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: 10536786
• 负责人: Cara Lorene Loomis
• 金额: $ 4.68万
• 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/10536786
• 关键词: Active Sites; Adrenodoxin; Affinity; Aldosterone; Binding; Biochemical; Biological Assay; CYP11A1 gene; CYP11B1 gene; Cardiovascular system; Catalysis; Clinical; Complex; Cortodoxone; Crystallization; Cytochrome P450; Data; Development; Disease; Distant; Drug Design; 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; protein protein interaction; 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.

建议总结