The saintly side of Smac/DIABLO: giving anticancer drug-induced apoptosis a boost

A number of agents that target components of the apoptotic pathway or their regulators are currently undergoing preclinical and clinical testing as potential drugs.¹ In the field of cancer chemotherapy, antisense oligonucleotides that target Bcl-2² or Bcl-x_L as well as inhibitors of the antiapoptotic kinase Akt³ are currently in development. Results presented over the past year raise the possibility that anticancer drug-induced apoptosis might also be enhanced by peptides that mimic the action of second mitochondrial activator of caspases (Smac)/direct IAP binding protein with low PI (DIABLO), a mitochondrial polypeptide identified scarcely 3 years ago. Here we summarize the rationale and current state of development of these Smac-based peptides.

Previous studies (reviewed in Kaufmann and Earnshaw⁴ and Herr and Debatin⁵) have demonstrated that many anticancer drugs induce apoptosis by activating the mitochondrial or intrinsic cell death pathway (Figure 1). This pathway involves a series of still poorly understood changes that result in translocation or release of a number of proapoptotic polypeptides from the intermembrane space of mitochondria to the cytoplasm.^6,7 One of these polypeptides, cytochrome c, induces a conformational change in the C-terminal domain of the cytoplasmic adaptor protein apoptotic protease-activating factor 1 (Apaf-1). This exposes the N-terminal caspase recruitment domain of Apaf-1, which complexes with and activates procaspase-9.^6,8 The resulting multimeric complex, termed the apoptosome, then proteolytically activates caspase-3 and possibly caspase-7.

Figure 1

A two-signal model for mitochondrial initiation of caspase activation. Upon receiving an apoptotic stimulus, mitochondria release a variety of polypeptides that initiate downstream signaling. At least two of these participate in caspase activation. As indicated in the text, one signal contributing to caspase activation consists of cytochrome c-mediated initiation of apoptosome formation. A second signal provided by Smac/DIABLO and polypeptides with similar motifs modulates the ability of IAP proteins to bind and inhibit susceptible forms of caspase-3, -7 and -9. Both of these signals are required for optimal caspase activation. Under conditions where the action of Smac/DIABLO is deficient, Smac-based peptides can replace the second signal

Caspases, like other proteases, are tightly regulated. Inhibitor of apoptosis (IAP) proteins, a conserved family of polypeptides found in baculoviruses and lower eukaryotic organisms as well as mammals, directly bind and inhibit caspases.⁹ The defining features of these IAP proteins are two structural motifs, the baculovirus inhibitor repeat (BIR) and really interesting new gene (RING) domains. BIR domains are ∼80 amino-acid C3H zinc-finger domains present in 1–3 homologous copies per IAP protein. Structural analyses have indicated that BIR domains bind to target caspases in such a way that amino acids upstream of the BIRs are draped across the caspase-active sites in a reverse orientation relative to normal substrates. The resulting occlusion of the caspase-active sites inhibits their activity. Most mammalian IAP proteins also contain a single C-terminal RING domain, a zinc-finger motif with E3 ubiquitin ligase activity. The RING fingers present in IAP proteins have been shown to ubiquitinate activated caspases in vitro and in intact cells, thereby facilitating their proteasome-mediated degradation.

The inhibition of caspases by IAP proteins is itself subject to regulation. Along with cytochrome c, at least four polypeptides capable of binding XIAP are released from mitochondria during the course of apoptosis.¹⁰ The most extensively studied of these is Smac/DIABLO.^10,11 After synthesis in the cytoplasm, this 27 kDa polypeptide is imported into mitochondria, where the N-terminal 53 amino acids are proteolytically removed to yield the mature polypeptide beginning with the sequence AVPI. By a process that is understood only at the most superficial level, apoptotic stimuli induce release of mature Smac to the cytoplasm, where it binds to IAP proteins¹⁰ and disrupts their ability to bind caspases.^12,13 X-ray crystallography of Smac bound to the third BIR domain of XIAP¹⁴ and high-resolution NMR of a 9-amino-acid Smac peptide bound to the same XIAP domain¹⁵ have demonstrated that the N-terminal AVPI sequence of mature Smac binds in a shallow groove in BIR3, where it assumes an extended conformation with a kink at the proline. The amino group of the Smac N-terminal alanine is hydrogen bonded to Glu314 of XIAP, while the methyl and carbonyl groups on the same alanine interact with the indole ring of Trp323. The importance of these interactions is underscored by the observation that Smac peptides containing propionic acid, glycine or methionine in place of alanine fail to bind to BIR3.^15,16 Additional hydrogen bonds and van der Waals forces also stabilize interactions between the next three amino acids of Smac and BIR3. By occupying this site, Smac competitively inhibits the binding of BIR3 to the p12 subunit of partially processed caspase-9.¹² The same domain of Smac binds to BIR2 of XIAP, albeit ∼10-fold less tightly,¹⁵ and inhibits the binding of BIR2 to caspase-3.^16,17,18

Implicit in descriptions of Smac/DIABLO as an XIAP antagonist is the concept that the mitochondrial pathway is a two-signal system.¹⁶ In addition to cytochrome c, which induces apoptosome assembly, full caspase activation requires a second mediator that relieves the intrinsic inhibition by IAP proteins when they are present (Figure 1). This model suggests that a deficiency of either signal will inhibit apoptosis. Consistent with this model, many cells fail to undergo apoptosis after microinjection of cytochrome c¹⁹ or transfection with Smac alone.^{11,20,21,22,23}

This model also predicts that XIAP gene deletion should enhance apoptosis and Smac deletion should inhibit it. In accord with these predictions, disruption of the Drosophila homologues of these genes results in the expected changes in apoptosis during fly development.²⁴ In mice, on the other hand, targeted deletion of either XIAP²⁵ or Smac²⁶ fails to produce an obvious alteration in apoptosis. These latter observations have not only raised questions about the importance of IAP proteins and their antagonists as apoptotic regulators in mammalian cells, but also have been difficult to reconcile with reports that XIAP overexpression inhibits^9,27 and Smac transfection enhances^{11,20,21,22,23,28} the proapoptotic effects of a variety of stimuli in mammalian tissue culture cells.

Before the phenotype of the Smac knockout mouse was reported, several groups began to study the effect of Smac-based peptides on chemotherapy-induced apoptosis. Reports that XIAP, cIAP1 and/or cIAP2 are overexpressed in a number of malignancies^27,29,30 provided the rationale for these studies. Four groups have now reported that the N-terminal 4–8 amino acids of Smac tethered to carrier peptides (e.g., the penetratin sequence from the Drosophila transcription factor antennapaedia) increase the number of cells undergoing apoptosis after treatment with a variety of conventional and experimental chemotherapeutics, including TRAIL, taxanes, epothilones, topoisomerase poisons and antimetabolites, in tissue culture.^21,22,31,32 Particularly exciting is the demonstration that these peptides enhance the proapoptotic effects of drug doses that are themselves minimally toxic to cancer cells.^21,31 Moreover, these peptides enhance the long-term antiproliferative effects of chemotherapeutic agents rather than merely accelerating apoptosis in cells that are destined to die anyway.³¹ Two of these studies have gone on to demonstrate the success of these peptides in improving the efficacy of anticancer therapy in mouse models. In one, high concentrations (1 mM) of Smac-derived peptides tethered to the carrier peptide TAT enhanced the ability of TRAIL to reduce the size of glioblastomas in situ.²¹ In the other, the N-terminal seven amino acids of Smac tethered to an arginine-rich penetration sequence enhanced chemotherapy-induced suppression of H460 lung cancer cells grown as flank xenografts.³²

Although this striking Smac peptide-induced therapeutic enhancement seems to be at odds with the limited effects of deleting the murine XIAP and Smac genes,^25,26 several points must be kept in mind. First, gene disruptions involve chronic alterations and provide a potential opportunity for compensatory changes in other regulatory components of apoptotic pathways.^25,26 In contrast, treatment with Smac-based peptides might produce acute changes in the function of IAP proteins without the opportunity for compensation. Second, it has been suggested that XIAP might be more active in cancer cells than normal cells,³² although this conclusion must be viewed as tentative because it is based on a comparison of normal fibroblasts with epithelium-derived tumor cells. If it is true that XIAP is more active in cancer cells, however, the lessons learned from XIAP disruption experiments in normal cells might not apply to cancer.

Additional observations have begun to provide a framework for understanding the ability of Smac peptides to enhance the efficacy of anticancer drugs. Several groups have reported that cleaved caspases are present in enzymatically inactive aggregates in the cytoplasm of carcinoma cells undergoing apoptosis.^33,34 The demonstration that XIAP is also present in this fraction³⁴ raises the possibility that Smac-mediated displacement of caspases from XIAP might be deficient in some cells. Consistent with this hypothesis, two groups have reported the absence of Smac release during apoptosis in certain cancer cell lines.^31,32 These observations suggest that one of the two signals contributing to activation of the intrinsic pathway might be weak or absent under some circumstances. An explanation for this deficiency is currently lacking, but will likely be found by following up recent studies suggesting that Smac release and cytochrome c release are independently regulated processes.^35,36,37

What is the evidence that Smac peptides are actually enhancing the effects of chemotherapy by providing the second signal for caspase activation? Pull-down studies have established that biotinylated Smac-based peptides bind to IAP proteins in situ.³¹ Importantly, the Smac peptides displace cleaved caspases from the cytoplasmic aggregates described above and enhance the activity of the cleaved caspases.³¹ All of these observations are consistent with a model in which Smac peptides replace endogenous Smac in providing a second signal for igniting the caspase-9 pathway (Figure 1).

With four publications showing promising effects of Smac peptides in vitro and in vivo, one might conclude that Smac-based therapies are ready to enter the clinic. For a number of reasons, however, further studies are required prior to clinical testing. First, the peptides described to date are not very potent, with high micromolar concentrations required for efficacy in vitro. This makes them expensive for laboratory studies and prohibitively expensive for clinical testing. Second, the existing studies have only reported efficacy when Smac peptides are administered directly to the tumor site. This approach is not usually feasible in the clinical setting, where cancers are rarely as well circumscribed as the xenograft models tested. Third, the stability, distribution, penetration and clearance of the existing Smac peptides and their analogs require further study. While the recent reports present a “test of concept”, attention to mundane matters that determine how well the peptides penetrate tumor cells and how long they stay there will have a huge impact on how well these agents perform when they undergo clinical testing. Finally, the possibility that these peptides inhibit other targets requires further scrutiny. Although it has been shown that these molecules bind XIAP, cIAP1 (and probably cIAP2) as well as ML-IAP in intact cells,^31,38 an interaction with other BIR proteins or even unanticipated targets has not been ruled out.

In summary, recent studies have suggested that Smac-based peptides are capable of enhancing the cytotoxicity of chemotherapeutic agents in a variety of tumor cells in vitro and in vivo. These studies also suggest that Smac activity might be deficient in tumor cells, providing an explanation for the efficacy of these peptides. Identification of more potent and more permeant Smac mimetics will likely be required to translate these findings into strategies that can be tested in the clinic.