RESUMO
Adrenergic alpha(1), alpha(2) and beta receptors are members of the G-protein-coupled receptor families (GPCRs) mediating physiological responses to adrenaline (epinephrine) and noradrenaline (norepinephrine). Since GPCRs are major targets for potential therapeutic agents, development of robust, reliable and cost effective functional screening methods for these receptors is in the focus of pharmacological research. For this reason, the aim of the present study was to develop an intracellular calcium assay for investigating the pharmacology of the alpha(2C) type of adrenergic receptors (alpha(2C)-AR). Although activation of alpha(2C)-AR is not linked to calcium mobilization, co-expression of these receptors with the chimeric Galpha(qi5) protein, containing the five carboxyl-terminal amino acids from G(i), or promiscuosus Galpha(16) protein can divert receptor signaling to the G(q) pathway generating Ca(2+) release from intracellular stores. In order to assess the functional potency of alpha(2)-AR agonists and antagonists, we established a fluorometric Ca(2+) assay using cell lines stably and constitutively co-expressing alpha(2C)-AR and Galpha(qi5) or Galpha(16) proteins (Galpha(qi5)/alpha(2C) and Galpha(16)/alpha(2C)). As part of the pharmacological characterization, we measured the changes in cytoplasmic Ca(2+) levels due to activation of the chimeric Galpha(qi5) or Galpha(16) coupled recombinant alpha(2C) receptors as a function of increasing concentration of several agonists (noradrenaline, brimonidine, oxymetazoline, clonidine, moxonidine) and antagonists (MK912, yohimbine). The binding affinities of alpha(2)-AR agonist and antagonists and the inhibition of the forskolin-stimulated cAMP accumulation in alpha(2C)-AR expressing cells were also measured. These results confirmed that the Galpha(qi5)/alpha(2C) and Galpha(16)/alpha(2C) recombinant systems can be useful for modelling the native G(i)-coupled system. Our results indicate that a plate-reader based fluorometric Ca(2+) assay may be suitable in high-throughput screening for alpha(2C)-AR ligands as well.
Assuntos
Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Receptores Adrenérgicos alfa 2/efeitos dos fármacos , Receptores Adrenérgicos alfa 2/genética , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Agonistas alfa-Adrenérgicos/farmacologia , Animais , Tartarato de Brimonidina , Células CHO , Cálcio/metabolismo , Colforsina/farmacologia , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Fluorometria , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/biossíntese , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Humanos , Quinoxalinas/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ensaio Radioligante , Ratos , Proteínas Recombinantes/biossíntese , TransfecçãoRESUMO
A novel series of arylsulfonamides was prepared either by automated parallel or by traditional solution-phase synthesis. Several members of this compound library were identified as high-affinity dopamine D3 and D2 receptor ligands. The most interesting representative, compound 2, showed potent antipsychotic behaviour coupled with a beneficial cognitive and EPS profile.
Assuntos
Antipsicóticos/síntese química , Antipsicóticos/farmacologia , Cognição/efeitos dos fármacos , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/metabolismo , Sulfonamidas/síntese química , Sulfonamidas/farmacologia , Animais , Antipsicóticos/metabolismo , Apomorfina/farmacologia , Aprendizagem da Esquiva/efeitos dos fármacos , Ligação Competitiva/efeitos dos fármacos , Disponibilidade Biológica , Encéfalo/metabolismo , Catalepsia/induzido quimicamente , Humanos , Indicadores e Reagentes , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos da Memória/induzido quimicamente , Relação Quantitativa Estrutura-Atividade , Ratos , Ratos Wistar , Comportamento Estereotipado/efeitos dos fármacos , Sulfonamidas/metabolismoRESUMO
A novel series of indole-2-carboxamidine derivatives was prepared and identified as NR2B selective NMDA receptor antagonists. The influence of the substituents on the indole skeleton as well as the substitution of the benzyl moiety on the biological activity of the compounds was studied. Compound 5a was po active in the formalin test in mouse.