The Schematic representation of approach used to derive non-peptide SMAC mimics.

(A) Cell based activity of compounds AVPI and compound 3. MDA-MB-231 breast cancer cells were cultured in 10 % FBS DMEM media in 96-well plates overnight and followed by dosed with AVPI, compound 3, or vehicle at varying concentrations in serum free DMEM media. After 48 hours treatment, the effects on cells proliferation were evaluated by the ATPLite assay. Cell survival was expressed as percentage of control ± s.e.m., compared to DMSO control, from two independent experiments, each having fours wells per drug concentration. (B) Similar amounts of wild type and XIAP knockout (−/−) mouse embryonic fibroblasts (MEF) cells were cultured 24 hours in 10 % FBS RMPI-1640 media in white 96-well plate before treated by compound 3 or DMSO (control) at varying concentrations. After 24 hours treatment in 10 % FBS RMPI-1640 media, the effects on cells proliferation were evaluated by the ATPLite assay. Cell survival was expressed as percentage of control ± s.e.m., compared to DMSO control, from three independent experiments, each having fours wells per drug concentration. (C) Caspase-3 activation assay. After cultured in 10 % FBS DMEM media overnight, MDA-MB 231 breast cancer cells were treated by compound 3 at various concentrations, DMSO vehicle, or staurosporine 100 nM. After 24 hours culture, the cells were lysed and caspase-3 activities of the lysates were detected by meso scale discovery (MSD) caspase-3 assay kit detecting cleaved versus total caspase-3. Staurosporine was used to indicate the efficiency of the assay. (D) Growth inhibition of MDAMB-231 cells by compound 3 alone or in combination with TRAIL. Cells were cultured in 10 % FBS DMEM media in white 96-well plates overnight and than treated with TRAIL or compound 3 at indicated concentrations alone for 20 and 24 hours, respectively. When used together, compound 3 was added 4 hours before TRAIL (total incubation time, 24 hours). Cell viability (%) was determined by ATPLite assay and was expressed as percentage of control ± s.e.m., compared to DMSO control, from two independent experiments, each having fours wells per drug concentration.

(A) Superposition between the X-ray structure of AVPI (magenta) in complex with the Bir3 domain of XIAP (surface representation) and the docked conformation of compound 3; The Ala and Val residues occupy the first of two sub-pockets (P1 and P2) on the surface of the domain, while the Ile occupies the second. (B) Chemical shift mapping data with ¹⁵N Bir3 and compound 3. The surface of the Bir3 domain of XIAP is colored according to the shifts induced by compound 3: red > orange> yellow ≫ white = no shifts). (C) Fractional changes (p) of chemical shifts in Bir3 as a function of ligand concentration. The experimental data were fitted to the non-linear equation as described in the methods resulting in K_d = 1.2 µM. (D) Calorimetric titration of Bir3 with compound 3. The top panel shows raw data in power versus time. The bottom panel shows data after peak integration, subtraction of blank titration data, and concentration normalization. The solid line is the fit to a single binding site model. K_d ~ 2.5 µM.