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

Kas Steuten,Matthew Bogyo,Oguz Bolgi,Scott Lovell,Christoph Peters,John C Widen,Berati Cerikan,Mirko Cortese,Ruth Geiss-Friedlander,Ryan K Muir,Heeyoung Kim,Christopher J Neufeldt,Brett M Babin,Ouma Onguka,John M Bennett,Ralf Bartenschlager

doi:10.1021/acsinfecdis.0c00815

Kas Steuten, Matthew Bogyo + Show 14 more

Open Access

https://doi.org/10.1021/acsinfecdis.0c00815

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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 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.

Highlights

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
To identify potential inhibitors of main protease (Mpro) and papain-like protease (PLpro), we developed fluorogenic substrate assays that allowed us to screen a focused library of cysteine reactive molecules
For our two lead Mpro inhibitors, we found that their apparent EC50 values dropped by 2−3-fold upon expression of transmembrane protease serine 2 (TMPRSS2) (Figure 3C)

Summary

■ RESULTS AND DISCUSSION

To identify potential inhibitors of Mpro and PLpro, we developed fluorogenic substrate assays that allowed us to screen a focused library of cysteine reactive molecules. As further validation of the off-target activity of the two lead molecules, we tested the compounds for their ability to inhibit purified Cats B and L enzymes These results confirmed that both are relatively potent inhibitors of cathepsins with IC50 values in the low micromolar range (Figure 4A,B). Recently, has inhibition of processing of a genetically expressed Mpro substrate or labeling of active Mpro enzyme been established as a measure of Mpro activity in cells.[7,42] In this work, we describe our efforts to screen a library of approximately 650 diverse covalent inhibitor scaffolds against the two primary SARS-CoV-2 cysteine proteases, Mpro and PLpro.

MS systems used were either a Thermo Fisher Finnigan

■ ACKNOWLEDGMENTS

■ REFERENCES