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Distal protein-protein interactions contribute to nirmatrelvir resistance

Author

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  • Eric M. Lewandowski

    (University of South Florida)

  • Xiujun Zhang

    (University of South Florida)

  • Haozhou Tan

    (the State University of New Jersey)

  • Aiden Jaskolka-Brown

    (University of South Florida)

  • Navita Kohaal

    (University of South Florida)

  • Aliaksandra Frazier

    (University of South Florida)

  • Jesper J. Madsen

    (University of South Florida)

  • Lian M. C. Jacobs

    (University of South Florida)

  • Jun Wang

    (the State University of New Jersey)

  • Yu Chen

    (University of South Florida)

Abstract

SARS-CoV-2 main protease, Mpro, is responsible for processing the viral polyproteins into individual proteins, including the protease itself. Mpro is a key target of anti-COVID-19 therapeutics such as nirmatrelvir (the active component of Paxlovid). Resistance mutants identified clinically and in viral passage assays contain a combination of active site mutations (e.g., E166V, E166A, L167F), which reduce inhibitor binding and enzymatic activity, and non-active site mutations (e.g., P252L, T21I, L50F), which restore the fitness of viral replication. To probe the role of the non-active site mutations in fitness rescue, here we use an Mpro triple mutant (L50F/E166A/L167F) that confers nirmatrelvir drug resistance with a viral fitness level similar to the wild-type. By comparing peptide and full-length Mpro protein as substrates, we demonstrate that the binding of Mpro substrate involves more than residues in the active site. Particularly, L50F and other non-active site mutations can enhance the Mpro dimer-dimer interactions and help place the nsp5-6 substrate at the enzyme catalytic center. The structural and enzymatic activity data of Mpro L50F, L50F/E166A/L167F, and others underscore the importance of considering the whole substrate protein in studying Mpro and substrate interactions, and offers important insights into Mpro function, resistance development, and inhibitor design.

Suggested Citation

  • Eric M. Lewandowski & Xiujun Zhang & Haozhou Tan & Aiden Jaskolka-Brown & Navita Kohaal & Aliaksandra Frazier & Jesper J. Madsen & Lian M. C. Jacobs & Jun Wang & Yu Chen, 2025. "Distal protein-protein interactions contribute to nirmatrelvir resistance," Nature Communications, Nature, vol. 16(1), pages 1-8, December.
  • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-56651-x
    DOI: 10.1038/s41467-025-56651-x
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