PGRMC1: A Regulator of CYP3A Activity

PGRMC1：CYP3A 活性的调节因子

• 批准号: 7708535
• 负责人: ERIN G SCHUETZ
• 金额: $ 25.2万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-16 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7708535
• 关键词: Adenoviruses; Adrenal Glands; Affect; Antibodies; Binding; CYP3A4 gene; Catalysis; Cells; Cytochrome P450; Cytochromes b5; Data; Development; Endoplasmic Reticulum; Genotype; Goals; Heme; Hemeproteins; Hepatic; Human; In Vitro; Intestines; Liver; Liver Microsomes; Mediating; Metabolic; Metabolism; Midazolam; Mutate; NADPH-Ferrihemoprotein Reductase; Patients; Pharmaceutical Preparations; Play; Role; Subfamily lentivirinae; System; Testing; Testosterone; Tissues; Xenobiotics; Yeasts; improved; kidney cell; mRNA Expression; novel; oxidation; public health relevance; reconstitution

DESCRIPTION (provided by applicant): The long-term objectives of this proposal are to determine the magnitude of PGRMC1's effect on CYP3A4 activity and if this effect is substrate specific. PGRMC1 is a hemoprotein with a cytochrome b5-like domain. PGRMC1 enhances CYP activity in yeast and in mammalian adrenal cells but it is unknown how or if it interacts and affects activity of drug metabolizing cytochromes P450. The central hypothesis to be tested is that PGRMC1 modulates CYP3A4 catalysis. We propose this novel idea because both PGRMC1 and CYP3A4 co-localize in the hepatic endoplasmic reticulum, and because PGRMC1 physically associates with CYP3A4. Nonetheless, PGRMC1's role in modulating CYP3A4 catalytic activity remains unexplored. Our preliminary data further reveal a relationship between PGRMC1 and CYP3A4 activity by showing, among 530 human livers, a significant correlation between CYP3A4 mediated metabolism of both midazolam and testosterone and PGRMC1 mRNA expression, and a PGRMC1 associated genotype. We propose to test the hypothesis that PGRMC1 modulates CYP3A4 activity in three specific aims. In Aim 1, the extent of PGRMC1's effect on CYP3A4 activity will be quantified. CYP3A4 mediated metabolism of testosterone and midazolam will be compared between (a) CYP3A4/POR (human NADPH-P450 reductase) supersomes with and without expressed PGRMC; (b) CYP3A4/POR supersomes reconstituted with purified PGRMC1; and (c) pooled human liver microsomes in which PGRMC1 activity is decreased with PGRMC1 inhibitory antibodies. To determine if the effect is tissue specific, in Aim 2 testosterone and midazolam oxidation will be compared among Adenovirus-CYP3A4/POR transfected human liver, intestine and kidney cells in which PGRMC1 expression is increased with AdV-PGRMC1 or depleted with PGRMC1 ShRNA lentivirus. In Aim 3 the requirement for PGRMC1's heme binding domain will be determined by mutating it and examining the effects on PGRMC1's ability to interact with CYP3A4 and modulate its activity. In total the results should provide novel and important information about the contribution of PGRMC1 to CYP3A catalytic activity in liver, intestine and kidney cells. PUBLIC HEALTH RELEVANCE: CYP3A4 oxidatively metabolizes over half of the orally effective drugs in use today. Despite the fact that PGRMC1 is required for yeast CYP activity, modulates mammalian adrenal CYP activity, and has been shown to directly interact with CYP3A4, its contribution to CYP3A4 activity has never been tested. A clearer understanding of the role PGRMC1 plays in modulating CYP3A4 activity should enhance the development of safer medications. Specifically, gaining an understanding of the role of PGRMC1 in modulating CYP3A4 catalytic activity will increase our ability to accurately predict the metabolic rate and metabolite profiles of drugs in patients using improved in vitro systems. Moreover, it is likely that modulation of CYP3A4 activity by PGRMC1 will be a paradigm for its interactions with other xenobiotic metabolizing CYPs.

描述（由申请人提供）：该提案的长期目标是确定 PGRMC1 对 CYP3A4 活性的影响程度以及该影响是否具有底物特异性。 PGRMC1 是一种具有细胞色素 b5 样结构域的血红素蛋白。 PGRMC1 增强酵母和哺乳动物肾上腺细胞中的 CYP 活性，但尚不清楚它如何或是否相互作用并影响药物代谢细胞色素 P450 的活性。要测试的中心假设是 PGRMC1 调节 CYP3A4 催化。我们提出这个新想法是因为 PGRMC1 和 CYP3A4 共同定位于肝内质网，并且因为 PGRMC1 与 CYP3A4 物理上相关。尽管如此，PGRMC1 在调节 CYP3A4 催化活性中的作用仍有待探索。我们的初步数据进一步揭示了 PGRMC1 和 CYP3A4 活性之间的关系，在 530 个人类肝脏中，CYP3A4 介导的咪达唑仑和睾酮代谢与 PGRMC1 mRNA 表达以及 PGRMC1 相关基因型之间存在显着相关性。我们建议测试 PGRMC1 在三个特定目标中调节 CYP3A4 活性的假设。在目标 1 中，将量化 PGRMC1 对 CYP3A4 活性的影响程度。将比较 (a) 有和没有表达 PGRMC 的 CYP3A4/POR（人 NADPH-P450 还原酶）超体之间的 CYP3A4 介导的睾酮和咪达唑仑代谢； (b) 用纯化的 PGRMC1 重建 CYP3A4/POR 超体； (c) 合并的人肝微粒体，其中 PGRMC1 活性因 PGRMC1 抑制抗体而降低。为了确定效果是否具有组织特异性，在 Aim 2 中，将在腺病毒-CYP3A4/POR 转染的人肝、肠和肾细胞中比较睾酮和咪达唑仑的氧化，其中 PGRMC1 表达随 AdV-PGRMC1 增加或用 PGRMC1 ShRNA 慢病毒减少。在目标 3 中，对 PGRMC1 血红素结合域的要求将通过对其进行突变并检查对 PGRMC1 与 CYP3A4 相互作用和调节其活性的能力的影响来确定。总的来说，这些结果应该提供有关 PGRMC1 对肝、肠和肾细胞中 CYP3A 催化活性的贡献的新颖且重要的信息。公共健康相关性：CYP3A4 氧化代谢当今使用的口服有效药物的一半以上。尽管事实上 PGRMC1 是酵母 CYP 活性所必需的，可调节哺乳动物肾上腺 CYP 活性，并且已被证明可与 CYP3A4 直接相互作用，但其对 CYP3A4 活性的贡献从未经过测试。更清楚地了解 PGRMC1 在调节 CYP3A4 活性中的作用应该会促进更安全药物的开发。具体来说，了解 PGRMC1 在调节 CYP3A4 催化活性中的作用将提高我们使用改进的体外系统准确预测患者药物代谢率和代谢物谱的能力。此外，PGRMC1 对 CYP3A4 活性的调节可能将成为其与其他外源代谢 CYP 相互作用的范例。