Objective

Indoleamine 2’3'-dioxygenase (IDO1) is upregulated during tumour development, providing cancer cells with a route to evade the immune system. Here we use inhibitors of this promising immuno-oncology target as an illustrative example to demonstrate a robust paradigm for hit profiling. Commercially available IDO1 inhibitor screening kits assessed compound effects on the activity of purified human IDO1 and IDO1 in SKOV-3 carcinoma cells. The Fluorescent Thermal Shift Assay monitored the effect of compounds on the thermal stability of purified IDO1 whilst a Cellular Thermal Shift Assay (CETSA^®) monitored cellular target engagement. We show qualitative agreement between a tailored set of biophysical, biochemical and cellular techniques with rank order of compounds matching those observed in the literature. Differences in inhibitor potency were observed across assays with some compounds significantly more potent in cells than in in vitro IDO1 enzyme assays. These data highlight that although correlating target engagement and efficacy adds value to hit identification, to fully understand a compound’s pharmacology a tailored screening cascade is required.