Our goal is to provide a physical rationale for small molecule-associated allosteric inhibition in glutamate racemase (GR), which has emerged as an antimicrobial drug target of the highest order. From a structure based drug design perspective, GR suffers from large scale, often inexplicable, idiosyncratic ligand-associated structural changes. The current proposal includes data that represents a breakthrough in our understanding of how and why GR is so reactive, and describes the optimization of a new class of antimicrobial agents that exploits this reactivity by forming reversible covalent bonds selectively with the catalytic machinery of GR. We have shown that these GR inhibitors have remarkable antimicrobial activity against S. aureus, which surpass even some ?-lactam antibiotics. These slow acting, reversible inhibitors provide an unparalleled opportunity to study a critical enzymatic activation process, which we believe is at the heart of designing effective allosteric inhibitors. Here we combine a fresh approach to studying ligation of GR by developing an automated surface plasmon resonance assay. Importantly, our preliminary results invalidate the previously published theories for how small molecule allosteric drug lead compounds inhibit the GR from the H. pylori, the causative agent of gastric cancer. We present a novel theory that specifies how allosteric inhibition results from dampening the native flexibility of GR enzymes, which prevents a key GR activation process. An array of computational and experimental methods are employed, which support this model of GR inhibition. The hypothesis concerning GR allosteric inhibition via dampened enzyme motion due to drug binding will be validated by our group's recent development of a MD-informed placement of non-natural fluorescent amino acid, L-(7-hydroxycoumarin-4-yl) ethylglycine (7HC) into an allosterically controlled region of GR. Additionally, we have solved the H. pylori-D-glu X-ray crystal structure to 1.9 resolution, which will allow us to capture the covalent interactions with a family of slow acting reversible Michael acceptor antimicrobial agents.
The specific aims are:
Aim 1 : Determine the mechanism of small molecule allosteric inhibition of H. pylori glutamate racemase at the atomistic level;
Aim 2 : Determine the global structural changes that occur in glutamate racemases in solution due to small molecule binding using a biosynthesized GR with a site specifically incorporated non-natural amino acid, L-(7- hydroxycoumarin-4-yl) ethylglycine (7HC);
Aim 3 : Exploiting the link between enzyme dynamics and catalytic power of GR to design novel classes of slow acting reversible Michael acceptors, which undergo reaction with the activated form of GR: realizing the goal of stable GR inhibitors with ?tunable? electrophilicity. Upon successful completion of the proposed specific aims, not only will we learn why GR needs to be so flexible, but we will understand how the remote binding of certain allosteric drug lead compounds damage this catalytic power, at the atomistic (and even the electronic) level.

Public Health Relevance

Upon successful completion of the proposed project, not only will we learn why glutamate racemase requires its significant structural plasticity, but we will also understand how the remote binding of certain allosteric drug compounds damages its catalytic power. The knowledge gained from these studies will provide a powerful and novel type of structure-activity relationship for allosteric drug discovery and optimization. The highly potent glutamate racemase inhibitors that have been discovered as part of the proposed project have the potential to open up a new epoch in targeting bacterial cell wall biosynthesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM097373-06
Application #
9381976
Study Section
Macromolecular Structure and Function D Study Section (MSFD)
Program Officer
Fabian, Miles
Project Start
2012-09-01
Project End
2021-08-31
Budget Start
2017-09-01
Budget End
2018-08-31
Support Year
6
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Iowa
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
062761671
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Li, Quinn; Gakhar, Lokesh; Ashley Spies, M (2018) Determinants of human glucokinase activation and implications for small molecule allosteric control. Biochim Biophys Acta Gen Subj 1862:1902-1912
Li, Quinn; Folly da Silva Constantino, Laura; Spies, M Ashley (2018) Integrating Experimental and In Silico HTS in the Discovery of Inhibitors of Protein-Nucleic Acid Interactions. Methods Enzymol 601:243-273
Vance, Nicholas R; Gakhar, Lokesh; Spies, M Ashley (2017) Allosteric Tuning of Caspase-7: A Fragment-Based Drug Discovery Approach. Angew Chem Int Ed Engl 56:14443-14447
Hengel, Sarah R; Spies, M Ashley; Spies, Maria (2017) Small-Molecule Inhibitors Targeting DNA Repair and DNA Repair Deficiency in Research and Cancer Therapy. Cell Chem Biol 24:1101-1119
Hengel, Sarah R; Malacaria, Eva; Folly da Silva Constantino, Laura et al. (2016) Small-molecule inhibitors identify the RAD52-ssDNA interaction as critical for recovery from replication stress and for survival of BRCA2 deficient cells. Elife 5:
Dean, Sondra F; Whalen, Katie L; Spies, M Ashley (2015) Biosynthesis of a Novel Glutamate Racemase Containing a Site-Specific 7-Hydroxycoumarin Amino Acid: Enzyme-Ligand Promiscuity Revealed at the Atomistic Level. ACS Cent Sci 1:364-373
Subramanyam, Shyamal; Jones, William T; Spies, Maria et al. (2013) Contributions of the RAD51 N-terminal domain to BRCA2-RAD51 interaction. Nucleic Acids Res 41:9020-32
Whalen, Katie L; Spies, M Ashley (2013) Flooding enzymes: quantifying the contributions of interstitial water and cavity shape to ligand binding using extended linear response free energy calculations. J Chem Inf Model 53:2349-59
Spies, M Ashley (2013) Nexus between protein-ligand affinity rank-ordering, biophysical approaches, and drug discovery. ACS Med Chem Lett 4:895-7
Whalen, Katie L; Chau, Anthony C; Spies, M Ashley (2013) In silico optimization of a fragment-based hit yields biologically active, high-efficiency inhibitors for glutamate racemase. ChemMedChem 8:1681-9