SB1317

Kinase experiment:

The recombinant enzymes (CDK2/cyclin A, JAK2, and FLT3) are used. All assays are carried out in 384-well white microtiter plates using the PKLight assay system. This assay platform is a luminometric assay for the detection of ATP in the reaction using a luciferase-coupled reaction. The compounds are tested at eight concentrations prepared from 3- or 4-fold serial dilution starting at 10 μM. For CDK2/cyclin A assay, the reaction mixture consisted of the following components in 25 μL of assay buffer (50 mM Hepes, pH 7.5, 10 mM MgCl2, 5 mM MnCl2, 5 mM BGP, 1 mM DTT, 0.1 mM sodium orthovanadate), 1.4 μg/mL of CDK2/cyclin A complex, 0.5 μM RbING substrate, and 0.5 μM ATP. The mixture is incubated at room temperature for 2 h. Then 13 μL of PKLight ATP detection reagent is added and the mixture is incubated for 10 min. Luminescence signals are detected on a multilabel plate reader. The analytical software Prism 5.0 is used to generate IC50 values from the data[1].

Cell experiment:

All cell lines are obtained from the American Type Culture Collection and cultured. For proliferation assays in 96-well plates, 20 000 cells are seeded in 100 μL of medium and treated the following day with compounds (e.g., SB1317 ) (in triplicate) at concentrations up to 10 μM for 48 h. Cell viability is monitored using the CellTiter-96 Aqueous One solution cell proliferation assay. Dose-response curves are plotted to determine IC50 values for the compounds using the XL-fit software[1].

Animal experiment:

Mice and Rats[1] Male BALB/c mice (aged ~10-12 weeks and weighing 17-22 g), male Beagle dogs (~6-7 months of age, weighing 10-14 kg), and male Wistar rats (aged 6-8 weeks, weighing 239-249 g) are used in this study. The oral doses for mice, dogs, and rats are 75, 10, and 10 mg/kg, respectively. The doses are administered by gavage as suspensions (0.5% methylcellulose and 0.1% Tween 80) to mice and rats, and as gelatin capsules (12 Torpac) to dogs. Following oral dosing serial blood samples are collected (cardiac puncture in mice, jugular vein in dogs, and superior vena cava in rats) at different time points (0-24 h) in tubes containing K3EDTA as anticoagulant, centrifuged, and plasma is separated and stored at -70°C until analysis. Plasma samples are processed and analyzed by LC-MS/MS. Pharmacokinetic parameters are estimated by noncompartmental methods using WinNonlin.

References:

[1]. William AD, et al. Discovery of kinase spectrum selective macrocycle (16E)-14-methyl-20-oxa-5,7,14,26-tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8(27),9,11,16,21,23-decaene (SB1317/TG02), a potent inhibitor of cyclin dependent kina
[2]. Pasha MK, et al. Preclinical metabolism and pharmacokinetics of SB1317 (TG02), a potent CDK/JAK2/FLT3 inhibitor. Drug Metab Lett. 2012 Mar;6(1):33-42.