Structure and Function of Human Cytochrome P450 11B Enzymes Involved in Cushing’s Disease and Hypertension

人体血压和应激/免疫反应受类固醇控制 激素分别为醛固酮和皮质醇。在中国有多种疾病状态 这些荷尔蒙是过度生产的，而选择性抑制剂是 对临床有益。然而，醛固酮选择性抑制剂的研究进展 细胞色素P450 11B2的产生和细胞色素的皮质醇的产生 P450 11B1(CYP11B1)的氨基酸序列同源性达93%。 目前的提案试图通过确定结构上的 细胞色素P11B1与细胞色素P11B2在功能上的不同。中环 假设结构和功能研究将揭示独特的细胞色素P11B1 可被利用来支持异构体选择性药物设计的特征。 为了探索CYP11B酶与配体相互作用的差异，我们将测定X光 细胞色素P11B1及其底物的结构和目前正在研究的几种抑制剂 发展，但由于选择性差，并不是最优的。初步数据显示 与现有相应的CYP11B2结构相比的结构差异， 可用于临床的区别(目标1)。初步数据还表明， 共享的必需氧化还原伙伴肾上腺素与两个CYP11B的相互作用不同 酶，这可能提供一种正交的方法来选择性抑制-一种 不专注于活动站点。因此，X射线结构也将被确定为 细胞色素P11B1通过融合蛋白途径与肾上腺素相互作用(目标2)。 最终目的是探索细胞色素P11B1可能存在的不同构象 溶液和这种蛋白质通常被包埋的膜中。所有膜 用X射线结晶学和堆积法测定了P450的结构 相互作用通过膜结合表面发生，配体通过该表面进入/退出。 因此，虽然这些蛋白质被认为是灵活和动态的，但X射线结晶学 通常只显示一种形式，这可能不是最合适的构象 阻滞剂设计。冷冻EM在CYP11B1(目标3)中的应用是一种正交方法 以确定该酶更相关的构象。因此，拟议的研究将 确定结构并定义控制CYP11B1活性的新的相互作用。 随后可以将这一新信息与CYP11B2进行对比，以便于 针对每种酶的临床相关分子抑制剂的开发。

Human blood pressure and stress/immune responses are controlled by the steroid hormones aldosterone and cortisol, respectively. There are multiple disease states in which these hormones are overproduced and for which selective inhibitors would be clinically beneficial. However, the development of selective inhibitors of aldosterone production by cytochrome P450 11B2 (CYP11B2) and cortisol production by cytochrome P450 11B1 (CYP11B1) has been frustrated by their 93% amino acid sequence identity. The current proposal seeks to rectify this gap in knowledge by determining structural features of CYP11B1 that underlie functional differences from CYP11B2. The central hypothesis is that structural and functional studies will reveal unique CYP11B1 characteristics that could be harnessed to support the design of isoform-selective drugs. To probe differences in CYP11B enzyme interactions with ligands, we will determine Xray structures of CYP11B1 with its substrate and several inhibitors that currently under development but are non-optimal because of poor selectivity. Preliminary data suggests structural differences compared to the existing corresponding CYP11B2 structure, distinctions that might be exploited clinically (aim 1). Preliminary data also suggests that the shared required redox partner adrenodoxin interacts differently with the two CYP11B enzymes, which may provide an orthogonal approach to selective inhibition—one that doesn’t focus on the active site. Thus an X-ray structure will also be determined for CYP11B1 interacting with adrenodoxin by employing a fusion protein approach (aim 2). The final aim probes different conformations of CYP11B1 that are likely to exist in solution and in the membrane where this protein is normally embedded. All membrane P450 structures have been determined by X-ray crystallography and packing interactions occur via the membrane-binding surface through which ligands enter/exit. Thus, while these proteins are known to be flexible and dynamic, X-ray crystallography often only reveals one form, which may not be the most appropriate conformation for inhibitor design. Application of cryo-EM to CYP11B1 (aim 3) is an orthogonal approach to define more relevant conformations of this enzyme. Thus, the proposed research will determine the structures and define new interactions controlling the activity of CYP11B1. Contrasting this new information with CYP11B2 can subsequently be used to facilitate the development of clinically-relevant molecules inhibitors specific for each enzyme.