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 lupus-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 mediators of Bz-423-induced apoptosis. We have also demonstrated that Bz-423 provokes apoptosis by inhibiting the mitochondria! F1 Fo-ATPase and have identified the oligomycin sensitivity conferring protein (OSCP) subunit of this enzyme as its molecular target. Bz-423 is the first F1 Fo-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 F1 FoATPase-Bz-423 interactions, the mechanism of F1 Fo-ATPase inhibition by Bz-423, and early mitochondria! signals that arise as a result of target binding/F1Fo-ATPase inhibition. 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 F1 Fo-ATPase, and help advance Bz-423 (or a derivative) into the clinic. Lupus is an autoimmune disease that afflicts up to 1,000,000 Americans and at least 1,000 people in the U.S. die each year from this disorder. New drugs for lupus have not been developed in over 40 years. This research will provide critical data needed for the development and testing of new, effective lupus drugs.
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