IMP Dehydrogenase and the Hydra of Cancer Chemotherapy

描述(由申请人提供)：肌氨酸-5‘-单磷酸脱氢酶(IMPDH，IMP：NAD氧化还原酶，例如1.2.1.14)催化鸟嘌呤核苷酸生物合成的限速步骤，即IMP氧化为黄嘌呤单磷酸(XMP)。IMPDH在DNA和RNA合成、G蛋白介导的信号转导和中间代谢中起着核心作用。IMPDH是抗癌、抗病毒、免疫抑制和抗微生物化疗抑制剂的靶标，但也是其他药物的激活剂。动力学、光谱和遗传学方法的综合运用将被用来理解催化机制，包括蛋白质灵活性的贡献，以及未知功能的进化保守亚域的代谢作用。 人IMPDH的催化机理将通过动力学和紫外光谱方法进行表征。重点介绍抗癌剂6-硫代嘌呤核苷(6MPRT)的反应，IMPDH对该反应具有重要的代谢激活作用。6MPRT如何影响结合在NAD位点的临床使用的抑制剂的抑制作用将被确定。将研究IMPDH反应的反向，以深入了解XMP和共价酶-XMP*中间体的相互转化。无论是单价阳离子活化剂K+和NAD结合位点抑制剂，激活XMP与水之间的氧同位素交换，还是限制水的可及性，都将加深对这一反应的理解。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度的粘度相关性来确定。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。该亚区的代谢作用将从大肠杆菌模式生物染色体内亚区缺失的体内后果中确定。一项补充研究将在结构域的晶体结构和代谢物结构之间使用计算对接屏幕。与潜在配体的相互作用将通过核磁共振进行测试，并对IMPDH活性进行调节。这些结果将揭示一种关键酶的内部工作原理，以及是否有希望通过组合已知药物或发现新的亚区配体来提高临床疗效。

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.