Exploiting new approaches for selective inhibition of trypsin-like serine proteases

PROJECT SUMMARY/ABSTRACT Serine proteases of the S1 (trypsin-like) family control digestion, immunity, coagulation, and tissue remodeling; their dysregulation drives pancreatitis, cancer progression, and coagulopathies. Because the catalytic clefts of these enzymes are nearly identical, most small-molecule inhibitors lack the selectivity needed for clear mechanistic studies or safe therapeutic use. Our recent work revealed that mesotrypsin, a cancer-promoting trypsin isoform, transiently adopts an autoinhibited conformation that exposes a previously unknown cryptic pocket adjacent to the active site; a first-generation small molecule that binds this pocket confirmed the feasibility of allosteric inhibition via conformational stabilization. Building on this insight, the present project will integrate structure-guided compound design, enhanced molecular-dynamics ensemble modeling, graph-based pocket prediction, high-throughput virtual screening, x-ray crystallography, and detailed enzyme kinetics to achieve three goals: optimize potent, selective allosteric inhibitors of mesotrypsin; discover and exploit analogous cryptic pockets in pancreatic trypsins 1 and 2; and extend cryptic-pocket targeting to additional clinically important S1 proteases, thrombin and hepatocyte growth-factor activator. Through the coordinated application of computational prediction and experimental validation, we will illuminate fundamental links between conformational landscapes and protease regulation, furnish isoform-selective chemical probes to interrogate protease biology, and deliver lead compounds that could inform future therapy in cancer, pancreatitis, and thrombosis. More broadly, the project establishes a generalizable platform for exploiting hidden allosteric sites in structurally conserved enzyme families.