Investigation of Substrate Specificity, Mechanism, and Inhibition of IGPS

项目摘要/摘要 胰岛素样生长因子合成酶(IGP Synthase，IGPS)是一种很有前景的抗生素新药靶点。这种酶起着至关重要的作用 在细菌的色氨酸生物合成中，但在人类中不存在。这项提案的总体目标是 是对各种底物-IGPS相互作用有了新的理解，并开发了新的IGPS抑制剂 它可能有潜在的抗感染作用。因此，这项工作与理解和 治疗顽固且广泛耐药的细菌。 我们将使用结核分枝杆菌的IGPS(MtIGPS)作为模型系统。新型MtIPGS衬底 类似物将以这样一种方式进行战略性设计，即它们相对于酶的行为将 回答有关IGPS结构-功能关系和催化的问题。一系列新型基板 具有改变或移除的官能团和芳环取代的类似物将被合成。 类似物结合并作为MtIGPS底物的能力将被确定。这些数据将是 系统地与用天然底物和无活性还原得到的结果进行比较 基片模拟。这些实验将确定底物功能的作用和重要性 分组并显示哪些必须保留，哪些可以修改以用于抑制 优化。 几个怀疑在配体结合中发挥作用的MtIGPS活性位点残基将被替换 使用诱变技术。这些突变对动力学参数的影响将为深入了解 每个残基在结合和催化中的作用。这些数据将揭示出具体问题的重要性 IGPS配体和活性中心之间的分子间相互作用，它们补充了数据 用底物类似物获得。 人们普遍接受的IGPS催化机制始于分子内吸热。 底物的环化和脱芳构化。MtIGPS可能通过强烈绑定或 构象上围绕所产生的中间体或结构相似的先前转变而坍塌 州政府。新的MtIGPS抑制剂设计用于在没有酶的情况下将其转化为过渡态类似物 翻转形成的产品将被合成。这些新化合物的抑制性能和效果 将确定IGPS动力学路径上的化合物。

Project Summary / Abstract IGP Synthase (IGPS) is a promising new antibiotic drug target. This enzyme plays a critical role in tryptophan biosynthesis in bacteria but is not present in humans. The overall goals of this proposal are to gain new understanding of various substrate-IGPS interactions and to develop new IGPS inhibitors which may have potential as anti-infective agents. Thus, this work is highly relevant to understanding and treating intractable and widespread resistant bacteria. We will use IGPS from M. tuberculosis (MtIGPS) as a model system. New MtIPGS substrate analogs will be strategically designed in such a way that their behavior with respect to the enzyme will answer questions about IGPS structure-function relationships and catalysis. A series of new substrate analogs with altered or removed functional groups and aromatic ring substitutions will be synthesized. The analogs' ability to bind and act as substrates for MtIGPS will be determined. These data will be systematically compared to those obtained with the natural substrate and an unreactive reduced substrate analog. These experiments will determine the role and importance of the substrate functional groups and reveal which ones must be preserved and which ones can be modified for inhibitor optimization. Several MtIGPS active site residues suspected to play a role in ligand binding will be replaced using mutagenesis. The effects of these mutations on kinetic parameters will provide insights into the role of each residue in binding and catalysis. These data will reveal the importance of specific intermolecular interactions between IGPS ligands and the active site, and they complement the data obtained with substrate analogs. The generally-accepted mechanism for IGPS catalysis begins with an endothermic intramolecular cyclization and de-aromatization of the substrate. MtIGPS likely facilitates this step by binding strongly or conformationally collapsing around the resulting intermediate or a structurally-similar preceding transition state. New MtIGPS inhibitors designed to be converted enzymatically into transition state analogs without turning over to form products will be synthesized. The inhibitory properties and the effects of these new compounds on the IGPS kinetic pathway will be determined.