Kinase experiment:

The linear phase of each kinetic reaction is defined at the respective enzyme amount (0.05, 0.1, 0.12 and 1 μg/mL for PI3Kα, PI3β, PI3δ and PI3γ, respectively) and reaction time (20 min). PI3K activity is assayed at various concentrations of ATP (5, 10, 25, 50, 100 μM) in the presence of increasing concentrations of ZSTK474. A Lineweaver-Burk plot is developed by plotting 1/v (the inverse of v, where v is obtained by subtracting the HTRF signal of the kinase test sample from the HTRF signal of the minus-enzyme control) versus 1/[ATP] (the inverse of the ATP concentration). For the minus-enzyme control, PIP2 is incubated with ATP in the absence of kinase. To determine the Ki value (inhibition constant) of ZSTK474 for each PI3K isoform, the slope of the respective Lineweaver-Burk plot is replotted against the ZSTK474 concentration. The Ki values are calculated by analysis using GraphPad Prism 4[1].

Animal experiment:

Mice[2] Mice are randomly assigned to receive different doses of ZSTK474 (50, 100, 200, and 300 mg/kg) to determine the optimum dose; in our experiment, the optimum dose is 200 mg/kg. Then mice are randomly assigned to one of three groups: a sham-operated group (phosphate-buffered saline, PBS); a control group (MCAO+PBS); a ZSTK474-treated group (MCAO+ZSTK474). In the ZSTK474-treated group, the mice are given the optimum dose of 200 mg/kg ZSTK474. In the sham-operated group and control group, mice are given an equivalent volume of PBS. All mice receive that same dose daily via oral gavage beginning at 6 h after the onset of focal ischemia and continuing for two more days, i.e., for a total of 3 days.

ZSTK474 strongly inhibits the growth of tumor cells by direct inhibition activity of PI3K, which includes all isoforms of class I PI3K with IC50 values of 16 nM, 44 nM, 49 nM, and 4.6 nM for PI3Kα, PI3-Kβ, PI3-Kγ, PI3-Kδ, respectively.[1]
PI3Ks are a family of enzymes, which phosphorylate the 3‘- OH position of the inositol ring of phosphoinositides.PI3Ks are divided into three classes based on the structural features and in vitro lipid substrate specificity. The three class-Ia PI3-ks (p110 α / β / δ ) and the sole class-Ib PI3K (p110 γ ) couple growth factor receptors and G-protein-coupled receptors, respectively, to a wide range of downstream pathways. Signal transduction via the PI3K/Akt pathway is essential for regulating cellular activities, such as proliferation, survival, migration, motility and tumorigenesis, in diverse cell types.[2]
ZSTK474 is a potent ATP-competitive pan class I PI3K inhibitor, which has high selectivity over other classes of PI3K and protein kinases. ZSTK474 also inhibited the bone-resorbing activity of mature osteoclasts. Experiments revealed that 0.1μM of ZSTK474 completely prevented pit formation by osteoclasts. Moreover, the IC50 values for PI3Kα, PI3-Kβ, PI3-Kγ, PI3-Kδ are 16 nM, 44 nM,49 nM, and 4.6 nM, respectively.[1]
Melanoma cells injected BDF 1 mice were orally administrated ZSTK474 daily at 100, 200, or 400 mg/kg of body weight from days 0 to 13. By measuring the volume of tumor, the outcomes showed that ZSTK474 administered orally to mice had strong antitumor activity against human cancer xenografts without toxic effects in vital organs. Akt phosphorylation was decreased in xenograft tumors after oral administration of ZSTK474. [3]
References:
1.Toyama S, Tamura N, Haruta K, et al. Research article Inhibitory effects of ZSTK474, a novel phosphoinositide 3-kinase inhibitor, on osteoclasts and collagen-induced arthritis in mice[J]. 2010.
2.Marone R, Cmiljanovic V, Giese B, et al. Targeting phosphoinositide 3-kinase—moving towards therapy[J]. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 2008, 1784(1): 159-185.
3.Yaguchi S, Fukui Y, Koshimizu I, et al. Antitumor activity of ZSTK474, a new phosphatidylinositol 3-kinase inhibitor[J]. Journal of the National Cancer Institute, 2006, 98(8): 545-556.