RESUMO
Opioid receptors (ORs) convert extracellular messages to signaling events by coupling to the heterotrimeric G proteins, Gαâ¢ßγ Classic pharmacological methods, such as [35S]GTPγS binding and inhibition of cyclic AMP production, allow for general opioid characterization, but they are subject to the varying endogenous Gα proteins in a given cell type. Bioluminescence resonance energy transfer (BRET) technology offers new insight by allowing the direct observation of Gα subunit-specific effects on opioid pharmacology. Using a Venus-tagged Gßγ and nanoluciferase-tagged truncated G protein receptor kinase 3, an increase in BRET signal correlated with OR activation mediated by a specific Gα protein. The magnitude of the BRET signal was normalized to the maximum response obtained with 10 µM 2-(3,4-dichlorophenyl)-N-methyl-N-[(1R,2R)-2-pyrrolidin-1-ylcyclohexyl]acetamide (U50,488) for the kappa OR (KOR). Opioids reached equilibrium with the KOR, and concentration-response curves were generated. Although the full agonists U50,488, salvinorin A, nalfurafine, and dynorphin peptides were equally efficacious regardless of the Gα subunit present, the concentration-response curves were leftward shifted when the KOR was signaling through Gαz compared with other Gαi/o subunits. In contrast, the Gα subunit distinctly affected both the efficacy and potency of partial kappa agonists, such as the benzomorphans, and the classic mu opioid antagonists, naloxone, naltrexone, and nalmefene. For example, (-)pentazocine had EC50 values of 7.3 and 110 nM and maximal stimulation values of 79% and 35% when the KOR signaled through Gαz and Gαi1, respectively. Together, these observations suggest KOR pharmacology varies based on the specific Gα subunit coupled to the KOR. SIGNIFICANCE STATEMENT: Opioid receptors couple to various heterotrimeric Gαßγ proteins to convert extracellular cues to precise intracellular events. This paper focuses on how the various inhibitory Gα subunits influence the pharmacology of full and partial agonists at the kappa opioid receptor. Using a bioluminescent assay, the efficacy and potency of kappa opioids was determined. Opioid signaling was more potent through Gαz compared with other Gα proteins. These observations suggest that Gαz may impact opioid pharmacology and cellular physiology more than previously thought.
Assuntos
Analgésicos Opioides/farmacologia , Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Receptores Opioides kappa/metabolismo , (trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida/farmacologia , Diterpenos Clerodânicos/farmacologia , Dinorfinas/farmacologia , Células HEK293 , Humanos , Morfinanos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Compostos de Espiro/farmacologiaRESUMO
A combination of buprenorphine (BUP) and samidorphan (SAM) at a 1:1 (mg/mg) fixed-ratio dose is being investigated as an adjunctive treatment of major depressive disorder (BUP/SAM, ALKS 5461). Both [3H]BUP and [3H]SAM bound to the µ-, κ-, and δ-opioid receptors (MOR, KOR, and DOR, respectively) with Kd values of 3 nM or less. [3H]BUP dissociated from the MOR more slowly than [3H]SAM did. In the [35S]GTPγS assay, BUP was a partial agonist at the MOR, KOR, and DOR. SAM was an antagonist at the MOR and a partial agonist at the KOR and DOR. The pharmacology of the combination of SAM and BUP was characterized at ratios like the molar ratios of both compounds at steady state in humans. In all assessments, SAM reduced the efficacy of BUP at the MOR without altering its potency. At the KOR, SAM had no significant effect on the activity of BUP. In bioluminescent resonance energy transfer assays, SAM, naltrexone, and naloxone were partial agonists when the MOR was coupled to the Gα oB and Gα z, and were antagonists when coupled to Gα i At the KOR, SAM was a partial agonist activating Gα oA and Gα oB and a full agonist in stimulating Gα z SAM inhibited BUP's recruitment of ß-arrestin to the MOR, suggesting an attenuation of BUP's efficacy in activating G proteins correlated with an inhibition of ß-arrestin recruitment. The collective data suggest that SAM attenuates the efficacy of BUP under all conditions tested at the MOR and DOR but had little effect on BUP activity at the KOR.
Assuntos
Buprenorfina/farmacologia , Transtorno Depressivo Maior/tratamento farmacológico , Naltrexona/análogos & derivados , Animais , Células CHO , Linhagem Celular , Cricetulus , Combinação de Medicamentos , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Naloxona/farmacologia , Naltrexona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Receptores Opioides delta/metabolismo , Receptores Opioides mu/metabolismo , beta-Arrestinas/metabolismoRESUMO
Patients receiving the cytokine immunotherapy, interferon-alpha (IFN-α) frequently present with neuropsychiatric consequences and cognitive impairments. Patients (25-80%) report symptoms of depression, including, anhedonia, irritability, fatigue and impaired motivation. Our lab has previously demonstrated treatment (170,000IU/kg sc, 3 times per week for 4weeks) of the pro-inflammatory cytokine, IFN-α, induced a depressive phenotype in rats in the forced swim test (FST). Here, we examine the biological mechanisms underlying behavioral changes induced by IFN-α, which may be reflective of mechanisms underlying inflammation associated depression. We also investigate the potential of 3-carboxamido seco-nalmefene (3CS-nalmefene), a novel opioid modulator (antagonist at mu and partial agonist at kappa and delta opioid receptors in vitro), to reverse IFN-α induced changes. In vitro radioligand receptor binding assays and the [35S] GTPγS were performed to determine the affinity of 3CS-nalmefene for the mu, kappa and delta opioid receptors. IFN-α treatment increased circulating and central markers of inflammation and hypothalamic-pituitaryadrenal (HPA) axis activity (IL-6, IL-1ß and corticosterone) while increasing immobility in the FST, impairing of object displacement learning in the object exploration task (OET), and decreasing neuronal proliferation and brain-derived neurotrophic factor (BDNF) in the hippocampus. Treatment with 3CS-nalmefene (0.3mg/kg/sc twice per day, 3 times per week for 4weeks) prevented IFN-α-induced immobility in the FST and impaired object displacement learning. In addition, 3CS-nalmefene prevented IFN-α-induced increases in inflammation and hyperactivity of the HPA-axis, the IFN-α-induced reduction in both neuronal proliferation and BDNF expression in the hippocampus. Overall, these preclinical data would support the hypothesis that opioid receptor modulation is a relevant target for treatment of depression.
Assuntos
Antidepressivos/administração & dosagem , Transtorno Depressivo/tratamento farmacológico , Naltrexona/análogos & derivados , Antagonistas de Entorpecentes/administração & dosagem , Receptores Opioides/agonistas , Animais , Ansiedade/induzido quimicamente , Ansiedade/tratamento farmacológico , Comportamento Animal/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proliferação de Células/efeitos dos fármacos , Depressão/induzido quimicamente , Depressão/tratamento farmacológico , Transtorno Depressivo/induzido quimicamente , Modelos Animais de Doenças , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Interferon-alfa/administração & dosagem , Masculino , Naltrexona/administração & dosagem , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos WistarRESUMO
Derivatives of the lead compound N-BPE-8-CAC (1) where each CH of the biphenyl group was individually replaced by N were prepared in hopes of identifying high affinity ligands with improved aqueous solubility. Compared to 1, binding affinities of the five possible pyridinyl derivatives for the µ opioid receptor were between threefold lower to fivefold higher with the Ki of the most potent compound being 0.064 nM. Docking of 8-CAC (2) into the unliganded binding site of the mouse µ opioid receptor (pdb: 4DKL) revealed that 8-CAC and ß-FNA (from 4DKL) make nearly identical interactions with the receptor. However, for 1 and the new pyridinyl derivatives 4-8, binding is not tolerated in the 8-CAC binding mode due to the steric constraints of the large N-substituents. Either an alternative binding mode or rearrangement of the protein to accommodate these modifications may account for their high binding affinity.
Assuntos
Compostos de Bifenilo/farmacologia , Ciclopropanos/farmacologia , Piridinas/farmacologia , Receptores Opioides/química , Animais , Compostos de Bifenilo/síntese química , Compostos de Bifenilo/química , Ciclopropanos/síntese química , Ciclopropanos/química , Relação Dose-Resposta a Droga , Humanos , Ligantes , Camundongos , Modelos Moleculares , Estrutura Molecular , Piridinas/síntese química , Piridinas/química , Receptores Opioides/metabolismo , Relação Estrutura-AtividadeRESUMO
The Kv1.3 channel is a recognized target for pharmaceutical development to treat autoimmune diseases and organ rejection. ShK-186, a specific peptide inhibitor of Kv1.3, has shown promise in animal models of multiple sclerosis and rheumatoid arthritis. Here, we describe the pharmacokinetic-pharmacodynamic relationship for ShK-186 in rats and monkeys. The pharmacokinetic profile of ShK-186 was evaluated with a validated high-performance liquid chromatography-tandem mass spectrometry method to measure the peptide's concentration in plasma. These results were compared with single-photon emission computed tomography/computed tomography data collected with an ¹¹¹In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-conjugate of ShK-186 to assess whole-blood pharmacokinetic parameters as well as the peptide's absorption, distribution, and excretion. Analysis of these data support a model wherein ShK-186 is absorbed slowly from the injection site, resulting in blood concentrations above the Kv1.3 channel-blocking IC50 value for up to 7 days in monkeys. Pharmacodynamic studies on human peripheral blood mononuclear cells showed that brief exposure to ShK-186 resulted in sustained suppression of cytokine responses and may contribute to prolonged drug effects. In delayed-type hypersensitivity, chronic relapsing-remitting experimental autoimmune encephalomyelitis, and pristane-induced arthritis rat models, a single dose of ShK-186 every 2 to 5 days was as effective as daily administration. ShK-186's slow distribution from the injection site and its long residence time on the Kv1.3 channel contribute to the prolonged therapeutic effect of ShK-186 in animal models of autoimmune disease.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Canal de Potássio Kv1.3/antagonistas & inibidores , Proteínas/farmacologia , Linfócitos T/efeitos dos fármacos , Absorção/efeitos dos fármacos , Absorção/imunologia , Animais , Artrite/tratamento farmacológico , Artrite/imunologia , Artrite/metabolismo , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Humanos , Concentração Inibidora 50 , Canal de Potássio Kv1.3/imunologia , Canal de Potássio Kv1.3/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Macaca fascicularis , Bloqueadores dos Canais de Potássio/imunologia , Bloqueadores dos Canais de Potássio/farmacocinética , Bloqueadores dos Canais de Potássio/farmacologia , Proteínas/farmacocinética , Ratos , Ratos Sprague-Dawley , Saimiri , Linfócitos T/imunologia , Linfócitos T/metabolismo , Distribuição Tecidual/efeitos dos fármacos , Distribuição Tecidual/imunologiaRESUMO
Because of increased opioid misuse, there is a need to identify new targets for minimizing opioid tolerance, and physical and psychological dependence. Previous studies showed that fibroblast growth factor 21 (FGF21) decreased alcohol and sweet preference in mice. In this study, FGF21-transgenic (FGF21-Tg) mice, expressing high FGF21 serum levels, and wildtype (WT) C57BL/6J littermates were treated with morphine and saline to determine if differences exist in their physiological and behavioral responses to opioids. FGF21-Tg mice displayed reduced preference for morphine in the conditioned place preference assay compared to WT littermates. Similarly, FGF21-Tg mice had an attenuation of the magnitude and rate of acute morphine antinociceptive tolerance development, and acute and chronic morphine physical dependence, but exhibited no change in chronic morphine antinociceptive tolerance. The ED50 values for morphine-induced antinociception in the 55 °C hot plate and the 55 °C warm-water tail withdrawal assays were similar in both strains of mice. Likewise, FGF21-Tg and WT littermates had comparable responses to morphine-induced respiratory depression. Overall, FGF21-Tg mice had a decrease in the development of acute analgesic tolerance, and the development of physical dependence, and morphine preference. FGF21 and its receptor have therapeutic potential for reducing opioid withdrawal symptoms and craving, and augmenting opioid therapeutics for acute pain patients to minimize tolerance development.
Assuntos
Tolerância a Medicamentos/genética , Fatores de Crescimento de Fibroblastos/sangue , Fatores de Crescimento de Fibroblastos/fisiologia , Dependência de Morfina/genética , Morfina/efeitos adversos , Nociceptividade/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Insuficiência Respiratória/induzido quimicamente , Insuficiência Respiratória/genética , Síndrome de Abstinência a Substâncias/genética , Síndrome de Abstinência a Substâncias/terapiaRESUMO
A series of bivalent hydroxy ether butorphan ligands were prepared and their binding affinities at the opioid receptors determined. Addition of a hydroxy group to a hydrocarbon chain can potentiate binding affinity up to 27- and 86-fold at the mu and kappa opioid receptors, respectively. Two bivalent ligands with sub-nanomolar binding affinity at the mu and kappa opioid receptors were discovered.
Assuntos
Analgésicos/química , Ligantes , Morfinanos/química , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/metabolismo , Analgésicos/síntese química , Analgésicos/farmacologia , Animais , Células CHO , Cricetinae , Cricetulus , Morfinanos/síntese química , Morfinanos/farmacologia , Ligação Proteica , Receptores Opioides kappa/antagonistas & inibidores , Receptores Opioides mu/antagonistas & inibidoresRESUMO
We synthesized several hydrophobic esters and ethers of butorphanol and assessed their affinities at opioid receptors in CHO cell membranes. Tested compounds displayed moderate to high affinities to the mu and kappa receptors. The findings accord with previous evidence of a lipophilic binding pocket in the opioid receptors that can be accessed to afford good binding affinity without the need for a phenolic hydrogen-bond donor group. The most potent (K(i)=61 pM at mu and 48 pM at kappa) novel agent was (-)-N-cyclobutylmethylmorphinan-3-yl-14-ol phenoxyacetate (4d).
Assuntos
Butorfanol/química , Butorfanol/síntese química , Química Farmacêutica/métodos , Antagonistas de Entorpecentes , Receptores Opioides/química , Animais , Células CHO , Cricetinae , Cricetulus , Desenho de Fármacos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Químicos , Fenol , Ligação Proteica , TemperaturaRESUMO
Our investigation of bivalent ligands at mu, delta, and kappa opioid receptors is focused on the preparation of ligands containing kappa agonist and mu agonist/antagonist pharmacophores at one end joined by a chain containing the mu antagonist pharmacophores (naltrexone, naloxone, or nalbuphine) at the other end. These ligands were evaluated in vitro by their binding affinity at mu, delta, and kappa opioid receptors and their relative efficacy in the [35S]GTPgammaS assay.
Assuntos
Morfinanos/síntese química , Nalbufina/análogos & derivados , Naloxona/análogos & derivados , Naltrexona/análogos & derivados , Receptores Opioides delta/efeitos dos fármacos , Receptores Opioides kappa/efeitos dos fármacos , Receptores Opioides mu/efeitos dos fármacos , (trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida/farmacologia , Animais , Células CHO , Cricetinae , Cricetulus , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Humanos , Ligantes , Morfinanos/química , Morfinanos/farmacologia , Nalbufina/síntese química , Nalbufina/farmacologia , Naloxona/síntese química , Naloxona/farmacologia , Naltrexona/síntese química , Naltrexona/farmacologia , Ensaio Radioligante , Receptores Opioides delta/agonistas , Receptores Opioides delta/antagonistas & inibidores , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/antagonistas & inibidores , Receptores Opioides mu/agonistas , Receptores Opioides mu/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
To further extend the structure-activity relationships of levorphanol, two series of novel morphinans were prepared by incorporation of an indole or aminothiazole fragment to the hexyl ring (ring C) in levorphanol. Such morphinans differed from previously reported ligands in that such indole- or aminothiazole-containing morphinans displayed enhanced binding affinity to the delta opioid receptor, while the affinity to kappa and micro receptors was slightly reduced.
Assuntos
Analgésicos Opioides/síntese química , Indóis/síntese química , Levorfanol/análogos & derivados , Levorfanol/síntese química , Tiazóis/síntese química , Analgésicos Opioides/farmacologia , Animais , Células CHO , Cricetinae , Cricetulus , Indóis/farmacologia , Levorfanol/farmacologia , Ensaio Radioligante , Relação Estrutura-Atividade , Tiazóis/farmacologiaRESUMO
Dysregulation of enzymes involved in prostaglandin biosynthesis plays a critical role in influencing the biological behavior and clinical outcome of several tumors. In human gliomas, overexpression of cyclooxygenase-2 has been linked to increased aggressiveness and poor prognosis. In contrast, the role of prostaglandin E synthase in influencing the biological behavior of human gliomas has not been established. We report that constitutive expression of the microsomal prostaglandin E synthase-1 (mPGES-1) is associated with increased prostaglandin E(2) (PGE(2)) production and stimulation of growth in the human astroglioma cell line U87-MG compared with human primary astrocytes. Consistently, pharmacologic and genetic inhibition of mPGES-1 activity and expression blocked the release of PGE(2) from U87-MG cells and decreased their proliferation. Conversely, exogenous PGE(2) partially overcame the antiproliferative effects of mPGES-1 inhibition and stimulated U87-MG cell proliferation in the absence of mPGES-1 inhibitors. The EP2/EP4 subtype PGE(2) receptors, which are linked to stimulation of adenylate cyclase, were expressed in U87-MG cells to a greater extent than in human astrocytes. PGE(2) increased cyclic AMP levels and stimulated protein kinase A (PKA) activity in U87-MG cells. Treatment with a selective type II PKA inhibitor decreased PGE(2)-induced U87-MG cell proliferation, whereas a selective type I PKA inhibitor had no effect. Taken together, these results are consistent with the hypothesis that mPGES-1 plays a critical role in promoting astroglioma cell growth via PGE(2)-dependent activation of type II PKA.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dinoprostona/metabolismo , Glioma/enzimologia , Oxirredutases Intramoleculares/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/enzimologia , Proliferação de Células/efeitos dos fármacos , Proteína Quinase Tipo II Dependente de AMP Cíclico , Dinoprostona/farmacologia , Glioma/patologia , Humanos , Oxirredutases Intramoleculares/genética , Microssomos/enzimologia , Prostaglandina-E Sintases , Células Tumorais CultivadasRESUMO
A series of homo- and heterodimeric ligands containing kappa agonist and mu agonist/antagonist pharmacophores joined by a linker chain of varying lengths was synthesized and evaluated in vitro by their binding affinity at mu, delta, and kappa opioid receptors. The functional activities of these compounds were measured in the [(35)S]GTPgammaS binding assay. The data suggest that the stereochemistry of the pharmacophores, the N-substituents of the pharmacophore, ester linkages, and the spacer length were crucial factors for optimum interactions of such ligands at opioid receptor binding sites. These novel ligands as well as their pharmacological properties will serve as the basis for our continuing investigation of such bivalent ligands as probes of the opioid receptor oligomerization phenomena and for in vivo studies as analgesics.
Assuntos
Morfinanos/síntese química , Morfinanos/farmacologia , Receptores Opioides delta/antagonistas & inibidores , Receptores Opioides kappa , Receptores Opioides mu , Animais , Células CHO , Cricetinae , Avaliação Pré-Clínica de Medicamentos , Humanos , Técnicas In Vitro , Ligantes , Conformação Molecular , Morfinanos/química , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/antagonistas & inibidores , Receptores Opioides mu/agonistas , Receptores Opioides mu/antagonistas & inibidores , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Here, we report the synthesis of a designed multi-pharmacophore ligand derived from the linkage of a delta selective peptide antagonist (Dmt-Tic) and a mu/kappa morphinan agonist butorphan (MCL 101) through a two methylene spacer. The new compound MCL 450 maintains the same characteristics as those the two reference compounds. MCL 450 represents a useful starting point for the synthesis of other multiple opioid ligands endowed with analgesic properties with low tolerance and dependence.
Assuntos
Dipeptídeos/síntese química , Morfinanos/síntese química , Antagonistas de Entorpecentes , Receptores Opioides/agonistas , Tetra-Hidroisoquinolinas/síntese química , Animais , Células CHO , Cricetinae , Cricetulus , Dipeptídeos/química , Dipeptídeos/farmacologia , Ligantes , Morfinanos/química , Morfinanos/farmacologia , Ensaio Radioligante , Receptores Opioides delta/antagonistas & inibidores , Receptores Opioides kappa/agonistas , Receptores Opioides mu/agonistas , Relação Estrutura-Atividade , Tetra-Hidroisoquinolinas/química , Tetra-Hidroisoquinolinas/farmacologiaRESUMO
Angiogenesis plays a crucial role in tumor development and growth. The present investigation was undertaken to test the potential involvement of the cyclooxygenase-2 (COX-2) pathway in the regulation of angiogenesis and growth in pancreatic cancer. We compared the angiogenic characteristics of a COX-2-positive human pancreatic tumor cell line, BxPC-3, with those of a COX-2-negative pancreatic tumor cell line, AsPC-1. Cultured BxPC-3 cells promoted a marked increase of endothelial cell migration in comparison with migration that occurred in the absence of cancer cells. Furthermore, BxPC-3 cell culture supernatants induced endothelial cell capillary morphogenesis in vitro and neovascularization in vivo. In contrast, cultured AsPC-1 cells elicited a modest effect on endothelial cell migration and neovascularization in vivo. Pretreatment of BxPC-3 cells with the selective COX-2 inhibitor NS-398 (50 micro M) dramatically decreased angiogenic responses of endothelial cells. NS-398 (25-100 micro M) caused inhibition of BxPC-3 cell proliferation but had no effect on AsPC-1 cell growth. SC-560, a selective COX-1 inhibitor, had no effect on growth of either cell lines. These results suggest an involvement of COX-2 in the control of tumor-dependent angiogenesis and growth in certain pancreatic cancers and provide the rational for inhibition of the COX pathway as an effective therapeutic approach for pancreatic tumors.
Assuntos
Inibidores de Ciclo-Oxigenase/farmacologia , Isoenzimas/fisiologia , Neovascularização Patológica/enzimologia , Nitrobenzenos/farmacologia , Neoplasias Pancreáticas/irrigação sanguínea , Neoplasias Pancreáticas/patologia , Prostaglandina-Endoperóxido Sintases/fisiologia , Sulfonamidas/farmacologia , Capilares/efeitos dos fármacos , Capilares/patologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Dinoprostona/metabolismo , Fatores de Crescimento Endotelial/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Homeostase , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Linfocinas/metabolismo , Proteínas de Membrana , Neovascularização Patológica/prevenção & controle , Neoplasias Pancreáticas/enzimologia , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandina-Endoperóxido Sintases/metabolismo , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Glycopeptides related to ß-endorphin penetrate the blood-brain barrier (BBB) of mice to produce antinociception. Two series of glycopeptides were assessed for opioid receptor binding affinity. Attempts to alter the mu-selectivity of [D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin (DAMGO)-related glycopeptides by altering the charged residues of the amphipathic helical address were unsuccessful. A series of pan-agonists was evaluated for antinociceptive activity (55 °C tail flick) in mice. A flexible linker was required to maintain antinociceptive activity. Circular dichroism (CD) in H2O, trifluoroethanol (TFE), and SDS micelles confirmed the importance of the amphipathic helices (11s â 11sG â 11) for antinociception. The glycosylated analogues showed only nascent helices and random coil conformations in H2O. Chemical shift indices (CSI) and nuclear Overhauser effects (NOE) with 600 MHz NMR and CD confirmed helical structures in micelles, which were rationalized by molecular dynamics calculations. Antinociceptive studies with mice confirm that these glycosylated endorphin analogues are potential drug candidates that penetrate the BBB to produce potent central effects.
Assuntos
Sistema Nervoso Central/efeitos dos fármacos , Glicopeptídeos/farmacologia , Peptídeos Opioides/farmacologia , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Glicopeptídeos/química , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos Opioides/química , Conformação Proteica , Receptores Opioides mu/efeitos dos fármacosRESUMO
A novel series of morphinans were synthesized, and their binding affinity at and functional selectivity for micro, delta, and kappa opioid receptors were evaluated. These dimeric ligands can be viewed as dimeric morphinans, which were formed by coupling two identical morphinan pharmacophores (cyclorphan (1) or MCL 101 (2)) with varying connecting spacers. Ligands 6 and 7 with alkyl spacers on the nitrogen position and ligands 8 and 9 in which the two morphinan pharmacophores were coupled by ether moieties at the 3-hydroxyl positions showed significant decrease in affinity at all three opioid receptors. An improvement in the affinity was achieved by introducing an ester moiety as the spacer in the dimeric morphinans. It was observed that the affinity of these ligands was sensitive to the character and length of the spacer. Compound 13 (MCL-139) with a 4-carbon ester spacer, compound 17 (MCL-144) containing a 10-carbon spacer, and compound 19 (MCL-145) with the conformationally constrained fumaryl spacer were the most potent ligands in this series, displaying excellent affinities at micro and kappa receptors (K(i) = 0.09-0.2 nM at micro and K(i) = 0.078-0.049 nM at kappa), which were comparable to the parent compound 2. Ligand 12, a compound containing only one morphinan pharmacophore and a long-chain ester group, had affinity at both micro and kappa receptors almost identical to that of the parent ligand 2. In the [(35)S]GTPgammaS binding assay, ligands 13, 17, and 19 and their parent morphinans 1 and 2 stimulated [(35)S]GTPgammaS binding mediated by the micro and kappa receptors. Compounds 13 and 17 were full kappa agonists and partial micro agonists, while compound 19 was a partial agonist at both micro and kappa receptors. These novel ligands, as well as their interesting pharmacological properties, will serve as the basis for our continuing investigation of the dimeric ligands as potential probes for the pharmacotherapy of cocaine abuse and may also open new avenues for the characterization of opioid receptors.
Assuntos
Morfinanos/síntese química , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/antagonistas & inibidores , Receptores Opioides mu/agonistas , Receptores Opioides mu/antagonistas & inibidores , Animais , Células CHO , Cricetinae , Dimerização , Desenho de Fármacos , Humanos , Ligantes , Morfinanos/química , Morfinanos/farmacologia , Ensaio Radioligante , Relação Estrutura-AtividadeRESUMO
A series of 10-ketomorphinan analogues were synthesized, and their binding affinity at all three opioid receptors was investigated. In most cases, high affinity at micro and kappa receptors, and lower affinity at delta receptor was observed, resulting in good selectivity for micro and kappa receptors. A wide range of substituents can be accommodated on the nitrogen position. The N-(S)-tetrahydrofurfuryl analogue 11 displayed the highest affinity at all three receptors. The N-cyclobutylmethyl analogue 13 gave both high affinity and selectivity at kappa receptor, and N-2-phenylethyl analogue 18 exhibited good affinity and selectivity at micro receptor. Further modifications of the 3-substituent indicated that one H-bond donor was an essential requirement for good affinity at micro and kappa receptors. Similar modifications were investigated at the 3-OH group of morphinans: levorphanol (2a), cyclorphan (2b), and MCL-101 (2c) lacking the 10-keto group. The 3-amino bioisosteric analogues (40 and 41) displayed reasonably good affinity at micro and kappa receptors. The 3-carboxamido replacement (compounds 46-48) in the morphinan subseries resulted in similar affinities comparable to their corresponding 3-OH congeners. The high affinity of these carboxamido analogues, along with their greater lipophilicity and metabolic stability, make them promising candidates for further pharmacological investigation.
Assuntos
Morfinanos/síntese química , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/metabolismo , Animais , Encéfalo/metabolismo , Células CHO , Cricetinae , Cobaias , Ligação de Hidrogênio , Técnicas In Vitro , Ligantes , Modelos Moleculares , Conformação Molecular , Morfinanos/química , Morfinanos/farmacologia , Ensaio Radioligante , Relação Estrutura-AtividadeRESUMO
A series of levo- and dextromorphinan pairs have been synthesized and evaluated for their affinities to the mu, kappa, and delta opioid receptors, the N-methyl-D-aspartate (NMDA) channel, and sigma 1 and 2 receptors. It was found that levo isomers tended to have higher affinities at the opioid receptors and moderate to high affinities to the NMDA and sigma receptors, while dextro isomers tended to have lower affinities to the opioid receptors but comparatively higher affinities to the NMDA and sigma receptors. This series of compounds have interesting and complex pharmacological profiles, and merit further investigation as potential therapies for drug abuse treatment.
Assuntos
Morfinanos/farmacologia , N-Metilaspartato/efeitos dos fármacos , Receptores Opioides/efeitos dos fármacos , Receptores sigma/efeitos dos fármacos , Animais , Humanos , Masculino , Morfinanos/síntese química , Ratos , Ratos Sprague-DawleyRESUMO
Glycosylated ß-endorphin analogues of various amphipathicity were studied in vitro and in vivo in mice. Opioid binding affinities of the O-linked glycopeptides (mono- or disaccharides) and unglycosylated peptide controls were measured in human receptors expressed in CHO cells. All were pan-agonists, binding to µ-, δ-, or κ-opioid receptors in the low nanomolar range (2.2-35 nM K(i)'s). The glycoside moiety was required for intravenous (i.v.) but not for intracerebroventricular (i.c.v.) activity. Circular dichroism and NMR indicated the degree of helicity in H2O, aqueous trifluoroethanol, or micelles. Glycosylation was essential for activity after i.v. administration. It was possible to manipulate the degree of helicity by the alteration of only two amino acid residues in the helical address region of the ß-endorphin analogues without destroying µ-, δ-, or κ-agonism, but the antinociceptive activity after i.v. administration could not be directly correlated to the degree of helicity in micelles.