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

描述(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的合成，包括激素。我们目前对这些膜酶的结构知识是基于X射线结构的，但我们对这些酶在溶液中或与不形成可结晶的紧密络合物的配体之间的行为知之甚少。因此，人类P450酶功能的重要结构方面尚未在溶液中进行监测。我们的长期目标是引入和开发溶液核磁共振作为一种工具，在溶液中的原子水平上询问P450的功能。我们的近期目标是应用2D溶液核磁共振研究人类雄激素产生细胞色素P450 17A1(CYP17A1)中的配体结合，以指导药物设计工作。由于今年将有24万美国男性被诊断出患有前列腺癌，而前列腺癌是对雄激素的反应而增殖，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执行两种酶反应(羟基酶和裂解酶)，仅抑制其中一种反应对于前列腺癌的治疗是可取的，而且没有严重的副作用。由于缺乏对酶如何发挥作用的了解，限制了对反应选择性CYP17A1抑制剂的寻找。尽管我们产生了一种结合了两种反应的新药抑制剂(阿比特龙)的CYP17A1的X射线晶体结构，但事实证明，每种反应的天然底物都很难结晶。在这项研究中，我们将使用2D核磁共振(核磁共振)来生成关于天然底物和新的抑制剂在活性部位的位置和方向的详细信息，以指导后续用于前列腺癌治疗的裂解酶选择性抑制剂的开发。我们的中心假设是，不同的底物与类固醇抑制剂阿比特龙的结合位置和方向相同，但在羟基酶和裂解酶反应中的参与程度略有不同。我们将通过两个特定的目标来验证这一假设：1)比较羟基酶底物和阿比特龙的位置和方向；2)确定裂解酶底物和仅裂解酶抑制剂的位置和方向。以往核磁共振技术在膜细胞色素P450酶研究中的应用受到技术障碍的限制，这些技术障碍涉及其相对较大的尺寸(55-60 kDa)、疏水性的膜结合表面以及在同位素标记所需的最小介质中的过度表达。我们已经克服了这些困难，生产了足够数量的同位素标记蛋白质，在收集核磁共振数据的同时确定了保持膜蛋白溶解性的条件，并产生了选择性标记的CYP17A1来简化这个55.6 kDa酶的核磁共振谱。这些成功首次实现了对人类P450酶的多维核磁共振研究。因此，这项工作不仅直接为新型CYP17A1抑制剂的设计提供了依据，也为将该方法应用于许多其他人类细胞色素P450酶奠定了基础，可能会影响未来对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.