Structural Requirements for Sterol 14alpha-Demethylases

 描述（由申请人提供） 甾醇14 β-脱甲基酶（CYP 51）是一种单加氧酶，催化独特的三步反应（14 β-甲基-14 β-醇-14 β-醛-14 β-脱甲基产物加甲酸），从环化甾醇前体中除去14 β-甲基。CYP 51被认为是目前存在的P450超家族所有成员的可能进化祖先。CYP 51反应是人体胆固醇、真核微生物人类病原体（如真菌和原生动物）中麦角固醇和植物中各种植物甾醇生物合成所必需的。绝大多数固醇用于形成有活力的真核细胞膜（流动性、渗透性、整合膜蛋白的多种功能的调节），而一些固醇用作细胞生长、发育和增殖所必需的许多调节分子的前体。在单细胞生物中阻断甾醇生物合成是致命的。CYP 51抑制剂（唑类）是临床上主要的抗真菌药物和农用杀菌剂。其中两种药物（泊沙康唑和雷武康唑）目前正在进行临床试验，用于治疗由原生动物病原体克氏锥虫（查加斯病）引起的致命人类感染。 目前使用的所有唑类都是凭经验发现的，即通过筛选它们对真菌细胞生长的影响，并且在效率、安全性、副作用和对抗性的敏感性方面远非理想。我们研究的长期目标是了解CYP 51功能保守、药物作用和耐药性的分子基础。在这里，我们建议应用在以前的资助周期中获得的关于CYP 51结构/功能的信息，以合理化CYP 51靶向药物发现范式。我们将对高效、无毒的实验性VNI支架进行原生动物/真菌/人CYP 51结构指导的衍生化，以使药理学优化的候选药物适用于恰加斯病的临床试验（包括由 寄生虫的天然耐药菌株）和内脏利什曼病（Aim 1a），以及开发新的定制设计的真菌CYP 51抑制剂（Aim 1b）。目的二是评价人CYP 51作为胆固醇相关疾病（包括癌症和阿尔茨海默病）的潜在药物靶点。目的3是确定CYP 51与其电子供体伙伴NADPH-细胞色素P450还原酶复合物的结构，这将揭示参与蛋白质-蛋白质相互作用的关键残基，并可能导致开发新型CYP 51抑制剂。这项研究的结果将指导未来的药物发现方法，从而能够创造出旨在挽救数百万人生命的合理设计的以物种为导向的候选药物。

 DESCRIPTION (provided by applicant) Sterol 14-demethylase (CYP51) is the monooxygenase that catalyzes a unique three step reaction (14- methyl 14-alcohol14-aldehyde14-demethylated product plus formic acid), removing the 14- methyl group from the cyclized sterol precursors. CYP51 is regarded as a possible evolutionary ancestor to all members of the currently existing P450 superfamily. The CYP51 reaction is required for biosynthesis of cholesterol in humans, ergosterol in eukaryotic microbial human pathogens (such as fungi and protozoa), and various phytosterols in plants. The vast majority of the sterols are utilized for the formation of viable eukaryotic membranes (fluidity, permeability, modulation of multiple functions of integrated membrane proteins), while some sterols serve as precursors for a number of regulatory molecules essential for cell growth, development, and multiplication. Blocking sterol biosynthesis in unicellular organisms is lethal. CYP51 inhibitors (azoles) are used as the major clinical antifungal drugs and agricultural fungicides. Two of these drugs (posaconazole and ravuconazole) are presently in clinical trials for the deadly human infection caused by the protozoan pathogen Trypanosoma cruzi (Chagas disease). All of the azoles currently in use were discovered empirically, i.e. by screening their effects on fungal cell growth, and are far from ideal in terms of efficiency, safet, side effects, and sensitivity to resistance. The long-term goal of our research is to understand the molecular basis for the CYP51 functional conservation, drug action, and drug resistance. Here we propose to apply the information on the CYP51 structure/function that was gained during the previous cycles of funding to rationalize the CYP51-targeting drug discovery paradigm. We will perform protozoan/fungal/human CYP51 structure-guided derivatization of the highly potent, non-toxic experimental VNI scaffold to make pharmacologically-optimized drug candidates suitable for clinical trials for Chagas disease (including the infections caused by naturally resistant strains of the parasite) and visceral leishmaniasis (Aim 1a), as well as to develop new custom-designed inhibitors of fungal CYP51s (Aim 1b). Aim 2 will evaluate human CYP51 as a potential drug target for cholesterol- related human diseases, including cancer and Alzheimer's disease. Aim 3 of this proposal is to determine the structure of CYP51 in complex with its electron donor partner, NADPH-cytochrome P450 reductase, which will reveal the key residues involved in the protein-protein interaction, and may lead to the development of a novel type of CYP51 inhibitors. The results of this research will direct future approaches in drug discovery, enabling the creation of rationally designed species-oriented drug candidates directed at saving millions of human lives.