title: New challenges and opportunities in drug discovery targeting glutamate racemases
title: a structure-based perspective
titleType: Subtitle
Creators: name: Cooling, Grant
Publisher: name: University of Iowa
Year: 2025
Subjects: public abstract

description: Complex biological processes necessary for life are largely conducted by enzymes. The limelight has yet to fully capture how integral and the impact enzymes play in the development of medicines. Recently, science has started to fully grasp the significance that enzyme flexibility and dynamics play in structure and function. Two case studies will be presented in this thesis in developing small molecule inhibitors for a flexible enzyme, and consequently evaluate their inhibitory mechanisms of action. Both case studies delve into inhibiting the hyper flexible enzyme, glutamate racemase. This enzyme is responsible for production of D-glutamate necessary for the cross-linking of peptidoglycan present in all bacteria. Without properly crosslinked peptidoglycan bacterial cells are subject to lysis via osmotic pressure.
description: The first case study focuses on covalent inhibition of this dynamic enzyme, glutamate racemase. This case study elucidates the mechanism of action for inhibiting glutamate racemases with a model system more friendly to structural techniques. There has been a resurgence in the interest in covalent inhibitors within drug discovery programs in recent years. In order to improve their potency and residence time, a significant number of resources have been put into developing better covalent inhibitors with reduced off-target effects. Enzyme kinetics, mass spectrometry, surface-plasmon resonance, and X-ray crystallography techniques were used to elucidate structural insights into ligand recognition and identify covalent ligand adducts. Molecular dynamics and quantum mechanics-molecular mechanics geometry optimizations reveal that the catalytic machinery is poised for reaction with these ligands. One compound was discovered initially as an alanine racemase inhibitor but was found to also be a Mycobacterium tuberculosis glutamate racemase inhibitor. This study resolves the mechanism of inhibition of glutamate racemase by β-chloro-D-alanine (BCDA).
description: The second case study, broken into two segments, delves into the cryptic allosteric pocket present in Helicobacter pylori glutamate racemase. Infections of Helicobacter pylori have been linked to gastric ulcers and cancer. The first portion of this case study involved scaffold hopping from a known inhibitor, Compound A, to novel chemical space, based on shape similarity. An in silico workflow was employed resulting in a series of compounds predicted to allosterically bind to H. pylori glutamate racemase. Attempts to characterize these compounds utilized enzyme kinetic assays, surface-plasmon resonance, and X-ray crystallography. The screening purposes here served to lay the foundation for expanding the chemical space of known binders to glutamate racemase making future endeavors more tractable and viable. A fragment-based screening campaign against H. pylori glutamate racemase resulted in the identification of several fragments capable of activating and inhibiting glutamate racemase. These fragments were characterized by biophysical techniques, including Differential Scanning Fluorimetry, surface-plasmon resonance and X-ray crystallography, and co-crystal structure with an activating fragment is described.