Elucidating the Role of Organic Solute Transporter (OST) Alpha/Beta in Bile Acid Transport and Drug Interactions

项目概要/摘要 有机溶质转运蛋白（OST）α/β是一种定位于肝基底外侧膜的异聚体蛋白， 肠和肾上皮细胞，是一种重要的转运蛋白，参与胆汁酸（BAs）的稳态， 和其他类固醇激素。OSTα/β缺陷小鼠表现出BA吸收不良的特征。的肝表达 OSTα/β在阻塞性胆汁淤积和原发性胆汁性胆管炎中显著增加，这表明 转运蛋白为胆汁淤积性肝病中的BA提供排泄途径。OSTα/β过表达于 非酒精性脂肪性肝炎患者，并且已经发现这种转运蛋白的遗传缺陷之间存在联系。 以及先天性腹泻和胆汁淤积，这意味着OSTα/β在健康和疾病中的关键作用。失调 BA处置，特别是通过药物介导的肝外排转运蛋白抑制，可导致肝细胞 导致肝毒性的亲脂性BA蓄积。这是药物致肝的重要机制 损伤（DILI）是药物开发中的主要安全性问题。然而，关于OSTα/β在以下方面的作用知之甚少： BA介导的DILI。一些BA和药物是已知的OSTα/β底物和/或抑制剂，但系统研究 OSTα/β介导的BA转运缺乏。因此，鉴定哪些BA是OSTα/β底物， 与OSTα/β介导的BA转运相互作用的药物，并阐明对BA至关重要的OSTα/β残基 转运对于理解该转运蛋白在人类健康、疾病和DILI中的作用至关重要。具体目标 将检验以下假设：1）BA对OSTα/β的亲和力是BA亲脂性/肝毒性的函数; 2）药物- OSTα/β介导的BA转运的诱导改变依赖于BA种类; 3）进化上 OSTα/β跨膜结构域中的保守、带电和亲水/极性残基对于 OSTα/β介导的BA转运。使用稳定的OSTα/β过表达人细胞系和临床试验， 在拟议的项目中，相关的体外培养的人肝细胞模型，最普遍的亲水和 将分析亲脂性/肝毒性BA，以鉴定OSTα/β底物并阐明药物相互作用 OSTα/β介导的这些BA底物的转运。在本项目的目标1中，OSTα/β介导的转运 将通过OSTα/β的过表达或诱导来研究特异性BA的动力学。在目标2中，药物诱导 将阐明OSTα/β介导的BA处置的改变。为了进一步揭示OSTα/β介导的BA 通过对OSTα/β跨膜结构域中的关键氨基酸残基的研究， (Aim 3）。拟议的研究结果将提供基本的，和机制的信息， OSTα/β在人类健康和疾病中的作用，以及DILI相关药物对OSTα/β介导的BA的影响 运输这些数据将使用计算建模改善BA介导的DILI的预测。结果 预计将提供有关OSTα/β功能障碍的生物标志物候选物的新见解， BA相关病症的治疗靶点（例如，非酒精性脂肪肝、肥胖症、糖尿病）。

Project Summary/Abstract Organic solute transporter (OST) α/β, a heteromeric protein localized on the basolateral membrane of hepatic, intestinal and kidney epithelial cells, is an important transporter involved in the homeostasis of bile acids (BAs) and other steroid hormones. OSTα/β-deficient mice exhibit features of BA malabsorption. Hepatic expression of OSTα/β is significantly increased in obstructive cholestasis and primary biliary cholangitis, suggesting that this transporter provides an excretion pathway for BAs in cholestatic liver disease. OSTα/β is overexpressed in patients with nonalcoholic steatohepatitis, and a link has been found between genetic defects in this transporter and congenital diarrhea and cholestasis, implying a key role for OSTα/β in health and disease. Dysregulation of BA disposition, particularly by drug-mediated inhibition of hepatic efflux transporters, can lead to hepatocellular accumulation of lipophilic BAs resulting in hepatotoxicity. This is an important mechanism of drug-induced liver injury (DILI), a major safety issue in drug development. However, very little is known about the role of OSTα/β in BA-mediated DILI. A few BAs and drugs are known OSTα/β substrates and/or inhibitors, but a systematic study of OSTα/β-mediated BA transport is lacking. Therefore, identifying which BAs are OSTα/β substrates, identifying drugs that interact with OSTα/β-mediated BA transport, and elucidating OSTα/β residues that are crucial for BA transport is vital to understanding the role of this transporter in human health, disease and DILI. Specific aims will test the following hypotheses: 1) BA affinity for OSTα/β is a function of BA lipophilicity/hepatotoxicity; 2) drug- induced alterations in OSTα/β-mediated BA transport are dependent on the BA species; and 3) evolutionarily conserved, charged and hydrophilic/polar residues in the transmembrane domains of OSTα/β are crucial for OSTα/β-mediated BA transport. Using a stable, OSTα/β-overexpressing human cell line and the clinically relevant sandwich-cultured human hepatocyte model in the proposed project, the most prevalent hydrophilic and lipophilic/hepatotoxic BAs will be analyzed to identify OSTα/β substrates and elucidate the interaction of drugs with OSTα/β-mediated transport of these BA substrates. In Aim 1 of this project, OSTα/β-mediated transport kinetics of specific BAs will be studied by overexpression or induction of OSTα/β. In Aim 2, drug-induced alterations in OSTα/β-mediated BA disposition will be elucidated. To further reveal OSTα/β-mediated BA transport mechanisms, critical amino acid residues in OSTα/β’s transmembrane domains will be investigated (Aim 3). The results of the proposed studies will provide fundamental, and mechanistic information about the role of OSTα/β in human health and disease, and the effect of DILI-associated drugs on OSTα/β-mediated BA transport. These data will improve predictions of BA-mediated DILI using computational modeling. The results are anticipated to provide novel insights regarding biomarker candidates of OSTα/β dysfunction, and novel therapeutic targets for BA-related disorders (e.g., nonalcoholic fatty liver disease, obesity, diabetes).