Senior Scientist Pelago Bioscience AB, Stockholms Lan, Sweden
Validating target engagement of compounds utilizing a high-throughput CETSA® approach can generate valuable leads, discover novel chemistry and drive SAR with the benefit that hits are on the endogenous target within a physiological environment. The development of high-throughput CETSA® assays can be expediated by employing ‘off-the-shelf’ AlphaLISA® SureFire® Ultra kits for detection in a 384-well assay format. Previously, setting up optimal assay conditions for these kits has been limited to single or few targets. This study was conceived to tackle a large-scale kit validation approach, allowing for rapid assessment of many targets investigating whether the proteins are detectable, meltable and shiftable using commercially available tool molecules. The resultant findings allow for a wide overview of which target classes, proteins and tools are amenable ‘off the shelf’ for target engagement assessment. We first set out to determine the optimal cell type(s) in which most targets are expressed. Therefore, all targets were cross-referenced with their expression levels in all available cell lines of the Human Protein Atlas. This resulted in the selection of two cell types, THP1 and U2OS cells, and 46 intracellular proteins to be examined in both cell types in six-point temperature melting curves. The selected proteins belong to diverse classes and are localized in different cellular compartments. 41 proteins demonstrated good detectability and qualified for full twelve-point temperature melt curves in a single cell line. Having pinpointed the optimal melting range, the proteins were examined for their ability to shift upon target engagement with up to three commercially available tool compounds. A thermal shift was observed for most targets using standard conditions, and a few selected examples were further followed-up with lysate experiments and thermal proteome profiling.
In summary, we demonstrate an approach that allows for rapid validation of large batches of AlphaLISA® SureFire® kits to test individual proteins for their ability to be detected, melted and shifted in high-throughput CETSA®. Further, the newly validated kits were employed to focus on a few selected proteins illustrating the effects of compounds on pathways, and follow-up of selected compounds via thermal proteome profiling.
