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J Biomol Screen ; 11(2): 194-207, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16490772

RESUMO

Screening assays using target-based affinity selection coupled with high-sensitivity detection technologies to identify small-molecule hits from chemical libraries can provide a useful discovery approach that complements traditional assay systems. Affinity selection-mass spectrometry (AS-MS) is one such methodology that holds promise for providing selective and sensitive high-throughput screening platforms. Although AS-MS screening platforms have been used to discover small-molecule ligands of proteins from many target families, they have not yet been used routinely to screen integral membrane proteins. The authors present a proof-of-concept study using size exclusion chromatography coupled to AS-MS to perform a primary screen for small-molecule ligands of the purified muscarinic M2 acetylcholine receptor, a G-protein-coupled receptor. AS-MS is used to characterize the binding mechanisms of 2 newly discovered ligands. NGD-3350 is a novel M2-specific orthosteric antagonist of M2 function. NGD-3366 is an allosteric ligand with binding properties similar to the allosteric antagonist W-84, which decreases the dissociation rate of N-methyl-scopolamine from the M2 receptor. Binding properties of the ligands discerned from AS-MS assays agree with those from in vitro biochemical assays. The authors conclude that when used with appropriate small-molecule libraries, AS-MS may provide a useful high-throughput assay system for the discovery and characterization of all classes of integral membrane protein ligands, including allosteric modulators.


Assuntos
Espectrometria de Massas/métodos , Antagonistas Muscarínicos/farmacologia , Ligação Proteica/efeitos dos fármacos , Receptor Muscarínico M2/química , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Acetilcolina , Regulação Alostérica , Sítio Alostérico , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Cobaias , Insetos , Ligantes , Estrutura Molecular , Ensaio Radioligante , Receptor Muscarínico M2/isolamento & purificação
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