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Updated: Apr 6, 2026
Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays
Published on: September 19, 2018
Multicolored, Tb³⁺-Based Antibody-Free Detection of Multiple Tyrosine Kinase Activities
Andrew M Lipchik1, Minervo Perez1, Wei Cui1
1Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy and Purdue Center for Cancer Research, Purdue University, 201 S. University Street, West Lafayette, Indiana 47907, United States.
Abstract:
Kinase signaling is a major mechanism driving many cancers. While many inhibitors have been developed and are employed in the clinic, resistance due to crosstalk and pathway reprogramming is an emerging problem. High-throughput assays to detect multiple pathway kinases simultaneously could better model these complex relationships and enable drug development to combat this type of resistance. We developed a strategy to take advantage of time-resolved luminescence of Tb(3+)-chelated phosphotyrosine-containing peptides, which facilitated efficient energy transfer to small molecule fluorophores conjugated to the peptides to produce orthogonally colored biosensors for two different kinases. This enabled multiplexed detection with high signal-to-noise in a high-throughput-compatible format. This proof-of-concept study provides a platform that could be applied to other lanthanide metal and fluorophore combinations to achieve even greater multiplexing without the need for phosphospecific antibodies.
Insights
Developing novel biosensors for kinase signaling pathways can combat cancer drug resistance. This new method allows simultaneous detection of multiple kinases, improving drug development for complex cancers.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Kinase signaling pathways are crucial in cancer development and progression.
- Drug resistance, often caused by pathway crosstalk and reprogramming, poses a significant challenge in cancer therapy.
- Current methods for monitoring kinase activity may not fully capture complex signaling networks.
Purpose of the Study:
- To develop a high-throughput assay for simultaneous detection of multiple kinase activities.
- To create a novel biosensor platform for studying kinase signaling in cancer.
- To provide a tool for developing drugs that overcome resistance mechanisms.
Main Methods:
- Utilized time-resolved luminescence of terbium(Tb(3+))-chelated phosphotyrosine-containing peptides.
- Engineered biosensors for two different kinases using energy transfer to fluorophores.
- Achieved multiplexed detection in a high-throughput-compatible format.
Main Results:
- Demonstrated multiplexed detection of kinases with high signal-to-noise ratio.
- Developed orthogonally colored biosensors for simultaneous kinase activity monitoring.
- Established a proof-of-concept for a versatile biosensing platform.
Conclusions:
- The developed biosensor platform enables high-throughput, multiplexed detection of kinase signaling.
- This approach offers a strategy to combat drug resistance by modeling complex pathway reprogramming.
- The platform can be extended with different lanthanide metals and fluorophores for enhanced multiplexing capabilities.
