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Biological Activity
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Description
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PF-04691502 is an ATP-competitive PI3K/mTOR dual inhibitor with IC50 of 32 nM. |
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Targets
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mTORC1 |
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IC50 |
32 nM [1] |
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In Vitro
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PF-04691502 potently inhibits recombinant class I PI3K and mTOR in biochemical assays and suppresses transformation of avian fibroblasts mediated by wild-type PI3K γ, δ, or mutant PI3Kα. In PIK3CA-mutant and PTEN-deleted cancer cell lines, PF-04691502 reduces phosphorylation of AKT T308 and AKT S473 (IC(50) of 7.5-47 nM and 3.8-20 nM, respectively) and inhibits cell proliferation (IC(50) of 179-313 nM). PF-04691502 inhibits mTORC1 activity in cells as measured by PI3K-independent nutrient stimulated assay, with an IC(50) of 32 nM and inhibits the activation of PI3K and mTOR downstream effectors including AKT, FKHRL1, PRAS40, p70S6K, 4EBP1, and S6RP. Short-term exposure to PF-04691502 predominantly inhibits PI3K, whereas mTOR inhibition persists for 24 to 48 hours. PF-04691502 induces cell cycle G(1) arrest, concomitant with upregulation of p27 Kip1 and reduction of Rb. [1] |
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In Vivo
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Antitumor activity of PF-04691502 is observed in U87 (PTEN null), SKOV3 (PIK3CA mutation), and gefitinib- and erlotinib-resistant non-small cell lung carcinoma xenografts. [1] PF-04691502 inhibits tumor growth at 7 days by 72%. FDG-PET imaging revealed that PF-04691502 reduces glucose metabolism dramatically. Tissue biomarkers of PI3K/mTOR pathway activity, p-AKT (S473), and p-RPS6 (S240/244), are also dramatically inhibited following PF-04691502 treatment. [2] |
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Clinical Trials
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PF-04691502 is currently in Phase II clinical trials in patients with recurrent endometrial cancer and Breast Cancer. A Phase II clinical trials in patients with solid tumors has been completed. |
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Features
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Combination Therapy
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Description
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Combination with PF-04691502, PD-0325901 induces striking tumor regression, apoptosis associated with upregulation of Bim and downregulation of Mcl-1, and greatly improves duration of survival. [2] PD-0325901 at 1.5 mg/kg SID and in combination with PF-04691502 (7.5 mg/kg; SID) significantly inhibits growth of H460 (carry Kras and PIK3CA mutations) orthotopic lung tumors. [3] |
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Protocol
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Kinase Assay
[1]
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| Kinase Assay |
The fluorescence polarization assay for ATP competitive inhibition is done as follows: mPI3Kα dilution solution (90 nM) is prepared in fresh assay buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 5 mM DTT, 0.05% CHAPS) and kept on ice. The enzyme reaction contains 0.5 nM mouse PI3Kα (p110α/p85α complex purified from insect cells), 30 μM PIP2, PF-04691502 (0, 1, 4, and 8 nM), 5 mM MgCl2, and 2-fold serial dilutions of ATP (0–800 μM). Final dimethyl sulfoxide is 2.5%. The reaction is initiated by the addition of ATP and terminated after 30 minutes with 10 mM EDTA. In a detection plate, 15 uL of detector/probe mixture containing 480 nM GST-Grp1PH domain and 12 nM TAMRA tagged fluorescent PIP3 in assay buffer is mixed with 15 uL of kinase reaction mixture. The plate is shaken for 3 minutes, and incubated for 35 to 40 minutes before reading on an LJL Analyst HT. |
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Cell Assay
[1]
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| Cell Lines |
BT20, U87MG, and SKOV3 cells |
| Concentrations |
0-3 mM |
| Incubation Time |
3 days |
| Methods |
BT20, U87MG, and SKOV3 cells are plated at 3,000 cell/well in 96-well culture plates in growth medium with 10% FBS. Cells are incubated overnight and treated with DMSO (0.1% final) or serial diluted compound for 3 days. Resazurin is added to 0.1 mg/mL. Plates are incubated at 37 °C in 5% CO2 for 3 hours. Fluorescence signals are read as emission at 590 nm after excitation at 530 nm. IC50 values are calculated by plotting fluorescence intensity to drug concentration in nonlinear curves. |
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Animal Study
[2]
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| Animal Models |
LSL-KrasG12D heterozygous mice (B6.129-Kras tm4Tyj) and Ptendel mice (c;129S4-Pten tm1Hwu/J), Orthotopic transplant of ovarian tumors |
| Formulation |
0.5% methylcellulose |
| Doses |
daily at either 7.5 or 10 mg/kg |
| Administration |
Administered via oral gavage |
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References |
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[1] Yuan J, Mol Cancer Ther, 2011, 10(11), 2189-2199
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[2] Kinross KM, Mol Cancer Ther, 2011, 10(8), 1440-1449
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[3] Simmons BH, Cancer Chemother Pharmacol, 2012, 70(2), 213-220
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