title: A fluorescence polarization-based assay for the identification and evaluation of calmodulin antagonists.
abstract: A fluorescence polarization (FP) assay was developed to identify calmodulin (CaM) antagonists. A fluorescent tracer was newly designed by covalently labeling N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), which is a well-known CaM antagonist, with the Cy5 dye. In the FP assay, the tracer (Cy5-W-7) was bound to CaM with a dissociation constant (K(d)) of 6.5 microM and demonstrated efficient competitive activity with other CaM antagonists, including W-7, chlorpromazine, trifluoperazine, W-5, and clozapine, indicating that Cy5-W-7 binds to the ligand-binding site of CaM in a specific manner. The inhibitory activities of Cy5-W-7 and CaM antagonists were subsequently measured by the CaM-dependent calcineurin phosphatase assay, and the results were confirmed with those of the FP assays. In addition, assay optimization for high-throughput screening was performed, and a Z' factor of 0.7 was achieved in a 1536-well format. The FP assay was found to be a simple and reliable alternative to conventional assays for evaluating CaM antagonists.
authors.affiliation: Medicinal Chemistry Laboratory, Mitsubishi Tanabe Pharma, 2-2-50 Kawagishi, Toda-shi, Saitama 335-8505, Japan. arai.tomonori@me.mt-pharma.co.jp
authors.forename: Tomonori
authors.initials: T
authors.lastname: Arai
authors.forename: Mai
authors.initials: M
authors.lastname: Yatabe
authors.forename: Megumi
authors.initials: M
authors.lastname: Furui
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Akatsuka
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Uehata
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Kamiyama
mesh_terms: D001665:Binding Sites; D002147:Calmodulin / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D002232:Carbocyanines / Q000737:chemistry* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D007700:Kinetics; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D013449:Sulfonamides / Q000737:chemistry* / Q000378:metabolism
chemical_list: D002147:Calmodulin; D002232:Carbocyanines; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D013449:Sulfonamides; C085321:cyanine dye 5; C017967:W 7; C120817:calcineurin phosphatase; D010744:Phosphoric Monoester Hydrolases