Leiden University Scholarly Publications

Yang, X.; Michiels, T.J.M.; Jong, C. de; Soethoudt, M.; Dekker, N.; Gordon, E.; ... ; IJzerman, A.P. (2018)

An Affinity-Based Probe for the Human Adenosine A(2A) Receptor

Article / Letter to editor

Using activity-based protein profiling (ABPP), functional proteins can be interrogated in their native environment. Despite their pharmaceutical relevance, G protein-coupled receptors (GPCRs) have been difficult to address through ABPP. In the current study, we took the prototypical human adenosine A 2A receptor (hA(2A)R) as the starting point for the construction of a chemical toolbox allowing two-step affinity-based labeling of GPCRs. First, we equipped an irreversibly binding hA(2A)R ligand with a terminal alkyne to serve as probe. We showed that our probe irreversibly and concentration-dependently labeled purified hA(2A)R Click-ligation with a sulfonated cyanine-3 fluorophore allowed us to visualize the receptor on SDS-PAGE. We further demonstrated that labeling of the purified hA(2A)R by our probe could be inhibited by selective antagonists. Lastly, we showed successful labeling of the receptor in cell membranes overexpressing hA(2A)R, making our probe a promising affinity...Show moreUsing activity-based protein profiling (ABPP), functional proteins can be interrogated in their native environment. Despite their pharmaceutical relevance, G protein-coupled receptors (GPCRs) have been difficult to address through ABPP. In the current study, we took the prototypical human adenosine A 2A receptor (hA(2A)R) as the starting point for the construction of a chemical toolbox allowing two-step affinity-based labeling of GPCRs. First, we equipped an irreversibly binding hA(2A)R ligand with a terminal alkyne to serve as probe. We showed that our probe irreversibly and concentration-dependently labeled purified hA(2A)R Click-ligation with a sulfonated cyanine-3 fluorophore allowed us to visualize the receptor on SDS-PAGE. We further demonstrated that labeling of the purified hA(2A)R by our probe could be inhibited by selective antagonists. Lastly, we showed successful labeling of the receptor in cell membranes overexpressing hA(2A)R, making our probe a promising affinity-based tool compound that sets the stage for the further development of probes for GPCRs.Show less

All authors

Yang, X.; Michiels, T.J.M.; Jong, C. de; Soethoudt, M.; Dekker, N.; Gordon, E.; Stelt, M. van der; Heitman, L.H.; Es, D. van der; IJzerman, A.P.

Date

2018-09-13

Journal

Journal of Medicinal Chemistry

Volume

61

Issue

17

Pages

7892 - 7901

DOI

doi:10.1021/acs.jmedchem.8b00860