Utilization of substrate-induced quenching for screening targets promoting NADH and NADPH consumption.
Oxidation of reduced nicotinamide adenine dinucleotides is a common event for many biochemical reactions. However, its exploitation for ultrahigh-throughput screening purposes is not an easy task and is affected by various drawbacks. It is known that such nucleotides induce quenching on the fluorescence of several dyes and that this quenching disappears with oxidation of the nucleotide. We have made use of this property to develop an assay for high-throughput screening with NADH and NADPH-dependent reductases. Full screening campaigns have been run with excellent assay quality parameters, and interesting hits have been identified. The method is amenable to miniaturization and allows easy identification of false positives without needing extra secondary assays. Although it is based on monitoring substrate consumption, it is demonstrated that the effect of fractional conversion on assay sensitivity is negligible.
Vázquez
María Jesús
MJ
Assay Development, Glaxo SmithKline, Madrid, Spain.
Ashman
Stephen
S
Ramón
Fernando
F
Calvo
David
D
Bardera
Ana
A
Martín
J Julio
JJ
Rüdiger
Martin
M
Tew
David
D
Domínguez
Juan Manuel
JM
D001681:Biological Assay; D004396:Coloring Agents; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D020128:Inhibitory Concentration 50; D008027:Light; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D010640:Phenothiazines / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000037:antagonists & inhibitors / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors
D004396:Coloring Agents; D010640:Phenothiazines; D009243:NAD; D009249:NADP; D010744:Phosphoric Monoester Hydrolases; D010751:Phosphopyruvate Hydratase; C009469:thionine