Transmissible SARS-CoV-2 variants with resistance to clinical protease inhibitors

First-generation vaccines and drugs have helped reduce disease severity and blunt the spread of SARS-CoV-2. However, ongoing virus transmission and evolution and increasing selective pressures have the potential to yield viral variants capable of resisting these interventions. Here, we investigate the susceptibility of natural variants of the main protease (Mpro/3CLpro) of SARS-CoV-2 to protease inhibitors. Multiple single amino acid changes in Mpro confer resistance to nirmatrelvir (the active component of Paxlovid). An additional inhibitor in clinical development, ensitrelvir, shows a different resistance mutation profile. Importantly, phylogenetic analyses indicate that nirmatrelvir-resisting variants have pre-existed the introduction of this drug into the human population and are capable of spreading. A similarly strong argument can be made for ensitrelvir. These results caution against broad administration of protease inhibitors as stand-alone therapies and encourage the development of additional protease inhibitors and other antiviral drugs with different mechanisms of action and resistance profiles. One Sentence SummaryResistance to protease inhibitor drugs, nirmatrelvir (Paxlovid) and ensitrelvir, exists in SARS-CoV-2 variants in the human population.

SARS-CoV-2 3CLpro mutations confer resistance to Paxlovid (nirmatrelvir/ritonavir) in a VSV-based, non-gain-of-function system

Protease inhibitors are among the most powerful antiviral drugs. A first protease inhibitor against the SARS-CoV-2 protease 3CLpro, Paxlovid (nirmatrelvir / ritonavir), has recently been authorized by the U.S. FDA for emergency use (EUA 105 Pfizer Paxlovid). To find resistant mutants against the protease-inhibitor-component of Paxlovid, nirmatrelvir, we engineered a chimeric Vesicular Stomatitis Virus (VSV). By replacing an intergenic region, which is essential for separate gene transcription, with 3CLpro, this chimeric VSV became dependent on the protease to process two of its genes. We then applied selective pressure with nirmatrelvir to induce mutations. The effect of those mutants was confirmed by re-introduction in the 3CLpro and testing with a recently developed cellular assay. Furthermore, we found that mutations predicted by our method already exist in SARS-CoV-2 sequence depositions in NCBI and GISAID data bases. These may represent emerging resistant virus variants or a natural heterogeneity in the susceptibility to nirmatrelvir. One-Sentence SummaryMutations of the main protease of SARS-CoV-2 result in resistance against licensed drugs such as Paxlovid (nirmatrelvir / ritonavir). Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/495455v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1d539d3org.highwire.dtl.DTLVardef@1c76534org.highwire.dtl.DTLVardef@1c54c4corg.highwire.dtl.DTLVardef@14355d_HPS_FORMAT_FIGEXP M_FIG C_FIG