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Assay Drug Dev Technol. 2016 Apr;14(3):207-12. doi: 10.1089/adt.2016.706.

Diaphorase Coupling Protocols for Red-Shifting Dehydrogenase Assays.

Author information

1
NCATS Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health , Rockville, Maryland.

Abstract

Dehydrogenases are an important target for the development of cancer therapeutics. Dehydrogenases either produce or consume NAD(P)H, which is fluorescent but at a wavelength where many compounds found in chemical libraries are also fluorescent. By coupling dehydrogenases to diaphorase, which utilizes NAD(P)H to produce the fluorescent molecule resorufin from resazurin, the assay can be red-shifted into a spectral region that reduces interference from compound libraries. Dehydrogenases that produce NAD(P)H, such as isocitrate dehydrogenase 1 (IDH1), can be read in kinetic mode. Dehydrogenases that consume NAD(P)H, such as mutant IDH1 R132H, can be read in endpoint mode. Here, we report protocols for robust and miniaturized 1,536-well assays for WT IDH1 and IDH1 R132H coupled to diaphorase, and the counterassays used to further detect compound interference with the coupling reagents. This coupling technique is applicable to dehydrogenases that either produce or consume NAD(P)H, and the examples provided here can act as guidelines for the development of high-throughput screens against this enzyme class.

PMID:
27078681
PMCID:
PMC4840998
DOI:
10.1089/adt.2016.706
[Indexed for MEDLINE]
Free PMC Article

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