Structural Requirements for Sterol 14alpha-demethylase

甾醇 14α-脱甲基酶的结构要求

• 批准号: 7383002
• 负责人: Galina I Lepesheva
• 金额: $ 34.59万
• 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 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7383002
• 关键词: Active Sites; Affinity; Amino Acid Sequence; Amino Acids; Antiparasitic Agents; Attention; Azoles; Bacteria; Binding; Biochemical; Biological; Biology; Candida albicans; Catalysis; Cells; Chagas Disease; Clinical; Crystallization; Cytochrome P450; Development; Drug Delivery Systems; Drug resistance; Effectiveness; Enzymes; Eukaryota; Eukaryotic Cell; Family; Funding; Gene Family; Genes; Goals; Grant; Heme; Heme Iron; Human; In Vitro; Infection; Laboratories; Lead; Membrane; Microbe; Mixed Function Oxygenases; Modeling; Mutate; Mutation; Mycobacterium tuberculosis; Mycoses; Nature; Nitrogen; Numbers; Organism; Orthologous Gene; Pathway interactions; Pharmaceutical Preparations; Process; Progress Reports; Protozoa; Range; Rattus; Reaction; Resolution; Rodent Model; Roentgen Rays; Role; Sequence Alignment; Specificity; Sterol Biosynthesis Pathway; Sterols; Structure; Structure-Activity Relationship; Substrate Specificity; Testing; Trypanosoma; Trypanosoma brucei brucei; Trypanosoma cruzi; Trypanosomatina; Tuberculosis; Yeasts; analog; base; demethylation; enzyme structure; enzyme substrate; fungus; high throughput screening; inhibitor/antagonist; insight; interest; member; mouse model; novel; pathogen; protein structure; small molecule libraries; three dimensional structure

DESCRIPTION (provided by applicant): The cytochrome P450 superfamily contains more than 6,500 genes constituting at least 711 gene families. Only one of these gene families, CYP51, is found in all biological kingdoms, bacteria to humans. CYP51 catalyzes an essential step in sterol biosynthesis, the sterol 141-demethylation reaction which is the first step in the postsqualene portion of the pathway. This competing renewal will continue studies of CYP51 with a long term goal of developing detailed understanding of inhibition of this important drug target. We will determine the high resolution X-ray structure of this enzyme from different eukaryotes, including rat, Candida albicans and trypanosomes (T. brucei and T. cruzi). The more than 100 CYP51 sequences known throughout biology contain 29 conserved amino acids, the CYP51 signature. When certain of these amino acids are mutated in the soluble form of CYP51 from bacteria (M. tuberculosis) and compared to the same mutation in membrane bound eukaryotic forms (human, trypanosomes), significant functional differences are observed. Therefore we believe that 3D structure including functional conformational dynamics in the prokaryotic CYP51 may be quite different from those in eukaryotic forms emphasizing the need to determine eukaryotic CYP51 structure to compare with the bacterial structure, leading to better understanding of the general principles of CYP51 conservation. CYP51 is a well known drug target for pathogenic yeast/fungal infections and azole CYP51 inhibitors are used to treat such infections. However, it is challenging to develop drugs which are specific for the pathogen and do not target host (human) P450s. We will investigate two types of inhibitors for their effectiveness in inhibition of CYP51 from different organisms, particularly those from trypanosomes. Both substrate-based and nitrogen-based inhibitors will be examined because they bind differently in the P450 active site region. Also we will carry out high throughput screening of a 160,000 chemical library to search for tight binding molecules which might also be inhibitors of CYP51. Having identified potent inhibitors in vitro we will co-crystallize them with CYP51 orthologs (emphasis on trypanosomal forms) in order to understand in detail the structural basis of inhibition. This study will provide insight into how the CYP51 protein structure influences tight binding of both types of inhibitors and will suggest what structural features of catalytic inhibitors of CYP51 are most important for this inhibition process. We will also study the effect of the most potent inhibitors in cellular forms of trypanosomes and subsequently in well established mouse models for T. cruzi infection. These studies will lead to a detailed understanding of the structure/function relationship of this essential P450, and also will establish a detailed paradigm for discovery of specific drugs for the infectious protozoa, T. brucei and T. cruzi, and perhaps information on potential lead compounds for treatment of such infections. It can be expected that a paradigm for development of specific inhibitors of sterol 141-demethylase will arise from these studies. These inhibitors will be specific for the enzymes in pathogenic protozoa such as trypansomes and have little or no effect on the host (human) enzyme. As a result of this inhibition, the protozoa will not be able to synthesize sterols and can not survive.

描述（由申请人提供）：细胞色素P450超家族包含超过6500个基因，构成至少711个基因家族。这些基因家族中只有一个CYP51存在于所有生物王国中，从细菌到人类。CYP51催化甾醇生物合成的一个重要步骤，即甾醇141-去甲基化反应，这是该途径后角鲨烯部分的第一步。这一竞争性更新将继续CYP51的研究，其长期目标是详细了解这一重要药物靶点的抑制作用。我们将从不同的真核生物中测定该酶的高分辨率x射线结构，包括大鼠、白色念珠菌和锥虫（布氏体和克氏体）。在整个生物学中已知的超过100个CYP51序列包含29个保守氨基酸，即CYP51的特征。当这些氨基酸中的某些在细菌（结核分枝杆菌）的CYP51可溶性形式中发生突变，并与在膜结合的真核生物形式（人，锥虫）中发生相同突变时，观察到显著的功能差异。因此，我们认为原核CYP51的三维结构包括功能构象动力学可能与真核形式的CYP51有很大不同，强调需要确定真核CYP51的结构以与细菌结构进行比较，从而更好地了解CYP51的一般保护原理。CYP51是众所周知的致病性酵母菌/真菌感染的药物靶点，唑类CYP51抑制剂用于治疗此类感染。然而，开发针对病原体特异性且不针对宿主（人类）p450的药物是具有挑战性的。我们将研究两种类型的抑制剂对来自不同生物体的CYP51的抑制效果，特别是来自锥虫的CYP51。底物抑制剂和氮基抑制剂都将被检查，因为它们在P450活性位点区域的结合方式不同。此外，我们还将对16万个化学文库进行高通量筛选，以寻找可能也是CYP51抑制剂的紧密结合分子。在体外鉴定出有效的抑制剂后，我们将把它们与CYP51同源物（重点是锥虫体形式）共结晶，以便详细了解抑制的结构基础。本研究将深入了解CYP51蛋白结构如何影响两种抑制剂的紧密结合，并将提示CYP51催化抑制剂的哪些结构特征对这种抑制过程最重要。我们还将研究最有效的抑制剂对细胞形式的锥虫的影响，并随后在完善的克氏锥虫感染小鼠模型中研究。这些研究将导致对这一重要P450的结构/功能关系的详细了解，也将为发现针对感染性原生动物，布鲁氏t和克氏t的特异性药物建立一个详细的范例，并可能为治疗这类感染的潜在先导化合物提供信息。可以预期，从这些研究中将出现一种开发甾醇141-去甲基酶特异性抑制剂的范例。这些抑制剂对致病性原生动物（如锥虫）中的酶具有特异性，对宿主（人）的酶几乎没有影响。由于这种抑制，原生动物将无法合成甾醇，无法生存。