Elucidating Novel Strategies for Inhibiting Dihydroneopterin Aldolase Using Potent Antibacterial Agents

Abstract

Dihydroneopterin aldolase (DHNA) plays a critical role in the production of tetrahydrofolate in the cell. Lacking a mammalian counterpart, DHNA is a potentially targetable enzyme for novel antibacterial discovery, and this research seeks to validate several aspects of its viability. However, the complex octameric nature of DHNA paired with its promiscuousness in reactivity has made it difficult to study using primarily experimental methods, with conformational changes made up of small loop movements unlikely to be seen in purely structural studies. This thesis details not only the foundation for targeting DHNA using previously underutilized methods, specifically Molecule Dynamics and Quantum Mechanics/Molecule Mechanics simulations, but also a tool designed to make the analysis of enzyme kinetic data straightforward, reproducible, and efficient. Utilizing a combination of experimental methods, including enzyme kinetics, protein purification, and computational techniques in the form of quantum mechanical and molecular dynamics simulations, novel mechanistic insight into both substrate binding and catalysis allows for targeting DHNA in both noncompetitive and covalent means. We have shown that DHNA is cooperative in nature, depended on the conformation of a flexible loop and the key catalytic residues and their function. Additionally, we present a compound synthesized for competitive covalent inhibition that shows high potency in vitro. By combining experimental and computational methods in a synergistic method, this work provides the necessary basis for targeting DHNA using new methods, allowing for the future development of potentially potent inhibitors of DHNA.