Abstract
The emergence of SARS-CoV-2 variants and drug-resistant mutants calls for additional oral antivirals. The SARS-CoV-2 papain-like protease (PL(pro)) is a promising but challenging drug target. In this study, we designed and synthesized 85 noncovalent PL(pro) inhibitors that bind to the newly discovered Val70(Ub) site and the known BL2 groove pocket. Potent compounds inhibited PL(pro) with inhibitory constant K(i) values from 13.2 to 88.2 nM. The co-crystal structures of PL(pro) with eight leads revealed their interaction modes. The in vivo lead Jun12682 inhibited SARS-CoV-2 and its variants, including nirmatrelvir-resistant strains with EC(50) from 0.44 to 2.02 μM. Oral treatment with Jun12682 significantly improved survival and reduced lung viral loads and lesions in a SARS-CoV-2 infection mouse model, suggesting PL(pro) inhibitors are promising oral SARS-CoV-2 antiviral candidates.