Organic anion-transporting polypeptides, OATP1B1, OATP1B3, and OATP2B1 are multispecific membrane proteins mediating the hepatocellular uptake of structurally diverse endo- and exogenous compounds, including various kinds of drugs. Co-administration of OATP1B/2B1 substrates may lead to altered pharmacokinetics or even toxicity. Therefore, the study of the interaction with these OATPs is essential in drug development and is recommended by international regulatory agencies, the FDA, EMA, and PMDA. In general, radiolabeled indicators are used to measure drug interactions of OATPs, and, lately, fluorescent probes are also gaining wider application in OATP tests. However, all of the currently available methods (either radioactive or fluorescence-based) comprise multiple steps, including the removal of the indicator in the end of the experiment. Hence, they are not ideally suited for high-throughput screening. In the current study, in order to find an indicator allowing real-time assessment of hepatic OATP function, we searched for an activatable fluorogenic OATP substrate. Here, we show that 8-acetoxypyrene-1,3,6-trisulfonate (Ace), a fluorogenic derivative of the hepatic OATP substrate pyranine (8-hydroxypyrene-1,3,6-trisulfonate) enters the cells via OATP1B1/3 or OATP2B1 function. In living cells, Ace is then converted into highly fluorescent pyranine, allowing “no-wash” measurement of OATP function and drug interactions. Furthermore, we demonstrate that Ace can be used in an indirect assay termed as competitive counterflow suitable to distinguish between transported substrates and inhibitors of OATP1B1. The fluorescence-based methods described here are unique and open the way toward high-throughput screening of interactions between new molecular entities and OATPs.

中文翻译：

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8-乙酰氧基-三磺芘作为第一个可激活的荧光探针，用于有机阴离子转运多肽、OATP1B1、OATP1B3 和 OATP2B1 的添加和读取评估

有机阴离子转运多肽 OATP1B1、OATP1B3 和 OATP2B1 是多特异性膜蛋白，可介导肝细胞对结构多样的内源性和外源性化合物（包括各种药物）的摄取。OATP1B/2B1 底物的共同给药可能导致药代动力学改变甚至毒性。因此，研究与这些 OATP 的相互作用对于药物开发至关重要，并得到国际监管机构、FDA、EMA 和 PMDA 的推荐。通常，放射性标记的指示剂用于测量 OATP 的药物相互作用，最近，荧光探针在 OATP 测试中也得到了更广泛的应用。然而，所有当前可用的方法（基于放射性或基于荧光的）都包含多个步骤，包括在实验结束时去除指示剂。因此，它们不适合高通量筛选。在目前的研究中，为了找到一个可以实时评估肝脏 OATP 功能的指标，我们寻找了一种可激活的荧光 OATP 底物。在这里，我们展示了 8-乙酰氧基芘-1,3,6-三磺酸盐 (Ace)，一种肝脏 OATP 底物吡喃（8-羟基芘-1,3,6-三磺酸盐）的荧光衍生物，通过 OATP1B1/3 或OATP2B1 功能。在活细胞中，Ace 然后被转化为高荧光的吡喃，从而可以“免洗”测量 OATP 功能和药物相互作用。此外，我们证明 Ace 可用于称为竞争性逆流的间接测定，适用于区分运输的底物和 OATP1B1 抑制剂。