IMP Dehydrogenase and the Hydra of Cancer Chemotherapy

说明（由申请人提供）：肌苷-5 '-单磷酸脱氢酶（IMPDH，IMP：NAD氧化还原酶，E.G. 1.2.1.14）催化鸟嘌呤核苷酸生物合成中的限速步骤，IMP氧化为黄嘌呤核苷单磷酸（XMP）。IMPDH在DNA和RNA合成、G-蛋白介导的信号转导和中间代谢中具有中心作用。IMPDH是抗癌、抗病毒、免疫抑制和抗微生物化疗抑制剂的靶标，但也是其它药物的活化剂。动力学，光谱和遗传学方法的综合组合将被用来了解催化机制，包括蛋白质的灵活性的贡献，和未知功能的进化保守子域的代谢作用。 人IMPDH的催化机制将通过动力学和UV光谱方法表征。将强调抗癌剂6-巯基嘌呤核苷酸（6 MPRT）的反应，IMPDH催化必需的代谢活化。将确定6 MPRT如何影响在NAD位点结合的临床使用的抑制剂的抑制。将研究IMPDH反应的逆过程，以深入了解XMP和共价酶-XMP * 中间体的相互转化。一价阳离子激活剂K+和NAD结合位点抑制剂是否激活XMP和水之间的氧同位素交换或限制水的可及性将增强对该反应的理解。蛋白质动力学对这种柔性酶的催化效率的贡献将由反应速率的粘度依赖性来确定。配体如何调节蛋白质的灵活性将通过定点自旋标记和EPR光谱进行评估。该亚结构域的代谢作用将从大肠杆菌染色体内亚结构域缺失的体内结果确定。coli模式生物。一项补充研究将采用计算对接屏幕之间的结构域和代谢物结构的晶体结构。将通过NMR测试与潜在配体的相互作用并用于IMPDH活性的调节。这些结果将揭示一种必需酶的内部工作，以及是否有希望通过已知药物的组合或新的亚结构域配体的发现来提高临床疗效。

DESCRIPTION (provided by applicant): lnosine-5'-monophosphate dehydrogenase (IMPDH, IMP:NAD oxidoreductase, E.G. 1.2.1.14) catalyzes the rate-limiting step in guanine nucleotide biosynthesis, the oxidation of IMP to xanthosine monophosphate (XMP). IMPDH has a central role in DNA and RNA syntheses, in G-protein mediated signal transduction, and in intermediary metabolism. IMPDH is a target for anticancer, anti-viral, immunosuppressive and anti-microbial chemotherapeutic inhibitors, but is an activator of other drugs. An integrated combination of kinetic, spectroscopic and genetic methods will be used to understand the catalytic mechanism, including the contribution of protein flexibility, and the metabolic role of the evolutionary conserved subdomain of unknown function. The catalytic mechanism of human IMPDH will be characterized by kinetic and UV spectroscopic methods. The reaction of the anticancer agent 6-mercaptopurine ribotide (6MPRT), for which IMPDH catalyzes an essential metabolic activation, will be emphasized. How 6MPRT affects inhibition by clinically used inhibitors that bind at the NAD site will be determined. The reverse of the IMPDH reaction will be investigated to provide insight into the interconversion of XMP and the covalent enzyme-XMP* intermediate. Whether the monovalent cation activator K+, and NAD binding site inhibitors, activate oxygen isotope exchange between XMP and water or restrict water accessibility will enhance the understanding of this reaction. The contribution of protein dynamics to the catalytic efficiency of this flexible enzyme will be determined from the viscosity dependence of the reaction rates. How ligands modulate the flexibility of the protein will be assessed by site-directed spin labeling and EPR spectroscopy. The metabolic role of the subdomain will be determined from the in vivo consequences of deletion of the subdomain within the chromosome of the E. coli model organism. A complementary study will employ a computational docking screen between the crystal structure of the domain and metabolite structures. Interactions with potential ligands will be tested by NMR and for modulation of IMPDH activity. These results will reveal the inner working of an essential enzyme and whether there is promise for enhancing clinical efficacy through combinations of known drugs or the discovery of novel subdomain ligands.