MS Binding Assays for D1 and D5 Dopamine Receptors.
MS Binding Assays are a label-free alternative to radioligand binding assays. They provide basically the same capabilities as the latter, but an unlabeled reporter ligand is used instead of a radioligand. The study presented herein describes the development of MS Binding Assays that address D1 and D5 dopamine receptors. A highly sensitive, rapid and robust LC-ESI-MS/MS quantification method for the selective D1 dopamine receptor antagonist SCH23390 ((5R)-8-chloro-3-methyl-5-phenyl-1,2,4,5-tetrahydro-3-benzazepin-7-ol) was established and validated, using its 8-bromo analogue SKF83566 as an internal standard. This quantification method proved to be suitable for the characterization of SCH23390 binding to human D1 and D5 receptors. Following the concept of MS Binding Assays, saturation experiments for D1 and D5 receptors were performed, as well as competition experiments for D1 receptors. The results obtained are in good agreement with results from radioligand binding assays and therefore indicate that the established MS Binding Assays addressing D1 and D5 receptors are well-suited substitutes for radioligand binding assays, the technique that has so far dominated affinity determinations toward these targets.
Department für Pharmazie-Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377, München, Germany.
Patrick
P
Neiens
Department für Pharmazie-Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377, München, Germany.
Georg
G
Höfner
Department für Pharmazie-Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377, München, Germany. Klaus.Wanner@cup.uni-muenchen.de.
Klaus Theodor
KT
Wanner
D001552:Benzazepines / Q000032:analysis* / Q000378:metabolism* / Q000494:pharmacology; D001667:Binding, Competitive; D002853:Chromatography, Liquid; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D011869:Radioligand Assay; D017447:Receptors, Dopamine D1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D050641:Receptors, Dopamine D5 / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
D001552:Benzazepines; D008024:Ligands; D017447:Receptors, Dopamine D1; C534628:SCH 23390; D050641:Receptors, Dopamine D5
binding assays; liquid chromatography; mass spectrometry; neurotransmitters; receptors