Structure and Function of Cytochrome P450 17A1

描述(申请人提供)：细胞色素P450 17A1(细胞色素P450 17A1)是一种双功能单加氧酶，在人类类固醇合成中起关键作用。由于细胞色素P17A1对雄激素和雌激素的产生是必不可少的，了解该酶的功能在生殖生物学、激素反应性化疗以及理解由细胞色素P17A1缺陷引起的疾病方面具有实用价值。到目前为止，由于缺乏该膜蛋白的实验结构信息，对该蛋白的研究一直受到限制。第一批结构现在表明，抑制剂结合的方式与建议的截然不同，并为在更详细的水平上评估CYP17A1的结合、催化和抑制提供了新的机会。这项建议的目的是通过聚合的结构、合成和功能方法来了解控制CYP17A1的多功能反应的机制。我们的中心假设是，类固醇底物以类似于在新结构中观察到的抑制剂的整体取向结合，但对配体位置和质子输送的严格空间控制将底物导向羟化或裂解酶反应。具体地说，我们将通过1)产生确定底物结合方向和与CYP17A1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所提出的羟基酶与裂解酶反应的机制进行功能评估，以及3)通过设计、合成和评估新的探针底物和抑制剂来测试我们对CYP17A1功能的理解。预期的结果是详细了解控制天然CYP17A1底物对这两种催化反应的结合和催化的结构特征。拟议的研究产生了大量的知识库来指导设计、开发和改进更有效的药物抑制剂，提高了对CYP17A1及其裂解酶活性的选择性。通过探索激素生物合成中的一种重要酶，这些结果达到了NIH的目标，该酶可能被操纵用于治疗雄激素敏感型和雌激素反应型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.