Objective

The aim of this work was to develop a cell-based target engagement assay for our target PI5P4K Gamma, a lipid kinase implicated in autophagy and a potential neurodegeneration target. The assay was based on the InCELL Pulse enzyme fragment complementation system developed by DiscoverX. Following the steps set out by DiscoverX, activity of four constructs generated with the reporter fused on the target at four locations was confirmed, and expression confirmed by western blot. The next phase involved performing thermal shifts on the constructs transfected into cells to determine optimal temperatures for the third phase, from this data one construct was selected for the next phase. The final phase generated concentration-response curves of compounds in isothermal conditions determined in the previous phase. Known inhibitors of the target showed stabilisation of the construct while a negative control showed no stabilisation at a constant temperature. Through this work, we successfully developed a cellular target engagement assay which will generate informative data on target engagement and cell penetrance of compounds.