Synergistic Inhibitor Binding to the Papain-Like Protease of Human SARS Coronavirus – Mechanistic and Inhibitor Design Implications

Lee, Hyun; Cao, Shuyi; Hevener, Kirk E.; Truong, Lena; Gatuz, Joseph L.; Patel, Kavankumar; Ghosh, Arun K.; Johnson, Michael E.

We have previously developed two potent chemical classes that inhibit the essential papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus (SARS-CoV). In this study, we applied a novel approach to identify small fragments that act synergistically with these inhibitors. A fragment library was screened in combination with four previously developed lead inhibitors by fluorescence-based enzymatic assays. Several fragment compounds synergistically enhanced the inhibitory activity of the lead inhibitors by approximately an order of magnitude. Surface plasmon resonance (SPR) measurements showed that three fragments bind specifically to the PLpro enzyme. Mode of inhibition, computational solvent mapping, and molecular docking studies suggest that these fragments bind adjacent to the binding site of the lead inhibitors and further stabilize the inhibitor-bound state. We propose potential next generation compounds based upon a computational, fragment-merging approach. This approach provides an alternative strategy for lead optimization in cases where direct co-crystallization is difficult.