Thiol esters as chemical warheads of SARS-CoV-2 main protease (3CLpro) peptide-like inhibitors

Peptide-like 3CLpro covalent binding inhibitors are the most effective antiviral drugs for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Their covalent warheads were designed based on the addition reaction activity of the aldehyde (ketone) carbonyl or its derivative structures. These addition reactions between the warheads and the thiol of the 3CLpro are reversible, and the resulting hemimonothioacetals are chemically unstable. Herein, after DFT calculation, we designed thiol ester warheads using the principle of ester exchange reaction. Then, the warhead fluorescence probe binding experiment suggested these adducts of thiol ester warheads and 3CLpro protein are more stable than the hemimonothioacetals mentioned earlier. Therefore, new 3CLpro inhibitors were subsequently designed through a structure-based drug design method employing those thiol ester warheads. Those 3CLpro inhibitors demonstrated potent 3CLpro inhibitory activities and anti-coronavirus HCoV-OC43 activities. Among them, B16 stands out as the most promising, demonstrating not only the strongest anti-coronavirus HCoV-OC43 activity but also being a moderate inhibitor of CYP3A4, suggesting that B16 does not require co-administration with ritonavir in the treatment of SARS-CoV-2 infection. This work demonstrates the significant potential of thiol esters as novel chemical warheads in designing covalent binding inhibitors for 3CLpro and beyond.