Structure and Function of Cytochrome P450 17A1

描述（由申请人提供）：细胞色素P450 17A1 （CYP17A1）是一种双功能单加氧酶，在人类类固醇生成中起关键作用。由于CYP17A1对雄激素和雌激素的产生至关重要，因此了解该酶的功能在生殖生物学、激素反应性化疗以及CYP17A1缺陷引起的疾病的理解方面具有实用价值。迄今为止，由于缺乏这种膜蛋白的实验结构信息，对CYP17A1的研究受到限制。第一个结构现在表明，抑制剂的结合与之前提出的非常不同，这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。本研究的目的是通过结构、合成和功能的方法来了解CYP17A1多功能反应的控制机制。我们的中心假设是，甾体底物的结合方向与新结构中观察到的抑制剂相似，但对附着位置和质子传递有严格的空间控制，引导底物进行羟基化或裂解反应。具体来说，我们将通过以下方法来验证这一假设：1)生成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的功能评估，以及羟基化酶与裂解酶反应的建议机制；3)通过设计、合成和评估新型探针底物和抑制剂来测试我们对CYP17A1功能的理解。预期的结果是对两种催化反应中控制天然CYP17A1底物结合和催化的结构特征的详细了解。拟议的研究产生了大量的知识基础，以指导设计，开发和改进更有效的药物抑制剂，提高CYP17A1的选择性及其裂解酶活性。通过探测激素生物合成中的一种重要酶，这些结果符合NIH的目标，这种酶可能被用于治疗雄激素敏感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.