Structural Requirements for Sterol 14alpha-Demethylases

项目摘要 甾醇14α-脱甲基酶是在所有生物界中发现的细胞色素P450酶， 它们的低（22-35%）序列同一性在整个基因组，分为一个家庭（CYP 51），因为他们的 严格的功能保护。从细菌到人类，它们都催化着同样不寻常的三步反应 从五种环化甾醇前体（14α-甲基）中的一种或多种氧化除去14α-甲基的方法 甲基→14α-醇→14α-醛→14α-脱甲基产物加甲酸）。真核微粒体 膜结合CYP 51使用NADPH-细胞色素P450还原酶（CPR）作为其氧化还原伴侣，而 水溶性细菌直向同源物接受来自铁氧还蛋白和/或黄素蛋白的电子。CYP 51反应 甾醇是真核生物膜生物发生所必需的， 作为参与细胞生长、发育和增殖的多种调节分子的前体， 分裂过程（激素、维生素、核受体等）。50多年来，CYP 51反应 作为临床抗真菌药物和农业杀真菌剂（咪唑类、三唑类或 有时是吡啶），但酶本身尚未被包括在药物发现范式中， 处理的困难。 我们的长期目标是了解是什么使CYP 51成为CYP 51，以及这种CYP 51的结构特征 P450可用于进行合理设计的、有效的和功能上不可逆的物种选择性 抑制剂的我们已经发现，当结合外源配体（唑类、吡啶类，甚至是 底物类似物）CYP 51保持在其静息、无配体样状态，调节生理活性。 底物引起大规模的构象转换，涉及活性位点的骨架和 与电子供体配偶体相互作用的表面，制备用于催化的酶。 当前更新申请的目的是：1）通过结合冷冻电子显微镜和 X射线晶体学，底物结合的CYP 51/CPR和底物的复合物的结构- 结合荚膜甲基球菌CYP 51/铁氧还蛋白融合; 2）使用计算结构生物学更好地 了解CYP 51分子动力学; 3）评估我们的两种VNI衍生物的疗效， 查加斯病（由三种天然耐药菌株引起）小鼠模型中的药代动力学 的克氏锥虫）和昏睡病（布氏锥虫）的小鼠模型中，分析我们的 针对真菌新型隐球菌（隐球菌脑膜炎）CYP 51抑制剂的内部文库， 两种自由生活的致病性阿米巴，卡氏阿米巴（致盲性角膜炎）和福氏纳氏阿米巴 （原发性阿米巴脑膜脑炎），并测试我们的两个有效的功能不可逆的抑制剂的人类 癌细胞系和巨细胞病毒感染的人细胞中的CYP 51。

PROJECT SUMMARY Sterol 14α-demethylases are the cytochrome P450 enzymes found in all biological kingdoms and, regardless of their low (22-35%) sequence identity across phylogeny, grouped into one family (CYP51) because of their strict functional conservation. From bacteria to humans, they all catalyze the same unusual three-step reaction of the oxidative removal of the 14α-methyl group from one or more of five cyclized sterol precursors (14α- methyl →14α-alcohol→14α-aldehyde→14α-demethylated product plus formic acid). Eukaryotic microsomal membrane-bound CYP51s use NADPH-cytochrome P450 reductase (CPR) as their redox partner, while water-soluble bacterial orthologs accept electrons from ferredoxins and/or flavodoxins. The CYP51 reaction is required for biosynthesis of sterols, which are essential for eukaryotic membrane biogenesis and also serve as precursors for a variety of regulatory molecules that are involved in cellular growth, development, and division processes (hormones, vitamins, nuclear receptors, etc.). For more than 50 years, the CYP51 reaction has served as the target for clinical antifungal drugs and agricultural fungicides (imidazoles, triazoles, or sometimes pyridines), yet the enzyme per se has not been included in the drug discovery paradigm because of the difficulties of its handling. Our long-term goal is to understand what makes/keeps a CYP51 a CYP51 and what structural features of this P450 can be used to make rationally designed, potent, and functionally irreversible species-selective inhibitors. We have found that while upon binding of exogenous ligands (azoles, pyridines, and even a substrate analog) CYP51s remain in their resting, ligand-free-like state, accommodation of the physiological substrate causes a large-scale conformational switch that involves the backbone of the active site and the surface of interaction with the electron donor partner, preparing the enzyme for catalysis. The aims of the current renewal application are 1) to determine, by combining cryo-electron microscopy and X-ray crystallography, the structures of the complex of the substrate-bound CYP51/CPR and the substrate- bound Methylococcus capsulatus CYP51/ferredoxin fusion; 2) to use computational structural biology to better understand CYP51 molecular dynamics; 3) to evaluate the efficacy of our two VNI derivatives with optimized pharmacokinetics in the mouse models of Chagas disease (caused by three naturally drug resistant strains of Trypanosoma cruzi) and in the mouse model of sleeping sickness (Trypanosoma brucei), to analyze our in-house library of CYP51 inhibitors against a fungus Cryptococcus neoformans (cryptococcal meningitis) and two free-living pathogenic amoebas, Acanthamoeba castellanii (blinding keratitis) and Nagleria fowleri (primary amebic meningoencephalitis), and to test our two potent functionally irreversible inhibitors of human CYP51 in cancer cell lines and in cytomegalovirus infected human cells.