Structural Requirements for Sterol 14alpha-Demethylases

甾醇 14α-脱甲基酶的结构要求

• 批准号: 8604398
• 负责人: Galina I Lepesheva
• 金额: $ 34.38万
• 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 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8604398
• 关键词: Active Sites; Address; Alcohols; Aldehydes; Animal Model; Animals; Antifungal Agents; Aspergillus; Aspergillus fumigatus; Azole resistance; Azoles; Binding; Biochemical; Biochemistry; Biological; Biophysics; Candida; Candida albicans; Catalysis; Cell division; Cells; Cessation of life; Chagas Disease; Characteristics; Chemicals; Complex; Cytochrome P450; Development; Distant; Drug Design; Drug Targeting; Drug resistance; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Family; Formic Acids; Funding; Future; Goals; Grant; Growth and Development function; Health; Heme Iron; Human; Immunocompromised Host; Immunologic Deficiency Syndromes; Infection; Investigation; Knowledge; Lanosterol; Lead; Learning; Leishmania; Leishmaniasis; Ligands; Mediating; Membrane; Minor; Mixed Function Oxygenases; Modification; Molds; Molecular; Mycoses; Organism; Pharmaceutical Preparations; Phylogeny; Predisposition; Progress Reports; Protozoa; Reaction; Research; Resistance; Roentgen Rays; Site-Directed Mutagenesis; Sterol Biosynthesis Pathway; Sterols; Structure; System; Testing; Trypanosoma; Trypanosoma brucei brucei; Trypanosoma cruzi; Trypanosomatina; Trypanosomiasis; Yeasts; analog; base; cholesterol biosynthesis; design; drug candidate; drug development; fungus; global health; human migration; in vivo; inhibitor/antagonist; killings; member; membrane biogenesis; methyl group; mouse model; mutant; novel; pathogen; preference; pyridine; resistant strain; scaffold

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.

说明(申请人提供)：固醇14？-脱甲基酶(CYP51)是分布最广的，也可能是迄今为止已知的13,000个P450中最古老的。这种单加氧酶催化了一个独特的三步反应(14？甲基-14？醇-14？醛-14？脱甲基产物和甲酸)，从甾醇生物合成的初始环化中间体，即胆固醇生物合成中的羊毛甾醇中去除了14？甲基。在真核病原体中，CYP51是一个药物靶点，因为它催化的反应是膜形成所必需的，因此这种活性的丧失是致命的。在酵母和丝状真菌中，它被作为药物靶标进行了广泛的研究，在这些地方，与CYP血红素铁结合的氮唑被发现是非常有效的药物。其他真核人类病原体还没有得到非常详细的研究，我们已经开始详细分析锥虫和利什曼病中的CYP51，包括它是否可以作为杀死这些生物的药物靶标，这些生物每年在第三世界造成超过1亿人的死亡，并正在成为一个严重的全球问题，主要是由于人类迁徙和日益严重的免疫缺陷。我们对锥虫科的CYP51的详细研究在这笔赠款的前两个资助周期中得到了支持，在当前的这个周期中，我们还表征了新的化学支架，我们相信这些支架是非常有效的原生动物CYP51抑制剂的基础。这一相互竞争的续签申请由三个具体目标组成。首先，是基于结构的原生动物CYP51s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(唑类、吡啶类和底物类)的衍生物，并在四个测试系统中分析这些衍生物的最佳性能：原虫本身、人类细胞、感染原虫的人类细胞和恰加斯病的小鼠模型。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、烟曲霉和黄曲霉感染的潜在药物。这些真菌在人类中具有高度致病性，特别是在免疫功能低下的患者中，在通过生物化学和生物物理(包括X射线结构)对其CYP51进行表征后，我们将确定哪些天蚕素及其衍生物将最有效地用于抗真菌药物设计。第三，我们将比较来自三个不同生物界(原生动物/真菌/人)的CYP51的结构和功能特征，以建立详细的基础，我们可以在此基础上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CYP51相关的唑类耐药性的药物。总体而言，这些研究的结果将指导未来的药物开发方法，这将对全球卫生具有重大意义。