Nirmatrelvir, an orally active Mpro inhibitor, is a potent inhibitor of SARS-CoV-2 Variants of Concern

Abstract

New variants of SARS-CoV-2 with potential for enhanced transmission, replication, and immune evasion capabilities continue to emerge causing reduced vaccine efficacy and/or treatment failure. As of January 2021, the WHO has defined five variants of concern (VOC): B.1.1.7 (Alpha, α), B.1.351 (Beta, β), P.1 (Gamma, γ), B.1.617.2 (Delta, δ), and B.1.1.529 (Omicron, o). To provide a therapeutic option for the treatment of COVID-19 and variants, Nirmatrelvir, the antiviral component of PAXLOVIDTM, an oral outpatient treatment recently authorized for conditional or emergency use treatment of COVID-19, was developed to inhibit SARS-CoV-2 replication. Nirmatrelvir (PF-07321332) is a specific inhibitor of coronavirus main protease (Mpro, also referred to as 3CLpro), with potent antiviral activity against several human coronaviruses, including SARS-CoV-2, SARS-CoV, and MERS (Owen et al, Science 2021. doi: 10.1126/science.abl4784). Here, we evaluated PF-07321332 against the five SARS-CoV-2 VOC (α, β, γ, δ, o) and two Variants of Interest or VOI, C.37 (λ) and B.1.621 (μ), using qRT-PCR in VeroE6 cells lacking the P-glycoprotein (Pgp) multidrug transporter gene (VeroE6 P-gp knockout cells). Nirmatrelvir potently inhibited USA-WA1/2020 strain, and α, β, γ, λ, δ, μ, and o variants in VeroE6 P-gp knockout cells with mean EC50 values 38.0 nM, 41.0 nM, 127.2 nM, 24.9 nM, 21.2 nM, 15.9 nM, 25.7 nM and 16.2 nM, respectively. Sequence analysis of the Mpro encoded by the variants showed ~100% identity of active site amino acid sequences, reflecting the essential role of Mpro during viral replication leading to ability of Nirmatrelvir to exhibit potent activity across all the variants.

Structural basis for Nirmatrelvir in vitro efficacy against SARS-CoV-2 variants

“…These observations are consistent with recently reported biochemical characterization with similar kcat/Km and nirmatrelvir potency values (10,11). Nirmatrelvir also appears to retain its in vitro antiviral efficacy against Omicron relative to wildtype (12)(13)(14)(15).…”

Genetic surveillance of SARS-CoV-2 Mpro reveals high sequence and structural conservation prior to the introduction of protease inhibitor Paxlovid

“…In late 2021, P132H became the most prevalent M pro mutation with its frequency rapidly jumping from 0.012% to 6.15% after the Omicron surge, although this mutation does not necessarily offer any selective advantage on viral fitness, or alter inhibitor potency of nirmatrelvir (50). As expected, nirmatrelvir maintains antiviral activity against all five VOCs and two VOIs, including Omicron, Beta and Lambda which carry P132H, K90R and G15S mutations, respectively, in M pro (51–55). This, however, may change with widespread use of nirmatrelvir, which, not unlike the antibodies against the S protein, may exert selective pressure on its target, leading to a reduction of potency.…”

COVID-19 at a Glance: An Up-to-Date Overview on Variants, Drug Design and Therapies

“…Nirmatrelvir, molnupiravir, and remdesivir were tested in vitro against a panel of SARS-CoV-2 variants in live-virus antiviral assays. It was demonstrated that nirmatrelvir, as well as other clinically relevant antivirals, maintains its activity against all variants tested, including Omicron [ 147 ], confirming what was previously reported [ 148 , 149 ]. The isolation of the Omicron variant was reported for the first time by Yadav et al (2022) [ 150 ] by using an in vivo followed by in vitro method of SARS-CoV-2 virus culture; more specifically, it was obtained from infected Syrian hamsters and the subsequent passage into Vero CCL-81 cells, since the Omicron variant’s isolation in Vero CCL-81 from the clinical specimens of COVID-19 cases failed.…”