Peptide biosensors for the electrochemical measurement of protein kinase activity.
The kinase activities are elucidated using the novel redox-active cosubstrate adenosine 5'-[gamma-ferrocene] triphosphate (Fc-ATP), which enables the kinase-catalyzed transfer of a redox active gamma-phosphate-Fc to a hydroxyamino acid. In this report, a versatile electrochemical biosensor is developed for monitoring the activity and inhibition of a serine/threonine kinase, casein kinase 2 (CK2), and protein tyrosine kinases, Abl1-T315I and HER2, in buffered solutions and in cell lysates. The method is based on the labeling of a specific phosphorylation event with Fc, followed by electrochemical detection. The electrochemical response obtained from the "ferrocenylated" peptides enables monitoring the activity of the kinase and its substrate, as well as the inhibition of small molecule inhibitors on protein phosphorylation. Kinetic information was extracted from the electrochemical measurements for the determination of K(m) and V(m) values, which were in agreement with those previously reported. Kinase reactions were also performed in the presence of well-defined inhibitors of CK2, 4,5,6,7-tetrabromo-2-azabenzimidazole, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, and E-3-(2,3,4,5-tetrabromophenyl)acrylic acid as well as the nonspecific kinase inhibitors, staurosporine and N-benzoylstaurosporine. On the basis of the dependency of the Fc signal on inhibitor concentration, K(i) of the inhibitors was estimated, which were also in agreement with the literature values. The performance of the biosensor was optimized including the kinase reaction, incubation with Fc-ATP, and the small molecule inhibitors. Peptide modified electrochemical biosensors are promising candidates for cost-effective in vitro kinase activity and inhibitor screening assays.
Kerman
Kagan
K
Department of Chemistry, Faculty of Science, The University of Western Ontario, London, Ontario, Canada N6A 5B7.
Song
Haifeng
H
Duncan
James S
JS
Litchfield
David W
DW
Kraatz
Heinz-Bernhard
HB
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D001562:Benzimidazoles / Q000494:pharmacology; D015374:Biosensing Techniques; D001859:Bone Neoplasms / Q000201:enzymology* / Q000473:pathology; D047390:Casein Kinase II / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002384:Catalysis; D004563:Electrochemistry; D005296:Ferrous Compounds / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D000075163:Metallocenes; D009154:Mutation / Q000235:genetics; D012516:Osteosarcoma / Q000201:enzymology / Q000473:pathology; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D016315:Proto-Oncogene Proteins c-abl / Q000235:genetics / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D019311:Staurosporine / Q000031:analogs & derivatives / Q000494:pharmacology; D014230:Triazoles / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D051941:fms-Like Tyrosine Kinase 3 / Q000235:genetics / Q000378:metabolism
D000255:Adenosine Triphosphate; D001562:Benzimidazoles; D047390:Casein Kinase II; D005296:Ferrous Compounds; D000075163:Metallocenes; D010446:Peptide Fragments; D011493:Protein Kinase C; D047428:Protein Kinase Inhibitors; D016315:Proto-Oncogene Proteins c-abl; D018719:Erb-b2 Receptor Tyrosine Kinases; D019311:Staurosporine; D014230:Triazoles; D051941:fms-Like Tyrosine Kinase 3; C492322:2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole; C103118:4,5,6,7-tetrabromo-2-azabenzimidazole; C508053:ERBB2 protein, human; C497970:FLT3 protein, human; C059539:midostaurin; C004998:ferrocene