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的小分子相关变构抑制提供物理基本原理 种族酶（GR）已成为最高级的抗菌药物靶标。来自基于结构的 药物设计的角度，gr遭受大规模的，通常是不明显的，特质配体相关的 结构变化。当前的提案包括代表我们理解的突破的数据 GR如何和为什么如此反应性，并描述了一种新的抗菌剂的优化 通过与GR的催化机制选择性形成可逆的共价键来利用这种反应性。我们 已经表明，这些GR抑制剂具有针对金黄色葡萄球菌的显着抗菌活性， 甚至是一些-内酰胺抗生素。这些缓慢的表演，可逆抑制剂为您提供了无与伦比的机会 研究关键的酶促激活过程，我们认为这是设计有效变构的核心 抑制剂。在这里，我们通过开发自动表面结合了研究GR连接的新方法 等离子体共振测定。重要的是，我们的初步结果使先前发表的理论无效 小分子变构铅化合物如何抑制幽门螺杆菌的GR， 胃癌。我们提出了一种新颖的理论，该理论指定了变构抑制如何导致 GR酶的天然灵活性，可防止关键GR激活过程。一系列计算和 采用实验方法，支持这种GR抑制模型。关于GR的假设 通过该诅咒酶运动，由于药物结合引起的变构抑制作用将由我们小组的最新验证 开发非天然荧光氨基酸的MD信息放置，L-（7-羟基丙糖蛋白-4-基） 乙基甘氨酸（7HC）进入GR的变构控制区域。此外，我们已经解决了幽门螺杆菌-D-GLU X射线晶体结构至1.9Å分辨率，这将使我们能够捕获与一个家庭的共价相互作用 慢速可逆的迈克尔受体抗菌剂。具体目的是：目标1：确定 小分子变构抑制幽门螺杆菌种族酶在原子水平上的机理；目标2： 确定由于小分子而导致的溶液中谷氨酸种族酶发生的全局结构变化 使用生物合成的GR与特异性掺入非天然氨基酸的位点结合，l-（7- 羟基丙糖-4-基）乙基甘氨酸（7HC）;目标3：利用酶动力学与催化之间的联系 GR的力量设计出新颖的慢速行动可逆迈克尔受体的班级，并与之反应 GR的激活形式：实现具有“可调”亲电性的稳定GR抑制剂的目标。 成功完成拟议的特定目标后，我们不仅会了解为什么GR需要如此灵活，还需要 但是我们将了解某些变构药物铅的远程结合如何损害这种催化 功率，在原子（甚至电子）水平上。