AT13148

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°C 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.

References:

[1]. Yap TA, et al. 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.
[2]. Xi Y, et al. AT13148, a first-in-class multi-AGC kinase inhibitor, potently inhibits gastric cancer cells both in vitro and in vivo. Biochem Biophys Res Commun. 2016 Sep 9;478(1):330-6.