A high-throughput cell-based assay to identify specific inhibitors of transcription factor AP-1
A high-throughput cell-based assay to identify specific inhibitors of transcription factor AP-1
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DOI：
10.1177/1087057106296686
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发表时间：
2007-02-01
影响因子：
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通讯作者：
Henrich, Curtis J.
中科院分区：
文献类型：
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作者：
Ruocco, Katie M.;Goncharova, Ekaterina I.;Henrich, Curtis J.
致癌转录因子AP-1（激活蛋白-1）是肿瘤促进和进展所必需的。新的特异性AP-1抑制剂的鉴定将有利于癌症的预防和治疗。作者开发了一种高通量检测方法，用于筛选合成和天然产物库中丝裂原激活AP-1活性的非细胞毒性抑制剂。基于细胞的高通量筛选在384孔格式中使用荧光共振能量转移（FRET）底物进行，以定量在AP-1依赖性启动子控制下的β-内酰胺酶报告基因的活性。比率FRET读数使该测定非常稳健和可重复，特别是用于天然产物提取物。为了消除由于细胞杀伤引起的假阳性，结合了细胞毒性测定。用位于AP-1上游的激酶抑制剂和已知的AP-1天然产物抑制剂（去甲二氢愈创木酸和姜黄素）验证AP-1 β-内酰胺酶报告基因。该测定能够鉴定其他已知的AP-1抑制剂和蛋白激酶C调节剂，以及许多来自天然产物库的具有未知作用机制的化学上不同的化合物。应用天然产物提取物确定命中从一系列分类组。合成化合物和天然产物的筛选应确定可能用于预防和治疗癌症的新型AP-1抑制剂。
The oncogenic transcription factor AP-1 (activator protein-1) is required for tumor promotion and progression. Identification of novel and specific AP-1 inhibitors would be beneficial for cancer prevention and therapy. The authors have developed a high-throughput assay to screen synthetic and natural product libraries for noncytotoxic inhibitors of mitogen-activated AP-1 activity. The cell-based high-throughput screen is conducted in a 384-well format using a fluorescent resonance energy transfer (FRET) substrate to quantify the activity of a beta-lactamase reporter under the control of an AP-1-dependent promoter. The ratiometric FRET readout makes this assay extremely robust and reproducible, particularly for use with natural product extracts. To eliminate false positives due to cell killing, a cytotoxicity assay was incorporated. The AP-1 beta-lactamase reporter was validated with inhibitors of kinases located upstream of AP-1 and with known natural product inhibitors of AP-1 (nordihydroguaiaretic acid and curcumin). The assay was able to identify other known AP-1 inhibitors and protein kinase C modulators, as well as a number of chemically diverse compounds with unknown mechanisms of action from natural products libraries. Application to natural product extracts identified hits from a range of taxonomic groups. Screening of synthetic compounds and natural products should identify novel AP-1 inhibitors that may be useful in the prevention and treatment of cancers.