N-terminal guanidinylation of TIPP (Tyr-Tic-Phe-Phe) peptides results in major changes of the opioid activity profile

Derivatives of peptides of the TIPP (Tyr-Tic-Phe-Phe; Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) family containing a guanidino (Guan) function in place of the N-terminal amino group were synthesized in an effort to improve their blood–brain barrier permeability. Unexpectedly, N-terminal amidination significantly altered the in vitro opioid activity profiles. Guan-analogues of TIPP-related δ opioid antagonists showed δ partial agonist or mixed δ partial agonist/μ partial agonist activity. Guanidinylation of the mixed μ agonist/δ antagonists H-Dmt-Tic-Phe-Phe-NH₂ (DIPP-NH₂) and H-Dmt-TicΨ[CH₂NH]Phe-Phe-NH₂ (DIPP-NH₂[Ψ]) converted them to mixed μ agonist/δ agonists. A docking study revealed distinct positioning of DIPP-NH₂ and Guan-DIPP-NH₂ in the δ receptor binding site. Lys³-analogues of DIPP-NH₂ and DIPP-NH₂[Ψ] (guanidinylated or non-guanidinylated) turned out to be mixed μ/κ agonists with δ antagonist-, δ partial agonist- or δ full agonist activity. Compounds with some of the observed mixed opioid activity profiles have therapeutic potential as analgesics with reduced side effects or for treatment of cocaine addiction.     

Synthesis and receptor binding of opioid peptide analogues containing β3‐homo‐amino acids

β‐Amino acids containing hybrid peptides and β‐peptides show great potential as peptidomimetics. In this paper we describe the synthesis and affinity toward the µ‐ and δ‐opioid receptors of β‐peptides, analogues of Leu‐enkephalin, deltorphin I, dermorphin and α,β‐hybrides, analogues of deltorphin I. Substitution of α‐amino acid residues with β³‐homo‐amino acid residues, in general resulted in decrease of affinity to opioid receptors. However, the incorporation β³h‐D ‐Ala in position 2 or β³hPhe in position 3 of deltorphin I resulted in potent and selective ligand for δ‐opioid receptor. The NMR studies of β‐deltorphin I analogue suggest that conformational motions in the central part of the peptide backbone are partially restricted and some conformational preferences can be expected. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Buprenorphine‐elicited alteration of adenylate cyclase activity in human embryonic kidney 293 cells coexpressing κ‐, μ‐opioid and nociceptin receptors

Buprenorphine, a maintenance drug for heroin addicts, exerts its pharmacological function via κ‐ (KOP), μ‐opioid (MOP) and nociceptin/opioid receptor‐like 1 (NOP) receptors. Previously, we investigated its effects in an in vitro model expressing human MOP and NOP receptors individually or simultaneously (MOP, NOP, and MOP+NOP) in human embryonic kidney 293 cells. Here, we expanded this cell model by expressing human KOP, MOP and NOP receptors individually or simultaneously (KOP, KOP+MOP, KOP+NOP and KOP+MOP+NOP). Radioligand binding with tritium‐labelled diprenorphine confirmed the expression of KOP receptors. Immunoblotting and immunocytochemistry indicated that the expressed KOP, MOP and NOP receptors are N‐linked glycoproteins and colocalized in cytoplasmic compartments. Acute application of the opioid receptor agonists— U‐69593, DAMGO and nociceptin— inhibited adenylate cyclase (AC) activity in cells expressing KOP, MOP and NOP receptors respectively. Buprenorphine, when applied acutely, inhibited AC activity to ~90% in cells expressing KOP+MOP+NOP receptors. Chronic exposure to buprenorphine induced concentration‐dependent AC superactivation in cells expressing KOP+NOP receptors, and the level of this superactivation was even higher in KOP+MOP+NOP‐expressing cells. Our study demonstrated that MOP receptor could enhance AC regulation in the presence of coexpressed KOP and NOP receptors, and NOP receptor is essential for concentration‐dependent AC superactivation elicited by chronic buprenorphine exposure.     

Identification of phosphorylation sites in the COOH‐terminal tail of the μ‐opioid receptor

Phosphorylation is considered a key event in the signalling and regulation of the μ opioid receptor (MOPr). Here, we used mass spectroscopy to determine the phosphorylation status of the C‐terminal tail of the rat MOPr expressed in human embryonic kidney 293 (HEK‐293) cells. Under basal conditions, MOPr is phosphorylated on Ser³⁶³ and Thr³⁷⁰, while in the presence of morphine or [D‐Ala2, NMe‐Phe4, Gly‐ol5]‐enkephalin (DAMGO), the COOH terminus is phosphorylated at three additional residues, Ser³⁵⁶, Thr³⁵⁷ and Ser³⁷⁵. Using N‐terminal glutathione S transferase (GST) fusion proteins of the cytoplasmic, C‐terminal tail of MOPr and point mutations of the same, we show that, in vitro, purified G protein‐coupled receptor kinase 2 (GRK2) phosphorylates Ser³⁷⁵, protein kinase C (PKC) phosphorylates Ser³⁶³, while CaMKII phosphorylates Thr³⁷⁰. Phosphorylation of the GST fusion protein of the C‐terminal tail of MOPr enhanced its ability to bind arrestin‐2 and ‐3. Hence, our study identifies both the basal and agonist‐stimulated phospho‐acceptor sites in the C‐terminal tail of MOPr, and suggests that the receptor is subject to phosphorylation and hence regulation by multiple protein kinases.     

Single‐nucleotide polymorphism (A118G) in exon 1 of OPRM1 gene causes alteration in downstream signaling by mu‐opioid receptor and may contribute to the genetic risk for addiction

The opioid receptor mu1 (OPRM1) mediates the action of morphine. Although genetic background plays an important role in the susceptibility toward abuse of drugs as evident from familial, adoption and twin studies, association of specific single‐nucleotide polymorphisms of OPRM1 gene with narcotic addiction is to be established. Here, we demonstrate the involvement of A118G polymorphism of exon1 of human OPRM1 gene (hOPRM1), with heroin and alcohol addiction, in a population in eastern India. Statistical analysis exhibited a significant association of G allele with both heroin and alcohol addiction with a risk factor of P_trend < 0.05. The functional significance of G allele in A118G single‐nucleotide polymorphisms was evaluated by studying the regulation of protein kinase A (PKA), pCREB, and pERK1/2 by morphine in Neuro 2A cells, stably transfected with either wild type or A118G mutant hOPRM1. Unlike acute morphine treatment, both chronic morphine exposure and withdrawal precipitated by naloxone were differentially regulated by A118 and G118 receptor isoforms when both PKA and pERK1/2 activities were compared. Results suggest that the association of A118G polymorphism to heroin and alcohol addiction may be because of the altered regulation of PKA and pERK1/2 during opioid and alcohol exposures.     

Differential Agonist Regulation of the Human κ-Opioid Receptor

Abstract: Opiates are potent analgesics used clinically in the treatment of pain. A significant drawback to the chronic use and clinical effectiveness of opiates is the development of tolerance. To investigate the cellular mechanisms of tolerance, the cloned human κ-opioid receptor was stably expressed in human embryonic kidney (HEK 293) cells, and the effects of opioid agonist treatment were examined. The receptor-expressing cells showed specific high-affinity membrane binding for a κ-selective opioid, ³H-labeled (+)-(5α,7α,8β)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4,5]dec-8-yl]benzeneacetamide ([³H]U69,593), and a nonselective opioid antagonist, [³H]diprenorphine. Pretreatment with pertussis toxin or guanosine 5′-O-(3-thiotriphosphate) reduced [³H]69,593 binding, indicating that the human κ receptor coupled to G proteins of the G_i or G_o families in HEK 293 cells. The receptor-mediated inhibition of adenylyl cyclase was abolished by pertussis toxin pretreatment and was blocked by a κ-selective antagonist, norbinal-torphimine. A 3-h pretreatment with a κ-selective agonist, (±)-trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide (U50,488), caused receptor down-regulation, whereas no receptor down-regulation was found after levorphanol pretreatment. U50,488 or dynorphin A_1–17 pretreatments (3 h) desensitized the ability of U50,488 or dynorphin A_1–17 to inhibit cyclic AMP accumulation, as evidenced by a decrease in functional potency. Also, U50,488 pretreatment desensitized the ability of levorphanol to inhibit forskolin-stimulated cyclic AMP accumulation. In contrast, pretreatment of cells with either levorphanol or a potent nonselective opioid, etorphine, resulted in no apparent receptor desensitization. Taken together, these results demonstrate that the human κ receptor is differentially regulated by selective and nonselective opioid agonists, with selective agonists able to desensitize the receptor.     

First selective agonist of the neuropeptide Y1‐receptor with reduced size

Selective NPY analogues are potent tools for tumour targeting. Their Y₁‐receptors are significantly over‐expressed in human breast tumours, whereas normal breast tissue only expresses Y₂‐receptors. The endogenous peptide consists of 36 amino acids, whereas smaller peptides are preferred because of better labelling efficiencies. As Y₁‐receptor agonists enhance the tumour to background ratio compared to Y₁‐receptor antagonists, we were interested in the development of Y₁‐receptor selective agonists. We designed 19 peptides containing the C‐terminus of NPY (28–36) with several modifications. By using competition receptor binding affinity assays, we identified three NPY analogues with high Y₁‐receptor affinity and selectivity. Metabolic stability studies in human blood plasma of the N‐terminally 5(6)‐carboxyfluorescein (CF) labelled peptides resulted in half‐lives of several hours. Furthermore, the degradation pattern revealed proteolytic degradation of the peptides by amino peptidases. The most promising peptide was further investigated in receptor activation and internalization studies. Signal transduction assays revealed clear agonistic properties, which could be confirmed by microscopy studies that showed clear Y₁‐receptor internalization. For the first time, here we show the design and characterization of a small Y₁‐receptor selective agonist. This agonist might be a useful novel ligand for NPY‐mediated tumour diagnostics and therapeutics. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Cyclic biphalin analogues with a novel linker lead to potent agonist activities at mu,delta, and kappa opioid receptors

In an effort to improve biphalin’s potency and efficacy at the µ-(MOR) and δ-opioid receptors (DOR), a series of cyclic biphalin analogues 1–5 with a cystamine or piperazine linker at the C-terminus were designed and synthesized by solution phase synthesis using Boc-chemistry. Interestingly, all of the analogues showed balanced opioid agonist activities at all opioid receptor subtypes due to enhanced κ-opioid receptor (KOR) activity. Our results indicate that C-terminal flexible linkers play an important role in KOR activity compared to that of the other cyclic biphalin analogues with a hydrazine linker. Among them, analogue 5 is a potent (Ki?=?0.27, 0.46, and 0.87?nM; EC₅₀?=?3.47, 1.45, and 13.5?nM at MOR, DOR, and KOR, respectively) opioid agonist with high efficacy. Based on the high potency and efficacy at the three opioid receptor subtypes, the ligand is expected to have a potential synergistic effect on relieving pain and further studies including in vivo tests are worthwhile.     

Kappa opioids promote the proliferation of astrocytes via Gβγ and β‐arrestin 2‐dependent MAPK‐mediated pathways

GTP binding regulatory protein (G protein)‐coupled receptors can activate MAPK pathways via G protein‐dependent and ‐independent mechanisms. However, the physiological outcomes correlated with the cellular signaling events are not as well characterized. In this study, we examine the involvement of G protein and β‐arrestin 2 pathways in kappa opioid receptor‐induced, extracellular signal‐regulated kinase 1/2 (ERK1/2)‐mediated proliferation of both immortalized and primary astrocyte cultures. As different agonists induce different cellular signaling pathways, we tested the prototypic kappa agonist, U69593 as well as the structurally distinct, non‐nitrogenous agonist, C(2)‐methoxymethyl salvinorin B (MOM‐Sal‐B). In immortalized astrocytes, U69593, activated ERK1/2 by a rapid (min) initial stimulation that was sustained over 2 h and increased proliferation. Sequestration of activated Gβγ subunits attenuated U69593 stimulation of ERK1/2 and suppressed proliferation in these cells. Furthermore, small interfering RNA silencing of β‐arrestin 2 diminished sustained ERK activation induced by U69593. In contrast, MOM‐Sal‐B induced only the early phase of ERK1/2 phosphorylation and did not affect proliferation of immortalized astrocytes. In primary astrocytes, U69593 produced the same effects as seen in immortalized astrocytes. MOM‐Sal‐B elicited sustained ERK1/2 activation which was correlated with increased primary astrocyte proliferation. Proliferative actions of both agonists were abolished by either inhibition of ERK1/2, Gβγ subunits or β‐arrestin 2, suggesting that both G protein‐dependent and ‐independent ERK pathways are required for this outcome.     

The impact of backbone N‐methylation on the structure‐activity relationship of Leu10‐teixobactin

Antimicrobial resistance is a serious threat to global human health; therefore, new anti‐infective therapeutics are required. The cyclic depsi‐peptide teixobactin exhibits potent antimicrobial activity against several Gram‐positive pathogens. To study the natural product's mechanism of action and improve its pharmacological properties, efficient chemical methods for preparing teixobactin analogues are required to expedite structure‐activity relationship studies. Described herein is a synthetic route that enables rapid access to analogues. Furthermore, our new N‐methylated analogues highlight that hydrogen bonding along the N‐terminal tail is likely to be important for antimicrobial activity.     

Analogues of deltorphin I containing conformationally restricted amino acids in position 2: structure and opioid activity

New analogues of deltorphin I (DT I, Tyr‐d ‐Ala‐Phe‐Asp‐Val‐Val‐Gly‐NH₂), with the d ‐Ala residue in position 2 replaced by α‐methyl‐β‐azido(amino, 1‐pyrrolidinyl, 1‐piperidinyl or 4‐morpholinyl)alanine, were synthesized by a combination of solid‐phase and solution methods. All ten new analogues were tested for receptor affinity and selectivity to μ‐ and δ‐opioid receptors. The affinity of analogues containing (R) or (S)‐α‐methyl‐β‐azidoalanine in position 2 to δ‐receptors strongly depended on the chirality of the α,α‐disubstituted residue. Peptide II , containing (S)‐α‐methyl‐β‐azidoalanine in position 2, displayed excellent δ‐receptor selectivity with its δ‐receptor affinity being only three times lower than that of DT I. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and Evaluation of Anticonvulsant Activity of Some Schiff Bases of 7‐Amino‐1,3‐dihydro‐2H‐1,4‐benzodiazepin‐2‐one

A variety of 1,3‐dihydro‐2H‐1,4‐benzodiazepin‐2‐one azomethines and 1,3‐dihydro‐2H‐1,4‐benzodiazepin‐2‐one benzamide were prepared, characterized and evaluated for the anticonvulsant activity in the rat using picrotoxin‐induced seizure model. The prepared 1,3‐dihydro‐2H‐1,4‐benzodiazepin‐2‐one azomethine derivatives emerged potentially anticonvulsant molecular scaffolds exemplified by compounds, 7‐{(E)‐[(4‐nitrophenyl)methylidene]amino}‐5‐phenyl‐1,3‐dihydro‐2H‐1,4‐benzodiazepin‐2‐one, 7‐[(E)‐{[4‐(dimethylamino)phenyl]methylidene}amino]‐5‐phenyl‐1,3‐dihydro‐2H‐1,4‐benzodiazepin‐2‐one, 7‐{(E)‐[(4‐bromo‐2,6‐difluorophenyl)methylidene]amino}‐5‐phenyl‐1,3‐dihydro‐2H‐1,4‐benzodiazepin‐2‐one and 7‐[(E)‐{[3‐(4‐fluorophenyl)‐1‐phenyl‐1H‐pyrazol‐4‐yl]methylidene}amino]‐5‐phenyl‐1,3‐dihydro‐2H‐1,4‐benzodiazepin‐2‐one. All these four compounds have shown substantial decrease in the wet dog shake numbers and grade of convulsions with respect to the standard drug diazepam. The most active compound, 7‐[(E)‐{[4‐(dimethylamino)phenyl]methylidene}amino]‐5‐phenyl‐1,3‐dihydro‐2H‐1,4‐benzodiazepin‐2‐one, exhibited 74 % protection against convulsion which was higher than the standard drug diazepam. Furthermore, to identify the binding mode of the interaction amongst the target analogs and binding site of the benzodiazepine receptor, molecular docking study and molecular dynamic simulation were carried out. Additionally, in silico pharmacokinetic and toxicity predictions of target compounds were carried out using AdmetSAR tool. Results of ADMET studies suggest that the pharmacokinetic parameters of all the target compounds were within the acceptable range to become a potential drug candidate as antiepileptic agents.     

Molecular modeling study of the opioid receptor interactions with series of cyclic deltorphin analogues

In this study, ten tetra‐ and heptapeptide analogues of deltorphin containing the urea bridges between residues 2 and 4 have been docked into the δ‐ and µ‐opioid receptors to explain their different biological activities. The important factors explaining particular ligand activity such as free energy of binding, conformation of the ligand, its location inside the binding pocket as well as the number and strength of the receptor–ligand interactions have been discussed. Several different binding modes for investigated ligands have been proposed. It appears that the binding site is not identical even for very similar ligands. Results of this study help to explain the differences in biological activity of the deltorphin analogues, their interaction with the opioid receptors at the molecular level and support designing a new generation of potent opioid drugs with improved selectivity. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Inhibition of EGF-induced ERK/MAP kinase-mediated astrocyte proliferation by μ opioids: integration of G protein and β-arrestin 2-dependent pathways

Although μ, κ, and δ opioids activate extracellular signal‐regulated kinase (ERK)/mitogen‐activated protein (MAP) kinase, the mechanisms involved in their signaling pathways and the cellular responses that ensue differ. Here we focused on the mechanisms by which μ opioids rapidly (min) activate ERK and their slower (h) actions to inhibit epidermal growth factor (EGF)‐induced ERK‐mediated astrocyte proliferation. The μ‐opioid agonists ([d‐ ala², mephe⁴, gly‐ol⁵] enkephalin and morphine) promoted the phosphorylation of ERK/MAP kinase within 5 min via G_i/o protein, calmodulin (CaM), and β‐arrestin2‐dependent signaling pathways in immortalized and primary astrocytes. This was based on the attenuation of the μ‐opioid activation of ERK by pertussis toxin (PTX), the CaM antagonist, W‐7, and siRNA silencing of β‐arrestin2. All three pathways were shown to activate ERK via an EGF receptor transactivation‐mediated mechanism. This was disclosed by abolishment of μ‐opioid‐induced ERK phosphorylation with the EGF receptor‐specific tyrosine phosphorylation inhibitor, AG1478, and μ‐opioid‐induced reduction of EGF receptor tyrosine phosphorylation by PTX, and β‐arrestin2 targeting siRNA in the present studies and formerly by CaM antisense. Long‐term (h) treatment of primary astrocytes with [d ‐ala²,mephe⁴,gly‐ol⁵] enkephalin or morphine, attenuated EGF‐induced ERK phosphorylation and proliferation (as measured by 5′‐bromo‐2′‐deoxy‐uridine labeling). PTX and β‐arrestin2 siRNA but not W‐7 reversed the μ‐opioid inhibition. Unexpectedly, β‐arrestin‐2 siRNA diminished both EGF‐induced ERK activation and primary astrocyte proliferation suggesting that this adaptor protein plays a novel role in EGF signaling as well as in the opioid receptor phase of this pathway. The results lend insight into the integration of the different μ‐opioid signaling pathways to ERK and their cellular responses.     

Arrestin‐dependent but G‐protein coupled receptor kinase‐independent uncoupling of D2‐dopamine receptors

We reconstituted D2 like dopamine receptor (D2R) and the delta opioid receptor (DOR) coupling to G‐protein gated inwardly rectifying potassium channels (K_ir3) and directly compared the effects of co‐expression of G‐protein coupled receptor kinase (GRK) and arrestin on agonist‐dependent desensitization of the receptor response. We found, as described previously, that co‐expression of a GRK and an arrestin synergistically increased the rate of agonist‐dependent desensitization of DOR. In contrast, only arrestin expression was required to produce desensitization of D2R responses. Furthermore, arrestin‐dependent GRK‐independent desensitization of D2R‐K_ir3 coupling could be transferred to DOR by substituting the third cytoplasmic loop of DOR with that of D2R. The arrestin‐dependent GRK‐independent desensitization of D2R desensitization was inhibited by staurosporine treatment, and blocked by alanine substitution of putative protein kinase C phosphorylation sites in the third cytoplasmic loop of D2R. Finally, the D2R construct in which putative protein kinase C phosphorylation sites were mutated did not undergo significant agonist‐dependent desensitization even after GRK co‐expression, suggesting that GRK phosphorylation of D2R does not play an important role in uncoupling of the receptor.

     

Novel delta opioid receptor agonists exhibit differential stimulation of signaling pathways

A novel family of 1,3,5-trisubstituted 1,2,4-triazoles was discovered as potent and selective ligands for the δ opioid receptor by rational design. Compound 5b exhibited low-nanomolar in vitro binding affinity (IC₅₀ = 5.8 nM), excellent selectivity for the δ opioid receptor over the alternative μ and κ opioid receptors, full agonist efficacy in receptor down-regulation and MAP kinase activation assays, and low-efficacy partial agonist activity in stimulation of GTPγS binding. The apparent discrepancy observed in these functional assays may stem from different signaling pathways involved in each case, as found previously for other G-protein coupled receptors. More biological studies are underway to better understand the differential stimulation of signaling pathways by these novel compounds.     

Demonstration of κ3-Opioid Receptors in the SH-SY5Y Human Neuroblastoma Cell Line

Abstract: In addition to the μ- and δ-opioid receptors previously reported, the SH-SY5Y human neuroblastoma cell line has high levels of κ₃ receptors, accounting for 40% of total opioid binding, as measured with [³H]-diprenorphine binding. Competition studies reveal binding profiles for all three receptor classes that are similar to those observed in brain membranes. Differentiation with retinoic acid increases the levels of opioid receptor binding in the cell line, with the largest elevations in κ₃ binding. Fully 75% of the increased binding corresponds to κ₃ sites, which represent 50% of total opioid receptor binding in differentiated cells. Morphine inhibits forskolin-stimulated cyclic AMP accumulation, and this effect is readily blocked by the μ antagonist d -Phe-Cys-Tyr-d -Trp-Arg-Thr-Pen-Thr-NH₂ (CTAP). Naloxone benzoylhydrazone, a κ₃ agonist, inhibits forskolin-stimulated cyclic AMP accumulation more potently than morphine and is not reversed by CTAP. These studies indicate that SH-SY5Y cells contain high levels of functional κ₃ receptors.     

Profiling two indole‐2‐carboxamides for allosteric modulation of the CB1 receptor

Allosteric modulation of G‐protein coupled receptors (GPCRs) represents a novel approach for fine‐tuning GPCR functions. The cannabinoid CB1 receptor, a GPCR associated with the CNS, has been implicated in the treatment of drug addiction, pain, and appetite disorders. We report here the synthesis and pharmacological characterization of two indole‐2‐carboxamides:5‐chloro‐3‐ethyl‐1‐methyl‐N‐(4‐(piperidin‐1‐yl)phenethyl)‐1H‐indole‐2‐carboxamide (ICAM‐a) and 5‐chloro‐3‐pentyl‐N‐(4‐(piperidin‐1‐yl)phenethyl)‐1H‐indole‐2‐carboxamide (ICAM‐b). Although both ICAM‐a and ICAM‐b enhanced CP55, 940 binding, ICAM‐b exhibited the strongest positive cooperativity thus far demonstrated for enhancing agonist binding to the CB1 receptor. Although it displayed negative modulatory effects on G‐protein coupling to CB1, ICAM‐b induced β‐arrestin‐mediated downstream activation of extracellular signal‐regulated kinase (ERK) signaling. These results indicate that this compound represents a novel class of CB1 ligands that produce biased signaling via CB1.