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The properties ofa proapoptotic 1,4-benzodiazepine, Bz-423, identified through combinatorial chemistry and phenotype screening are described. Bz-423 rapidly generated superoxide (O^sub 2-^) in transformed Ramos B cells. This O^sub 2-^ response originated from mitochondria prior to mitochondrial transmembrane gradient collapse and opening of the permeability transition pore. Bz-423-induced 02- 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^sub 2-^ or scavenge free radicals attenuated the death cascade, which demonstrated that cell killing by Bz-423 depends on O^sub 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^sub 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.
Nonstandard abbreviations used: systemic lupus erythematosus (SLE); superoxide (O^sub 2^ ); reactive oxygen species (ROS); germinal center (GC); (NZB x NZW)F1 (NZB/W); tacrolimus (FK506); N-benzoylcarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD); dihydroethidium (DHE); 3,3'-dihexyloxacarbocyanine iodide (DiOC^sub 6^[3]) carboxyfluorescein-Val-Ala-Asp-fluoromethylketone (FAM-VAD-fmk); manganese(III)meso-tetrakis(4-benzoic acid)porphyrin (MnTBAP); propidium iodide (PI); mitochondrial transmembrane potential (Delta(Psi)^sub m^); carbonyl cyanide m-- chlorophenylhydrazone (CCCP); 2',7'-dichlorodihydrofluorescin diacetate (DCFH-DA); 2',7'-dichlorodihydrofluorescin (DCFH); 2',7'-dichlorofluorescein (DCF); peripheral benzodiazepine receptor (PBR); 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-- 3-isoquinolinecarboxamide (PK11195); mitochondrial permeability transition (MPT); mitochondrial respiratory chain (MRC); B cell receptor (BCR).
J. Clin. Invest. 110:1123-1132 (2002). doi:10.1172/JCI200216029.
Introduction
Cytotoxicity is an important mechanism for treating autoimmune diseases like systemic lupus erythematosus (SLE) (1) and is the basis for nearly all cancer treatments (2). The selectivity of many cytotoxic agents is limited and generally relies on the differential ability of diseased and healthy cells to tolerate and repair drug-induced cellular damage. Compounds that regulate disease-specific targets clearly have the potential to be better drugs - the development of imatinib mesylate, which inhibits the oncogenic kinase Bcr-Abl, is a recent example of such a molecule (3). However,...