Kinase experiment:

AT13148 is assayed against 40 kinases and the percentage inhibition at 10 μM of AT13148 is determined. Individual IC50 values are measured for selected kinases using ATP concentrations equivalent to the Km for each enzyme.

Cell experiment:

Cells are seeded onto 96-well micro-plates at a density of 1×104 cells per well. After treatment, MTT solution (0.5 mg/mL) is added for 2-3 h. The MTT-purple formazan productions are dissolved in 0.1 N hydrochloric acid, and optical density (OD) is obtained through the micro-plate reader at 570 nm wavelength.

Animal experiment:

For pharmacokinetic analysis, male athymic BALB/c mice are obtained from Harlan. AT13148 is formulated in 10% DMSO, 1% Tween-20, and 89% saline and administered at 5 mg/kg i.v. or p.o. Duplicate samples of heparinized whole blood are collected by cardiac puncture at 1, 2, 4, 6, 8, 16, 24, and 72 hours after dosing. Plasma and tissues (liver, kidney, spleen, and muscle are also taken) are prepared and frozen at –20℃ until analysis. AT13148 is extracted from plasma and tissues using acetonitrile containing an internal standard and quantified using a liquid chromatography tandem mass spectrometry (LC-MS/MS) method and appropriate standard curves. Pharmacokinetic parameters are determined using WinNonLin software version 5.2.

AT13148 is a novel, oral and ATP-competitive inhibitor of multi-AGC Kinase with IC50 values of 38nM, 8nM, 3nM, 63nM and 4nM for AKT, p70S6 kinase, PKA, SGK and Rho kinase, respectively [1].

In vitro studies, AT13148 has been reported to inhibit activity of AGC kinases which including AKT, p70S6 kinase, PKA, SGK and Rho kinase with the IC50 values of 38nM, 8nM, 3nM, 63nM and 4nM, respectively. In addition, AT13148 has been revealed to inhibit proliferation with GI50 values of 1.54μM, 1.59μM, 1.82μM, 2.65μM and 3.77μM in MES-SA, BT474, HCT-116, A549 and SK-OV-3 cell lines, respectively.

In vivo studies, AT13148 has shown the antitumor activity in multiple human tumor xenograft models including PTEN-deficient MES-SA human uterine sarcoma, HER2-positive, PIK3CA-mutant BT474 human breast cancer xenografts [1].

References:
[1] Yap TA1, Walton MI, Grimshaw KM, Te Poele RH, Eve PD, Valenti MR, de Haven Brandon AK, Martins V, Zetterlund A, Heaton SP, Heinzmann K, Jones PS, Feltell RE, Reule M, Woodhead SJ, Davies TG, Lyons JF, Raynaud FI, Eccles SA, Workman P, Thompson NT, Garrett MD. AT13148 is a novel, oral multi-AGC kinase inhibitor with potent pharmacodynamic and antitumor activity. Clin Cancer Res. 2012 Jul 15;18(14):3912-23.