Investigation of small molecule inhibitors of the SARS-CoV-2 papain-like protease by all-atom microsecond modelling, PELE Monte Carlo simulations, and in vitro activity inhibition

[Display omitted] •The SARS-CoV-2 PLpro is a critical target for development of potential antivirals.•PLpro has protease as well as deubiquitinating (DUB) and deISGylating activities.•Inhibitors such as GRL0617 and hypericin interact with the naphthalene (NP) pocket.•MD (µs) and PELE Monte Carlo simulations highlight favorable binding in the NP site.•GRL0617 and hypericin inhibit PLpro protease and DUB activities (µM range) in vitro. The SARS-CoV-2 papain-like (PLpro) protease is essential for viral replication. We investigated potential antiviral effects of hypericin relative to the well-known noncovalent PLpro inhibitor GRL-0617. Molecular dynamics and PELE Monte Carlo simulations highlight favourable binding of hypericin and GRL-0617 to the naphthalene binding pocket of PLpro. Although not potent as GRL-0617 (45.8 vs 1.6 µM for protease activity, respectively), in vitro fluorogenic enzymatic assays with hypericin show concentration-dependent inhibition of both PLpro protease and deubiquitinating activities. Given its use in supplementations and the FDA conditional approval of a synthetic version, further evaluation of hypericin as a potential SARS-CoV-2 antiviral is warranted.