As part of efforts to identify new cytotoxic agents, we probed a combinatorial benzodiazepine library against lymphoid cells for pro-apoptotic members. The ultimate goal of this work was to identify leads that may prove useful as mechanistic tools and therapeutic agents for autoimmune disorders like lupus. These experiments yielded a family of novel cytotoxic molecules, the lead compound of which we have designated Bz-423. Administering Bz-423 to two different strains of hpus-prone mice induced splenic lymphocyte apoptosis that afforded significant attenuation of disease progression (kidney inflammation) and improved survival. Unlike current drugs to treat lupus, Bz-423 suppressed the autoimmune response without adverse non-specific toxicity. Based on these data, the first project period of this grant studied the cell types targeted by Bz-423 and the cellular mediators of Bz-423-induced apoptosis. In addition to completing this work, we have demonstrated that Bz-423 provokes apoptosis by inhibiting the mitochondrial F1Fo-ATPase and have identified the oligomycin sensitivity conferring protein (OSCP) subunit of this enzyme as the molecular target of Bz-423. To our knowledge, Bz-423 is the first F1Fo-ATPase inhibitor that functions by interacting with the OSCP. We have also discovered additional therapeutic properties of Bz--423 linked to target binding and have demonstrated significant activity in an accepted animal model of rheumatoid arthritis. As a result of these findings, studies during the next project period will focus on exploring the structural basis of OSCpoBz - 423 binding, the mechanism FIFo-ATPase inhibition by Bz-423, early mitochondrial signals that arise as a result of target binding/F1Fo-ATPase inhibition, and the molecular basis for the unique selectivity displayed by Bz-423 in vivo. These studies should reveal fundamental new information on early pro-apoptotic signaling events in mitochondria, enhance our understanding of the function of the OSCP within the F1Fo- ATPase, and help advance Bz-423 (or a derivative) into the clinic.
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