Related Experiment Video
Updated: Aug 2, 2026
Using High Content Imaging to Quantify Target Engagement in Adherent Cells
Published on: November 29, 2018
Drug Target Engagement Using Coupled Cellular Thermal Shift Assay-Acoustic Reverse-Phase Protein Array
Adrien Herledan1, Marine Andres1,2, Aurore Lejeune-Dodge3
1University of Lille, Inserm, Institut Pasteur de Lille, U1177-Drugs and Molecules for Living Systems, Lille, France.
Abstract:
In the last 5 years, cellular thermal shift assay (CETSA), a technology based on ligand-induced changes in protein thermal stability, has been increasingly used in drug discovery to address the fundamental question of whether drug candidates engage their intended target in a biologically relevant setting. To analyze lysates from cells submitted to increasing temperature, the detection and quantification of the remaining soluble protein can be achieved using quantitative mass spectrometry, Western blotting, or AlphaScreen techniques. Still, these approaches can be time- and cell-consuming. To cope with limitations of throughput and protein amount requirements, we developed a new coupled assay combining the advantages of a nanoacoustic transfer system and reverse-phase protein array technology within CETSA experiments. We validated the technology to assess engagement of inhibitors of insulin-degrading enzyme (IDE), an enzyme involved in diabetes and Alzheimer's disease. CETSA-acoustic reverse-phase protein array (CETSA-aRPPA) allows simultaneous analysis of many conditions and drug-target engagement with a small sample size, in a rapid, cost-effective, and biological material-saving manner.
Insights
A new assay combines nanoacoustics and protein arrays for faster, cost-effective drug target engagement analysis. This cellular thermal shift assay (CETSA) method requires fewer cells and resources, accelerating drug discovery.
Area of Science:
- Biochemistry
- Pharmacology
- Biotechnology
Background:
- Cellular thermal shift assay (CETSA) measures drug-target engagement by detecting ligand-induced protein thermal stability changes.
- Traditional CETSA analysis methods (mass spectrometry, Western blotting, AlphaScreen) are often time-consuming and require substantial sample amounts.
- Limitations in throughput and protein requirements hinder the widespread application of CETSA in drug discovery.
Purpose of the Study:
- To develop a novel, high-throughput, and resource-efficient method for analyzing drug-target engagement using CETSA.
- To overcome the limitations of existing CETSA detection techniques regarding time, cell usage, and sample quantity.
- To validate a new coupled assay for assessing the engagement of drug candidates with their intended targets in a cellular context.
Main Methods:
- Development of a coupled assay integrating a nanoacoustic transfer system with reverse-phase protein array (RPPA) technology for CETSA.
- Application of the CETSA-acoustic reverse-phase protein array (CETSA-aRPPA) to analyze protein thermal stability changes in response to drug treatment.
- Validation of the CETSA-aRPPA technology using inhibitors targeting insulin-degrading enzyme (IDE), a key enzyme in diabetes and Alzheimer's disease.
Main Results:
- The developed CETSA-aRPPA enables simultaneous analysis of numerous conditions and drug-target interactions.
- The assay significantly reduces the required sample size compared to conventional methods.
- Demonstrated rapid, cost-effective, and biological material-saving analysis of drug-target engagement.
Conclusions:
- CETSA-aRPPA offers a significant advancement in drug discovery by enhancing the efficiency and reducing the resource demands of target engagement studies.
- This novel technology facilitates rapid assessment of drug candidates' interaction with their targets in a biologically relevant manner.
- The CETSA-aRPPA method holds promise for accelerating the identification and optimization of novel therapeutics for diseases like diabetes and Alzheimer's.
