Functional and Structural Characterization of Treatment-Emergent Nirmatrelvir Resistance Mutations at Low Frequencies in the Main Protease (Mpro) Reveals a Unique Evolutionary Route for SARS-CoV-2 to Gain Resistance - Report - MDSpire
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Functional and Structural Characterization of Treatment-Emergent Nirmatrelvir Resistance Mutations at Low Frequencies in the Main Protease (Mpro) Reveals a Unique Evolutionary Route for SARS-CoV-2 to Gain Resistance
This study identified novel low-frequency mutations in the SARS-CoV-2 main protease (Mpro) from patients treated with nirmatrelvir-ritonavir (NIR-RIT) who remained SARS-CoV-2 positive. These mutations confer complete resistance to NIR without compromising Mpro enzymatic activity, revealing a distinct evolutionary pathway for antiviral resistance.
Background
Nirmatrelvir-ritonavir (NIR-RIT) targets the SARS-CoV-2 main protease (Mpro), essential for viral replication by cleaving polyproteins. Resistance mutations in Mpro have been reported primarily from in vitro studies, often affecting the active site and reducing drug susceptibility. However, clinical data on resistance mutations, especially low-frequency variants emerging after treatment, remain limited. Understanding these mutations is critical to preserving antiviral efficacy and guiding treatment strategies.
Disruption of substrate binding site dynamics and protein stability
Key Findings
Two novel clinical Mpro variants (D48D/L58F/P132H and D48D/L67V/K90R/P132H) were identified in patients treated with NIR-RIT who remained SARS-CoV-2 positive.
These mutations localize to the N-terminal domain, away from the catalytic site, yet confer complete resistance to nirmatrelvir.
Mpro enzymatic activity remains intact despite these mutations, indicating resistance arises without loss of protease function.
Structural and thermal analyses suggest these mutations alter substrate binding site dynamics and reduce protein stability, potentially affecting substrate binding or dimerization.
These resistance mutations were not previously observed in cell culture studies, indicating a distinct in vivo evolutionary pathway.
Cross-resistance to other Mpro inhibitors, such as C5a, was also observed, highlighting broader implications for antiviral therapy.
Clinical Implications
Clinicians should be aware that resistance to nirmatrelvir-ritonavir can emerge in treated patients via mutations outside the Mpro active site, which may not compromise viral protease function but reduce drug efficacy. Monitoring for low-frequency resistance variants in clinical samples is important, especially in patients with persistent infection post-treatment. These findings underscore the need for ongoing surveillance and development of alternative or combination antiviral strategies to mitigate resistance.
Conclusion
This study uncovers novel low-frequency Mpro mutations conferring nirmatrelvir resistance through a unique evolutionary mechanism distinct from previously characterized active site mutations. These insights are critical for guiding future antiviral development and resistance management in SARS-CoV-2.
References
Author/Source/Year -- Analysis of Low-Frequency Nirmatrelvir Resistance Mutations in the Main Protease (Mpro)
by Natalie M Deschenes, Jimena Pérez-Vargas, Zoe Zhong, Merrilee Thomas, Calem Kenward, Wesley A Mosimann, Liam J Worrall, Nicholas Waglechner, Angel XinLiu Li, Finlay Maguire, Patryk Aftanas, Jason R Smith, Jared Lim, Robert N Young, Artem Cherkasov, Lubna Farooqi, Adnan Moinuddin, Lina Siddiqi, Imaan Malik, Maxime Lefebvre, Mark Paetzel, Natalie C J Strynadka, François Jean, Allison McGeer, Robert A Kozak
