Structural basis for the in vitro efficacy of nirmatrelvir against SARS-CoV-2 variants

The COVID-19 pandemic continues to be a public health threat with emerging variants of SARS-CoV-2. Nirmatrelvir (PF-07321332) is a reversible, covalent inhibitor targeting the main protease (M-pro) of SARS-CoV-2 and the active protease inhibitor in PAXLOVID (nirmatrelvir tablets and ritonavir tablets). However, the efficacy of nirmatrelvir is underdetermined against evolving SARS-CoV-2 variants. Here, we evaluated the in vitro catalytic activity and potency of nirmatrelvir against the M-pro of prevalent variants of concern (VOCs) or variants of interest (VOIs): Alpha (alpha, B.1.1.7), Beta (beta, B.1.351), Delta (delta, B1.617.2), Gamma (gamma, P.1), Lambda (lambda, B.1.1.1.37/C37), Omicron (omicron, B.1.1.529), as well as the original Washington or wildtype strain. These VOCs/VOIs carry prevalent mutations at varying frequencies in the M-pro specifically for alpha, beta, gamma (K90R), lambda (G15S), and omicron (P132H). In vitro biochemical enzymatic assay characterization of the enzyme kinetics of the mutant M-pros demonstrates that they are catalytically comparable to wildtype. We found that nirmatrelvir has similar potency against each mutant M-pro including P132H that is observed in the Omicron variant with a Ki of 0.635 nM as compared to a Ki of 0.933 nM for wildtype. The molecular basis for these observations were provided by solution-phase structural dynamics and structural determination of nirmatrelvir bound to the omicron, lambda, and beta M-pro at 1.63 to 2.09 angstrom resolution. These in vitro data suggest that PAXLOVID has the potential to maintain plasma concentrations of nirmatrelvir many-fold times higher than the amount required to stop the SARS-CoV-2 VOC/VOI, including Omicron, from replicating in cells.

Automated summary

The efficacy and potency of nirmatrelvir against different variants of SARS-CoV-2 were evaluated. The results showed that nirmatrelvir has similar potency against mutant M-pros, including Omicron, and can maintain high concentrations in cells to inhibit the replication of these variants.