Regulation of hepatic uptake transporters by tyrosine kinases

摘要 肝脏药物摄取转运蛋白是全身清除和患者结局的主要调节剂，然而， 介导这些转运蛋白功能的翻译后修饰的表征仍然存在 研究得还很不够。因此，与本提案相关的长期目标是确定 翻译后介导的重要药物转运蛋白调节的意义，如 OATP1B1。本提案的总体目标是描述酪氨酸激酶抑制剂的后果- (TKI)介导的OATP 1B 1抑制。中心假设是特定的酪氨酸激酶是必需的 OATPB 1的调节剂，干扰这一过程将减少肝脏蓄积， OATP 1B 1底物的血浆浓度，导致药代动力学（PK）和药效学（PD） 药物相互作用（DDI）。该项目的基本原理是阐明监管机制， 影响OATP 1B 1功能将有助于改善迄今为止不幸 产生了各种意想不到的DDI介导的危及生命的毒性，其中包括他汀类药物诱导的 横纹肌溶解中心假设将通过追求两个具体目标进行测试：1）识别和阐明 参与OATP 1B 1功能的调节激酶的作用;和2）研究调节激酶的体内作用。 激酶对OATP 1B 1活性的影响在第一个目标下，基于LC/MS/MS的蛋白质组学将用于测量 激酶抑制剂在体外和离体调节OATP 1B 1磷酸化的能力， 综合激酶抑制或结合试验将用于鉴定 OATP 1B 1功能。在第二个目标下，人转基因OATP 1B 1，以及转运蛋白和激酶， 缺陷型体内小鼠模型将用于评估OATP 1B 1底物在存在下的PK/PD变化 或无TKI。这将为酪氨酸激酶在调节OATP 1B 1中的作用提供机制性的见解。 介导的肝处理底物。目标2的集体研究结果也将用于制定 基于生理学的PK/PD模型，以预测TKI-OATP 1B 1底物相互作用的结局。研究 本申请中提出的方法是创新的，因为它代表了对现状的实质性偏离， 探索酪氨酸激酶活性作为OATP 1B 1功能的调节剂和作为全身水平的介导剂， 内源性生物标志物和外源性物质。这项研究意义重大，因为它有望 为OATP 1B 1功能所必需的调节机制提供了重要的新见解， 调节这种转运蛋白的激酶途径，以及它们在肝脏处理许多相关的 内源性和外源性底物。最终，这些知识有望为基金会做出贡献。 预防危及生命的DDI的方法。

ABSTRACT Hepatic drug uptake transporters are major regulators of systemic clearance and patient outcome, however, characterization of post-translational modifications that mediate the function of these transporters have remained greatly under studied. Therefore, the long-term goal associated with this proposal is to define the extent and significance of post-translational-mediated regulation of pharmacologically important drug-transporters, such as OATP1B1. The overall objective of this proposal is to delineate the consequences of tyrosine kinase inhibitor- (TKI) mediated inhibition of OATP1B1. The central hypothesis is that specific tyrosine kinase(s) are essential regulators of OATPB1, and that interference with this process will decrease hepatic accumulation and increase plasma concentrations of OATP1B1 substrates, resulting in pharmacokinetic (PK) and pharmacodynamic (PD) drug-drug interactions (DDIs). The rationale for this project is that elucidation of the regulatory mechanisms that affect OATP1B1 function will contribute to refinement of therapeutic strategies that have unfortunately to date yielded a variety of unexpected DDI-mediated life-threatening toxicities, which includes statin-induced rhabdomyolysis. The central hypothesis will be tested by pursuing two specific aims: 1) Identify and elucidate the role of regulatory kinases involved in OATP1B1 function; and 2) Investigate the in vivo effects of regulatory kinases on OATP1B1 activity. Under the first aim, LC/MS/MS-based proteomics will be used to measure the ability of kinase inhibitors to modulate OATP1B1 phosphorylation in vitro and ex vivo, while genetic manipulation and comprehensive kinase inhibition or binding assays will be utilized to identify the regulatory kinase of OATP1B1 function. Under the second aim, human transgenic OATP1B1, as well as transporter- and kinase- deficient in vivo mouse models will be used to assess PK/PD changes of OATP1B1 substrates in the presence or absence of TKIs. This will provide mechanistic insight into the role of tyrosine kinases in regulating OATP1B1- mediated hepatic handling of substrates. The collective findings of Aim 2 will also be utilized to develop physiologically-based PK/PD models to predict outcome of TKI-OATP1B1 substrate interactions. The research proposed in this application is innovative because it represents a substantive departure from the status quo by exploring tyrosine kinase activity as a regulator of OATP1B1 function and as a mediator of systemic levels of both endogenous biomarkers and xenobiotics. The proposed research is significant because it is expected to provide important new insights into regulatory mechanisms essential to the function of OATP1B1, the molecular kinase pathways modulating this transporter, and their role in the hepatic handling of the many relevant endogenous and xenobiotic substrates. Ultimately, such knowledge is expected to contribute to the foundation of preventing life-threatening DDIs in patients.