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PLoS One. 2014 Aug 5;9(8):e103836. doi: 10.1371/journal.pone.0103836. eCollection 2014.

A high-throughput assay for small molecule destabilizers of the KRAS oncoprotein.

Author information

1
Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America.
2
Division of Preclinical Innovation, National Institutes of Health Chemical Genomics Center, National Center for Advancing Translational Sciences, Bethesda, Maryland, United States of America.

Abstract

Mutations in the Ras family of small GTPases, particularly KRAS, occur at high frequencies in cancer and represent a major unmet therapeutic need due to the lack of effective targeted therapies. Past efforts directed at inhibiting the activity of the Ras oncoprotein have proved difficult. We propose an alternative approach to target Ras by eliminating Ras protein from cells with pharmacological means. In this study, we developed a cell-based, high-content screening platform to identify small molecules that could promote the degradation of the KRAS oncoprotein. We generated an EGFP-KRASG12V fluorescence reporter system and implemented it for automated screening in 1536-well plates using high-throughput cellular imaging. We screened a library of clinically relevant compounds at wide dose range and identified Ponatinib and AMG-47a as two candidate compounds that selectively reduced the levels of EGFP-KRASG12V protein but did not affect EGFP protein in cells. This proof-of-principle study demonstrates that it is feasible to use a high-throughput screen to identify compounds that promote the degradation of the Ras oncoprotein as a new approach to target Ras.

PMID:
25093678
PMCID:
PMC4122376
DOI:
10.1371/journal.pone.0103836
[Indexed for MEDLINE]
Free PMC Article

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