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

Abstract

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 (IC 50 = 0.1−0.3 μM) and antiviral activity (EC 50 = 0.09−0.96 μM). Fumaramide inhibitors Jun13567 (15), Jun13728 (16), and Jun13714 (18) showed favorable in vivo pharmacokinetic properties with intraperitoneal injection. The X-ray crystal structure of PL pro with Jun13567 (15) 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.