Structural Requirements for Sterol 14alpha-demethylase

甾醇 14α-脱甲基酶的结构要求

• 批准号: 8022865
• 负责人: Galina I Lepesheva
• 金额: $ 32.75万
• 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/8022865
• 关键词: Active Sites; Amino Acids; Azoles; Bacteria; Binding; Biological; Biology; Candida albicans; Cytochrome P450; Development; Drug Delivery Systems; Effectiveness; Enzymes; Eukaryota; Gene Family; Genes; Goals; Human; In Vitro; Infection; Lead; Leishmania infantum; Membrane; Mutate; Mutation; Mycobacterium tuberculosis; Mycoses; Names; Nitrogen; Organism; Orthologous Gene; Pathway interactions; Pharmaceutical Preparations; Principal Investigator; Process; Protozoa; Rattus; Reaction; Research; Resolution; Roentgen Rays; Sterol Biosynthesis Pathway; Sterols; Structure; Structure-Activity Relationship; Trypanosoma; Trypanosoma brucei brucei; Trypanosoma cruzi; Yeasts; base; demethylation; enzyme structure; high throughput screening; inhibitor/antagonist; insight; mouse model; pathogen; programs; 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6，500个基因，构成至少711个基因家族。这些基因家族中只有一个，CYP 51，在所有生物王国中发现，从细菌到人类。CYP 51催化甾醇生物合成中的一个重要步骤，甾醇141-脱甲基化反应，这是该途径的后角鲨烯部分的第一步。这一竞争性更新将继续对CYP 51进行研究，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，包括大鼠，白色念珠菌和锥虫（T。brucei和T. cruzi）。整个生物学中已知的100多个CYP 51序列含有29个保守氨基酸，即CYP 51标签。当这些氨基酸中的某些在来自细菌的CYP 51的可溶形式中突变时（M.结核）中的相同突变，并且与膜结合真核形式（人、锥虫）中的相同突变相比，观察到显著的功能差异。因此，我们认为，三维结构，包括功能构象动力学在原核细胞的CYP 51可能是非常不同的，从那些在真核形式强调需要确定真核细胞的CYP 51结构，以比较与细菌的结构，导致更好地理解的一般原则的CYP 51的保护。CYP 51是致病性酵母菌/真菌感染的众所周知的药物靶标，唑类CYP 51抑制剂用于治疗此类感染。然而，开发特异性针对病原体且不靶向宿主（人）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不同生物体（特别是锥虫）的CYP 51抑制的有效性。将检查基于底物和基于氮的抑制剂，因为它们在P450活性位点区域的结合不同。我们还将对160，000个化学文库进行高通量筛选，以寻找可能也是CYP 51抑制剂的紧密结合分子。在体外鉴定出有效的抑制剂后，我们将使它们与CYP 51直向同源物（强调锥虫形式）共结晶，以详细了解抑制的结构基础。这项研究将深入了解CYP 51蛋白结构如何影响两种类型的抑制剂的紧密结合，并将表明CYP 51的催化抑制剂的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在细胞形式的锥虫中的作用，并随后在建立良好的小鼠T。克氏感染这些研究将有助于深入了解P450的结构与功能关系，并为发现针对感染性原虫T. brucei和T. Cruzi，以及关于用于治疗此类感染的潜在先导化合物的信息。可以预期，开发甾醇141-脱甲基酶特异性抑制剂的范例将从这些研究中产生。这些抑制剂对病原性原生动物如锥虫体内的酶具有特异性，对宿主（人）酶几乎没有或没有影响。由于这种抑制，原生动物将无法合成甾醇，无法生存。