PGRMC1: A Regulator of CYP3A Activity

PGRMC1：CYP3A 活性的调节因子

• 批准号: 7894981
• 负责人: ERIN G SCHUETZ
• 金额: $ 21万
• 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/7894981
• 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（由申请方提供）：本提案的长期目的是确定PGRMC 1对CYP 3A 4活性的影响程度，以及该影响是否具有底物特异性。PGRMC 1是一种具有细胞色素b5样结构域的血红素蛋白。PGRMC 1增强酵母和哺乳动物肾上腺细胞中的P450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。待检验的中心假设是PGRMC 1调节CYP 3A 4催化。我们提出这个新的想法，因为PGRMC 1和CYP 3A 4共同定位在肝内质网，因为PGRMC 1物理协会与CYP 3A 4。尽管如此，PGRMC 1在调节CYP 3A 4催化活性中的作用仍然未被探索。我们的初步数据进一步揭示了PGRMC 1和CYP 3A 4活性之间的关系，通过显示，在530人肝脏中，CYP 3A 4介导的咪达唑仑和睾酮代谢与PGRMC 1 mRNA表达之间的显著相关性，以及PGRMC 1相关基因型。我们建议测试的假设，PGRMC 1调节CYP 3A 4活性在三个特定的目标。在目的1中，将量化PGRMC 1对CYP 3A 4活性的影响程度。将比较（a）含和不含表达PGRMC的CYP 3A 4/POR（人NADPH-P450还原酶）supersomes;（B）用纯化PGRMC 1复溶的CYP 3A 4/POR supersomes;和（c）用PGRMC 1抑制性抗体降低PGRMC 1活性的合并人肝微粒体之间CYP 3A 4介导的睾酮和咪达唑仑代谢。为了确定该效应是否具有组织特异性，在Aim 2中，将在腺病毒-CYP 3A 4/POR转染的人肝、肠和肾细胞中比较睾酮和咪达唑仑氧化，其中用AdV-PGRMC 1增加PGRMC 1表达或用PGRMC 1 shRNA慢病毒消除PGRMC 1表达。在目的3中，将通过突变PGRMC 1的血红素结合结构域并检查对PGRMC 1与CYP 3A 4相互作用并调节其活性的能力的影响来确定对PGRMC 1的血红素结合结构域的需求。总的来说，这些结果应该提供新的和重要的信息，PGRMC 1的贡献CYP 3A催化活性在肝，肠和肾细胞。公共卫生相关性：CYP 3A 4氧化代谢目前使用的口服有效药物的一半以上。尽管事实上PGRMC 1是酵母细胞活性所必需的，调节哺乳动物肾上腺皮质激素活性，并已被证明与CYP 3A 4直接相互作用，但其对CYP 3A 4活性的贡献从未被测试过。更清楚地了解PGRMC 1在调节CYP 3A 4活性中的作用，应有助于开发更安全的药物。具体而言，了解PGRMC 1在调节CYP 3A 4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产物谱的能力。此外，PGRMC 1对CYP 3A 4活性的调节可能是其与其他异生物质代谢CYP相互作用的范例。