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.

人体血压和压力/免疫反应由类固醇控制 激素醛固酮和皮质醇。有多种疾病状态， 这些激素会过量产生，而选择性抑制剂 临床有益。然而，选择性醛固酮抑制剂的开发 细胞色素P450 11 B2（CYP 11 B2）产生和细胞色素P450 11 B2产生皮质醇 P450 11B 1（CYP 11B 1）因其93%的氨基酸序列同一性而受到挫折。 目前的建议试图通过确定结构性的， CYP 11B 1的特征是与CYP 11B 2的功能差异的基础。中央 假设结构和功能研究将揭示独特的CYP 11B 1 这些特征可以用来支持异构体选择性药物的设计。 为了探索CYP 11B酶与配体相互作用的差异，我们将测定X射线 CYP 11B 1及其底物和几种抑制剂的结构， 但由于选择性差而不是最佳的。初步数据提示 与现有相应CYP 11 B2结构相比的结构差异， 可用于临床的区别（目标1）。初步数据还表明， 共享的必需氧化还原配偶体肾上腺氧还蛋白与两种CYP 11B不同地相互作用 酶，这可能提供了一种正交的方法来选择性抑制-一种 不会集中在活性部位。因此，X射线结构也将被确定为 CYP 11B 1通过融合蛋白的方法与肾上腺素还蛋白相互作用（目的2）。 最终的目的是探索CYP 11B 1的不同构象，这些构象可能存在于 溶液和膜中，这种蛋白质通常嵌入。所有膜 通过X射线晶体学和堆积法测定了P450的结构 相互作用经由配体通过其进入/离开的膜结合表面发生。 因此，虽然这些蛋白质是已知的灵活和动态的，X射线晶体学， 通常只显示一种形式，这可能不是最适合的构象， 抑制剂设计cryo-EM应用于CYP 11B 1（目的3）是一种正交方法 来定义这种酶的更相关的构象。因此，拟议的研究将 确定结构并定义控制CYP 11B 1活性的新相互作用。 将该新信息与CYP 11B 2进行对比，随后可用于促进 开发对每种酶特异的临床相关分子抑制剂。