The Bcl-2-related survival proteins confer cellular resistance to a wide range of apoptosis-inducing agents. In work can-led out in our labs, a novel small molecular ligand to the Bcl-xL protein has been identified, which inhibits the molecular pore function of Bcl-xL and selectively kills Bcl-xL and, at higher doses, Bcl neg2-expressing cells. We made the initial observation that Bcl-xL-expressing hepatocyte cell lines are more sensitive than isogenic control cells to antimycin A (AA), a known inhibitor of mitochondrial electron transport. A 2-methoxy antimycin A analog lacking effects on mitochondrial respiration still exhibited selective toxicity for Bcl-xL plus cells and mitochondria. Computational molecular docking analysis predicted that antimycin A conforms to a conserved hydrophobic groove on the molecular surface of Bcl-xL. We confirmed this interaction by showing competitive binding of AA and its 2-methoxy derivative with a known hydrophobic groove ligand to recombinant Bcl-xL and Bcl-2 proteins, a BH3 domain peptide derived from the pro-apoptotic dimerization partner, Bak. Finally, we found that AA inhibits the pore-forming activity of Bcl-xL in synthetic Liposomes, demonstrating that this small ligand can directly inhibit the function of Bcl-2-related survival proteins.
Two aims of this application investigate the structural determinants of AA binding to the Bcl-xL hydrophobic pocket and mechanism of pore inhibition. Initial screening of human hematopoietic cell lines for cytotoxic effects of antimycin A indicates myeloma cell lines, including multi-drug resistant sublines, are sensitive to AA and 2-methoxy AA. Several published studies have shown that myeloma cell survival is predominantly dependent on Bcl-xL, despite the expression of several related anti-apoptotic proteins. We propose, using pre-clinical models, to test whether multiple myeloma is particularly susceptible to Bcl-xL -targeted therapies, and validate Bcl-xL as the relevant target of 2-methoxy AA in myeloma cells, The questions to be addressed In three specific alms are as follows:
Specific Aims : 1. Evaluate efficacy and toxicology of 2-methoxy antimycin A3 in mouse myeloma and hepatoma tumor models. 2. Characterize biochemical mechanism of antimycin A inhibition of Bcl-,xL pore-forming function, including analysis of mutations in Bcl-xL hydrophobic groove binding site; x-ray crystallography of antimycin complex with Bcl-xL; and membrane topology studies. 3. Determine the role of endogenous pro-apoptotic dimer partners of Eel-if in the cytotoxic mechanism of antimycin A.
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