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 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20 ℃ 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.

PP242, a novel potent and selective mTOR inhibitor, can inhibit the active site of mTOR kinase in both mTORC1 and mTORC2 with IC50 of 8 nM.
The mammalian target of rapamycin (mTOR), a serine-threonine kinase, is present in two protein complexes, mTORC1 and mTORC2, that have distinct subunit composition, substrates and mechanisms of activation.
Treatment of PP242 was shown to lead to the death of certain mouse and human leukemia cells [1]. In primary AML cells and CD34+ progenitor cells, PP242 can inhibit the activity of mTOR and their downstream targets, therefore inducing cell apoptosis [2].
The component has also been used to study the role of COX-2 in vivo. In models of acute leukemia harboring the Philadelphia chromosome (Ph) translocation, PP242 can delay the onset of leukemia and augment the effects of front-line tyrosine kinase inhibitors more effectively, thereby suppressing the expansion of leukemia [1]. In addition, PP242 was shown to suppresse leukemia progression in a murine leukemia model which was driven by mutated FLT3 with constitutive activation of mTOR [2].
References:
1.Janes MR, Limon JJ, So L, Chen J, Lim RJ, Chavez MA, et al. Effective and selective targeting of leukemia cells using a TORC1/2 kinase inhibitor. Nat Med 2010,16:205-213.
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.