The BCL-2 family proteins are key positive and negative regulators of apoptosis. Biochemical and subcellular fractionation studies localize the anti-apoptotic members, such as BCL-2 and BCL-X L , at outer mitochondrial membranes (OMM) 1 and, to a lesser extent, at endoplasmic reticulum and nuclear membranes. Pro-apoptotic factors either reside at the OMM or as soluble cytosolic proteins, as with BAX, BID, and BAD. Following an apoptotic signal, these factors are translocated to mitochondria. BCL-2 and BCL-X L cancel out the activity of pro-apoptotic members at the OMM, at least partly through direct interactions (1).The tertiary structures of pro-and anti-apoptotic BCL-2 proteins consist of all ␣-helical protein folds involving one or more conserved BCL-2-homology domains (BH1-4). Although all four BH domains are necessary for the proper functioning of BCL-2 and BCL-X L , the BH3 domains of pro-apoptotic members are themselves sufficient for apoptosis induction (2). BH3 domains from pro-apoptotic BH proteins also make heterodimer contacts with related anti-apoptotic partners. The structure of the complex of a soluble BCL-X L protein (BCL-X L ⌬C) and BAK BH3 peptide from nuclear magnetic resonance (NMR) spectroscopy showed peptide bound to a hydrophobic groove bounded by the BH1, BH2, and BH3 domains of BCL-X L (3).In addition to their ability to nullify pro-apoptotic binding partners, the anti-apoptotic BCL-2 family members may maintain cell survival through regulation of ion and metabolite gradients across mitochondrial membranes. BCL-X L and BCL-2 form pores in synthetic lipid vesicles and membranes. Interestingly, this intrinsic pore-forming activity is inhibited by a bound BAK BH3 peptide (4). It remains an unresolved question whether BCL-X L and BCL-2 affect OMM permeability directly or through interactions with other known mitochondrial channels. For example, BCL-X L may promote osmotic homeostasis by maintaining the voltage-dependent anion channel in an open state, allowing transit of metabolites across the OMM (5-7). BCL-X L also interacts with the adenine nucleotide translocator, the most abundant protein in the inner mitochondrial membrane and a major component of the permeability transition pore complex (8).Previously, we reported that AA, an inhibitor of mitochondrial electron transport at complex III, selectively kills murine hepatocyte cell lines that over-express BCL-X L or BCL-2 (4, 9). Mitochondrial electron transport inhibition is not correlated with differential killing of BCL-X L -expressing cells, as 2-methoxy AA (2-OMeAA) has similar effects despite a 1000-fold reduction in electron transport inhibition. Rather, a direct effect of AA on BCL-X L function(s) was proposed, based on fluorescent binding assays showing stoichiometric interaction of AA and BCL-X L (⌬C). Computational modeling using the BCL-X L (⌬C) NMR structure (3) predicted a binding site for AA along the long axis of the hydrophobic groove (Fig. 1). Finally, dosedependent inhibition of BCL-X L (⌬C) pores in synthetic liposome...
The BCL-2 family proteins are attractive targets for drug design. As pivotal regulators of apoptotic cell death, the logic of manipulating BCL-2 functions for anti-tumor effects is perhaps the strongest for any of the molecular targets proposed for cancer therapeutics. Moreover, elevated levels of anti-apoptotic proteins have been demonstrated in virtually every type of human cancer. BCL2-specific antisense oligonucleotides have shown broad anti-cancer activities in pre-clinical models and are currently in several phase III trials. Rational drug design to manipulate the functions of these proteins has been hampered by the lack of a clear understanding of biochemical or molecular functions. Initial efforts have been centered on disrupting protein-protein interactions within the BCL-2 homology (BH) family. Substantial progress in this task has been made using molecular modeling and drug leads.
Overexpression of Bcl-x L in multiple cancers correlates with resistance to chemotherapy and radiation therapy, and provides a rationale for development of small-molecule Bcl-x L inhibitors. Based on knockout studies, nonneoplastic cells also require Bcl-x L survival functions, particularly when challenged with cytotoxic agents. We analyze the selective cytotoxicity of one Bcl-x L inhibitor, 2-methoxy antimycin A, toward cells with excess exogenous Bcl-x L in isogenic cell line pairs. This selectivity, characteristic of a gain-offunction mechanism, is not shared by other known Bcl-x L inhibitors, including BH3I-2, HA14-1, ABT-737, gossypol, or the stapled BH3 helical peptide SAHB-BID. We show that Bcl-x L overexpression induces a shift in energy metabolism from oxidative phosphorylation to glycolysis. Treatment with 2-methoxy antimycin A acutely reverses the metabolic effects of Bcl-x L , causing mitochondrial hyperpolarization and a progressive increase in mitochondrial NAD(P)H. We identify an additional small-molecule Bcl-x L inhibitor, NSC 310343, establishing a class of Bcl-x L inhibitors with gain-of-function activity. In contrast to other Bcl-x L inhibitors, combining gain-of-function Bcl-x L inhibitors with a standard inducer of apoptosis, staurosporine, enhances selective cytotoxicity toward Bcl-x Loverexpressing cells. These results provide an example of the intersection of bioenergetic metabolism and Bcl-x L functions and suggest a metabolic basis for the gain-offunction mechanism of
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