Inflammation, PXR Modification and Drug Disposition

描述(申请人提供)：我的实验室的长期目标是提高我们对控制药物处置和药物代谢的基本调控机制的理解。人们清楚地认识到，在受潜在全身炎症影响的患者中，药物不良反应(ADR)的频率会增加。在这些个体中，可变的药物反应并不一定与遗传多态有关，而是由于编码重要药物代谢酶(DME)和药物转运蛋白的关键基因的表达迅速和显著减少。在肝脏中，孕烷x受体(PXR)是主要的异源“传感器”。PXR的配体依赖激活增加了大量外源物质和处方药的整体摄取、代谢、转运和最终消除。基于强大的初步数据，我们假设利福平治疗后患者发生的免疫抑制效应是通过PXR蛋白的翻译后修饰而发生的。我们提供的证据表明，肿瘤坏死因子α促进连接的PXR的多聚总甲基化，以反馈抑制核因子-kappa B靶基因。目的1利用一种新颖的、创新的基于质谱学的方法，在体外、在培养细胞中、然后在肝细胞中依次确定配体激活的PXR SUMO化的特定位点。目的2用生化方法验证选择性抑制炎症反应的机制是由于SUMO修饰的PXR阻止核受体辅阻遏子/核因子-kappaB复合体的清除。了解PXR如何从肝脏DMES的正调控因子转化为肝组织炎症转录抑制因子的生化细节和分子机制将为理解药物介导的肝炎症反应抑制提供新的分子范式。有关PXR SUMO化生物学的新的分子见解将为开发解决ADRs的新药理学策略提供新的机会，并最终将有助于识别将用于治疗炎症性肝病的小分子。

DESCRIPTION (provided by applicant): The long-term objectives of my laboratory are to improve our understanding of the basic regulatory mechanisms that govern drug disposition and drug metabolism. There is a clear recognition that the frequency of adverse drug reactions (ADRs) is increased in patients affected with an underlying systemic inflammation. In these individuals, variable drug responses are not necessarily related to genetic polymorphisms but are due to the rapid and dramatic reduction in the expression of key genes that encode important drug metabolizing enzymes (DMEs) and drug transporter proteins. In liver, the pregnane x receptor (PXR) is the master- xenobiotic 'sensor'. Ligand-dependent activation of PXR increases the overall uptake, metabolism, transport and eventual elimination of a myriad of xenobiotics and prescription drugs. Based upon strong preliminary data, we hypothesize that the immunosuppressive effects that occur in patients following Rifampicin therapy occurs via the post-translational modification of the PXR protein. We provide evidence that TNFalpha promotes poly-sumoylation of liganded-PXR to feedback repress NF-kappa B-target genes. Aim 1 will define the specific sites of ligand-activated PXR SUMOylation sequentially in vitro, in cultured cells, and then in hepatocytes using a novel and innovative mass spectrometry-based approach. Aim 2 will test the hypothesis that the mechanism of selective repression of the inflammatory response is due to SUMO-modified PXR preventing clearance of nuclear receptor corepressor/NF-kappa B complexes using biochemical methods. Understanding the biochemical details and molecular mechanisms of how PXR is converted from a positive regulator of hepatic DMEs into a transcriptional suppressor of inflammation in liver tissue will provide a new molecular paradigm for understanding drug-mediated repression of the hepatic inflammatory response. New molecular insights regarding the biology of PXR SUMOylation will provide new opportunities to develop novel pharmacological strategies for addressing ADRs, and will eventually help to identify small molecules that will be used to treat inflammatory liver diseases.