High-Throughput Screening and Mechanistic Characterization of Enzyme Inhibitors as Tools for Rational Drug Discovery

Abstract

Rational design of enzyme inhibitors is a cornerstone of drug discovery. However, progress is limited by inefficient enzyme activity assays and unclear determinants of enzyme-inhibitor interactions. In this thesis, I developed tools for the development of inhibitors targeting Cps2L, a pathogenic bacterial thymidylyltransferase, and glucose-6-phosphate dehydrogenase (G6PD), linked to cancer and inflammation. For Cps2L, I developed a NADPH-coupled assay that monitors real-time enzyme activity, improving the precision and efficiency of existing methods. Using this platform, I characterized thymidine diphosphate-L-rhamnose as a non-covalent Cps2L inhibitor. For G6PD, I evaluated structure-reactivity-activity relationships of ortho-substituted aryl aldehydes as imine-forming inhibitors. I showed that aldehyde reactivity is controlled by intramolecular interactions formed by ortho-substituents. Reactivity trends correlated with inhibition potency, but not reversibility. Diverse binding mechanisms and novel G6PD inhibitors were identified within the aldehyde panel. Together, this work demonstrates how assay innovation and electrophile-focused inhibitor profiling advances rational drug design across diverse targets.