Abstract 1954 Deciphering Selectivity in Mesotrypsin Inhibition: Validating an allosteric mechanism through mutation and enzyme kinetics

Introduction: Mesotrypsin, a significant member of the S1 serine protease family, is increasingly recognized for its role in the progression of cancers, particularly in the prostate and lung. Our research aims to develop selective and potent inhibitors of mesotrypsin, thus improving treatment options for cancers associated with mesotrypsin. The development of targeted inhibitors for mesotrypsin is challenging due to its close homology with the cationic (trypsin 1) and anionic (trypsin 2) isoforms. Our group has crystallized unliganded mesotrypsin and discovered a formerly unknown autoinhibited conformation and a novel allosteric inhibitory site with sequence variability across trypsin isoforms. Specifically, mesotrypsin is characterized by the presence of histidine at residue 217, where trypsin 1 has aspartic acid and trypsin 2 possesses tyrosine. Additionally, mesotrypsin has tryptophan at residue 221a, which is a glutamine for both other trypsins. We have identified a small-molecule selective inhibitor, mesotrypsin allosteric inhibitory ligand (MAIL), that is predicted by molecular docking to bind at this allosteric site. The goal of the present study is to validate the predicted binding site of MAIL and to identify key binding interactions that shape inhibitory specificity by using site-directed mutagenesis and enzyme kinetics approaches. Methods: We conducted molecular docking of MAIL with the autoinhibited conformation of mesotrypsin using Glide software by Schrödinger to predict key residues involved in binding interactions. We performed site-directed mutagenesis of mesotrypsin H217 and W221a using the QuikChange mutagenesis approach to substitute the residues in mesotrypsin with the amino acids present at these positions in trypsin 1. We carried out enzyme kinetics experiments on WT and mutant enzymes using the colorimetric peptide substrate Z-Gly-Pro-Arg-p-nitroanilide to evaluate the impact of mutations on kinetic constants and susceptibility to inhibition by MAIL. Results: Molecular docking studies predicted the binding mode of MAIL in the allosteric inhibitory site with the interactions involving pi stacking with W221a, Y215 , Y172, hydrogen bonding with Q192, G219 and cation-pi interaction with R224. Mutations H217D and W221aQ were successfully accomplished and confirmed by DNA sequencing. Enzyme kinetics assays demonstrated that the mesotrypsin W221aQ mutant in particular had a 5-fold higher inhibition constant Ki than WT mesotrypsin, recapitulating the weaker susceptibility to inhibition of trypsin 1, which possesses a native Q221a residue. Our experiments provide validation of the predicted binding mode of MAIL in the allosteric site and highlight the vital role of W221a in mesotrypsin selectivity. Conclusion: This study demonstrates the viability of allosteric inhibition as a method for the selective targeting of mesotrypsin in cancer therapy. Our enzyme kinetics studies validate the allosteric binding site and identify key determinants of MAIL inhibition and specificity. These insights will be useful during ongoing and future work to optimize and refine MAIL to boost its selectivity and potency, ultimately working towards novel treatment options for patients with mesotrypsin-driven cancers. The authors would like to acknowledge funding support under NIH R01GM144393, as well as synchrotron access from Lawrence Berkeley National Laboratory under ALS-11671.