Background
CEP-33779, is a highly selective, orally active inhibitor of Janus kinase 2 (JAK2). When evaluate against the other members of the JAK family, CEP-33779 demonstrates varying degrees of selectivity from >40-fold versus JAK1 to >800-fold against TYK2. In a cellular system, CEP-33779 is shown to inhibit JAK2 in irf-bla TF-1 cells utilizing the GeneBLAzer reporter assay. It is also able to mitigate several immune parameters associated with SLE advancement, including the protection and treatment of mice with lupus nephritis. The ability of CEP-33779 to suppress growth of colorectal tumors by inhibiting the IL-6/JAK2/STAT3 signaling suggests a potential therapeutic utility of JAK2 inhibitors in multiple tumors types, particularly those with a strong inflammatory component.
Reference
[1].Kristine L Stump, Lily D Lu, Pawel Dobrzanski, Cynthia Serdikoff, Diane E Gingrich, Ben J Dugan, Thelma S Angeles, Mark S Albom, Mark A Ator, Bruce D Dorsey, Bruce A Ruggeri, Matthew M Seavey. A highly selective, orally active inhibitor of Janus kinase 2, CEP-33779, ablates disease in two mouse models of rheumatoid arthritis. Arthritis Research & Therapy 2011, 13:R68.
[2].Lily D. Lu, Kristine L. Stump, Nate H. Wallace, Pawel Dobrzanski, Cynthia Serdikoff, Diane E. Gingrich, Benjamin J. Dugan, Thelma S. Angeles, Mark S. Albom, Jennifer L. Mason, Mark A. Ator, Bruce D. Dorsey, Bruce A. Ruggeri, Matthew M. Seavey. Depletion of Autoreactive Plasma Cells and Treatment of Lupus Nephritis in Mice Using CEP-33779, a Novel, Orally Active, Selective Inhibitor of JAK2. The Journal of Immunology October 1, 2011 vol. 187 no. 7 3840-3853.
[3].Matthew M. Seavey, Lily D. Lu, Kristine L. Stump, Nate H. Wallace, William Hockeimer, Teresa M. O'Kane, Bruce A. Ruggeri, Pawel Dobrzanski. Therapeutic Efficacy of CEP-33779, a Novel Selective JAK2 Inhibitor, in a Mouse Model of Colitis-Induced Colorectal Cancer. Mol Cancer Ther April 2012 11; 984.
Protocol
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]. |
参考文献: [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. |
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