Defects in apoptosis mechanisms commonly occur in cancers, preventing cell eradication by blocking activation of Caspase-family cell death proteases. XIAP (X-linked Inhibitor of Apoptosis Protein) is an endogenous inhibitor of Caspases that contributes to chemoresistance and that is associated with poor treatment outcomes in patients with certain malignancies. Antisense and peptide-inhibitor experiments have validated the concept that overcoming the cytoprotective effects of XIAP could be useful for restoring apoptosis-sensitivity to tumor cells. We developed a high-throughput enzyme derepression assay, based on the ability of recombinant XIAP to repress active Caspase-3 activity in vitro, and we screened mixture-based combinatorial chemical libraries for compounds that reversed XIAP-mediated inhibition of Caspase-3, identifying a class of poly-phenylureas with XIAP-inhibitory activity. Characterization of the biochemical mechanism of these compounds revealed that they target the BIR2 but not BIR3 domain of XIAP, overcoming XIAP-mediated suppression of Caspase-3 and –7, but not Caspase-9, and lacking the ability compete with SMAC peptides for binding to XIAP. These compounds, but not inactive structural analogues, stimulated increases in Caspase activity and directly induced apoptosis of a wide variety of human tumor cell lines in culture at low micromolar concentrations (1-10 uM); they also suppressed clonogenic survival and sensitized cancer cells to chemotherapeutic drugs and to the apoptosis-inducing cytokine TRAIL. The doses of phenylurea XIAP-antagonists required to induce apoptosis were modulated in predictable manners by either over-expressing or knocking-out XIAP expression. Apoptosis induction by active compounds was suppressed by a pharmacological inhibitor of Caspases (zVAD-fmk), but not by Bcl-2 or Bcl-XL. XIAP-antagonists were considerably less toxic to cultured normal cells compared with tumor and leukemia cells, and mice tolerated the compounds well. Active compounds also suppressed growth of established tumors in xenograft models in mice, including Caspase activation and tumor cell apoptosis in vivo. Thus, these IAP-inhibitory compounds could serve as prototypes for development of novel therapeutic agents based on the concept of restoring Caspase activity in cancers.

[Proc Amer Assoc Cancer Res, Volume 45, 2004]