New affinity probe targeting VEGF receptors for kinase inhibitor selectivity profiling by chemical proteomics.
Solid tumors are dependent for growth on nutrients and the supply of oxygen, which they often acquire via neoangiogenesis. Vascular endothelial growth factors and the corresponding receptors (VEGFRs) play central roles in this process, and consequently, the blockade of this pathway is one therapeutic strategy for cancer treatment. A number of small molecules inhibiting VEGFR inhibitors have been developed for clinical use, and a comprehensive view of target selectivity is important to assess the therapeutic as well as risk potential of a drug molecule. Recent advances in mass spectrometry-based chemical proteomics allow analyses of drug-target interactions under close-to-physiological conditions, and in this study, we report on the design, synthesis, and application of a small molecule affinity probe as a tool for the selectivity profiling of VEGFR and other kinase inhibitors. The probe is capable of binding >132 protein kinases, including angiokinases such as VEGFRs, PDGFRs, and c-KIT from lysates of cancer cell lines or human placenta tissue. Combining the new probe with Kinobeads in competitive binding assays, we were able to identify nanomolar off-targets of the VEGFR/PDGFR inhibitors pazopanib and axitinib. Because of its broad binding spectrum, the developed chemical tool can be generically used for the discovery of kinase inhibitor targets, which may contribute to a more comprehensive understanding of the mechanisms of action of such drugs.
Chair for Proteomics and Bioanalytics, Technische Universität München , Emil Erlenmeyer Forum 5, 85354 Freising, Germany.
Xin
X
Ku
Stephanie
S
Heinzlmeir
Dominic
D
Helm
Guillaume
G
Médard
Bernhard
B
Kuster
D000595:Amino Acid Sequence; D000077784:Axitinib; D001667:Binding, Competitive; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D005260:Female; D006801:Humans; D007093:Imidazoles / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D007191:Indazoles / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D020014:K562 Cells; D008956:Models, Chemical; D062105:Molecular Docking Simulation / Q000379:methods; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D011743:Pyrimidines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D040262:Receptors, Vascular Endothelial Growth Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D054852:Small Molecule Libraries / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D013449:Sulfonamides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D053719:Tandem Mass Spectrometry
D007093:Imidazoles; D007191:Indazoles; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D054852:Small Molecule Libraries; D013449:Sulfonamides; C516667:pazopanib; D000077784:Axitinib; D011505:Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor