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

基本信息

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
项目类别：
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

项目摘要

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> 90％的药物代谢和包括激素在内的重要内源性化合物的合成。我们目前对这些膜酶的结构知识是基于X射线结构的，但是我们对溶液中酶的行为或没有形成不适合结晶的紧密复合物的配体的行为了解。因此，在溶液中尚未监测人P450酶功能的重要结构方面。我们的长期目标是引入和利用解决方案NMR作为在解决方案中原子水平上询问P450功能的工具。我们的直接目标是将2D溶液NMR应用于人类产生的雄激素细胞色素P450 17A1（CYP17A1）中的配体结合，以指导药物设计工作。由于今年将被诊断出24万名美国男性患有前列腺癌，而前列腺癌则会响应雄激素而增殖，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而没有严重的副作用。搜索反应选择性CYP17A1抑制剂受到对酶如何功能的了解的限制。尽管我们产生了与两种反应的新药物抑制剂结合的CYP17A1的X射线晶体结构（阿比罗酮），但对于每种反应而言，具有CYP17A1天然底物的晶体被证明是难以捉摸的。在这项研究中，我们将使用2D核磁共振（NMR）来生成有关活性部位中天然底物和新抑制剂的位置和方向的详细信息，从而指导了随后开发裂解裂解酶选择剂以进行前列腺癌处理。我们的中心假设是，不同的底物在与类固醇抑制剂阿比里酮的相同的一般位置和方向上结合，但较小的差异涉及它们参与羟化酶与裂解酶反应的参与。我们将使用两个特定目的检验这一假设：1）将羟化酶底物与阿比罗酮的位置和方向进行比较； 2）确定裂解酶底物的位置和方向和仅裂解酶抑制剂的位置和方向。 NMR先前在研究膜细胞色素P450酶的先前应用受到与其尺寸相对较大（55-60 kDa），疏水膜结合表面相关的技术障碍的限制，以及同位素标签所需的最小介质所需的最小培养基的挑战性过表达。我们已经克服了这些，以产生足够数量的同位素标记的蛋白质，确定在收集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光谱。这些成功允许通过多维NMR首次研究人类P450酶。因此，这项工作不仅直接为新型CYP17A1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，这可能会影响未来对人类药物代谢的研究。