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Updated: Jun 16, 2026
Bioluminescence Resonance Energy Transfer (BRET)-Based Assay for Measuring Interactions of CRAF with 14-3-3 Proteins in Live Cells
Published on: March 1, 2024
Fluorescent cascade and direct assays for characterization of RAF signaling pathway inhibitors
Kevin R Kupcho1, Rica Bruinsma, Tina M Hallis
1Invitrogen Corporation, 501 Charmany Drive, Madison, WI 53719, USA.
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.
Insights
Two new fluorescent assays distinguish RAF pathway inhibitors from direct RAF kinase inhibitors. These methods use Förster-resonance energy transfer (FRET) to analyze B-RAF, B-RAF V599E, and C-RAF activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- RAF kinases are crucial regulators of cell signaling pathways involved in growth, differentiation, and survival.
- Dysregulation of the RAF pathway presents a significant therapeutic target for various diseases.
- Developing selective inhibitors requires robust assays to differentiate pathway modulators from direct kinase inhibitors.
Purpose of the Study:
- To develop and validate novel homogeneous fluorescent assay formats.
- To differentiate between RAF pathway inhibitors and direct RAF kinase inhibitors.
- To characterize inhibitors targeting B-RAF, B-RAF V599E, and C-RAF.
Main Methods:
- Utilized Förster-resonance energy transfer (FRET) for assay development.
- Created cascade assays for RAF and MEK signaling pathways.
- Developed a direct ERK kinase assay to isolate inhibitor action.
- Employed terminally labeled peptide substrates sensitive to proteolysis.
- Implemented a time-resolved FRET assay with a fluorescently labeled MEK substrate.
Main Results:
- Successfully developed two homogeneous fluorescent assay formats.
- Demonstrated the ability to distinguish RAF pathway inhibitors from direct RAF kinase inhibitors.
- Validated assays using B-RAF, B-RAF V599E, and C-RAF.
- Cascade assays detected compounds affecting activated and unactivated kinases.
- Direct assays pinpointed the specific site of inhibitor action within the pathway.
Conclusions:
- The developed FRET-based assays provide a sensitive and specific method for profiling RAF pathway inhibitors.
- These assays are valuable tools for drug discovery and development targeting the RAF signaling pathway.
- The ability to distinguish between pathway and direct kinase inhibition facilitates the identification of more precise therapeutic agents.
