Proteins are dynamic molecules capable of performing complex biological functions necessary for life. The impact of protein dynamics in the development of medicines is often understated. Science is only now beginning to unravel the numerous consequences of protein flexibility on structure and function. This thesis will encompass two case studies in developing small molecule inhibitors targeting flexible enzymes, and provide a thorough evaluation of their inhibitory mechanisms of action.
The first case study focuses on caspases, a family of cysteine proteases responsible for executing the final steps of apoptosis. Consequently, they have been the subject of intense research due to the critical role they play in the pathogenesis of various cardiovascular and neurodegenerative diseases. A fragment-based screening campaign against human caspase-7 resulted in the identification of a novel series of allosteric inhibitors, which were characterized by numerous biophysical methods, including an X-ray co-crystal structure of an inhibitory fragment with caspase-7. The fragments described herein appear to have a significant impact on the substrate binding loop dynamics and the orientation of the catalytic Cys-His dyad, which appears to be the origin of their inhibition. This screening effort serves the dual purpose of laying the foundation for future medicinal chemistry efforts targeting caspase proteins, and for probing the allosteric regulation of this interesting class of hydrolases.
The second case study focuses on glutamate racemase, another dynamic enzyme responsible for the stereoinversion of glutamate, providing the essential function of D-glutamate production for the crosslinking of peptidoglycan in all bacteria. Herein, I present a series of covalent inhibitors of an antimicrobial drug target, glutamate racemase. The application of covalent inhibitors has experienced a renaissance within drug discovery programs in the last decade. To leverage the superior potency and drug target residence time of covalent inhibitors, there have been extensive efforts to develop highly specific covalent modifications to reduce off-target liabilities. A combination of enzyme kinetics, mass spectrometry, and surface-plasmon resonance experiments details a highly specific 1,4-conjugate addition of a small molecule inhibitor with the catalytic Cys74 of glutamate racemase. Molecular dynamics simulations and quantum mechanics-molecular mechanics geometry optimizations reveal, with unprecedented detail, the chemistry of the conjugate addition. Two compounds from this series of inhibitors display antimicrobial potency comparable to β-lactam antibiotics, with significant activity against methicillin-resistant S. aureus strains. This study elucidates a detailed chemical rationale for covalent inhibition and provides a platform for the development of antimicrobials with a novel mechanism of action.