The TIPP opioid peptide family: development of delta antagonists, delta agonists, and mixed mu agonist/delta antagonists

The discovery of the prototype delta opioid antagonists TIPP (H-Tyr-Tic-Phe-Phe-OH) and TIP (H-Tyr-Tic-Phe-OH) in 1992 was followed by extensive structure-activity relationship studies, leading to the development of analogues that are of interest as pharmacological tools or as potential therapeutic agents. Stable TIPP-derived delta opioid antagonists with subnanomolar delta receptor binding affinity and extraordinary delta receptor selectivity include TIPP[Psi] (H-Tyr-TicPsi[CH(2)NH]Phe-Phe-OH] and TICP[Psi] (H-Tyr-TicPsi[CH(2)NH]Cha-Phe-OH); Cha: cyclohexylalanine), which are widely used in opioid research. Theoretical conformational analyses in conjunction with the pharmacological characterization of conformationally constrained TIPP analogues led to a definitive model of the receptor-bound conformation of H-Tyr-Tic-(Phe-Phe)-OH-related delta opioid antagonists, which is characterized by all-trans peptide bonds. Further structure-activity studies revealed that the delta antagonist vs delta agonist behavior of TIP(P)-derived compounds depended on very subtle structural differences in diverse locations of the molecule and suggested a delta receptor model involving a number of different inactive receptor conformations. A further outcome of these studies was the identification of a new class of potent and very selective dipeptide delta agonists of the general formula H-Tyr-Tic-NH-X (X = arylalkyl), which are of interest for drug development because of their low molecular weight and lipophilic character. Most interestingly, TIPP analogues containing a C-terminal carboxamide group displayed a mixed mu agonist/delta antagonist profile, and thus were expected to be analgesics with a low propensity to produce tolerance and physical dependence. This turned out to be the case with the TIPP-derived mu agonist/delta antagonist DIPP-NH(2)[Psi] (H-Dmt-TicPsi[CH(2)NH]Phe-Phe-NH(2)); Dmt: 2',6'- dimethyltyrosine).     

Benzylideneoxymorphone: A new lead for development of bifunctional mu/delta opioid receptor ligands

Opioid analgesic tolerance remains a considerable drawback to chronic pain management. The finding that concomitant administration of delta opioid receptor (DOR) antagonists attenuates the development of tolerance to mu opioid receptor (MOR) agonists has led to interest in producing bifunctional MOR agonist/DOR antagonist ligands. Herein, we present 7-benzylideneoxymorphone (6, UMB 246) displaying MOR partial agonist/DOR antagonist activity, representing a new lead for designing bifunctional MOR/DOR ligands.     

Agonist and antagonist activities of ligands derived from naltrexone and oxymorphone.

The pharmacological profile of naltrindole (NTI) and three of its analogues, N-methyl-NTI (N-Me-NTI), oxymorphindole (OMI) and naltriben (NTB) were studied in antinociceptive assays. The compounds were found to have agonist activities that appear to be mediated mainly by kappa opioid receptors because norbinaltorphimine (nor-BNI), the selective kappa opioid receptor antagonist inhibited their effects significantly. All of the compounds, behaved as antagonists at doses that were lower than those that produced agonist effects and they possessed a profile that was very selective for inhibiting the antinociceptive activities of delta opioid receptor agonists. Differential antagonism by NTB of the activities of DSLET and DPDPE was demonstrated.     

Anticonvulsant effects of mu (DAGO) and delta (DPDPE) enkephalins in rats

The effects of highly selective mu and delta opioid peptide agonists were determined in two rat models of experimentally-induced convulsions, the flurothyl threshold test and the maximal electroshock test. Intracerebroventricular injections of the mu selective enkephalin DAGO (0.3-2.2 nmol) resulted in a dose-related protection in both seizure models. Pretreatment with a low dose of naloxone (29 nmol) or the irreversible mu antagonist beta-FNA (21 nmol), but not the delta opioid antagonist ICI 154,129 (50 nmol), antagonized the anticonvulsant actions of DAGO. Intracerebroventricular injections of the delta selective enkephalin DPDPE (70-140 nmol) also resulted in seizure protection. These effects were selectively antagonized by the delta antagonist ICI 174,864 (2.8 nmol), but not by pretreatment with beta-FNA. Thus, using agonists and antagonists highly selective for mu and delta opioid receptors, anticonvulsant actions of enkephalin have been described against chemically- and electrically-induced convulsions in rats.     

Reciprocal opioid-opioid interactions between the ventral tegmental area and nucleus accumbens regions in mediating mu agonist-induced feeding in rats

Feeding elicited by the mu-selective agonist, [D-Ala2, M-Phe4, Gly-ol5]-encephalin administered into the nucleus accumbens is blocked by accumbal pre-treatment with mu, delta1, delta2 and kappa, but not mu1 opioid antagonists. Correspondingly, mu-agonist-induced feeding elicited from the ventral tegmental area is blocked by ventral tegmental area pre-treatment with mu and kappa, but not delta opioid antagonists. A bi-directional opioid-opioid feeding interaction has been firmly established such that mu-agonist-induced feeding elicited from the ventral tegmental area is blocked by accumbal naltrexone, and that accumbal mu-agonist-induced feeding is blocked by naltrexone pre-treatment in the ventral tegmental area. To determine which opioid receptor subtypes mediate the regional bi-directional opioid-opioid feeding interactions between these two sites, the present study examined the dose-dependent ability of either general (naltrexone), mu (beta-funaltrexamine), kappa (nor-binaltorphamine) or delta (naltrindole) opioid antagonists administered into one site to block mu-agonist-induced feeding elicited from the other site. General, mu and kappa, but not delta opioid receptor antagonist pre-treatment in the ventral tegmental area dose-dependently reduced mu-agonist-induced feeding elicited from the nucleus accumbens. General, mu and delta, and to a lesser degree kappa, opioid receptor antagonist pre-treatment in the nucleus accumbens dose-dependently reduced mu-agonist-induced feeding elicited from the ventral tegmental area. Thus, multiple, but different opioid receptor subtypes are involved in mediating opioid-opioid feeding interactions between the nucleus accumbens and ventral tegmental area regions.     

High-affinity carbamate analogues of morphinan at opioid receptors

A series of carbamate analogues were synthesized from levorphanol (1a), cyclorphan (2a) or butorphan (3a) and evaluated in vitro for their binding affinity at mu, delta, and kappa opioid receptors. Functional activities of these compounds were measured in the [(35)S]GTPgammaS binding assay. Phenyl carbamate derivatives 2d and 3d showed the highest binding affinity for kappa receptor (K(i)=0.046 and 0.051 nM) and for mu receptor (K(i)=0.11 and 0.12 nM). Compound 1c showed the highest mu selectivity. The preliminary assay for agonist and antagonist properties of these ligands in stimulating [(35)S]GTPgammaS binding mediated by the kappa opioid receptor illustrated that all of these ligands were kappa agonists. At the mu receptor, compounds 1b, 1c, 2b, and 3b were agonists, while compounds 2c-e and 3c-e were mu agonists/antagonists.     

Switching agonist/antagonist properties of opiate alkaloids at the delta opioid receptor using mutations based on the structure of the orphanin FQ receptor

In an earlier study, we have demonstrated that by mutating five amino acid residues to those conserved in the opioid receptors, the OFQ receptor could be converted to a functional receptor that bound many opioid alkaloids with nanomolar affinities. Surprisingly, when the reciprocal mutations, Lys-214 --> Ala (TM5), Ile-277 --> Val/His-278 --> Gln/Ile-279 --> Val (TM6), and Ile-304 --> Thr (TM7), are introduced in the delta receptor, neither the individual mutations nor their various combinations significantly reduce the binding affinities of opioid alkaloids tested. However, these mutations cause profound alterations in the functional characteristics of the mutant receptors as measured in guanosine 5'-3-O-(thio)triphosphate binding assays. Some agonists become antagonists at some constructs as they lose their ability to activate them. Some alkaloid antagonists are transformed into agonists at other constructs, but their agonistic effects can still be blocked by the peptide antagonist TIPP. Even the delta inverse agonist 7-benzylidenenaltrexone becomes an agonist at the mutant containing both the Ile-277 --> Val/His-278 --> Gln/Ile-279 --> Val and Ile-304 --> Thr mutations. Thus, although the mutated residues are thought to be part of the binding pocket, they are critically involved in the control of the delta receptor activation process. These findings shed light on some of the structural bases of ligand efficacy. They are also compatible with the hypothesis that a ligand may achieve high affinity binding in several different ways, each having different effects on receptor activation.     

Effects of a novel opioid peptide antagonist on rat bladder motility in vivo

The agonist, and opioid antagonist, effects of intracerebroventricularly (ICV) given D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH₂ (CTP), a cyclic analogue of somatostatin octapeptide, were evaluated using the micturition reflex of the anesthetized rat as the endpoint. Antagonist effects were evaluated against equieffective doses of selective mu [D-Ala²,NMPhe⁴,Gly-ol]enkephalin (DAGO) and delta [D-Pen²,D-Pen⁵] enkephalin (DPDPE) opioid agonists. At low ICV doses, CTP preferentially antagonized DPDPE rather than DAGO; increasing the dose of CTP further effectively antagonized both mu and delta agonists, while even higher doses showed an agonist effect alone which was not blocked by adrenergic, cholinergic or opioid antagonists. Selective opioid antagonist doses of CTP failed to block the inhibition of the micturition reflex produced by pentobarbital. Possible residual somatostatin like properties of CTP were tested by using somatostatin as a possible antagonist of equieffective doses of DPDPE and DAGO; somatostatin did not antagonize these agonists. Repeated exposure to CTP resulted in the development of acute tolerance to the agonist effect, and also prevented the inhibition of the reflex by high doses of somatostatin, with the converse experiment showing a similar pattern; thus, repeated somatostatin resulted in tolerance and subsequent cross-tolerance to the agonist effects of CTP. In animals tolerant to somatostatin, CTP nevertheless behaved as an opioid antagonist. The present results indicate that CTP possesses opioid antagonist properties in vivo which are pharmacological in nature but nevertheless retains residual somatostatin-like activity at higher doses.     

Upregulation of opioid receptor subtypes correlates with potency changes of morphine and DADLE

Chronic treatment with opioid antagonists increases the potency of opioid agonists and produces an increase in brain opioid binding sites. In the present study, 8 day treatment with naltrexone blocked morphine and DADLE analgesia for the entire treatment period and increased mu 1, mu 2 and delta opioid receptor binding sites in mouse brain. mu 1 and mu 2 binding were increased by 81 and 67%, respectively, while delta binding was increased by 31%. Consistent with these binding changes, the potency of ICV morphine to produce analgesia was increased by over 3-fold, while the potency of ICV DADLE was increased by only 1.7. These findings indicate that relative increases in opioid receptor subtypes agree with pharmacodynamic studies on potency changes of opioid agonists.     

Pyrrolo- and pyridomorphinans: Non-selective opioid antagonists and delta opioid agonists/mu opioid partial agonists

Opioid ligands have found use in a number of therapeutic areas, including for the treatment of pain and opiate addiction (using agonists) and alcohol addiction (using antagonists such as naltrexone and nalmefene). The reaction of imines, derived from the opioid ligands oxymorphone and naltrexone, with Michael acceptors leads to pyridomorphinans with structures similar to known pyrrolo- and indolomorphinans. One of the synthesized compounds, 5e, derived from oxymorphone had substantial agonist activity at delta opioid receptors but not at mu and/or kappa opioid receptors and in that sense profiled as a selective delta opioid receptor agonist. The pyridomorphinans derived from naltrexone and naloxone were all found to be non-selective potent antagonists and as such could have utility as treatments for alcohol abuse.     

Conformation-activity relationships of opioid peptides with selective activities at opioid receptors

The discovery of endogenous opioid peptides 25 years ago opened up a new chapter in efforts to understand the origins and control of pain, its relationships to other biological functions, including inflammatory and other immune responses, and the relationships of opioid peptides and their receptors to a variety of undesirable or toxic side effects often associated with the nonpeptide opiates such as morphine including addiction, constipation, a variety of neural toxicities, tolerance, and respiratory depression. For these investigations the need for potent and highly receptor selective agonists and antagonists has been crucial since they in principle allow one to distinguish unequivocally the roles of the different opioid receptors (mu, delta, and kappa) in the various biological and pathological roles of the opioid peptides and their receptors. Conformational and topographical constraint of the linear natural endogenous opioid peptides has played a major role in developing peptide ligands with high selectivity for mu, delta, and kappa receptors, and in understanding the conformational, topographical, and stereoelectronic structural requirements of the opioid peptides for their interactions with opioid receptors. In turn, this had led to insights into the three-dimensional pharmacophore for opioid receptors. In this article we review and discuss some of the developments that have led to potent, selective, and stable peptide and peptidomimetic ligands that are highly potent and selective, and that have delta agonist, mu antagonist, and kappa agonist biological activities (other authors in this issue will discuss the development of other types of activities and selectivities). These have led to ligands that provide unique insight into opioid pharmacophores and the critical roles opioid ligands and receptor scan play in pain, addiction, and other human maladies.     

Inhibition of an opioid-evoked vagal reflex in rats by naloxone,SMS 201-995 and ICI 154,129

Intravenous injection of opioid agonists in rats evokes a vagal reflex resulting in a fall in heart rate and blood pressure. Three opioid antagonists, naloxone, SMS 201-995, and ICI 154,129 were used to assess the nature of the opioid receptors that mediate the vagal reflex. The agonists used were morphine, Tyr-Pro-NMePhe-d-Pro-NH₂ (PLO17), and d-Ala²-Leu⁵-enkephalin (DADL). At challenge doses of morphine, PLO17, and DADL at five times the ED50 for bradycardia, the naloxone ED50 for DADL was nine times greater than that for morphine and PLO17. The pA₂ value of naloxone against DADL was significantly less than that for morphine and PLO17. The antagonist properties of SMS 201-995 were similar to those of naloxone. ICI 154,129, a putative delta receptor antagonist, was not, however, selective in its antagonism of opioid bradycardia. Both SMS 201-995 and ICI 154,129, when injected alone, produced changes in heart rate and blood pressure. The cardiovascular actions of the peptide antagonists were not affected by naloxone hydrochloride at doses up to 4 mg/kg i.v.     

Modeling and simulation of the human delta opioid receptor

A model for the human delta opioid receptor has been generated via sequence alignment, structure building using the crystal structure of bovine rhodopsin as a template, and refinement by molecular dynamics simulation. The model building suggested that, in addition to the previously postulated interaction between D128 and Y308, an internal salt bridge also exists between residues D128 and R192, both of which are conserved in all the opioid receptors. The model and salt bridge were then shown to be stable during a 20-nsec simulation in a lipid bilayer. It is therefore proposed that both of these interactions play a role in stabilizing the inactive state of the receptor. The model is also used in an effort to rationalize many of the mutational studies performed on delta opioid receptors, and to suggest a plausible explanation for the differences between known delta opioid agonists and antagonists.     

NPY-induced feeding: pharmacological characterization using selective opioid antagonists and antisense probes in rats

The ability of neuropeptide Y to potently stimulate food intake is dependent in part upon the functioning of mu and kappa opioid receptors. The combined use of selective opioid antagonists directed against mu, delta or kappa receptors and antisense probes directed against specific exons of the MOR-1, DOR-1, KOR-1 and KOR-3/ORL-1 opioid receptor genes has been successful in characterizing the precise receptor subpopulations mediating feeding elicited by opioid peptides and agonists as well as homeostatic challenges. The present study examined the dose-dependent (5-80 nmol) cerebroventricular actions of general and selective mu, delta, and kappa1 opioid receptor antagonists together with antisense probes directed against each of the four exons of the MOR-1 opioid receptor gene and each of the three exons of the DOR-1, KOR-1, and KOR-3/ORL-1 opioid receptor genes upon feeding elicited by cerebroventricular NPY (0.47 nmol, 2 ug). NPY-induced feeding was dose-dependently decreased and sometimes eliminated following pretreatment with general, mu, delta, and kappa1 opioid receptor antagonists. Moreover, NPY-induced feeding was significantly and markedly reduced by antisense probes directed against exons 1, 2, and 3 of the MOR-1 gene, exons 1 and 2 of the DOR-1 gene, exons 1, 2, and 3 of the KOR-1 gene, and exon 3 of the KOR-3/ORL-1 gene. Thus, whereas the opioid peptides, beta-endorphin and dynorphin A(1-17) elicit feeding responses that are respectively more dependent upon mu and kappa opioid receptors and their genes, the opioid mediation of NPY-induced feeding appears to involve all three major opioid receptor subtypes in a manner similar to that observed for feeding responses following glucoprivation or lipoprivation.     

Role of delta opioid receptors and their ligands in the development of adaptive heart protection against arrhythmogenesis

It has been found that stimulation of delta-1 opioid receptors by intravenous administration of DPDPE (0.5 mg/kg) decreases the incidence of ischemic and reperfusion-induced arrhythmias and also increases myocardial tolerance to the arrhythmogenic action of epinephrine in rats. Pretreatment with a selective delta-2 agonist, DSLET, had no antiarrhythmic effect. The inhibition of the enzymatic breakdown of endogenous enkephalins by intravenous administration of acetorphan decreased the incidence of epinephrine-induced arrhythmias. Pretreatment with a selective delta opioid receptor antagonist, ICI-174.868, completely abolished this antiarrhythmic effect. Adaptation of rats to repeated immobilization stress during 12 days increased myocardial tolerance to the arrhythmogenic action of coronary artery occlusion (10 min) and reperfusion (10 min). Pretreatment with a selective delta opioid receptor antagonist, TIPP(Psy), did not abolish the antiarrhythmic effect of adaptation to immobilization stress. It seems that endogenous agonists of delta opioid receptors are not involved in the antiarrhythmic effect resulting from adaptation to stress.     

Tramadol reduces the 5-HTP-induced head-twitch response in mice via the activation of mu and kappa opioid receptors

Tramadol, an atypical opioid analgesic, stimulates both opiatergic and serotonergic systems. Here we have investigated the effect of tramadol in mice on 5-hydroxyptrytophan (5-HTP)-induced head twitch response (HTR), which is an animal model for the activation of the CNS 5-HT(2A) receptors in mice. Tramadol attenuated 5-HTP-induced HTR in a dose-dependent manner as morphine. Furthermore, the nonselective opioid receptor antagonists, naloxone and diprenorphine (M5050), reversed the effect of tramadol on 5-HTP-induced HTR dose-dependently. Interestingly, in contrast to the selective delta opioid receptor antagonist NTI, beta-FNA, a selective mu receptor antagonist, and nor-BNI, a selective kappa opioid receptor antagonist, antagonized the attenuation of 5-HTP-induced HTR by tramadol. In conclusion, administration of tramadol systemically inhibits 5-HTP-induced HTR in mice by activating opiatergic system in the CNS. Our findings show that mu and kappa opioid receptors, but not delta opioid receptor, play an important role in the regulation of serotonergic function in the CNS.     

Endogenous opioids and feeding behavior: A decade of further progress (2004–2014). A Festschrift to Dr. Abba Kastin

Functional elucidation of the endogenous opioid system temporally paralleled the creation and growth of the journal, Peptides, under the leadership of its founding editor, Dr. Abba Kastin. He was prescient in publishing annual and uninterrupted reviews on Endogenous Opiates and Behavior that served as a microcosm for the journal under his stewardship. This author published a 2004 review, “Endogenous opioids and feeding behavior: a thirty-year historical perspective”, summarizing research in this field between 1974 and 2003. The present review “closes the circle” by reviewing the last 10 years (2004–2014) of research examining the role of endogenous opioids and feeding behavior. The review summarizes effects upon ingestive behavior following administration of opioid receptor agonists, in opioid receptor knockout animals, following administration of general opioid receptor antagonists, following administration of selective mu, delta, kappa and ORL-1 receptor antagonists, and evaluating opioid peptide and opioid receptor changes in different food intake models.     

Nonselective coupling of the human mu-opioid receptor to multiple inhibitory G-protein isoforms.

The human mu-opioid receptor was expressed in Saccharomyces cerevisiae. Binding of [3H]diprenorphine to yeast spheroplasts was specific and saturable (Kd = 1 nm, Bmax = 0.2-1 pmol x mg-1 of membrane proteins). Inhibition of [3H]diprenorphine binding by antagonists and agonists with varying opioid selectivities (mu, delta and kappa) occurred with the same order of potency as in mammalian tissues. Affinities of antagonists were the same with yeast spheroplasts as in reference tissues whereas those of agonists, except etorphine and buprenorphine, were 10-fold to 100-fold lower. Addition of heterotrimeric Gi,o-proteins purified from bovine brain shifted the mu-opioid receptor into a high-affinity state for agonists. Using individually purified Galpha-subunits re-associated with betagamma-dimers, we showed that alphao1, alphao2, alphai1, alphai2 and alphai3 reconstituted high-affinity agonist binding with equal efficiency. This suggests that the structural determinants of the mu-opioid receptor responsible for G-protein coupling are not able to confer a high degree of specificity towards any member of the Gi,o family. The selective effects of opioid observed in specialized tissues upon opioid stimulation may be a result of regulation of G-protein activity by cell-specific factors which should conveniently be analysed using the reconstitution assay described here.     

14-O-Heterocyclic-substituted naltrexone derivatives as non-peptide mu opioid receptor selective antagonists: Design,synthesis, and biological studies

Mu opioid receptor antagonists have clinical utility and are important research tools. To develop non-peptide and highly selective mu opioid receptor antagonist, a series of 14-O-heterocyclic-substituted naltrexone derivatives were designed, synthesized, and evaluated. These compounds showed subnanomolar-to-nanomolar binding affinity for the mu opioid receptor. Among them, compound 1 exhibited the highest selectivity for the mu opioid receptor over the delta and kappa receptors. These results implicated an alternative ‘address’ domain in the extracellular loops of the mu opioid receptor.     

Opioid-Inhibited Adenylyl Cyclase in Rat Brain Membranes: Lack of Correlation with High-Affinity Opioid Receptor Binding Sites

Opioid agonists bind to GTP-binding (G-protein)-coupled receptors to inhibit adenylyl cyclase. To explore the relationship between opioid receptor binding sites and opioid-inhibited adenylyl cyclase, membranes from rat striatum were incubated with agents that block opioid receptor binding. These agents included irreversible opioid agonists (oxymorphone-p-nitrophenylhydrazone), irreversible antagonists [naloxonazine, beta-funaltrexamine, and beta-chlornaltrexamine (beta-CNA)], and phospholipase A2. After preincubation with these agents, the same membranes were assayed for high-affinity opioid receptor binding [3H-labeled D-alanine-4-N-methylphenylalanine-5-glycine-ol-enkephalin (mu), 3H-labeled 2-D-serine-5-L-leucine-6-L-threonine enkephalin (delta), and [3H]ethylketocylazocine (EKC) sites] and opioid-inhibited adenylyl cyclase. Although most agents produced persistent blockade in binding of ligands to high-affinity mu, delta, and EKC sites, no change in opioid-inhibited adenylyl cyclase was detected. In most treated membranes, both the IC50 and the maximal inhibition of adenylyl cyclase by opioid agonists were identical to values in untreated membranes. Only beta-CNA blocked opioid-inhibited adenylyl cyclase by decreasing maximal inhibition and increasing the IC50 of opioid agonists. This effect of beta-CNA was not due to nonspecific interactions with G(i), Gs, or the catalytic unit of adenylyl cyclase, as neither guanylylimidodiphosphate-inhibited, NaF-stimulated, nor forskolin-stimulated activity was altered by beta-CNA pretreatment. Phospholipase A2 decreased opioid-inhibited adenylyl cyclase only when the enzyme was incubated with brain membranes in the presence of NaCl and GTP. These results confirm that the receptors that inhibit adenylyl cyclase in brain do not correspond to the high-affinity mu, delta, or EKC sites identified in brain by traditional binding studies.