PerspectiveMedicine

Targeting Apoptotic Pathways in Cancer Cells

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Science  03 Sep 2004:
Vol. 305, Issue 5689, pp. 1411-1413
DOI: 10.1126/science.1102974

From cell division to programmed cell death, protein-protein interactions are a central regulatory feature of nearly all biological processes in a living organism. Hence, modulating or mimicking protein-protein interactions with biologically active peptides or chemical compounds offers an attractive strategy for therapeutic intervention in specific disease pathways. The ability to escape suicide (apoptosis) is a hallmark of most cancer cells and often correlates with tumor aggressiveness and resistance to traditional anticancer drug treatments (1). Consequently, academic and industrial laboratories are engaged in a Herculean effort to develop new molecules that reactivate the apoptotic program in tumor cells by specifically targeting protein-protein interactions (2). On pages 1466 and 1471 of this issue, Walensky et al. (3) and Li et al. (4) present two provocative approaches to inducing tumor-selective apoptosis. In each case, they have engineered an experimental therapeutic that mimics key interactions between proteins that belong to either the receptor-dependent (extrinsic) or mitochondrial-dependent (intrinsic) apoptotic pathways of normal cells.

There is much interest in exploiting biologically active peptides as pharmaceutical lead compounds. The use of peptides as therapeutics is, however, limited by their low bioavailability, their inefficiency in crossing cell membranes (due primarily to their size), and their poor metabolic stability in vivo. Efforts to overcome these limitations have led to the generation of synthetic peptides that contain nonnatural amino acids. These so-called “peptidomimetics” mimic the structural and functional properties of their native parental peptides and often have certain advantages. For example, they may be conformationally stable, resistant to degradation by enzymes, have an increased ability to penetrate cell membranes and, most important, can be engineered to specifically bind to the interaction surfaces of target proteins (5).

Death and the cancer cell.

Macromolecular peptidomimetic manipulation of apoptosis. Cells of higher eukaryotes contain extrinsic receptor pathways and intrinsic pathways that activate effector caspases and induce apoptosis. (Right) The extrinsic cell death pathway is mediated by a subgroup of the TNF receptor superfamily called the death receptors (TNFR1, FAS, and TRAIL). Receptor-mediated cell death is initiated by the recruitment of adaptor proteins, like FADD, which then bind to DED-containing procaspases to generate a death-inducing signaling complex (DISC) that leads to activation of caspase-8. Caspase-8 directly cleaves and activates caspase-3, the executioner enzyme of apoptosis. (Left) In the mitochondrial or intrinsic pathway, proapoptotic BCL2 family members BAX and BAK translocate to the mitochondria. The BH3-only protein BID activates BAX and BAK to mediate the release of cytochrome c in the cytosol. This triggers the assembly of the apoptosome (APAF1 and caspase-9) and subsequent activation of caspase-3 and cell death. The SAHB BH3 peptidomimetic designed by Walensky et al. (3) mimics the BH3 helix of BID and hence is able to activate BAX and BAK, resulting in cytochrome c release from mitochondria. Inhibitor of apoptosis (IAP) proteins bind directly to caspases and inhibit their enzymatic activity. The inhibitory function of IAPs is countered by the second mitochondria-derived activator of caspases (SMAC). Four amino acid residues in the amino terminus of SMAC interact with the Bir domain of IAPs. Compound 3 designed by Li et al. (4) mimics the four amino-terminal residues of SMAC that interact with IAPs. This new molecule acts synergistically with TRAIL or TNF-α to induce apoptosis of glioblastoma cells.

CREDIT: KATHARINE SUTLIFF/SCIENCE

In their study, Walensky et al. (3) generated a peptidomimetic of a critical BH3 (BCL-2 homology 3) protein-protein interaction domain to induce mitochondrial-dependent apoptosis (figure). Members of the BCL2 family of proteins are central regulators of mitochondrial integrity and apoptotic cell death (6). However, BCL2 family members exist in two distinct flavors: inhibitors of apoptosis (BCL2 and BCL-XL) and inducers of apoptosis (BAX, BAK, and BID). Proapoptotic members such as BID harbor a BH3 protein-protein interaction domain in the form of an amphipathic α helix that performs several essential functions. First, it interacts with a hydrophobic groove on antiapoptotic BCL2 family members and effectively blocks their function. Second, it induces oligomerization of proapoptotic members (BAX and BAK), resulting in release of cytochrome c from mitochondria, activation of the apoptosome, and subsequent induction of apoptosis (figure).

Walensky et al. (3) started with the parental BH3 peptide. Then, by substituting nonnatural amino acids on the surface opposite the protein-protein interaction surface using a chemical technique called hydrocarbon stapling, they were able to generate a covalent “staple” that stabilized the α-helical structure. The resulting BH3 peptidomimetic, called SAHB (stabilized α helix of BCL2 domains), has improved pharmacological properties relative to the native parental BH3 peptide. These properties include greater resistance to proteolytic degradation, and the ability to boost cytochrome c release and to induce apoptosis in a variety of leukemic cell lines. In vivo, SAHB (BH3) peptidomimetic treatment of mice bearing human leukemia xenografts substantially reduced the expansion and organ infiltration of leukemic cells without any detectable toxicity to normal organs.

The cell membrane is a formidable barrier that generally prevents passage of molecules greater than 500 daltons (roughly equivalent to the mass of five amino acids) (7). However, peptides encountered by the cell membrane are often larger than several thousand daltons. Walensky and co-workers showed that the SAHB (BH3) peptidomimetic (>2000 daltons) is taken up by a fluid-phase endocytic mechanism. Recent work on the transduction domain of the HIV-1 Tat protein has shown that poly(Arg)-containing peptides enter cells by fluid-phase macropinocytosis, a specialized form of endocytosis (8). However, the SAHB (BH3) peptidomimetic contains only three arginine residues, an insufficient number to induce macropinocytosis, suggesting that covalent stabilization of the α-helical structure by hydrocarbon stapling may confer the SAHB peptide with lipophilic properties that enable it to cross cellular membranes. The Walensky et al. results begin to open the experimental therapeutic door to entirely new types of therapies aimed at modulating specific protein-protein interactions.

In a complementary study, Li and co-workers (4) used a similar strategy of mimicking protein-protein interactions to induce tumor-selective apoptosis. However, they generated a synthetic compound that mimics the protein-protein interaction domain of an activator of apoptosis, SMAC. The key effectors of apoptosis are the caspase proteases that, when activated, result in the destruction of the cell's interior. Caspases are held in check, in part, by protein-protein interactions with IAPs, proteins that block apoptosis. Not surprisingly, IAPs undergo gene amplification and overexpression in human tumors (9). IAPs, in turn, are inhibited by protein-protein interactions with the amino-terminal four residues of proapoptotic SMAC. Following release from mitochondria after BAX/BAK activation, SMAC interacts with a groove in the Bir domain on the IAP surface (figure). Thus, inhibition of IAPs with SMAC molecular mimetics would be predicted to have valuable therapeutic potential for treating both cancer and inflammatory diseases. Indeed, Fulda et al. have previously shown that linkage of the native SMAC amino-terminal peptide to a protein transduction domain resulted in caspase activation and the killing of tumor cells in a mouse model of glioblastoma (10).

To enhance the pharmacological properties of the quadrimeric amino-terminal SMAC peptide, Li et al. started with the crystal structure of the SMAC-IAP interaction, then designed a 180-member peptidomimetic library harboring nonnatural amino acid replacements (4). The peptidomimetic library was screened to find molecules that could compete with the binding of the SMAC peptide to the Bir domain of different forms of IAP. After further chemical modification of a candidate molecule, Li et al. generated compound 3 that, like SMAC, has a high avidity for different forms of IAP including X-chromosome encoded IAP (XIAP), cellular IAP-1, and cellular IAP-2. Compound 3 blocked the interaction of XIAP with active caspase 9. In previous work, SMAC was shown to act synergistically with a death receptor called TRAIL to induce tumor-selective apoptosis (10). Impressively, treatment of glioblastoma cells with a combination of the ligand for the TRAIL receptor and compound 3 resulted in apoptosis of the tumor cells, whereas normal cells were not harmed. Li et al. (4) also demonstrated that compound 3 could potentiate apoptosis in cells treated with TNF-α (tumor necrosis factor-α) without activation of the nuclear transcription factor NF-κB. Because TNF-α mediates host responses in acute and chronic inflammatory conditions, these results suggest that compound 3 may have potential for treating inflammatory diseases (11). Although the efficacy of compound 3 was not evaluated in vivo, the authors are using compound 3 as a lead structure for the refinement of future therapeutic compounds with better pharmacological properties.

Peptidomimetics are only now emerging as a powerful solution for overcoming the limitations imposed by the physical properties of native peptides. Walensky et al. (3) and Li et al. (4) demonstrate provocative proof-of-concept approaches to the design of peptidomimetics that may have a decided impact on future therapeutics that target disease by modulating specific protein-protein interactions.

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