Fluorescent Cascade and Direct Assays for Characterization of RAF Signaling Pathway Inhibitors
Kevin R Kupcho, Rica Bruinsma, Tina M Hallis, David A Lasky, Richard L Somberg, Tammy Turek-Etienne, Kurt W Vogel, Kristin G Huwiler*
Identifiers and Pagination:Year: 2008
First Page: 43
Last Page: 53
Publisher Id: CCGTM-1-43
Article History:Received Date: 03/1/2008
Revision Received Date: 19/1/2008
Acceptance Date: 23/1/2008
Electronic publication date: 25/2/2008
Collection year: 2008
open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.5/), which permits unrestrictive use, distribution, and reproduction in any medium, provided the original work is properly cited.
RAF kinases are part of a conserved signaling pathway that impacts cell growth, differentiation, and survival, and RAF pathway dysregulation is an attractive target for therapeutic intervention. We describe two homogeneous fluorescent formats that distinguish RAF pathway inhibitors from direct RAF kinase inhibitors, using B-RAF, B-RAF V599E, and C-RAF. A Förster-resonance energy transfer (FRET) based method was used to develop RAF and MEK cascade assays as well as a direct ERK kinase assay. This method uses a peptide substrate, that is terminally labeled with a FRET-pair of fluorophores, and that is more sensitive to proteolysis relative to the phosphorylated peptide. A second time-resolved FRET-based assay using fluorescently labeled MEK substrate was used to detect direct inhibitors of RAF kinase activity. The cascade assays detect compounds that interact with activated and unactivated kinases within the recapitulated RAF pathway, and the direct assays isolate the point of action for an inhibitor.