Discovery of Fragment-Based Inhibitors of SARS-CoV-2 PL^Pro

SARS-CoV-2 papain-like protease (PL^Pro) plays a key role in viral replication and the host immune response and is a promising target for developing new antiviral treatments. We previously reported a fragment-based screen to identify hits that bind to SARS-CoV-2 PL^Pro. Here, we describe the discovery of potent PL^Pro inhibitors by optimizing one of these hits via extensive medicinal chemistry guided by multiple X-ray structures of cocomplexes. Lead compound 46 is shown to bind to the S3 and S4 pockets with nanomolar affinity (0.4 μM) and exhibits robust cellular activity and resistance to mutation. This novel class of PL^Pro inhibitors can potentially be used as a starting point for the development of inhibitors to combat the emergence of drug-resistant viral strains and future coronavirus outbreaks.