A bioluminescent method for measuring thymidylate kinase activity suitable for high-throughput screening of inhibitor.
Blocking human thymidylate kinase (TMPK) function has a chemosensitization effect in anticancer treatment. However, a rapid and sensitive TMPK activity assay method suitable for inhibitor screening has been lacking. We have designed a luciferase-coupled TMPK assay in which luminescence emission is proportional to the magnitude of TMPK inhibition. The advantages of using this new method over the conventional nicotinamide adenine dinucleotide (reduced form, NADH)-coupling method in screening inhibitor include low cost, low limit in detecting inhibitory signal, more accurate, and devoid of interference due to compound absorbance at 340 nm.
Hu
Chun-Mei
CM
Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Chang
Zee-Fen
ZF
D000255:Adenosine Triphosphate / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D057166:High-Throughput Screening Assays; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008163:Luminescent Measurements; D009703:Nucleoside-Phosphate Kinase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D015203:Reproducibility of Results
D004791:Enzyme Inhibitors; D000255:Adenosine Triphosphate; D004228:Dithionitrobenzoic Acid; D008156:Luciferases; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase