Objective

Target engagement is a critical driver of clinical efficacy, yet drug discovery suffers from a lack of technologies to measure intracellular target engagement in a physiologically relevant manner. This problem is exemplified by targets such as Androgen Receptor (AR), where recombinant protein production is challenging, and cellular effects are influenced by complex networks of signalling pathways and interactions. We developed a novel high throughput CETSA® assay (CETSA® HT) to measure AR target engagement in prostate cancer cells, where AR was thermally stabilised upon agonist binding, and stabilisation was reversed upon antagonist binding. Using this assay, we demonstrate that CETSA can exclusively identify direct AR binders where other cellular assays cannot. We also performed competition experiments using CETSA, whereby agonist and antagonist competition was determined by differential AR thermal stability to derive measures of Ki. We therefore demonstrate the first system where the label-free CETSA method can be used to determine an intracellular measure of apparent binding affinity, as quantified by target engagement in prostate cancer cells. The ability to confidently identify direct AR binders, and to derive physiologically relevant measures of intracellular binding affinity, has the potential to expedite the identification of novel AR antagonists for the treatment of prostate cancer. These observations might have broad application across early drug discovery.