Biological Activity
PF-04691502 is an ATP-competitive PI3K/mTOR dual inhibitor, which potently inhibited recombinant class I PI3K and mTOR in biochemical assays and suppressed transformation of avian fibroblasts mediated by wild-type PI3K γ, δ, or mutant PI3Kα. PF-04691502 inhibited mTORC1 activity in cells as measured by PI3K-independent nutrient stimulated assay, with an IC(50) of 32 nmol/L and inhibited the activation of PI3K and mTOR downstream effectors including AKT, FKHRL1, PRAS40, p70S6K, 4EBP1, and S6RP. PF-04691502 reduced phosphorylation of AKT T308 and AKT S473 (IC(50) of 7.5-47 nmol/L and 3.8-20 nmol/L, respectively) and inhibited cell proliferation (IC(50) of 179-313 nmol/L) in PIK3CA-mutant and PTEN-deleted cancer cell lines. PF-04691502 induced cell cycle G(1) arrest, concomitant with upregulation of p27 Kip1 and reduction of Rb. PF-04691502 has entered phase II clinical trials.
Product Citations
Customer Product Validations & Biological Datas
. pf-04691502, figure 7..jpg) |
Source |
Mol Ther Nucleic Acids.(2016). PF-04691502, Figure 7. (Abmole Bioscience, Houston, TX) |
| Method |
|
| Cell Lines |
A549 and H460 cell |
| Concentrations |
5 nmol/l to 5 µmol/l for A549, and 5 nmol/l to 100 µmol/l for H460 cells |
| Incubation Time |
72 h |
| Results |
In A549 cells PF0 and siRNA-V combination revealed a moderate synergism at 6 and 12 nmol/l siRNA-V concentration, and slight synergism at 18 nmol/l, with CI values between 0.62 and 0.88 (Figure 7a, Table 1). In H460 cells, PF0 and siRNA-V showed moderate synergism at 4 nmol/l siRNA-V concentration, synergism at 6 nmol/l, and slight synergism at 12 nmol/l, with CI values between 0.51 and 0.81 (Figure 7b, Table 1). |
. pf-04691502, figure 6..jpg) |
Source |
Mol Ther Nucleic Acids.(2016). PF-04691502, Figure 6. (Abmole Bioscience, Houston, TX) |
| Method |
tube formation assays |
| Cell Lines |
human umbilical vein endothelial cells (HUVEC) |
| Concentrations |
100 nmol/l |
| Incubation Time |
6 h |
| Results |
PF0 treatment showed poor organization of HUVEC tube-like structures and a nonsignificant (P < 0.05) decrease in segments length (75.63 ± 13.71%) compared to control (Figure 6e). |
. pf-04691502, figure 5..jpg) |
Source |
Mol Ther Nucleic Acids.(2016). PF-04691502, Figure 5. (Abmole Bioscience, Houston, TX) |
| Method |
wound healing assay |
| Cell Lines |
A549 and H460 cell |
| Concentrations |
|
| Incubation Time |
|
| Results |
A549 and H460 cells also showed significant (P < 0.05) differences between PF0 (200 nmol/l for A549 and 2,000 nmol/l for H460) treated and nontreated cells (Figure 5c,d). A549 cells showed that after 16 hours of 50 nmol/l siRNA-V and 200 nmol/l PF0 treatment wound closure was 18.48 ± 1.60% comparing with 47.24 ± 2.09 of the control treated cells. |
. pf-04691502, figure 4..jpg) |
Source |
Mol Ther Nucleic Acids.(2016). PF-04691502, Figure 4. (Abmole Bioscience, Houston, TX) |
| Method |
Inhibition of anchorage-dependent colony formation |
| Cell Lines |
A549 and H460 cells |
| Concentrations |
200 nmol/l for A549 and 2,000 nmol/l for H460 |
| Incubation Time |
|
| Results |
The reduction was more remarkable after PF0 was combined with siRNA-V showing a significant (P < 0.05) decrease in colony formation to a 29.83 ± 8.11% as compared with control. For A549 cells, the combination resulted in a significant (P < 0.05) colony formation reduction when compared with PF0 treatment (Figure 4c). The relative colony-forming ability of H460 cells was reduced after PF0 treatment to 61.86 ± 20.48% and after siRNA-V treatment to 73.21 ± 1.97% as compared with controls (Figure 4b,d). PF0 and siRNA-V combination showed a significant (P < 0.05) decrease in relative colony formation to 7.97 ± 9.20% as compared with control groups. |
. pf-04691502, figure 3..jpg) |
Source |
Mol Ther Nucleic Acids.(2016). PF-04691502, Figure 3. (Abmole Bioscience, Houston, TX) |
| Method |
VEGF silencing effect analysis by quantitative real-time PCR |
| Cell Lines |
A549 and H460 cells |
| Concentrations |
200 nmol/l for A549 and 2,000 nmol/l for H460 |
| Incubation Time |
48 h |
| Results |
In A549 cells, the siRNA-V treatment showed a significant (P < 0.05) decrease of 62.58 ± 16.60% in VEGF gene expression compared to control (Figure 3a). siRNAV and PF0 combination treatment showed a significantly (P < 0.01) decreased VEGF expression of 75.06 ± 13.21% compared to control. In H460 cells, we observed a significant (P < 0.001) decrease in the relative VEGF mRNA expression after siRNA-V transfection by 92.22 ± 3.10% as well as after the combination treatment 69.59 ± 7.71% compared to control cells (Figure 3b). |
. pf-04691502, figure 2..jpg) |
Source |
Mol Ther Nucleic Acids.(2016). PF-04691502, Figure 2. (Abmole Bioscience, Houston, TX) |
| Method |
siRNA-V and PF0 combination cytotoxicity assays |
| Cell Lines |
A549, H460 cells |
| Concentrations |
5 to 500 nmol/l for A549, and 10 to 5,000 nmol/l for H460 cells |
| Incubation Time |
24, 48, and 72 h |
| Results |
We observed a significant (P < 0.05) decrease in relative cell viability for the combination compared to singleagent treatments (Figure 2a,b). The combination cytotoxicity assays demonstrated that siRNA-V potentiates the anticancer activity of PF0 against A459 and H460 cells. |
. pf-04691502, figure 1..jpg) |
Source |
Mol Ther Nucleic Acids.(2016). PF-04691502, Figure 1. (Abmole Bioscience, Houston, TX) |
| Method |
Dose response cytotoxicity analysis |
| Cell Lines |
A549 cells |
| Concentrations |
5 to 5,000 nmol/l |
| Incubation Time |
24, 48, and 72 h |
| Results |
In H460 cells, PF0 (5 nmol/l to 200 µmol/l) or siRNA-V (5 to 200 nmol/l) cytotoxicity was also found to be dose and time dependent (Figure 1d,e and Supplementary Figure S2). IC50 values for PF0 in H460 cells were 1,965.5 ± 131.7 nmol/l; 1,080.1 ± 307.1 nmol/l; 936.7 ± 174.6 nmol/l after 24, 48, and 72 hours of treatment respectively (Figure 1d and Supplementary Figure S2a). |
Protocol (for reference only)
| Cell Experiment |
|---|
| Cell lines |
BT20, U87MG, and SKOV3 cells |
| Preparation method |
Cell proliferation assays.
BT20, U87MG, and SKOV3 cells were plated at 3,000 cell/well in 96-well culture plates in growth medium with 10% FBS. Cells were incubated overnight and treated with DMSO (0.1% final) or serial diluted compound for 3 days. Resazurin was added to 0.1 mg/mL. Plates were incubated at 37°C in 5% CO2 for 3 hours. Fluorescence signals were read as emission at 590 nm after excitation at 530 nm. IC50 values were calculated by plotting fluorescence intensity to drug concentration in nonlinear curves. |
| Concentrations |
0~1000 nM |
| Incubation time |
3 days |
| Animal Experiment |
|---|
| Animal models |
SKOV3, U87MG, or NSCLC cells tumour xenograft models in Female nu/nu mice (6–8 weeks old) |
| Formulation |
0.5% methylcellulose in water suspension |
| Dosages |
0.5, 1, 5, and 10 mg/kg once daily |
| Administration |
orally |
Chemical Information
| Molecular Weight |
425.48 |
| Formula |
C22H27N5O4 |
| CAS Number |
1013101-36-4
|
| Solubility (25°C) |
DMSO 13 mg/mL |
| Storage |
Powder -20°C 3 years ; 4°C 2 years
In solvent -80°C 6 months ; -20°C 1 month
|
Conversion of different model animals based on BSA (PMID: 27057123)
| Species |
Mouse |
Rat |
Rabbit |
Guinea pig |
Hamster |
Dog |
| Weight (kg) |
0.02 |
0.15 |
1.8 |
0.4 |
0.08 |
10 |
| Body Surface Area (m2) |
0.007 |
0.025 |
0.15 |
0.05 |
0.02 |
0.5 |
| Km factor |
3 |
6 |
12 |
8 |
5 |
20 |
| Animal A (mg/kg) = Animal B (mg/kg) multiplied by |
Animal B Km
|
| Animal A Km |
For example, to modify the dose of Compound A used for a mouse (20 mg/kg) to a dose based on the BSA for a rat, multiply 20 mg/kg by the Km factor for a mouse and then divide by the Km factor for a rat. This calculation results in a rat equivalent dose for Compound A of 10 mg/kg.
References
[1] Yuan J, et al. Mol Cancer Ther. PF-04691502, a potent and selective oral inhibitor of PI3K and mTOR kinases with antitumor activity.
