Ligand-Specific Regulation of the Endogenous Mu-Opioid Receptor by Chronic Treatment with Mu-Opioid Peptide Agonist

Since the discovery of the endomorphins (EM), the postulated endogenous peptide agonists of the mu-opioid receptors, several analogues have been synthesized to improve their binding and pharmacological profiles. We have shown previously that a new analogue, cis-1S,2R-aminocyclohexanecarboxylic acid2-endomorphin-2 (ACHC-EM2), had elevated mu-receptor affinity, selectivity, and proteolytic stability over the parent compound. In the present work, we have studied its antinociceptive effects and receptor regulatory processes. ACHC-EM2 displayed a somewhat higher (60%) acute antinociceptive response than the parent peptide, EM2 (45%), which peaked at 10 min after intracerebroventricular (icv) administration in the rat tail-flick test. Analgesic tolerance developed to the antinociceptive effect of ACHC-EM2 upon its repeated icv injection that was complete by a 10-day treatment. This was accompanied by attenuated coupling of mu-sites to G-proteins in subcellular fractions of rat brain. Also, the density of mu-receptors was upregulated by about 40% in the light membrane fraction, with no detectable changes in surface binding. Distinct receptor regulatory processes were noted in subcellular fractions of rat brains made tolerant by the prototypic full mu-agonist peptide, DAMGO, and its chloromethyl ketone derivative, DAMCK. These results are discussed in light of the recently discovered phenomenon, that is, the “so-called biased agonism” or “functional selectivity

Liquid Chromatography-Electrospray Linear Ion Trap Mass Spectrometry Analysis of Targeted Neuropeptides in Tac1-/- Mouse Spinal Cords Reveal Significant Lower Concentration of Opioid Peptides

Tachykinin and opioid peptides play a central role in pain transmission, modulation and inhibition. The treatment of pain is very important in medicine and many studies using NK1 receptor antagonists failed to show significant analgesic effects in humans. Recent investigations suggest that both pronociceptive tachykinins and the analgesic opioid systems are important for normal pain sensation. The analysis of opioid peptides in Tac1-/- spinal cord tissues offers a great opportunity to verify the influence of the tachykinin system on specific opioid peptides. The objectives of this study were to develop a HPLC–MS/MRM assay to quantify targeted peptides in spinal cord tissues. Secondly, we wanted to verify if the Tac1-/- mouse endogenous opioid system is hampered and therefore affect significantly the pain modulatory pathways. Targeted neuropeptides were analyzed by high performance liquid chromatography linear ion trap mass spectrometry. Our results reveal that EM-2, Leu-Enk and Dyn A were down-regulated in Tac1-/- spinal cord tissues. Interestingly, Dyn A was almost 3 fold down-regulated (p < 0.0001). No significant concentration differences were observed in mouse Tac1-/- spinal cords for Met-Enk and CGRP. The analysis of Tac1-/- mouse spinal cords revealed noteworthy decreases of EM-2, Leu-Enk and Dyn A concentrations which strongly suggest a significant impact on the endogenous pain-relieving mechanisms. These observations may have insightful impact on future analgesic drug developments and therapeutic strategies

A novel non-Opioid binding site for endomorphin-1

Endomorphins are natural amidated opioid tetrapeptides with the following structure: Tyr-Pro-Trp-Phe-NH2 (endomorphin-1),and Tyr-Pro-Phe-Phe-NH2 (endomorphin-2). Endomorphins interact selectively with the µ-opioid or MOP receptors and exhibit nanomolar or sub-nanomolar receptor binding affinities,therefore they suggested to be endogenous agonists for the µ-opioid receptors. Endomorphins mediate a number of characteristic opioid effects,such as antinociception,however there are several physiological functions in which endomorphins appear to act in a fashion that does not involve binding to and activation of the µ-opioid receptor. Our recent data indicate that a radiolabelled [3H]endomorphin-1 with a specific radioactivity of 2.35 TBq/mmol - prepared by catalytic dehalogenation of the diiodinated peptide precursor in the presence of tritium gas - is able to bind to a second,naloxone insensitive recognition site in rat brain membranes. Binding heterogeneity,i.e.,the presence of higher (Kd = 0.4 nM/Bmax = 120 fmol/mg protein) and lower (Kd = 8.2 nM/Bmax = 432 fmol/mg protein) affinity binding components is observed both in saturation binding experiments followed by Schatchard analysis,and in equilibrium competition binding studies. The signs of receptor multiplicity,e.g.,curvilinear Schatchard plots or biphasic displacement curves are seen only if the non-specific binding is measured in the presence of excess unlabeled endomorphin-1 and not in the presence of excess unlabeled naloxone. The second,lower affinity non-opioid binding site is not recognized by heterocyclic opioid alkaloid ligands,neither agonists such as morphine,nor antagonists such as naloxone. On the contrary,endomorphin-1 is displaced from its lower affinity,higher capacity binding site by several natural neuropeptides,including methionine-enkephalin-Arg-Phe,nociceptin-orphanin FQ,angiotensin and FMRF-amide. This naloxone-insensitive,consequently non-opioid binding site seems to be present in nervous tissues carrying low density or no µ-opioid receptors,such as rodent cerebellum,or brain of µ-opioid receptor deficient (MOPr-/-) transgenic or ‘knock-out’ (K.O.) mice. The newly described non-opioid binding component is not coupled to regulatory G-proteins,nor does it affect adenylyl cyclase enzyme activity. Taken together endomorphin-1 carries opioid and,in addition to non-opioid functions that needs to be taken into account when various effects of endomorphin-1 are evaluated in physiological or pathologic conditions.</p

Measuring ligand efficacy at the mu-opioid receptor using a conformational biosensor.

The intrinsic efficacy of orthosteric ligands acting at G-protein-coupled receptors (GPCRs) reflects their ability to stabilize active receptor states (R*) and is a major determinant of their physiological effects. Here, we present a direct way to quantify the efficacy of ligands by measuring the binding of a R*-specific biosensor to purified receptor employing interferometry. As an example, we use the mu-opioid receptor (µ-OR), a prototypic class A GPCR, and its active state sensor, nanobody-39 (Nb39). We demonstrate that ligands vary in their ability to recruit Nb39 to µ-OR and describe methadone, loperamide, and PZM21 as ligands that support unique R* conformation(s) of µ-OR. We further show that positive allosteric modulators of µ-OR promote formation of R* in addition to enhancing promotion by orthosteric agonists. Finally, we demonstrate that the technique can be utilized with heterotrimeric G protein. The method is cell-free, signal transduction-independent and is generally applicable to GPCRs

Substance P induces gastric mucosal protection at supraspinal level via increasing the level of endomorphin-2 in rats.

The aim of the present study was to analyze the potential role of substance P (SP) in gastric mucosal defense and to clarify the receptors and mechanisms that may be involved in it. Gastric ulceration was induced by oral administration of acidified ethanol in male Wistar rats. Mucosal levels of calcitonin gene-related peptide (CGRP) and somatostatin were determined by radioimmunoassay. For analysis of gastric motor activity the rubber balloon method was used. We found that central (intracerebroventricular) injection of SP (9.3-74pmol) dose-dependently inhibited the formation of ethanol-induced ulcers, while intravenously injected SP (0.37-7.4nmol/kg) had no effect. The mucosal protective effect of SP was inhibited by pretreatment with neurokinin 1-, neurokinin 2-, neurokinin 3- and mu-opioid receptor antagonists, while delta- and kappa-opioid receptor antagonists had no effect. Endomorphin-2 antiserum also antagonized the SP-induced mucosal protection. In the gastroprotective dose range SP failed to influence the gastric motor activity. Inhibition of muscarinic cholinergic receptors, or the synthesis of nitric oxide or prostaglandins significantly reduced the effect of SP. In addition, centrally injected SP reversed the ethanol-induced reduction of gastric mucosal CGRP content. It can be concluded, that SP may induce gastric mucosal protection initiated centrally. Its protective effect is likely to be mediated by endomorphin-2, and vagal nerve may convey the centrally initiated protection to the periphery, where both prostaglandins, nitric oxide and CGRP are involved in mediating this effect

A sztereoizoméria hatása peptidek térszerkezetére és bioaktivitására = The effect of stereoisomerism on the spatial structure and bioactivity of peptides

Kutatásom során tanulmányoztam a cisz-transz és L-D izomériának a peptidek térszerkezeti és konformációs tulajdonságaira, illetve bioaktivitására kifejtett hatásait. Az opioid peptidek összehasonlító konformáció-analízise alapján azonosítottam a sztereoizomerek karakterisztikus térszerkezeti sajátságait, illetve a peptidek közötti konformációs hasonlóságokat és különbségeket. A farmakofór elemek relatív térbeli elrendeződésének leírására használt módszer alkalmasnak bizonyult a sztereoizomerek egymástól való megkülönböztetésére, és a farmakofórok térbeli összefüggéseinek jellemzésére. Az antimikrobiális peptidek esetén meghatároztam a cisz-transz izomériának a sztereoizomerek konformációs tulajdonságainak kialakításában és a peptidek dinamikus viselkedésében betöltött szerepét. A bázikus aminosavakat tartalmazó alanin-alapú peptidekre vonatkozóan átfogóan feltérképeztük a folding folyamatokat, és a szimulációk alapján különböző módszerek alkalmazásával jellemeztük a hajtogatódási útvonalakat. Az antimikrobiális peptidek palindrom szekvenciái esetében elvégeztünk egy részletes térszerkezet-vizsgálatot, és azonosítottuk a peptidek jellegzetes térszerkezeti sajátságait. A peptid-micella/membrán rendszerek esetén tanulmányoztuk a sztereoizomer antimikrobiális peptidek micellához/membránhoz kötődésének folyamatát, illetve részletesen jellemeztük a sztereoizomerek micella- és membrán-kötött konformációit, valamint a peptidek és micellák/membránok között kialakuló kölcsönhatásokat. | In my project, the effects of cis-trans and L-D isomerisms on the structural and conformational properties, as well as on the bioactivity of peptides were studied. Based on the comparative conformational analysis of opioid peptides, the characteristic structural features of stereoisomers, as well as the conformational similarities and dissimilarities between the peptides were identified. The method, applied to describe the relative spatial arrangements of pharmacophore elements, proved to be suitable to distinguish the stereoisomers from one another, and to characterize the spatial relationships of pharmacophores. For the antimicrobial peptides, the role of cis-trans isomerism played in the formation of conformational properties of stereoisomers, and in the dynamic behaviour of peptides, was determined. The folding processes were comprehensively explored, regarding the alanine-based peptides containing basic amino acids, and based on the simulations, the folding pathways were characterized using various methods. A detailed structural investigation was performed on the palindromic sequences of antimicrobial peptides, and the typical structural features of peptides were identified. For the peptide-micelle/membrane systems, the binding processes of stereoisomeric antimicrobial peptides to micelle/membrane were studied, as well as the micelle- and membrane-bound conformations, and the interactions evolved between the peptides and micelles/membranes were characterized in detail