SARS-CoV-2 M^pro Protease Variants of Concern Display Altered Viral Substrate and Cell Host Target Galectin-8 Processing but Retain Sensitivity toward Antivirals

Abstract

The main protease of SARS-CoV-2 (M pro ) is the most promising drug target against coronaviruses due to its essential role in virus replication. With newly emerging variants there is a concern that mutations in M pro may alter the structural and functional properties of protease and subsequently the potency of existing and potential antivirals. We explored the effect of 31 mutations belonging to 5 variants of concern (VOCs) on catalytic parameters and substrate specificity, which revealed changes in substrate binding and the rate of cleavage of a viral peptide. Crystal structures of 11 M pro mutants provided structural insight into their altered functionality. Additionally, we show M pro mutations influence proteolysis of an immunomodulatory host protein Galectin-8 (Gal-8) and a subsequent significant decrease in cytokine secretion, providing evidence for alterations in the escape of host-antiviral mechanisms. Accordingly, mutations associated with the Gamma VOC and highly virulent Delta VOC resulted in a significant increase in Gal-8 cleavage. Importantly, IC50s of nirmatrelvir (Pfizer) and our irreversible inhibitor AVI-8053 demonstrated no changes in potency for both drugs for all mutants, suggesting M pro will remain a high-priority antiviral drug candidate as SARS-CoV-2 evolves.

Contributions of hyperactive mutations in Mprofrom SARS-CoV-2 to drug resistance

“…In purified form, T21I, L50F and L141R Mpro all showed elevated ac9vity (1.6, 1.7, and 1.9 fold increased enzyme proficiency compared to WT). The enzyme ac9vity of L50F and T21I have been reported by others, and also found to be increased by a small amount (1.6 fold and 1.3 fold respec9vely) compared to WT 14,15 . The ac9vity of L50F is somewhat controversial as two studies reported null ac9vity 11,12 and one report was unable to purify this variant 14 .…”

Differential specificity of SARS‐CoV‐2 main protease variants on peptide versus protein‐based substrates

“…This enhancement in activity resulted from an increase in both binding affinity (K M ) and turnover rate (k cat ). In optimal buffer, there were no substantial changes in the kinetic constants of the protease variants toward the peptide substrate QS1, suggesting that the mutations did not change the active site space enough to affect the enzymes' function [7]. In contrast, the protease variants were noticeably different when analyzed using a protein substrate.…”

“…The HCoV genome encodes several nonstructural proteins that are synthesized as two large polyproteins pp1a and pp1ab, which are cleaved by the virus proteases named papain-like protease and the main protease (M pro ; EC3.4.22.69) [2]. The protease's primary role is to process the polyproteins pp1a and pp1ab, which mediate the functions required for viral replication and transcription [1,[3][4][5], and possibly also cleavage of host proteins [6,7]. The enzyme has a very stringent cleavage specificity, recognizing preferentially the sequence Leu-Gln↓ (Ser, Ala, Gly) [3,8].…”

The Effect of Deuteration and Homologation of the Lactam Ring of Nirmatrelvir on Its Biochemical Properties and Oxidative Metabolism

“…Our previous report demonstrated the effect of mutations in M pro from four VOCs on protease activity against viral and host substrates and furthermore showed no change in potency for nirmatrelvir. 49 To ensure that modifications in the chemical structure of 1 did not affect its ability to inhibit M pro from VOCs, we chose several variants with mutations close to the binding site of the drug.…”