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

有机阴离子转运多肽(OATP)1B1和OATP1B3(简称OATP1B1/3)是肝脏- 特定的药物转运蛋白，介导从血液到肝脏的各种内源性 化合物、环境毒素和许多临床重要药物(如降脂他汀类和抗癌药物 代理)。OATP1B1/3是转运介导的药物相互作用(DDiS)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。失调症 OATP1B1/3对药物处置改变和药物不良事件有显著影响。我们的长期目标是 通过OATP1B1/3阐明药物/毒素处置的分子机制，并预测和 缓解OATP介导的药物与药物和药物与疾病的相互作用。尽管很明显，因素(药物、 衰老、疾病)调节OATP1B1/3功能可能导致药物与药物或药物与疾病的相互作用 不幸的是，由于缺乏OATP1B1/3底物，我们预测这种相互作用的能力受到阻碍 有关OATP1B1/3法规的信息。特别是，调节赖氨酸乙酰化，一种主要的翻译后 已知的可以改变许多靶蛋白功能的修饰，包括用于表观遗传调控的组蛋白，已经 尚未对OATP1B1/3进行研究。此应用程序的总体目标是确定分子 赖氨酸乙酰化调控OATP1B1/3的机制及赖氨酸的影响 脱乙酰酶(KDAC)抑制剂(KDACIs)对OATP1B1/3表达和功能的影响。我们的中心假设是 OATP1B1/3是赖氨酸乙酰化的蛋白质，其脱乙酰基涉及KDAC6；OATP1B1/3可以是 以组蛋白脱乙酰化为靶点的KDACIs在表观遗传水平上调控，在翻译后水平上通过 KDAC6抑制作用。我们新的初步数据显示：1)OATP1B1/3是赖氨酸乙酰化的；2)突变 模拟OATP1B1的超乙酰化和特异性KDAC6抑制显著降低OATP1B1/3 转运功能；以及3)FDA批准的PAN KDAC抑制剂(KDACIs)抑制组蛋白脱乙酰化诱导 OATP1B1/3的mRNA和蛋白水平。以初步结果为指导，概述了两个特定的目标。在AIM 1，我们将阐明KDAC6在调节OATP1B1/3赖氨酸乙酰化和转运蛋白功能中的作用。在……里面 目的2，我们将剖析PAN KDACI药物对OATP1B1/3的调节以及表观遗传学之间的相互作用 通过K-ac对OATP1B1/3进行翻译后调控。蛋白质组学、生物化学和遗传学的结合 工程学方法将用于细胞系和生理相关的三明治原代培养。 人肝细胞。这些实验的结果将阐明新的翻译后和表观遗传学 参与调控OATP1B1/3的机制(S)。从这些研究中获得的知识将是非常宝贵的 合理设计新药和抑制剂，在避免多余药物的同时优化药物治疗 互动。这项工作将增强我们预测赖氨酸脱乙酰酶改变OATP1B1/3功能的能力 调节剂(例如，作为HDAC抑制剂/激活剂和肝病状态的药物/候选药物)。

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).