Fragment-based design of SARS-CoV-2 PLpro inhibitors using molecular modeling techniques

Respiratory syndrome coronavirus type 2 (SARS-CoV-2) has so far represented the most severe pandemic of the twenty first century. Currently, the number of new cases and deaths produced by the most recent SARS-CoV-2 variants has been drastically reduced mainly due to the great deal of resources invested in vaccination campaigns. However, given the expected seasonal trend of SARS-CoV-2 there is a clear need of finding unexplored targets which could protect against future emergent variants. The papain-like protease (PLpro) moonlights as a potent suppressor of the host’s innate immune mechanisms due to its ability to cleave ubiquitin and ISG15 posttranslational modifications. The release of extracellular free ISG15 through the viral PLpro has been proven to promote the “cytokine storm” in COVID-19. Therefore, targeting PLpro represents a promising therapeutic strategy that would not only suppress SARS-CoV-2 replication but also restore antiviral immunity in the host and possibly alleviate the characteristic hyper-inflammation in COVID-19 patients. In 2022, Srinivasan et al. reported three single-digit micromolar inhibitors with a hydroxybenzene-like structure binding to an allosteric binding pocket in PLpro. This finding encouraged us to further explore hydroxybenzene based scaffolds with possibly higher inhibitory activity against PLpro. In this study, we developed a fragment-based hit discovery strategy using DOCK3.7 as the central compound screening engine and an enzymatic assay as the preliminary hit validation method. Large-scale docking of tranches of fragment-like compounds from the ZINC20-22 databases provided twenty docking hits. Further validation of the docking hits using a protease inhibition assay resulted in a two-digit micromolar fragment inhibitor, which is the most efficient fragment found so far in the research project.