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

doi:10.1074/mcp.M400124-MCP200

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

From Axxima Pharmaceuticals AG, 81377 München, Germany

‡To whom correspondence should be addressed: Axxima Pharmaceuticals AG, Max-Lebsche-Platz 32, 81377 München, Germany. Tel.: 49-89-550-65-356; Fax: 49-89-550-65-461; E-mail: henrik.daub{at}axxima.com

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 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.

Footnotes

↵* This work was supported by a grant from the German Bundesministerium für Bildung und Forschung.
Published, MCP Papers in Press, October 8, 2004, DOI 10.1074/mcp.M400124-MCP200
↵1 The abbreviations used are: CML, chronic myeloid leukemia; 16-BAC, 16-benzyldimethyl-n-hexadecylammonium chloride; ATF2, activating transcription factor 2; CSK, C-terminal Src kinase; DMF, dimethylformamide; EDC, N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride; EGF, epidermal growth factor; ERK, extracellular signal-regulated protein kinase; FAK, focal adhesion kinase; FBS, fetal bovine serum; FGFR, fibroblast growth factor receptor; GAK, cyclin G-associated kinase; HFF, human foreskin fibroblast; IFN-β, interferon-β; JNK, c-jun N-terminal kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MAPKAP-K2, MAPK-activated protein kinase 2; MBP, myelin basic protein; MEK1, MAPK/ERK kinase 1; MIP-1α, macrophage inflammatory protein-1α; MS, mass spectrometry; PBMC, peripheral blood mononuclear cell; PP58, pyrido[2,3-d]pyrimidine derivative 58; RICK, Rip-like interacting caspase-like apoptosis-regulatory protein kinase; Rsk1, ribosomal S6 protein kinase 1; SRPK1, SR protein-specific kinase 1; TNF-α, tumor necrosis factor-α.
- Received September 8, 2004.
- Revision received October 7, 2004.