Abstract Atazanavir (ATV) has already been considered as a potential repurposing drug to 2019 coronavirus disease (COVID-19), however, there are controversial reports on its mechanism of action and effectiveness as anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Through the pre-clinical chain of experiments: enzymatic, molecular docking, cell-based, and in vivo assays, it is demonstrated here that both SARS-CoV-2 B.1 lineage and variant of concern gamma are susceptible to this antiretroviral. Enzymatic assays and molecular docking calculations showed that SARS-CoV-2 main protease (M pro ) was inhibited by ATV, with Morrison’s inhibitory constant (Ki) 1.5-fold higher than boceprevir (GC376, a positive control). ATV was a competitive inhibition, increasing the M pro ’s Michaelis-Menten (K m ) more than 6-fold. Cell-based assays indicated that SARS-CoV-2 gamma is more susceptible to ATV than its predecessor strain B.1. Using oral administration of ATV in mice to reach plasmatic exposure similar to humans, transgenic mice expression in human angiotensin converting enzyme 2 (K18-hACE2) were partially protected against lethal challenge with SARS-CoV-2 gamma. Moreover, less cell death and inflammation were observed in the lung from infected and treated mice. Our studies may contribute to a better comprehension of the M pro /ATV interaction, which could pave the way to the development of specific inhibitors of this viral protease.

Atazanavir is a competitive inhibitor of SARS-CoV-2 Mpro, impairing variants replication in vitro and in vivo