Chemical proteomic analysis reveals alternative modes of action for pyrido[2,3-d]pyrimidine kinase inhibitors.
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.
Axxima Pharmaceuticals AG, 81377 München, Germany.
Josef
J
Wissing
Klaus
K
Godl
Dirk
D
Brehmer
Stephanie
S
Blencke
Martina
M
Weber
Peter
P
Habenberger
Matthias
M
Stein-Gerlach
Andrea
A
Missio
Matt
M
Cotten
Stefan
S
Müller
Henrik
H
Daub
D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D017209:Apoptosis; D001665:Binding Sites; D019556:COS Cells; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D016207:Cytokines / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D007249:Inflammation; D020128:Inhibitory Concentration 50; D008024:Ligands; D008070:Lipopolysaccharides / Q000737:chemistry; D013058:Mass Spectrometry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D040901:Proteomics / Q000379:methods*; D011725:Pyridines / Q000737:chemistry / Q000494:pharmacology*; D011743:Pyrimidines / Q000737:chemistry / Q000494:pharmacology*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013997:Time Factors
D000970:Antineoplastic Agents; D016207:Cytokines; D004791:Enzyme Inhibitors; D008024:Ligands; D008070:Lipopolysaccharides; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D011743:Pyrimidines; D011092:Polyethylene Glycols; D000255:Adenosine Triphosphate