title: Synthesis, binding affinity, and cross-linking of monodentate photoactive phenothiazines to calmodulin.
abstract: Various photoactive phenothiazines were synthesized that possessed a 2-azido, 3-azido, 2-benzoyl, or 1,3,4-trifluoro-2-azido functionality in combination with various modifications of the N-alkyl side chain. These phenothiazines were evaluated for their ability to inhibit the calmodulin-mediated activation of phosphodiesterase (PDE). All were active in inhibiting the action of calmodulin (CaM), but those possessing either a 3-azido and a 4-(4-methyl-1-piperazinyl)butyl side chain or a 2-benzoyl group and 3-(dimethylamino)propyl side chain proved to be most active (I50 = 14 +/- 3 microM and 7 +/- 1 microM, respectively) when compared to the known inhibitor, chlorpromazine (CPZ, I50 = 30 microM). Calmodulin was photolabeled with ca. 35% efficiency in a light- and calcium-dependent fashion using a radiolabeled analog, 3-azido-10-(4-(4-[14C]methyl-1-piperazinyl)butyl)phenothiazine, of one of these compounds. Competition studies using this radiolabeled analog and CPZ were consistent with binding to one or both of the hydrophobic binding pockets of CaM.
authors.affiliation: Department of Chemistry, University of Kentucky, Lexington 40506, USA.
authors.forename: M
authors.initials: M
authors.lastname: Golinski
authors.forename: P J
authors.initials: PJ
authors.lastname: DeLaLuz
authors.forename: R
authors.initials: R
authors.lastname: Floresca
authors.forename: T J
authors.initials: TJ
authors.lastname: Delcamp
authors.forename: T C
authors.initials: TC
authors.lastname: Vanaman
authors.forename: D S
authors.initials: DS
authors.lastname: Watt
mesh_terms: D000345:Affinity Labels / Q000138:chemical synthesis / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002147:Calmodulin / Q000737:chemistry*; D002417:Cattle; D003432:Cross-Linking Reagents; D004789:Enzyme Activation; D066298:In Vitro Techniques; D015394:Molecular Structure; D010640:Phenothiazines / Q000138:chemical synthesis* / Q000737:chemistry / Q000528:radiation effects; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010777:Photochemistry
chemical_list: D000345:Affinity Labels; D002147:Calmodulin; D003432:Cross-Linking Reagents; D010640:Phenothiazines; D010727:Phosphoric Diester Hydrolases