Abstract 1713 Shaping antiviral strategies with potent and selective non-covalent SARS-CoV-2 papain-like protease inhibitors

SARS-CoV-2 continues to elude containment efforts and remains a significant global health challenge. There is a critical need for drugs that employ multiple mechanisms of action, ensuring a more robust and versatile approach to effectively manage both current and emerging coronavirus infections. The papain-like protease (PLpro) domain of Nsp3 is essential for viral replication in human coronaviruses (HCoV). PLpro also contributes to the COVID-19-associated 'cytokine storm' by cleaving ubiquitin and interferon-stimulated gene 15 protein (ISG15) from host proteins and disrupting host immune responses. Consequently, PLpro is a promising target for small-molecule therapeutics. In this study, we have refined non-covalent inhibitors of SARS-CoV-1 PLpro and SARS-CoV-2 PLpro from primary literature, resulting in a lead inhibitor with improved cytotoxicity values and specificity towards SARS-CoV-1 and SARS-CoV-2 PLpro while improving antiviral activity. Selectivity studies show minimal off-target activity against the closest structural homologs of HCoV PLpro. Furthermore, lead inhibitors demonstrate good predicted aqueous solubility, lipophilicity, Caco-2 cell permeability, and physics-based permeability properties. Employing computational extra-precision docking, we observe tight binding and engagement of the BL2 and the BL2 groove of PLpro, effectively obstructing the active site from natural substrates. Moreover, our lead inhibitor interacts with residues across the BL2, termed here as the Alpa-cleft of PLpro. This blocking mechanism is further supported by binding energetics and kinetics of the ligand-receptor interactions derived from molecular dynamics (MD) simulations. These findings underscore the potential for advancing inhibitors targeting residues surrounding the deep and shallow binding pockets of SARS-CoV-2 PLpro, inhibiting its catalytic activity. We gratefully acknowledge the generous support of the Richard Barber Interdisciplinary Research Program for funding this project.