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Pro108Ser mutation of SARS-CoV-2 3CL^pro reduces the enzyme activity and ameliorates the clinical severity of COVID-19.

ABSTRACT: Recently, an international randomized controlled clinical trial showed that patients with SARS-CoV-2 infection treated orally with the 3-chymotrypsin-like protease (3CL^pro) inhibitor PF-07321332 within three days of symptom onset showed an 89% lower risk of COVID-19-related hospital admission/ death from any cause as compared with the patients who received placebo. Lending support to this critically important result of the aforementioned trial, we demonstrated in our study that patients infected with a SARS-Cov-2 sub-lineage (B.1.1.284) carrying the Pro108Ser mutation in 3CL^pro tended to have a comparatively milder clinical course (i.e., a smaller proportion of patients required oxygen supplementation during the clinical course) than patients infected with the same sub-lineage of virus not carrying the mutation. Characterization of the mutant 3CL^pro revealed that the Kcat/Km of the 3CL^pro enzyme containing Ser108 was 58% lower than that of Pro108 3CL^pro. Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) revealed that the reduced activity was associated with structural perturbation surrounding the substrate-binding region of the enzyme, which is positioned behind and distant from the 108th amino acid residue. Our findings of the attenuated clinical course of COVID-19 in patients infected with SARS-CoV-2 strains with reduced 3CL^pro enzymatic activity greatly endorses the promising result of the aforementioned clinical trial of the 3CL^pro inhibitor.

SUBMITTER: Abe K

PROVIDER: S-EPMC8789791 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The main protease, Mpro, of SARS-CoV-2 is required to cleave the viral polyprotein into precise functional units for virus replication and pathogenesis. Here, we report quantitative reporters for Mpro function in living cells in which protease inhibition by genetic or chemical methods results in robust signal readouts by fluorescence (enhanced green fluorescent protein [eGFP]) or bioluminescence (firefly luciferase). These gain-of-signal systems are scalable to high-throughput platforms for quantitative discrimination between Mpro mutants and/or inhibitor potencies as evidenced by validation of several reported inhibitors. Additional utility is shown by single Mpro amino acid variants and structural information combining to demonstrate that both inhibitor conformational dynamics and amino acid differences are able to influence inhibitor potency. We further show that a recent variant of concern (Omicron) has an unchanged response to a clinically approved drug, nirmatrelvir, whereas proteases from divergent coronavirus species show differential susceptibility. Together, we demonstrate that these gain-of-signal systems serve as robust, facile, and scalable assays for live cell quantification of Mpro inhibition, which will help expedite the development of next-generation antivirals and enable the rapid testing of emerging variants. IMPORTANCE The main protease, Mpro, of SARS-CoV-2 is an essential viral protein required for the earliest steps of infection. It is therefore an attractive target for antiviral drug development. Here, we report the development and implementation of two complementary cell-based systems for quantification of Mpro inhibition by genetic or chemical approaches. The first is fluorescence based (eGFP), and the second is luminescence based (firefly luciferase). Importantly, both systems rely upon gain-of-signal readouts such that stronger inhibitors yield higher fluorescent or luminescent signal. The high versatility and utility of these systems are demonstrated by characterizing Mpro mutants and natural variants, including Omicron, as well as a panel of existing inhibitors. These systems rapidly, safely, and sensitively identify Mpro variants with altered susceptibilities to inhibition, triage-nonspecific, or off-target molecules and validate bona fide inhibitors, with the most potent thus far being the first-in-class drug nirmatrelvir.

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Pro108Ser mutation of SARS-CoV-2 3CL^pro reduces the enzyme activity and ameliorates the clinical severity of COVID-19.

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OmicsDI is part of the ELIXIR infrastructure

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Dataset Information

Pro108Ser mutation of SARS-CoV-2 3CLpro reduces the enzyme activity and ameliorates the clinical severity of COVID-19.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Pro108Ser mutation of SARS-CoV-2 3CL^pro reduces the enzyme activity and ameliorates the clinical severity of COVID-19.