CCT128930

Kinase experiment:

Profiling against 50 different human kinases is carried out using 10 μM CCT128930 at an ATP concentration equivalent to the Km for each enzyme. All other enzyme assays are performed[1].

Cell experiment:

All cell lines are grown in their recommended culture medium, supplemented with 10% fetal bovine serum at 37°C in 5% CO2 and passaged for less than six months prior to replacement from early passage frozen stocks. CCT128930 and LY294002 are made up as 10mM stocks in DMSO. Cells are regularly screened for Mycoplasma using a PCR-based assay. Cells are seeded in 96-well plates and allowed to attach for 36 hours to ensure exponential growth prior to treatment. In vitro antiproliferative activity is determined using a 96-hour SRB assay, and GI50 values are derived[1].

Animal experiment:

Mice[1] PTEN-null U87MG human glioblastoma cells (2×106) are injected subcutaneously (s.c.) in the right flank of 6-8 weeks old female CrTacNCr-Fox1nu mice. For HER2-positive, PIK3CA-mutant BT474 human breast cancer xenografts, cells (5×106) are administered s.c. in medium supplemented with matrigel (1:1) into the mammary fat pads of female mice implanted s.c. with estradiol pellets (0.025 mg, 90 day release) 3 days previously. Animals are randomized and treatment is started with vehicle or CCT128930 when established tumors are ~100 mm3 in mean volume. Control mice receive vehicle only (10% DMSO, 5% Tween 20, 85% saline) and treated mice received 50 mg/kg CCT128930 intraperitoneally (i.p.) daily for 5 days (U87MG human glioblastoma xenografts) or 40 mg/kg CCT128930 i.p. twice daily for 5 days (BT474 human breast cancer xenografts). Tumor size and body weight are monitored three times a week. Tumor size is evaluated by measurement of 2 orthogonal diameters with calipers and volume is calculated. At the end of the study, tumors are excised and weighed.

References:

[1]. Yap TA et al. Preclinical pharmacology, antitumor activity, and development of pharmacodynamic markers for the novel, potent AKT inhibitor CCT128930. Mol Cancer Ther. 2011 Feb;10(2):360-71.