Challenges for targeting SARS-CoV-2 proteases as a therapeutic strategy for COVID-19

Two proteases produced by the SARS-CoV-2 virus, M ^pro and PL ^pro , 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 cathepsin 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.