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: 10368081
• 负责人: Emily E Scott
• 金额: $ 18.14万
• 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 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10368081
• 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; 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.

人类血压和压力/免疫反应由类固醇控制 醛固酮和皮质醇激素分别有多种疾病状态。 哪些激素过量产生以及哪些选择性抑制剂 然而，选择性醛固酮抑制剂的开发在临床上是有益的。 细胞色素 P450 11B2 (CYP11B2) 产生和细胞色素产生皮质醇 P450 11B1 (CYP11B1) 因其 93% 的氨基酸序列同一性而受到挫败。 当前的提案旨在通过确定结构性问题来纠正这一知识差距 CYP11B1 的特征是其与 CYP11B2 功能差异的基础。 假设结构和功能研究将揭示独特的 CYP11B1 可以利用这些特征来支持异构体选择性药物的设计。 为了探究 CYP11B 酶与配体相互作用的差异，我们将确定 X 射线 CYP11B1 的结构及其底物和目前正在研究的几种抑制剂 但由于初步数据表明选择性较差，因此并非最佳。 与现有相应CYP11B2结构相比的结构差异， 可能在临床上利用的区别（目标 1）。 共享所需的氧化还原伙伴肾上腺素与两个 CYP11B 的相互作用不同 酶，这可能提供一种选择性抑制的正交方法 不聚焦于活性位点，因此也将确定 X 射线结构。 CYP11B1 通过采用融合蛋白方法与肾上腺素相互作用（目标 2）。 最终目标是探测 CYP11B1 可能存在于的不同构象 溶液和该蛋白质通常嵌入的膜中。 P450 结构已通过 X 射线晶体学和堆积确定 相互作用通过配体进入/退出的膜结合表面发生。 因此，虽然这些蛋白质已知具有灵活性和动态性，但 X 射线晶体学 通常只揭示一种形式，这可能不是最适合的构象 抑制剂设计。冷冻电镜在 CYP11B1 中的应用（目标 3）是一种正交方法。 因此，拟议的研究将定义该酶的更多相关构象。 确定结构并定义控制 CYP11B1 活性的新相互作用。 随后可以将该新信息与 CYP11B2 进行对比，以促进 开发针对每种酶的临床相关分子抑制剂。