Design, Synthesis, and Unprecedented Interactions of Covalent Dipeptide-Based Inhibitors of SARS-CoV-2 Main Protease...

The main protease (M^pro) 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 M^pro inhibitors, 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 M^pro. 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 M^pro in complex with 8p revealed covalent binding to the enzyme's catalytic residue Cys145 and showed specific, unprecedented interactions within the substrate binding pocket. Compounds 10c and especially 8n were 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.