Unit: Pharmacology of Neuro-Endocrine Regulations

Director: Rougeot Catherine

Based on biochemical and pharmaco-chemical post-genomic approaches, our data provide the first evidence for the existence in mammals of a physiological inhibitor of enkephalin-inactivating metallo-ectopeptidases; it was characterized, previously in rat and presently in human, and named "Sialorphin". We have determined its significance and functional specificity in vitro and in vivo. In a remarkable discovery, it was found that Sialorphin is an efficient physiological dual antagonist of both enkephalin-catabolizing ectoenzymes, NEP (Neutral EndoPeptidase) and AP-N (AminoPeptidase-N); and, as an opiate agonist, it is a powerful inhibitor of pain perception in the rat.

In mammals, a number of zinc metallo-ectopeptidases located at the surface of cells in nervous and systemic tissues have been identified as ecto-enzymes catalyzing the post-secretory processing of neuropeptides and regulatory peptides which are physiologically involved in the regulation of important functions. Among the neuronal peptide-signals are enkephalins, which are potently and rapidly inactivated in vivo by both ecto-enzymes, NEP (Neutral EndoPeptidase) and AP-N (AminoPeptidase-N). Enkephalins are implicated in the receptor dependent modulation of behavioral adaptive responses to stressful environmental stimuli; notably, they regulate spinal processing of nociceptive information and analgesic mechanism, modulate mood-related states including emotional and/or motivational responses. Because of their physiological importance and the critical role of ecto-enzymes in modulating their functional potency it is essential to investigate and understand whether they can be protected by endogenous inhibitors. The data is also crucial from physio-pathological and therapeutic points of view.

From the gene to an integrated physiological function: In an integrative post-genomic approach (inverse pharmacology), we have established the molecular and functional bases providing the first evidence for the existence of an endogenous antagonist of metallo-ectopeptidase activity in mammals. Hence, we characterized the peptide-mediator sialorphin, which is the first physiological inhibitor of the membrane-anchored métallo-ectoendopeptidase NEP (Neutral EndoPeptidase) identified to date in rodent. And, this endocrine signal messenger of the adaptative response to stress is a powerful inhibitor of painful perception in rat.

For instance, in vivo analyses of behavioral pharmaco-chemistry have revealed that sialorphin displays potent analgesic activity in two behavioral rat models of peripheral injury-induced acute pain. The analgesia induced by sialorphin required the activation of Mu- and Delta-opioid receptors, consistent with the involvement of the endogenous enkephalinergic pathways which are specifically mediated by Mu- and Delta-opioid receptors; Indeed, these receptors are involved in the transmission of 1- the endogenous opiate signals such as the enkephalins, which are inactivated by both metallo-ectopeptidases NEP and AP-N and also, of 2- the alkaloid opiate, morphine.

Furthermore, in vivo sialorphin exerts psycho-stimulant activity in rat model of analysis of behavioral adaptative responses to socio-sexual or environmental stimuli. Hence, in a model of behavioral despair, sialorphin displays a dose-dependent antidepressant-like activity. Otherwise, in a model of analysis of socio-sexual behavior, we showed that sialorphin has the ability to modulate, at doses related to physiological circulating levels, the male rat sexual performance, while stimulating the apparent socio-sexual arousal or motivation.

Together the results obtained in vitro ex vivo and in vivo led us to propose that the physiological effects of sialorphin are associated with its capacity -to entirely protect the endogenous enkephalins from inactivation by the enkephalin-degrading ecto-enzymes, NEP and AP-N, thereby -to potentiate their physiological role, in particular the enkephalin-dependent anti-nociceptive and anti-depressive mechanisms, in vivo.

The physiological properties of rat sialorphin demonstrate that this endogenous inhibitor carries a structural signature remarkably adapted in vivo, in terms of selectivity, bioavailability and efficiency, to the topological and functional characteristics of its target-ectopeptidases and, as a consequence it is particularly efficient as an antinociceptive and an antidepressant molecule. This finding raises the question of the existence of such endogenous enkephalin-ectopeptidase inhibitor in human. Using a combined post-genomic and biochemical approach, we have presently characterized the endogenous functional homolog of rat sialorphin in humans, at the molecular and integrated functional level (on human cell and behavioral rat models) (Wisner et al, manuscrit submitted). To our knowledge, it is the first demonstration of the existence in humans of a physiological dual inhibitor of NEP and AP-N enkephalin-inactivating ecto-peptidases. This discovery is of crucial interest from a physiological and physio-pathological point of view when the extent of the functions mediated by the endogenous opioidergic pathways are considered; Indeed the endogenous opioid peptides, notably the enkephalins, have a pivotal role in the adaptative mechanisms of physiological regulation of the antinociceptive-pronociceptive balance and mood steady-state which are triggered during environmental changes.

From a physiological function to a candidate drug: Sialorphin is a natural bioactive molecule, discovered using a genomic approach and authenticated by post genomic and physio-pharmacological research; its important role and functions at the integrated physiological level have now been characterized and confirmed. And, also importantly, because of its in vivo properties, sialorphin may have therapeutic implications as a potential initiator of molecular pathways which could be exploited to develop new candidate drugs for the clinical management of pain relief and alleviation of affective disorders.

Keywords: Pharmaco-chemistry, Pain, Stress, motivation, Peptide hormone mediator, Opiates, Metallo-ectopeptidases

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