Structural Requirements for Sterol 14alpha-Demethylases

 描述(由申请人提供) 甾醇14甲基去甲基酶(14α-Domethylase，CYP51)是一种单加氧酶，它催化一个独特的三步反应(14-甲基-甲基-14-甲基-乙醇-14-乙醛-→-14-α-→14-α-脱甲基化产物和甲酸)，从环化的类固醇前体中去除14-α-甲基。CYP51被认为是目前存在的P450超家族所有成员的一个可能的进化祖先。人类胆固醇的生物合成、真核微生物人类病原体(如真菌和原生动物)中的麦角甾醇以及植物中各种植物甾醇的合成都需要细胞色素P51反应。绝大多数的甾醇被用来形成具有生命力的真核膜(流动性、通透性、调节整合的膜蛋白的多种功能)，而一些甾醇则是一些调节分子的前体，这些分子对细胞的生长、发育和增殖至关重要。阻止单细胞生物体中的类固醇生物合成是致命的。CYP51抑制剂(氮唑类)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，以治疗由原生动物病原体克氏锥虫(恰加斯病)引起的致命人类感染。目前使用的所有唑类药物都是根据经验发现的，即通过筛选它们对真菌细胞生长的影响而发现的，在效率、安全性、副作用和对耐药性的敏感性方面远不理想。我们研究的长期目标是了解CYP51功能保守、药物作用和耐药性的分子基础。在这里，我们建议应用在前几个资助周期中获得的关于CYP51结构/功能的信息，以使以CYP51为靶向的药物发现范例合理化。我们将对高效、无毒的实验性VNI支架进行原生动物/真菌/人类CYP51结构引导的衍生化，以使经过药物优化的候选药物适合恰加斯病的临床试验(包括由 这项研究的目的是开发一种新型的抗寄生虫药物(目标1a)和内脏利什曼病(目标1a)，并开发针对真菌细胞色素P51s的新的定制抑制剂(目标1b)。Aim 2将评估人类CYP51作为治疗与胆固醇相关的人类疾病的潜在药物靶点，包括癌症和阿尔茨海默病。本研究的目的3是确定细胞色素P450还原酶与其电子供体NADPH-细胞色素P450还原酶复合体的结构，以揭示蛋白质-蛋白质相互作用中的关键残基，并可能开发出新型的细胞色素P51抑制剂。这项研究的结果将指导未来的药物发现方法，使合理设计的以物种为导向的候选药物能够拯救数百万人的生命。

 DESCRIPTION (provided by applicant) Sterol 14α-demethylase (CYP51) is the monooxygenase that catalyzes a unique three step reaction (14α- methyl →14α-alcohol→14α-aldehyde→14α-demethylated product plus formic acid), removing the 14α- 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.