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PRAS40 phosphorylation in vivo coincided with inhibition of xeno-
graft tumor growth, albeit at high plasma concentrations where
inhibition of off-target kinases involved in cell proliferation or reg-
ulation may have contributed to tumor growth inhibition. As AKT
remains an attractive anti-cancer target, AKT inhibitors such as
the ones presented here may be important for further
development.
15.
Ishiyama, T.; Murata, M.; Miyaura, N. J. Org. Chem. 1995, 60, 7508.
16. U-87 MG cells were seeded on a 96-well cell culture plate at 6000 cells/well in
5% FBS, and treated with 36 in threefold serial dilutions for three days. Cell
viability was measured by alamarBlue cell staining (Invitrogen, DAL1100).
17. Lippa, B.; Pan, G.; Corbett, M.; Li, C.; Kauffman, G. S.; Pandit, J.; Robinson, S.;
Wei, L.; Kozina, E.; Marr, E. S.; Borzillo, G.; Knauth, E.; Barbacci-Tobin, E. G.;
Vincent, P.; Troutman, M.; Baker, D.; Rajamohan, F.; Kakar, S.; Clark, T.; Morris,
J. Bioorg. Med. Chem. Lett. 2008, 18, 3359.
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10. U-87 MG cells in 5% FBS were incubated with inhibitors in threefold serial
dilutions for 1 h at 37 °C. The cells were lysed and PRAS40 phosphorylation was
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20. Mice (n = 3) were dosed with compounds 9 or 36 (30 mg/kg po) and after 6 h,
AKT signaling was stimulated in mouse liver by treatment with HGF via tail
vein injection. Five minutes post-HGF treatment, the mice were sacrificed and
livers were harvested for quantitation of phospho- and total PRAS40 by ELISA.
pPRAS40 was normalized to total PRAS40 to calculate inhibition. Factorial
ANOVA followed by Dunnett’s post hoc test was used to determine statistical
significance. Data for 36 was from one of two experiments.
21. 10-Week-old female CD1 nude mice (Charles River Laboratories, Wilmington,
MA) were injected sc in the flank with 5 Â 10
6 U-87 MG cells in 0.2 mL DMEM.
On day 10, mice were randomized into groups of n = 10 with an initial tumor
volume of ꢀ200 mm3. Diet was supplemented. Tumor measurements and body
weights were recorded twice per week. Tumor volume was calculated as
length  width  height. Tumor measurements were analyzed by RMANOVA
using StatView version 5.0.1 (SAS Institute, Cary, NC). Scheffé’s post hoc test
was used to determine statistical significance. Data for 36 was from one of
three experiments.