Cellular thermal shift assay (CETSA) for determining the drug binding affinity using Ba/F3 clones stably expressing receptor pseudokinases
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Hanna Karvonen1, Juuli Raivola2, Daniela Ungureanu3
1Cancer Signaling, Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland.
Abstract:
The majority of drug screening approaches are performed using recombinant proteins, however, drug binding to its target(s) in cells should be also assessed, especially for drugs aimed at modulating intracellular signaling pathways. As a result, the development of a cellular thermal shift assay (CETSA) has become an important tool for determining the binding affinity of drugs to their intracellular targets. Cell lines, such as Ba/F3, are an excellent model system to stably express and study a target protein when this protein is not endogenously expressed or only present at low levels. Together with CETSA, Ba/F3 clones allow study of the transforming properties of the protein in question, its downstream intracellular signaling activation pathways, as well as its drug binding kinetics. This chapter describes in detail the establishment of Ba/F3 clones stably expressing receptor pseudokinases, such as receptor tyrosine kinase-like orphan receptors (ROR1, ROR2) and protein tyrosine kinase 7 (PTK7), and the use thereof to evaluate binding of small molecule inhibitors to their intracellular (pseudo)kinase domain by CETSA.
Insights
This study introduces a cellular thermal shift assay (CETSA) using Ba/F3 cell lines to assess drug binding affinity to intracellular targets, specifically receptor pseudokinases like ROR1, ROR2, and PTK7.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Drug screening often uses recombinant proteins, but intracellular target binding is crucial for pathway modulators.
- Cellular Thermal Shift Assay (CETSA) measures drug-target binding within cells.
- Ba/F3 cell lines enable stable expression of low-abundance or absent endogenous proteins for study.
Purpose of the Study:
- To detail the establishment of Ba/F3 clones expressing receptor pseudokinases (ROR1, ROR2, PTK7).
- To evaluate the binding kinetics of small molecule inhibitors to intracellular pseudokinase domains using CETSA.
- To demonstrate the utility of Ba/F3-CETSA for studying drug-target interactions in a cellular context.
Main Methods:
- Stable expression of receptor pseudokinases (ROR1, ROR2, PTK7) in Ba/F3 cells.
- Application of Cellular Thermal Shift Assay (CETSA) to assess drug binding.
- Analysis of intracellular signaling pathways and transforming properties.
Main Results:
- Successfully established Ba/F3 clones expressing specific receptor pseudokinases.
- Demonstrated the ability of CETSA to quantify small molecule inhibitor binding to intracellular pseudokinase domains.
- Validated the Ba/F3-CETSA system for drug discovery and characterization.
Conclusions:
- Ba/F3 cell lines are a robust model for studying receptor pseudokinases and their drug interactions.
- CETSA in conjunction with Ba/F3 clones provides a powerful method for assessing intracellular drug binding affinity.
- This approach facilitates the development of targeted therapeutics modulating intracellular signaling.
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