Chemical Proteomic Analysis Reveals Alternative Modes of Action for Pyrido[2,3-d]pyrimidine Kinase Inhibitors

• Published in: Molecular & cellular proteomics Vol. 3; no. 12; pp. 1181 - 1193
• Main Authors: Wissing, Josef, Godl, Klaus, Brehmer, Dirk, Blencke, Stephanie, Weber, Martina, Habenberger, Peter, Stein-Gerlach, Matthias, Missio, Andrea, Cotten, Matt, Müller, Stefan, Daub, Henrik
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 01.12.2004
• Subjects: Adenosine Triphosphate - chemistry; Amino Acid Sequence; Animals; Antineoplastic Agents - chemistry; Antineoplastic Agents - pharmacology; Apoptosis; Binding Sites; Cell Line, Tumor; Cells, Cultured; Chromatography, Liquid; COS Cells; Cytokines - metabolism; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors - chemistry; HeLa Cells; Humans; Inflammation; Inhibitory Concentration 50; Ligands; Lipopolysaccharides - chemistry; Mass Spectrometry; Models, Chemical; Molecular Sequence Data; Mutagenesis, Site-Directed; Plasmids - metabolism; Polyethylene Glycols - chemistry; Protein Kinase Inhibitors - chemistry; Protein Kinase Inhibitors - pharmacology; Proteomics - methods; Pyridines - chemistry; Pyridines - pharmacology; Pyrimidines - chemistry; Pyrimidines - pharmacology; Sequence Homology, Amino Acid; Signal Transduction; Time Factors
• ISSN: 1535-9476; 1535-9484; 1535-9484
• DOI: 10.1074/mcp.M400124-MCP200

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 p38α 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.