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

Abstract

SARS-CoV-2 continues to represent a global health emergency as a highly transmissible, airborne virus. An important coronaviral drug target for treatment of COVID-19 is the conserved main protease (Mpro). Nirmatrelvir is a potent Mpro inhibitor and the antiviral component of Paxlovid™. The significant viral sequencing effort during the ongoing COVID-19 pandemic represented a unique opportunity to assess potential nirmatrelvir escape mutations from emerging variants of SARS-CoV-2. To establish the baseline mutational landscape of Mpro prior to the introduction of Mpro inhibitors, Mpro sequences and its cleavage junction regions were retrieved from ~4,892,000 high-quality SARS-CoV-2 genomes in GISAID. Any mutations identified from comparison to the reference sequence (Wuhan-hu-1) were cataloged and analyzed. Mutations at sites key to nirmatrelvir binding and protease functionality (e.g., dimerization sites) were still rare. Structural comparison of Mpro also showed conservation of key nirmatrelvir contact residues across the extended Coronaviridae family (alpha-, beta-, and gamma-coronaviruses). Additionally, we showed that over time the SARS-CoV-2 Mpro enzyme remained under purifying selection and was highly conserved relative to the spike protein. Now, with the EUA approval of Paxlovid and its expected widespread use across the globe, it is essential to continue large-scale genomic surveillance of SARS-CoV-2 Mpro evolution. This study establishes a robust analysis framework for monitoring emergent mutations in millions of virus isolates, with the goal of identifying potential resistance to present and future SARS-CoV-2 antivirals.

The substitutions L50F, E166A and L167F in SARS-CoV-2 3CLpro are selected by a protease inhibitorin vitroand confer resistance to nirmatrelvir

“…A recent sequence analysis across 4.9 million global SARS-CoV-2 isolates, circulating prior to the introduction of Paxlovid, showed a high genetic conservation of the 3CLpro protein [31]. From the 305 amino acid positions, only 3 have a polymorphism in >1% of the samples and 13 have a change in 1% - 0.11% of the samples.…”

A ricin-based peptide BRIP from Hordeum vulgare inhibits Mpro of SARS-CoV-2

“…We focused on the mutations reported in current circulating lineages of SARS-CoV-2, i.e., Delta and Omicron. The mutations reported in M pro protein of the Delta variant are at positions 1, 4, 7, 12, 14, 122, 125, 166, 285, 286, and 298 42 . Among these, mutation of Glu166 could only have an impact on M pro -BRIP binding.…”

Recent changes in the mutational dynamics of the SARS-CoV-2 main-protease substantiate the danger of emerging resistance to antiviral drugs.

“…In-host evolution of a virus under pressure from an antiviral drug leads to mutations that reduce the effectiveness of said drug 36,44 . While this likely comes with a somewhat reduced natural function of the viral target protein (such as the SARS-CoV-2 M pro ), the net effect for the virus might still be advantageous.…”

“…P zer's recent M pro analyses demonstrated high sequence and structural conservation prior to the widespread use of nirmatrelvir. However, the authors emphasize the importance of continuous genetic surveillance of M pro due to the risk of drug-resistance development 36 .…”