Design, Synthesis, and Unprecedented Interactions of Covalent Dipeptide-Based Inhibitors of SARS-CoV-2 Main Protease and Its Variants Displaying Potent Antiviral Activity.

The main protease (M) 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 inhibitors, including compounds (IC = 0.0752 μM), (IC = 0.0887 μM), (IC = 0.0199 μM), (IC = 0.0376 μM), (IC = 0.0177 μM), and (IC = 0.0130 μM). Most of them additionally inhibited cathepsin L and were also active against SARS-CoV-1 and MERS-CoV M. In Calu-3 cells, several inhibitors, including , , and , displayed high antiviral activity in the nanomolar range without showing cellular toxicity. The cocrystal structure of SARS-CoV-2 M in complex with revealed covalent binding to the enzyme's catalytic residue Cys145 and showed specific, unprecedented interactions within the substrate binding pocket. Compounds and especially 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.