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.
Wissing
Josef
J
Axxima Pharmaceuticals AG, 81377 München, Germany.
Godl
Klaus
K
Brehmer
Dirk
D
Blencke
Stephanie
S
Weber
Martina
M
Habenberger
Peter
P
Stein-Gerlach
Matthias
M
Missio
Andrea
A
Cotten
Matt
M
Müller
Stefan
S
Daub
Henrik
H
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