Exploiting Enzyme Plasticity in Drug Discovery: application to glutamate racemase

在药物发现中利用酶可塑性：在谷氨酸消旋酶中的应用

• 批准号: 9381976
• 负责人: Michael Ashley Spies
• 金额: $ 30.86万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9381976
• 关键词: Active Sites; Amino Acids; Anabolism; Articular Range of Motion; Bacteria; Binding; Biological; Biological Assay; Biophysics; Cell Wall; Chemistry; Complex; Coupled; Covalent Interaction; Crystallization; D Glutamate; Data; Development; Drug Compounding; Drug Design; Drug Targeting; Electronics; Eligibility Determination; Enzyme Activation; Enzymes; Family; Gap Junctions; Glutamate racemase; Goals; Heart; Helicobacter pylori; Hybrids; Knowledge; Lead; Learning; Ligands; Ligation; Link; Metabolic; Methods; Modeling; Monobactams; Motion; Nature; Peptidoglycan; Pharmaceutical Preparations; Pharmacologic Substance; Process; Property; Proteins; Publishing; Racemases; Reaction; Regulation; Reporter; Research; Resolution; Rhodanine; Roentgen Rays; Sampling; Series; Shapes; Site; Source; Specific qualifier value; Staphylococcus aureus; Structure; Structure-Activity Relationship; Surface Plasmon Resonance; System; Testing; Time; Umbelliferones; Work; X-Ray Crystallography; analog; antimicrobial; antimicrobial drug; base; biophysical techniques; cofactor; covalent bond; design; drug discovery; flexibility; inhibitor/antagonist; malignant stomach neoplasm; novel; prevent; small molecule; theories; tool; virtual

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.

我们的目标是为谷氨酸小分子相关的变构抑制提供一个物理基础 消旋酶（GR），其已成为最高级别的抗微生物药物靶标。从一个基于 从药物设计的角度来看，GR患有大规模的，通常是无法解释的，特异质配体相关的 结构变化。目前的提案包括的数据代表了我们对以下问题的理解的突破： 如何以及为什么GR是如此的反应性，并描述了一类新的抗菌剂的优化， 通过与GR的催化机制选择性地形成可逆共价键， 已经表明这些GR抑制剂对S.金黄色葡萄球菌， 甚至是β-内酰胺类抗生素这些缓慢作用的可逆抑制剂提供了无与伦比的机会， 研究一个关键的酶激活过程，我们认为这是设计有效的变构 抑制剂的在这里，我们结合联合收割机一个新的方法来研究连接的GR通过开发一个自动表面 等离子体共振测定。重要的是，我们的初步结果推翻了先前发表的理论， 小分子变构药物先导化合物如何从H.幽门螺杆菌，病原体 胃癌我们提出了一个新的理论，说明如何变构抑制的结果，从阻尼 GR酶的天然灵活性，阻止了关键的GR激活过程。一系列计算和 采用支持GR抑制的这种模型的实验方法。关于GR的假设 通过药物结合抑制酶运动的变构抑制作用将通过我们小组最近的 非天然荧光氨基酸L-（7-羟基香豆素-4-基）的MD-知情放置的开发 乙基甘氨酸（7 HC）进入GR的变构控制区。幽门-D-葡萄糖 X射线晶体结构达到1.9 Å分辨率，这将使我们能够捕获与一系列分子的共价相互作用 缓慢作用的可逆迈克尔受体抗菌剂。具体目标是：目标1：确定 小分子变构抑制H.幽门螺杆菌谷氨酸消旋酶在原子水平;目标2： 确定溶液中谷氨酸消旋酶中由于小分子引起的整体结构变化 使用生物合成的GR与位点特异性掺入的非天然氨基酸L-（7- 目的3：利用酶动力学和催化活性之间的联系， GR设计新型慢作用可逆Michael受体的能力， GR的活化形式：实现具有“可调”亲电性的稳定GR抑制剂的目标。 在成功完成所提出的具体目标后，我们不仅将了解为什么GR需要如此灵活， 但我们将了解某些变构药物先导化合物的远程结合如何破坏这种催化作用， 在原子（甚至电子）层面上的力量。