Cellular thermal shift assay (CETSA) for determining the drug binding affinity using Ba/F3 clones stably expressing receptor pseudokinases.

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.