*4.2.1. Small-molecule IAP antagonists*

126 Apoptosis and Medicine

apoptotic pathways [126].

an inhibitor of procaspase-9 [130].

form a complex with Smac/DIABLO [131].

function as a direct therapeutic strategy [125].

XIAP and with the inhibition of mitochondrial and apoptosis-inducing factor-dependent

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

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

The expression and/or function of IAPs are deregulated in many human cancers because of genetic aberrations, an increase in their mRNA or protein expression or the loss of endogenous inhibitors such as Smac. The expression levels of IAPs and their antagonists have been correlated with clinical parameters and cancer prognosis in several retrospective trials. However, these results should be interpreted with caution due to the low numbers of samples studied in some reports and the limitations of currently available reagents for analyzing the expression of IAP proteins in tissue specimens. Therefore, additional studies

The issue of primary or acquired resistance to current chemotherapeutic-based treatments is a major impediment to effective cancer treatment. Although there are many genetic and biochemical alterations that occur in cancer cells, in vitro experiments demonstrate that the up-regulation of IAPs expression increases resistance to chemotherapy and radiation. The fundamental role of the IAPs in apoptosis regulation and their elevated expression in many tumour types suggest that there is value in exploiting the inhibition of IAP expression and

are required to evaluate the prognostic value of IAPs in human malignancies [10].

**4.2. Use of IAPs into clinic: prognostic and therapeutic values** 

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 repression of caspase-3 more potently than Smac peptides.

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].
