A fluorescence-based thiol quantification assay for ultra-high-throughput screening for inhibitors of coenzyme A production
A fluorescence-based thiol quantification assay for ultra-high-throughput screening for inhibitors of coenzyme A production
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DOI：
10.1089/adt.2007.105
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发表时间：
2008-06-01
影响因子：
1.8
通讯作者：
Takagi, Toshimitsu
中科院分区：
文献类型：
--
作者：
Chung, Christine C.;Ohwaki, Kenji;Takagi, Toshimitsu
在这里，我们报道了一种用于超高通量筛选(UHTS)肥胖和癌症两个潜在药物靶点的抑制剂的无细胞酶试验的发展和微型化：脂肪酸合成酶(Fas)和乙酰辅酶A(CoA)羧基酶(ACC)2。该方法检测Fas催化丙二酰辅酶A和乙酰辅酶A缩合的产物CoA。辅酶A的游离硫醇可以与7-二乙氨基-3-(4‘-马来酰亚胺基苯基)-4-甲基香豆素(CPM)反应，CPM是一种前荧光香豆素马来酰亚胺衍生物，与硫醇反应后变成荧光。Fas通过丙二酰辅酶A和乙酰辅酶A的缩合生成长链脂肪酸和辅酶A。在我们的Fas分析中，在Fas反应中释放的CoA与CPM形成荧光加合物，当激发波长为405 nm时，该加合物在530 nm处发射。利用这种辅酶A的检测方法，我们测量了脂肪酸合成途径中的顺序酶的活性，以建立ACC2/Fas偶联分析方法，其中ACC2由乙酰辅酶A合成丙二酰辅酶A。我们将Fas和ACC2/Fas分别小型化到3,456和1,536孔板格式，并完成了针对该酶系统小分子抑制剂的uHTSS。这份报告显示了这些潜在药物靶点的分析开发、小型化和抑制剂筛选的结果。
Here we report the development and miniaturization of a cell-free enzyme assay for ultra-high-throughput screening (uHTS) for inhibitors of two potential drug targets for obesity and cancer: fatty acid synthase (FAS) and acetyl-coenzyme A (CoA) carboxylase (ACC) 2. This assay detects CoA, a product of the FAS-catalyzed condensation of malonyl-CoA and acetyl-CoA. The free thiol of CoA can react with 7-diethylamino- 3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM), a profluorescent coumarin maleimide derivative that becomes fluorescent upon reaction with thiols. FAS produces long-chain fatty acid and CoA from the condensation of malonyl-CoA and acetylCoA. In our FAS assay, CoA released in the FAS reaction forms a fluorescence adduct with CPM that emits at 530 nm when excited at 405 nm. Using this detection method for CoA, we measured the activity of sequential enzymes in the fatty acid synthesis pathway to develop an ACC2/FAS-coupled assay where ACC2 produces malonyl-CoA from acetyl-CoA. We miniaturized the FAS and ACC2/FAS assays to 3,456- and 1,536-well plate format, respectively, and completed uHTSs for small molecule inhibitors of this enzyme system. This report shows the results of assay development, miniaturization, and inhibitor screening for these potential drug targets.