**4.1. Role of the IAPs in apoptosis**

As long as apoptosis pathway must be tightly regulated, the IAPs are the only known regulatory proteins that control the activity of both initiator and effectors caspases in the process. In this way, there are studies where XIAP was found to prevent caspase-3 processing in response to caspase-8 activation. So it is suggested that XIAP is able to inhibit this extrinsic apoptotic signaling by blocking the downstream effectors caspases, avoiding the direct interference of the caspase-8 activation [124].

On the other hand, other IAPs proteins are not potent inhibitors of caspases, such as c-IAP proteins. They are able to bind with Smac having high affinities to prevent it from blocking XIAP-mediated inhibition of caspases. These IAPs also show a E3 ubiquitin ligase activity, allowing the regulation of several cell death effectors and modulators, playing a key role in cellular survival. c-IAP1 and c-IAP2 are able to interact with TNFα-receptor-associated factors -1 and -2 (TRAFs) trough their association in a complex with TNFα receptor 2. The TNFα receptor can mediate survival and death cell signals. In this case, c-IAP1 and c-IAP2 have been proposed to reduce the level of caspase-8 activation and protect cells from apoptosis in a TNFα relationship [10].

124 Apoptosis and Medicine

of anti-apoptotic genes such as *Bcl-2*. In contrast, several pro-apoptotic genes related with the endoplasmic reticulum (ER)-stress-induced apoptosis, such as *BBC3* and *Noxa*, appeared upregulated. These results seem to show that the mechanism of action and selectivity of **16** was via the activation of the ER-stress-induced apoptosis. The linkage between the 5-FU moiety

*o*Ns OMe

<sup>N</sup> <sup>N</sup>

<sup>N</sup> <sup>N</sup>

N

R OMe

N

N H

O O

F

OH

**16**, R = SO2-C6H4-*o*NH2

and the chain of **16** is through the *N*-1 atom of the pyrimidine [20] (Figure 5).

**Figure 5.** Several *O,N*-acetalic purine derivatives reported by us [20,21]. Fmoc is the

N

OH

**4. Inhibitor of apoptosis protein (IAP) family as therapeutic target** 

Cl

**15**

In the apoptotic pathway, the balance between pro and anti-apoptotic proteins is tightly regulated, so an imbalance directed to the anti-apoptotic regulation involves the apparition of survival advantages onto the initiating cancerous cells. In this way, the role of the IAPs family proteins is important. This family was first identified in baculovirus and the main characteristic is the presence of a baculovirus IAP repeat (BIR) domain, that mediates interactions with a number of proteins, including caspases and also other structural domains such as a RING or caspase activation recruitment domain (CARD) [122] (Figure 6). In the IAP family we can find proteins involved in the inhibition of apoptosis through inhibition of caspases or modulation of the nuclear factor kB (NF-kB) signalling pathways. This family includes eight members in mammals (BIRC1– BIRC8), also known as neuronal apoptosis inhibitory protein (NAIP), cellular IAP1 (cIAP1) and IAP2 (cIAP2), Xchromosome linked IAP (XIAP), survivin, Apollon (BRUCE), livin (ML-IAP) and ILP2 [123] (Figure 6).

As long as apoptosis pathway must be tightly regulated, the IAPs are the only known regulatory proteins that control the activity of both initiator and effectors caspases in the process. In this way, there are studies where XIAP was found to prevent caspase-3 processing in response to caspase-8 activation. So it is suggested that XIAP is able to inhibit this extrinsic apoptotic signaling by blocking the downstream effectors caspases, avoiding

On the other hand, other IAPs proteins are not potent inhibitors of caspases, such as c-IAP proteins. They are able to bind with Smac having high affinities to prevent it from blocking XIAP-mediated inhibition of caspases. These IAPs also show a E3 ubiquitin ligase activity,

N

Cl

fluorenylmethyloxycarbonyl group; *o*Ns is the -SO2-C6H4-*o*NO2 group.

N N

N

**4.1. Role of the IAPs in apoptosis** 

the direct interference of the caspase-8 activation [124].

O

N Fmoc

**14**

**Figure 6.** Identifying some of the structural motifs of IAPs. The baculoviral IAP repeat (BIR) domains; 70–80-amino-acid cysteine- and histidine-rich domains that chelate zinc ions. The presence of at least one BIR domain is the defining characteristic of the IAP family. The number of BIR domains in a given IAP varies from one to three. Another motif is the really interesting new gene (RING) zinc finger, a caspase recruitment domain and ubiquitin-associated (UBA) domains are found in individual IAPs. Finally, the CARD motif (Caspase Recruitment Domain) is a protein–protein interaction domain that mediates oligomerization with other CARD-containing proteins and is found in a number of proteins involved in the regulation of cell death.

There is another family member of the IAPs, called Livin, which are expressed in high levels in melanoma and colon cancer, in embryonic tissues and transformed cells. The overexpression of Livin isoforms α and β blocks apoptosis induced via the extrinsic death receptor pathway [125].

Survivin is involved in the control of cell proliferation and cell death and gene expression is regulated in a cell cycle dependent manner in mitosis. The spliced transcription of the survivin gene gives rise to wild-type survivin, survivin-2α, survivin-2B, survivin-ΔEx-3 and survivin-3B. This protein is stabilized by phosphorylation thanks to a p34cdc2–cyclin B1 complex during mitosis. The anti-apoptotic function of survivin has been linked with its interaction with Smac/DIABLO, with the stabilization of XIAP protein through its binding to

XIAP and with the inhibition of mitochondrial and apoptosis-inducing factor-dependent apoptotic pathways [126].

Apoptosis as a Therapeutic Target in Cancer and Cancer Stem Cells: Novel Strategies and Futures Perspectives 127

Novel drugs have been developed and some of them are in current clinical trials. Several strategies have been chosen for anticancer drug development targeting IAP molecules: (1) small-molecule IAP antagonists, (2) antisense oligonucleotides (ASOs), (3) Smac mimetic molecules and others [132]. Small-molecule IAP antagonists and antisense oligonucleotides have garnered the most attention. Also, IAP antagonists have been extensively studied in combination with other cytotoxic agents including anticancer drugs, small-molecule signal transduction inhibitors, proteasome inhibitors and death receptor ligands as well as with

Owing to the differences in the mechanism of caspase inhibition by the BIR 2 and BIR 3, domain molecules have been developed to specically target the BIR 2 or BIR 3 region of XIAP. The structural data surrounding the interaction between the BIR 3 domain of XIAP and caspase-9, suggests that small molecules that bind the BIR 3 pocket of XIAP could mimic the action of Smac and inhibit the interaction between XIAP and caspase-9. These structural studies have facilitated a variety of chemical biology approaches including uorescent polarization, nuclear magnetic resonance, 'in silico' virtual screening and computer modelling to identify BIR 3 inhibitors [133]. The rst small-molecule XIAP inhibitors were reported by [134]. These inhibitors were identied using a high throughput enzymatic derepression assay in which recombinant XIAP was combined with active caspase-3 to inhibit caspase-mediated cleavage of the uorogenic peptide substrate. With this assay, they screened 160.000 compounds in 1536-well format and identied potent XIAP inhibitors including the compounds TWX006 and TWX024, aryl sulphonamides with exible acyclic diamines in the rst and third fragments. These compounds derepressed XIAP-mediated inhibition of caspase-3 more potently than Smac. In addition, these molecules bound the BIR 2-linker region of XIAP, and, in enzymatic assays, relieved the

Recently, small-molecule phenylurea-based chemical inhibitors of XIAP were identified by large-scale combinatorial library screening. Subsequent studies have confirmed that the active XIAP inhibitors, but not their inactive structural analogues, could induce apoptosis in a variety of human cancer cell lines and xenograft. Furthermore, it was determined that these XIAP inhibitors act by binding to its BIR-2 domain, resulting in elevated activity of the downstream caspase-3 and caspase-7. Thus, the action of these exogenous XIAP inhibitors was found to be mechanistically distinct from that of the endogenous inhibitor second modulator of apoptotic proteases, which predominantly binds to the BIR-3 domain [135].

The single-stranded antisense oligodeoxynucleotides (AS ODNs) are short stretches of synthetic DNA, approximately 12–30 nucleotides long, and are complementary to a specific mRNA strand. Hybridization of the AS ODNs to the mRNA by Watson–Crick base pairing prevents the target gene from being translated into protein, thereby blocking the action of

radiation therapy.

*4.2.1. Small-molecule IAP antagonists* 

repression of caspase-3 more potently than Smac peptides.

*4.2.2. Antisense oligonucleotides* 

Other IAPs is Bruce/Apollon which is a membrane bounded protein involved in protein ubiquination-mediated degradation by its ability to target proteins thanks to the presence of a C-terminus E2 motif. It is also shown that Bruce is able to bind to caspase -3, -7 and -9 [127]. In Bruce regulation are involved the E2 UbcH5 and the E3 Nrdp1, which ubiquitinate the epidermal growth factor receptor family member, ErbB3. It has been shown that a decrease in Bruce content by Nrdp1 over-expression induces apoptosis in different cell lines. These studies suggest that this IAP protein play a critical role in apoptosis inhibition in certain cell types avoiding pro-caspase-9 cleavage when it binds to this protein [128].

Naip has been studied because of the clinical relevance in tumors such as prostate or breast cancer. Naip has two different functions; in the first one it is involved in the inflammatory process by caspase-1, -4 and -5 activation, and the second one is via apoptosis regulation by caspase-3 and -7 inhibition [129]. Davoody *et al* showed the cleavage inhibition of procaspase-3 by apoptosome activated caspase-9 and the inhibition of the autocatalytic processing of procaspase-9 in the apoptosome complex. This fact indicates that unlike other IAPs, Naip is an inhibitor of procaspase-9 [130].

Finally, Ts-IAP, also known as ILP-2, is the product of a human testis-specific mRNA and is related to the C-ter region of another member of the IAPs protein family, XIAP. It is showed that this protein is a weak caspase-9 inhibitor, and also a highly unstable molecule. However, a stabilized form of this protein containing nine additional N-ter residues may form a complex with Smac/DIABLO [131].
