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The main protease (Mpro) of SARS-CoV-2 is a key drug target for the development of antiviral therapeutics. Here, we designed and synthesized a series of small-molecule peptidomimetics with various cysteine-reactive electrophiles. Several compounds were identified as potent SARS-CoV-2 Mproinhibitors, including compounds 8n(IC50= 0.0752 μM), 8p(IC50= 0.0887 μM), 8r(IC50= 0.0199 μM), 10a(IC50= 0.0376 μM), 10c(IC50= 0.0177 μM), and 10f(IC50= 0.0130 μM). Most of them additionally inhibited cathepsin L and were also active against SARS-CoV-1 and MERS-CoV Mpro. In Calu-3 cells, several inhibitors, including 8r, 10a, and 10c, displayed high antiviral activity in the nanomolar range without showing cellular toxicity. The cocrystal structure of SARS-CoV-2 Mproin complex with 8prevealed covalent binding to the enzyme’s catalytic residue Cys145 and showed specific, unprecedented interactions within the substrate binding pocket. Compounds 10cand especially 8nwere effective against a panel of naturally occurring nirmatrelvir-resistant mutants, particularly E166V, and showed metabolic stability and additional favorable pharmacokinetic properties, making it a suitable candidate for further preclinical development.

Matriptase and hepsin belong to the family of type II transmembrane serine proteases (TTSPs). Increased activity of these and the plasma protease, hepatocyte growth factor activator (HGFA), is associated with unregulated cell signaling and tumor progression through increased MET and RON kinase signaling pathways. These proteases are highly expressed in multiple solid tumors and hematological malignancies. Herein, we detail the synthesis and structure–activity relationships (SAR) of a dipeptide library bearing Arg α-ketobenozothiazole (kbt) warheads as novel inhibitors of HGFA, matriptase, and hepsin. We elucidated the substrate specificity for HGFA using positional scanning of substrate combinatorial libraries (PS-SCL), which was used to discover selective inhibitors of matriptase and hepsin. Using these selective inhibitors, we have clarified the specific role of hepsin in maintaining epithelial cell membrane integrity, known to be lost in breast cancer progression. These selective compounds are useful as chemical biology tools and for future drug discovery efforts.

Cathepsin L is a key host cysteine protease utilized by coronaviruses for cell entry and is a promising drug target for novel antivirals against SARS-CoV-2. The marine natural product gallinamide A and several synthetic analogues were identified as potent inhibitors of cathepsin L with IC50values in the picomolar range. Lead molecules possessed selectivity over other cathepsins and alternative host proteases involved in viral entry. Gallinamide A directly interacted with cathepsin L in cells and, together with two lead analogues, potently inhibited SARS-CoV-2 infection in vitro, with EC50values in the nanomolar range. Reduced antiviral activity was observed in cells overexpressing transmembrane protease, serine 2 (TMPRSS2); however, a synergistic improvement in antiviral activity was achieved when combined with a TMPRSS2 inhibitor. These data highlight the potential of cathepsin L as a COVID-19 drug target as well as the likely need to inhibit multiple routes of viral entry to achieve efficacy.