Irreversible photolabeling of active site of neutral endopeptidase- 24.11 "enkephalinase" by azidothiorphan and [14C]-azidothiorphan

A Beaumont, JF Hernandez, P Chaillet, P Crine and BP Roques

Departement de Chimie Organique, U 266 INSERM, UA 498 CNRS, Faculte de Pharmacie, Paris, France.

Azidothiorphan and its [14C]-labeled analogue have been developed as photoaffinity ligands for the active site of the neutral endopeptidase 24.11. In in vitro assays azidothiorphan inhibits the endopeptidase activity with a Ki of 0.75 nM. After ultraviolet irradiation the inhibitor binds irreversibly to the enzyme, and many factors suggest that the photolabeling occurs at the active site. The binding is accompanied by a loss of enzymatic activity, and the inclusion of the competitive inhibitor thiorphan protects the endopeptidase from this inactivation. In addition the binding of another competitive inhibitor [3H]N-[(R,S)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl]-glycine to the active site of endopeptidase-24.11 is inhibited after irradiation with azidothiorphan. Experiments with [14C]-azidothiorphan have shown that very little nonspecific binding of inhibitor to enzyme occurs and the the labeled probe remains bound under denaturing conditions. Azidothiorphan has also been found to produce a long-lasting naloxone- reversible analgesia after intracerebroventricular administration. The results show that azidothiorphan should prove useful both for structural studies and for investigations on the synthesis and turnover of the neutral endopeptidase-24.11.

Volume 32, Issue 5, pp. 594-599, 11/01/1987
Copyright © 1987 by American Society for Pharmacology and Experimental Therapeutics