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A High-Throughput Assay for Small Molecule Destabilizers of the KRAS Oncoprotein

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...

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Autores principales: Carver, Joseph, Dexheimer, Thomas S., Hsu, Dennis, Weng, Meng-Tzu, Smith, Jordan L., Guha, Rajarshi, Jadhav, Ajit, Simeonov, Anton, Luo, Ji
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4122376/
https://www.ncbi.nlm.nih.gov/pubmed/25093678
http://dx.doi.org/10.1371/journal.pone.0103836
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author Carver, Joseph
Dexheimer, Thomas S.
Hsu, Dennis
Weng, Meng-Tzu
Smith, Jordan L.
Guha, Rajarshi
Jadhav, Ajit
Simeonov, Anton
Luo, Ji
author_facet Carver, Joseph
Dexheimer, Thomas S.
Hsu, Dennis
Weng, Meng-Tzu
Smith, Jordan L.
Guha, Rajarshi
Jadhav, Ajit
Simeonov, Anton
Luo, Ji
author_sort Carver, Joseph
collection PubMed
description 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-KRAS(G12V) 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-KRAS(G12V) 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.
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spelling pubmed-41223762014-08-12 A High-Throughput Assay for Small Molecule Destabilizers of the KRAS Oncoprotein Carver, Joseph Dexheimer, Thomas S. Hsu, Dennis Weng, Meng-Tzu Smith, Jordan L. Guha, Rajarshi Jadhav, Ajit Simeonov, Anton Luo, Ji PLoS One Research Article 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-KRAS(G12V) 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-KRAS(G12V) 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. Public Library of Science 2014-08-05 /pmc/articles/PMC4122376/ /pubmed/25093678 http://dx.doi.org/10.1371/journal.pone.0103836 Text en © 2014 Carver et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Carver, Joseph
Dexheimer, Thomas S.
Hsu, Dennis
Weng, Meng-Tzu
Smith, Jordan L.
Guha, Rajarshi
Jadhav, Ajit
Simeonov, Anton
Luo, Ji
A High-Throughput Assay for Small Molecule Destabilizers of the KRAS Oncoprotein
title A High-Throughput Assay for Small Molecule Destabilizers of the KRAS Oncoprotein
title_full A High-Throughput Assay for Small Molecule Destabilizers of the KRAS Oncoprotein
title_fullStr A High-Throughput Assay for Small Molecule Destabilizers of the KRAS Oncoprotein
title_full_unstemmed A High-Throughput Assay for Small Molecule Destabilizers of the KRAS Oncoprotein
title_short A High-Throughput Assay for Small Molecule Destabilizers of the KRAS Oncoprotein
title_sort high-throughput assay for small molecule destabilizers of the kras oncoprotein
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4122376/
https://www.ncbi.nlm.nih.gov/pubmed/25093678
http://dx.doi.org/10.1371/journal.pone.0103836
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