title: A high-throughput assay for small molecule destabilizers of the KRAS oncoprotein.
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.
authors.affiliation: Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America.
authors.forename: Joseph
authors.initials: J
authors.lastname: Carver
authors.affiliation: Division of Preclinical Innovation, National Institutes of Health Chemical Genomics Center, National Center for Advancing Translational Sciences, Bethesda, Maryland, United States of America.
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Dexheimer
authors.affiliation: Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America.
authors.forename: Dennis
authors.initials: D
authors.lastname: Hsu
authors.affiliation: Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America.
authors.forename: Meng-Tzu
authors.initials: MT
authors.lastname: Weng
authors.affiliation: Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America.
authors.forename: Jordan L
authors.initials: JL
authors.lastname: Smith
authors.affiliation: Division of Preclinical Innovation, National Institutes of Health Chemical Genomics Center, National Center for Advancing Translational Sciences, Bethesda, Maryland, United States of America.
authors.forename: Rajarshi
authors.initials: R
authors.lastname: Guha
authors.affiliation: Division of Preclinical Innovation, National Institutes of Health Chemical Genomics Center, National Center for Advancing Translational Sciences, Bethesda, Maryland, United States of America.
authors.forename: Ajit
authors.initials: A
authors.lastname: Jadhav
authors.affiliation: Division of Preclinical Innovation, National Institutes of Health Chemical Genomics Center, National Center for Advancing Translational Sciences, Bethesda, Maryland, United States of America.
authors.forename: Anton
authors.initials: A
authors.lastname: Simeonov
authors.affiliation: Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America.
authors.forename: Ji
authors.initials: J
authors.lastname: Luo
mesh_terms: D000970:Antineoplastic Agents / Q000302:isolation & purification* / Q000494:pharmacology*; D002478:Cells, Cultured; D004354:Drug Screening Assays, Antitumor; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D055550:Protein Stability / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras); D054852:Small Molecule Libraries / Q000032:analysis*; D014162:Transfection; D018631:ras Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D054852:Small Molecule Libraries; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins
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