Structural Requirements for Sterol 14alpha-demethylase

项目摘要/摘要 细胞色素P450超家族包含6500多个基因，构成至少711个基因 家人。这些基因家族中只有一种叫CYP51，存在于所有生物界，从细菌到人类。 CYP51催化甾醇生物合成中必不可少的一步，即去甲基化反应。 途径的角鲨烯后部分的第一步。这一竞争性更新将继续研究CYP51 长期目标是详细了解对这一重要药物靶点的抑制。我们会 确定不同真核生物包括大鼠、念珠菌等的该酶的高分辨X射线结构 白念珠菌和锥虫(布氏锥虫和克鲁兹锥虫)。已知的100多个CYP51序列 生物含有29种保守的氨基酸，这是CYP51的特征。当这些氨基酸中的某些是 从细菌(结核分枝杆菌)中突变为可溶形式的CYP51，并与相同的突变在 膜结合的真核生物形式(人、锥虫)，观察到显著的功能差异。 因此，我们认为原核生物细胞色素P51的三维结构包括功能构象动力学 可能与那些强调需要确定真核细胞色素P51的真核形式截然不同 结构与细菌结构进行比较，从而更好地理解细菌的一般原理 细胞色素P51的保守性。CYP51是一种众所周知的针对致病性酵母/真菌感染和唑类的药物靶点 CYP51抑制剂被用来治疗这种感染。然而，开发具有挑战性的药物是很有挑战性的 特定于病原体，不以宿主(人)P450为目标。我们将研究两种类型的抑制剂 它们对来自不同生物，特别是来自锥虫的细胞色素P51有抑制作用。两者都有 将检查基于底物的和基于氮的抑制剂，因为它们在P450中结合的方式不同 活动站点区域。此外，我们还将对16万个化学库进行高通量筛选，以搜索 紧密结合的分子，可能也是CYP51的抑制剂。在体外发现了有效的抑制剂后，我们 将它们与CyP51同源基因(强调锥体形式)共结晶，以了解 详述抑制的结构基础。这项研究将提供对CYP51蛋白结构的洞察 影响两种类型的抑制剂的紧密结合，并将提示催化抑制剂的结构特征 在这一抑制过程中起着至关重要的作用。我们还将研究最有效的 锥虫细胞形式的抑制剂以及随后在已建立的克氏锥虫小鼠模型中的抑制剂 感染。这些研究将导致对这一结构/功能关系的详细理解 Essential P450，并将为发现治疗传染病的特定药物建立一个详细的范例 原生动物、布氏锥虫和克鲁兹锥虫，以及可能用于治疗此类疾病的潜在先导化合物的信息 感染。

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.