The interaction of cells with the environment is crucial for their function within the organism. These interchanges take place at the cell membrane. Extracellular signals are translated inside the cells through cell membrane proteins. Since the protein environment can influence their binding capabilities, it is imperative to study ligand target and off-target interactions within their natural microenvironment. Therefore, it is crucial to identify the targets of a ligand of interest on the living cells.
Understanding all the targets and off-targets a given ligand binds to on a living cell is pivotal to comprehend its function. In the development of small molecules, peptides, proteins and antibodies for clinical, diagnostic and research purposes it is essential to know all their binding partners at the cell surface.
To tackle this question Frei* et al. 2012 developed the TriCEPS molecule to enable ligand-based receptor capturing (LRC-TriCEPS) and further developed it also for the use of small molecules and N-, C., O, glycosylated targets, Sobotzki et al. 2018**. This conceptually new approach identifies the targets of a ligand on living cells. The key molecule of the technology is the trifunctional molecule TriCEPS.
In a first pretest step the optimal binding conditions of the ligand to the unknown targets (time, temperature, pH, best cell type, identify co-factors) is tested by Flow-TriCEPS using flow cytometry.
Then in the second step the targets and off-targets of the user's ligand of interest are identified on living cells using the LC-MS/MS based LRC-TriCEPS or LRC-HATRIC technology platform.
In the third step the identified targets of the ligand can be confirmed by siRNA knockdown combined with Flow-TriCEPS. Test by flow cytometry if the binding of the ligand is reduced when the identified target is knocked down.
Using LRC-TriCEPS it was shown that even low-expressed receptors of weak binding ligands can be identified.
The TriCEPS and Hatric technologies are tools to perform mode of action studies for target identification on the living cells for drug candidates/ligands such as peptides, antibodies, ADC’s, proteins and small molecules. They:
- Identify the best cell type to use in a target identification experiment
- Identify the optimal binding conditions on the living cells
- Identify co-factors needed for binding to the cells of drug candidates
- Perform functional assays with Flow-TriCEPS coupled drug candidates/ligands.
- Identify the targets and off-targets
- Confirm the results
Dualystems Biotech offers these exciting new technologies in a service for fee model for industry and academia.
References:
*A. P. Frei et al., Direct identification of ligand-receptor interactions on living cells and tissues. Nat. Biotechnol. 30, 997–1001 (2012).
**N. Sobotzki et al., HATRIC-based identification of receptors for orphan ligands NATURE COMMUNICATIONS | (2018) 9:1519
