Structural Requirements for Sterol 14alpha-Demethylases

甾醇 14α-脱甲基酶的结构要求

• 批准号: 10077559
• 负责人: Galina I Lepesheva
• 金额: $ 36.4万
• 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 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10077559
• 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; Blood - brain barrier anatomy; 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; 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; Growth and Development function; Health; Hormones; Human; Imidazole; Industrial fungicide; Keratitis; Knowledge; Lead; Libraries; Life; Ligands; Lipid Bilayers; Membrane; Methylococcus capsulatus; Minor; Molecular; Molecular Conformation; Motion; Mutagenesis; NADPH-Ferrihemoprotein Reductase; Naegleria fowleri; Nuclear Receptors; Orthologous Gene; Oxidants; Oxidation-Reduction; Pathogenicity; Pathway interactions; Pharmaceutical Preparations; Phylogeny; Physiological; Play; Process; Progress Reports; Proteins; Protons; Protozoan Infections; Publications; 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; base; cancer cell; cell growth; design; drug candidate; drug discovery; efficacy evaluation; electron donor; enzyme structure; flexibility; fungus; in vivo evaluation; inhibitor/antagonist; insight; membrane biogenesis; methyl group; molecular dynamics; mouse model; novel; posaconazole; preference; preservation; primary amebic meningoencephalitis; pyridine; 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.

项目概要 甾醇 14α-去甲基酶是在所有生物界中发现的细胞色素 P450 酶，无论 它们在整个系统发育过程中的序列同一性较低（22-35%），由于其 严格的功能保护。从细菌到人类，它们都会催化相同的不寻常的三步反应 从五种环化甾醇前体（14α- 甲基→14α-醇→14α-醛→14α-脱甲基产物加甲酸）。真核微粒体 膜结合 CYP51 使用 NADPH-细胞色素 P450 还原酶 (CPR) 作为其氧化还原伙伴，而 水溶性细菌直系同源物接受来自铁氧还蛋白和/或黄素氧还蛋白的电子。 CYP51反应 是甾醇生物合成所必需的，甾醇是真核细胞膜生物发生所必需的，也可用于 作为参与细胞生长、发育和发育的各种调节分子的前体 分裂过程（激素、维生素、核受体等）。 50 多年来，CYP51 反应 已作为临床抗真菌药物和农业杀菌剂（咪唑、三唑或 有时是吡啶），但酶本身并未包含在药物发现范式中，因为 其处理的困难。 我们的长期目标是了解是什么使 CYP51 成为/保持 CYP51 以及其结构特征 P450 可用于进行设计合理、有效且功能不可逆的物种选择性 抑制剂。我们发现，当结合外源配体（唑类、吡啶类，甚至 底物类似物）CYP51 保持静息、无配体样状态，适应生理学 底物引起大规模的构象转换，涉及活性位点的主链和 与电子供体伴侣相互作用的表面，准备用于催化的酶。 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和底物的复合物的结构 结合的荚膜甲基球菌 CYP51/铁氧化还原蛋白融合体； 2）利用计算结构生物学更好地 了解CYP51分子动力学； 3) 评估我们的两种经过优化的 VNI 衍生物的功效 恰加斯病（由三种天然耐药菌株引起）小鼠模型中的药代动力学 克氏锥虫）和昏睡病小鼠模型（布氏锥虫），以分析我们的 针对新型隐球菌（隐球菌脑膜炎）真菌的内部 CYP51 抑制剂库和 两种自由生活的致病性阿米巴原虫，卡氏棘阿米巴（致盲性角膜炎）和福氏耐格里阿米巴 （原发性阿米巴脑膜脑炎），并测试我们的两种有效的功能性不可逆的人类抑制剂 癌细胞系和巨细胞病毒感染的人类细胞中的 CYP51。