The group of Professor Bernd Wollscheid from ETH Zurich showed in their latest publication that HATRIC-LRC (HATRIC Ligand Receptor Capture) enables the identification of the targets of a small molecule at the cell membrane of living cells to elucidate the mode of action of the molecule. Further, it was demonstrated that the HATRIC-LRC technology platform needs less cells for target identification than the previously published LRC-TriCEPS method and it enables the identification of N-, C-, and O- glycosylated targets.
Target identification using HATRIC-LRC is based on the trifunctional cross linker HATRIC that contains an NHS ester to couple the small molecule, a hydrazone to covalently bind to the carbohydrates of the unknown target proteins at the cell membrane and an azide as pull down function.
The small molecule is coupled to HATRIC using a primary amine. Either your small molecule contains already a primary amine or a primary amine is added by derivatization. In the next step cells expressing the unknown targets and off-targets are mildly oxidized and the HATRIC coupled small molecule is added. The small molecule binds its target(s) at the cell membrane and the second arm of HATRIC covalently binds to the carbohydrates of the target protein(s). During that interaction the cells are still alive. Then the cells are lysed, processed and the third arm of HATRIC is used to pull down the target proteins. Proteins are identified through bottom-up proteomics, relatively quantified and compared to one or several controls. The target candidates are those proteins that are enriched in the ligand of interest samples compared to the control treated samples since the ligand target interaction increases the likelihood of the target carbohydrates to covalently bind to HATRIC compared to the average cell surface protein. Controls can be a positive control such as transferrin or a negative control for example samples treated with an excess of the small molecule devoid of HATRIC prior to adding the HATRIC small molecule conjugate (competition).
Link to Samedan Ltd. Pharmaceutical Publishers