Structural Requirements for Sterol 14alpha-demethylase

甾醇 14α-脱甲基酶的结构要求

基本信息

批准号：
8008964
负责人：
Galina I Lepesheva
金额：
$ 5.18万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-01-28 至 2010-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8008964
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 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 Organism Orthologous Gene Pathway interactions Pharmaceutical Preparations Process Progress Reports Protozoa Rattus Reaction Resolution Rodent Model Roentgen Rays Role Screening procedure Sequence Alignment Specificity Sterol Biosynthesis Pathway Sterols Structure Structure-Activity Relationship Substrate Specificity Testing Trypanosoma Trypanosoma brucei brucei Trypanosoma cruzi Trypanosomatina Yeasts abstracting 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

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 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 Organism Orthologous Gene Pathway interactions Pharmaceutical Preparations Process Progress Reports Protozoa Rattus Reaction Resolution Rodent Model Roentgen Rays Role Screening procedure Sequence Alignment Specificity Sterol Biosynthesis Pathway Sterols Structure Structure-Activity Relationship Substrate Specificity Testing Trypanosoma Trypanosoma brucei brucei Trypanosoma cruzi Trypanosomatina Yeasts abstracting 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

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项目摘要

PROJECT SUMMARY/ABSTRACT 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 14α-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.

项目摘要/摘要细胞色素P450超家族包含构成至少711个基因的6,500多个基因家庭。这些基因家族中只有一个CYP51在所有生物界，人类的细菌中都发现。 CYP51催化了固醇生物合成的重要步骤，即固醇14α-甲基化反应，这是路径的邮局部分中的第一步。这种竞争的更新将继续研究CYP51 长期目标是详细了解对这一重要药物靶标的抑制作用。我们将从不同的真核生物（包括大鼠，念珠菌）确定该酶的高分辨率X射线结构白色质体和锥虫（T. Brucei和T. Cruzi）。整个中已知的100多个CYP51序列生物学包含29个保守的氨基酸，即CYP51签名。当某些氨基酸是以细菌的CYP51的可溶性形式突变（结核分枝杆菌），并将膜结合的真核形式（人，锥虫）观察到显着的功能差异。因此，我们认为3D结构在原核生物CYP51中包括功能构象动力学可能与确定真核CYP51的需要的真核形式大不相同与细菌结构进行比较的结构，从而更好地理解 CYP51保护。 CYP51是致病性酵母/真菌感染和唑的众所周知的药物靶标 CYP51抑制剂用于治疗此类感染。但是，开发药物是具有挑战性的针对病原体，不靶向宿主（人）P450。我们将研究两种类型的抑制剂它们在抑制不同生物体的CYP51方面的有效性，尤其是来自锥虫的生物体。两个都基于底物和基于氮的抑制剂将被检查，因为它们在P450中的结合不同活性场所区域。我们还将对160,000个化学库进行高吞吐量筛查以搜索紧密结合分子也可能是CYP51的抑制剂。在体外确定了有效的抑制剂之后将与CYP51直系同源物（强调锥虫形式）共结合它们，以便理解详细说明抑制的结构基础。这项研究将洞悉CYP51蛋白结构如何影响两种类型抑制剂的紧密结合，并建议催化抑制剂的结构特征 CYP51的抑制过程最重要。我们还将研究最有效的效果锥虫细胞形式的抑制剂，随后在良好的Cruzi小鼠模型中感染。这些研究将导致对此的结构/功能关系有详细的理解必不可少的P450，还将建立一个详细的范式，以发现特定药物的传染性药物 Protozoa，T。Brucei和T. Cruzi，以及有关治疗的潜在铅化合物的信息感染。