Challenges for Targeting SARS-CoV-2 Proteases as a Therapeutic Strategy for COVID-19

Two proteases produced by the SARS-CoV-2 virus, the main protease and papain-like protease, are essential for viral replication and have become the focus of drug development programs for treatment of COVID-19. We screened a highly focused library of compounds containing covalent warheads designed to target cysteine proteases to identify new lead scaffolds for both M^pro and PL^pro proteases. These efforts identified a small number of hits for the M^pro protease and no viable hits for the PL^pro protease. Of the M^pro hits identified as inhibitors of the purified recombinant protease, only two compounds inhibited viral infectivity in cellular infection assays. However, we observed a substantial drop in antiviral potency upon expression of TMPRSS2, a transmembrane serine protease that acts in an alternative viral entry pathway to the lysosomal cathepsins. This loss of potency is explained by the fact that our lead M^pro inhibitors are also potent inhibitors of host cell cysteine cathepsins. To determine if this is a general property of M^pro inhibitors, we evaluated several recently reported compounds and found that they are also effective inhibitors of purified human cathepsins L and B and showed similar loss in activity in cells expressing TMPRSS2. Our results highlight the challenges of targeting M^pro and PL^pro proteases and demonstrate the need to carefully assess selectivity of SARS-CoV-2 protease inhibitors to prevent clinical advancement of compounds that function through inhibition of a redundant viral entry pathway.