Development and validation of a high-throughput screening assay for human long-chain fatty acid transport proteins 4 and 5.
Development and validation of a high-throughput screening assay for human long-chain fatty acid transport proteins 4 and 5.
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DOI：
10.1177/1087057110369700
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发表时间：
2010-06
影响因子：
--
通讯作者：
Xie XS
中科院分区：
文献类型：
--
作者：
Zhou W;Madrid P;Fluitt A;Stahl A;Xie XS
膳食长链脂肪酸（LCFA）跨细胞膜的摄取主要由脂肪酸转运蛋白（FATPs）介导。该转运蛋白的六种亚型在整个人类和啮齿动物体内差异表达。为了促进针对FATP亚型的药物发现，我们利用稳定表达重组人FATP 4和5的哺乳动物细胞系，并使用96孔荧光成像板读数器（FLIPR）开发了高通量筛选（HTS）测定。LCFA摄取信号与背景比为3 - 5倍。两种4-芳基-二氢嘧啶酮j3和j5分别以0.21 μ M和0.63 μ M的半数最大抑制浓度（IC 50）产生对FATP 4的抑制，并且显示出比FATP 5高约100倍的选择性。针对FATP 5筛选US Drug Collection文库。在Z'因子为0.6 ± 0.2的情况下观察到约0.4%的命中率。两个确认的命中物是胆汁酸、chenodiol和ursodiol，其IC 50分别为2.4和0.22 μ M。为了提高通量，使用Analyst HT开发了384孔格式的单时间点测量，结果与96孔格式相当。总之，基于FATP 4和5细胞的荧光测定适合于初级药物筛选，而分化的细胞系可用于次级药物筛选。
Dietary long-chain fatty acid (LCFA) uptake across cell membranes is mediated principally by fatty acid transport proteins (FATPs). Six subtypes of this transporter are differentially expressed throughout the human and rodent body. To facilitate drugs discovery against FATP subtypes, we utilized mammalian cell lines stably expressing the recombinant human FATP4 and 5, and developed a high-throughput screening (HTS) assay using a 96-well fluorometric imaging plate reader (FLIPR). LCFA uptake signal-to background ratios were between 3 and 5-fold. Two 4-aryl-dihydropyrimidinones, j3 and j5, produced inhibition of FATP4 with a half-maximal inhibitory concentration (IC50) of 0.21 uM, and 0.63 uM, respectively, and displayed approximately 100-fold selectivity over FATP5. The US Drug Collection library was screened against the FATP5. A hit rate of around 0.4% was observed with a Z’ factor of 0.6 ± 0.2. Two confirmed hits are bile acids, chenodiol and ursodiol with an IC50 of 2.4 and 0.22 uM, respectively. To increase throughput, a single-time-point measurement in 384-well format was developed using the Analyst HT and the results are comparable with 96-well format. In conclusion, the FATP4 and 5 cell-based fluorescence assays are suitable for a primary drug screen, while differentiated cell lines useful for a secondary drug screen.
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DOI：
10.1083/jcb.200207080
发表时间：
2003-06-23
期刊：
The Journal of cell biology
影响因子：
--
作者：
Herrmann T;van der Hoeven F;Grone HJ;Stewart AF;Langbein L;Kaiser I;Liebisch G;Gosch I;Buchkremer F;Drobnik W;Schmitz G;Stremmel W
通讯作者：
Stremmel W
影响因子：
4.8
作者：
Gimeno, RE;Hirsch, DJ;Stahl, A
通讯作者：
Stahl, A
DOI：
10.1152/ajpendo.2001.280.6.e1000
发表时间：
2001-06-01
影响因子：
5.1
作者：
Basu, A;Basu, R;Jensen, MD
通讯作者：
Jensen, MD
影响因子：
6.5
作者：
Richards, MR;Harp, JD;Schaffer, JE
通讯作者：
Schaffer, JE
影响因子：
6.5
作者：
Li, Hong;Black, Paul N.;DiRusso, Concetta C.
通讯作者：
DiRusso, Concetta C.