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. We designed and synthesized 85 noncovalent PL(pro) inhibitors that bind to a recently discovered ubiquitin binding site and the known BL2 groove pocket near the S4 subsite. Leads inhibited PL(pro) with the inhibitory constant K(i) values from 13.2 to 88.2 nanomolar. 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 micromolar. Oral treatment with Jun12682 improved survival and reduced lung viral loads and lesions in a SARS-CoV-2 infection mouse model, suggesting that PL(pro) inhibitors are promising oral SARS-CoV-2 antiviral candidates.