RESUMO
Pre-synaptic dopamine, norepinephrine and serotonin transporters (DAT, NET and SERT) terminate synaptic catecholamine transmission through reuptake of released neurotransmitter. Common approaches for studying these transporters involve radiolabeled substrates or inhibitors which, however, have several limitations. In this study we have used a novel neurotransmitter transporter uptake assay kit. The assay employs a fluorescent substrate that mimics the biogenic amine neurotransmitters and is taken up by the cell through the specific transporters, resulting in increased fluorescence intensity. In order to validate the assay, a variety of reference and proprietary neurotransmitter transporter ligands from a number of chemical and pharmacological classes were tested. The ability of these compounds to inhibit the selective transporter-mediated uptake demonstrated a similar rank order of potency and IC(50) values close to those obtained in radiolabeled neurotransmitter uptake assays. The described assay enables monitoring of dynamic transport activity of DAT, NET and SERT and is amenable for high-throughput screening and compound characterization.
Assuntos
Bioensaio/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Microscopia de Fluorescência/métodos , Neuroquímica/métodos , Neurotransmissores/agonistas , Proteínas de Transporte de Neurotransmissores/análise , Proteínas de Transporte de Neurotransmissores/efeitos dos fármacos , Psicotrópicos/farmacologia , Animais , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/fisiologia , Células CHO , Cricetinae , Cricetulus , Proteínas da Membrana Plasmática de Transporte de Dopamina/análise , Proteínas da Membrana Plasmática de Transporte de Dopamina/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Corantes Fluorescentes , Humanos , Ligantes , Estrutura Molecular , Proteínas de Transporte de Neurotransmissores/metabolismo , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/análise , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , Psicotrópicos/química , Psicotrópicos/isolamento & purificação , Proteínas da Membrana Plasmática de Transporte de Serotonina/análise , Proteínas da Membrana Plasmática de Transporte de Serotonina/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismoRESUMO
The 3-ethoxycarbonyl-4-quinolone compound 1 has previously been identified via a database search as an interesting lead compound for ligand binding at the benzodiazepine site of GABA(A) receptors (Kahnberg et al. J. Mol. Graphics Modelling 2004, 23, 253-261). Pharmacophore-guided optimization of this lead compound yielded a number of high-affinity ligands for the benzodiazepine site including compounds 20 and 23-25 displaying sub-nanomolar affinities. A few of the compounds have been tested on the alpha(1)beta(2)gamma(2S) and alpha(3)beta(2)gamma(2S) GABA(A) receptor subtypes, and two of the compounds (5 and 19) display selectivity for alpha(1)- versus alpha(3)-containing receptors by a factor of 22 and 27, respectively. This selectivity for alpha(1)beta(2)gamma(2S) is in the same range as that for the well-known alpha(1) subunit selective compound zolpidem.