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

提案摘要 人膜细胞色素P450（CYP）酶11B 1和11B 2催化皮质醇的最后步骤 和醛固酮合成。过量的皮质醇会引起库欣病，导致体重增加， 免疫抑制（2），而过量的醛固酮引起原发性醛固酮增多症，导致高血压 （三）、这两种疾病状态的治疗选择受到药物选择性差的限制，这是由于高剂量的 CYP 11B 1和CYP 11B 2之间的序列同源性，特别是在它们的活性位点。这项建议旨在 确定这些酶之间的结构和功能差异，可用于为开发提供信息 治疗库欣氏病和原发性醛固酮增多症的药物。CYP 11B之间的一个差异 酶的作用在于它们与它们共有的氧化还原配偶体肾上腺氧还蛋白的相互作用。我的CYP 11B 1初步数据 和之前Scott实验室对CYP 11B 2的研究表明，肾上腺素能变构调节CYP 11B和CYP 11B 酶，但对每种酶的影响不同（9）。这表明肾上腺素能调节 结合两种CYP 11B酶--这种差异可以作为开发药物的靶点， 一种酶。然而，肾上腺素还没有很好地表征变构作用的基础。这 一项提案试图通过在结构和功能上表征肾上腺皮质激素变构作用来填补这一空白。 CYP 11B酶。首先，X射线结构将确定形成CYP 11B 1/肾上腺素还蛋白的残基 用于与CYP 11B 2/肾上腺氧还蛋白复合物的现有结构进行比较的界面。第二，预稳定-- 使用停止流动的状态动力学将确定P450表面上的肾上腺皮质激素结合是否改变了P450表面上的肾上腺皮质激素。 P450通过主要改变配体结合或释放与远距离埋藏的活性位点中的配体相互作用。 最后，诱变研究将调查一个特定的环的作用，假设该环负责突变。 人类P450酶之间变构效应的差异。总的来说，这项研究将提供一个广泛的结构， 以及肾上腺素还蛋白对CYP 11B 1和CYP 11B 2的变构作用的功能表征。该详细 表征推进了我们对生化系统的理解，但也有可能揭示 这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

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.