(A) Schematic of the apoptotic pathway with structure and site of action of LBW242, which binds to the IAPs and prevents their neutralization of the caspases. (B) LBW242 competes with full-length Smac for occupancy of the XIAP-BIR3 surface groove, as assessed by a time-resolved fluorescence energy transfer assay. (C) LBW242 overcomes XIAP-BIR3–mediated repression of caspase-3 activity in a cell-free extract, resulting in activation of caspase-3 and cleavage of a fluorogenic substrate. (D) LN827 cells treated with the indicated concentrations of LBW242 for 4 hours reveal no change in cellular levels of XIAP or caspase-9 (input). Immunoprecipitation of caspase-9 followed by immunoblot analysis revealed dose-dependent decrease in associated XIAP (IP caspase-9). (E and F) Densitometric analysis with data expressed relative to vehicle controls. (G) LBW242 concentrations in plasma, brain, and tumor of 3 SK-OV-3 tumor–bearing nude mice after 14 days of daily parenteral dosing of LBW242 (50 mg/kg), measured 4 hours following the final dose. Data represent mean ± SEM for plasma and tumor, and the average of pooled samples for brain. (H) U87 cells were treated in triplicate with the indicated concentrations of LBW242. Relative cell numbers were assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay (see Methods). Data points represent the mean ± SEM.