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

Abstract

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.

Keywords: SARS-CoV-2; cathepsin cross-reactivity; main protease; papain-like protease; viral entry.

Figure 1.. Design of quenched-fluorescent M_pro substrates for the inhibitor screening assay.

A) Chemical structures of internally quenched M^pro substrates. B) Progress curves and C) initial velocities of M^pro substrates. 10 μM substrate was added to 100 nM M^pro immediately prior to fluorescence readout.

Figure 2.. Screening of covalent inhibitor library against SARS-CoV-2 M_pro and PL^pro.

Residual activity of A) M^pro and B) PL^pro after 30 minutes incubation with 20 μM of each compound measured by cleavage rate of M^pro substrate 2 and Ac-LRGG-ACC for PL^pro. C) Structures of M^pro hit compounds and D) their kinetic inhibition values measured without preincubation. Data are means ± SD of at least two replicate experiments

Figure 3.. Potency of M_pro hits in cellular SARS-CoV-2 infection assays.

A) Two out of six newly identified M^pro inhibitors are active in A549+ACE2 infection model. B) SARS-CoV-2 inhibition curves of Remdesivir, E64d and K11777 in A549+ACE2 cells with or without expression of TMPRSS2. C) SARS-CoV-2 inhibition curves of M^pro inhibitors JCP400 and JCP403 in A549+ACE2 cells with or without expression of TMPRSS2. Data are means ± SD of two replicate experiments.

Figure 4.. JCP400 and JCP403 inhibit cathepsin L and B.

A) JCP400 and JCP403 compete with covalent labeling of broad spectrum cathepsin ABP BMV109 in A549+ACE2 cells. Cells were incubated with each compound for 1 h prior to addition of BMV109 B) JCP400 and JCP403 inhibit substrate cleavage of recombinant cathepsin L and B. Data are means ± SD of two replicate experiments.

Figure 5.. Reported M^pro inhibitors cross react with cathepsin B and L.

A) Inhibition of recombinant cathepsins. Protease was incubated for 10 min with inhibitor prior to addition of substrate 6QC and fluorescent readout. Data are means ± SD of two replicate experiments. B) In-cell competition labeling with BMV109. A549+ACE2 cells were subjected to 1h treatment with inhibitor at indicated concentrations followed by 1h incubation with 1 μM BMV109. Cells were lysed and ran on SDS-PAGE gels that were scanned for in-gel fluorescence. Bar graphs represent relative densitometric quantification of two replicate experiments ± SD. C) Plots of EC₅₀ curves of reported M^pro inhibitors in A549+ACE2 cells +/− TMPRSS2. Data are means ± SD of two replicate experiments.