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Cellular thermal shift assay: an approach to identify and assess protein target engagement
Liying Zhang1, Yuchuan Wang1, Chang Zheng1
1Hebei Key Laboratory for Chronic Diseases, School of Basic Medical Sciences, North China University of Science and Technology, Tangshan, Hebei, China.
Introduction:
A comprehensive and global knowledge of protein target engagement is of vital importance for mechanistic studies and in drug development. Since its initial introduction, the cellular thermal shift assay (CETSA) has proven to be a reliable and flexible technique that can be widely applied to multiple contexts and has profound applications in facilitating the identification and assessment of protein target engagement.
Areas Covered:
This review introduces the principle of CETSA, elaborates on western blot-based CETSA and MS-based thermal proteome profiling (TPP) as well as the major applications and prospects of these approaches.
Expert Opinion:
CETSA primarily evaluates a given ligand binding to a particular target protein in cells and tissues with the protein thermal stabilities analyzed by western blot. When coupling mass spectrometry with CETSA, thermal proteome profiling allows simultaneous proteome-wide experiment that greatly increased the efficiency of target engagement evaluation, and serves as a promising strategy to identify protein targets and off-targets as well as protein-protein interactions to uncover the biological effects. The CETSA approaches have broad applications and potentials in drug development and clinical research.
Insights
Cellular Thermal Shift Assay (CETSA) is a key method for understanding protein target engagement in drug development. This review covers CETSA principles, western blot, and MS-based thermal proteome profiling applications.
Area of Science:
- Biochemistry
- Pharmacology
- Proteomics
Background:
- Protein target engagement knowledge is crucial for mechanistic studies and drug development.
- Cellular Thermal Shift Assay (CETSA) is a reliable and flexible technique for assessing protein target engagement.
- CETSA has broad applications in various biological contexts.
Purpose of the Study:
- To review the principle of CETSA.
- To elaborate on western blot-based CETSA and MS-based thermal proteome profiling (TPP).
- To discuss the major applications and future prospects of CETSA approaches.
Main Methods:
- Western blot-based CETSA for analyzing protein thermal stabilities.
- Mass spectrometry-based thermal proteome profiling (TPP) for proteome-wide analysis.
- Evaluation of ligand binding to target proteins in cells and tissues.
Main Results:
- CETSA effectively evaluates ligand binding and target engagement.
- TPP enables simultaneous, proteome-wide target engagement evaluation, increasing efficiency.
- CETSA approaches facilitate the identification of protein targets, off-targets, and protein-protein interactions.
Conclusions:
- CETSA and TPP are powerful tools for drug development and clinical research.
- These methods offer profound applications in identifying and assessing protein target engagement.
- CETSA approaches have significant potential for uncovering biological effects and guiding therapeutic strategies.
