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 Mpro and PLpro
proteases. These efforts identified a small number of hits for the Mpro
protease and no viable hits for the PLpro
protease. Of the Mpro 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 Mpro inhibitors are also potent inhibitors of host cell
cysteine cathepsins. To determine if this is a general property of Mpro 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 Mpro and PLpro
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.