Benzodiazepine-induced superoxide signals B cell apoptosis: mechanistic insight and potential therapeutic utility

The properties of a proapoptotic 1,4-benzodiazepine, Bz-423, identified through combinatorial chemistry and phenotype screening are described. Bz-423 rapidly generated superoxide (02) in transformed Ramos B cells. This O-2(-) response originated from mitochondria prior to mitochondrial transmembrane gradient collapse and opening of the permeability transition pore. Bz-423-induced O-2(-) functioned as an upstream signal that initiated an apoptotic program characterized by cytochrome c release, mitochondrial depolarization, and caspase activation. Pretreatment of cells with agents that either block the formation of Bz-423-induced O-2(-) or scavenge free radicals attenuated the death cascade, which demonstrated that cell killing by Bz-423 depends on O-2(-). Parallels between Ramos cells and germinal center B cells prompted experiments to determine whether Bz-423 had therapeutic activity in vivo. This possibility was tested using the (NZB x NZW)F-1 murine model of lupus, in which the pathologically enhanced survival and expansion of germinal center B cells mediate disease. Administration of Bz-423 for 12 weeks specifically controlled germinal center hyperplasia and reduced the histological evidence of glomerulonephritis. Collectively, these studies define a new structure-function relationship for benzodiazepines and point to a. new target and mechanism that could be of value for developing improved drugs to manage systemic lupus erythematosus and related disorders.