Exploiting Enzyme Plasticity in Drug Discovery: application to glutamate racemase

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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 Å 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.