Structural Basis and Inhibitor Development of SARS-CoV-2 Papain-like Protease

Papain-like protease (PLpro), a crucial functional domain of the SARS-CoV-2 non-structural protein 3 (nsp3), plays a dual role in both hydrolyzing viral polyprotein precursors and modulating host immune responses. These critical functions position PLpro as a key target in the ongoing development of antiviral therapies for SARS-CoV-2. This review analyzes more than 100 PLpro-ligand co-crystal structures and summarizes the major binding modes between these ligands and PLpro. Most of these ligands bind to sites analogous to those targeted by the classical non-covalent inhibitor GRL0617, primarily involving the P3 and P4 subsites and the BL2 loop. Based on these structural insights, optimized inhibitors have expanded targeting beyond the canonical binding site to auxiliary regions such as the BL2 groove and the Val70 site, and in some cases toward the catalytic Cys111 buried within a narrow pocket. Certain ligands identified through various screening approaches bind to non-canonical or allosteric regions, such as the S1 and S2 sites or the zinc-finger domain, engaging PLpro through distinct interaction modes and thereby offering additional opportunities for PLpro inhibitor design. The review also discusses potential strategies for future PLpro inhibitor development informed by recent structural advances. Taken together, these structural and functional insights support ongoing efforts in the structure-guided design and optimization of PLpro inhibitors.