Fluorescence-based assay for the interaction of small molecules with the human renal organic anion transporter 1
Fluorescence-based assay for the interaction of small molecules with the human renal organic anion transporter 1
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DOI：
10.1006/abio.2000.4633
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发表时间：
2000-07-15
影响因子：
2.9
通讯作者：
Ho, ES
中科院分区：
文献类型：
--
作者：
Cihlar, T;Ho, ES
将全身血液循环中的小分子分泌到尿液中是肾脏的生理基本功能之一。人体有机阴离子转运蛋白（hOAT1）是肾小管分泌负电荷分子的关键成分，包括多种重要的治疗药物。在某些情况下，与hOAT1相互作用的化合物可能诱导药代动力学药物相互作用或引起肾毒性。我们利用稳定表达hOAT1的CHO细胞开发了一种基于荧光的96孔格式检测方法，可以评估小分子与hOAT1之间的相互作用。该实验是基于对6-羧基荧光素运输的抑制，6-羧基荧光素是一种高亲和力的hOAT1底物（K-m = 3.9 μ M），被确定为几种荧光有机阴离子之一。使用基于6-羧基荧光素的抑制实验确定的各种已知hOAT1底物的相对抑制效力与其K-m值相关良好，表明荧光实验具有适当的特异性。该体外试验可用于评估肾脏清除有机阴离子的机制，评估潜在的药物-药物相互作用和/或各种治疗方法的肾毒性作用，并筛选可能作为有效肾保护剂的新型hOAT1抑制剂。(C) 2000年学术出版社。
Secretion of small molecules from the systemic blood circulation into urine is one of the physiologically essential functions of the kidney. The human organic anion transporter (hOAT1) is a key component in the renal tubular secretion of negatively charged molecules including a variety of important therapeutics. In some cases, compounds interacting with hOAT1 may induce pharmacokinetic drug-drug interactions or cause nephrotoxicity. We developed a fluorescence-based, 96-well format assay using CHO cells stably expressing hOAT1, which allows for the evaluation of interactions between small molecules and hOAT1. The assay is based on the inhibition of the transport of 6-carboxyfluorescein, a high-affinity hOAT1 substrate (K-m = 3.9 mu M), which was identified as one of several fluorescent organic anions. The relative inhibition potency of various known hOAT1 substrates determined using the 6-carboxyfluorescein-based inhibition assay correlated well with their K-m values, indicating that the fluorescent assay exhibits a proper specificity. This in vitro assay can be employed to evaluate the mechanism of renal clearance of organic anions, to assess potential drug-drug interactions and/or nephrotoxic effects of various therapeutics, and to screen for novel hOAT1 inhibitors that could serve as efficient nephroprotectants. (C) 2000 Academic Press.