IAPs (inhibitor of apoptosis proteins) comprise a family of well-conserved eukaryotic anti-apoptotic proteins. It has been shown that IAP overexpression is one of the mechanisms cancer cells employ to counteract the induction of apoptosis by agents such as ionizing radiation and chemotherapeutic agents. Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP binding protein with low pI) is a protein released from the mitochondria in response to apoptotic stimuli. Smac has been shown to promote apoptosis by directly binding to and displacing caspase-9 from XIAP (X-linked inhibitor of apoptosis protein) and other IAPs. Hence, Smac appears to function as an endogenous pro-apoptotic protein by directly inhibiting the function of IAPs. We have synthesized a series of rationally designed nonpeptide Smac mimetics which can effectively bind to the BIR3 domain of XIAP with high affinities. We present the ability of SH-122 and SH-130, two of our compounds, to sensitize 2LMP, a tumorgenic subclone of MDA-MB-231 human breast cancer cell line, to recombinant human TRAIL (TNF related apoptosis inducing ligand) as well as ionizing radiation. Our results show that both SH-122 and SH-130 can dramatically sensitize 2LMP cells to TRAIL in both 24 hour WST-8 assays as well as Annexin V/Propidium Iodide apoptosis assays. In addition, our Smac mimetics can enhance the extent of radiation induced cytotoxicity as seen in 48 hour WST-8 assays. Also a modest level of apoptosis enhancement was seen in the cells irradiated in the presence of our compounds. We propose that high affinity Smac mimetics may function as potent radio/chemo sensitizers by blocking the anti-apoptotic function of IAPs in human cancer cells. Such small molecule compounds may have the therapeutic potential to be developed as an entirely novel class of biologically targeted anticancer agents to be used in combination with ionizing radiation, conventional chemotherapeutic agents, or other agents that induce apoptosis.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]