Inhibition constants (K_i) for the antagonists are determined by addition of the IAP protein constructs to wells containing serial dilutions of the antagonists or the peptide AVPW, and the Hid-FAM probe or AVP-diPhe-FAM probe, as appropriate, in the polarization buffer. Samples are read after a 30-minute incubation. Fluorescence polarization values are plotted as a function of the antagonist concentration, and the IC₅₀ values are obtained by fitting the data to a 4-parameter equation using software. K_i values for the antagonists are determined from the IC₅₀ valued.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Detached cells are washed with phosphate-buffered saline (PBS) and are resuspended in assay media (MDA-MB-231 cells: RPMI1640 supplemented with 10% fetal bovine serum and 2 mM L-glutamine [GlutaMAX-1]) or culture media (HMECs: MEBM^® with MEGM SingleQuots^®). Cells are placed in tissue culture-treated, white-wall or black-wall, clear-bottom, 96-well plates at 1×10⁴ cells/well in a volume of 50 μL. The plates are incubated at 37°C and 5% CO₂ overnight, the media is removed, and GDC-0152 or it's enantiomer are added in assay media. Cells cultured in white-wall, clear-bottom plates are incubated at 37°C and 5% CO₂ for 3 days before cell viability is measured using the CellTiter-Glo^® luminescent cell viability assay kit.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Cells are resuspended in PBS and the cell suspension is mixed 1:1 with Matrigel. The cells (1.5×10⁷) are then implanted subcutaneously into the right flank of 130 female nude mice aged 6-8 weeks. Tumor volumes are calculated. Ten mice with the appropriate mean tumor volume are assigned randomLy to each of six groups. The mean tumor volume±the standard error of the mean (SEM) for all six groups is 168±3 mm³ at the initiation of treatment (Day 0). Mice are dosed 1 or vehicle (PBS) by oral gavage with a dose volume of 4.0 mL/kg. The mice are observed on each day of the study, and tumor volumes and body weights are measured twice each week. Percent tumor growth inhibition is calculated using the formula %TGI=100× (1- Tumor Volumedose/Tumor Volumevehicle).

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Flygare JA, et al. Discovery of a potent small-molecule antagonist of inhibitor of apoptosis (IAP) proteins and clinical candidate for the treatment of cancer (GDC-0152). J Med Chem. 2012 May 10;55(9):4101-13.

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