CEP-33779

Kinase experiment:

The kinase activity of baculovirus-expressed human JAK1, JAK2, or JAK3 is measured. Each 96-well Costar high binding plate is coated with 100 μL/well of 10 μg/mL neutravidin in TBS at 37 °C for 2 h, followed by 100 μL/well of 1 μg/mL 15-mer peptide substrate at 37 °C for 1 h. The kinase assay mixture (total volume=100 μL/well) consisting of 20 mM HEPES (pH 7.2), ATP (0.2 μM ATP for JAK1 and JAK2 and 0.1 μM ATP for JAK3), 1 mM MnCl2, 0.1% BSA, and CEP-33779 (diluted in DMSO, 2.5% DMSO final in assay) is added to the assay plate. Enzyme is added and the reaction is allowed to proceed for 20 min at room temperature. Detection of the phosphorylated product is performed by adding 100 μL/well of diluted Eu-N1 labeled PY100 antibody. Samples are incubated at RT for 1 h, followed by addition of 100 μL enhancement solution. Plates are agitated for 10 min, and the fluorescence of the resulting solution is measured. IC50 values are determined[1].

Animal experiment:

Mice: Nude mice bearing CWR22 xenografts are dosed orally with 55 mg/kg of CEP-33779 or a vehicle (PEG400). At 2, 6, and 24 h after dosing animals (3/group) are sacrificed, tumors are excised and plasma samples are prepared. Tumor extracts are prepared using Triton-based extraction buffer supplemented with inhibitors of proteases and phosphatases. Equal amounts of extracts are resolved on SDS-PAGE gels and STAT3 phosphorylation and expression are analyzed by Western blot using specific antibodies[1].

References:

[1]. Dugan BJ, et al. A selective, orally bioavailable 1,2,4-triazolo[1,5-a]pyridine-based inhibitor of Janus kinase 2 for use in anticancer therapy: discovery of CEP-33779. J Med Chem. 2012 Jun 14;55(11):5243-54.
[2]. Seavey MM, et al. Therapeutic efficacy of CEP-33779, a novel selective JAK2 inhibitor, in a mouse model of colitis-induced colorectal cancer. Mol Cancer Ther. 2012 Apr;11(4):984-93.
[3]. Tang SJ, et al. CEP-33779 antagonizes ATP-binding cassette subfamily B member 1 mediated multidrug resistance by inhibiting its transport function. Biochem Pharmacol. 2014 Sep 15;91(2):144-56.