In RD-ES and A673 Ewing sarcoma cells, ZSTK474 treatment reduced AKT/S6 phosphorylation and increased cleaved PARP. EWSR1-FLI1 protein expression did not attenuate, so the observed response should not be described as fusion-protein depletion. [2]
Class I PI3K inhibition and ATP competition
Biochemical assay · Human PI3Kα, PI3Kβ, PI3Kδ and PI3Kγ enzyme preparations in a PIP2-to-PIP3 HTRF assay [1]
Kinetic analysis supported ATP-competitive inhibition of all four isoforms. PI3Kδ was most potently inhibited. IC50 values are enzyme-assay endpoints measured at 10 µM ATP, not cell-treatment concentrations.
Literature-Reported Treatment Conditions
- Treatment concentration
- Kinetic inhibitor series: α 0, 5, 10, 20 nM; β 0, 10, 20, 40 nM; δ 0, 2.5, 5, 10 nM; γ 0, 5, 10, 25 nM (Figure 1). Separate concentration-response series for IC50 determination.
- Exposure duration
- 20 min enzyme reaction at room temperature; 14 h detection incubation
- Method
- HTRF assay with 10 µM PIP2; ATP 5, 10, 25, 50, 100 µM for kinetics; 10 µM ATP for IC50 determination
- Reported limits
- PI3K assay inhibitor vehicle not specified in the read methods.
Methods and source locations
Compound reported in the paper: ZSTK474
Compound-level literature evidence; does not establish Adooq lot identity, purity or potency.
Materials and Methods: PI3K HTRF assay, enzyme kinetics and IC50 determination; Results: competitive inhibition and isoform comparison; Figures 1–2 and Table 1
Inhibition constants for PI3Kα, β, δ and γ: Competitive kinetic fitting in GraphPad Prism 4; minus-enzyme background control; triplicate experiments, representative of two or three independent experiments (Figure 1 caption). — Results: competitive inhibition; Figure 1
PI3Kα, β, δ and γ activity inhibition at 10 µM ATP: Logistic concentration-response fit. Plus-enzyme/no-inhibitor and minus-enzyme controls; LY294002 comparator. Figure 2: mean ± SD, three independent experiments each in triplicate; Table 1 point estimates have no ± values. — Table 1; Figure 2; Methods: IC50 determination
Weak direct mTOR inhibition in a counterassay
Biochemical assay · Rat brain-derived mTOR; p70S6K–GST phosphorylation at Thr389 [1]
ZSTK474 inhibited mTOR activity by less than 40% even at 100 µM. The study supports weaker direct mTOR inhibition than PI3K inhibition under the reported assay conditions, rather than complete absence of mTOR activity.
Literature-Reported Treatment Conditions
- Treatment concentration
- 0.1, 1, 10 and 100 µM ZSTK474
- Exposure duration
- 30 min at 30°C
- Method
- K-LISA ELISA of p70S6K Thr389 phosphorylation; Figure 3 reports 100 µM ATP
- Vehicle
- DMSO control
- Reported limits
- Methods describe mixing enzyme and ATP-containing buffer volumes; final ATP concentration is not independently clarified beyond the Figure 3 caption report.
Methods and source locations
Compound reported in the paper: ZSTK474
Compound-level literature evidence; does not establish Adooq lot identity, purity or potency.
Methods: K-LISA mTOR assay; Results: inhibition of mTOR; Figure 3
Direct mTOR activity inhibition: 450 nm absorbance minus 595 nm background, normalized to DMSO; 5 µM wortmannin positive control. Mean ± SD of three independent experiments carried out in triplicate. — Results: inhibition of mTOR; Figure 3 and caption
Signaling and PARP cleavage in Ewing sarcoma cells
Cell-based study · RD-ES and A673 human Ewing sarcoma cells [2]
ZSTK474 reduced AKT and S6 phosphorylation and increased cleaved PARP in dose/time experiments. EWSR1-FLI1 protein expression did not attenuate, limiting claims that the effect requires loss of the fusion protein.
Literature-Reported Treatment Conditions
- Treatment concentration
- 0, 1, 3 and 10 µM
- Exposure duration
- 6, 24 and 48 h; baseline 0 h also shown
- Method
- Immunoblotting against phosphorylated and total signaling proteins, cleaved PARP and EWSR1-FLI1
- Reported limits
- ZSTK474 stock solvent, final solvent fraction and biological replicate count for Figure 5A/B are not specified in the read methods/caption.
Methods and source locations
Compound reported in the paper: ZSTK474
Compound-level literature evidence; does not establish Adooq lot identity, purity or potency.
Results: induction of apoptosis and antitumor effects in Ewing sarcoma; effect on EWSR1-FLI1; Figure 5A/B; Methods: Drugs, cell culture and immunoblot analysis
AKT Ser473/Thr308 and S6 Ser235/236 phosphorylation; cleaved PARP: Untreated lanes at each time, total AKT/S6 and α-tubulin loading controls. No band-intensity percentages inferred. — Figure 5A/B; Results: Ewing sarcoma subsection
EWSR1-FLI1 protein expression: Immunoblot comparison with untreated cells and α-tubulin loading control. — Figure 5A/B; Results: effect of ZSTK474 on EWSR1-FLI1
