GDC-0349 

The kinase activity of mTOR enzyme is assessed by incubating purified recombinant enzyme (mTOR(1360-2549)+GBL, prepared in-house) in a reaction mixture containing ATP, MnCl₂, and a fluorescently labeled mTOR substrate, e.g., GFP-4E-BP1. The reaction is stopped by an addition of a Terbium-labeled phospho-specific antibody, e.g., Tb-labeled anti-p4E-BP1 T37/T46, EDTA, and TR-FRET buffer solution. Product formation is detected by way of time-resolved fluorescence resonance energy transfer (TR-FRET), which occurs when the phosphorylated substrate and labeled antibody are in close proximity due to phosphospecific binding. Enzymatic activity is measured as an increase in TR-FRET signal using a Perkin Elmer Envision plate reader. The assay is performed in a 384-well Proxiplate Plus using the following protocol: Compound activity is tested in 10 point dose curves starting at the highest final concentration of 10 uM. They are serially diluted in 100% DMSO prior to further dilution with assay buffer. The reaction mixture (8μL) containing 0.25 nM mTOR+GBL enzyme, 400 nM GFP-4E-BP1, 8 uM ATP, 50 mM Hepes pH 7.5, 0.01% Tween 20, 10 mM MnCl₂, 1 mM EGTA, 1 mM DTT, 1% DMSO (±compound) is incubated at room temperature for 30 minutes. 8 μL of solution containing 2 nM Tb-anti-p4E-BP1 antibody & 10 mM EDTA diluted TR-FRET buffer is then added and incubated for 30 minutes to stop the reaction. The plate is scanned with the Envision plate reader. K_i values are calculated in Assay Explorer using the Morrison ATP-competitive tight binding equation for K_i apparent determination^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[1]
Human breast cancer cells (MCF7 neo/HER2; modified ATCC variant) are implanted subcutaneously into the mammary fat pad of female NCR nude mice (5×10⁶ cells/100 uL of 1:1 mixture of Hank’s Balanced Salt Solution (HBSS)/Matrigel). To support estrogen dependent growth, recipient animals are pre-implanted with 0.36 mg estrogen pellets. Tumors are monitored until they reached a mean tumor volume of approximately 200-225 mm³, then similarly sized tumors are randomly assigned to treatment cohorts (n=5-10). Human 786-O renal adenocarcinoma cells are implanted subcutaneously into the right hind flank of female nu/nu mice (1×10⁷ cells/200 uL in 1:1 PBS/Matrigel). Tumors are monitored until they reached a mean tumor volume of approximately 205 mm³, then similarly sized tumors are randomly assigned to treatment cohorts (n=10). Human prostate cancer NCI-PC3 cells are resuspended in Hank’s Balanced Salt Solution and implanted subcutaneously into the right hind flanks of 120 female NCR nude mice. Each mouse is injected with 5×10⁶ cells. Tumors are monitored until they reached a mean tumor volume of approximately 200-250 mm³. The dimesylate salt of GDC-0349 is dosed daily or every third day by oral gavage (100 uL dose /25 gm animal) for 14-21 days.Tumor volume and body weight measurements are collected twice weekly. Tumor volumes are calculated.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Pei Z, et al. Discovery and Biological Profiling of Potent and Selective mTOR Inhibitor GDC-0349. ACS Med Chem Lett. 2012 Nov 29;4(1):103-7.  [Content Brief]