Structural Requirements for Sterol 14alpha-Demethylases

性状（由申请人提供）：甾醇14？-脱甲基酶（CYP 51）是迄今已知的> 13，000种P450中分布最广泛并且可能是最古老的。该单加氧酶催化独特的三步反应（14？-甲基->14？-酒精->14？-醛->14？-脱甲基产物加甲酸）除去14？-来自甾醇生物合成中初始环化中间体的甲基，即胆固醇生物合成中的羊毛甾醇。CYP 51是真核病原体中的药物靶标，因为它催化的反应对于膜形成是必需的，因此该活性的丧失是致命的。它已被广泛研究，作为酵母和丝状真菌中的药物靶标，其中已发现与血红素铁结合的唑类是非常有效的药物。其他真核人类病原体尚未进行详细研究，我们已经开始对锥虫和利什曼原虫中的CYP 51进行详细分析，包括它是否可以作为杀死这些生物体的药物靶标，这些生物体每年在“第三世界”造成一亿多人死亡，并正在成为一个严重的全球性问题，主要是由于人类迁移和日益严重的免疫缺陷。我们对来自锥虫科的CYP 51的详细研究在该资助的前两个资助周期中得到了支持，在当前的资助周期中，我们还表征了新型化学支架，我们认为这些支架是非常有效的原生动物CYP 51抑制剂的基础。这种竞争性的续期申请包括三个具体目标。首先，是基于结构的原生动物CYP 51选择性抑制剂的开发。在这里，我们将合成我们发现的三种原始支架（基于唑、吡啶和底物）的衍生物，并在四个测试系统中分析这些衍生物中最好的：原生动物本身、人类细胞、感染原生动物的人类细胞和小鼠模型恰加斯病。其次，我们将测试这些支架及其衍生物作为治疗白色念珠菌和烟曲霉（A）和A。黄的这些真菌在人类中具有高致病性，特别是在免疫功能低下的患者中，并且在通过其CYP 51的生物化学和生物物理学（包括X射线结构）表征之后，我们将确定哪些scofids和衍生物将是最有效的抗真菌药物设计。第三，我们将比较来自三种不同生物界（原生动物/真菌/人类）的CYP 51的结构-功能特征，以详细建立我们可以确定酶和抑制剂的哪些特征将导致合理设计病原体选择性药物和有效治疗CYP 51相关唑类耐药性的药物的基础。总的来说，这些研究的结果将指导未来的药物开发方法，这对全球健康具有重大意义。

DESCRIPTION (provided by applicant): Sterol 14?-demethylase (CYP51) is the most widely distributed and perhaps the oldest of the > 13,000 P450s known to date. This monooxygenase 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 initial cyclized intermediate in sterol biosynthesis, i.e. lanosterol in cholesterol biosynthesis. CYP51 is a drug target in eukaryotic pathogens because the reaction it catalyzes is essential for membrane formation and therefore loss of this activity is lethal. It has been studied extensively as a drug target in yeast and filamentous fungi where azoles that bind to the CYP heme iron have been found to be very effective drugs. Other eukaryotic human pathogens have not been studied in great detail and we have begun a detailed analysis of CYP51 in trypanosomes and leishmania, including whether it can serve as a drug target for killing these organisms which cause more than one hundred million deaths each year in the 'third world' and is becoming a serious global problem, mainly due to human migration and growing immunodeficiency. Our detailed investigation of CYP51 from Trypanosomatidae has been supported during the first two funding cycles of this grant, and during this current cycle we have also characterized novel chemical scaffolds which we believe are the basis of very efficient inhibitors of CYP51 from protozoa. This competing renewal application consists of three Specific Aims. First, is structure-based development of selective inhibitors for protozoan CYP51s. Here we will synthesize derivatives of the three original scaffolds we discovered (azole, pyridine and substrate based) and analyze the best of these derivatives in four test systems: protozoa themselves, human cells, human cells infected with protozoa and mouse models for Chagas disease. Second, we will test these scaffolds and their derivatives as potential drugs for treatment of infection by Candida albicans and Aspergillus (A) fumigatus and A. flavus. These fungi are highly pathogenic in humans, especially in immunocompromised patients, and after characterization by biochemistry and biophysics (including X-ray structure) of their CYP51s we will establish which scoffids and derivatives will be most effective for antifungal drug design. Third, we will compare structure-function characteristics of CYP51s from three different biological kingdoms (protozoa/fungi/human) to establish in detail the basis on which we can identify what features of both the enzymes and the inhibitors will lead to rational design of pathogen-selective drugs and drugs effective for CYP51-related azole resistance. Overall, the results arising from these studies will direct future approaches for drug development which will have major importance in global health.