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Updated: Oct 26, 2025
Using High Content Imaging to Quantify Target Engagement in Adherent Cells
Published on: November 29, 2018
Sensitive Measurement of Drug-Target Engagement by a Cellular Thermal Shift Assay with Multiplex Proximity Extension
Rasel A Al-Amin1, Caroline J Gallant1, Phathutshedzo M Muthelo1
1Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala SE-751 08, Sweden.
Abstract:
The ability to monitor target engagement in cellular contexts is a key for successful drug discovery and also valuable in clinical routine. A cellular thermal shift assay (CETSA) provides realistic information about drug binding in cells and tissues, revealing drug-target engagement in clinically relevant samples. The CETSA combined with mass spectrometry (MS) detection can be applied in the early hit identification phase to generate target engagement data for large sets of proteins. However, the analysis is slow, requires substantial amounts of the sample material, and often misses proteins of specific interest. Here, we combined the CETSA and the multiplex proximity extension assay (PEA) for analysis of target engagement of a set of 67 proteins from small amounts of the sample material treated with kinase inhibitors. The results were concordant with the corresponding analyses read out via MS. Our approach allows analyses of large numbers of specific target proteins at high sensitivity in limited sample aliquots. Highly sensitive multiplex CETSA-PEA assays are therefore promising for monitoring drug-target engagement in small sample aliquots in the course of drug development and potentially in clinical settings.
Insights
We developed a multiplex assay combining cellular thermal shift assay (CETSA) and proximity extension assay (PEA) for drug target engagement. This method efficiently analyzes many specific proteins from small samples with high sensitivity.
Area of Science:
- Pharmacology
- Biochemistry
- Assay Development
Background:
- Monitoring drug-target engagement is crucial for drug discovery and clinical applications.
- Cellular thermal shift assay (CETSA) provides insights into drug binding in cells and tissues.
- Traditional CETSA with mass spectrometry (MS) is time-consuming and requires large sample volumes.
Purpose of the Study:
- To develop a sensitive and efficient method for analyzing drug-target engagement.
- To overcome limitations of traditional CETSA, such as speed and sample requirements.
- To enable multiplexed analysis of specific protein targets from limited sample amounts.
Main Methods:
- Combined cellular thermal shift assay (CETSA) with multiplex proximity extension assay (PEA).
- Analyzed target engagement for 67 proteins using kinase inhibitors.
- Utilized small sample amounts for the multiplexed assay.
Main Results:
- Achieved concordant results compared to traditional CETSA with MS detection.
- Demonstrated high sensitivity in analyzing specific target proteins.
- Enabled analysis of numerous proteins from limited sample aliquots.
Conclusions:
- The multiplex CETSA-PEA assay is a sensitive and efficient method for monitoring drug-target engagement.
- This approach is suitable for analyzing small sample volumes in drug development.
- The assay holds promise for clinical applications requiring precise target engagement monitoring.
