SYNTHESIS, BINDING-AFFINITY, AND CROSS-LINKING OF MONODENTATE PHOTOACTIVE PHENOTHIAZINES TO CALMODULIN

Various photoactive phenothiazines were synthesized that possessed a 2 -azido, 3-azido, 2-benzoyl, or 1,3,4-trifluoro-2-azido functionality i n combination with various modifications of the N-alkyl side chain. Th ese phenothiazines were evaluated for their ability to inhibit the cal modulin-mediated activation of phosphodiesterase (PDE). All were activ e in inhibiting the action of calmodulin (CaM), but those possessing e ither 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 mo st active (I-50 = 14 +/- 3 mu M and 7 +/- 1 mu M, respectively) when c ompared to the known inhibitor, chlorpromazine (CPZ, I-50 = 30 mu M). Calmodulin was photolabeled with ca. 35% efficiency in a light- and ca lcium-dependent fashion using a radiolabeled analog, 3-azido-10-(4-(4- [C-14]methyl-1-piperazinyl)butyl) phenothiazine, of one of these compo unds. Competition studies using this radiolabeled analog and CPZ were consistent with binding to one or both of the hydrophobic binding pock ets of CaM.