The effective binding of a ligand to a protein can be monitored by following the induced shift in thermal stability of the protein. This shift in stability is a well-known phenomenon and assays measuring such thermal shifts have been used extensively on purified proteins to detect interactions and estimate relative affinities of ligands to proteins. Here we present the implementation of high throughput screening protocol to a thermal shift assay in a cellular format (called the cellular thermal shift assay (CETSA)). The method allows studies of target engagement of drug candidates in a cellular context.
The CETSA process can be divided into three main steps; firstly, an incubation step where cells are treated with compound(s) of interest. Secondly, heating is applied to denature proteins with the assumption that protein with bound ligand will have a different melting temperature from that of the control sample. In the third step, precipitated protein is separated from soluble protein and the amount of remaining soluble target protein in the lysed cells is evaluated by means of specific antibodies.
We have studied p38α kinase as an example model system to demonstrate two separate protocols for CETSA, one approach with extensive sample workup and detection using Western blots and the second using a homogeneous AlphaScreen® SureFire™ detection format. The latter protocol has been worked out to allow an increased throughput, as putative applications require large number of samples to be handled.
To illustrate the feasibility of applying the homogeneous assay for screening purposes we have also tested how the homogeneous assay protocol responds to the presence of a small set of test compounds taken from a diversity library.