N-acyl-N-alkyl sulfonamide probes for ligand-directed covalent labeling of GPCRs: the adenosine A2B receptor as case study

Beerkens, B.L.H.; Andrianopoulou, V.; Wang, X.; Liu, R.; Westen, G.J.P. van; Jespers, W.; IJzerman, A.P.; Heitman, L.H.; Es, D. van der

doi:doi:10.1021/acschembio.4c00210

Small molecular tool compounds play an essential role in the study of G protein-coupled receptors (GPCRs). However, tool compounds most often occupy the orthosteric binding site, hampering the study of GPCRs upon ligand binding. To overcome this problem, ligand-directed labeling techniques have been developed that leave a reporter group covalently bound to the GPCR, while allowing subsequent orthosteric ligands to bind. In this work, we applied such a labeling strategy to the adenosine A(2B) receptor (A(2B)AR). We have synthetically implemented the recently reported N-acyl-N-alkyl sulfonamide (NASA) warhead into a previously developed ligand and show that the binding of the A(2B)AR is not restricted by NASA incorporation. Furthermore, we have investigated ligand-directed labeling of the A(2B)AR using SDS-PAGE, flow cytometric, and mass spectrometry techniques. We have found one of the synthesized probes to specifically label the A(2B)AR, although detection was hindered by...Show moreSmall molecular tool compounds play an essential role in the study of G protein-coupled receptors (GPCRs). However, tool compounds most often occupy the orthosteric binding site, hampering the study of GPCRs upon ligand binding. To overcome this problem, ligand-directed labeling techniques have been developed that leave a reporter group covalently bound to the GPCR, while allowing subsequent orthosteric ligands to bind. In this work, we applied such a labeling strategy to the adenosine A(2B) receptor (A(2B)AR). We have synthetically implemented the recently reported N-acyl-N-alkyl sulfonamide (NASA) warhead into a previously developed ligand and show that the binding of the A(2B)AR is not restricted by NASA incorporation. Furthermore, we have investigated ligand-directed labeling of the A(2B)AR using SDS-PAGE, flow cytometric, and mass spectrometry techniques. We have found one of the synthesized probes to specifically label the A(2B)AR, although detection was hindered by nonspecific protein labeling most likely due to the intrinsic reactivity of the NASA warhead. Altogether, this work aids the future development of ligand-directed probes for the detection of GPCRs.Show less

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N-acyl-N-alkyl sulfonamide probes for ligand-directed covalent labeling of GPCRs: the adenosine A2B receptor as case study