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 Summary Resistance to protease inhibitor drugs, nirmatrelvir (Paxlovid) and ensitrelvir, exists in SARS-CoV-2 variants in the human population. ### Competing Interest Statement DvL is founder of ViraTherapeutics GmbH and serves as a scientific advisor to Boehringer Ingelheim and Pharma KG. The other authors have no competing interests to declare.