RESUMEN
The structures of three nitro-substituted phenothiazines [1,3,4-trifluoro-2-nitrophenothiazine, 10-(4-chlorobutyl)-1,3,4-trifluoro-2-nitrophenothiazine and 10-(4-chlorobutyl)-3-nitrophenothiazine] have been determined. The first of these red compounds forms infinite stacks in the solid state, in which donor and acceptor regions of the approximately planar molecules alternate. The molecules of the other two compounds, which have folded, or 'butterfly', conformations in the solid state, do not form stacks, presumably because the bulky chlorobutyl substituents cannot be accommodated. The very dark color of solid 3-nitrophenothiazine suggests the presence of extended molecular stacks, but crystals suitable for a structure determination could not be obtained.
Asunto(s)
Fenotiazinas/química , Cristalización , Cristalografía por Rayos X , Conformación Molecular , Estructura MolecularRESUMEN
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.
Asunto(s)
Calmodulina/química , Fenotiazinas/síntesis química , Marcadores de Afinidad/síntesis química , Marcadores de Afinidad/química , Animales , Sitios de Unión , Unión Competitiva , Bovinos , Reactivos de Enlaces Cruzados , Activación Enzimática , Técnicas In Vitro , Estructura Molecular , Fenotiazinas/química , Fenotiazinas/efectos de la radiación , Hidrolasas Diéster Fosfóricas/metabolismo , FotoquímicaRESUMEN
The biotinylated probe, 3-azido-10-(4-(4-biotinyl-1-piperazinyl)butyl)phenothiazine, was used to examine the phenothiazine binding domains in calmodulin (CaM) by photolabeling. This phenothiazine, synthesized from 3-azido-10-(4-(1-piperazinyl)butyl)phenothiazine and d-biotinyl tosylate, inhibited the CaM-mediated activation of phosphodiesterase (PDE) with an I50 of 12.5 (+/- 2.8) microM. Photolabeling of CaM produced covalent adducts in excellent yield (32%) in a light- and Ca2+-dependent manner. Studies performed over a range of drug concentrations suggested a 2:1 stoichiometry for the binding of the phenothiazine probe to CaM. Limited trypsin digestion and purification of the resulting fragments by either SDS-PAGE or HPLC provided two principal phenothiazine-containing peptides. Amino acid composition and sequence analyses performed on these two peptides established that both the N- and C-terminal domains in CaM, particularly the regions amino terminal to Ca2+-binding loops 1 and 3, were modified by the photoactivated phenothiazine derivative. These data, particularly for the C-terminal domain, are in excellent agreement with the X-ray structure analysis of a 1:1 CaM-trifluoperazine complex.
Asunto(s)
Marcadores de Afinidad/síntesis química , Calmodulina/química , Fenotiazinas/síntesis química , Marcadores de Afinidad/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Biotina/química , Calmodulina/genética , Calmodulina/metabolismo , Bovinos , Activación Enzimática , Técnicas In Vitro , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Fenotiazinas/química , Hidrolasas Diéster Fosfóricas/metabolismo , FotoquímicaRESUMEN
The development of targeted, bidentate photoaffinity reagents for mapping the interacting domains of calmodulin (CaM) with the enzymes that it regulates required the synthesis and evaluation of the binding affinity of various phenothiazines. These photoaffinity reagents would possess a photoactive 3-azidophenothiazine group for cross-linking the hydrophobic binding domain of CaM, a second photoactive benzophenone group that would be activated at a different wavelength than the 3-azidophenothiazine group, and a suitable radiolabel. Difficulties were encountered in identifying those structural features that would be compatible with the introduction of a benzophenone group, with the solubility of these benzophenone-substituted phenothiazines, and with the ability of these phenothiazines to inhibit the calmodulin-mediated activation of phosphodiesterase. Solutions to this problem involved the preparation of phenothiazines possessing a quaternary ammonium salt, a zwitterionic amino acid, or a carbohydrate moiety. The phenothiazines that possessed photoactive 3-azido and benzophenone groups and in which one of the piperazine nitrogens in the side chain was converted to a quaternary, N-methylammonium iodide inhibited the calmodulin-mediated activation of phosphodiesterase at a level comparable to that of chlorpromazine.