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活性的影响的大小，以及该效果是否特定。 PGRMC1是具有细胞色素B5样结构域的血蛋白。 PGRMC1增强了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的细胞色素P450的活性。要测试的中心假设是PGRMC1调节CYP3A4催化。我们提出了这种新颖的想法，因为PGRMC1和CYP3A4在肝内质网中共定位，并且PGRMC1与CYP3A4物理相关。尽管如此，PGRMC1在调节CYP3A4催化活性中的作用仍未探索。我们的初步数据进一步揭示了PGRMC1和CYP3A4活性之间的关系，通过在530种人体肝中，CYP3A4介导的咪达唑仑与睾丸激素和PGRMC1 mRNA表达的代谢与PGRMC1相关的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AIM 1中，将量化PGRMC1对CYP3A4活性的影响的程度。 CYP3A4介导的睾丸激素和咪达唑仑的代谢将进行比较（a）CYP3A4/POR（人NADPH-P450还原酶），具有和没有表达的PGRMC； （b）用纯化的PGRMC1重构CYP3A4/POR SUPERSOM； （c）PGRMC1抑制性抗体降低PGRMC1活性的合并人肝微粒体。为了确定该作用是否特异性组织，在AIM 2睾丸激素和咪达唑仑氧化中，将在腺病毒-CYP3A4/POR转染的人肝，肠和肾细胞中进行比较，其中PGRMC1表达与PGRMC1 SHRNA Lentivirus相比增加了PGRMC1的表达。在AIM 3中，PGRMC1的血红素结合结构域的要求将通过突变并检查对PGRMC1与CYP3A4相互作用并调节其活性的影响来确定。总体而言，结果应提供有关PGRMC1对CYP3A肝脏，肠和肾细胞中CYP3A催化活性的贡献的新颖和重要信息。公共卫生相关性：CYP3A4氧化代谢当今使用的一半以上的口服有效药物。尽管酵母CYP活性需要PGRMC1，但调节了哺乳动物肾上腺CYP活性，并且已证明与CYP3A4直接相互作用，但其对CYP3A4活性的贡献从未进行过测试。对PGRMC1在调节CYP3A4活性中的作用的更清晰了解应增强更安全的药物的发展。具体而言，了解PGRMC1在调节CYP3A4催化活性中的作用将提高我们使用改善体外系统的患者准确预测药物的代谢率和代谢物概况的能力。此外，PGRMC1对CYP3A4活性的调节可能会成为其与其他异种生物代谢CYP的相互作用的范式。