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Journal of Biomolecular Screening
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What's this?

Identification of Inhibitors of the Kinase Activity of Oncogenic V600EBRAF in an Enzyme Cascade High-Throughput Screen

Yvette Newbatt

Cancer Research UK Centre for Cancer Therapeutics, Haddow Laboratories, The Institute of Cancer Research, Sutton, UK

Samantha Burns

Cancer Research UK Centre for Cancer Therapeutics, Haddow Laboratories, The Institute of Cancer Research, Sutton, UK

Robert Hayward

Cancer Research UK Centre for Cell and Molecular Biology, Chester Beatty Laboratories, The Institute of Cancer Research, London, UK

Steven Whittaker

Cancer Research UK Centre for Cell and Molecular Biology, Chester Beatty Laboratories, The Institute of Cancer Research, London, UK

Ruth Kirk

Cancer Research UK Centre for Cell and Molecular Biology, Chester Beatty Laboratories, The Institute of Cancer Research, London, UK

Christopher Marshall

Cancer Research UK Centre for Cell and Molecular Biology, Chester Beatty Laboratories, The Institute of Cancer Research, London, UK

Caroline Springer

Cancer Research UK Centre for Cancer Therapeutics, Haddow Laboratories, The Institute of Cancer Research, Sutton, UK

Edward Mcdonald

Cancer Research UK Centre for Cancer Therapeutics, Haddow Laboratories, The Institute of Cancer Research, Sutton, UK

Cancer Genome Project

Richard Marais

Cancer Research UK Centre for Cell and Molecular Biology, Chester Beatty Laboratories, The Institute of Cancer Research, London, UK

Paul Workman

Cancer Research UK Centre for Cancer Therapeutics, Haddow Laboratories, The Institute of Cancer Research, Sutton, UK

Wynne Aherne

Cancer Research UK Centre for Cancer Therapeutics, Haddow Laboratories, The Institute of Cancer Research, Sutton, UK

The Cancer Genome Project has identified several oncogenic mutations in BRAF that represent important opportunities for cancer drug discovery. The V600EBRAF mutation accounts for approximately 90% of the mutations identified. A strong case has emerged from molecular, cellular, and structural studies for the identification and development of inhibitors of this mutated BRAF protein. The authors have developed and run a high-throughput screen to find inhibitors of V600EBRAF using an enzyme cascade assay in which oncogenic BRAF activates MEK1, which in turn activates ERK2, which then phosphorylates the transcription factor ELK1. A phosphospecific antibody, Europium-labeled secondary antibody, and a time-resolved fluorescent readout were used to measure phosphorylation of ELK1. Overall assay variation was 12.4%. The assay was used to screen 64,000 compounds with an overall Z' factor of 0.58 ± 0.12. A series of 3,5, di-substituted pyridines were identified as inhibitors of the cascade assay. These compounds did not inhibit a shortened activated MEK1 to ELK1 cascade but were active (0.5-27.9 µM) in a V600EBRAF assay and represent a potential starting point for future drug discovery and development.

Key Words: cancer • V600EBRAF • kinase inhibitors • DELFIA • high-throughput screen

This version was published on March 1, 2006

Journal of Biomolecular Screening, Vol. 11, No. 2, 145-154 (2006)
DOI: 10.1177/1087057105283584
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