High throughput screening for stimulators of apoptosis

The involvement of the "Inhibitor of Apoptosis Proteins" (IAP) family has been strongly implicated in the resistance of various cancers to apoptosis (click here for our analysis of the IAP family and related pharmaceutical/therapeutic opportunities). The X-linked inhibitor of apoptosis protein (XIAP) is one particularly well-defined member of the IAP family. XIAP expression is elevated in 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 recent reports that low dose gamma-irradiation upregulates XIAP in non-small cell lung carcinoma cells and as a result resistance to radiation-induced apoptosis was enhanced. 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.

A second well-known IAP is survivin. Although not observed in adult differentiated tissue, this IAP is present in most transformed cell lines and cancers tested to date. Even more importantly, survivin expression appears to be homogenous within the tumor environment, a feature unusual in a disease characterized by spontaneous mutation. Survivin has been shown to inhibit caspase directly and apoptosis in general. It has recently been suggested that survivin expression may predict the response to radiochemotherapy and perhaps more importantly, targeting survivin may increase the efficacy of such therapies. In our IAP dossier we analyzed pharmaceutical opportunities for the targeting of this class of protein. One strategy that was discussed involves the development of Smac/DIABLO mimics. Smac/DIABLO is an endogenous protein that is released from the mitochondrial intermembrane space during mitochondria-induced apoptosis and binds to a number of IAPs including both XIAP and survivin. This results in the disruption of IAP binding and inactivation of caspases thereby promoting apoptosis.

In their recent Analytical Biochemistry paper Glover et al have developed a high-throughput fluorescence polarization assay able to screen for Smac/DIABLO mimics. Utilizing a fluorescein-labeled peptide similar to the "IAP binding" domain of Smac N terminus complexed with the BIR3 domain of X-linked IAP (XIAP) this assay was used to identify small-molecule mimics of the action of Smac.

This group has already screened the National Cancer Institute "Training Set" of 230 compounds, with well-defined biological actions, and the "Diversity Set" of 2000 chemically diverse structures for compounds that significantly reduced fluorescence polarization. Further use of this robust assay under high-throughput screening conditions will hopefully lead to the discovery of novel compounds able to simulate the action of Smac/DIABLO.