PP242

In vitro mTOR (FRAP1) kinase assay

Recombinant mTOR was incubated with PP242 at 2-fold dilutions over a concentration range of 50 ~ 0.001 μM in an assay containing 50 mM HEPES, pH 7.5, 1 mM EGTA, 10 mM MgCl2, 0.01% Tween, 10 μM ATP (2.5 μCi of γ-32P-ATP), and 3 μg/mL BSA. Rat recombinant PHAS-1/4EBP1 (2 mg/mL) was used as a substrate. Reactions were terminated by spotting onto nitrocellulose, which was washed with 1 M NaCl/1% phosphoric acid (approximately 6 times, 5 ~ 10 mins each). Sheets were dried and the transferred radioactivity was quantitated by phosphorimaging. IC50 value was calculated by fitting the data to a sigmoidal dose-response curve using the Prism software package.

Cell lines

AML cells

Preparation method

The solubility of this compound in DMSO is ≥61.6mg/mL. General tips for obtaining a higher concentration: Please warm the tube at 37 °C for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20 °C for several months.

Reaction Conditions

0.1, 0.2, 0.6, 1.7 or 5.0 μM; 48 hrs

Applications

In primary AML cells cultured alone or cocultured with stromal cells, PP242 dose-dependently induced apoptosis. In addition, PP242 induced apoptosis in CD34＋ AML progenitor cells cultured under the above-mentioned conditions.

Animal models

Ba/F3-ITD/luc/GFP mouse model of leukemia

Dosage form

60 mg/kg, p.o.; every other day

Applications

At the dose of 60 mg/kg, PP242 reduced leukemia burden. In addition, The anti-leukemia effect of PP242 was greater than that of Rapamycin at the dose of 0.5 mg/kg (the tolerable dose that was previously shown to inhibit mTOR signaling).

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

References:

[1]. Apsel B, Blair JA, Gonzalez B, Nazif TM, Feldman ME, Aizenstein B, Hoffman R, Williams RL, Shokat KM, Knight ZA. Targeted polypharmacology: discovery of dual inhibitors of tyrosine and phosphoinositide kinases. Nat Chem Biol. 2008 Nov;4(11):691-9.

[2]. Zeng Z, Shi YX, Tsao T, Qiu Y, Kornblau SM, Baggerly KA, et al. Targeting of mTORC1/2 by the mTOR kinase inhibitor PP242 induces apoptosis in AML cells under conditions mimicking the bone marrow microenvironment. Blood 2012,120:2679-2689.