Structural Requirements for Sterol 14alpha-demethylase

描述(申请人提供)：细胞色素P450超家族包含6500多个基因，构成至少711个基因家族。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催化甾醇生物合成的一个重要步骤，即甾醇141去甲基化反应，这是该途径的角鲨烯后部分的第一步。这一竞争性的更新将继续对CYP51的研究，长期目标是开发对这一重要药物靶点的抑制的详细了解。我们将从不同的真核生物中确定该酶的高分辨X射线结构，包括大鼠、白色念珠菌和锥虫(布氏锥虫和克鲁兹锥虫)。生物学上已知的100多个CYP51序列包含29个保守的氨基酸，这是CYP51的特征。当这些氨基酸中的某些氨基酸以来自细菌(结核分枝杆菌)的可溶形式的CYP51突变，并与膜结合的真核形式(人、锥虫)中的相同突变进行比较时，观察到显著的功能差异。因此，我们认为原核生物中包含功能构象动力学的3D结构可能与真核细胞中的不同，强调需要确定真核细胞的CYP51结构以与细菌结构进行比较，从而更好地理解CYP51保守的一般原理。CYP51是一种众所周知的致病性酵母菌/真菌感染的药物靶点，而唑类CYP51抑制剂可用于治疗此类感染。然而，开发针对病原体的、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，特别是来自锥虫的细胞色素P51的抑制效果。将对底物抑制剂和氮基抑制剂进行检查，因为它们在P450活性部位区域结合的方式不同。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。在体外确定了有效的抑制剂后，我们将把它们与CYP51同系物(重点是锥体形式)共结晶，以详细了解抑制的结构基础。这项研究将深入了解CYP51的蛋白质结构如何影响这两种类型的抑制剂的紧密结合，并将提示CYP51的催化抑制剂的结构特征对这一抑制过程最重要。我们还将研究最有效的抑制剂在锥虫细胞形式中的作用，以及随后在已建立的克氏锥虫感染小鼠模型中的效果。这些研究将导致对这一基本P450的结构/功能关系的详细了解，并将建立一个详细的范例，用于发现针对传染性原虫布氏支原体和克氏支原体的特定药物，以及可能用于治疗此类感染的潜在先导化合物的信息。可以预期，从这些研究中将产生开发甾醇141-脱甲基酶的特定抑制剂的范例。这些抑制剂将专用于致病原虫中的酶，如锥虫，对宿主(人类)酶几乎没有影响。由于这种抑制，原生动物将无法合成类固醇，无法生存。

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.