Structure-Based Design of Covalent SARS-CoV-2 Papain-like Protease Inhibitors

The COVID-19 pandemic is caused by SARS-CoV-2, a highly transmissible and pathogenic RNA betacoronavirus. Like other RNA viruses, SARS-CoV-2 continues to evolve with or without drug selection pressure, and many variants have emerged since the beginning of the pandemic. The papain-like protease, PL^pro, is a cysteine protease that cleaves viral polyproteins as well as ubiquitin and ISG15 modifications from host proteins. Leveraging our recently discovered Val70^Ub binding site in PL^pro, we designed covalent PL^pro inhibitors by connecting cysteine reactive warheads to the biarylphenyl PL^pro inhibitors via flexible linkers. Several leads displayed potent enzymatic inhibition (IC₅₀ = 0.1-0.3 μM) and antiviral activity (EC₅₀ = 0.09-0.96 μM). Fumaramide inhibitors <b>Jun13567 (15)</b>, <b>Jun13728 (16)</b>, and <b>Jun13714 (18)</b> showed favorable <i>in vivo</i> pharmacokinetic properties with intraperitoneal injection. The X-ray crystal structure of PL^pro with <b>Jun13567 (15)</b> validated our design strategy, revealing covalent conjugation between the catalytic Cys111 and the fumaramide warhead. The results suggest these covalent PL^pro inhibitors are promising SARS-CoV-2 antiviral drug candidates.