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

溶液 NMR 在（人）膜细胞色素 P450 17A1 中的应用

• 批准号: 8573554
• 负责人: David Fernando Estrada
• 金额: $ 5.33万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-11-18 至 2014-11-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8573554
• 关键词: Accounting; Active Sites; Adopted; Adverse effects; Affinity; Amino Acids; Androgens; Antineoplastic Agents; Behavior; Binding; Biochemical; Biochemical Reaction; Carbon; Catalysis; Chemicals; Complex; Crystallization; Cytochrome P450; Data; Development; Diagnosis; Drug Design; Drug Targeting; Enzymes; Future; Goals; Heme; Hormones; Human; Investigation; Knowledge; Label; Ligand Binding; Ligands; Lobe; Location; Lyase; Malignant neoplasm of prostate; Maps; Measures; Membrane; Membrane Proteins; Metabolism; Metastatic Prostate Cancer; Minor; Mixed Function Oxygenases; Modeling; Molecular Conformation; Monitor; NMR Spectroscopy; Nuclear Magnetic Resonance; Outcome; Pharmaceutical Preparations; Physiological Processes; Pregnenolone; Progesterone; Proliferating; Proteins; Public Health; Reaction; Relative (related person); Research; Roentgen Rays; Solubility; Solutions; Structure; Surface; Techniques; Testing; Time; Vitamins; Work; Xenobiotics; abiraterone; analog; base; cancer therapy; design; drug metabolism; drug synthesis; enzyme structure; inhibitor/antagonist; insight; men; novel; public health relevance; response; success; tool

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.

描述（由申请人提供）：人类细胞色素 P450 酶负责超过 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了解是基于 X 射线结构，但我们对这些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利用溶液 NMR 作为在溶液中原子水平询问 P450 功能的工具。我们的近期目标是应用 2D 溶液 NMR 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。由于今年将有超过 240,000 名美国男性被诊断出患有前列腺癌，并且前列腺癌会因雄激素而增殖，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 执行两种酶反应（羟化酶和裂解酶），并且仅抑制其中一种对于前列腺癌治疗来说是理想的，而且不会产生严重的副作用。由于缺乏对该酶功能的了解，对反应选择性 CYP17A1 抑制剂的研究受到限制。尽管我们制备了 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，但具有每种反应的 CYP17A1 天然底物的晶体已被证明是难以捉摸的。在本研究中，我们将使用二维核磁共振（NMR）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，不同的底物与类固醇抑制剂阿比特龙的结合位置和方向相同，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) 确定裂合酶底物和仅裂合酶抑制剂的位置和方向。以前应用 NMR 研究膜细胞色素 P450 酶受到技术障碍的限制，这些技术障碍涉及其相对较大的尺寸 (55-60 kDa)、疏水性膜结合表面以及同位素标记所需的最小介质中具有挑战性的过度表达。我们已经克服了这些问题，产生了足够数量的同位素标记蛋白质，确定了在收集 NMR 数据时保持膜蛋白溶解度的条件，并生成选择性标记的 CYP17A1 以简化这种 55.6 kDa 酶的 NMR 谱。这些成功首次允许通过多维 NMR 对人类 P450 酶进行研究。因此，这项工作不仅直接为新型 CYP17A1 抑制剂的设计提供信息，而且还为将该方法用于许多其他人类细胞色素 P450 酶提供了基础，可能会影响人类药物代谢的未来研究。