Regulation of hepatic uptake transporters by tyrosine kinases

摘要 然而，肝脏药物摄取转运体是全身清除和患者预后的主要调节因素。 调节这些转运蛋白功能的翻译后修饰的特征仍然存在 在很大程度上被研究。因此，与这项提议相关的长期目标是界定范围和 翻译后调节重要的药物转运体的意义，如 OATP1B1。这项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OATP1B1抑制作用。中心假设是特定的酪氨酸激酶(S)是必不可少的 OATPB1的调节因子，干扰这一过程将减少肝脏蓄积并增加 OATP1B1底物的血浆浓度，导致药代动力学(PK)和药效学(PD) 药物-药物相互作用(DDIS)。该项目基本原理是阐明了 影响OATP1B1功能将有助于改进迄今为止不幸的治疗策略 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 横纹肌溶解症。核心假设将通过追求两个具体目标来检验：1)确定和阐明 调节激酶在OATP1B1功能中的作用；以及2)研究调节蛋白在体内的作用。 激酶对OATP1B1活性的影响。在第一个目标下，将使用基于LC/MS/MS的蛋白质组学来测量 在基因操作过程中，激酶抑制剂在体外和体外调节OATP1B1磷酸化的能力 并将利用全面的激酶抑制或结合分析来鉴定调节性激酶 OATP1B1功能。在第二个目标下，人类转基因OATP1B1以及转运蛋白和激酶- 体内缺陷小鼠模型将被用来评估OATP1B1底物在存在时PK/PD的变化 或缺少TKI。这将为酪氨酸激酶在调节OATP1B1中的作用提供机械性的见解。 介导的底物的肝脏处理。目标2的集体调查结果也将被用来开发 基于生理的PK/PD模型预测TKI-OATP1B1底物相互作用的结果。这项研究 在本申请中提出的建议是创新的，因为它代表了对现状的实质性偏离 探讨酪氨酸激酶活性作为OATP1B1功能的调节因子和全身水平的调节因子 既有内源生物标志物，也有外源生物。这项拟议的研究意义重大，因为预计它将 为OATP1B1分子功能所必需的调控机制提供重要的新见解 调节这一转运蛋白的激酶通路，以及它们在肝脏处理中的作用 内源和异源底物。最终，这样的知识有望为基金会做出贡献。 预防危及患者生命的DDIS。

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.