Abstract
Nirmatrelvir was the first protease inhibitor (PI) specifically developed against the SARS-CoV-2 main protease (3CLpro/Mpro) and licensed for clinical use. As SARS-CoV-2 continues to spread, variants resistant to nirmatrelvir and other currently available treatments are likely to arise. This study aimed to identify and characterize mutations that confer resistance to nirmatrelvir. To safely generate Mpro resistance mutations, we passaged a previously developed chimeric vesicular stomatitis virus (VSV-Mpro) with increasing, yet suboptimal concentrations of nirmatrelvir, using Wuhan-1 and Omicron Mpro variants, and selected a large set of mutants. Some mutations are frequently present in GISAID, suggesting their relevance in SARS-CoV-2. The resistance phenotype of a subset of mutations was characterized against clinically available PIs (nirmatrelvir and ensitrelvir) with cell-based and biochemical assays. Moreover, we showed the putative molecular mechanism of resistance based on in silico molecular modelling. These findings will help to understand SARS-CoV-2 protease-inhibitor-resistance mechanisms, the relevance of specific in the clinic and thereby inform treatment decisions.
Teaser Understanding how SARS-CoV-2 could counter the antiviral drug nirmatrelvir and what it means for the future of COVID-19 treatment.
Competing Interest Statement
D.v.L. is founder of ViraTherapeutics GmbH. D.v.L serves as a scientific advisor to Boehringer Ingelheim and Pharma KG. E.H. and D.v.L have received an Austrian Science Fund (FWF) grant in the special call "SARS urgent funding". E.H. is a registered consultant at Guidepoint. D. Bante holds stocks of Pfizer Inc. and Oxford Nanopore Technologies plc. All other authors declare they have no competing interests. S.A.M and R.S.H. are inventors on the patent application "Live cell assay for protease inhibition", application number WO/2022/094463.
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