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

基本信息

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
项目类别：
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

项目摘要

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中小分子相关的变构抑制提供物理原理消旋酶（GR），已成为最高级别的抗菌药物靶点。从药物设计的角度来看，GR 遭受大规模的、通常难以解释的、特殊的配体相关当前的提案包含的数据代表了我们对结构性变化的理解的突破。 GR 如何以及为何如此具有反应性，并描述了新型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来利用这种反应性。研究表明，这些 GR 抑制剂对金黄色葡萄球菌具有显着的抗菌活性，其活性超过甚至一些 β-内酰胺抗生素，这些缓慢作用、可逆的抑制剂也提供了无与伦比的机会。研究关键的酶激活过程，我们认为这是设计有效变构的核心在这里，我们通过开发自动化表面结合了一种新的方法来研究 GR 连接。重要的是，我们的初步结果使之前发表的理论无效。小分子变构药物先导化合物如何抑制幽门螺杆菌（幽门螺杆菌的病原体）的 GR 我们提出了一种新的理论，详细说明了变构抑制是如何通过抑制胃癌而产生的。 GR 酶的天然灵活性，阻止了关键的 GR 激活过程。采用的实验方法支持了有关 GR 抑制的假设。由于药物结合而抑制酶运动而产生的变构抑制将通过我们小组最近的研究得到验证开发基于 MD 的非天然荧光氨基酸 L-(7-羟基香豆素-4-基) 布局此外，我们还解决了幽门螺杆菌-D-glu 的问题。 X 射线晶体结构分辨率达到 1.9 Å，这将使我们能够捕获与一系列物质的共价相互作用慢效可逆迈克尔受体抗菌剂的具体目标是：目标 1：确定原子水平上小分子变构抑制幽门螺杆菌谷氨酸消旋酶的机制；目标2：确定溶液中谷氨酸消旋酶由于小分子而发生的整体结构变化使用生物合成的 GR 与专门掺入非天然氨基酸的位点进行结合，L-(7- 羟基香豆素-4-基)乙基甘氨酸 (7HC)；目标 3：利用酶动力学和催化之间的联系 GR 设计新型缓慢作用可逆迈克尔受体的能力，这些受体与 GR的激活形式：实现亲电性“可调”的稳定GR抑制剂的目标。成功完成所提出的具体目标后，我们不仅会了解为什么 GR 需要如此灵活，但我们将了解某些变构药物的远程结合如何导致损伤复合这种催化作用原子（甚至电子）水平的能量。