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

Abstract

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 (VOC) or variants of interest (VOI): Alpha (α, B.1.1.7), Beta (β, B.1.351), Delta (δ, B1.617.2), Gamma (γ, P.1), Lambda (λ, B.1.1.1.37/C37), Omicron (ο, B.1.1.529) as well as the original Washington or wildtype strain. These VOC/VOI carry prevalent mutations at varying frequencies in the M ^pro specifically for: α, β, γ (K90R), λ (G15S) and ο (P132H). In vitro biochemical enzymatic assay characterization of the enzyme kinetics of the mutant M ^pros demonstrate 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 bases for these observations were provided by solution-phase structural dynamics and structural determination of nirmatrelvir bound to the ο, λ and β M ^pro at 1.63 - 2.09 Å 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.