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; 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催化皮质醇的最终步骤 和醛固酮合成。多余的皮质醇会导致库欣氏病，导致体重增加和 免疫抑制（2），而超过醛固酮会引起原发性醛固酮含量，导致高血压 （3）。两种疾病状态的治疗选择受到药物选择性不佳的限制，这是由于较高的 CYP11B1和CYP11B2之间的序列同源性，尤其是在其活性位点。该建议旨在 确定这些酶之间可用于开发信息的结构和功能差异 用于库欣病和原发性醛固酮症的选择性药物。 CYP11b之间的一个区别 酶在于与共享氧化还原伴侣肾上腺素毒素的相互作用。我使用CYP11B1的初步数据 Scott实验室对CYP11B2的研究表明，肾上腺素毒素会适应两个CYP11B 酶，但对每种酶产生不同的影响（9）。这表明肾上腺素毒素的潜在差异 结合两种CYP11b酶 - 差异性，可以针对开发仅有选择性阻止的药物 一种酶。但是，肾上腺素毒素变构作用的基础尚未得到充分表现。这 提案旨在通过结构和功能表征肾上腺素毒素变构作用来填补这一空白 每个CYP11B酶。首先，X射线结构将定义形成CYP11B1/肾上腺素的残差 与CYP11B2/肾上腺素毒素复合物的现有结构进行比较的界面。第二，稳定 - 使用停止流量的状态动力学将决定肾上腺素毒素在P450表面的结合是否改变 P450与配体在远处建造的活性位点的配体相互作用，通过主要改变配体结合或释放。 最后，诱变研究将研究假设是负责的特定循环的效果 人P450酶之间变构作用的差异。总体而言，这项研究将提供广泛的结构 肾上腺素毒素变构作用对CYP11B1和CYP11B2的功能表征。这是详细的 表征我们对生化系统的理解，但也有可能揭示 两种酶之间的差异有助于开发库欣氏病的选择性药物治疗 和主要的醛固酮主义。