Functional selectivity of EM-2 analogs at the mu-opioid receptor.

The mu opioid receptor agonists are the most efficacious pain controlling agents but their use is accompanied by severe side effects. More recent developments indicate that some ligands can differentially activate receptor downstream pathways, possibly allowing for dissociation of analgesia mediated through the G protein from the opioid-related side effects mediated by ß-arrestin pathway. In an effort to identify such biased ligands, here we present a series of thirteen endomorphin-2 (EM-2) analogs with modifications in positions 1, 2, and/or 3. All obtained analogs behaved as mu receptor selective agonists in calcium mobilization assay carried out on cells expressing opioid receptors and chimeric G proteins. A Bioluminescence Resonance Energy Transfer (BRET) approach was employed to determine the ability of analogs to promote the interaction of the mu opioid receptor with G protein or ß-arrestin 2. Nearly half of the developed analogs showed strong bias towards G protein, in addition four compounds were nearly inactive towards ß-arrestin 2 recruitment while blocking the propensity of EM-2 to evoke mu-ß-arrestin 2 interaction. The data presented here contribute to our understanding of EM-2 interaction with the mu opioid receptor and of the transductional propagation of the signal. In addition, the generation of potent and selective mu receptor agonists strongly biased towards G protein provides the scientific community with novel tools to investigate the in vivo consequences of biased agonism at this receptor.

Synthesis, Biological Activity and Molecular Docking of Chimeric Peptides Targeting Opioid and NOP Receptors.

Recently, mixed opioid/NOP agonists came to the spotlight for their favorable functional profiles and promising outcomes in clinical trials as novel analgesics. This study reports on two novel chimeric peptides incorporating the fragment Tyr-c[D-Lys-Phe-Phe]Asp-NH2 (RP-170), a cyclic peptide with high affinity for µ and κ opioid receptors (or MOP and KOP, respectively), conjugated with the peptide Ac-RYYRIK-NH2, a known ligand of the nociceptin/orphanin FQ receptor (NOP), yielding RP-170-RYYRIK-NH2 (KW-495) and RP-170-Gly3-RYYRIK-NH2 (KW-496). In vitro, the chimeric KW-496 gained affinity for KOP, hence becoming a dual KOP/MOP agonist, while KW-495 behaved as a mixed MOP/NOP agonist with low nM affinity. Hence, KW-495 was selected for further in vivo experiments. Intrathecal administration of this peptide in mice elicited antinociceptive effects in the hot-plate test; this action was sensitive to both the universal opioid receptor antagonist naloxone and the selective NOP antagonist SB-612111. The rotarod test revealed that KW-495 administration did not alter the mice motor coordination performance. Computational studies have been conducted on the two chimeras to investigate the structural determinants at the basis of the experimental activities, including any role of the Gly3 spacer.

Harnessing the Anti-Nociceptive Potential of NK2 and NK3 Ligands in the Design of New Multifunctional µ/δ-Opioid Agonist-Neurokinin Antagonist Peptidomimetics.

Opioid agonists are well-established analgesics, widely prescribed for acute but also chronic pain. However, their efficiency comes with the price of drastically impacting side effects that are inherently linked to their prolonged use. To answer these liabilities, designed multiple ligands (DMLs) offer a promising strategy by co-targeting opioid and non-opioid signaling pathways involved in nociception. Despite being intimately linked to the Substance P (SP)/neurokinin 1 (NK1) system, which is broadly examined for pain treatment, the neurokinin receptors NK2 and NK3 have so far been neglected in such DMLs. Herein, a series of newly designed opioid agonist-NK2 or -NK3 antagonists is reported. A selection of reported peptidic, pseudo-peptidic, and non-peptide neurokinin NK2 and NK3 ligands were covalently linked to the peptidic µ-opioid selective pharmacophore Dmt-DALDA (H-Dmt-d-Arg-Phe-Lys-NH2) and the dual µ/δ opioid agonist H-Dmt-d-Arg-Aba-ßAla-NH2 (KGOP01). Opioid binding assays unequivocally demonstrated that only hybrids SBL-OPNK-5, SBL-OPNK-7 and SBL-OPNK-9, bearing the KGOP01 scaffold, conserved nanomolar range µ-opioid receptor (MOR) affinity, and slightly reduced affinity for the δ-opioid receptor (DOR). Moreover, NK binding experiments proved that compounds SBL-OPNK-5, SBL-OPNK-7, and SBL-OPNK-9 exhibited (sub)nanomolar binding affinity for NK2 and NK3, opening promising opportunities for the design of next-generation opioid hybrids.

Synthesis, Pharmacological Evaluation, and Computational Studies of Cyclic Opioid Peptidomimetics Containing ß3-Lysine.

Our formerly described pentapeptide opioid analog Tyr-c[D-Lys-Phe-Phe-Asp]NH2 (designated RP-170), showing high affinity for the mu (MOR) and kappa (KOR) opioid receptors, was much more stable than endomorphine-2 (EM-2) in the rat brain homogenate and displayed remarkable antinociceptive activity after central (intracerebroventricular) and peripheral (intravenous ) administration. In this report, we describe the further modification of this analog, which includes the incorporation of a ß3-amino acid, (R)- and (S)-ß3-Lys, instead of D-Lys in position 2. The influence of such replacement on the biological properties of the obtained analogs, Tyr-c[(R)-ß3-Lys-Phe-Phe-Asp]NH2 (RP-171) and Tyr-c[(S)-ß3-Lys-Phe-Phe-Asp]NH2, (RP-172), was investigated in vitro. Receptor radiolabeled displacement and functional calcium mobilization assays were performed to measure binding affinity and receptor activation of the new analogs. The obtained data revealed that only one of the diastereoisomeric peptides, RP-171, was able to selectively bind and activate MOR. Molecular modeling (docking and molecular dynamics (MD) simulations) suggests that both compounds should be accommodated in the MOR binding site. However, in the case of the inactive isomer RP-172, fewer hydrogen bonds, as well as instability of the canonical ionic interaction to Asp147, could explain its very low MOR affinity.

Pharmacological Characterization of µ-Opioid Receptor Agonists with Biased G Protein or ß-Arrestin Signaling, and Computational Study of Conformational Changes during Receptor Activation.

In recent years, G protein vs. ß-arrestin biased agonism at opioid receptors has been proposed as an opportunity to produce antinociception with reduced adverse effects. However, at present this approach is highly debated, a reason why more information about biased ligands is required. While the practical relevance of bias in the case of µ-opioid receptors (MOP) still needs to be validated, it remains important to understand the basis of this bias of MOP (and other GPCRs). Recently, we reported two cyclopeptides with high affinity for MOP, the G protein biased Dmt-c[d-Lys-Phe-pCF3-Phe-Asp]NH2 (F-81), and the ß-arrestin 2 biased Dmt-c[d-Lys-Phe-Asp]NH2 (C-33), as determined by calcium mobilization assay and bioluminescence resonance energy transfer-based assay. The biased character of F-81 and C-33 has been further analyzed in the [35S]GTPγS binding assay in human MOP-expressing cells, and the PathHunter enzyme complementation assay, used to measure ß-arrestin 2 recruitment. To investigate the structural features of peptide-MOP complexes, we performed conformational analysis by NMR spectroscopy, molecular docking, and molecular dynamics simulation. These studies predicted that the two ligands form alternative complexes with MOP, engaging specific ligand-receptor contacts. This would induce different displays of the cytosolic side of the seven-helices bundle, in particular by stabilizing different angulations of helix 6, that could favor intracellular coupling to either G protein or ß-arrestin.

Design, Synthesis and Functional Analysis of Cyclic Opioid Peptides with Dmt-Tic Pharmacophore.

The opioid receptors are members of the G-protein-coupled receptor (GPCR) family and are known to modulate a variety of biological functions, including pain perception. Despite considerable advances, the mechanisms by which opioid agonists and antagonists interact with their receptors and exert their effect are still not completely understood. In this report, six new hybrids of the Dmt-Tic pharmacophore and cyclic peptides, which were shown before to have a high affinity for the µ-opioid receptor (MOR) were synthesized and characterized pharmacologically in calcium mobilization functional assays. All obtained ligands turned out to be selective antagonists of the δ-opioid receptor (DOR) and did not activate or block the MOR. The three-dimensional structural determinants responsible for the DOR antagonist properties of these analogs were further investigated by docking studies. The results indicate that these compounds attach to the DOR in a slightly different orientation with respect to the Dmt-Tic pharmacophore than Dmt-TicΨ[CH2-NH]Phe-Phe-NH2 (DIPP-NH2[Ψ]), a prototypical DOR antagonist peptide. Key pharmacophoric contacts between the DOR and the ligands were maintained through an analogous spatial arrangement of pharmacophores, which could provide an explanation for the predicted high-affinity binding and the experimentally observed functional properties of the novel synthetic ligands.

The search for opioid analgesics with limited tolerance liability.

Reducing the well-known side effects of opioids prescribed to treat chronic pain remains unresolved, despite extensive research in this field. Among several options to tackle this problem the synthesis of multifunctional compounds containing hybridized structures gained a lot of interest. Recently, extensively investigated are combinations of opioid agonist and antagonist pharmacophores embodied in a single molecule. To this end, agonism at the µ opioid receptor (MOR) with simultaneous antagonism at the δ opioid receptor (DOR) emerged as a promising avenue to obtaining novel analogs devoid of serious adverse effects associated with morphine-based analgesics. In this review we covered up-to-date research on the synthesis of peptide-based ligands with MOR agonist/DOR antagonist profile.

Pharmacological Profile and Molecular Modeling of Cyclic Opioid Analogues Incorporating Various Phenylalanine Derivatives.

Peptide-based agonists of the µ opioid receptor (µOR) are promising therapeutic candidates for pain relief with reduced side effects compared to morphine. A deep understanding of µOR-ligand interactions is necessary for future design of peptide-based opioid analgesics. To explore the requirements of the µOR binding pocket, eight new analogues of our cyclic peptide Tyr-c[d-Lys-Phe-Phe-Asp]NH2 displaying high µOR affinity were synthesized, in which Phe in either the third or fourth position was replaced by various derivatives of this amino acid (ß3 -Phe, homoPhe, ß3 -homoPhe and PhGly). The aim of this research was to examine the structural effects of such modifications on the bioactivity, and both experimental and theoretical methods were used. The binding of the cyclic analogues to all three OR types (µ, δ, κ) was assessed by radioligand competitive binding assay, and their functional activity was determined in a calcium mobilization assay. In order to provide structural hypotheses explaining the obtained experimental affinities, the complexes of the cyclic peptides with µOR were subjected to molecular modeling.

Biased Agonism as an Emerging Strategy in the Search for Better Opioid Analgesics.

Morphine and related drugs that act through activating opioid receptors are the most effective analgesics for the relief of severe pain. They have been used for decades, despite the range of unwanted side effects that they produce, as no alternative has been found so far. The major goal of opioid research is to understand the mechanism of action of opioid receptor agonists and to improve the therapeutic utility of opioid drugs. In the search for safer and more potent analgesics, analogs with mixed opioid receptor profile gained a lot of interest. However, recently the concept of biased agonism, that highlights the fact that some ligands are able to differentially activate receptor downstream pathways, became a new approach in the design of novel drug candidates for clinical application. In this review, we summarize current knowledge on the development of opioid ligands of peptide and nonpeptide structure, showing how much opioid pharmacology evolved in recent years.

Synthesis and Pharmacological Evaluation of Hybrids Targeting Opioid and Neurokinin Receptors.

Morphine, which acts through opioid receptors, is one of the most efficient analgesics for the alleviation of severe pain. However, its usefulness is limited by serious side effects, including analgesic tolerance, constipation, and dependence liability. The growing awareness that multifunctional ligands which simultaneously activate two or more targets may produce a more desirable drug profile than selectively targeted compounds has created an opportunity for a new approach to developing more effective medications. Here, in order to better understand the role of the neurokinin system in opioid-induced antinociception, we report the synthesis, structure-activity relationship, and pharmacological characterization of a series of hybrids combining opioid pharmacophores with either substance P (SP) fragments or neurokinin receptor (NK1) antagonist fragments. On the bases of the in vitro biological activities of the hybrids, two analogs, opioid agonist/NK1 antagonist Tyr-[d-Lys-Phe-Phe-Asp]-Asn-d-Trp-Phe-d-Trp-Leu-Nle-NH2 (2) and opioid agonist/NK1 agonist Tyr-[d-Lys-Phe-Phe-Asp]-Gln-Phe-Phe-Gly-Leu-Met-NH2 (4), were selected for in vivo tests. In the writhing test, both hybrids showed significant an antinociceptive effect in mice, while neither of them triggered the development of tolerance, nor did they produce constipation. No statistically significant differences in in vivo activity profiles were observed between opioid/NK1 agonist and opioid/NK1 antagonist hybrids.

Endomorphin-2 analogs containing modified tyrosines: Biological and theoretical investigation of the influence on conformation and pharmacological profile.

New analogs of the endogenous opioid agonist endomorphin-2 (EM-2, H-Tyr-Pro-Phe-Phe-NH2) have been obtained by introducing modified tyrosines at the position 1 of the sequence. For all analogs, the cis/trans conformation ratio about the tyramine-Pro amide bond, lipophilicity, receptor affinities, and functional activities, have been determined. Among the novel derivatives, [Dmt(3'-Cl)]1EM-2 (4) stood out for its subnanomolar µ-opioid receptor affinity and potent agonist activity, superior to that of the parent peptide EM-2. Hybrid quantum mechanics/molecular mechanics docking computations supported the cis tyramine-Pro bioactive conformation, and allowed us to analyze the contribution of the substituents of the "message" tyramine to binding, highlighting the role of halogen-bonding in the higher receptor affinity of peptide 4.

In vitro and in vivo activity of cyclopeptide Dmt-c[d-Lys-Phe-Asp]NH2, a mu opioid receptor agonist biased toward ß-arrestin.

Morphine and related drugs, which are the most effective analgesics for the relief of severe pain, act through activating opioid receptors. The endogenous ligands of these receptors are opioid peptides which cannot be used as antinociceptive agents due to their low bioactivity and stability in biological fluids. The major goal of opioid research is to understand the mechanism of action of opioid receptor agonists in order to improve therapeutic utility of opioids. Analgesic effects of morphine are mediated mostly through activation of the mu opioid receptor. However, in the search for safer and more effective drug candidates, analogs with mixed opioid receptor profile gained a lot of interest. Recently, the concept of biased agonists able to differentially activate GPCR downstream pathways, became a new approach in the design of novel drug candidates. It is hypothesized that compounds promoting G-protein signaling may produce analgesia while ß-arrestin recruitment may be responsible for opioid side effects. In this report we showed that replacement of the tyrosine residue in the mu-selective ligand Tyr-c[d-Lys-Phe-Asp]NH2 with 2',6'-dimethyltyrosine (Dmt) produced a cyclopeptide Dmt-c[d-Lys-Phe-Asp]NH2 with mu/delta opioid receptor agonist profile. This analog showed improved antinociception in the hot-plate test, probably due to the simultaneous activation of mu and delta receptors but also significantly inhibited the gastrointestinal transit. Using the bioluminescence resonance energy transfer (BRET) assay it was shown that this analog was a mu receptor agonist biased toward ß-arrestin. ß-Arrestin-dependent signaling is most likely responsible for the observed inhibition of gastrointestinal motility exerted by the novel cyclopeptide.

Antinociceptive potency of a fluorinated cyclopeptide Dmt-c[D-Lys-Phe-p-CF3-Phe-Asp]NH2.

Opioid peptides and opiate drugs such as morphine, mediate their analgesic effects, but also undesired side effects, mostly through activation of the mu opioid receptor. However, delta- and kappa-opioid receptors can also contribute to the analgesic effects of opioids. Recent findings showed that simultaneous activation of multiple opioid receptors may result in additional analgesia with fewer side effects. Here, we evaluated the pharmacological profile of our formerly developed mixed mu/kappa-opioid receptor ligands, Dmt-c[D-Lys-Phe-Phe-Asp]NH2 (C-36) and Dmt-c[D-Lys-Phe-p-CF3-Phe-Asp]NH2 (F-81). The ability of these peptides to cross the blood-brain barrier was tested in the parallel artificial membrane permeability (PAMPA) assay. On the basis of the hot-plate test in mice after central and peripheral administration, analog F-81 was selected for the anti-nociceptive and anti-inflammatory activity assessment after peripheral administration.

Cyclopeptide Dmt-[D-Lys-p-CF3-Phe-Phe-Asp]NH2, a novel G protein-biased agonist of the mu opioid receptor.

Opioid peptides and alkaloid drugs such as morphine, mediate their analgesic effects, but also undesired side effects, mostly through activation of the mu opioid receptor which belongs to the G protein-coupled receptor (GPCR) family. A new important pharmacological concept in the field of GPCRs is biased agonism. Two mu receptor ligands, Dmt-c[D-Lys-Phe-Phe-Asp]NH2 (C-36) and Dmt-c[D-Lys-Phe-p-CF3-Phe-Asp]NH2 (F-81), were evaluated in terms of their ability to promote or block mu receptor/G protein and mu receptor/ß-arrestin interactions. Using the bioluminescence resonance energy transfer (BRET) assay it was shown that C-36 activated both, G protein and ß-arrestin pathways. Incorporation of trifluoromethyl group into the aromatic ring of phenylalanine in the sequence of F-81 led to activation of G-protein pathway rather than ß-arrestin recruitment. Opioid cyclopeptide F-81 turned out to be a biased G protein mu receptor agonist. Such biased ligands are able to separate the biological actions of an activated receptor and have the potential to become more effective drug candidates with fewer side effects.

Synthesis, receptor binding studies, optical spectroscopic and in silico structural characterization of morphiceptin analogs with cis-4-amino-L-proline residues.

Three novel morphiceptin analogs, in which Pro in position 2 and/or 4 was replaced by cis-4-aminoproline connected with the preceding amino acid through the primary amino group, were synthesized. The opioid receptor affinities, functional assay results, enzymatic degradation studies and experimental and in silico structural analysis of such analogs are presented. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Synthesis and activity of opioid peptidomimetics with ß2- and ß3-amino acids.

Morphiceptin (Tyr-Pro-Phe-Pro-NH2) is a selective ligand of the mu opioid receptor, an important target in pain regulation. In this study, morphiceptin was modified at positions 2 or 3 by introduction of ß2- or ß3-amino acids and additionally in position 1 by replacing Tyr by Dmt (2',6'-dimethyltyrosine), which resulted in obtaining enzymatically stable analogs with mixed opioid receptor affinity profiles. An analog of the sequence Dmt-d-Ala-(R)-ß2-1-Nal-Pro-NH2 [Nal=3-(1-naphthyl)-alanine] showed very high activity at the mu and delta receptors in the calcium mobilization functional test but did not cross the artificial membrane imitating the blood-brain barrier. In the in vivo test this analog induced strong antinociceptive effect in the writhing test in mice after intraperitioneal but also oral administration and inhibited diarrhea similarly to loperamide. Therefore, it may become an interesting lead compound in the development of peripherally restricted drugs for the treatment of gastrointestinal disorders.