Avoiding Fluorescence Assay Interference-The Case for Diaphorase

Overview

Journal Assay Drug Dev Technol

Specialties Chemistry
Pharmacology

Date 2016 Apr 15

PMID 27078679

Citations 13

Authors

Matthew D Hall

Anton Simeonov

Mindy I Davis

Affiliations

Soon will be listed here.

Abstract

Fluorescence is utilized as the output for a range of assay formats used in high-throughput screening (HTS). Interference with these assays from the compounds in libraries utilized in HTS is a well-recognized phenomenon, particularly for assays relying on UV excitation such as for direct detection of the oxidoreductase cofactors NADH or NADPH. In this study, we discuss these interference challenges and highlight the specific case of the diaphorase/resazurin system that can be coupled to enzymes utilizing NADH or NADPH. We review the utilization of this assay system in the literature and argue that the diaphorase/resazurin system is underutilized in assay development. It is the authors' hope that this Perspective and the accompanying Technical Brief in this issue will stimulate interest in a robust and sensitive coupling system to avoid assay fluorescence interference.

Citing Articles

Machine Learning-Driven Data Valuation for Optimizing High-Throughput Screening Pipelines.

Hesse J, Boldini D, Sieber S J Chem Inf Model. 2024; 64(21):8142-8152.

PMID: 39440790 PMC: 11558681. DOI: 10.1021/acs.jcim.4c01547.

Machine Learning Assisted Hit Prioritization for High Throughput Screening in Drug Discovery.

Boldini D, Friedrich L, Kuhn D, Sieber S ACS Cent Sci. 2024; 10(4):823-832.

PMID: 38680560 PMC: 11046457. DOI: 10.1021/acscentsci.3c01517.

Determination of pharmacological inhibition of ALDH2 by ethanol clearance in mice.

Haenisch M, Paik J, Kim A, Goldstein A, Amory J Toxicol Appl Pharmacol. 2024; 483:116801.

PMID: 38181938 PMC: 10881113. DOI: 10.1016/j.taap.2023.116801.

A Versatile Continuous Fluorometric Enzymatic Assay for Targeting Nicotinate Phosphoribosyltransferase.

Minazzato G, Marangoni E, Fortunato C, Petrelli R, Cappellacci L, Del Bello F Molecules. 2023; 28(3).

PMID: 36770640 PMC: 9919730. DOI: 10.3390/molecules28030961.

Development of a High-throughput Agar Colony Formation Assay to Identify Drug Candidates against Medulloblastoma.

Sedeeq M, Maklad A, Gueven N, Azimi I Pharmaceuticals (Basel). 2020; 13(11).

PMID: 33167547 PMC: 7694510. DOI: 10.3390/ph13110368.

References

Ippolito J, Piwnica-Worms D . A fluorescence-coupled assay for gamma aminobutyric acid (GABA) reveals metabolic stress-induced modulation of GABA content in neuroendocrine cancer. PLoS One. 2014; 9(2):e88667. PMC: 3923810. DOI: 10.1371/journal.pone.0088667. View

Simeonov A, Jadhav A, Thomas C, Wang Y, Huang R, Southall N . Fluorescence spectroscopic profiling of compound libraries. J Med Chem. 2008; 51(8):2363-71. DOI: 10.1021/jm701301m. View

Bembenek M, Kuhn E, Mallender W, Pullen L, Li P, Parsons T . A fluorescence-based coupling reaction for monitoring the activity of recombinant human NAD synthetase. Assay Drug Dev Technol. 2005; 3(5):533-41. DOI: 10.1089/adt.2005.3.533. View

Preuss J, Richardson A, Pinkerton A, Hedrick M, Sergienko E, Rahlfs S . Identification and characterization of novel human glucose-6-phosphate dehydrogenase inhibitors. J Biomol Screen. 2012; 18(3):286-97. DOI: 10.1177/1087057112462131. View

Teichert A, Arnold L, Otieno S, Oda Y, Augustinaite I, Geistlinger T . Quantification of the vitamin D receptor-coregulator interaction. Biochemistry. 2009; 48(7):1454-61. PMC: 2654718. DOI: 10.1021/bi801874n. View

Croce A, Bottiroli G . Autofluorescence spectroscopy and imaging: a tool for biomedical research and diagnosis. Eur J Histochem. 2015; 58(4):2461. PMC: 4289852. DOI: 10.4081/ejh.2014.2461. View

Dahl R, Bravo Y, Sharma V, Ichikawa M, Dhanya R, Hedrick M . Potent, selective, and orally available benzoisothiazolone phosphomannose isomerase inhibitors as probes for congenital disorder of glycosylation Ia. J Med Chem. 2011; 54(10):3661-8. PMC: 3437750. DOI: 10.1021/jm101401a. View

Auld D, Southall N, Jadhav A, Johnson R, Diller D, Simeonov A . Characterization of chemical libraries for luciferase inhibitory activity. J Med Chem. 2008; 51(8):2372-86. DOI: 10.1021/jm701302v. View

Rees M, Davis M, Shen M, Titus S, Raimondo A, Barrett A . A panel of diverse assays to interrogate the interaction between glucokinase and glucokinase regulatory protein, two vital proteins in human disease. PLoS One. 2014; 9(2):e89335. PMC: 3929664. DOI: 10.1371/journal.pone.0089335. View

10.

Davis M, Shen M, Simeonov A, Hall M . Diaphorase Coupling Protocols for Red-Shifting Dehydrogenase Assays. Assay Drug Dev Technol. 2016; 14(3):207-12. PMC: 4840998. DOI: 10.1089/adt.2016.706. View

11.

Dahlin J, Nissink J, Strasser J, Francis S, Higgins L, Zhou H . PAINS in the assay: chemical mechanisms of assay interference and promiscuous enzymatic inhibition observed during a sulfhydryl-scavenging HTS. J Med Chem. 2015; 58(5):2091-113. PMC: 4360378. DOI: 10.1021/jm5019093. View

12.

Popovici-Muller J, Saunders J, Salituro F, Travins J, Yan S, Zhao F . Discovery of the First Potent Inhibitors of Mutant IDH1 That Lower Tumor 2-HG in Vivo. ACS Med Chem Lett. 2014; 3(10):850-5. PMC: 4025665. DOI: 10.1021/ml300225h. View

13.

Horie S . Advances in research on DT-diaphorase--catalytic properties, regulation of activity and significance in the detoxication of foreign compounds. Kitasato Arch Exp Med. 1990; 63(1):11-30. View

14.

LOWRY O, ROBERTS N, KAPPHAHN J . The fluorometric measurement of pyridine nucleotides. J Biol Chem. 1957; 224(2):1047-64. View

15.

Guilbault G, Kramer D . FLUOROMETRIC PROCEDURE FOR MEASURING THE ACTIVITY OF DEHYDROGENASES. Anal Chem. 1965; 37:1219-21. DOI: 10.1021/ac60229a011. View

16.

Betzi S, Alam R, Martin M, Lubbers D, Han H, Jakkaraj S . Discovery of a potential allosteric ligand binding site in CDK2. ACS Chem Biol. 2011; 6(5):492-501. PMC: 3098941. DOI: 10.1021/cb100410m. View

17.

Kumar A, Zhang M, Zhu L, Liao R, Mutai C, Hafsat S . High-throughput screening and sensitized bacteria identify an M. tuberculosis dihydrofolate reductase inhibitor with whole cell activity. PLoS One. 2012; 7(6):e39961. PMC: 3386958. DOI: 10.1371/journal.pone.0039961. View

18.

Burghardt T, Lyke J, Ajtai K . Fluorescence emission and anisotropy from rhodamine dimers. Biophys Chem. 1996; 59(1-2):119-31. DOI: 10.1016/0301-4622(95)00118-2. View

19.

Davis M, Gross S, Shen M, Straley K, Pragani R, Lea W . Biochemical, cellular, and biophysical characterization of a potent inhibitor of mutant isocitrate dehydrogenase IDH1. J Biol Chem. 2014; 289(20):13717-25. PMC: 4022846. DOI: 10.1074/jbc.M113.511030. View

20.

Duveau D, Yasgar A, Wang Y, Hu X, Kouznetsova J, Brimacombe K . Structure-activity relationship studies and biological characterization of human NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase inhibitors. Bioorg Med Chem Lett. 2013; 24(2):630-5. PMC: 3970110. DOI: 10.1016/j.bmcl.2013.11.081. View