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Given that μ opioid (MOP) andcanabinoid (CB1)receptors are colocalized in various regions of the central nervoussystem and have been reported to associate as heteromer (MOP-CB1) in cultured cells, the possibility of functional, endogenousMOP-CB1in nociception and other pharmacologic effectshas been raised. As a first step in investigating this possibility,we have synthesized a series of bivalent ligands 1–5that contain both μ agonist and CB1antagonistpharmacophores for use as tools to study the functional interactionbetween MOP and CB1receptors in vivo. Immunofluorescentstudies on HEK293 cells coexpressing both receptors suggested 5(20-atom spacer) to be the only member of the series thatbridges the protomers of the heteromer. Antinociceptive testing inmice revealed 5to be the most potent member of the series.As neither a mixture of monovalent ligands 9+ 10nor bivalents 2–5producedtolerance in mice, MOR-CB1apparently is not an importanttarget for reducing tolerance. [ABSTRACT FROM AUTHOR]

MDAN-21, 7'-{2-[(7-{2-[({(5α,6α)-4,5-Epoxy-3,14-dihydroxy-17-methylmorphin-6-yl}-aminocarbonyl)metoxy]-acetylamino} -heptylaminocarbonyl)-methoxy]-acetylamino}-naltrindole, a bivalent opioid ligand containing a mu-opioid receptor agonist (derived from oxymorphone) linked to the delta-opioid receptor antagonist (related to naltrindole) by a spacer of 21 atoms, was reported to have potent analgesic properties in mice. Tolerance, physical dependence, and conditioned place preference were not evident in that species. The finding that bivalent ligands in this series, with spacers 19 atoms or greater, were devoid of tolerance and dependence led to the proposal that MDAN-21 targets heteromeric mu-delta-opioid receptors. The present study focused on its effects in nonhuman primates (Macaca mulatta), a species with a physiology and behavioral repertoire not unlike humans. With regard to opioids, this species usually better predicts clinical outcomes. MDAN-21 substituted for morphine in morphinedependent monkeys in the remarkably low dose range 0.006-0.032 mg/kg, subcutaneously. Although MDAN-21 failed to produce reliable thermal analgesia in the dose range 0.0032-0.032 mg/kg, intramuscularly, it was active in the same dose range and by the same route of administration, in the capsaicin-induced thermal allodynia assay. The results suggest that MDAN-21 may be useful in the treatment of opioid dependence and allodynia. The data provide additional evidence that opioid withdrawal is associated with sensitized pain. [ABSTRACT FROM AUTHOR]

CCK2 receptor antagonists potentiate pain relief by MOP receptor agonists. In an attempt to enhance this effect, we prepared bivalent ligands incorporating CCK2 receptor antagonist and MOP receptor agonist pharmacophores. Ligands with 16- to 22-atom spacers could simultaneously bind both receptors but provided no advantage in activity over individual ligands. We now examine the effect of these ligands on receptor internalization as a mechanism of receptor regulation. We prepared CHO cell lines expressing nonfluorescent halves (YN and YC) of yellow fluorescent protein attached to each receptor. Spatial approximation of constructs was needed to yield fluorescence. Monovalent MOP agonist 1 signaled normally and internalized the MOP receptor. Monovalent CCK2 antagonist 2 did not stimulate receptor internalization. In the dual receptor-bearing cells, bivalent ligands 3a−c capable of simultaneously binding both receptors resulted in cell surface fluorescence and internalization of the fluorescent complex in a time- and temperature-dependent manner. Bivalent ligand 4 with spacer too short to occupy both receptors simultaneously yielded no signal. Receptor tethering with appropriate bivalent ligands can down-regulate signaling by moving a nonactivated receptor into the endocytic pathway. [ABSTRACT FROM AUTHOR]

We have synthesized the three Schiff-base ligands H2L1-H2L3 and their CoII, FeIII and RuIII metal complexes. All compounds have been characterized by analytical and spectroscopic methods. Oxidation of cyclohexane has been done by the metal complexes in CH3CN using H2O2 and/or t-butylhydroperoxide (TBHP) as a co-catalyst. The keto-enol tautomeric forms of the ligands have been studied in polar and non-polar organic solvents. Electrochemical properties of the complexes have been studied at different scan rates. Thermal studies were carried out for the compounds. The ligands H2L1-H2L3 were mutagenic on Salmonella Typhimurium TA 98 strain in the presence and/or absence of S9 mix. While the ligands H2L1 and H2L2 showed mutagenic activity on the strain TA 100 with and without S9 mix, the ligand H2L3 was not mutagenic for TA 100. Antimicrobial activity studies of the compounds have also been carried out. [ABSTRACT FROM AUTHOR]

A novel Schiff base ligand was synthesized by the condensation reaction of 2,6-diformylpyridine and 4-aminoantipyrine in MeOH and characterised by its melting point, elemental analysis, FT-IR, 1 H, 13 C NMR and mass spectroscopic studies. Molecular structure of the ligand was determined by single crystal X-ray diffraction technique. The electrochemical properties of the Schiff base ligand were studied in different solvents at various scan rates. Sensor ability of the Schiff base ligand was investigated by colorimetric and fluorometric methods. Visual colour change of the ligand was investigated in MeOH solvent in presence of various metal ions Na + , Mg 2+ , Al 3+ , K + , Cr 3+ , Mn 2+ , Fe 3+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ , Cd 2+ , Hg 2+ and Pb 2+ . Upon addition of Al 3+ ion into a MeOH solution of the ligand, an orange colour developed which is detectable by naked eye. Fluorescence emission studies showed that the ligand showed single emission band at 630–665 nm upon excitation at 560 nm. Addition of metal ions Na + , Mg 2+ , K + , Cr 3+ , Mn 2+ , Fe 3+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ , Cd 2+ , Hg 2+ and Pb 2+ (1:1 M ratio) cause fluorescence quenching, however addition of Al +3 resulted in an increase in fluorescence intensity. No significant variation was observed in the fluorescence intensity caused by Al 3+ in presence of other metal ions. Therefore, the Schiff base ligand can be used for selective detection of Al 3+ ions in the presence of the other metal ions studied. [ABSTRACT FROM AUTHOR]

Allosteric binding pockets in peptide-binding G protein-coupled receptors create opportunities for the development of small molecule drugs with substantial benefits over orthosteric ligands. To gain insights into molecular determinants for this pocket within type 1 and 2 cholecystokinin receptors (CCK1R and CCK2R), we prepared a series of receptor constructs in which six distinct residues in TM2, -3, -6, and -7 were reversed. Two novel iodinated CCK1R- and CCK2R-selective 1,4-benzodiazepine antagonists, differing only in stereochemistry at C3, were used. When all six residues within CCK1R were mutated to corresponding CCK2R residues, benzodiazepine selectivity was reversed, yet peptide binding selectivity was unaffected. Detailed analysis, including observations of gain of function, demonstrated that residues 6.51, 6.52, and 7.39 were most important for binding the CCK1R-selective ligand, whereas residues 2.61 and 7.39 were most important for binding CCK2Rselective ligand, although the effect of substitution of residue 2.61 was likely indirect. Ligand-guided homology modeling was applied to wild type receptors and those reversing benzodiazepine binding selectivity. The models had high predictive power in enriching known receptor-selective ligands from related decoys, indicating a high degree of precision in pocket definition. The benzodiazepines docked in similar poses in both receptors, with C3 urea substituents pointing upward, whereas different stereochemistry at C3 directed the C5 phenyl rings and N1 methyl groups into opposite orientations. The geometry of the binding pockets and specific interactions predicted for ligand docking in these models provide a molecular framework for understanding ligand selectivity at these receptor subtypes. Furthermore, the strong predictive power of these models suggests their usefulness in the discovery of lead compounds and in drug development programs. [ABSTRACT FROM AUTHOR]

Pain is among the most common symptoms in cancer and approximately 90% of patients experience end-stage cancer pain. The management of cancer pain is challenging due to the significant side effects associated with opioids, and novel therapeutic approaches are needed. MMG22 is a bivalent ligand containing MOR agonist and mGluR 5 antagonist pharmacophores joined by a 22-atom spacer. MMG22 exhibited extraordinary analgesia following intrathecal administration in a mouse model of bone cancer pain. Here, we assessed the effectiveness of systemic administration of MMG22 in reducing cancer pain and evaluated whether MMG22 displays side effects associated with opioids. Fibrosarcoma cells were injected into and around the calcaneus bone in C3H mice. Mechanical hyperalgesia was defined as an increase in the paw withdrawal frequencies (PWFs) evoked by application of a von Frey monofilament (3.9 mN bending force) applied to the plantar surface of the hind paw Subcutaneous (s.c.), intramuscular (i.m.), and oral (p.o.) administration of MMG22 produced robust dose-dependent antihyperalgesia, whose ED 50 was orders of magnitude lower than morphine. Moreover, the ED 50 for MMG22 decreased with disease progression. Importantly, s.c. administration of MMG22 did not produce acute (24 h) or long-term (9 days) tolerance, was not rewarding (conditioned place preference test), and did not produce naloxone-induced precipitated withdrawal or alter motor function. A possible mechanism of action of MMG22 is discussed in terms of inhibition of spinal NMDAR via antagonism of its co-receptor, mGluR 5 , and concomitant activation of neuronal MOR. We suggest that MMG22 may be a powerful alternative to traditional opioids for managing cancer pain. This article is part of the Special Issue entitled 'New Vistas in Opioid Pharmacology'. • Systemic administration OF MMG22 targets a MOR-mGluR5 heteromer and potently reduces bone cancer pain in mice. • MMG22 does not possess adverse effects associated with traditional opioids. • MMG22 activates MOR and antagonizes mGluR5. [ABSTRACT FROM AUTHOR]

Cisplatin and other widely employed platinum-based anticancer agents produce chemotherapy-induced peripheral neuropathy (CIPN) that often results in pain and hyperalgesia that are difficult to manage. We investigated the efficacy of a novel bivalent ligand, MCC22, for the treatment of pain arising from CIPN. MCC22 consists of mu opioid receptor (MOR) agonist and chemokine receptor 5 (CCR5) antagonist pharmacophores connected through a 22-atom spacer and was designed to target a putative MOR-CCR5 heteromer localized in pain processing areas. Mice received once daily intraperitoneal (i.p.) injections of cisplatin (1 mg/kg) for seven days and behavior testing began 7 days later. Cisplatin produced mechanical hyperalgesia that was decreased dose-dependently by MCC22 given by intrathecal (ED 50 = 0.004 pmol) or i.p. (3.07 mg/kg) routes. The decrease in hyperalgesia was associated with decreased inflammatory response by microglia in the spinal cord. Unlike morphine, MCC22 given daily for nine days did not exhibit tolerance to its analgesic effect and its characteristic antihyperalgesic activity was fully retained in morphine-tolerant mice. Furthermore, MCC22 did not alter motor function and did not exhibit rewarding properties. Given the exceptional potency of MCC22 without tolerance or reward, MCC22 has the potential to vastly improve management of chronic pain due to CIPN. This article is part of the Special Issue entitled 'New Vistas in Opioid Pharmacology'. • Chemotherapy often causes peripheral neuropathy that can be chronic and painful. • The bivalent ligand, MCC22, consists of a mµ opioid receptor agonist and a CCR5 receptor antagonist. • MCC22 potently reduced chemotherapy-induced hyperalgesia produced by cisplatin in mice. • MCC22 did not produce tolerance or reward. [ABSTRACT FROM AUTHOR]

This report describes the unique pharmacological profile of FBNTI, a potent DOR antagonist that acts as a MOR agonist via an allosteric mechanism. Binding of FBNTI to opioid receptors expressed in HEK 293 cells revealed a 190-fold greater affinity for DOR ( K i = 0.84 nM) over MOR ( K i = 160 nM). In mice, intrathecal FBNTI produced potent antinociception (ED 50 = 46.9 pmol/mouse), which was antagonized by selective MOR antagonists (CTOP, β-FNA). Autoantagonism of the MOR agonism by FBNTI was observed above the ED 75 dose, suggesting antagonism of activated MOR. That FBNTI is devoid of agonism in DOR knockout mice is consistent with allosteric activation of the MOR protomer via FBNTI bound to within a MOR-DOR heteromer. This proposed mechanism is supported by calcium mobilization assays, which indicate that FBNTI selectively activates the MOR-DOR heteromer and functionally antagonizes the MOR protomer at >ED 75 . The unprecedented mode of MOR activation by FBNTI may be responsible for the lack of tolerance after intrathecal (i.t.) administration. FBNTI was highly effective upon topical administration to the ipsolateral hind paw in the Hargreaves assay (EC 50 = 0.17 ± 0.08 μM) and without significant contralateral activity, suggesting a lack of systemic exposure.

It is proposed that two types of opioid receptor heteromers exist: a) those that are constitutive and b) those that are induced by bivalent ligands. Mu opioid agonists interact with constitutive MOR-DOR heteromer to mediate tolerance and dependence. Bivalent ligand, MDAN21, is devoid of these adverse effects by virtue of its DOR antagonist pharmacophore. We propose that bivalent ligands MMG22 and MCC22 induce colocalized receptors to form heteromers (MOR-mGluR5 and MOR-CCR5, respectively) that do not occur naturally, thereby promoting unique pharmacology. Heteromer induction with bivalent ligands offers a general approach to unique pharmacology that complements traditional SAR.

Chemokine release promotes cross-talk between opioid and chemokine receptors that in part leads to reduced efficacy of morphine in the treatment of chronic pain. On the basis of the possibility that a MOR-CCR5 heteromer is involved in such cross-talk, we have synthesized bivalent ligands (MCC series) that contain mu opioid agonist and CCR5 antagonist pharmacophores linked through homologous spacers (14-24 atoms). When tested on lipopolysaccharide-inflamed mice, a member of the series (MCC22; 3e) with a 22-atom spacer exhibited profound antinociception (i.t. ED50 = 0.0146 pmol/mouse) that was 2000× greater than morphine. Moreover, MCC22 was ~3500× more potent than a mixture of mu agonist and CCR5 antagonist monovalent ligands. These data strongly suggest that MCC22 acts by bridging the protomers of a MOR-CCR5 heteromer having a TM5,6 interface. Molecular simulation studies are consistent with such bridging. This study supports the MOR-CCR5 heteromer as a novel target for the treatment of chronic pain., Competing Interests: Notes “The authors declare no competing financial interest.”

Radiolabeled antagonists of specific peptide receptors identify a higher number of receptor binding sites than agonists and may thus be preferable for in vivo tumor targeting. In this study, two novel radioiodinated 1,4-benzodiazepines, (S)-1-(3-iodophenyl)-3-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)urea (9) and (R)-1-(3-iodophenyl)-3-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)urea (7), were developed. They were characterized in vitro as high affinity selective antagonists at cholecystokinin types 1 and 2 (CCK(1) and CCK(2)) receptors using receptor binding, calcium mobilization, and internalization studies. Their binding to human tumor tissues was assessed with in vitro receptor autoradiography and compared with an established peptidic CCK agonist radioligand. The (125)I-labeled CCK(1) receptor-selective compound 9 often revealed a substantially higher amount of CCK(1) receptor binding sites in tumors than the agonist (125)I-CCK. Conversely, the radioiodinated CCK(2) receptor-selective compound 7 showed generally weaker tumor binding than (125)I-CCK. In conclusion, compound 9 is an excellent radioiodinated nonpeptidic antagonist ligand for direct and selective labeling of CCK(1) receptors in vitro. Moreover, it represents a suitable candidate to test antagonist binding to CCK(1) receptor-expressing tumors in vivo.

Both mu-opioid (MOP) and type 2 cholecystokinin (CCK2) receptors are present in areas of the central nervous system that are involved in modulation of pain processing. We conducted bioluminescence resonance energy transfer (BRET) studies on COS cells coexpressing MOP and CCK2 receptors to determine whether receptor heterodimerization is involved in such modulation. These studies revealed the absence of constitutive or monovalent ligand-induced heterodimerization. Heterodimerization of MOP and CCK2 receptors therefore is unlikely to be responsible for the opposing effects between morphine and CCK in the CNS. However, association was induced, as indicated by a positive BRET signal, on exposure of the cells to bivalent ligands containing mu-opioid agonist and CCK2 receptor antagonist pharmacophores linked through spacers containing 16-22 atoms but not with a shorter (9-atom) spacer. These studies demonstrate for the first time that an appropriately designed bivalent ligand is capable of inducing association of G-protein-coupled receptors. The finding that opioid tolerance studies with these ligands in mice showed no correlation with the BRET data is consistent with the absence of association of MOP and CCK2 receptors in vivo.