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

Wissing, Josef; Godl, Klaus; Brehmer, Dirk; Blencke, Stephanie; Weber, Martina; Habenberger, Peter; Stein-Gerlach, Matthias; Missio, Andrea; Cotten, Matthew; Müller, Stefan; Daub, Henrik

doi:10.1074/mcp.m400124-mcp200

Cited by 85 publications

(90 citation statements)

References 40 publications

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“…Binding assays were done as previously described (10,12). For subsequent immunoblotting, the following antibodies were used: rabbit anti-PDGFR type B, rabbit anti-AMP-activated protein kinase (AMPK; both from Upstate Biotechnology, Lake Placid, NY), mouse anti-TBK1 (Merck, Darmstadt, Germany), mouse anti-p53 (Oncogene Research Products, San Diego, CA), mouse anti-Yes, mouse anti-Aurora A (both from BD Transduction Laboratories, Lexington, KY), goat antiribosomal protein S6 kinase 3 (RSK3), mouse anti-Lyn (both from Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-FLAG antibody and mouse anti-a-tubulin antibody (both from Sigma, St. Louis, MO).…”

Section: Methodsmentioning

confidence: 99%

“…SU6668 was synthesized as described and covalently coupled to EAH Sepharose 4B (Amersham Biosciences, Buckinghamshire, United Kingdom) using carbodiimide coupling chemistry (5,12). Lysis of frozen HeLa cell pellets (1 Â 10 9 cells, Cilbiotech, Mons, Belgium) was done with 15 mL of buffer containing 20 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 0.25% Triton X-100, 1 mmol/L EDTA, 1 mmol/L EGTA plus additives (10 mmol/L sodium fluoride, 1 mmol/L orthovanadate, 10 Ag/mL aprotinin, 10 Ag/mL leupeptin, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L DTT).…”

Section: Methodsmentioning

confidence: 99%

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Proteomic Characterization of the Angiogenesis Inhibitor SU6668 Reveals Multiple Impacts on Cellular Kinase Signaling

Godl¹,

Gruß

Eickhoff³

et al. 2005

Cancer Research

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102

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Knowledge about molecular drug action is critical for the development of protein kinase inhibitors for cancer therapy. Here, we establish a chemical proteomic approach to profile the anticancer drug SU6668, which was originally designed as a selective inhibitor of receptor tyrosine kinases involved in tumor vascularization. By employing immobilized SU6668 for the affinity capture of cellular drug targets in combination with mass spectrometry, we identified previously unknown targets of SU6668 including Aurora kinases and TANK-binding kinase 1. Importantly, a cell cycle block induced by SU6668 could be attributed to inhibition of Aurora kinase activity. Moreover, SU6668 potently suppressed antiviral and inflammatory responses by interfering with TANK-binding kinase 1-mediated signal transmission. These results show the potential of chemical proteomics to provide rationales for the development of potent kinase inhibitors, which combine rather unexpected biological modes of action by simultaneously targeting defined sets of both serine/threonine and tyrosine kinases involved in cancer progression. (Cancer Res 2005; 65(15): 6919-26)

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Proteomic Characterization of the Angiogenesis Inhibitor SU6668 Reveals Multiple Impacts on Cellular Kinase Signaling

Godl¹,

Gruß

Eickhoff³

et al. 2005

Cancer Research

Self Cite

102

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show abstract

“…To identify native targets of dasatinib in CML cells, we used a chemical proteomics approach (16)(17)(18)(19)(20). We immobilized dasatinib and used the resultant resin as an affinity reagent to identify binding proteins from cell lysates by SDS/PAGE and liquid chromatography coupled to tandem MS (LC-MSMS).…”

mentioning

confidence: 99%

The Btk tyrosine kinase is a major target of the Bcr-Abl inhibitor dasatinib

Hantschel

Rix

Schmidt

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

263

224

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“…PP58 is one of the pyrido [2,3-d]pyrimidines which are small molecule broad kinase inhibitors that inhibit SRC kinase among other tyrosine and serine/threonine kinases by competitive inhibition at the ATP binding site. 4 PP58 was used because it retains its activity when attached to solid substrates and it has a wide range of target kinases. 2,4 SRC kinase was chosen as a model analyte since the literature shows that it binds to PP58 on beads, 4 it is an important cytoplasmic tyrosine kinase involved in regulation of cellular functions such as proliferation and differentiation, 5 and it is implicated in some breast and colon cancers.…”

mentioning

confidence: 99%

“…4 PP58 was used because it retains its activity when attached to solid substrates and it has a wide range of target kinases. 2,4 SRC kinase was chosen as a model analyte since the literature shows that it binds to PP58 on beads, 4 it is an important cytoplasmic tyrosine kinase involved in regulation of cellular functions such as proliferation and differentiation, 5 and it is implicated in some breast and colon cancers. 6 In the work presented here, buffer solutions were spiked with various concentrations of SRC kinase, and the changes of transistor electrical characteristics were measured following exposure to the SRC solutions.…”

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confidence: 99%

Kinase detection with gallium nitride based high electron mobility transistors

Makowski

Bryan

Sitar

et al. 2013

Applied Physics Letters

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A label-free kinase detection system was fabricated by the adsorption of gold nanoparticles functionalized with kinase inhibitor onto AlGaN/GaN high electron mobility transistors (HEMTs). The HEMTs were operated near threshold voltage due to the greatest sensitivity in this operational region. The Au NP/HEMT biosensor system electrically detected 1 pM SRC kinase in ionic solutions. These results are pertinent to drug development applications associated with kinase sensing. V C 2013 AIP Publishing LLC. [http://dx.doi.org/10.1063/1.4812987] Quantification of cellular or tissue kinome activity is important in basic cell biology research 1 and drug development. 2 Label-free methods of kinome activity profiling are ideal due to elimination of artifacts caused by changes to kinase characteristics with labeling. 3 An important label-free method of kinome activity measurement is the kinase affinity assay. In this method, cell lysates are exposed to beads functionalized with small molecule kinase inhibitors that bind kinases in the cell lysate. The kinases are removed from the beads by elution, and the kinase profile is identified by mass spectrometry. The factors that affect kinome activity profiles are the amount of kinase present in the cell lysate and the kinase affinity to the inhibitor beads. 2 Affinity is in turn affected by kinase activation state as regulated by phosphorylation. 2,3 Basic science and clinical applications exist for the kinase affinity assay. An example basic science application was the monitoring of cell cycle kinase phosphorylation status. 1 An example clinical application was a triple negative breast cancer (TNBC) drug screening study. 2 This study monitored changes in the kinome activity profile of TNBC cells with exposure to a MEK inhibitor drug. The kinome activity data guided selection of a combination of kinase inhibitor drugs in conjunction with the MEK inhibitor that resulted in decreased drug resistance compared to using any of the drugs alone. 2 Monitoring of the entire kinome gave a better overall view of drug efficacy than by monitoring the activity of a single kinase where drug resistance often develops by compensatory upregulation of uninhibited kinases. 2 Another clinical application of the kinase affinity assay was in drug development through determining kinase inhibitor drug targets. 3 Cell lysates were treated with soluble kinase inhibitors, and the lysates were exposed to the affinity beads. The soluble drug bound its target kinases, and the remaining kinases had their ATP binding sites available to bind the beads of the affinity assay. 3 The kinases with a decreased representation based on mass spectrometry following drug treatment were identified as the drug targets. 3 In this proof-of-concept report, we have demonstrated label-free detection of a kinase by a nanoparticle (NP)/high electron mobility transistor (HEMT) biosensor system. This detection method excluded the complication of a sequential kinase affinity assay followed by mass spectrometry analysis. Gold (Au) NPs w...

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

Cited by 85 publications

References 40 publications

Proteomic Characterization of the Angiogenesis Inhibitor SU6668 Reveals Multiple Impacts on Cellular Kinase Signaling

Proteomic Characterization of the Angiogenesis Inhibitor SU6668 Reveals Multiple Impacts on Cellular Kinase Signaling

The Btk tyrosine kinase is a major target of the Bcr-Abl inhibitor dasatinib

Kinase detection with gallium nitride based high electron mobility transistors

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