GNE-317

PI3K; mTOR

GNE-317 is not a substrate of P-gp or BCRP transporter in transfected Madin-Darby canine kidney (MDCK) cells. In mouse plasma, GNE-317's binding to plasma proteins shows a 14.9% free fraction, while its binding to brain tissues is higher at 5.4% free. Cytostasis is visible in GNE-317, but U87 cells do not die. [1]

GNE-317 (40 mg/kg, p.o.) significantly inhibits the PI3K pathway in the mouse brain, 40% to 90% of the pAkt and pS6 signals being suppressed up to 6 hours after administration. In the U87 and GS2 orthotopic models, GNE-317 (40 mg/kg, p.o.) effectively inhibits tumor growth by 90% and 50%, respectively. GNE-317 (30 mg/kg, p.o.; 40 mg/kg the first two weeks) increases the lifespan of mice from a median of 55.5 to 75 days in the GBM10 tumor model. [1]

Compounds are serially diluted (3-fold in 100% DMSO) across a 384-well polypropylene mother plate from column 1 to column 12 and column 13 to column 24, to yield 11 concentrations for GSK2126458. Columns 6 and 18 contain only DMSO. Once titrations are made, 0.05μL is transferred to a 384-well low-volume assay plate. This assay plate contains three pharmacological controls (known PI3K inhibitors) and 3 assay controls: (1) Enzyme without inhibitor; (2) Buffer minus enzyme, and (3) Buffer minus enzyme plus native PIP3. DMSO is stamped into all wells of columns 6 and 18. PIP3 is added at 40 μM in 1X Reaction buffer (1μL of 200 μM PIP3) to alternating rows of column 18 (wells 18 B, D, F, H, J, L, N, P). The no-enzyme control reactions are run in wells 18 A, C, E, G, I, K, M, O (0.1μL of 100% DMSO). The PI3-Kinase profiling assay is optimized using the HTRF kit. The assay kit contains seven reagents: 1) 4X Reaction Buffer; 2) native PIP2 (substrate); 3) Stop A (EDTA); 4) Stop B (Biotin-PIP3); 5) Detection Mix A (Streptavidin-APC); 6) Detection Mix B (Eu-labeled Anti-GST plus GST-tagged PHdomain); 7) Detection Mix C (KF). PI3Kinase Reaction Buffer is prepared by diluting the stock 1:4 with de-ionized water. Freshly prepared DTT is added at a final concentration of 5 mM on the day of use. Enzyme addition and compound pre-incubation are initiated by the addition of 2.5μL of PI3K (at twice its final concentration) in 1X reaction buffer to all wells using a Multidrop Combi. Plates are incubated at room temperature for 15 minutes. Reactions are initiated by addition of 2.5μL of 2X substrate solution (PIP2 and ATP in 1X reaction buffer) using a Multidrop Combi. Plates are incubated at room temperature for one hour. Reactions are quenched by the addition of 2.5μL of stop solution (Stop A and Stop B pre-mixed at a ratio of 5:1, respectively) to all wells using the Multidrop Combi. The quenched reactions are then processed to detect product formation by adding 2.5μL of Detection Solution to all wells using the Mulitdrop Combi (Detection mix C, Detection mix A, and Detection mix B combined together in an 18:1:1 ratio, i.e.: for a 6000 μL total volume, mix 5400 μL Detection mix C, 300μL Detection mix A, and 300 μL Detection mix B. Note: this solution should be prepared 2 hours prior to use). Following a one hour incubation in the dark, the HTRF signal is measured on the Envision plate reader set for 330nm excitation and dual emission detection at 620nm (Eu) and 665nm (APC).

GL261 is an aggressive C57BL/6J-derived glioma line. Green fluorescent protein (GFP) and luciferase (Luc), which come from different plasmids, are both transfected into this cell line. The resulting monoclonal GL261-GFP-Luc cells are cultured at 5% oxygen and kept alive in Dulbecco's modified Eagle's medium supplemented with 10% FBS and Penicillin/Streptomycin (100 U/mL). For cell selection, 4 mg/mL G418 and 4 mg/mL Puromycin were used. A 96-well format is used for the cellular viability assays, with 2000 cells plated in each well under culture conditions. A 48-hour incubation period with the drug or a vehicle is followed by an MTS assay to determine the viability of the cells. A Synergy Mx automated plate reader is used to measure absorbance at 490 nm to assess viability and at 650 nm to take background into account. The percent survival is calculated by normalizing the numerical data from drug-treated wells to the data from vehicle-treated wells[1].

U87 and GS2 orthotopic tumor-bearing mice
40 mg/kg
p.o.

[1]. Clin Cancer Res. 2012 Nov 15;18(22):6239-48.

C19H22N6O3S

414.4814

414.1474

C, 55.06; H, 5.35; N, 20.28; O, 11.58; S, 7.73

1394076-92-6

1394076-92-6

Solid powder

CC1=C(SC2=C1N=C(N=C2N3CCOCC3)C4=CN=C(N=C4)N)C5(COC5)OC

XOZLHJMDLKDZAL-UHFFFAOYSA-N

InChI=1S/C19H22N6O3S/c1-11-13-14(29-15(11)19(26-2)9-28-10-19)17(25-3-5-27-6-4-25)24-16(23-13)12-7-21-18(20)22-8-12/h7-8H,3-6,9-10H2,1-2H3,(H2,20,21,22)

5-[6-(3-methoxyoxetan-3-yl)-7-methyl-4-morpholin-4-ylthieno[3,2-d]pyrimidin-2-yl]pyrimidin-2-amine

GNE317; GNE-317; GNE 317

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~47 mg/mL warming (~113.4 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL