Regulation of OATP1B1 and OATP1B3 by lysine acetylation and lysine deacetylase inhibitors

有机阴离子转运多肽（OATP）1B 1和OATP 1B 3（简称OATP 1B 1/3）是肝- 特异性药物转运蛋白介导多种内源性药物从血液进入肝脏的摄取， 化合物、环境毒素和许多临床上重要的药物（例如，降脂他汀类药物和抗癌 代理商）。OATP 1B 1/3是转运介导的药物相互作用（DDI）的重要决定因素， 严重的副作用，如他汀类药物引起的横纹肌溶解，有时是致命的肌肉毒性。功能障碍 OATP 1B 1/3显著导致药物分布改变和药物不良事件。我们的长期目标是 通过OATP 1B 1/3描述药物/毒素处置的分子机制，并预测和 减轻OATP介导的药物-药物和药物-疾病相互作用。虽然很明显，因素（药物， 衰老、疾病）调节OATP 1B 1/3功能可能导致药物-药物或药物-疾病相互作用， OATP 1B 1/3底物，不幸的是，我们预测这种相互作用的能力受到阻碍，由于缺乏 关于OATP 1B 1/3法规的信息。特别是，调节赖氨酸乙酰化，这是一个主要的翻译后修饰。 已知改变许多靶蛋白功能的修饰，包括用于表观遗传调节的组蛋白， 未对OATP 1B 1/3进行研究。本申请的总体目标是确定分子量。 通过赖氨酸乙酰化调控OATP 1B 1/3的机制，并评估赖氨酸 脱乙酰酶（KDAC）抑制剂（KDACI）对OATP 1B 1/3表达和功能的影响。我们的核心假设是， OATP 1B 1/3是赖氨酸乙酰化蛋白，其脱乙酰化涉及KDAC 6; OATP 1B 1/3可以是 在表观遗传水平上由靶向组蛋白去乙酰化的KDACI调节，在翻译后水平上由 KDAC 6抑制。我们的新的初步数据表明：1）OATP 1B 1/3是赖氨酸乙酰化的; 2）诱变 模拟OATP 1B 1的超乙酰化和特异性KDAC 6抑制可显著降低OATP 1B 1/3 3）FDA批准的泛KDAC抑制剂（KDACI）抑制组蛋白脱乙酰化诱导 OATP 1B 1/3的mRNA和蛋白水平。在初步结果的指导下，概述了两个具体目标。在Aim中 1，我们将阐明KDAC 6在调节OATP 1B 1/3赖氨酸乙酰化和转运蛋白功能中的作用。在 目的2，我们将分析泛KDACI药物对OATP 1B 1/3的调节以及表观遗传学和细胞凋亡之间的相互作用。 以及通过K-Ac对OATP 1B 1/3的翻译后调节。结合蛋白质组学、生物化学和遗传学 工程方法将用于细胞系和生理学相关的体外培养的原代 人肝细胞。这些实验的结果将阐明新的翻译后和表观遗传 参与调节OATP 1B 1/3的机制。从这些研究中获得的知识将是无价的 合理设计新药和抑制剂，优化药物治疗，同时避免不需要的药物 交互.这项工作将提高我们预测赖氨酸脱乙酰酶改变OATP 1B 1/3功能的能力 调制器（例如，作为HDAC抑制剂/激活剂的药物/候选物和肝病状态）。

Organic anion transporting polypeptides (OATP)1B1 and OATP1B3 (abbreviated as OATP1B1/3) are liver- specific drug transport proteins that mediate uptake, from blood into the liver, of a diverse array of endogenous compounds, environmental toxins, and many clinically important drugs (e.g., lipid-lowering statins and anticancer agents). OATP1B1/3 are important determinants of transport-mediated drug-drug interactions (DDIs) resulting in severe side effects, such as statin-induced rhabdomyolysis, a sometimes-fatal muscle toxicity. Dysfunction of OATP1B1/3 significantly contributes to altered drug disposition and adverse drug events. Our long-term goal is to delineate the molecular mechanisms underlying drug/toxin disposition through OATP1B1/3, and to predict and mitigate OATP-mediated drug-drug and drug-disease interactions. Although it is evident that factors (drugs, aging, disease) modulating OATP1B1/3 function could cause drug-drug or drug-disease interactions with OATP1B1/3 substrates, unfortunately, our ability to predict such interactions is hampered due the dearth of information on OATP1B1/3 regulation. In particular, modulating lysine acetylation, a major post-translational modification known to alter function of numerous target proteins, including histone for epigenetic regulation, has not been investigated for OATP1B1/3. The overall objective of this application is to determine the molecular mechanisms governing the regulation of OATP1B1/3 by lysine acetylation, and to evaluate the impact of lysine deacetylase (KDAC) inhibitors (KDACIs) on OATP1B1/3 expression and function. Our central hypothesis is that OATP1B1/3 are lysine-acetylated proteins, deacetylation of which involves KDAC6; OATP1B1/3 can be regulated at the epigenetic level by KDACIs targeting histone deacetylation and at the post-translational level by KDAC6 inhibition. Our novel preliminary data show that 1) OATP1B1/3 are lysine-acetylated; 2) mutagenesis mimicking hyper-acetylation of OATP1B1 and specific KDAC 6 inhibition significantly reduces OATP1B1/3 transport function; and 3) FDA-approved pan KDAC inhibitors (KDACIs) inhibiting histone deacetylation induce mRNA and protein levels of OATP1B1/3. Guided by preliminary results, two specific Aims are outlined. In Aim 1, we will elucidate the role of KDAC6 in regulation of OATP1B1/3 lysine acetylation and transporter function. In Aim 2, we will dissect the regulation of OATP1B1/3 by pan KDACI drugs and the interplay between epigenetic and posttranslational regulation of OATP1B1/3 via K-Ac. A combination of proteomics, biochemical, and genetic engineering approaches will be utilized in cell lines and in the physiologically relevant sandwich-cultured primary human hepatocytes. The outcomes of these experiments will elucidate novel posttranslational and epigenetic mechanism(s) involved in regulating OATP1B1/3. The knowledge gained from these studies will be invaluable toward the rational design of novel drugs and inhibitors to optimize drug therapy while avoiding unwanted drug interactions. This work will enhance our ability to predict altered OATP1B1/3 function by lysine-deacetylase modulators (e.g., drugs/candidates that are HDAC inhibitors/activators and liver disease states).