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 (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 Mpro 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 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 Mpro 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.