The development of novel small molecule XIAP inhibitors with anti-cancer activity

The treatment of cancer represents a major unmet market. This field is highly segmented both in terms of cancer sub-types and the various pharmacological classes available to oncologists (for a full analysis of the potential of different patient groups and various anti-cancer strategies in development or on the market click here for our feature “The Cancer Market Outlook to 2008”).

The efficacy and adverse effect profile of many current gold-standard treatments of cancer is sub-optimal and hence heavy R&D investment is driving innovative compounds in clinical development. Apoptosis stimulators have emerged as one group of key targets for the control of cancer. This therapeutic class has, however, remained predominantly experimental since early generation apoptosis stimulators lacked specificity, efficacy and were susceptibility to drug resistance.

From a molecular point of view the development of apoptosis stimulators has concentrated heavily on the caspases and endogenous inhibitors of apoptosis, predominantly Bcl-2 proteins. Over the past few years a considerable amount of research has been conducted and our view of apoptosis has changed dramatically. Major advances have included the emergence of the IAP ("Inhibitor of Apoptosis Proteins") family and to meet the development of this field LeadDiscovery has recently produced a full analysis of the IAPs and how they can be targeted for the treatment of cancer (Click here for this report). This field has grown exponentially since 1995 and continues to do so. Although XIAP and survivin remain the better known members of this family, numerous human IAPs have now been identified. Members of this family represent key regulators of apoptosis.

The three main apoptotic pathways identified thus far (the mitochondrial, endoplasmic reticulum (ER) stress, and death receptor pathways) are activated by caspase-9, -12, and -8, respectively. The mitochondrial pathway may also serve as a point of convergence for the other two pathways. The down-stream caspase-3 is the primary executioner caspase.

The X-linked inhibitor of apoptosis protein (XIAP) is one particularly well-defined member of the IAP family. XIAP expression is elevated in various cancers for example non-small cell lung cancer and acute myelogenous leukemia. With respect to the latter there is a strong correlation between expression and survival. A convincing body of evidence supports a direct role of XIAP in the resistance of cancer cells to radiation and chemotherapeutic intervention. Perhaps most convincing are reports that low dose gamma-irradiation upregulates XIAP in non-small cell lung carcinoma cells inducing resistance to radiation-induced apoptosis. On the other hand XIAP antisense sensitized cells to low dose gamma-irradiation. A similar approach was used to sensitize ovarian carcinoma cells to cisplatin-induced apoptosis.

XIAP has been shown to attenuate pro-caspase-3 cleavage and caspase-3 activity and in prostate cancer cells this leads to the inhibition of taxol-induced apoptosis. This inhibitory activity as well as the inhibition of caspase-7 is dependent on the BIR2 domain and its flanking regions within XIAP. A second BIR domain, BIR3, suppresses caspase-9.

While antisense technology has been used to establish a proof of concept for inhibiting XIAP, small molecule inhibitors of this protein are more sought after and in their recent Cancer Cell paper, Schimmer et al have made the important breakthrough describing the identification of such inhibitors.

Using a caspase-3 enzyme derepression assay (the assay was based on a mixture of recombinant caspase-3 repressed by XIAP) to screen eleven mixture-based small molecule combinatorial libraries (constituting approximately one million compounds), and then individual molecules purified from mixtures displaying activity in the assay, Schimmer et al identified 36 hits that increased caspase-3 activity. Of these 8 came from a library of nearly 100,000 polyphenylureas. Each of these molecules also restored caspase-7 activity but had no effect on caspase-9 suggesting that they acted at BIR2 or its flanking region. This was confirmed using a second assay in which XIAP was repressed by BIR2.

The potency of the polyphenylureas was in the micromolar range, and with a similar potency these molecules induced apoptosis in Jurkat cells. The active polyphenylureas were selective for XIAP and furthermore cytotoxicity was specifically induced in fibroblasts expressing XIAP suggesting that in contrast to conventional chemotherapeutic molecules the cytotoxicity of these XIAPs would be restricted to cancer cells.

The anti-cancer activity of the XIAP inhibitors was then investigated further against the NCI’s panel of 60 cancer cell lines, each of which expresses XIAP. For 4 representative XIAP inhibitors the mean cytotoxic activity across this panel was approximately 1-20 micromolar, 10-fold lower than doxorubicin and similar to that of etopiside. When investigated clonogenic survival was also reduced within this concentration range. Furthermore the XIAP inhibitors also potentiated the effects of a variety of anti-cancer drugs. As well as being effective against multiple cell lines, these molecules were also cytotoxic to primary cultures of chronic lymphocytic and myolegenous leukemia cells. This not only established efficacy under conditions closer to those of the clinic but also demonstrated that cell death could be produced in both slowly dividing primary cultures as well as more rapidly dividing cell lines.

In a final study, two representative polyphenylureas were shown to reduce the size of established colon or prostate cancer xenografts by over 50% at 30mg/kg ip. Even at doses ten-fold higher than this no adverse effects were observed.

This important study shows for the first time that molecules able to relieve endogenous inhibition of the caspases can directly kill cancer cells as opposed to only being able to synergize with other cytotoxic agents. In contrast non-cancerous cells were insensitive to the apoptotic effects of the polyphenylureas. These findings are important on many different levels. Firstly, it demonstrates that cancer cells differ from their non-cancerous counterparts in that although caspase levels are elevated in tumors cells, apoptosis is in a state of chronic repression as a result of IAP over-expression. This means that overcoming this repression represents a highly selective approach to the treatment of cancer. Second, it establishes and explains the lack of adverse effects seen following treatment with the polyphenylureas suggesting that this new class of drug will have a broad therapeutic window. Thirdly, although the polyphenylureas may be effective as a monotherapy, they have been shown to enhance the cytotoxic effects of a range of anti-cancer agents. Further development of this class towards the clinic is therefore eagerly awaited.

Entry date Tuesday, June 08, 2004

Adapted from Schimmer et al, Cancer Cell. 2004 Jan;5(1):25-35.