Abstract
Small molecule inhibitors belonging to the pyrido[2,3-d]pyrimidine class of compounds were developed as antagonists of protein tyrosine kinases implicated in cancer progression. Derivatives from this compound class are effective against most of the imatinib mesylate-resistant BCR-ABL mutants isolated from advanced chronic myeloid leukemia patients. Here, we established an efficient proteomics method employing an immobilized pyrido[2,3-d]pyrimidine ligand as an affinity probe and identified more than 30 human protein kinases affected by this class of compounds. Remarkably, in vitro kinase assays revealed that the serine/threonine kinases Rip-like interacting caspase-like apoptosis-regulatory protein kinase (RICK) and p38alpha were among the most potently inhibited kinase targets. Thus, pyrido[2,3-d]pyrimidines did not discriminate between tyrosine and serine/threonine kinases. Instead, we found that these inhibitors are quite selective for protein kinases possessing a conserved small amino acid residue such as threonine at a critical site of the ATP binding pocket. We further demonstrated inhibition of both p38 and RICK kinase activities in intact cells upon pyrido[2,3-d]pyrimidine inhibitor treatment. Moreover, the established functions of these two kinases as signal transducers of inflammatory responses could be correlated with a potent in vivo inhibition of cytokine production by a pyrido[2,3-d]pyrimidine compound. Thus, our data demonstrate the utility of proteomic methods employing immobilized kinase inhibitors for identifying new targets linked to previously unrecognized therapeutic applications.
Highlights
Small molecule inhibitors belonging to the pyrido[2,3d]pyrimidine class of compounds were developed as antagonists of protein tyrosine kinases implicated in cancer progression
Inhibitors belonging to the pyrido[2,3-d]pyrimidine class of compounds were known to act on several protein kinases, all of these previously described targets belonged to the tyrosine kinase family and it appeared that pyrido[2,3-d]pyrimidine inhibitors are selective for this particular group of the human kinome (6 –9)
The hydrophobic pocket does not interact with any part of a kinase-bound ATP molecule, explaining why the substitution of the critical threonine with a larger residue such as methionine sterically interferes with inhibitor binding, but does not abrogate protein kinase activity
Summary
Reagents and Plasmids—Cell culture media and LipofectAMINE were obtained from Invitrogen (San Diego, CA). For in vitro association with inhibitor affinity beads, HeLa cells or transfected COS-7 cells were lysed in buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, 1 mM EGTA plus additives. Affinity Chromatography and Preparative Gel Electrophoresis— Frozen HeLa cells (2.5 ϫ 109; Cilbiotech, Mons, Belgium) were lysed in 30 ml of the same Triton X-100-containing buffer plus additives used for sample preparation prior to the analytical in vitro association experiments. PP58 inhibition of Abl, Src, and EphB4 activities were assayed for 30 min at 30 °C in a reaction buffer containing 20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0.8 mM MnCl2, 1 mM DTT, 0.1 mM EGTA, 100 M sodium orthovanadate, 50 M ATP, 2 Ci [␥-32P]ATP, and 0.2 mg/ml GST-Crk, 0.4 mg/ml myelin basic protein (MBP), or 0.2 mg/ml glyceraldehyde-3-phosphate dehydrogenase as kinase substrates, respectively. Primer sequences for IFN- were: 5Ј-GACATCCCTGAGGAGATTAAGCA-3Ј (forward), reverse: 5Ј-GGAGCATCTCATAGATGGTCAATG-3Ј (reverse), 5Ј-FAM-CGTCCTCCTTCTGGAACTGCTGCAG-TAMRA-3Ј (TaqMan probe)
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