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There is both theoretical and therapeutic interest in establishing whether the signals conveyed by the enkephalins are turned off under the action of a specific peptidase which might, in this case, represent a target for a new class of psychoactive agents. Enkephalinase, a dipeptidyl carboxypeptidase cleaving the Gly3-Phe4 bond of enkephalins and distinct fropm angiotensin coverting enzyme (ACE), might be selectively involved in enkephalinergic transmission. It is a membrane-bound enzyme whose localization in the vicinity of opiate receptors in the central nervous system is suggested by parallel regional and subcellular distributions as well as by the effects of lesions. Such a role is further supported by the ontogenetic development of enkephalinase, its substrate specificity accounting for the increased biological activity of several enkephalin analogues and its adaptive increase following chronic treatment with morphine. To investigate the functional role of this enzyme further, we have designed a potent and specific enkephalinase inhibitor. We report here that this compound, thiorphan [(DL-3-mercapto-2-benzylpropanoyl)-glycine; patent no. 8008601] protects the enkephalins from the action of enkephalinase in vitro in nanomolar concentration and in vivo after either intracerebroventricular or systemic administration. In addition, thiorphan itself displays antinociceptive activity which is blocked by naloxone, an antagonist of opiate receptors.

The main properties and localisation of enkephalin-dipeptidylcarboxypeptidase (“enkephalinase”) a membrane-bound enzyme cleaving the enkephalins at the Gly3-Phe4 amide bond are described. Its involvement in the inactivation of synaptically-released enkephalins is discussed in the light of criteria proposed to identify “neuropeptidases”.

Fluorescent dansylated derivatives of enkephalins were prepared, with the dansyl group either at the N- or C-terminal part of methionine-enkephalin (Met-E) or its peptidase-resistant analogue D-Ala2-Met-E. An energy transfer, from the Tyr to the dansyl group, was found for all compounds and spectral properties were dependent upon the environment polarity. Met-E-(CH2)2 dansyl II and D-Ala2-Met-E-(CH2)2 dansyl III exhibit biological activities on guinea-pig ileum and binding affinities on striatal membranes, similar to Met-E. III exhibits significant analgesic activity in mice after i.v. administration (about 35 % that of morphine). Fluorescent enkephalins potentially represent useful probes for the exploration of opiate receptors, studies of ligand-receptor interactions and evaluation of enkephalins cleaving peptidases in various tissues.