Chelerythrine and Sanguinarine Dock at Distinct Sites on BclXL that are Not the Classic BH3 Binding Cleft

The ratio of the levels of pro-survival and pro-apoptotic members of the Bcl-2 protein family is thought to be an important regulatory factor for determining the sensitivity of the mammalian cells to apoptotic stimuli. High levels of expression of pro-survival members such as BclXL in human cancers were frequently found to be a good prognostic indicator predicting poor response to chemotherapy. The pro-survival members of the Bcl-2 family mediate their effects through heterodimerization with the BH3 region of the pro-apoptotic members. Structural analyses of the binding complex of the BH3 peptide and BclXL showed that a hydrophobic groove termed the BH3 binding cleft is the docking site for the BH3 region. Chemical mimetics of the BH3 region such as BH3I-1 that target the BH3 binding cleft indeed exhibit pro-apoptotic activities. Chelerythrine (CHE) and sanguinarine (SAN) are natural benzophenanthridine alkaloids that are structurally homologous to each other. CHE was previously identified as an inhibitor of BclXL function from a high-throughput screen of natural products, but its mode of interaction with BclXL is not known. By determining the effect of site-directed mutagenesis on ligand binding and using saturation transfer difference (STD) NMR experiments, we have verified locations of these docked ligands. Surprisingly, CHE and SAN bind separately at the BH groove and BH1 region of BclXL respectively, different from the BH3 binding cleft where other known inhibitors of BclXL target. Interestingly, certain residues on the flexible loop between helices α1 and α2 of BclXL are also perturbed upon CHE, but not SAN or BH3I-1 binding. Although CHE and SAN are similarly effective as BH3I-1 in displacing bound BH3 peptide, they are much more effective in inducing apoptosis, raising the possibility that CHE and SAN might be able to antagonize other pro-survival mechanisms in addition to the one that involves BH3 region binding.