Chasing allosteric inhibition of the SARS-CoV-2 PLpro via Molecular Dynamics Simulations with Flooding Fragments (MDFFr)

Abstract The SARS-CoV-2 papain-like protease (PLpro) represents a crucial therapeutic target due to its dual role in viral polyprotein processing and suppression of host immune responses through de-ubiquitination and de-ISGylation activities. To identify novel allosteric druggable sites on PLpro, we developed a molecular dynamics approach with flooding fragments (MDFFr), advancing previous Molecular Dynamics (MD) flooding simulation methods. Using MDFFr, we evaluated interactions of known phenolic inhibitors with SARS-CoV-2 PLpro and discovered multiple allosteric druggable sites. Our simulations not only validated experimentally known binding sites but also revealed previously unidentified hotspots, including protein-protein interaction sites for ubiquitin and ISG-15 (Interferon-Stimulated Gene 15). The MDFFr approach demonstrates robust capability for physics-based druggability assessment of biological targets using only protein 3D structure, while providing detailed insights into fragment-protein interactions at both druggable sites and protein-protein interfaces. These findings unveil new opportunities for allosteric inhibition of PLpro, potentially advancing therapeutic strategies against SARS-CoV-2 and other coronavirus-related diseases.