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

建议书摘要 人膜细胞色素P450(CYP)酶11B1和11B2催化皮质醇的最后步骤 和醛固酮的合成。皮质醇过量会导致库欣病，导致体重增加和 免疫抑制(2)，而过量的醛固酮会导致原发性醛固酮增多症，导致高血压 (3)。这两种疾病的治疗选择都受到药物选择性差的限制，这是由于 CYP11B1和CYP11B2的序列同源性，特别是在它们的活性位点上。这项建议旨在 确定这些酶之间的结构和功能差异，这些差异可以用来指导发育 治疗库欣病和原发性醛固酮增多症的选择性药物。CYP11B的一个区别是 酶在于它们与共同的氧化还原伙伴肾上腺素的相互作用。我与CYP11B1的初步数据 之前斯科特实验室对CYP11B2的研究表明，肾上腺素变构调节两种细胞色素P11B 酶，但对每种酶的影响不同(9)。这暗示了肾上腺素如何 结合两种CYP11B酶--这一差异可能被用于开发仅选择性阻断的药物 一种酶。然而，肾上腺素变构作用的基础还没有得到很好的描述。这 一项提案试图通过从结构和功能上表征肾上腺素变构效应来填补这一空白 每种细胞色素P11B酶。首先，X射线结构将确定形成CYP11B1/肾上腺素的残基 与CYP11B2/肾上腺素复合体的现有结构进行比较的界面。第二，稳定前-- 使用停流的状态动力学将确定肾上腺素与P450表面的结合是否改变 P450主要通过改变配体的结合或释放与远距离埋藏的活性部位的配体相互作用。 最后，突变研究将调查假设负责的特定环路的影响。 人类P450酶之间变构效应的差异。总体而言，这项研究将提供广泛的结构性 以及肾上腺素变构对细胞色素P11B1和细胞色素P11B2的作用的功能特性。这是详细的 表征促进了我们对生化系统的理解，但也有可能揭示 两种酶在开发两种库欣病选择性药物治疗中的差异 和原发性醛固酮增多症。

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.