Search and prove of efficient inhibitors against papain-like protease from SARS-CoV-2

Papain-like protease (PLpro) is a key protease encoded by SARS-CoV-2, essential for viral polyprotein processing, replicase-transcriptase complex assembly, and interference with host immune responses. New COVID-19 cases continue to emerge, and WHO has identified coronaviruses with similar PLpro structures as potential pandemic threats. Using molecular modeling, docking, and protease activity assays, we identified four potent inhibitors of SARS-CoV-2 /PLpro with IC₅₀ values of 6.96-20.21 µM. Their binding affinities, determined by fluorescence titration, yielded dissociation constants (KD) of 4.18-13.06, supporting their inhibitory activity. In silico toxicity studies suggest that most inhibitors have LD₅₀ values comparable to GRL0617, a known PLpro inhibitor. Structural analysis revealed that all inhibitors interact with key residues Asp¹⁶⁴, Pro²⁴⁸, Tyr²⁶⁴, Tyr²⁶⁸, and Gln²⁶⁹ of PLpro, but differ in specific hydrogen bonding and hydrophobic interactions. Notably, compound P636-0664 exhibits the most extensive interactions, forming 2 hydrogen bonds and 13 hydrophobic contacts. These findings provide a structural basis for the optimization of inhibitors targeting the SARS-CoV-2 PLpro, contributing directly to anti-coronavirus drug discovery.