Current Chemical Genomics and Translational Medicine

2008, 1 : 43-53
Published online 2008 February 25. DOI: 10.2174/1875397300801010043
Publisher ID: CCGTM-1-43

RESEARCH ARTICLE
 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 and Kristin G Huwiler, *
Invitrogen Corporation, 501 Charmany Drive, Madison, WI 53719, USA

* Address correspondence to this author at the Invitrogen Corporation, 501 Charmany Drive, Madison, WI 53719, USA; Tel: 608-204-5428; Fax: 608-204-5433; E-mail: kristin.huwiler@invitrogen.com

ABSTRACT

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.

Keywords:

Cascade assay, Direct Assay, C-RAF, B-RAF, B-RAF V599E, MEK1, ERK2, FRET, TR-FRET.