Exploiting Enzyme Plasticity in Drug Discovery: application to glutamate racemase

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基础。 消旋酶(GR)，已成为最高级别的抗菌药物靶标。从基于结构的 从药物设计的角度来看，GR患有大规模的、往往难以解释的、特殊的配基相关的疾病 结构性变化。目前的提案包括的数据代表了我们对 GR是如何以及为什么如此活跃的，并描述了一类新的抗菌剂的优化 通过利用GR的催化机制选择性地形成可逆的共价键来利用这种反应性。我们 结果表明，这些GR抑制剂对金黄色葡萄球菌具有显著的抗菌活性，超过了金黄色葡萄球菌。 甚至一些-内酰胺类抗生素。这些慢作用、可逆的抑制剂提供了一个无与伦比的机会 研究一个关键的酶激活过程，我们认为这是设计有效变构的核心 抑制剂。在这里，我们结合了一种新的方法来研究GR的结扎，开发了一个自动化的表面 等离子激元共振分析。重要的是，我们的初步结果使先前发表的理论无效 小分子变构药物先导化合物如何抑制幽门螺杆菌的GR 胃癌。我们提出了一种新的理论，说明了变构抑制是如何通过抑制 GR酶的天然灵活性，防止了关键的GR激活过程。一组计算和 实验方法支持这一GR抑制模型。关于GR的假说 由于药物结合而抑制的酶运动导致的变构抑制将通过我们小组最近的 分子标记非天然荧光氨基酸L-(7-羟基香豆素-4-基)的研究 乙基甘氨酸(7HC)进入GR的变构控制区。此外，我们还解决了H.Pylori-D-Glu X-射线晶体结构到1.9？分辨率，这将使我们能够捕获与一族 慢作用可逆迈克尔受体抗菌剂。具体目标是：目标1：确定 小分子变构抑制幽门螺杆菌谷氨酸外消旋酶的原子水平机制；目的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与特定结合的非天然氨基酸结合，L-(7- 羟基香豆素-4-乙基甘氨酸(7HC)；目标3：利用酶动力学和催化之间的联系 GR设计新型慢作用可逆Michael受体的能力，该受体与 GR的活化形式：实现稳定的GR抑制剂的目标，具有可调的亲电性。 在成功完成拟议的具体目标后，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是如何破坏这种催化作用的 力量，在原子(甚至电子)层面上。

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.