Inflammation, PXR Modification and Drug Disposition

描述（由申请人提供）：我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节机制的理解。有一个明确的认识，即药物不良反应（ADR）的频率增加，受影响的患者与潜在的全身炎症。在这些个体中，可变的药物反应不一定与遗传多态性相关，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表达的快速和急剧减少。在肝脏中，胆甾烷X受体（PXR）是异生物质的主“传感器”。PXR的配体依赖性激活增加了大量外源性物质和处方药的总体摄取、代谢、运输和最终消除。基于强有力的初步数据，我们假设利福平治疗后患者的免疫抑制作用是通过PXR蛋白的翻译后修饰发生的。我们提供的证据表明，TNF α促进配体PXR的聚类小泛素化，以反馈抑制NF-κ B靶基因。目的1将确定特定的网站配体激活PXR SUMO化顺序在体外，在培养细胞，然后在肝细胞中使用一种新的和创新的质谱为基础的方法。目的2将使用生物化学方法来检验选择性抑制炎症反应的机制是由于SUMO修饰的PXR阻止核受体辅抑制物/NF-κ B复合物的清除的假设。了解PXR如何从肝DME的正调节因子转化为肝组织炎症的转录抑制因子的生物化学细节和分子机制，将为了解药物介导的肝脏炎症反应抑制提供新的分子范式。关于PXR SUMO化生物学的新分子见解将为开发解决ADR的新药理学策略提供新的机会，并最终有助于确定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.