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1.
Bioorg Med Chem Lett ; 25(12): 2488-92, 2015 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-25981685

RESUMO

Antagonism of orexin receptors has shown clinical efficacy as a novel paradigm for the treatment of insomnia and related disorders. Herein, molecules related to the dual orexin receptor antagonist filorexant were transformed into compounds that were selective for the OX2R subtype. Judicious selection of the substituents on the pyridine ring and benzamide groups led to 6b; which was highly potent, OX2R selective, and exhibited excellent development properties.


Assuntos
Antagonistas dos Receptores de Orexina/química , Receptores de Orexina/química , Piperidinas/química , Triazóis/química , Animais , Cães , Meia-Vida , Camundongos , Antagonistas dos Receptores de Orexina/farmacocinética , Antagonistas dos Receptores de Orexina/uso terapêutico , Receptores de Orexina/metabolismo , Piperidinas/farmacocinética , Piperidinas/uso terapêutico , Ligação Proteica , Pirimidinas/química , Ratos , Distúrbios do Início e da Manutenção do Sono/tratamento farmacológico , Distúrbios do Início e da Manutenção do Sono/veterinária , Relação Estrutura-Atividade , Triazóis/farmacocinética , Triazóis/uso terapêutico
2.
J Biol Chem ; 286(33): 28867-28875, 2011 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-21700703

RESUMO

Schizophrenia is a highly heritable neuropsychiatric disorder affecting ∼1% of the world's population. Linkage and association studies have identified multiple candidate schizophrenia susceptibility genes whose functions converge on the glutamatergic neurotransmitter system. One such susceptibility gene encoding D-amino acid oxidase (DAO), an enzyme that metabolizes the NMDA receptor (NMDAR) co-agonist D-serine, has the potential to modulate NMDAR function in the context of schizophrenia. To further investigate its cellular regulation, we sought to identify DAO-interacting proteins that participate in its functional regulation in rat cerebellum, where DAO expression is especially high. Immunoprecipitation with DAO-specific antibodies and subsequent mass spectrometric analysis of co-precipitated proteins yielded 24 putative DAO-interacting proteins. The most robust interactions occurred with known components of the presynaptic active zone, such as bassoon (BSN) and piccolo (PCLO). The interaction of DAO with BSN was confirmed through co-immunoprecipitation assays using DAO- and BSN-specific antibodies. Moreover, DAO and BSN colocalized with one another in cultured cerebellar granule cells and in synaptic junction membrane protein fractions derived from rat cerebellum. The functional consequences of this interaction were studied through enzyme assay experiments, where DAO enzymatic activity was significantly inhibited as a result of its interaction with BSN. Taking these results together, we hypothesize that synaptic D-serine concentrations may be under tight regulation by a BSN-DAO complex. We therefore predict that this mechanism plays a role in the modulation of glutamatergic signaling through NMDARs. It also furthers our understanding of the biology underlying this potential therapeutic entry point for schizophrenia and other psychiatric disorders.


Assuntos
D-Aminoácido Oxidase/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Terminações Pré-Sinápticas/metabolismo , Serina/metabolismo , Membranas Sinápticas/metabolismo , Animais , Cerebelo/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , D-Aminoácido Oxidase/genética , Humanos , Masculino , Proteínas do Tecido Nervoso/genética , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Esquizofrenia/genética , Esquizofrenia/metabolismo , Serina/genética , Membranas Sinápticas/genética
3.
Artigo em Inglês | MEDLINE | ID: mdl-20936132

RESUMO

MrgD, a member of the Mas-related gene family, is expressed exclusively in small diameter IB4(+) neurons in the dorsal root ganglion. This unique expression pattern, the presence of a single copy of MrgD in rodents and humans, and the identification of a putative ligand, beta-alanine, make it an experimentally attractive therapeutic target for pain with limited likelihood of side effects. We have devised a high throughput calcium mobilization assay that enables identification of both agonists and antagonists from a single screen for MrgD. Screening of the Library of Pharmacologically Active Compounds (LOPAC) validated this assay approach, and we identified both agonists and antagonists active at micromolar concentrations in MrgD expressing but not in parental CHO-DUKX cell line. Further characterization was performed using a subset of these screening hits. Our results demonstrated that the dual agonist/antagonist assay format is feasible and likely can be extended to most GPCRs with known agonist.


Assuntos
Descoberta de Drogas/métodos , Fluorometria/métodos , Receptores Acoplados a Proteínas G , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Humanos , Cinética , Nociceptores , Ratos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequenas
4.
Bioorg Med Chem Lett ; 20(9): 2983-6, 2010 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-20347298

RESUMO

A dihydroquinolinone moiety was found to be a potent serotonin reuptake inhibitor pharmacophore when combined with certain amines. This fragment was coupled with selected D(2) ligands to prepare a series of dual acting compounds with attractive in vitro profiles as dopamine D(2) partial agonists and serotonin reuptake inhibitors. Structure-activity studies revealed that the linker plays a key role in contributing to D(2) affinity, function, and SRI activity.


Assuntos
Antipsicóticos/química , Agonistas de Dopamina/química , Quinolonas/química , Esquizofrenia/tratamento farmacológico , Inibidores Seletivos de Recaptação de Serotonina/química , Animais , Antipsicóticos/síntese química , Antipsicóticos/uso terapêutico , Modelos Animais de Doenças , Agonistas de Dopamina/síntese química , Agonistas de Dopamina/uso terapêutico , Quinolonas/síntese química , Quinolonas/uso terapêutico , Receptor 5-HT1A de Serotonina/química , Receptor 5-HT1A de Serotonina/metabolismo , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D2/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/síntese química , Inibidores Seletivos de Recaptação de Serotonina/uso terapêutico , Relação Estrutura-Atividade
5.
J Pharmacol Exp Ther ; 332(1): 190-201, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19828876

RESUMO

The preclinical characterization of WS-50030 [7-{4-[3-(1H-inden-3-yl)propyl]piperazin-1-yl}-1,3-benzoxazol-2(3H)-one] is described. In vitro binding and functional studies revealed highest affinity to the D(2) receptor (D(2L) K(i), 4.0 nM) and serotonin transporter (K(i), 7.1 nM), potent D(2) partial agonist activity (EC(50), 0.38 nM; E(max), 30%), and complete block of the serotonin transporter (IC(50), 56.4 nM). Consistent with this in vitro profile, WS-50030 (10 mg/kg/day, 21 days) significantly increased extracellular 5-HT in the rat medial prefrontal cortex, short-term WS-50030 treatment blocked apomorphine-induced climbing (ID(50), 0.51 mg/kg) in a dose range that produced minimal catalepsy in mice and induced low levels of contralateral rotation in rats with unilateral substantia nigra 6-hydroxydopamine lesions (10 mg/kg i.p.), a behavioral profile similar to that of the D(2) partial agonist aripiprazole. In a rat model predictive of antipsychotic-like activity, WS-50030 and aripiprazole reduced conditioned avoidance responding by 42 and 55% at 10 mg/kg, respectively. Despite aripiprazole's reported lack of effect on serotonin transporters, long-term treatment with aripiprazole or WS-50030 reversed olfactory bulbectomy-induced hyperactivity at doses that did not reduce activity in sham-operated rats, indicating antidepressant-like activity for both compounds. Despite possessing serotonin reuptake inhibitory activity in addition to D(2) receptor partial agonism, WS-50030 displays activity in preclinical models predictive of antipsychotic- and antidepressant efficacy similar to aripiprazole, suggesting potential efficacy of WS-50030 versus positive and negative symptoms of schizophrenia, comorbid mood symptoms, bipolar disorder, major depressive disorder, and treatment-resistant depression. Furthermore, WS-50030 provides a tool to further explore how combining these mechanisms might differentiate from other antipsychotics or antidepressants.


Assuntos
Antidepressivos/farmacologia , Antipsicóticos/farmacologia , Benzoxazóis/farmacologia , Agonistas de Dopamina/farmacologia , Indenos/farmacologia , Receptores de Dopamina D2/agonistas , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Animais , Antidepressivos/química , Antipsicóticos/química , Aprendizagem da Esquiva/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Benzoxazóis/química , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Células CHO , Cricetinae , Cricetulus , Dopamina/metabolismo , Agonistas de Dopamina/química , Avaliação Pré-Clínica de Medicamentos , Humanos , Indenos/química , Masculino , Camundongos , Camundongos Endogâmicos , Microdiálise , Atividade Motora/efeitos dos fármacos , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Serotonina/metabolismo , Antagonistas do Receptor 5-HT1 de Serotonina , Antagonistas do Receptor 5-HT2 de Serotonina , Inibidores Seletivos de Recaptação de Serotonina/química , Transfecção
6.
Assay Drug Dev Technol ; 8(1): 106-13, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19922239

RESUMO

The 5-hydroxytryptamine 2C (5-HT(2C)) receptor is a member of the serotonin 5-HT(2) subfamily of G-protein-coupled receptors signaling predominantly via the phospholipase C (PLC) pathway. Stimulation of phosphoinositide (PI) hydrolysis upon 5-HT(2C) receptor activation is traditionally assessed by measuring inositol monophosphate (IP(1)) using time-consuming and labor-intensive anion exchange radioactive assays. In this study, we have developed and optimized a cellular IP(1) assay using homogeneous time-resolved fluorescence (HTRF), a fluorescence resonance energy transfer (FRET)-based technology (Cisbio; Gif sur Yvette, France). The measurement is simple to carry out without the cumbersome steps associated with radioactive assays and may therefore be used as an alternative tool to evaluate PI hydrolysis activated by 5-HT(2C) agonists. In Chinese hamster ovary (CHO) cells stably expressing 5-HT(2C) receptors, characterization of 5-HT(2C) agonists with the HTRF platform revealed a rank order of potency (EC(50), nM) comparable to that from intracellular calcium mobilization studies measured by the fluorometric imaging plate reader (FLIPR). A similar rank order of potency was seen with conventional radioactive PI assay with the exception of 5-HT. Lastly, the new assay data correlated better with agonist-induced calcium responses in FLIPR (R(2) = 0.78) than with values determined by radioactive IP(1) method (R(2) = 0.64). Our study shows that the HTRF FRET-based assay detects IP(1) with good sensitivity and may be streamlined for high-throughput (HTS) applications.


Assuntos
Fosfatos de Inositol/metabolismo , Receptor 5-HT2C de Serotonina/fisiologia , Animais , Células CHO , Cálcio/metabolismo , Cricetinae , Cricetulus , Transferência Ressonante de Energia de Fluorescência , Humanos , Ligantes
7.
Bioorg Med Chem Lett ; 19(19): 5552-5, 2009 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-19720528

RESUMO

A 5-fluoro-tetrahydrocarbazole serotonin reuptake inhibitor (SRI) building block was combined with a variety of linkers and dopamine D2 receptor ligands in an attempt to identify potent D2 partial agonist/SRI molecules for treatment of schizophrenia. This approach has the potential to treat a broader range of symptoms compared to existing therapies. Selected compounds in this series demonstrate high affinity for both targets and D2 partial agonism in cell-based and in vivo assays.


Assuntos
Carbazóis/química , Agonistas de Dopamina/química , Receptores de Dopamina D2/agonistas , Esquizofrenia/tratamento farmacológico , Inibidores Seletivos de Recaptação de Serotonina/química , Antagonistas do Receptor 5-HT1 de Serotonina , Animais , Carbazóis/síntese química , Carbazóis/farmacologia , Modelos Animais de Doenças , Agonistas de Dopamina/síntese química , Agonistas de Dopamina/farmacologia , Ratos , Receptor 5-HT1A de Serotonina/metabolismo , Receptores de Dopamina D2/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/síntese química , Inibidores Seletivos de Recaptação de Serotonina/farmacologia
8.
Eur J Pharmacol ; 605(1-3): 53-6, 2009 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-19168056

RESUMO

In functional assay assessments using the five muscarinic receptor subtypes, a second generation of muscarinic M(1)-preferring receptor agonists [AC-42 (1), AC-260584 (2), 77-LH-28-1 (3) and LY-593039 (4)] was shown to have higher selectivity for muscarinic M(1) over M(3) receptor as compared to historical agonists [talsaclidine (8), sabcomeline (10), xanomeline (11), WAY-132983 (12), cevimeline (9) and NGX-267 (6)]. Another striking difference of these more recent compounds is their affinities for the dopamine D(2) and 5-HT(2B) receptors. Taken together, these results suggest that the newer compounds may have a greater clinical safety profile, especially with regard to muscarinic M(3) receptor-mediated events, than the historical agonists, but their affinities for other receptors may still compromise their use to validate the therapeutic potential of muscarinic M(1) receptor agonists.


Assuntos
Agonistas Muscarínicos/farmacologia , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M3/agonistas , Ligantes , Agonistas Muscarínicos/efeitos adversos , Ligação Proteica , Receptor 5-HT2B de Serotonina/efeitos dos fármacos , Receptor 5-HT2B de Serotonina/metabolismo , Receptores de Dopamina D2/efeitos dos fármacos , Receptores de Dopamina D2/metabolismo
9.
Bioorg Med Chem Lett ; 18(21): 5789-91, 2008 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-18849166

RESUMO

We identified small molecule NTS1R agonist compounds through virtual screening of the corporate database using a ROCS approach that searches multi-conformer representations efficiently. As a starting point for the ROCS search, we used the known NTS1R selective antagonist, SR-48527, based on the hypothesis that NT agonists and antagonists might share similar binding regions. Conformations were expanded and selected as database search queries based on a cluster analysis. The search provided us with virtual hits that were tested in intracellular calcium mobilization assays of NTS1R agonist and antagonist activities measured in FLIPR format as well as in [(3)H]NT competition binding studies. The results indicated that two initial hits produced partial agonist activity with potency in the moderate micromolar range.


Assuntos
Receptores de Neurotensina/agonistas , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Ligantes , Modelos Moleculares
10.
J Pharmacol Exp Ther ; 327(3): 827-39, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18753411

RESUMO

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGlu5) enhance N-methyl-d-aspartate receptor function and may represent a novel approach for the treatment of schizophrenia. ADX47273 [S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-piperidin-1-yl}-methanone], a recently identified potent and selective mGlu5 PAM, increased (9-fold) the response to threshold concentration of glutamate (50 nM) in fluorometric Ca(2+) assays (EC(50) = 170 nM) in human embryonic kidney 293 cells expressing rat mGlu5. In the same system, ADX47273 dose-dependently shifted mGlu5 receptor glutamate response curve to the left (9-fold at 1 microM) and competed for binding of [(3)H]2-methyl-6-(phenylethynyl)pyridine (K(i) = 4.3 microM), but not [(3)H]quisqualate. In vivo, ADX47273 increased extracellular signal-regulated kinase and cAMP-responsive element-binding protein phosphorylation in hippocampus and prefrontal cortex, both of which are critical for glutamate-mediated signal transduction mechanisms. In models sensitive to antipsychotic drug treatment, ADX47273 reduced rat-conditioned avoidance responding [minimal effective dose (MED) = 30 mg/kg i.p.] and decreased mouse apomorphine-induced climbing (MED = 100 mg/kg i.p.), with little effect on stereotypy or catalepsy. Furthermore, ADX47273 blocked phencyclidine, apomorphine, and amphetamine-induced locomotor activities (MED = 100 mg/kg i.p.) in mice and decreased extracellular levels of dopamine in the nucleus accumbens, but not in the striatum, in rats. In cognition models, ADX47273 increased novel object recognition (MED = 1 mg/kg i.p.) and reduced impulsivity in the five-choice serial reaction time test (MED = 10 mg/kg i.p.) in rats. Taken together, these effects are consistent with the hypothesis that allosteric potentiation of mGlu5 may provide a novel approach for development of antipsychotic and procognitive agents.


Assuntos
Regulação Alostérica/efeitos dos fármacos , Antipsicóticos/farmacologia , Cognição/efeitos dos fármacos , Oxidiazóis/farmacologia , Piperidinas/farmacologia , Receptores de Glutamato Metabotrópico/efeitos dos fármacos , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Química Encefálica/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Hipocampo/metabolismo , Humanos , Córtex Pré-Frontal/metabolismo , Ratos , Receptor de Glutamato Metabotrópico 5
11.
J Neurosci ; 27(16): 4492-6, 2007 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-17442834

RESUMO

The recently identified Mas-related gene (Mrg) family of G-protein-coupled receptors is expressed almost exclusively in dorsal root ganglion (DRG) neurons. The expression of one family member, MrgD, is even further confined to IB4+, nonpeptidergic, small-diameter nociceptors. Although the functional consequences of MrgD activation are not known, this expression profile provides intriguing potential for a role in pain sensation or modulation. In a recombinant cell line, we first assessed the functional significance of MrgD activation by coexpressing MrgD with the KCNQ2/3 potassium channel, a channel implicated in pain. Whole-cell voltage-clamp recordings revealed that bath application of the ligand for MrgD, beta-alanine, resulted in robust inhibition of KCNQ2/3 activity. Pharmacological blockade of G(i/o) and phospholipase C signaling revealed a partial and complete block of the response, respectively. We extended these observations to dissociated DRG neuron cultures by examining MrgD modulation of M-currents (carried primarily by KCNQ2/3). Here too, beta-alanine-induced activation of endogenous MrgD inhibited M-currents, but primarily via a pertussis toxin-sensitive pathway. Finally, we assessed the consequence of beta-alanine-induced activation of MrgD in phasic neurons. Phasic neurons that fired a single action potential (AP) before beta-alanine application fired multiple APs during beta-alanine exposure. In sum, we provide evidence for a novel interaction between MrgD and KCNQ/M-type potassium channels that contributes to an increase in excitability of DRG neurons and thus may enhance the signaling of primary afferent nociceptive neurons.


Assuntos
Gânglios Espinais/metabolismo , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/metabolismo , Neurônios/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Potenciais de Ação/fisiologia , Animais , Células CHO , Cricetinae , Cricetulus , Masculino , Técnicas de Patch-Clamp , Ratos , Ratos Long-Evans , Ratos Sprague-Dawley
12.
Biochim Biophys Acta ; 1770(6): 890-901, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17363172

RESUMO

GPR26 and GPR78 are orphan GPCRs (oGPCRs) that share 51% amino acid sequence identity and are widely expressed in selected tissues of the human brain as well as the developing and adult mouse brain. Investigation of the functional activity of GPR26 and GPR78 via expression in HEK293 cells showed that both proteins are constitutively active and coupled to elevated cAMP production. Accordingly, in yeast, GPR26 demonstrated apparent agonist-independent coupling to a chimeric Gpa1 protein in which the 5 C-terminal amino acids were from Galphas. A comparison of the proteins revealed an atypical glutamine residue in GPR78 in place of the conserved arginine residue (R3.50) in the so-called DRY box. Site-directed mutants R3.50 in GPR26 were constructed and retained their constitutive activity suggesting that these 2 receptors activate G proteins in a manner that is distinct from other group 1 GPCRs.


Assuntos
Encéfalo/metabolismo , Receptores Acoplados a Proteínas G/biossíntese , Animais , Sequência de Bases , Encéfalo/citologia , Linhagem Celular , AMP Cíclico/biossíntese , Chaperona BiP do Retículo Endoplasmático , Subunidades alfa de Proteínas de Ligação ao GTP/biossíntese , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos
13.
Proc Natl Acad Sci U S A ; 104(12): 5163-8, 2007 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-17360345

RESUMO

We evolved muscarinic receptors in yeast to generate a family of G protein-coupled receptors (GPCRs) that are activated solely by a pharmacologically inert drug-like and bioavailable compound (clozapine-N-oxide). Subsequent screening in human cell lines facilitated the creation of a family of muscarinic acetylcholine GPCRs suitable for in vitro and in situ studies. We subsequently created lines of telomerase-immortalized human pulmonary artery smooth muscle cells stably expressing all five family members and found that each one faithfully recapitulated the signaling phenotype of the parent receptor. We also expressed a G(i)-coupled designer receptor in hippocampal neurons (hM(4)D) and demonstrated its ability to induce membrane hyperpolarization and neuronal silencing. We have thus devised a facile approach for designing families of GPCRs with engineered ligand specificities. Such reverse-engineered GPCRs will prove to be powerful tools for selectively modulating signal-transduction pathways in vitro and in vivo.


Assuntos
Evolução Molecular , Modelos Moleculares , Receptores Acoplados a Proteínas G/metabolismo , Animais , Linhagem Celular Transformada , Clozapina/análogos & derivados , Clozapina/farmacologia , Drogas Desenhadas , Epitopos , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Hipocampo/efeitos dos fármacos , Humanos , Hidrólise/efeitos dos fármacos , Ligantes , Proteínas Mutantes/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fosfatidilinositóis/metabolismo , Engenharia de Proteínas , Artéria Pulmonar/citologia , Artéria Pulmonar/efeitos dos fármacos , Ratos , Receptor Muscarínico M3/metabolismo , Receptor Muscarínico M4/metabolismo , Saccharomyces cerevisiae
14.
Eur J Pharmacol ; 552(1-3): 36-45, 2006 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-17056032

RESUMO

The pharmacology of aplindore (DAB-452) was characterized in CHO-K1 cells stably transfected with the human dopamine D(2) receptor short isoform (CHO-D(2s)) and in a behavioral model for post-synaptic agonism in rats. In [(3)H]-spiperone competition binding studies, aplindore showed high affinity for dopamine D(2) and D(3) receptors and low affinity for the dopamine D(4), serotonin (5-HT)(1A), 5-HT(2) receptors and the alpha1-adrenoceptor. The high potency partial agonist activity of aplindore was demonstrated in [(35)S]guanosine 5'-O-(3-thiotriphosphate) ([(35)S]GTPgammaS) binding, extracellular signal-regulated kinase (ERK)-phosphorylation and intracellular calcium flux assay using fluorometric plate reader ([Ca(2+)](i)-FLIPR) format. The [Ca(2+)](i)-FLIPR assay was conducted with CHO-D(2S) receptor cells also stably expressing chimeric G(alphaq/o)-proteins. In all assay modalities, the potencies and intrinsic activities of aplindore were lower than dopamine and higher than aripiprazole. In contrast to the [(35)S]GTPgammaS binding and ERK-phosphorylation assays, the [Ca(2+)](i)-FLIPR assay was able to detect the low partial agonist activity of SDZ 208-912. In unilaterally 6-hydroxydopamine (6-OHDA) lesioned rats, aplindore induced contralateral turning, which was blocked by the dopamine D(2) receptor antagonist raclopride. The dopamine D(2) receptor selective partial agonist profile of aplindore suggests that it should be effective for the treatment of dopaminergic-based disorders, such as schizophrenia and Parkinson's disease.


Assuntos
Agonistas de Dopamina/farmacologia , Indóis/farmacologia , Receptores de Dopamina D2/agonistas , Animais , Ligação Competitiva , Células CHO , Cálcio/metabolismo , Cricetinae , Cricetulus , Agonistas de Dopamina/metabolismo , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Indóis/metabolismo , Masculino , Atividade Motora/efeitos dos fármacos , Oxidopamina/toxicidade , Fosforilação/efeitos dos fármacos , Quimpirol/farmacologia , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Receptor 5-HT1A de Serotonina/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D4/metabolismo , Receptores 5-HT2 de Serotonina/metabolismo , Substância Negra/efeitos dos fármacos , Substância Negra/patologia , Substância Negra/fisiopatologia
15.
Brain Res ; 1087(1): 1-14, 2006 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-16647048

RESUMO

This report describes the identification and characterization of the murine orphan GPCR, Gpr101. Both human and murine genes were localized to chromosome X. Similar to its human ortholog, murine Gpr101 mRNA was detected predominantly in the brain within discrete nuclei. A knowledge-restricted hidden Markov model-based algorithm, capable of accurately predicting G-protein coupling selectivity, indicated that both human and murine GPR101 were likely coupled to Gs. This prediction was supported by the elevation of cyclic AMP levels and the activation of a cyclic AMP response element-luciferase reporter gene in HEK293 cells over-expressing human GPR101. Consistent with this, over-expression of human GPR101 in a yeast-based system yielded an elevated, agonist-independent reporter gene response in the presence of a yeast chimeric Galphas protein. These results indicate that GPR101 participates in a potentially wide range of activities in the CNS via modulation of cAMP levels.


Assuntos
Proteínas de Ligação ao GTP/fisiologia , Expressão Gênica/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Animais , Northern Blotting/métodos , Encéfalo/metabolismo , Linhagem Celular , Mapeamento Cromossômico/métodos , Clonagem Molecular/métodos , AMP Cíclico/metabolismo , Biblioteca Gênica , Genes Reporter/fisiologia , Testes Genéticos/métodos , Humanos , Hibridização In Situ/métodos , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Transfecção/métodos , Técnicas do Sistema de Duplo-Híbrido
16.
Biochem Biophys Res Commun ; 324(1): 171-7, 2004 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-15464998

RESUMO

DNA sequences encoding the murine ortholog of the human P2Y12 receptor were cloned. The human and mouse P2Y12 receptors were expressed in a yeast cell-based GPCR expression technology containing chimeric yeast Galpha protein (Gpa1) constructs in which the 5 C-terminal amino acids were identical to corresponding sequences from mammalian Galphai/o proteins. LacZ reporter gene assays of agonist-induced activation of the G protein-coupled mating signal transduction pathway revealed murine P2Y12 functional pharmacological properties that closely resembled those exhibited by the human P2Y12 receptor. In NIH3T3 cells, the mouse P2Y12 stimulated calcium uptake monitored in FLIPR via coupling to a Galphaq/i3 chimeric protein. Murine P2Y12 mRNA was expressed at high levels in the brain and at lower levels in a variety of peripheral tissues. In situ hybridization analysis indicated glia-specific expression within the brain.


Assuntos
Proteínas de Membrana/metabolismo , Receptores Purinérgicos P2/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Cálcio/metabolismo , Clonagem Molecular , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Genes Reporter , Humanos , Hibridização In Situ , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Ratos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y12 , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Alinhamento de Sequência
17.
Bioinformatics ; 20(18): 3490-9, 2004 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-15297294

RESUMO

MOTIVATION: Determining the coupling specificity of G-protein coupled receptors (GPCRs) is important for understanding the biology of this class of pharmacologically important proteins. Currently available in silico methods for predicting GPCR-G-protein coupling specificity have high error rate. METHOD: We introduce a new approach for creating hidden Markov models (HMMs) based on a first guess about the importance of various residues. We call these knowledge restricted HMMs to emphasize the fact that the state space of the HMM is restricted by the application of a priori knowledge. Specifically, we use only those amino acid residues of GPCRs which are likely to interact with G-proteins, namely those that are predicted to be in the intra-cellular loops. Furthermore, we concatenate these predicted loops into one sequence rather than considering them as four disparate units. This reduces the HMM state space by drastically decreasing the sequence length. RESULTS: Our knowledge restricted HMM based method to predict GPCR-G-protein coupling specificity has an error rate of <1%, when applied to a test set of GPCRs with known G-protein coupling specificity. AVAILABILITY: Academic users can get the data set mentioned herein and HMMs from the authors.


Assuntos
Proteínas de Ligação ao GTP/química , Modelos Químicos , Mapeamento de Interação de Proteínas/métodos , Receptores Acoplados a Proteínas G/química , Alinhamento de Sequência/métodos , Análise de Sequência de Proteína/métodos , Sítios de Ligação , Simulação por Computador , Cadeias de Markov , Modelos Estatísticos , Ligação Proteica , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade
18.
J Biol Chem ; 278(48): 47466-76, 2003 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-14507916

RESUMO

G protein-coupled receptor kinases (GRKs) specifically bind and phosphorylate the agonist-occupied form of G protein-coupled receptors. To further characterize the mechanism of GRK/receptor interaction, we developed a yeast-based bioassay using strains engineered to functionally express the somatostatin receptor subtype 2 and exhibit agonist-dependent growth. Here, we demonstrate that agonist-promoted growth was effectively inhibited by co-expression with either wild type GRK2 or GRK5, whereas catalytically inactive forms of these kinases were without effect. In an effort to identify residues involved in receptor interaction, we generated a pool of GRK5 mutants and then utilized the bioassay to identify mutants selectively deficient in inhibiting agonist-promoted growth. This resulted in the identification of a large number of mutants, several of which were expressed, purified, and characterized in more detail. Two of the mutants, GRK5-L3Q/K113R and GRK5-T10P, were defective in receptor phosphorylation and also exhibited a partial defect in phospholipid binding and phospholipid-stimulated autophosphorylation of the kinase. In contrast, these mutants had wild type activity in phosphorylating the non-receptor substrate tubulin. To further characterize the function of the NH2-terminal region of GRK5, we generated a deletion mutant lacking residues 2-14 and found that this mutant was also severely impaired in receptor phosphorylation and phospholipid-promoted autophosphorylation. In addition, an NH2-terminal 14-amino acid peptide from GRK5 selectively inhibited receptor phosphorylation by GRK5 but had minimal effect on GRK2 activity. Based on these findings, we propose a model whereby the extreme NH2 terminus of GRK5 mediates phospholipid binding and is required for optimal receptor phosphorylation.


Assuntos
Bioensaio/métodos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Células COS , Catálise , Divisão Celular , Linhagem Celular , Relação Dose-Resposta a Droga , Quinase 5 de Receptor Acoplado a Proteína G , Vetores Genéticos , Humanos , Insetos , Cinética , Lipídeos/química , Mutagênese , Mutação , Peptídeos/química , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Fosforilação , Plasmídeos/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Estrutura Terciária de Proteína , Receptores Adrenérgicos beta 2/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Tempo , Quinases de Receptores Adrenérgicos beta
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