Defining Structural and Functional Differences Between Cytochrome P450 11B1 and 11B2 Interactions with Redox Partner Adrenodoxin for Developing Cushing’s Disease and Primary Aldosteronism Treatments

定义细胞色素 P450 11B1 和 11B2 与氧化还原伙伴肾上腺素的相互作用在库欣病和原发性醛固酮增多症治疗中的结构和功能差异

• 批准号: 10685280
• 负责人: Cara Lorene Loomis
• 金额: $ 4.77万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-29 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10685280
• 关键词: Active Sites; Adrenodoxin; Affinity; Aldosterone; Binding; Biochemical; Biological Assay; CYP11A1 gene; CYP11B1 gene; Cardiovascular system; Catalysis; Clinical; Complex; Cortodoxone; Cytochrome P450; Data; Development; Disease; Distant; Drug Design; Enzyme Interaction; Enzymes; FDA approved; Future; Goals; Human; Hydrocortisone; Hyperaldosteronism; Hypertension; Immunosuppression; Investigation; Kinetics; Ligand Binding; Ligands; Literature; Membrane; Metabolic; Mutagenesis; Outcome; Oxidation-Reduction; Pharmaceutical Preparations; Pharmacotherapy; Pituitary-dependent Cushing' s disease; Proteins; Research; Roentgen Rays; Role; Sequence Homology; Structure; Structure-Activity Relationship; Surface; System; Weight Gain; design; improved; inhibitor; insight; novel strategies; prevent; therapy design

PROPOSAL SUMMARY Human membrane cytochrome P450 (CYP) enzymes 11B1 and 11B2 catalyze the final steps in cortisol and aldosterone synthesis, respectively. Excess cortisol causes Cushing’s disease, leading to weight gain and immune suppression (2), whereas excess aldosterone causes primary aldosteronism, leading to hypertension (3). Treatment options for both disease states have been limited by poor drug selectivity, resulting from the high sequence homology between CYP11B1 and CYP11B2, especially in their active sites. This proposal aims to identify structural and functional differences between these enzymes that could be used to inform development of selective drugs for Cushing’s disease and primary aldosteronism. One difference between the CYP11B enzymes lies in their interaction with their shared redox partner, adrenodoxin. My preliminary data with CYP11B1 and previous Scott lab studies on CYP11B2 demonstrate that adrenodoxin allosterically modulates both CYP11B enzymes, but with different impacts on each enzyme (9). This suggests potential differences in how adrenodoxin binds the two CYP11B enzymes--differences that could be targeted to develop drugs that selectively block only one enzyme. However, the basis for adrenodoxin’s allosteric effect has not been well-characterized. This proposal seeks to fill this gap by structurally and functionally characterizing the adrenodoxin allosteric effect on each CYP11B enzyme. First, an X-ray structure will define the residues forming the CYP11B1/adrenodoxin interface for comparison with an existing structure of the CYP11B2/adrenodoxin complex. Second, pre steady-- state kinetics using stopped flow will determine whether adrenodoxin binding on the P450 surface changes the P450 interactions with ligands in the distant buried active site by primarily altering ligand binding or release. Finally, mutagenesis studies will investigate the effect of a specific loop hypothesized to be responsible for the differences in the allosteric effect among human P450 enzymes. Overall, this study will provide a broad structural and functional characterization of the adrenodoxin allosteric effect on CYP11B1 and CYP11B2. This detailed characterization advances our understanding of the biochemical system but also has the potential to reveal differences between the two enzymes useful in developing selective drug treatments for both Cushing’s disease and primary aldosteronism.

提案摘要 人膜​​细胞色素 P450 (CYP) 酶 11B1 和 11B2 催化皮质醇的最后步骤 和醛固酮合成过多会导致库欣病，导致体重增加和 免疫抑制（2），而醛固酮过多会导致原发性醛固酮增多症，导致高血压 (3) 两种疾病状态的治疗选择均因药物选择性差而受到限制。 CYP11B1 和 CYP11B2 之间的序列同源性，特别是在它们的活性位点上。 确定这些酶之间的结构和功能差异，可用于为发育提供信息 库欣病和原发性醛固酮增多症的选择性药物的一个区别。 酶的作用在于它们与共享的氧化还原伙伴肾上腺素的相互作用。我与 CYP11B1 的初步数据。 Scott 实验室之前对 CYP11B2 的研究表明，肾上腺素还可以变构调节 CYP11B 酶，但对每种酶的影响不同 (9)。 结合两种 CYP11B 酶——可以针对这些差异开发选择性阻断药物 然而，肾上腺素还蛋白变构作用的基础尚未得到充分表征。 该提案旨在通过从结构和功能上表征肾上腺素还蛋白变构效应来填补这一空白。 首先，X 射线结构将定义形成 CYP11B1/肾上腺素的残基。 与 CYP11B2/肾上腺素复合物的现有结构进行比较的界面 其次，预稳定。 使用停止流动的状态动力学将确定 P450 表面上的肾上腺素还蛋白结合是否会改变 P450 通过主要改变配体结合或释放与远处埋藏活性位点中的配体相互作用。 最后，诱变研究将调查特定循环的影响，该循环负责导致 总体而言，这项研究将提供广泛的结构。 以及肾上腺素还蛋白对 CYP11B1 和 CYP11B2 变构作用的功能表征。 表征增进了我们对生化系统的理解，同时也有可能揭示 两种酶之间的差异可用于开发库欣病的选择性药物治疗 和原发性醛固酮增多症。