Identification of inhibitors of the kinase activity of oncogenic V600EBRAF in an enzyme cascade high-throughput screen
Identification of inhibitors of the kinase activity of oncogenic V600EBRAF in an enzyme cascade high-throughput screen
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DOI：
10.1177/1087057105283584
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发表时间：
2006-03-01
影响因子：
--
通讯作者：
Aherne, W
中科院分区：
文献类型：
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作者：
Newbatt, Y;Burns, S;Aherne, W
癌症基因组计划已经确定了 BRAT 中的几种致癌突变，这代表了癌症药物发现的重要机会。 (V600E)BRAF 突变约占已识别突变的 90%。分子、细胞和结构研究为鉴定和开发这种突变 BRAF 蛋白的抑制剂提供了强有力的案例。作者开发并运行了一种高通量筛选，使用酶级联测定来寻找 (V600E)BRAF 抑制剂，其中致癌 BRAF 激活 MEK1，MEK1 反过来激活 ERK2，然后磷酸化转录因子 ELK1。使用磷酸特异性抗体、铕标记的二抗和时间分辨荧光读数仪来测量 ELK1 的磷酸化。总体检测变异为 12.4%。该测定用于筛选 64,000 种化合物，总体 Z' 因子为 0.58 +/- 0.12。一系列 3,5,二取代吡啶被鉴定为级联测定的抑制剂。这些化合物不会抑制缩短的活化 MEK1 至 ELK1 级联，但在 (V600E)BRAF 测定中具有活性 (0.5-27.9 μM)，代表了未来药物发现和开发的潜在起点。
The Cancer Genome Project has identified several oncogenic mutations in BRAT that represent important opportunities for cancer drug discovery. The (V600E)BRAF mutation accounts for approximately 90% of the mutations identified. A strong case has emerged from molecular, cellular, and structural studies for the identification and development of inhibitors of this mutated BRAF protein. The authors have developed and run a high-throughput screen to find inhibitors of (V600E)BRAF using an enzyme cascade assay in which oncogenic BRAF activates MEK1, which in turn activates ERK2, which then phosphorylates the transcription factor ELK1. A phosphospecific antibody, Europium-labeled secondary antibody, and a time-resolved fluorescent readout were used to measure phosphorylation of ELK1. Overall assay variation was 12.4%. The assay was used to screen 64,000 compounds with an overall Z' factor of 0.58 +/- 0.12. A series of 3,5,di-substituted pyridines were identified as inhibitors of the cascade assay. These compounds did not inhibit a shortened activated MEK1 to ELK1 cascade but were active (0.5-27.9 mu M) in a (V600E)BRAF assay and represent a potential starting point for future drug discovery and development.