Abstract
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.
Highlights
IntroductionThe use of assays that report in the “down” or “off” direction following drug exposure often results in a limited signal-to-noise ratio leading to false positives, which can misdirect drug discovery resources [1]
Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Washington School of Medicine, Seattle, WA, USA 5 Seattle Children’s Hospital, Seattle, WA, USAEffective high-throughput screening (HTS) campaigns can establish a solid foundation upon which successful drug discovery efforts are built
To test the hypothesis that high-specificity HTS-compatible reporters of oncogenic function can be rapidly generated without a priori identification of pathway-specific transcripts and mapping of promoter elements, we constructed a gene trap vector called SABRE (Splice Acceptor Bifunctional REporter)
Summary
The use of assays that report in the “down” or “off” direction following drug exposure often results in a limited signal-to-noise ratio leading to false positives, which can misdirect drug discovery resources [1]. To help address these issues, we set out to develop an improved reporter platform, initially focused on cancer drug development, with the following criteria in mind: First, the platform had to be HTS compatible, capable of testing thousands of samples in a reasonable time frame and cost-effective format. The readout must have a robust signal-to-noise ratio, ideally reporting an increase in signal upon pathway inhibition. The platform had to be flexible so that the technology can be employed to interrogate any transcriptionally active pathway in any cancer model
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