Protective effect of a mitochondria-targeting peptide against paclitaxel-induced peripheral neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major side effect of several anticancer agents including paclitaxel, a chemotherapeutic drug widely used in cancer treatment. CIPN deteriorates patients' quality of life and compromises cancer treatment. Dysfunction or injury of mitochondria has been suggested to be involved in the induction of this neuropathy. SS-20 is a tetrapeptide that targets mitochondria and restores mitochondrial bioenergetics. This study was aimed to examine the protective effect of SS-20 against paclitaxel-induced peripheral neuropathy using a murine model. Repeated administration of paclitaxel to mice induced peripheral neuropathy as demonstrated by the presence of mechanical allodynia and the loss of intraepidermal nerve fibers in the hind paw. Concomitant administration of SS-20 protected against the development of the neuropathy. Our results suggest that SS-20 may be a drug candidate for the prevention of CIPN.

Improving the Utility of a Dynorphin Peptide Analogue Using Mannosylated Glycoliposomes.

Peptide therapeutics offer numerous advantages in the treatment of diseases and disorders of the central nervous system (CNS). However, they are not without limitations, especially in terms of their pharmacokinetics where their metabolic lability and low blood-brain barrier penetration hinder their application. Targeted nanoparticle delivery systems are being tapped for their ability to improve the delivery of therapeutics into the brain non-invasively. We have developed a family of mannosylated glycoliposome delivery systems for targeted drug delivery applications. Herein, we demonstrate via in vivo distribution studies the potential of these glycoliposomes to improve the utility of CNS active therapeutics using dynantin, a potent and selective dynorphin peptide analogue antagonist of the kappa opioid receptor (KOR). Glycoliposomal entrapment protected dynantin against known rapid metabolic degradation and ultimately improved brain levels of the peptide by approximately 3-3.5-fold. Moreover, we linked this improved brain delivery with improved KOR antagonist activity by way of an approximately 30-40% positive modulation of striatal dopamine levels 20 min after intranasal administration. Overall, the results clearly highlight the potential of our glycoliposomes as a targeted delivery system for therapeutic agents of the CNS.

Mannosylated glycoliposomes for the delivery of a peptide kappa opioid receptor antagonist to the brain.

Dynantin is a potent and selective synthetic polypeptide kappa opioid receptor antagonist which has potential antidepressant and anxiolytic-like therapeutic applications, however its clinical development has been hampered by plasma stability issues and poor penetration of the blood brain barrier. Targeted liposome delivery systems represent a promising and non-invasive approach to improving the delivery of therapeutic agents across the blood brain barrier. As part of our work focused on targeted drug delivery, we have developed a novel mannosylated liposome system. Herein, we investigate these glycoliposomes for the targeted delivery of dynantin to the central nervous system. Cholesterol was tested and optimized as a formulation excipient, where it improved particle stability as measured via particle size, entrapment and ex vivo plasma stability of dynantin. The in vitro PRESTO-TANGO assay system was used to confirm that glycoliposomal entrapment did not impact the affinity or activity of the peptide at its receptor. Finally, in vivo distribution studies in mice showed that the mannosylated glycoliposomes significantly improved delivery of dynantin to the brain. Overall, the results clearly demonstrate the potential of our glycoliposomes as a targeted delivery system for therapeutic agents of the central nervous system.

Novel µ opioid antagonists derived from the µ opioid agonists endomorphin and [Dmt1 ]DALDA (H-Dmt-D-Arg-Phe-Lys-NH2 ).

Hybrid analogues of the µ opioid agonists endomorphin and [Dmt1 ]DALDA (H-Dmt-D-Arg-Phe-Lys-NH2 , Dmt = 2',6'-dimethyltyrosine) containing cis-4-amino-Pro, trans-4-amino-Pro, cis-4-aminoethyl-Pro or cis-4-guanidinylethyl-Pro in the 2 position of the peptide sequence were synthesized. None of the compounds retained high µ opioid agonist activity and, unexpectedly, substitution of cis-4-amino-Pro resulted in a novel class of potent µ opioid antagonists. In particular, the compound H-Dmt-cis-4-amino-Pro-Trp-Lys-NH2 (CZ-1) turned out to be a highly selective µ opioid antagonist with ~1 nM µ receptor binding affinity.

Mast Cell Degranulation and Fibroblast Activation in the Morphine-induced Spinal Mass: Role of Mas-related G Protein-coupled Receptor Signaling.

BACKGROUND: As the meningeally derived, fibroblast-rich, mass-produced by intrathecal morphine infusion is not produced by all opiates, but reduced by mast cell stabilizers, the authors hypothesized a role for meningeal mast cell/fibroblast activation. Using the guinea pig, the authors asked: (1) Are intrathecal morphine masses blocked by opiate antagonism?; (2) Do opioid agonists not producing mast cell degranulation or fibroblast activation produce masses?; and (3) Do masses covary with Mas-related G protein-coupled receptor signaling thought to mediate mast cell degranulation? METHODS: In adult male guinea pigs (N = 66), lumbar intrathecal catheters connected to osmotic minipumps (14 days; 0.5 µl/h) were placed to deliver saline or equianalgesic concentrations of morphine sulfate (33 nmol/h), 2',6'-dimethyl tyrosine-(Tyr-D-Arg-Phe-Lys-NH2) (abbreviated as DMT-DALDA; 10 pmol/h; µ agonist) or PZM21 (27 nmol/h; biased µ agonist). A second pump delivered subcutaneous naltrexone (25 µg/h) in some animals. After 14 to 16 days, animals were anesthetized and perfusion-fixed. Drug effects on degranulation of human cultured mast cells, mouse embryonic fibroblast activation/migration/collagen formation, and Mas-related G protein-coupled receptor activation (PRESTO-Tango assays) were determined. RESULTS: Intrathecal infusion of morphine, DMT-DALDA or PZM21, but not saline, comparably increased thermal thresholds for 7 days. Spinal masses proximal to catheter tip, composed of fibroblast/collagen type I (median: interquartile range, 0 to 4 scale), were produced by morphine (2.3: 2.0 to 3.5) and morphine plus naltrexone (2.5: 1.4 to 3.1), but not vehicle (1.2: 1.1 to 1.5), DMT-DALDA (1.0: 0.6 to 1.3), or PZM21 (0.5: 0.4 to 0.8). Morphine in a naloxone-insensitive fashion, but not PZM21 or DMT-DALDA, resulted in mast cell degranulation and fibroblast proliferation/collagen formation. Morphine-induced fibroblast proliferation, as mast cell degranulation, is blocked by cromolyn. Mas-related G protein-coupled receptor activation was produced by morphine and TAN67 (∂-opioid agonist), but not by PZM21, TRV130 (mu biased ligand), or DMT-DALDA. CONCLUSIONS: Opiates that activate Mas-related G protein-coupled receptor will degranulate mast cells, activate fibroblasts, and result in intrathecal mass formation. Results suggest a mechanistically rational path forward to safer intrathecal opioid therapeutics.

Solid-Phase Azopeptide Diels-Alder Chemistry for Aza-pipecolyl Residue Synthesis To Study Peptide Conformation.

Solid-phase chemistry for the synthesis and Diels-Alder reaction of Fmoc-protected azopeptides has been developed and used to construct aza-pipecolyl (azaPip) peptides. Considering their ability to induce electronic and structural constraints that favor cis-amide isomer geometry and type VI ß-turn conformation in model peptides, azaPip residues have now been introduced into biologically relevant targets by this enabling synthetic method. Turn conformers were shown to be important for receptor affinity, selectivity, and activity by employing azaPip residues to study the conformational requirements of opioid and cluster of differentiation 36 receptor peptide ligands.

Characterization of Analgesic Actions of the Chronic Intrathecal Infusion of H-Dmt-D-Arg-Phe-Lys-NH2 in Rat.

OBJECTIVES: DMT-DALDA (H-Dmt-D-Arg-Phe-Lys-NH2; Dmt = 2',6'-dimethyltyrosine) is a selective mu opioid agonist. We sought to characterize efficacy, tolerance, dependence and side-effect profile when given by continuous intrathecal infusion. MATERIALS AND METHODS: Adult male Sprague Dawley rats were prepared with chronic intrathecal catheters and osmotic mini-pumps to deliver vehicle (saline), DMT-DALDA or morphine. Hind paw thermal escape latencies were assessed. In addition, effects upon intraplantar formalin-evoked flinching and withdrawal after 14 days of infusion were examined. The flare response after intradermal delivery was examined in the canine model. RESULTS: 1) Intrathecal infusion of 0.3 to 30 pmol/µL/hour of DMT-DALDA or 37.5 nmol/µL/hour of morphine more than 7 or 14 days resulted in a dose-dependent increase in thermal escape latency. The maximum antinociceptive effect was observed between 1 and 4 days after start of infusion with preserved cornea, blink, placing and stepping. By days 12 to 14, response latencies were below baseline. 2) On days 2 to 4 of DMT-DALDA infusion, the pan opioid receptor antagonist naloxone (Nx), but not the delta-preferring antagonist naltrindole, antagonized the analgesic effects. 3) Assessment of formalin flinching on day 1 following IT DMT-DALDA Infusion showed significant analgesia in phases 1 and 2. On day 6 of infusion there was minimal effect, while on day 13, there was an increase in flinching. 4) On days 7 and 14 of infusion Nx resulted in prominent withdrawal signs indicating dependence and withdrawal. 5) Intradermal morphine and DMT-DALDA both yield a naltrexone-insensitive, cromolyn-sensitive flare in the canine model at similar concentrations. CONCLUSIONS: These data suggest that DMT-DALDA is a potent, spinally active agonist with a propensity to produce tolerance dependence and mast cell degranulation. While it was equiactive to morphine in producing mast cell degranulation, it was >1000 fold more potent in producing analgesia, suggesting a possible lower risk in producing a spinal mass at equianalgesic doses.

Equipotent enantiomers of cyclic opioid peptides at µ opioid receptor.

Head-to-tail cyclized analogues of the µ opioid receptor (MOR) agonist tetrapeptides DALDA (H-Tyr-D-Arg-Phe-Lys-NH2 and [Dmt1]DALDA (H-Dmt-D-Arg-Phe-Lys-NH2; Dmt = 2',6'-dimethyltyrosine) and their enantiomers (mirror-image isomers) were synthesized and pharmacologically characterized in vitro. Three pairs of enantiomeric cyclic peptides with both mirror-image isomers having equipotent MOR binding affinities but different binding affinities at the δ and κ opioid receptors were identified. The cyclic peptide enantiomers c[-D-Arg-Phe-Lys-Tyr-] (1) and c[-Arg-D-Phe-D-Lys-D-Tyr-] (2) showed nearly identical MOR binding affinity (1 - 2 nM) and equipotent MOR antagonist activity. The results of a MOR docking study indicated a very similar binding mode of the two enantiomers with nearly complete spatial overlap of the peptide ring structures and side chain interactions with the same MOR residues. Compounds 1 and 2 represent the first pair of enantiomeric G-protein-coupled receptor (GPCR) ligands having multiple chiral centers, with both optical antipodes showing equal, low nanomolar receptor binding affinity.

Characterization of the antinociceptive effects of intrathecal DALDA peptides following bolus intrathecal delivery.

Background and aims We systematically characterized the potency and side effect profile of a series of small opioid peptides with high affinity for the mu opioid receptor. Methods Male Sprague Dawley rats were prepared with intrathecal (IT) catheters, assessed with hind paw thermal escape and evaluated for side effects including Straub tail, truncal rigidity, and pinnae and corneal reflexes. In these studies, DMT-DALDA (dDAL) (H-Dmt-D-Arg-Phe-Lys-NH2 MW=981), dDALc (H-Dmt-Cit-Phe-Lys-NH2 MW=868), dDALcn (H-Dmt-D-Cit-Phe-Nle-NH2 MW=739), TAPP (H-Tyr-D-Ala-Phe-Phe-NH2 MW=659), dDAL-TICP ([Dmt1]DALDA-(CH2)2-NH-TICP[psi]; MW=1519), and dDAL-TIPP (H-Dmt-D-Arg-Phe-Lys(Nε-TIPP)-NH2 were examined. In separate studies, the effects of approximately equiactive doses of IT DMT DALDA (10 pmol), morphine (30 nmol) and fentanyl (1 nmol) were examined on formalin-induced flinching at different pretreatment intervals (15 min - 24 h). Results (1) All agents resulted in a dose-dependent reversible effect upon motor function (Straub Tail>Truncal rigidity). (2) The ordering of analgesic activity (%MPE) at the highest dose lacking reliable motor signs after bolus delivery was: DMT-DALDA (80%±6/3 pmol); dDALc (75%±8/1 pmol); dDALcn (84%±10/300 pmol); TAPP (56%±12/10 nmol); dDAL-TICP (52%±27/300 pmol). (3) All analgesic effects were reversed by systemic (IP) naloxone (1 mg/kg). Naltrindole (3 mg/kg, IP) had no significant effect upon the maximum usable peptide dose. (4) Tolerance and cross-tolerance development after 5 daily boluses of DMT-DALDA (3 pmol) and morphine (30 nmol) revealed that both agents displayed a progressive decline over 5 days. (5) Cross-tolerance assessed at day 5 revealed a reduction in response to morphine in DMT-DALDA treated animal but not DMT-DALDA in the morphine treated animal, indicating an asymmetric cross-tolerance. (6) IT DMT-DALDA, morphine and fentanyl resulted in significant reductions in phase 1 and phase 2 flinching. With a 15 min pretreatment all drugs resulted in comparable reductions in flinching. However, at 6 h, the reduction in flinching after DMT-DALDA and morphine were comparably reduced while fentanyl was not different from vehicle. All effects on flinching were lost by 24 h. Conclusions These results emphasize the potent mu agonist properties of the DALDA peptidic structure series, their persistence similar to morphine and their propensity to produce tolerance. The asymmetric cross-tolerance between equiactive doses may reflect the relative intrinsic activity of morphine and DMT-DALDA. Implications These results suggest that the DALDA peptides with their potency and duration of action after intrathecal delivery suggest their potential utility for their further development as a spinal therapeutic to manage pain.

Strengthening peptide-based drug activity with novel glyconanoparticle.

The therapeutic application of peptide-based drugs is significantly limited by the rapid proteolytic degradation that occurs when in blood. Encapsulation of these peptide structures within a delivery system, such as liposomes, can greatly improve both stability and target delivery. As part of our work focused on novel ambiphilic mannosylated neoglycolipids as targeted drug delivery systems, we have developed a C14-alkyl-mannopyranoside that forms self-assembled monodisperse liposomes. Herein, these glycoliposomes are investigated as a potential method to improve the plasma stability of peptide-based drugs. Reversed phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry (MS) methods were developed to assess the in vitro plasma stability of two structurally diverse peptides, including the kappa opioid receptor selective antagonist dynantin, and the NOD2 innate immune receptor ligand muramyl dipeptide (MDP). The RP-HPLC methods developed were able to resolve the peptides from background plasma contaminants and provided suitable response levels and linearity over an appropriate concentration range. Both compounds were found to be significantly degraded in rat plasma. Increasing degrees of both entrapment and stabilization were noted when dynantin was combined with the C14-alkyl-mannopyranoside in increasing peptide:glycoside ratios. The combination of MDP with the glycolipid also led to peptide entrapment, which greatly improved the plasma stability of the peptide. Overall, the results clearly indicate that the stability of peptide-based structures, which are subject to degradation in plasma, can be greatly improved via entrapment within C14-alkyl-mannopyranoside-bearing glycoliposomes.

Chemical space screening around Phe3 in opioid peptides: Modulating µ versus δ agonism by Suzuki-Miyaura cross-couplings.

In this study, affinities and activities of derivatized analogues of Dmt-dermorphin[1-4] (i.e. Dmt-d-Ala-Phe-GlyNH2, Dmt = 2',6'-dimethyl-(S)-tyrosine) for the µ opioid receptor (MOP) and δ opioid receptor (DOP) were evaluated using radioligand binding studies, functional cell-based assays and isolated organ bath experiments. By means of solid-phase or solution-phase Suzuki-Miyaura cross-couplings, various substituted regioisomers of the phenylalanine moiety in position 3 of the sequence were prepared. An 18-membered library of opioid tetrapeptides was generated via screening of the chemical space around the Phe3 side chain. These substitutions modulated bioactivity, receptor subtype selectivity and highly effective ligands with subnanomolar binding affinities, contributed to higher functional activities and potent analgesic actions. In search of selective peptidic ligands, we show here that the Suzuki-Miyaura reaction is a versatile and robust tool which could also be deployed elsewhere.

A Genetically Encoded Biosensor Reveals Location Bias of Opioid Drug Action.

Opioid receptors (ORs) precisely modulate behavior when activated by native peptide ligands but distort behaviors to produce pathology when activated by non-peptide drugs. A fundamental question is how drugs differ from peptides in their actions on target neurons. Here, we show that drugs differ in the subcellular location at which they activate ORs. We develop a genetically encoded biosensor that directly detects ligand-induced activation of ORs and uncover a real-time map of the spatiotemporal organization of OR activation in living neurons. Peptide agonists produce a characteristic activation pattern initiated in the plasma membrane and propagating to endosomes after receptor internalization. Drugs produce a different activation pattern by additionally driving OR activation in the somatic Golgi apparatus and Golgi elements extending throughout the dendritic arbor. These results establish an approach to probe the cellular basis of neuromodulation and reveal that drugs distort the spatiotemporal landscape of neuronal OR activation.

Protective Effect of a Mitochondria-Targeted Peptide against the Development of Chemotherapy-Induced Peripheral Neuropathy in Mice.

Several chemotherapeutic agents used for cancer treatment induce dose-limiting peripheral neuropathy that compromises patients' quality of life and limits cancer treatment. Recently, mitochondrial dysfunction has been shown to be involved in the mechanism of chemotherapy-induced peripheral neuropathy. SS-20 is a mitochondria-targeted peptide that promotes mitochondrial respiration and restores mitochondrial bioenergetics. In the present study, we examined the protective effect of SS-20 against the development of chemotherapy-induced peripheral neuropathy utilizing a murine model of peripheral neuropathy induced by oxaliplatin, a first-line chemotherapy agent for colon cancer. Weekly administrations of oxaliplatin induced peripheral neuropathy as demonstrated by the development of neuropathic pain and loss of intraepidermal nerve fibers in the hind paw. Continuous administration of SS-20 protected against the development of oxaliplatin-induced neuropathic pain and mitigated the loss of intraepidermal nerve fibers to normal levels. Our findings suggest that SS-20 may be a drug candidate for the prevention of chemotherapy-induced peripheral neuropathy.

χ-Space Screening of Dermorphin-Based Tetrapeptides through Use of Constrained Arylazepinone and Quinolinone Scaffolds.

Herein, the synthesis of novel conformationally constrained amino acids, 4-amino-8-bromo-2-benzazepin-3-one (8-Br-Aba), 3-amino-3,4-dihydroquinolin-2-one, and regioisomeric 4-amino-naphthoazepinones (1- and 2-Ana), is described. Introduction of these constricted scaffolds into the N-terminal tetrapeptide of dermorphin (i.e., H-Tyr-d-Ala-Phe-Gly-NH2) induced significant shifts in binding affinity, selectivity, and in vitro activity at the µ- and δ-opioid receptors (MOP and DOP, respectively). A reported constrained µ-/δ-opioid lead tetrapeptide H-Dmt-d-Arg-Aba-Gly-NH2 was modified through application of various constrained building blocks to identify optimal spatial orientations in view of activity at the opioid receptors. Interestingly, when the aromatic moieties were turned toward the C-terminus of the peptide sequences, (partial) (ant)agonism at MOP and weak (ant)agonism at DOP were noticed, whereas the incorporation of the 1-Ana residue led toward balanced low nanomolar MOP/DOP binding and in vitro agonism.

Analgesic Properties of Opioid/NK1 Multitarget Ligands with Distinct in Vitro Profiles in Naive and Chronic Constriction Injury Mice.

The lower efficacy of opioids in neuropathic pain may be due to the increased activity of pronociceptive systems such as substance P. We present evidence to support this hypothesis in this work from the spinal cord in a neuropathic pain model in mice. Biochemical analysis confirmed the elevated mRNA and protein level of pronociceptive substance P, the major endogenous ligand of the neurokinin-1 (NK1) receptor, in the lumbar spinal cord of chronic constriction injury (CCI)-mice. To improve opioid efficacy in neuropathic pain, novel compounds containing opioid agonist and neurokinin 1 (NK1) receptor antagonist pharmacophores were designed. Structure-activity studies were performed on opioid agonist/NK1 receptor antagonist hybrid peptides by modification of the C-terminal amide substituents. All compounds were evaluated for their affinity and in vitro activity at the mu opioid (MOP) and delta opioid (DOP) receptors, and for their affinity and antagonist activity at the NK1 receptor. On the basis of their in vitro profiles, the analgesic properties of two new bifunctional hybrids were evaluated in naive and CCI-mice, representing models for acute and neuropathic pain, respectively. The compounds were administered to the spinal cord by lumbar puncture. In naive mice, the single pharmacophore opioid parent compounds provided better analgesic results, as compared to the hybrids (max 70% MPE), raising the acute pain threshold close to 100% MPE. On the other hand, the opioid parents gave poor analgesic effects under neuropathic pain conditions, while the best hybrid delivered robust (close to 100% MPE) and long lasting alleviation of both tactile and thermal hypersensitivity. The results presented emphasize the potential of opioid/NK1 hybrids in view of analgesia under nerve injury conditions.

A Cyclic Tetrapeptide ("Cyclodal") and Its Mirror-Image Isomer Are Both High-Affinity µ Opioid Receptor Antagonists.

Head-to-tail cyclization of the µ opioid receptor (MOR) agonist [Dmt1]DALDA (H-Dmt-d-Arg-Phe-Lys-NH2 (9; Dmt = 2',6'-dimethyltyrosine) resulted in a highly active, selective MOR antagonist, c[-d-Arg-Phe-Lys-Dmt-] (1) ("cyclodal"), with subnanomolar binding affinity. A docking study of cyclodal using the crystal structure of MOR in the inactive form showed a unique binding mode with the two basic residues of the ligand forming salt bridges with the Asp127 and Glu229 receptor residues. Cyclodal showed high plasma stability and was able to cross the blood-brain barrier to reverse morphine-induced, centrally mediated analgesia when given intravenously. Surprisingly, the mirror-image isomer (optical antipode) of cyclodal, c[-Arg-d-Phe-d-Lys-d-Dmt-] (2), also turned out to be a selective MOR antagonist with 1 nM binding affinity, and thus, these two compounds represent the first example of mirror image opioid receptor ligands with both optical antipodes having high binding affinity. Reduction of the Lys-Dmt peptide bond in cyclodal resulted in an analogue, c[-d-Arg-Phe-LysΨ[CH2NH]Dmt-] (8), with MOR agonist activity.

Prorenin/renin receptor blockade promotes a healthy fat distribution in obese mice.

OBJECTIVE: Administration of the handle region peptide (HRP), a (pro)renin receptor blocker, decreases body weight gain and visceral adipose tissue (VAT) in high-fat/high-carbohydrate (HF/HC) diet-fed mice. The objective of this study was to elucidate potential mechanisms implicated in these observations. METHODS: Mice were given a normal or a HF/HC diet along with saline or HRP for 10 weeks. RESULTS: In HF/HC-fed mice, HRP increased the expression of several enzymes implicated in lipogenesis and lipolysis in subcutaneous fat (SCF) while the expression of the enzyme implicated in the last step of lipogenesis decreased in VAT. A reduction was also observed in circulating free fatty acids in these animals which was accompanied by normalized adipocyte size in VAT and increased adipocyte size in SCF. ''Beiging'' is the evolution of a white adipose tissue toward a brown-like phenotype characterized by an increased mitochondrial density and small lipid droplets. HRP increased the expression of' "beiging" markers in SCF of HF/HC diet-fed mice. CONCLUSIONS: HRP treatment may favor healthy fat storage in SCF by activating a triglyceride/free fatty acid cycling and "beiging," which could explain the body weight and fat mass reduction.

Molecular Pharmacology of δ-Opioid Receptors.

Opioids are among the most effective analgesics available and are the first choice in the treatment of acute severe pain. However, partial efficacy, a tendency to produce tolerance, and a host of ill-tolerated side effects make clinically available opioids less effective in the management of chronic pain syndromes. Given that most therapeutic opioids produce their actions via µ-opioid receptors (MOPrs), other targets are constantly being explored, among which δ-opioid receptors (DOPrs) are being increasingly considered as promising alternatives. This review addresses DOPrs from the perspective of cellular and molecular determinants of their pharmacological diversity. Thus, DOPr ligands are examined in terms of structural and functional variety, DOPrs' capacity to engage a multiplicity of canonical and noncanonical G protein-dependent responses is surveyed, and evidence supporting ligand-specific signaling and regulation is analyzed. Pharmacological DOPr subtypes are examined in light of the ability of DOPr to organize into multimeric arrays and to adopt multiple active conformations as well as differences in ligand kinetics. Current knowledge on DOPr targeting to the membrane is examined as a means of understanding how these receptors are especially active in chronic pain management. Insight into cellular and molecular mechanisms of pharmacological diversity should guide the rational design of more effective, longer-lasting, and better-tolerated opioid analgesics for chronic pain management.

[Dmt(1)]DALDA analogues modified with tyrosine analogues at position 1.

Analogues of [Dmt(1)]DALDA (H-Dmt-d-Arg-Phe-Lys-NH2; Dmt=2',6'-dimethyltyrosine), a potent µ opioid agonist peptide with mitochondria-targeted antioxidant activity were prepared by replacing Dmt with various 2',6'-dialkylated Tyr analogues, including 2',4',6'-trimethyltyrosine (Tmt), 2'-ethyl-6'-methyltyrosine (Emt), 2'-isopropyl-6'-methyltyrosine (Imt) and 2',6'-diethyltyrosine (Det). All compounds were selective µ opioid agonists and the Tmt(1)-, Emt(1) and Det(1)-analogues showed subnanomolar µ opioid receptor binding affinities. The Tmt(1)- and Emt(1)-analogues showed improved antioxidant activity compared to the Dmt(1)-parent peptide in the DPPH radical-scavenging capacity assay, and thus are of interest as drug candidates for neuropathic pain treatment.