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Multiplexing terbium- and europium-based TR-FRET readouts to increase kinase assay capacity
Robert A Horton1, Kurt W Vogel
1Life Technologies Corporation, Madison, WI 53719, USA.
Abstract:
Identification and characterization of kinase inhibitor potency and selectivity is often an iterative process in which a library of compounds is first screened against a single kinase, and hits from that screen are then profiled against other kinases to determine specificity. By developing kinase assays that employ either a terbium- or a europium-based time-resolved fluorescence resonance energy transfer (TR-FRET) readout, one can take advantage of the distinct emission properties of these labels to develop assays for 2 kinases that can be performed simultaneously in the same well. This not only increases the information content provided per assay well but can immediately provide information on compound specificity. The authors have applied this strategy to the development of multiplexed assays for 2 examples systems: EGFR and IKKbeta, as well as lipid kinase family members mTOR and PIK3C3. They demonstrate the ability of these multiplexed assays to characterize selective kinase inhibitors in a dose-response mode, with no difference in results obtained from traditional single kinase assays performed separately.
Insights
Multiplexed kinase assays using time-resolved fluorescence resonance energy transfer (TR-FRET) enable simultaneous profiling of two kinases. This approach efficiently characterizes kinase inhibitor potency and selectivity, streamlining drug discovery.
Area of Science:
- Biochemistry
- Pharmacology
- Drug Discovery
Background:
- Kinase inhibitor profiling is typically iterative, involving single-kinase screening followed by specificity testing.
- Current methods can be time-consuming and resource-intensive.
Purpose of the Study:
- To develop and validate multiplexed kinase assays for simultaneous profiling of two kinases in a single well.
- To enhance the efficiency of characterizing kinase inhibitor potency and selectivity.
Main Methods:
- Utilized terbium- and europium-based time-resolved fluorescence resonance energy transfer (TR-FRET) assays.
- Developed multiplexed assays for kinase pairs: EGFR/IKKbeta and mTOR/PIK3C3.
- Performed dose-response analyses to assess inhibitor characterization.
Main Results:
- Successfully demonstrated simultaneous assaying of two kinases per well.
- Achieved comparable results to traditional single-kinase assays.
- Validated the ability to characterize selective kinase inhibitors efficiently.
Conclusions:
- Multiplexed TR-FRET assays offer a powerful strategy to increase assay information content and immediately assess compound specificity.
- This method streamlines the iterative process of kinase inhibitor identification and characterization.
