NDLI: Bioluminescent Cell-Based NAD(P)/NAD(P)H Assays for Rapid Dinucleotide Measurement and Inhibitor Screening

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Bioluminescent Cell-Based NAD(P)/NAD(P)H Assays for Rapid Dinucleotide Measurement and Inhibitor Screening

Content Provider	PubMed Central
Author	Vidugiriene, Jolanta Donna, Leippe Sobol, Mary Vidugiris, Gediminas Zhou, Wenhui Meisenheimer, Poncho Gautam, Prson Wennerberg, Krister Cali, James J.
Copyright Year	2014
Abstract	The central role of nicotinamide adenine dinucleotides in cellular energy metabolism and signaling makes them important nodes that link the metabolic state of cells with energy homeostasis and gene regulation. In this study, we describe the implementation of cell-based bioluminescence assays for rapid and sensitive measurement of those important redox cofactors. We show that the sensitivity of the assays (limit of detection ∼0.5 nM) enables the selective detection of total amounts of nonphosphorylated or phosphorylated dinucleotides directly in cell lysates. The total amount of NAD+NADH or NADP+NADPH levels can be detected in as low as 300 or 600 cells/well, respectively. The signal remains linear up to 5,000 cells/well with the maximum signal-to-background ratios ranging from 100 to 200 for NAD+NADH and from 50 to 100 for NADP+NADPH detection. The assays are robust (Z′ value >0.7) and the inhibitor response curves generated using a known NAD biosynthetic pathway inhibitor FK866 correlate well with the reported data. More importantly, by multiplexing the dinucleotide detection assays with a fluorescent nonmetabolic cell viability assay, we show that dinucleotide levels can be decreased dramatically (>80%) by FK866 treatment before changes in cell viability are detected. The utility of the assays to identify modulators of intracellular nicotinamide adenine dinucleotide levels was further confirmed using an oncology active compound library, where novel dinucleotide regulating compounds were identified. For example, the histone deacetylase inhibitor entinostat was a potent inhibitor of cellular nicotinamide adenine dinucleotides, whereas the selective estrogen receptor modulator raloxifene unexpectedly caused a twofold increase in cellular nicotinamide adenine dinucleotide levels.
Related Links	http://dx.doi.org/10.1089/adt.2014.605
Ending Page	526
Page Count	13
Starting Page	514
File Format	PDF
ISSN	15578127
e-ISSN	15578127
Journal	Assay and Drug Development Technologies
Issue Number	9-10
Volume Number	12
Language	English
Publisher	Mary Ann Liebert, Inc.
Publisher Date	2014-12-01
Access Restriction	Open
Rights Holder	Mary Ann Liebert, Inc.
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Drug Discovery Molecular Medicine

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