Development and Validation of a Fast and Homogeneous Cell-Based Fluorescence Screening Assay for Divalent Metal Transporter 1 (DMT1/SLC11A2) Using the FLIPR Tetra
Development and Validation of a Fast and Homogeneous Cell-Based Fluorescence Screening Assay for Divalent Metal Transporter 1 (DMT1/SLC11A2) Using the FLIPR Tetra
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DOI：
10.1177/1087057114521663
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发表时间：
2014-07-01
影响因子：
--
通讯作者：
Hediger, Matthias A.
中科院分区：
文献类型：
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作者：
Montalbetti, Nicolas;Simonin, Alexandre;Hediger, Matthias A.
二价金属离子转运蛋白 1 (DMT1) 是一种质子偶联的 Fe2+ 转运蛋白，对于肠细胞中的铁摄取以及许多其他细胞类型中转铁蛋白相关的内体铁转运至关重要。 DMT1 功能障碍与多种疾病有关，例如铁超载障碍和神经退行性疾病。目前工作的主要目标是开发和验证 DMT1 调节剂的基于荧光的筛选测定法。我们发现使用 FLIPR Tetra 荧光酶标仪可以可靠地监测负载钙 5 的 DMT1 表达 HEK293 细胞中的 Fe2+ 或 Cd2+ 流入。 DMT1 介导的金属转运显示出取决于细胞外底物浓度的饱和动力学，Fe2+ 和 Cd2+ 的 K-0.5 值分别为 1.4 μM 和 3.5 μM。此外，使用Cd2+作为DMT1的底物，我们发现属于苄基异硫脲家族的化合物(2-(3-甲脒基硫烷基甲基-苄基)异硫脲)的K-i值为2.1μM，该化合物已被鉴定为DMT1抑制剂。使用该化合物作为参考的优化筛选方法显示 Z' 因子为 0.51。总之，我们开发并验证了一种灵敏且可重复的基于细胞的荧光测定法，适用于鉴定特异性调节 DMT1 转运活性的化合物。
Divalent metal ion transporter 1 (DMT1) is a proton-coupled Fe2+ transporter that is essential for iron uptake in enterocytes and for transferrin-associated endosomal iron transport in many other cell types. DMT1 dysfunction is associated with several diseases such as iron overload disorders and neurodegenerative diseases. The main objective of the present work is to develop and validate a fluorescence-based screening assay for DMT1 modulators. We found that Fe2+ or Cd2+ influx could be reliably monitored in calcium 5-loaded DMT1-expressing HEK293 cells using the FLIPR Tetra fluorescence microplate reader. DMT1-mediated metal transport shows saturation kinetics depending on the extracellular substrate concentration, with a K-0.5 value of 1.4 mu M and 3.5 mu M for Fe2+ and Cd2+, respectively. In addition, Cd2+ was used as a substrate for DMT1, and we find a K-i value of 2.1 mu M for a compound (2-(3-carbamimidoylsulfanylmethyl-benzyl)isothiourea) belonging to the benzylisothioureas family, which has been identified as a DMT1 inhibitor. The optimized screening method using this compound as a reference demonstrated a Z' factor of 0.51. In summary, we developed and validated a sensitive and reproducible cell-based fluorescence assay suitable for the identification of compounds that specifically modulate DMT1 transport activity.