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

描述（由申请人提供）：人细胞色素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，但我们对酶在溶液中或与不形成适合结晶的紧密复合物的配体的行为了解甚少。因此，尚未在溶液中监测人P450酶功能的重要结构方面。我们的长期目标是引入和利用溶液NMR作为在溶液中原子水平上询问P450功能的工具。我们的近期目标是应用二维溶液NMR研究配体结合在人类雄激素产生细胞色素P450 17 A1（CYP 17 A1），直接药物设计的努力。由于今年将有超过240，000名美国男性被诊断患有前列腺癌，并且前列腺癌会对雄激素产生反应，因此抑制细胞色素P450（CYP 17 A1）是治疗转移性前列腺癌的有效方法。然而，CYP 17 A1进行两种酶促反应（羟化酶和裂解酶），并且仅抑制其中一种对于前列腺癌治疗是期望的，而没有严重的副作用。对反应选择性CYP 17 A1抑制剂的研究受到缺乏对酶功能的了解的限制。虽然我们产生了与两种反应的新药抑制剂（阿比特龙）结合的CYP 17 A1的X射线晶体结构，但已证明每个反应的CYP 17 A1天然底物的晶体是难以捉摸的。在这项研究中，我们将使用二维核磁共振（NMR）来生成有关活性位点中天然底物和新抑制剂的位置和方向的详细信息，指导随后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，不同的底物在与甾体抑制剂阿比特龙相同的一般位置和方向上结合，但在参与羟化酶与裂解酶反应方面存在微小差异。我们将使用两个特定目的来检验该假设：1）比较羟化酶底物与阿比特龙的位置和方向;和2）鉴定裂解酶底物和仅裂解酶抑制剂的位置和方向。先前应用NMR研究膜细胞色素P450酶受到技术障碍的限制，这些技术障碍与其相对较大的尺寸（55-60 kDa）、疏水性膜结合表面以及在同位素标记所需的最小培养基中具有挑战性的过度表达有关。我们已经克服了这些，以产生足够量的同位素标记的蛋白质，确定条件，以保持膜蛋白的溶解度，同时收集NMR数据，并产生选择性标记的CYP 17 A1，以简化这种55.6 kDa的酶的NMR光谱。这些成功允许，第一次，调查人类P450酶的多维NMR。因此，这项工作不仅直接为新型CYP 17 A1抑制剂的设计提供了信息，而且还为许多其他人类细胞色素P450酶使用这种方法提供了基础，可能会影响人类药物代谢的未来研究。

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.