Mechanisms of a pro-apoptotic benzodiazepine.

Abstract

Bz-423 is a immunomodulatory 1,4-benzodiazepine that exerts its therapeutic effects in autoimmune mice by selectively inducing apoptosis of pathogenic B and T cells. The overall goal of the studies presented in this dissertation is to understand the cellular and molecular basis for the selectivity observed <italic> in vivo</italic>. Not only will the findings of these experiments be useful in helping advance Bz-423 to the clinic, but will also provide one of the first comprehensive outlines of the apoptotic signaling pathways induced by a small molecule. Structure-activity studies identified the aromatic side chain at C2 and the C'4 position phenolic hydroxyl group are key elements for target binding and cytotoxic activity. Bz-423 induces an early, rapid production of superoxide (O₂<super>-</super>) from mitochondria, and this response is the critical signal initiating apoptosis. The oligomycin sensitivity-conferring protein (OSCP) component of the mitochondrial F₁F₀-ATPase is the physiologically relevant target for both O₂<super>-</super> production and subsequent cell death. JNK is a key molecule involved in Bz-423-induced apoptosis in fibroblasts. JNK activation occurs by a MAPK signaling cascade involving sequential activation of ASK1/MEKK/JNK resulting in activation of the transcription factor, c-Jun. JNK activation causes increased expression of BH3-only proteins triggering the activation of the multidomain pro-apoptotic proteins Bax and Bak. These results provide a detailed description of events during Bz-423-induced apoptosis, from initial O₂<super>- </super> production to cytochrome <italic>c</italic> release and caspase activation. Bz-423 induces apoptosis in a cellular model of pathogenic lupus through a mechanism unique from that observed in fibroblasts. In all cell types examined apoptotic signaling pathways induced by Bz-423 converge on the Bcl-2 family of proteins resulting in activation of Bax and Bak by BH3-only proteins culminating in cytochrome <italic>c</italic> release from the mitochondria. However, Bz-423-induced apoptosis in pathogenic lupus cells is independent of MAPK signaling and <italic> de novo</italic> protein synthesis. These differences result in increased kinetics of cell death. The difference in kinetics of Bz-423-induced cell death in different cell lines coupled with its pharmacokinetic profile could help explain why Bz-423 is selective to pathogenic B cells <italic>in vivo </italic>.