title: Radiolabelled calmodulin ligands: their low affinity and high lipophilicity may lead to artefacts in binding studies.
abstract: In order to characterize the interaction site of a series of putative calmodulin antagonists of the diphenylalkylamine type with calmodulin (CaM), a representative member of this chemical class was radiolabelled. The binding of the selected compound, [3H]-VUF 4576, to calmodulin was studied according to a recently described technique using CaM agarose. However, some peculiar results were obtained: the tight binding of [3H]-VUF 4576 increased in presence of cold VUF 4576, resulting in a high non-specific binding. The unexpected results could readily be explained by a high binding capacity of the labelled compound and the cold ligands to the walls of the test tubes used. Such results were also found when [3H]-chlorpromazine ([3H]-CPZ) was applied. In literature comparable findings have been published. To explain such results the influence of positive cooperativity or irreversible binding has been suggested. We suppose that not only in our study, but also in other published investigations, binding to glass of the radioligand and/or the cold compounds may have had a strong influence. We suggest, therefore, that care should be taken in interpreting non-classical displacement data obtained with ligands which combine a rather low affinity and a high degree of lipophilicity, not only for binding to calmodulin, but for other systems as well.
authors.affiliation: Department of Pharmacochemistry, Vrije Universiteit, Amsterdam, The Netherlands.
authors.forename: P
authors.initials: P
authors.lastname: Caldirola
authors.forename: B H
authors.initials: BH
authors.lastname: Schmidt
authors.forename: H
authors.initials: H
authors.lastname: Timmerman
mesh_terms: D001559:Benzhydryl Compounds / Q000378:metabolism; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000737:chemistry*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002746:Chlorpromazine / Q000032:analysis; D002846:Chromatography, Affinity; D003543:Cysteamine / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008055:Lipids / Q000737:chemistry; D011485:Protein Binding
chemical_list: D001559:Benzhydryl Compounds; D002147:Calmodulin; D008024:Ligands; D008055:Lipids; C080506:N-(2-(bis(4-fluorophenyl)methylthio)ethyl)-N-methyl-N-(2-phenyl)ethylamine; D003543:Cysteamine; D002746:Chlorpromazine