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All Studies   All Outcomes    Recent:   

Distal Protein-Protein Interactions Contribute to SARS-CoV-2 Main Protease Substrate Binding and Nirmatrelvir Resistance

Lewandowski et al., bioRxiv, doi:10.1101/2024.04.01.587566
Apr 2024  
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In Vitro and crystallographic study reveals that the L50F mutation in SARS-CoV-2 main protease (Mpro) can restore the reduced enzymatic activity caused by nirmatrelvir resistance mutations E166A/L167F through enhanced protein-protein interactions outside the active site. The L50F/E166A/L167F triple mutant exhibited near wild-type activity with Mpro protein substrates, suggesting resistance to paxlovid. The study highlights the importance of considering distal mutations when assessing drug resistance and the need for comprehensive variant surveillance.
Lewandowski et al., 2 Apr 2024, multiple countries, preprint, 10 authors. Contact: ychen1@usf.edu, junwang@pharmacy.rutgers.edu.
In Vitro studies are an important part of preclinical research, however results may be very different in vivo.
This PaperPaxlovidAll
Distal Protein-Protein Interactions Contribute to SARS-CoV-2 Main Protease Substrate Binding and Nirmatrelvir Resistance
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
doi:10.1101/2024.04.01.587566
SARS-CoV-2 main protease, M pro , is responsible for the processing of the viral polyproteins into individual proteins, including the protease itself. M pro 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. Although the mechanism of resistance for the active site mutations is apparent, the role of the non-active site mutations in fitness rescue remains elusive. In this study, we use the model system of a M pro triple mutant (L50F/E166A/L167F) that confers not only nirmatrelvir drug resistance but also a similar fitness of replication compared to the wild-type both in vitro and in vivo. By comparing peptide and full-length M pro protein as substrates, we demonstrate that the binding of M pro substrate involves more than residues in the active site. In particular, L50F and other non-active site mutations can enhance the M pro dimer-dimer interactions and help place the nsp5-6 substrate at the enzyme catalytic center. The structural and enzymatic activity data of M pro L50F, L50F/E166A/L167F, and others underscore the importance of considering the whole substrate protein in studying M pro and substrate interactions, and offers important insights into M pro function, resistance development, and inhibitor design.
Author Contributions: E.M.L, J.W, and Y.C wrote the manuscript; X.Z, A.F, and L.M.C.J carried out the gel-assay; H.T. performed the FRET enzymatic assay; E.M.L and A.J.B crystallized proteins; E.M.L and N.K was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Competing Interests: The authors declare no competing financial interests.
References
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Sanner, Olson, Spehner, Methods: Fluorescence gel assay fusion protein construct and purification The M pro C145A (C145A/L50F) and C. difficile PBP3 42-554 fusion protein was inserted into the pETGSTSumo vector. The cleavage site, SAVKRT, was inserted between M pro and PBP3 42-554. The expression constructs were transformed into Rosetta (DE3) pLysS cells. A single colony was picked and grew in LB (Luria-Bertani) media supplemented with 50 µg/mL kanamycin and 35 µg/mL chloramphenicol at 37 °C overnight. The overnight culture was then added into 1 L media at 1:100 and incubated at 37 °C until the OD600 reached 0.4. Protein expression was induced using, Biopolymers
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In this study, ' 'we use the model system of a M<jats:sup>pro</jats:sup>triple mutant (L50F/E166A/L167F) that ' 'confers not only nirmatrelvir drug resistance but also a similar fitness of replication ' 'compared to the wild-type both in vitro and in vivo. By comparing peptide and full-length ' 'M<jats:sup>pro</jats:sup>protein as substrates, we demonstrate that the binding of ' 'M<jats:sup>pro</jats:sup>substrate involves more than residues in the active site. In ' 'particular, L50F and other non-active site mutations can enhance the ' 'M<jats:sup>pro</jats:sup>dimer-dimer interactions and help place the nsp5-6 substrate at the ' 'enzyme catalytic center. 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