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)：这项建议的长期目标是确定PGRMC1‘S对细胞色素P3A4活性的影响的幅度，以及这种影响是否是底物特有的。PGRMC1是一种含细胞色素b5结构域的血红蛋白。PGRMC1增强酵母和哺乳动物肾上腺细胞中的CYP活性，但它如何或是否相互作用并影响药物代谢细胞色素P450的活性尚不清楚。需要检验的中心假设是PGRMC1调节CYP3A4的催化作用。我们提出这一新的想法是因为PGRMC1和CYP3A4共同定位于肝脏内质网，并且因为PGRMC1与CYP3A4在物理上存在联系。然而，PGRMC1的S在调节细胞色素P3A4催化活性中的作用尚不清楚。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个人的肝脏中，CYP3A4介导的咪达唑仑和睾酮的代谢与PGRMC1的mRNA表达和PGRMC1相关的基因显著相关。我们建议对PGRMC1在三个特定目的中调节CYP3A4活性的假设进行检验。在目标1中，将量化PGRMC1对CYP3A4活性的S影响的程度。(A)具有和不具有表达的PGRMC的CYP3A4/POR(人NADPH-P450还原酶)超体体；(B)由纯化的PGRMC1重组的CYP3A4/POR超体体；以及(C)用PGRMC1抑制抗体降低PGRMC1活性的混合人肝微体。为了确定这种作用是否具有组织特异性，在AIM 2中，比较了腺病毒-CYP3A4/POR转染人肝、肠和肾细胞中的睾酮和咪达唑仑氧化作用，其中人肝、肠和肾细胞中PGRMC1的表达被AdV-PGRMC1上调或被PGRMC1 shRNA慢病毒耗尽。在目的3中，通过突变PGRMC1的S血红素结合结构域，并检测其对PGRMC1的S与细胞色素P450 3A4相互作用和调节其活性的影响，来确定对该结构域的需求。总之，这些结果应该提供关于PGRMC1对肝脏、肠道和肾脏细胞中CYP3A催化活性的贡献的新的和重要的信息。与公共卫生相关：目前使用的口服有效药物中，有一半以上是由细胞色素P3A4氧化代谢的。尽管PGRMC1是酵母CYP活性所必需的，调节哺乳动物肾上腺CYP活性，并且已被证明直接与CYP3A4相互作用，但它对CYP3A4活性的贡献从未被测试过。更清楚地了解PGRMC1在调节CYP3A4活性中所起的作用，应该会促进更安全药物的开发。具体地说，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活性的调节很可能成为其与其他外源代谢的细胞色素P450相互作用的范例。