Facilitation of μ-opioid receptor activity by preventing δ-opioid receptor-mediated codegradation

δ-opioid receptors (DORs) form heteromers with μ-opioid receptors (MORs) and negatively regulate MOR-mediated spinal analgesia. However, the underlying mechanism remains largely unclear. The present study shows that the activity of MORs can be enhanced by preventing MORs from DOR-mediated codegradation. Treatment with DOR-specific agonists led to endocytosis of both DORs and MORs. These receptors were further processed for ubiquitination and lysosomal degradation, resulting in a reduction of surface MORs. Such effects were attenuated by treatment with an interfering peptide containing the first transmembrane domain of MOR?(MOR(TM1)), which interacted with DORs and disrupted the MOR/DOR interaction. Furthermore, the systemically applied fusion protein consisting of MOR(TM1) and TAT at the C terminus could disrupt the MOR/DOR interaction in the mouse spinal cord, enhance the morphine analgesia, and reduce the antinociceptive tolerance to morphine. Thus, dissociation of MORs from DORs in the cell membrane is?a potential strategy to improve opioid analgesic therapies.     

Design,synthesis and biological evaluation of novel tetrahydroisoquinoline quaternary derivatives as peripheral κ-opioid receptor agonists

A novel series of tetrahydroisoquinoline quaternary derivatives 4 were synthesized as peripheral κ-opioid receptor agonists. All the target compounds were evaluated in κ-opioid receptor binding assays, and compounds 4l, 4m, and 4n exhibited high affinity for κ-opioid receptor. Furthermore, compound 4l (κK_i = 0.94 nM) produced potent antinociceptive activity in the mouse acetic acid-induced writhing assay, with lower sedative side effects than the parent compound MB-1c.     

Membrane partitioning of various δ-opioid receptor forms before and after agonist activations: The effect of cholesterol

Lipid rafts depicted as densely packed and thicker membrane microdomains, based on the dynamic clustering of cholesterol and sphingolipids, may help as platforms involved in a wide variety of cellular processes. The reasons why proteins segregate into rafts are yet to be clarified. The human delta opioid receptor (hDOR) reconstituted in a model system has been characterised after ligand binding by an elongation of its transmembrane part, inducing rearrangement of its lipid microenvironment [Alves, Salamon, Hruby, and Tollin (2005) Biochemistry 44, 9168-9178]. We used hDOR to understand better the correlation between its function and its membrane microdomain localisation. A fusion protein of hDOR with the Green Fluorescent Protein (DOR?) allows precise receptor membrane quantification. Here we report that (i) a fraction of the total receptor pool requires cholesterol for binding activity, (ii) G-proteins stabilize a high affinity state conformation which does not seem modulated by cholesterol. In relation to its distribution, and (iii) a fraction of DOR? is constitutively associated with detergent-resistant membranes (DRM) characterised by an enrichment in lipids and proteins raft markers. (iv) An increase in the quantity of DOR? was observed upon agonist addition. (v) This DRM relocation is prevented by uncoupling the receptor-G-protein interaction.     

Design,synthesis and biological evaluation of a bivalent μ opiate and adenosine A1 receptor antagonist

The cross talk between different membrane receptors is the source of increasing research. We designed and synthesized a new hetero-bivalent ligand that has antagonist properties on both A₁ adenosine and μ opiate receptors with a K_i of 0.8 ± 0.05 and 0.7 ± 0.03 μM, respectively. This hybrid molecule increases cAMP production in cells that over express the μ receptor as well as those over expressing the A₁ adenosine receptor and reverses the antalgic effects of μ and A₁ adenosine receptor agonists in animals.     

Design and synthesis of new prostaglandin D₂ receptor antagonists

To identify new cost-effective prostaglandin D? (DP) receptor antagonists, a series of novel 3-benzoylaminophenylacetic acids were synthesized and biologically evaluated. Among those tested, some representative compounds were found to be orally available. Receptor selectivity and rat PK profiles were also evaluated. The structure-activity relationship (SAR) study is presented.     

The role of δ-opioid receptor subtypes in cocaine- and methamphetamine-induced place preferences

The effects of δ-receptor antagonists on cocaine- and methamphetamine-induced place preferences were examined in rats. Cocaine- and methamphetamine-induced place preferences were significantly attenuated by naltrindole (NTI: a non-selective δ-opioid receptor antagonist). Furthermore, naltriben (NTB: a selective δ₂-opioid receptor antagonist), but not 7-benzylidenenaltrexone (BNTX: a selective δ₁-opioid receptor antagonist), attenuated the cocaine- and methamphetamine-induce place preferences. These results suggest that δ-opioid receptors, particularly δ₂-opioid receptors, may be involved in the reinforcing effects of cocaine and methamphetamine.     

Design and synthesis of N-alkyl-N′-substituted 2,4-dioxo-3,4-dihydropyrimidin-1-diacylhydrazine derivatives as ecdysone receptor agonist

Based on the discovery of thymine as an ecdysteroid agonist, a series of 1,4-disubstituted diacylhydrazine derivatives with a thymine moiety were designed and synthesized. The activities of these compounds against Spodoptera litura (Fabricius) were evaluated by the insect immersion method. Results showed that compound 2h with an N-cyclohexylmethyl substituent exhibits the most potent agonist activity with a median lethal concentration of 23.21 μg/mL. This compound also caused malformation of molting larvae and adults. Compound 2h was further demonstrated as an ecdysteroid agonist by reporter gene assay on the Spodoptera frugiperda cell line (Sf9 cells). A molecular docking study indicated that hydrophobic interactions and the formation of hydrogen bonds between the compounds and the ecdysone receptor play critical roles in promoting the binding affinity of the compound. The structure of compound 2h may serve as a favorable template for the development of new ecdysteroid agonists with a pyrimidinedione moiety.     

ßarrestin1-biased agonism at human δ-opioid receptor by peptidic and alkaloid ligands

We have previously reported on the differential regulation of the human δ-opioid receptor (hDOR) by alkaloid (etorphine) and peptidic (DPDPE and deltorphin I) ligands, in terms of both receptor desensitization and post-endocytic sorting. Since ßarrestins are well known to regulate G protein-coupled receptors (GPCRs) signaling and trafficking, we therefore investigated the role of ßarrestin1 (the only isoform expressed in our cellular model) in the context of the hDOR. We established clonal cell lines of SK-N-BE cells over-expressing ßarrestin1, its dominant negative mutant (ßarrestin1^319-418), and shRNA directed against endogenous ßarrestin1. Interestingly, both binding and confocal microscopy approaches demonstrated that ßarrestin1 is required for hDOR endocytosis only when activated by etorphine. Conversely, functional experiments revealed that ßarrestin1 is exclusively involved in hDOR desensitization promoted by the peptides. Taken together, these results provide substantial evidence for a ßarrestin1-biased agonism at hDOR, where ßarrestin1 is differentially involved during receptor desensitization and endocytosis depending on the ligand.     

2,4-Diaminopyridine δ-opioid receptor agonists and their associated hERG pharmacology

A number of libraries were produced to explore the potential of 2,4-diaminopyridine lead 1. The resulting diaminopyridines proved to be potent and selective δ-opioid receptor agonists. Several rounds of lead optimisation using library chemistry identified compound 17 which went on to show efficacy in an electromyography model of neuropathic pain. The structure–activity relationship of the series against the hERG ion channel proved to be a key selectivity hurdle for the series.     

Design,synthesis, and evaluation of potent novel peroxisome proliferator-activated receptor γ indole partial agonists

Peroxisome Proliferator-Activated Receptor γ (PPARγ) is a nuclear receptor important for glucose homeostasis and insulin sensitivity. The anti-diabetic drugs thiazolidinediones improve insulin sensitivity by blocking PPARγ phosphorylation at S273; however, their full agonism on PPARγ also causes significant unwanted side effects. The indole derivative UHC1 displays insulin-sensitizing effect by acting as a partial agonist through the inhibition of PPARγ S273 phosphorylation, but without full agonist-associated side effects; however, its potency leaves much to be desired. Herein we report the design and synthesis of potent indole analogs as partial PPARγ agonists via the structure-activity relationship studies. Our studies revealed that vanillylamine and piperonyl benzylamine at Site 1 are favored to bind PPARγ with either biphenyl or 3-trifluoromethyl benzyl group at Site 2. In particular, compound WO91A with vanillylamine at Site 1 displays highly potent PPARγ binding affinity (IC₅₀ = 16.7 nM), over 30-fold more potent than the parental compound UHC1, yet with less side effect-associated transactivation activity.     

Design and synthesis of quinolinopropellane derivatives with selective δ opioid receptor agonism

Indolopropellane 2 was reported to show almost no binding affinity to the δ opioid receptor (DOR) in spite of the fact that 2 has both the propellane fundamental skeleton (message part) with binding ability to the opioid receptors and a possible DOR address structure (indole moiety). We developed the working hypothesis that almost no binding affinity of 2 to the DOR would be derived from its possibly stable bent conformer. To enable the propellane skeleton to adopt an extended conformation which would reasonably interact with the DOR, quinolinopropellanes 3a–d were designed which had an additional pharmacophore, quinoline nitrogen. The calculated binding free energies of ligand–DOR complexes strongly supported our working hypothesis. The synthesized quinolinopropellane 3a was a selective DOR full agonist, confirming our working hypothesis and the results of in silico investigation.     

Differential mediation of cold water swim stress-induced antinociception by δ-opioid receptor subtypes in diabetic mice

The involvement of δ-opioid receptor subtypes in cold water swim stress (CWSS)-induced antinociception in diabetic mice was compared with that in non-diabetic mice. Three-minute swim stress produced significant antinociception in both diabetic and non-diabetic mice as determined by the tail-pinch test. However, the extent of CWSS-induced antinociception in diabetic mice was significantly greater than that in non-diabetic mice. Pretreatment with naltriben, a selective δ₂-opioid receptor antagonist, significantly attenuated CWSS-induced antinociception in both non-diabetic and diabetic mice. In contrast, although 7-benzylidenenaltrexone, a selective δ₁-opioid receptor antagonist, significantly attenuated CWSS-induced antinociception in diabetic mice, it had no effect in non-diabetic mice. These results suggest that CWSS-induced antinociceotion in non-diabetic mice is mediated by δ₂-opioid receptors, whereas CWSS-induced antinociception in diabetic mice is mediated by both δ₁- and δ₂-opioid receptors. Furthermore, the enhanced CWSS-induced antinociception in diabetic mice may be due to the activation of δ₁-opioid receptors.     

Determination of δ-opioid receptor molecules mobility in living cells plasma membrane by novel method of FRAP analysis

Fluorescence recovery after photobleaching (FRAP) is the preferred method for analyzing the lateral mobility of fluorescently-tagged proteins in the plasma membranes (PMs) of live cells. FRAP experiments are described as being easy to perform; however, the analysis of the acquired data can be difficult. The evaluation procedure must be properly combined with the imaging setup of the confocal microscope to provide unbiased results.With the aim of increasing the accuracy of determining the diffusion coefficient (D) and mobile fraction (M_f) of PM proteins, we developed a novel method for FRAP analysis in the equatorial plane of the cell. This method is based on the calculation of photobleaching characteristics, derived from the light intensity profile and optical parameters of the confocal microscope, and on the model of fluorescent molecule diffusion in PM regions outside of the focal plane. Furthermore, cell movement artifacts in the FRAP data are ameliorated by using a region of interest, which is not fixed but instead moves adaptively in coordination with the movement of cells.When this method was used to determine the mobility of the δ-opioid receptor-eYFP in HEK293 cells, a highly significant decrease in receptor mobility was detected in cholesterol-depleted cells. This decrease was fully reversible by the replenishment of cholesterol levels. Our results demonstrate the crucial role played by cholesterol in the dynamic organization of δ-opioid receptors in the PM under in vivo conditions. Our method may be applied for the determination of the D and M_f values of other PM proteins.     

Enzymic synthesis of argininosuccinate catalyzed by δ-crystallin

Summary A fast and practical procedure has been developed for the synthesis of argininosuccinate using immobilized duck lens -crystallin as catalyst.     

Design, synthesis, and evaluation of a novel class of 2,3-disubstituted-tetrahydro-β-carboline derivatives

Several novel tetrahydro-β-carboline derivatives with amino acid residues at the 2-position and a glucosamine group at the 3-position of the tetrahydro-β-carboline nucleus were synthesized from a readily available starting material, tryptophane, and were evaluated for their anti-inflammatory activity in the present study. Our results showed that all of the derivatives tested exhibited a significant inhibition of xylene-induced inflammation in mice.     

Design,synthesis, structure–activity relationships,and docking studies of pyrazole-containing derivatives as a novel series of potent glucagon receptor antagonists

Glucagon receptor antagonists possess a great potential for treatment of type 2 diabetes mellitus. A series of pyrazole-containing derivatives were designed, synthesized and evaluated by biological assays as glucagon receptor antagonists. Most of the compounds exhibited good in vitro efficacy. Two of them, compounds 17f and 17k, displayed relatively potent antagonist effects on glucagon receptors with IC₅₀ values of 3.9 and 3.6 μM, respectively. The possible binding modes of 17f and 17k with the cognate receptor were explored by molecular docking simulation.     

Design and synthesis of a hybrid potentiator–corrector agonist of the cystic fibrosis mutant protein ΔF508-CFTR

A developing therapy of cystic fibrosis caused by the ΔF508 mutation in CFTR employs correction of defective CFTR chloride channel gating by a ‘potentiator’ and of defective CFTR protein folding by a ‘corrector’. Based on SAR data for phenylglycine-type potentiators and bithiazole correctors, we designed a hybrid molecule incorporating an enzymatic hydrolysable linker to deliver the potentiator (PG01) fragment 2 and the corrector (Corr-4a) fragment 13. The hybrid molecule 14 contained PG01-OH and Corr-4a–linker–CO₂H moieties, linked with an ethylene glycol spacer through an ester bond. The potentiator 2 and corrector 13 fragments (after cleavage) had low micromolar potency for restoration of ΔF508-CFTR channel gating and cellular processing, respectively. Cleavage of hybrid molecule 14 by intestinal enzymes under physiological conditions produced the active potentiator 2 and corrector fragments 13, providing proof-of-concept for small-molecule potentiator–corrector hybrids as a single drug therapy for CF caused by the ΔF508 mutation.     

Design, synthesis, and pharmacological evaluation of novel tetrahydroprotoberberine derivatives: selective inhibitors of dopamine D₁ receptor

A series of new tetrahydroprotoberberine (THPB) derivatives were designed, synthesized, and tested for their binding affinity towards dopamine (D(1) and D(2)) and serotonin (5-HT(1A) and 5-HT(2A)) receptors. Many of the THPB compounds exhibited high binding affinity and activity at the dopamine D(1) receptor, as well as high selectivity for the D(1) receptor over the D(2), 5-HT(1A), and 5-HT(2A) receptors. Among these, compound 19c exhibited a promising D(1) receptor binding affinity (K(i)=2.53nM) and remarkable selectivity versus D(2)R (inhibition=81.87%), 5-HT(1A)R (inhibition=61.70%), and 5-HT(2A)R (inhibition=24.96%). Compared with l-(S)-stepholidine (l-SPD) (D(1)K(i)=6.23nM, D(2)K(i)=56.17nM), compound 19c showed better binding affinity for the D(1) receptor (2.5-fold higher) and excellent D(2)/D(1) selectivity. Functional assays found compounds 18j, 18k, and 19c are pure D(1) receptor antagonists. These results indicate that removing the C10 hydroxy group and introducing a methoxy group at C11 of the pharmacophore of l-SPD can reverse the function of THPB compounds at the D(1) receptor. These results are in accord with molecular docking studies.     

Chemical coding of neurons expressing δ- and κ-opioid receptor and type I vanilloid receptor immunoreactivities in the porcine ileum

Opioid drugs have profound antidiarrheal and constipating actions in the intestinal tract and are effective in mitigating abdominal pain. Mediators of intestinal inflammation and allergy produce increased mucosal secretion, altered bowel motility and pain due to their ability to evoke enteric secretomotor reflexes through primary afferent neurons. In this study, the distribution of delta- and kappa-opioid receptor (DOR and KOR, respectively) immunoreactivities in chemically identified neurons of the porcine ileum was compared with that of the capsaicin-sensitive type 1 vanilloid receptor (VR1). DOR and VR1 immunoreactivities were observed to be highly localized in choline acetyltransferase (ChAT)- and calcitonin gene-related peptide (CGRP)-positive neurons and nerve fibers of the submucosal and myenteric plexuses and both receptors exhibited frequent colocalization. In the inner submucosal plexus, they also were colocalized in substance P (SP)-positive neurons. Neurons in the outer submucosal plexus expressed DOR immunoreactivity alone or in combination with VR1. KOR-immunoreactive neurons were found only in the myenteric plexus; these cells coexpressed immunoreactivity to ChAT, CGRP, vasoactive intestinal peptide (VIP) or nitric oxide synthase (NOS). In addition, some KOR-positive neurons coexpressed immunoreactivities to DOR and VR1. Based on their neurochemical coding, opioid and vanilloid receptor-immunoreactive neurons in the submucosal and myenteric plexuses may include primary afferents and constitute novel therapeutic targets for the palliation of painful intestinal inflammatory, hypersensitivity and dysmotility states.