The Opiorphin Team (PI: Catherine Rougeot)

The research group is focused on the study of the molecular mechanisms of  intercellular regulations and specifically molecules involved in mu- and delta-opioïd pathways

Combining a multi-disciplinary approach including genetic, biochemistry, molecular pharmacology and behavioral pharmacology, we discovered the existence of physiological inhibitors of the enkephalin-inactivating Zn-ectopeptidases, NEP and AP-N in mammals. Rat Sialorphin (QHNPR-pentapeptide) was identified using integrative post-genomic, and Human Opiorphin (QRFSR-pentapeptide) was later characterized using functional biochemistry. Integrative pharmaco-chemical studies allowed to elucidate their structure and function relationship.




Fig. 1: In silico representation of the 2D structure of QRFSR-peptide Opiorphin.
Blue: Glutamine (Q); red: Arginine, (R); green: Phenylalanine (F); yellow: Serine (S).



Major results and objectives


In mammals, Zinc metallo-ectopeptidases control the activity of several neuroendocrine peptide mediators which, in turn, co-ordinate the adaptative responses of the organism to environmental changes. Enkephalins are potent opioid neuropeptides, which are secreted in response to physical or psychological stress. They play a crucial role in the dynamic control of pain transmission and in the modulation of cerebral activity governing motivation and in the adaptive equilibrium of emotional states. Their action is specifically transmitted, like that of morphine (an alkaloid vegetal opiate), via mu- and delta-opioïd membrane receptors. However in vivo, circulating enkephalins are inactivated in few seconds by two metallo-ectopeptidases, NEP (Neutral-EndoPeptidase) and AP-N (AminoPeptidase-N) (Fig. 2).


Fig. 2: Schematic representation of the primary structure of enkephalins, YGGFM
and YGGFL and their specific sites of cleavage by NEP and AP-N
.
 

 


Rat Sialorphin: a ligand and a physiological regulator of NEP-enkephalinase


Sialorphin is a hormonal peptide messenger of intercellular communication in the rat that was identified using a pharmaco-biochemical post-genomic approach. Its functional characterization was based on the molecular identification of membrane receptor sites that bind circulating Sialorphin, in vivo (Rougeot et al., 1994, Eur. J. Biochem., 219: 765; Rougeot et al., 1997, Am. J. Physiol., 273: R1309; Rougeot et al., 2000, Peptides, 21: 443) (Fig. 3).


 

Fig. 3: Visualization of the main systemic targets for circulating Sialorphin in the adult male rat. Red grains, organs or tissues; black grains, cells.

 


We discovered that Sialorphin is a physiological ligand of rat NEP and a competitive inhibitor of membrane bound NEP.


Using two behavioral models of peripheral injury-induced acute pain in the adult male rat, we demonstrated that Sialorphin elicits a powerful anti-nociceptive activity. The analgesic effects induced by Sialorphin require the specific activation of endogenous mu- and delta-opioïd receptors, which are involved in the transmission of enkephalinergic signals. Altogether, ex vivo and in vivo data suggest that by protecting endogenous enkephalins released in response to pain stimuli from inactivation by the ectoenkephalinases, Sialorphin potentiates their physiological actions, and in particular anti-nociception (Rougeot et al., 2003, PNAS, 100: 8549).

Furthermore, because androgen-regulated Sialorphin is rapidly secreted in response to environmental stress in sexually mature male rats, we postulated that it might play a role linked to reproduction. Our data show that Sialorphin has the ability to modulate the male rat mating pattern, i.e., stimulating the sexual performance and the socio-sexual arousal and/or motivation (Messaoudi et al., 2004, Horm. & Behavior, 46: 684; Davies et al., 2007, British J. Urol. Int., 99: 431).
 

Human Opiorphin: a natural inhibitor of enkephalin-inactivating Zn-ectopeptidases NEP and AP-N, and a novel modulator of enkephalin-dependant opioid pathways.

A functional biochemical approach allowed us to discover the existence in human of a pentapeptide with similar properties to rat Sialorphin as a physiological inhibitor of the enkephalin-inactivating Zn-ectopeptidases. Indeed, using molecular pharmacology (recombinant pure hNEP-hAP-N) and cellular pharmacology approaches (human cell lines expressing hNEP and hAP-N), we demonstrated that Opiorphin is a dual inhibitor of both hNEP and hAP-N ectopeptidases and that it protects Met-enkephalin from degradation by these two ectoenzymes.


The behavioral pharmacological responses induced, in vivo in the rat, by human Opiorphin were then studied, especially those involving the nociceptive neurotransmission pathways which are under the negative feedback control of enkephalins. Using a behavioral model of mechanical acute pain (the Pin Pain test), we demonstrated that Opiorphin displays pain-suppressive potency comparable to morphine (Wisner et al., 2006, Proc. Natl. Acad. Sci. USA, 103: 17974; Rougeot et al., 2007, Med. Sci., 23: 33; Rougeot et al., 2008, Proc. Int. Peptide Symp., 1-3) (Fig. 4).


Fig.4: Comparison of the behavioral responses obtained after administration of Opiorphin or morphine. ***P=0.0002 vs vehicle by Mann-Whitney U-test.
 

 

 


Furthermore, we demonstrated that Opiorphin elicits minimal adverse morphine-associated effects at systemically active doses that produce a comparable analgesic potency in several experimental models of pain in rats. The analgesic responses induced by Opiorphin, which require activation of endogenous mu-opioïd pathways, is comparable to that induced by the morphine mu-opioïd agonist, in terms of effective doses as well as of potency. However, in contrast to morphine, Opiorphin does not develop significant abuse liability, anti-nociceptive drug tolerance or anti-peristaltism (Rougeot et al., 2010, J. Physiol. and Pharmacol., 61: 483).


Human Opiorphin is the first identified physiological inhibitor of enkephalin-inactivating ectopeptidases that induces anti-nociception in rat model by activating endogenous opioid pathways. This discovery is of crucial importance from a physiological and physio-pathological point of view, given the extent of the functions mediated by the endogenous opioidergic pathways.

In a physiological scenario based on its mechanism of action, human Opiorphin would be involved in the process of adaptation mediated by enkephalins and in particular in the regulation of the homeostatic equilibrium: pronociception-antinociception and depression-motivation. To explore the profile of secretion and distribution of Opiorphin in healthy human biological media, a clinical research protocol, which received agreement of the Paris-Cochin ethic committee, was established. The results of this project, obtained in physiological situations, will provide the basis for a further approach to identify human pathological states up- or down-regulating the levels of circulating Opiorphin.

Opiorphin may have important therapeutic applications as a potential initiator of molecular pathways, which could be exploited to develop new drug-candidates, notably for the clinical management of pain relief. Programs are currently in progress to design conformational restricted Opiorphin mimetics, which would retain the activity profile of the native peptide and present increased bioavailability properties.

Technical know-how: Biochemistry, Molecular and Integrative Pharmaco-Chemistry
  • Protein and small peptide purifications (HPLC).
  • Quantitative Immuno-assays (ELISA, RIA…).
  • Pharmacokinetics and biodistribution analyses (in vitro, ex vivo and in vivo).
  • FRET-based peptidase assays.