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Updated: May 10, 2026
Using High Content Imaging to Quantify Target Engagement in Adherent Cells
Published on: November 29, 2018
Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay
Daniel Martinez Molina1, Rozbeh Jafari, Marina Ignatushchenko
1Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
Abstract:
The efficacy of therapeutics is dependent on a drug binding to its cognate target. Optimization of target engagement by drugs in cells is often challenging, because drug binding cannot be monitored inside cells. We have developed a method for evaluating drug binding to target proteins in cells and tissue samples. This cellular thermal shift assay (CETSA) is based on the biophysical principle of ligand-induced thermal stabilization of target proteins. Using this assay, we validated drug binding for a set of important clinical targets and monitored processes of drug transport and activation, off-target effects and drug resistance in cancer cell lines, as well as drug distribution in tissues. CETSA is likely to become a valuable tool for the validation and optimization of drug target engagement.
Insights
We developed a cellular thermal shift assay (CETSA) to monitor drug binding to target proteins within cells and tissues. This method validates drug engagement, crucial for optimizing therapeutic efficacy and understanding drug behavior in biological systems.
Area of Science:
- Biophysics
- Pharmacology
- Molecular Biology
Background:
- Drug efficacy relies on target binding, but monitoring this interaction within cells is difficult.
- Current methods lack the ability to directly assess drug-target engagement in cellular environments.
Purpose of the Study:
- To develop a novel method for evaluating drug binding to target proteins directly within cells and tissue samples.
- To validate the utility of this assay for monitoring various aspects of drug action and distribution.
Main Methods:
- Development of the cellular thermal shift assay (CETSA).
- Utilizing the principle of ligand-induced thermal stabilization of proteins.
- Application of CETSA to validate drug binding, monitor drug transport, activation, off-target effects, resistance, and tissue distribution.
Main Results:
- Successfully validated drug binding for key clinical targets using CETSA.
- Demonstrated the assay's capability to monitor drug transport, activation, and off-target effects in cancer cell lines.
- Showcased the ability of CETSA to assess drug distribution within tissue samples.
Conclusions:
- The cellular thermal shift assay (CETSA) provides a robust method for evaluating drug target engagement in cellular and tissue contexts.
- CETSA is a valuable tool for drug discovery and development, aiding in validation and optimization processes.
- This assay facilitates a deeper understanding of drug behavior, including resistance and distribution, within biological systems.
