ABSTRACT
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.
Subject(s)
Calcium Signaling/genetics , Calcium Signaling/physiology , Receptors, Adrenergic, alpha-2/drug effects , Receptors, Adrenergic, alpha-2/genetics , Receptors, G-Protein-Coupled/drug effects , Adrenergic alpha-Agonists/pharmacology , Animals , Brimonidine Tartrate , CHO Cells , Calcium/metabolism , Colforsin/pharmacology , Cricetinae , Cricetulus , Cyclic AMP/metabolism , Fluorometry , GTP-Binding Protein alpha Subunits, Gq-G11/biosynthesis , GTP-Binding Protein alpha Subunits, Gq-G11/genetics , GTP-Binding Proteins/metabolism , Humans , Quinoxalines/pharmacology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Radioligand Assay , Rats , Recombinant Proteins/biosynthesis , TransfectionABSTRACT
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.
Subject(s)
Antipsychotic Agents/chemical synthesis , Antipsychotic Agents/pharmacology , Cognition/drug effects , Receptors, Dopamine D2/metabolism , Receptors, Dopamine D3/metabolism , Sulfonamides/chemical synthesis , Sulfonamides/pharmacology , Animals , Antipsychotic Agents/metabolism , Apomorphine/pharmacology , Avoidance Learning/drug effects , Binding, Competitive/drug effects , Biological Availability , Brain/metabolism , Catalepsy/chemically induced , Humans , Indicators and Reagents , Male , Maze Learning/drug effects , Memory Disorders/chemically induced , Quantitative Structure-Activity Relationship , Rats , Rats, Wistar , Stereotyped Behavior/drug effects , Sulfonamides/metabolismABSTRACT
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.
