While advances in laboratory automation has dramatically increased throughout of compound screening efforts, development of robust cell-based assays in relevant disease models remain resource-intensive and time-consuming, presenting a bottleneck to drug discovery campaigns. To address this issue, we present a modified gene trap approach to efficiently generate pathway-specific reporters that result in a robust "on" signal when the pathway of interest is inhibited. In this proof-of-concept study, we used vemurafenib and trametinib to identify traps that specifically detect inhibition of the mitogen-activated protein kinase (MAPK) pathway in a model of BRAFV600E driven human malignant melanoma. We demonstrate that insertion of our trap into particular loci results in remarkably specific detection of MAPK pathway inhibitors over compounds targeting any other pathway or cellular function. The accuracy of our approach was highlighted in a pilot screen of ~6000 compounds where 40 actives were detected, including 18 MEK, 10 RAF, and 3 ERK inhibitors along with a few compounds representing previously under-characterized inhibitors of the MAPK pathway. One such compound, bafetinib, a second generation BCR/ABL inhibitor, reduced phosphorylation of ERK and when combined with trametinib, both in vitro and in vivo, reduced growth of vemurafenib resistant melanoma cells. While piloted in a model of BRAF-driven melanoma, our results set the stage for using this approach to rapidly generate reporters against any transcriptionally active pathway across a wide variety of disease-relevant cell-based models to expedite drug discovery efforts.

中文翻译：

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一种改进的基因陷阱方法，用于改进高通量癌症药物的发现。


虽然实验室自动化的进步在整个化合物筛选工作中显着增加，但在相关疾病模型中开发强大的基于细胞的检测方法仍然需要大量资源且耗时，这给药物发现活动带来了瓶颈。为了解决这个问题，我们提出了一种改进的基因陷阱方法，可以有效地生成特定于途径的报告基因，当感兴趣的途径受到抑制时，该报告基因会产生强大的“开启”信号。在这项概念验证研究中，我们使用维罗非尼和曲美替尼来鉴定特异性检测 BRAFV600E 驱动的人类恶性黑色素瘤模型中丝裂原激活蛋白激酶 (MAPK) 通路抑制的陷阱。我们证明，将我们的陷阱插入特定位点，可以比针对任何其他途径或细胞功能的化合物显着特异性地检测 MAPK 途径抑制剂。我们的方法的准确性在约 6000 种化合物的试点筛选中得到了强调，其中检测到了 40 种活性物质，包括 18 种 MEK、10 种 RAF 和 3 种 ERK 抑制剂，以及一些代表以前未充分表征的 MAPK 途径抑制剂的化合物。其中一种化合物巴非替尼 (bafetinib) 是第二代 BCR/ABL 抑制剂，可减少 ERK 的磷酸化，当与曲美替尼 (Trametinib) 联合使用时，在体外和体内均可减少维莫非尼 (vemurafenib) 耐药性黑色素瘤细胞的生长。在 BRAF 驱动的黑色素瘤模型中进行试点时，我们的结果为使用这种方法快速生成针对各种疾病相关细胞模型中任何转录活性途径的报告基因奠定了基础，以加快药物发现工作。