Human Opiorphin, a natural antinociceptive modulator of opioid-dependent pathways

Abstract

Mammalian zinc ectopeptidases play important roles in turning off neural and hormonal peptide signals at the cell surface, notably those processing sensory information. We report here the discovery of a previously uncharacterized physiological inhibitor of enkephalin-inactivating zinc ectopeptidases in humans, which we have named Opiorphin. It is a QRFSR peptide that inhibits two enkephalin-catabolizing ectoenzymes, human neutral ecto-endopeptidase, hNEP (EC 3.4.24.11), and human ecto-aminopeptidase, hAP-N (EC 3.4.11.2). Opiorphin displays potent analgesic activity in chemical and mechanical pain models by activating endogenous opioid-dependent transmission. Its function is closely related to the rat sialorphin peptide, which is an inhibitor of pain perception and acts by potentiating endogenous mu- and delta-opioid receptor-dependent enkephalinergic pathways. Here we demonstrate the functional specificity in vivo of human Opiorphin. The pain-suppressive potency of Opiorphin is as effective as morphine in the behavioral rat model of acute mechanical pain, the pin-pain test. Thus, our discovery of Opiorphin is extremely exciting from a physiological point of view in the context of endogenous opioidergic pathways, notably in modulating mood-related states and pain sensation. Furthermore, because of its in vivo properties, Opiorphin may have therapeutic implications.

Fig. 1.

Human Opiorphin identified in salivary secretions. (a–c) Percentage inhibition of SP breakdown by human cell-surface endopeptidases. Salivary fractions were analyzed for their potency to inhibit the endoproteolysis of the NEP-sensitive natural substrate, SP, by human cells expressing membrane-anchored hNEP (bars). (a) CE-HPLC profile of salivary methanol acid extracts obtained from 45 ml of human saliva. The dotted line represents the percentage of ammonium acetate buffer (1 M). (b) RP-HPLC profile of the major CE-HPLC active fractions (black bars in a). (c) Final RP-HPLC elution profile of the major RP-HPLC active fractions (black bars in b) and for their absorbance at 264 nm (black line). (d Left) SELDI-TOF MS analysis of the major RP-HPLC active fraction of the last purification step (black bar in c Right). (d Right) SELDI-TOF MS analysis of the reference synthetic QRFSR peptide.

Fig. 2.

Human Opiorphin demonstrates functional activity in vitro. (a) Concentration-dependent inhibition by Opiorphin QRFSR peptide of SP endoproteolysis, mediated by hNEP expressed at the surface of LNCaP cells. Each point (white circle) represents the percentage of intact ³H-SP recovered (percentage of velocity without inhibitor − velocity in presence of inhibitor/velocity without inhibitor), which was measured in the absence or in the presence of various concentrations of QRFSR peptide (in μM). (b) Specific concentration-dependent inhibition by Opiorphin QRFSR peptide of SP endoproteolysis by pure recombinant hNEP. Each point (black circle) represents the percentage of intact ³H-SP recovered (measured and calculated as in a). (c) The breakdown of SP by recombinant hDPPIV in the absence (black bar) or in the presence (white bars) of Opiorphin QRFSR peptide or in the presence of synthetic DPPIV-inh2 (gray bar). The values represent the mean ± SD (n = 3) of the percentage of specific ³H-SP hydrolysis by hDPPIV. (d) Specific concentration-dependent inhibition by Opiorphin QRFSR peptide of Met-enkephalin cleavage by purified soluble pAP-M. Each point (white square) represents (mean of two independent experiments) the percentage of Met-enkephalin hydrolysis by pAP-M analyzed in the absence or in the presence of various concentrations of QRFSR peptide (in μM) by RP-HPLC.

Fig. 3.

Opiorphin-derived peptide displays potent analgesic activity in vivo in rat pain model. (a–b) Evaluation of the pain response of rats to noxious chemical stimuli after administration of the Opiorphin-derived peptide YQRFSR. (a) Effects of YQRFSR peptide (black diamond; 1 mg/kg) compared with morphine (crossed circle; 3 mg/kg i.p. given 15 min before test) and vehicle (white circle) in the absence or presence of the opioid antagonist naloxone (gray diamond; 3 mg/kg s.c. given 30 min before test) on the number of body tremors during the six 10-min periods of the formalin test. (b) Pain index calculated from results shown in a by the AUCI method described in Materials and Methods. The values represent the mean ± SEM of eight animals for each condition: pain index based on paw licking duration (gray-striped bar, vehicle; black-striped bar, YQRFSR peptide); pain index based on body tremor number (white bar, vehicle; black bar, YQRFSR peptide; gray bar, YQRFSR peptide plus naloxone; hatched bar, morphine). ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗; P < 0.001 by Dunnett's t test.