Investigation of Fluorescein Derivatives as Substrates of Organic Anion Transporting Polypeptide (OATP) 1B1 To Develop Sensitive Fluorescence-Based OATP1B1 Inhibition Assays
Investigation of Fluorescein Derivatives as Substrates of Organic Anion Transporting Polypeptide (OATP) 1B1 To Develop Sensitive Fluorescence-Based OATP1B1 Inhibition Assays
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DOI：
10.1021/acs.molpharmaceut.5b00664
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发表时间：
2016-02-01
影响因子：
4.9
通讯作者：
Sugiyama, Yuichi
中科院分区：
文献类型：
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作者：
Izumi, Saki;Nozaki, Yoshitane;Sugiyama, Yuichi
有机阴离子转运多肽（OATP）IBI在多种药物的肝摄取中起重要作用。由于OATPIB 1是药物相互作用（DDI）的位点，因此在药物开发期间需要评价候选药物对OATP 1B 1的抑制潜力。为了建立一个高灵敏度、高通量的基于荧光的OATP 1B 1抑制试验系统，本研究重点研究了荧光素（FL）及其衍生物，并使用表达转运蛋白的人胚肾293细胞评价了它们通过OATP 1B 1以及OATP 1B 3和OATP 2B 1的摄取。我们鉴定2 '，7'-二氯荧光素（DCF）、4 '，5'-二溴荧光素（DBF）和俄勒冈州绿色（OG）为良好的OATPIBI底物，Km值分别为5.29、4.16和54.1 μ M，V-max值分别为87.9、48.1和187 pmol/min/mg蛋白。除FL外，还将fluo-3和8-荧光素-cAMP、OG和DBF鉴别为OATP 1B 3底物。FL、OG、DCF和DBF被鉴定为OATP 2B 1底物。在FL衍生物中，DCF显示最高的OATP 1B 1介导的摄取。以DCF为探针测定的14种化合物对OATP 1B 1的Ki值与以[H-3]雌二醇-17 β-葡萄糖醛酸苷（E（2）G）为底物测定的结果一致，而所有抑制剂的[3 H]雌酮-3-硫酸盐和[H-3]磺基溴酞的Ki值均高于DCF。在DCF和E2 G之间观察到的相互竞争性抑制表明，它们在OATP 1B 1上具有相同的结合位点。因此，DCF和E2 G可用作体外OATP 1B 1抑制试验的敏感探针，这将有助于降低底物依赖性Ki变化可能导致的假阴性DDI预测的风险。
Organic anion transporting polypeptide (OATP) IBI plays an important role in the hepatic uptake of various drugs. Because OATPIB1 is a site of drug drug interactions (DDIs), evaluating the inhibitory potential of drug candidates on OATP1B1 is required during drug development. For establishing a highly sensitive, high-throughput fluorescence-based OATP1B1 inhibition assay system, the present study focused on fluorescein (FL) and its derivatives and evaluated their uptake via OATP1B1 as well as OATP1B3 and OATP2B1 using the transporter expressing human embryonic kidney 293 cells. We identified 2',7'-dichlorofluorescein (DCF), 4',5'-dibromofluorescein (DBF), and Oregon green (OG) as good OATPIBI substrates with Km values of 5.29, 4.16, and 54.1 mu M and V-max values of 87.9, 48.1, and 187 pmol/min/mg protein, respectively. In addition to FL, fluo-3, and 8-fluorescein-cAMP, OG, and DBF were identified as OATP1B3 substrates. FL, OG, DCF, and DBF were identified as OATP2B1 substrates. Among the FL derivatives, DCF displayed the highest OATP1B1-mediated uptake. The K-i values of 14 compounds on OATP1B1 determined with DCF as a probe exhibited good agreement with those obtained using [H-3]estradio1-17 beta-glucuronidq (E(2)G) as a substrate, whereas [3H]estrone-3-sulfate and [H-3]sulfobromophthalein yielded higher K-i values for all inhibitors than DCF. Mutually competitive inhibition observed between DCF and E2G suggested that they share the same binding site on OATP1B1. Therefore, DCF as well as E2G can be used as sensitive probes for in vitro OATP1B1 inhibition assays, which will help mitigate the risk of false negative DDI predictions potentially caused by substrate-dependent K-i variations.