title: Identification of inhibitors of the kinase activity of oncogenic V600E BRAF in an enzyme cascade high-throughput screen.
abstract: The Cancer Genome Project has identified several oncogenic mutations in BRAF that represent important opportunities for cancer drug discovery. The V600E BRAF 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 V600E BRAF 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 microM) in a V600E BRAF assay and represent a potential starting point for future drug discovery and development.
authors.affiliation: Cancer Research UK Centre for Cancer Therapeutics, Haddow Laboratories, The Institute of Cancer Research, Sutton, UK.
authors.forename: Yvette
authors.initials: Y
authors.lastname: Newbatt
authors.forename: Samantha
authors.initials: S
authors.lastname: Burns
authors.forename: Robert
authors.initials: R
authors.lastname: Hayward
authors.forename: Steven
authors.initials: S
authors.lastname: Whittaker
authors.forename: Ruth
authors.initials: R
authors.lastname: Kirk
authors.forename: Christopher
authors.initials: C
authors.lastname: Marshall
authors.forename: Caroline
authors.initials: C
authors.lastname: Springer
authors.forename: Edward
authors.initials: E
authors.lastname: McDonald
authors.forename: Richard
authors.initials: R
authors.lastname: Marais
authors.forename: Paul
authors.initials: P
authors.lastname: Workman
authors.forename: Wynne
authors.initials: W
authors.lastname: Aherne
mesh_terms: D004353:Drug Evaluation, Preclinical; D048369:MAP Kinase Kinase 1 / Q000378:metabolism*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000737:chemistry*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics* / Q000378:metabolism; D011725:Pyridines; D015398:Signal Transduction; D013379:Substrate Specificity; D050807:ets-Domain Protein Elk-1 / Q000378:metabolism*
chemical_list: D047428:Protein Kinase Inhibitors; D011725:Pyridines; D050807:ets-Domain Protein Elk-1; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D048369:MAP Kinase Kinase 1