A cell-based radioligand binding assay for farnesyl: protein transferase inhibitors
Robert B Lobell1, Joseph P Davide, Nancy E Kohl
1Department of Cancer Research, West Point, PA 19486, USA. rob_lobell@merck.com
Abstract:
Farnesyl:protein transferase (FPTase) catalyzes the covalent addition of the isoprenyl moiety of farnesylpyrophosphate to the C-terminus of the Ras oncoprotein and other cellular proteins. Inhibitors of FPTase (FTIs) have been developed as potential anticancer agents, and several compounds have been evaluated in clinical trials. To facilitate the identification of cell-active FTIs with high potency, the authors developed a method that uses a radiolabeled FTI that serves as a ligand in competitive displacement assays. Using high-affinity [(3)H]-labeled or [(125)I]-labeled FTI radioligands, they show that specific binding to FPTase can be detected in intact cells. Binding of these labeled FTI radioligands can be competed with a variety of structurally diverse FTIs, and the authors show that inhibition of FTI radioligand binding correlates well with inhibition of FPTase substrate prenylation in cells. This method provides a rapid and quantitative means of assessing FTI potency in cells and is useful for guiding the discovery of potent, novel inhibitors of FPTase. Similar methods could be employed in the optimization of inhibitors for other intracellular drug targets.
Insights
Researchers developed a new method using radiolabeled inhibitors to measure the potency of farnesyltransferase inhibitors (FTIs) in intact cells. This technique aids in discovering effective anticancer agents targeting farnesyltransferase (FPTase).
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Farnesyltransferase (FPTase) is crucial for prenylating proteins like Ras, making it a target for anticancer drug development.
- Farnesyltransferase inhibitors (FTIs) are investigated as potential anticancer agents, necessitating methods to assess their cell-based potency.
- Existing methods for evaluating FTIs may not fully capture their activity within intact cellular environments.
Purpose of the Study:
- To develop a novel, cell-based assay for quantifying the potency of farnesyltransferase inhibitors (FTIs).
- To utilize radiolabeled FTIs as ligands for competitive binding assays in intact cells.
- To establish a reliable method for identifying and optimizing potent FTI anticancer agents.
Main Methods:
- Development of radiolabeled farnesyltransferase inhibitors (FTIs) using isotopes like tritium ((3)H) and iodine-125 ((125)I).
- Application of competitive displacement assays using these radioligands in intact cells to detect specific binding to FPTase.
- Correlation of FTI radioligand binding inhibition with the inhibition of FPTase substrate prenylation within cells.
Main Results:
- Demonstrated specific binding of radiolabeled FTIs to FPTase in intact cells.
- Showed that diverse FTIs can compete with radioligand binding, confirming assay specificity.
- Established a strong correlation between FTI radioligand binding inhibition and cellular FPTase activity inhibition.
Conclusions:
- The developed radioligand binding assay provides a rapid and quantitative method for assessing FTI potency in cell-based systems.
- This assay is valuable for guiding the discovery and optimization of novel, potent farnesyltransferase inhibitors for cancer therapy.
- The methodology can potentially be adapted for optimizing inhibitors targeting other intracellular enzymes.
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