Antitumor Activity of ZSTK474, a New Phosphatidylinositol 3-Kinase Inhibitor
J. Natl. Cancer Inst. Yaguchi et al.
98: 545
Supplementary Data
Files in this Data Supplement:
- Supplementary Table 1 -
Effect of ZSTK474 on 139 protein kinases. The concentration of ZSTK474 used was 30 μM. ATP was present at 10 μM in all assays. Results are shown as percentage of the kinase activity in control reaction mixtures without ZSTK474. The assay protocol, cited at http://www.upstate.com/features/kp_protocols.asp, is shown below.
- Supplementary Fig. 1 -
Effect of ZSTK474 on PI3Kβ, PI3Kγ, and PI3Kδ activity. PI3K assay for recombinant p110, the catalytic subunit of PI3K, was described previously by Gray et al. (1). In a reaction volume of 20 μL, human p110 isoforms β, -γ, or -δ (Upstate) were incubated with 10 μM phosphatidylinositol-4,5-bisphosphate and 10 μM ATP in assay buffer for 30 minutes at room temperature. Stop buffer (5 μL) containing EDTA and biotinylated PIP3 was added, followed by 5 μL of detection buffer containing europium-labeled anti–glutathione S-transferase (GST) monoclonal antibody, GST-tagged general receptor for phosphoinositides (GRP1) PH domain, and streptavidin allophycocyanin. Plus and minus kinase control wells were also included. The plate was read in the time-resolved fluorescence mode, and the homogenous time-resolved fluorescence (HTRF) signal was determined according to the following formula: HTRF = 10,000 × (emission at 665 nm/emission at 620 nm). The PI3Kβ, PI3Kγ, or PI3Kδ activity was inhibited by ZSTK474 in a dose-dependent manner. The concentrations that inhibited 50% of PI3K activity (IC50) of ZSTK474 against PI3Kβ, PI3Kγ, or PI3Kδ were 17, 53, and 6 nM, respectively.
- (1) Gray A, Olsson H, Batty IH, Priganica L, Peter Downes C. Nonradioactive methods for the assay of phosphoinositide 3-kinases and phosphoinositide phosphatases and selective detection of signaling lipids in cell and tissue extracts. Anal Biochem 2003;313:234–45.
- Supplementary Protocol 1
