Structure and Function of Cytochrome P450 17A1

描述（由申请方提供）：细胞色素P450 17 A1（CYP 17 A1）是一种双功能单加氧酶，在人类固醇生成中起关键作用。由于CYP 17 A1对雄激素和雌激素的产生至关重要，因此了解这种酶的功能在生殖生物学，药物反应性化疗以及了解CYP 17 A1缺陷引起的疾病方面具有实用价值。迄今为止，由于缺乏这种膜蛋白的实验结构信息，对CYP 17 A1的研究受到限制。第一个结构现在表明，抑制剂的结合非常不同，从建议，并提供了一个新的机会，以评估CYP 17 A1的结合，催化和抑制在一个实质上更详细的水平。本提案的目的是通过收敛的结构，合成和功能的方法来了解控制CYP 17 A1的多功能反应的机制。我们的中心假设是，甾体底物结合在一个整体的方向类似于观察到的抑制剂在新的结构，但与紧密的空间控制的配体位置和质子传递直接基板对羟基化或裂解酶反应。具体而言，我们将通过以下方式检验这一假设：1）生成确定底物结合方向和与CYP 17 A1相互作用的X射线结构; 2）对底物结合和催化中的关键氨基酸以及羟化酶与裂解酶反应的拟议机制进行功能评价; 3）通过设计、合成和评价新型探针底物和抑制剂来检验我们对CYP 17 A1功能的理解。预期的结果是详细了解控制天然CYP 17 A1底物结合和催化两种催化反应的结构特征。拟议的研究产生了大量的知识库，以指导设计，开发和改进更有效的药物抑制剂，提高对CYP 17 A1及其裂解酶活性的选择性。这些结果通过探测激素生物合成中的一种重要酶来满足NIH的目标，这种酶可能被操纵用于治疗雄激素敏感性和雌激素反应性癌症以及其他类固醇相关疾病。

DESCRIPTION (provided by applicant): Cytochrome P450 17A1 (CYP17A1) is a dual-function monooxygenase with a key role in human steroidogenesis. Since CYP17A1 is essential for androgen and estrogen production, understanding how this enzyme functions has practical value in reproductive biology, hormone-responsive chemotherapy, and the understanding of diseases resulting from CYP17A1 defects. To date, investigations of CYP17A1 have been limited by the absence of experimental structural information of this membrane protein. The first structures now show that inhibitors bind very differently from proposed and provide a new opportunity to evaluate CYP17A1 binding, catalysis, and inhibition at a substantially more detailed level. The objective of this proposal is to understand the mechanisms controlling the multifunctional reactions of CYP17A1 through convergent structural, synthetic, and functional approaches. Our central hypothesis is that steroidal substrates bind in an overall orientation similar to that observed for inhibitors in the new structures, but with tight spatial control of liand position and proton delivery directing substrates toward either hydroxylation or lyase reactions. Specifically we will test this hypothesis by 1) generation of X-ray structures that determine substrate binding orientations and interactions with CYP17A1, 2) functional evaluation of key amino acids in substrate binding and catalysis and of proposed mechanisms for hydroxylase vs. lyase reactions, and 3) testing our understanding of CYP17A1 function via the design, synthesis, and evaluation of novel probe substrates and inhibitors. The expected outcome is a detailed understanding of the structural features that control binding and catalysis of native CYP17A1 substrates for both catalytic reactions. The proposed research generates a substantial knowledgebase to guide the design, development, and improvement of more effective pharmaceutical inhibitors with improved selectivity for CYP17A1 and its lyase activity. These outcomes meet NIH goals by probing an important enzyme in hormone biosynthesis that can potentially be manipulated for the treatment of androgen-sensitive and estrogen-responsive cancers, as well as other steroid-related diseases.