Utilisation of the InCELL Hunter™ Target Engagement Cell-Based Assay for Profiling of ULK1 Inhibitors
Drug efficacy and cellular target engagement studies have traditionally been monitored by evaluation of downstream cellular responses, such as detection of substrate phosphorylation by upstream kinases for example. Alternative biochemical techniques, based on measuring the thermal stability of a recombinant target protein in response to binding of accessory proteins or small molecules by assessing changes in the thermal shift (Tm) have been widely used in a variety of formats. More recently, this approach has been applied to the study of molecular stabilisation of target proteins expressed endogenously or exogenously in cells, animals or human samples using the Cellular Thermal Shift Assay (CETSA®). These approaches rely on melt curve analysis following interaction with drug or compound and subsequent detection of the target protein by immunoblotting. The levels of target protein detected at different temperatures can be compared between control and treated samples as a measure of target engagement.
Commercially-available assays such as the InCELL Hunter™ Target Engagement Assay (TEA; DiscoveRx® Corporation), based on Enzyme Fragment Complementation (EFC) between two inactive b-galactosidase fragments, an enhanced ProLabel® (ePL) and an Enzyme Acceptor (EA) allow the study of protein stability in live intact cells without the need to perform thermal shift studies. Here, we describe the application of this assay for the profiling of a panel of small molecule inhibitors to a key kinase in the autophagy pathway ULK1 (Unc-51 like autophagy activating kinase 1). Data output were compared to other assays measuring indirect target engagement through assessment of a downstream ULK1 substrate, Atg13 using a phospho-specific antibody and a phenotypic autophagy LC3 tandem reporter assay.