Circadian molecular regulation of the xenobiotic response

描述（由申请人提供）：最近，生物钟被认为是多种生理过程的调节剂，包括葡萄糖稳态、血压调节以及药物代谢和毒性。几种药物，包括抗高血压他汀类药物（如立普妥）和抗糖尿病药物二甲双胍（如格华止），建议在一天中的特定时间服用。然而，这些偏好的分子基础还没有得到很好的理解。该提议的基本假设是，治疗药物的细胞转运和代谢可以在转录水平上通过昼夜节律阻遏物调节，昼夜节律阻遏物改变对异生物质配体响应的核激素受体的功能。推进我们对这些受体和阻遏物相互作用的功能理解可能会突出治疗疾病的新治疗策略。例如，定义异生素转录网络的昼夜调节可以预测现有和新型治疗化合物的最佳治疗时间。此外，更深入地了解昼夜调节核激素受体途径，调节异生物质转录反应可能会导致新的策略，用于调节药物的吸收，代谢和/或毒性。我们先前的研究鉴定了生物钟组分隐花色素（Cry 1和Cry 2）作为营养响应性转录调节因子，这是由于它们对AMP活化蛋白激酶（AMPK）磷酸化的敏感性和它们调节糖皮质激素受体依赖性转录的能力。我们还通过驱动昼夜表达，确定了肝脏昼夜节律钟在葡萄糖稳态中的重要作用。 肝酶和转运蛋白，包括AMPK。在这些研究的过程中，我们已经产生了独特的工具和专业知识，使我们能够使用生物化学，遗传学，分子和生理学方法来揭示生物钟和生物钟阻遏物Cry 1和Cry 2的作用，特别是在控制药物转运和代谢的核激素受体途径中，具体目标如下：1）表征Cry 1和Cry 2与异生物素受体PXR和CAR的相互作用（定义隐花色素-异生物素受体相互作用的物种和同种型特异性和配体依赖性;鉴定相互作用所需结构域和序列），2）确定Cry 1和Cry 2在PXR/CAR介导的异生物质代谢调节中的作用（鉴定由隐花色素调节的PXR和CAR的特异性转录靶标，测量隐花色素耗尽后药物转运和代谢活性的变化），3）检查生物钟、PXR和CAR在二甲双胍治疗功效中的作用。

DESCRIPTION (provided by applicant): Circadian clocks have recently become recognized as modulators of a wide array of physiological processes, including glucose homeostasis, blood pressure modulation, and drug metabolism and toxicity. Several drugs, including the anti-hypertensive statins (e.g. Lipitor) and the anti-diabetic drug metformin (e.g. Glucophage), are recommended to be taken at specific times of day. However, the molecular basis for these preferences is not well understood. The underlying hypothesis of this proposal is that the cellular transport and metabolism of therapeutic drugs can be modulated at the transcriptional level by circadian repressors altering the function of nuclear hormone receptors that respond to xenobiotic ligands. Advancing our functional understanding of these receptor and repressor interactions may highlight new therapeutic strategies for treating disease. For example, defining the diurnal regulation of the xenobiotic transcriptional network could enable the prediction of optimal treatment times for existing and novel therapeutic compounds. In addition, a deeper understanding of the diurnal regulation of nuclear hormone receptor pathways that modulate the xenobiotic transcriptional response may lead to new strategies for modulating drug absorption, metabolism and/or toxicities. Our previous studies identified the circadian clock component cryptochromes (Cry1 and Cry2) as nutrient- responsive transcriptional regulators by virtue of their susceptibility to phosphorylation by AMP-activated protein kinase (AMPK) and their ability to modulate glucocorticoid receptor dependent transcription. We have also established an important role for the liver circadian clock in glucose homeostasis, via driving diurnal expression of hepatic enzymes and transporters, including AMPK. In the course of those studies, we have generated unique tools and expertise that enables us to use biochemical, genetic, molecular and physiological approaches to uncover the roles of circadian clocks and of the circadian repressors Cry1 and Cry2 specifically in nuclear hormone receptor pathways governing the control of drug transport and metabolism, in the following specific aims: 1) Characterize the interactions of Cry1 and Cry2 with the xenobiotic receptors PXR and CAR (define the species and isoform specificity and ligand dependence of cryptochrome-xenobiotic receptor interactions; identify domains and sequences required for interaction), 2) Define the role of Cry1 and Cry2 in the regulation of PXR/CAR-mediated xenobiotic metabolism (identify specific transcriptional targets of PXR and CAR that are regulated by cryptochromes, measure changes in drug transport and metabolizing activities upon cryptochrome depletion), 3) Examine the roles of circadian clocks, PXR and CAR in the efficacy of metformin treatment.