PF-04691502

Kinase experiment:

The biochemical protein kinase assays for class I PI3K and mTOR are assessed. 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 contained 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 DMSO 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[1].

Cell experiment:

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. U87MG and SKOV3 cells are plated in 96-well plates overnight and caspase-3/caspase-7 activity is assessed with the Caspase-Glo 3/7 Assay Kit[1].

Animal experiment:

Mice[1] Female nu/nu mice (6-8 weeks old) are used. Tumor cells for implantation are harvested and resuspended in serum-free medium mixed with matrigel (1:1). SKOV3, U87MG, or NSCLC cells (2.5-4×106) are implanted subcutaneously into the hind flank region. Treatment started when average tumor size is 100 to 200 mm3. PF-04691502 is formulated in 0.5% methylcellulose in water suspension and given orally once a day. Animal body weights and tumor volumes are measured every 2 to 3 days. Tumor volume is determined with Vernier calipers and calculated. Percentage of tumor growth inhibition (TGI) is calculated. Data are presented as mean±SE. Comparisons between treatment groups and vehicle group are done using 1-way ANOVA by Dunnett's tests. Student's t test is used to determine the P value for the comparison of 2 groups.

References:

[1]. Yuan J, et al. PF-04691502, a potent and selective oral inhibitor of PI3K and mTOR kinases with antitumor activity. Mol Cancer Ther. 2011 Nov;10(11):2189-99.