Specific nonpeptide photoprobes as tools for the structural study of the angiotensin II AT(1) receptor

The aim of this work was to obtain photoactivatable nonpeptide antagonists of the angiotensin II AT(1) receptor, Based on structure-function relations hips, two chemical structures as well as appropriate synthetic schemes were chosen as a frame for the design of radiolabeled azido probes, The feasibi lity of the strategy was first assessed by the synthesis of two tritiated l igands 21 and 22 possessing a high affinity for the AT(1) receptor and a lo w nonspecific binding to membrane or cell preparations. We then prepared tw o unlabeled azido derivatives 7 and 14 which retained a fairly high affinit y for the AT(1) receptor. The latter compound proved to be suitable for rec eptor irreversible labeling and was prepared in its tritiated form 28. This tritiated azido nonpeptide probe displayed a K-d value of 11.8 nM and a la w nonspecific binding. It was suitable for specific and efficient covalent labeling of the recombinant AT(1A) receptor stably expressed in CHO cells. The electrophoretic pattern of the specifically labeled entity was strictly identical to that of purified receptor photolabeled with a biotinylated pe ptidic photoactivatable probe. This new tool should be useful for the mappi ng of the nonpeptide receptor binding site. These potential applications ar e discussed in light of the current knowledge of molecular mechanisms of G- protein coupled receptor activation and inactivation.