-59922-9.
Haozhou Tan  #  1 ,  Qianru Zhang  #  2 ,  Kyriakos Georgiou  3 ,  Siyu Zhang  2 ,  Kan Li  1 ,  George Lambrinidis  3 ,  Antonios Kolocouris  3 ,  Xufang Deng  4  5 ,  Jun Wang  6
Affiliations
  • 1. Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, the State University of New Jersey, Brunswick, NJ, USA.
  • 2. Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK, USA.
  • 3. Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, National and Kapodistrian University of Athens, Athens, Greece.
  • 4. Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK, USA. [email protected].
  • 5. Oklahoma Center for Respiratory and Infectious Diseases, Oklahoma State University, Stillwater, OK, USA. [email protected].
  • 6. Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, the State University of New Jersey, Brunswick, NJ, USA. [email protected].
  • # Contributed equally.
PMID: 40379662 DOI: 10.1038/s41467-025-59922-9
Abstract

The SARS-CoV-2 papain-like protease (PLpro) is a cysteine protease that cleaves viral polyproteins and antagonizes the host immune response during viral replication. Jun12682 and PF-07957472 are the first-in-class PLpro inhibitors showing potent in vivo Antiviral efficacy in mouse models. In this study, we characterize naturally occurring mutations at residues located at the drug-binding site of Jun12682. The results reveal several PLpro mutants showing significant drug resistance while maintaining comparable enzymatic activity as the wild-type PLpro. The physiological relevance of the identified drug-resistant mutants, including E167G and Q269H, is validated through independent serial viral passage experiments. Molecular dynamics simulations and perturbative free energy calculations show that drug-resistant PLpro mutants weaken hydrogen bonding and π-π stacking interactions. Collectively, this study identifies E167, Y268, and Q269 as drug-resistant hotspots for PLpro inhibitors that bind to the BL2 loop and groove region, which are valuable in informing the design of the next-generation PLpro inhibitors.

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