Structural Requirements for Sterol 14alpha-Demethylases

项目总结 甾醇14α去甲基酶是一种细胞色素P450酶，存在于所有生物界，无论 在系统发育过程中，它们的序列同源性很低(22-35%)，归入一个家族(CYP51)，因为它们的 严格功能守恒。从细菌到人类，它们都能催化同样不同寻常的三步反应 从五个环化的甾醇前体中的一个或多个氧化去除14α-甲基(14α- 甲基→14α-醇→14α-醛→14α-脱甲基化产物加甲酸)。真核微粒体体 膜结合的细胞色素P51s使用NADPH-细胞色素P450还原酶(CPR)作为其氧化还原伙伴，而 水溶性细菌同系物接受铁氧还蛋白和/或黄毒素的电子。CYP51反应 是生物合成甾醇所必需的，这是真核膜生物发生所必需的，也是 作为各种调节分子的前体，这些分子参与细胞的生长、发育和 分裂过程(激素、维生素、核受体等)。在50多年的时间里，CYP51反应 已成为临床抗真菌药物和农用杀菌剂(咪唑、三氮唑或 有时是吡啶)，但这种酶本身并没有包括在药物发现范例中，因为 对于它的处理的困难。 我们的长期目标是了解是什么使/保持了一个CyP51成为一个CyP51，以及它的什么结构特征 P450可用于使设计合理、有效且在功能上不可逆转的物种选择性 抑制剂。我们已经发现，当结合外源配体(氮唑、吡啶，甚至是 底物类似物)CYP51保持在其静止的、无配体的类似状态，调节生理的 底物引起大规模的构象转换，涉及活性中心的主干和 表面与电子供体伙伴相互作用，准备用于催化的酶。 目前更新申请的目的是1)通过结合冷冻电子显微镜和 X-射线结晶学研究表明，底物结合的CyP51/CPR与底物结合的络合物的结构。 结合的甲基球菌被囊细胞色素P51/铁还蛋白融合；2)利用计算结构生物学更好地 了解CYP51分子动力学；3)评估我们的两种VNI衍生物的疗效 三株自然耐药株所致恰加斯病小鼠模型的药代动力学 克氏锥虫)和昏睡病(布氏锥虫)小鼠模型中，分析我们的 针对一种真菌新生隐球菌(隐球菌性脑膜炎)的CYP51抑制剂的内部库和 两种自由生活的致病性阿米巴，卡氏棘阿米巴(致盲性角膜炎)和福氏纳格勒 (原发性阿米巴脑膜脑炎)，并测试我们的两种有效的人类功能不可逆抑制物 细胞色素P51在癌细胞系和巨细胞病毒感染的人细胞中表达。

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.