Functional Analyses of a Small Molecule Bcl-XL Inhibitor
The Bcl-2 related survival proteins confer cellular resistance to a wide range of agents. In work carried out by the Hockenbery and Zhang labs, a novel small molecular ligand to the Bcl-XL and Bcl-2 proteins has been identified, which inhibits the molecular pore function of Bcl-XL and selectively kills Bcl-XL/Bcl-2 expressing cells. Bcl-XL expressing hepatocyte cell lines are more sensitive than isogenic control cells to antimycin A, a known inhibitor of mitochondrial electron transport. A 2-methoxy antimycin A analog fails to inhibit mitochondrial respiration, yet retains selective toxicity for Bcl-XL+ cells and mitochondria. Computational molecular docking analysis predicted that antimycin A conformed to a conserved hydrophobic groove on the molecular surface of Bcl-XL. This interaction was verified by showing competitive binding of antimycin A to recombinant Bcl-2 protein using a known hydrophobic groove ligand, a BH3 domain peptide derived from the pro-apoptotic dimerization partner, Bak. Finally, antimycin A 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.
Under the dual mentorship of Drs. Hockenbery and Zhang, I propose to characterize the molecular interaction between antimycin A and Bcl-XL in detail. A panel of antimycin A analogs will be used to construct a structure-activity relationship dataset. These data will be compared with Bcl-XL-dependent cellular sensitivity to the analogs. From a series of Bcl-XL proteins with single amino acid substitutions in the predicted hydrophobic groove docking site, several reduce or eliminate sensitivity to antimycin A without compromising the anti-apoptotic functions of Bcl-XL. Recombinant bacterial versions of these proteins will be expressed and tested for binding of antimycin A. An unaddressed question is how antimycin A inhibits pore forming activity of Bcl-XL. There may be allosteric or conformational changes subsequent to antimycin binding and this possibility will be investigated using membrane conformation-sensitive biophysical assays. It will be of particular interest to identify mutations in Bcl-XL that confer resistance to antimycin A, yet still bind antimycin A.