Structure and Function of Human Cytochrome P450 11B Enzymes Involved in Cushing’s Disease and Hypertension

参与库欣病和高血压的人类细胞色素 P450 11B 酶的结构和功能

• 批准号: 10194557
• 负责人: Emily E Scott
• 金额: $ 19.18万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10194557
• 关键词: Active Sites; Adrenodoxin; Aldosterone; Aldosterone Antagonists; Amino Acid Sequence; Amino Acids; Anabolism; Binding; Biochemistry; Biological Process; Blood Pressure; CYP11B1 gene; CYP11B2 gene; Characteristics; Chimeric Proteins; Clinical; Complex; Coupled; Cryoelectron Microscopy; Cytochrome P450; Data; Development; Disease; Drug Design; Electrons; Enzyme Interaction; Enzymes; Goals; Heart Diseases; Hormones; Human; Hydrocortisone; Hypertension; Immune; Immune response; Impairment; Kidney Diseases; Knowledge; Ligands; Membrane; Membrane Proteins; Metabolic; Mixed Function Oxygenases; Molecular Conformation; Mutagenesis; Oxidation-Reduction; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Pituitary-dependent Cushing' s disease; Production; Protein Isoforms; Proteins; Publishing; Research; Roentgen Rays; Role; Stress; Structure; Structure-Activity Relationship; Surface; Techniques; Work; X-Ray Crystallography; clinical development; clinically relevant; design; enzyme structure; experience; flexibility; human disease; improved; inhibitor/antagonist; novel strategies; prevent; protein protein interaction; response; side effect; small molecule; steroid hormone

Human blood pressure and stress/immune responses are controlled by the steroid hormones aldosterone and cortisol, respectively. There are multiple disease states in which these hormones are overproduced and for which selective inhibitors would be clinically beneficial. However, the development of selective inhibitors of aldosterone production by cytochrome P450 11B2 (CYP11B2) and cortisol production by cytochrome P450 11B1 (CYP11B1) has been frustrated by their 93% amino acid sequence identity. The current proposal seeks to rectify this gap in knowledge by determining structural features of CYP11B1 that underlie functional differences from CYP11B2. The central hypothesis is that structural and functional studies will reveal unique CYP11B1 characteristics that could be harnessed to support the design of isoform-selective drugs. To probe differences in CYP11B enzyme interactions with ligands, we will determine Xray structures of CYP11B1 with its substrate and several inhibitors that currently under development but are non-optimal because of poor selectivity. Preliminary data suggests structural differences compared to the existing corresponding CYP11B2 structure, distinctions that might be exploited clinically (aim 1). Preliminary data also suggests that the shared required redox partner adrenodoxin interacts differently with the two CYP11B enzymes, which may provide an orthogonal approach to selective inhibition—one that doesn’t focus on the active site. Thus an X-ray structure will also be determined for CYP11B1 interacting with adrenodoxin by employing a fusion protein approach (aim 2). The final aim probes different conformations of CYP11B1 that are likely to exist in solution and in the membrane where this protein is normally embedded. All membrane P450 structures have been determined by X-ray crystallography and packing interactions occur via the membrane-binding surface through which ligands enter/exit. Thus, while these proteins are known to be flexible and dynamic, X-ray crystallography often only reveals one form, which may not be the most appropriate conformation for inhibitor design. Application of cryo-EM to CYP11B1 (aim 3) is an orthogonal approach to define more relevant conformations of this enzyme. Thus, the proposed research will determine the structures and define new interactions controlling the activity of CYP11B1. Contrasting this new information with CYP11B2 can subsequently be used to facilitate the development of clinically-relevant molecules inhibitors specific for each enzyme.

人血压和应激/免疫反应由类固醇控制 骑马醛固酮和皮质醇分别。有多种疾病状态 这些骑马的生产过多，并且选择性抑制剂将是 临床上有益。但是，醛固酮选择性抑制剂的发展 细胞色素P450 11B2（CYP11B2）和细胞色素生产的生产 P450 11B1（CYP11B1）对其93％的氨基酸序列身份感到沮丧。 当前的提议试图通过确定结构性来纠正知识的差距 CYP11B1的特征是与CYP11B2的功能差异的基础。中央 假设是结构和功能研究将揭示独特的CYP11B1 可以利用的特征来支持同工型选择药物的设计。 为了探测CYP11B酶相互作用与配体的差异，我们将确定XRARE CYP11B1的结构及其底物和几种抑制剂 发展但由于选择性差而不是最佳的。初步数据建议 与现有的相应CYP11B2结构相比，结构差异 可能在临床上探索的区别（AIM 1）。初步数据还表明 共享所需的氧化还原伴侣肾上腺素与两个CYP11b的相互作用不同 酶，可以提供一种正交抑制方法的酶 - 一种 不专注于活动网站。还将确定X射线结构 CYP11B1通过采用融合蛋白方法与肾上腺素毒素相互作用（AIM 2）。 最终目标问题的不同考虑的CYP11B1可能存在 溶液和通常嵌入该蛋白的膜中。所有膜 P450结构已通过X射线晶体学和包装确定 相互作用通过配体进入/退出的膜结合表面发生。 虽然已知这些蛋白质是柔性和动态的，但X射线晶体学 通常只揭示一种形式，这可能不是最合适的构象 抑制剂设计。将冷冻EM应用于CYP11B1（AIM 3）是一种正交方法 定义该酶的更相关的构象。拟议的研究将 确定结构并定义控制CYP11B1活性的新相互作用。 将这些新信息与CYP11B2进行对比，随后可以用于促进 针对每种酶特异的临床分子抑制剂的发展。