Structural Requirements for Sterol 14alpha-Demethylases

甾醇 14α-脱甲基酶的结构要求

• 批准号: 10540692
• 负责人: Galina I Lepesheva
• 金额: $ 36.46万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10540692
• 关键词: Acanthamoeba castellanii; Active Sites; African Trypanosomiasis; Agriculture; Alcohols; Aldehydes; Amoeba genus; Animal Model; Antifungal Agents; Antiprotozoal Agents; Area; Azole resistance; Azoles; Bacteria; Binding; Biochemical; Biological; Brain; Cancer cell line; Catalysis; Cells; Chagas Disease; Chemotherapy-Oncologic Procedure; Chimeric Proteins; Cholesterol; Clinical; Clinical Trials; Complex; Cryoelectron Microscopy; Cryptococcal Meningitis; Cryptococcus neoformans; Crystallization; Cyclization; Cytochrome P450; Cytomegalovirus; Cytomegalovirus Infections; Data; Development; Disease; Drug Kinetics; Drug Targeting; Drug resistance; Eating; Electron Transport; Electrons; Enzymes; Ergosterol; Evolution; Excision; Family; Family member; Ferredoxin; Flavodoxin; Formic Acids; Freezing; Goals; Health; Hormones; Human; Imidazole; Keratitis; Knowledge; Libraries; Life; Ligands; Lipid Bilayers; Membrane; Methylococcus capsulatus; Microsomes; Minor; Molecular; Molecular Conformation; Motion; Mutagenesis; NADPH-Ferrihemoprotein Reductase; Naegleria fowleri; Nuclear Receptors; Orthologous Gene; Oxidation-Reduction; Pathogenicity; Pathway interactions; Pharmaceutical Preparations; Phylogeny; Physiological; Play; Process; Progress Reports; Proteins; Protons; Protozoan Infections; Publications; Rationalization; Reaction; Resistance; Rest; Roentgen Rays; Role; Solvents; Step Tests; Sterol Biosynthesis Pathway; Sterols; Structure; Substrate Specificity; Surface; System; Testing; Triazoles; Trypanosoma brucei brucei; Trypanosoma cruzi; Variant; Vertebral column; Vitamins; Water; Work; X-Ray Crystallography; analog; blood-brain barrier penetration; cancer cell; cell growth; demethylation; design; drug candidate; drug discovery; efficacy evaluation; electron donor; enzyme structure; flexibility; fungicide; fungus; in vivo evaluation; inhibitor; insight; membrane biogenesis; methyl group; molecular dynamics; mouse model; novel; posaconazole; preference; preservation; primary amebic meningoencephalitis; pyridine; rational design; resistant strain; scaffold; structural biology; virtual

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.

项目摘要 固醇14α-甲基酶是在所有生物学界中发现的细胞色素P450酶，无论如何 由于其在系统发育中的低（22-35％）序列身份，因此分为一个家族（CYP51） 严格的功能保护。从细菌到人类，它们都催化了相同的不寻常的三步反应 从五个环化的立体前体中的一个或多个从一个或多个中去除14α-甲基的氧化物（14α- 甲基→14α-醇→14α-醛醛→14α-二甲基化产物加甲酸）。真核微粒体 膜结合的CYP51使用NADPH-CYTOCHROME P450降低（CPR）作为其氧化还原伙伴，而 水溶性细菌直系同源物接受来自铁蛋白和/或黄酮毒素的电子。 CYP51反应 是立体观念的生物合成所必需的，这对于真核膜生物发生至关重要，也可以使用 作为参与细胞生长，发育和 分裂过程（激素，维生素，核接收器等）。 50多年来，CYP51反应 已成为临床抗真菌药物和农业杀菌剂（咪唑，三唑或 有时是吡啶），但是酶本身尚未包含在药物发现范式中，因为 其处理困难。 我们的长期目标是了解是什么使CYP51成为CYP51以及此结构特征 P450可用于使理性设计，潜在和功能性不可逆的选择性选择性 抑制剂。我们发现，在结合外源配体时（偶氮，吡啶，甚至是 基板类似物）CYP51保持其静止，不含配体的状态，适合生理 底物引起大规模构象开关，该开关涉及活性位点的主干和 与电子供体伴侣相互作用的表面，准备催化酶。 当前更新应用的目的是1）通过结合冷冻电子显微镜和 X射线晶体学，底物结合的CYP51/CPR的复合物和底物的结构 结合的甲基环球菌CYP51/铁氧还蛋白融合； 2）使用计算结构生物学来更好 了解CYP51分子动力学； 3）评估我们两个通过优化的VNI衍生物的效率 木塔疾病小鼠模型中的药代动力学（由三种天然抗药性菌株引起 crypanosoma cruzi）和鼠标疾病模型（Brucei锥虫），以分析我们 CYP51抑制剂的内部图书馆，针对真菌加密赛（隐脑膜炎）和 两种自由生活的致病性变形虫，阿斯塔米巴·卡斯泰拉尼（Acanthamoeba Castellanii）（盲目角膜炎）和纳格利亚·福勒里（Nagleria Fowleri） （原发性amebic脑膜脑炎），并测试我们的两个潜在的功能性不可逆抑制剂 癌细胞系和巨细胞病毒感染的人类细胞中的CYP51。