Application of solution NMR to (human) membrane cytochrome P450 17A1

描述（由申请人提供）：人类细胞色素P450酶负责90%的药物代谢和重要内源性化合物（包括激素）的合成。我们目前对这些膜酶的结构知识是基于x射线结构的，但我们对酶在溶液中的行为或与不形成适合结晶的紧密配合体的配体的行为知之甚少。因此，人类P450酶功能的重要结构方面尚未在溶液中监测。我们的长期目标是引入和利用溶液核磁共振作为一种工具，在溶液的原子水平上询问P450功能。我们的直接目标是应用二维溶液核磁共振来研究人类雄激素产生细胞色素P450 17A1 （CYP17A1）的配体结合，以指导药物设计工作。由于今年美国将有2440,000名男性被诊断为前列腺癌，并且前列腺癌在雄激素的作用下会增殖，因此抑制细胞色素P450 （CYP17A1）是治疗转移性前列腺癌的有效方法。然而，CYP17A1进行两种酶促反应（羟化酶和裂解酶），仅抑制其中一种酶促反应是治疗前列腺癌的理想选择，而不会产生严重的副作用。由于缺乏对酶功能的了解，对反应选择性CYP17A1抑制剂的研究受到限制。尽管我们制作了CYP17A1与两种反应的新药物抑制剂（阿比特龙）结合的x射线晶体结构，但CYP17A1与每种反应的天然底物的晶体已被证明是难以捉摸的。在这项研究中，我们将使用二维核磁共振（NMR）来生成有关天然底物和新抑制剂在活性部位的位置和方向的详细信息，指导后续开发用于前列腺癌治疗的裂解酶选择性抑制剂。我们的中心假设是，不同的底物与甾体抑制剂阿比特龙在相同的一般位置和方向结合，但在羟化酶和裂解酶反应中的参与存在微小差异。我们将通过两个具体目标来检验这一假设：1)比较羟化酶底物与阿比特龙的位置和方向；2)确定裂解酶底物和裂解酶抑制剂的位置和取向。以前应用核磁共振技术来研究膜细胞色素P450酶受到技术障碍的限制，这些技术障碍与它们相对较大的尺寸（55-60 kDa）、疏水的膜结合表面以及在同位素标记所需的最小介质中过度表达有关。我们克服了这些问题，生产了足够数量的同位素标记蛋白，确定了在收集核磁共振数据时保持膜蛋白溶解度的条件，并生成了选择性标记的CYP17A1，以简化这种55.6 kDa酶的核磁共振谱。这些成功使得首次通过多维核磁共振研究人类P450酶成为可能。因此，这项工作不仅直接指导了新型CYP17A1抑制剂的设计，而且为将这种方法用于许多其他人类细胞色素P450酶提供了基础，可能影响未来人类药物代谢的研究。

DESCRIPTION (provided by applicant): Human cytochrome P450 enzymes are responsible for >90% of drug metabolism and synthesis of important endogenous compounds including hormones. Our current structural knowledge of these membrane enzymes is based on X-ray structures, but we understand little of the enzymes' behavior in solution or with ligands that do not form tight complexes amenable to crystallization. Therefore, important structural aspects of human P450 enzyme function have not been monitored in solution. Our long-term goal is to introduce and exploit solution NMR as a tool to interrogate P450 function at the atomic level in solution. Our immediate objective is to apply 2D solution NMR to study ligand binding in the human androgen-producing cytochrome P450 17A1 (CYP17A1) to direct drug design efforts. Since >240,000 U.S. men will be diagnosed with prostate cancer this year and prostate cancer proliferates in response to androgens, inhibiting cytochrome P450 (CYP17A1) is an effective way to treat metastatic prostate cancer. However, CYP17A1 performs two enzymatic reactions (hydroxylase and lyase) and inhibition of only one of them is desirable for prostate cancer treatment without serious side effects. The search for reaction-selective CYP17A1 inhibitors is limited by a lack of understanding regarding how the enzyme functions. Although we produced an X-ray crystal structure of CYP17A1 bound to a new drug inhibitor of both reactions (abiraterone), crystals with the natural substrates of CYP17A1 for each reaction have proven elusive. In this study, we will use 2D Nuclear Magnetic Resonance (NMR) to generate detailed information regarding the location and orientation of both natural substrates and new inhibitors in the active site, guiding the subsequent development of lyase-selective inhibitors for prostate cancer treatment. Our central hypothesis is that different substrates bind in the same general location and orientation as the steroidal inhibitor abiraterone, but with minor differences accounting for their participation in the hydroxylase vs. lyase reactions. We will test this hypothesis using two specific aims: 1) comparing the location and orientation of hydroxylase substrates with abiraterone; and 2) identifying the location and orientation of lyase substrates and a lyase-only inhibitor. Previous application of NMR to study membrane cytochrome P450 enzymes has been limited by technical obstacles related to their relatively large size (55-60 kDa), hydrophobic membrane-binding surfaces, and challenging over-expression in minimal media needed for isotopic labeling. We have overcome these to produce sufficient quantities of isotopically labeled protein, determine conditions to maintain membrane protein solubility while collecting NMR data, and generate selectively labeled CYP17A1 to simplify the NMR spectrum of this 55.6 kDa enzyme. These successes permit, for the first time, investigation of human P450 enzymes by multidimensional NMR. Therefore this work not only directly informs the design of novel CYP17A1 inhibitors, but also provides the groundwork to use this approach for many other human cytochrome P450 enzymes, likely impacting the future study of human drug metabolism. PUBLIC HEALTH RELEVANCE: The proposed work is relevant to public health in two important ways. First, this study will provide biochemical insight into the function of the current prostate cancer drug target, CYP17A1, thus allowing for the design of novel drugs for the treatment of metastatic prostate cancer. Secondly, the biochemical technique we intend to use, multi-dimensional solution NMR spectroscopy, will be applied for the first time to study a human P450 enzyme, thereby opening the door for the future use of this technique in the study of other human enzymes involved in the breakdown of medications.