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Diaphorase Coupling Protocols for Red-Shifting Dehydrogenase Assays
Mindy I Davis1, Min Shen1, Anton Simeonov1
1NCATS 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.
Insights
Developing novel cancer therapeutics targeting dehydrogenases is crucial. This study presents a robust assay using diaphorase coupling to reduce compound interference, enabling high-throughput screening for this important enzyme class.
Area of Science:
- Biochemistry
- Enzymology
- Drug Discovery
Background:
- Dehydrogenases are critical targets for cancer therapeutics.
- NAD(P)H fluorescence overlaps with compound libraries, hindering assays.
- A red-shifted assay is needed to reduce interference.
Purpose of the Study:
- To develop miniaturized, robust 1,536-well assays for dehydrogenases.
- To couple dehydrogenases to diaphorase for reduced compound interference.
- To provide guidelines for high-throughput screening of dehydrogenases.
Main Methods:
- Coupling dehydrogenases (WT IDH1, mutant IDH1 R132H) to diaphorase.
- Utilizing resazurin to resorufin conversion for fluorescence detection.
- Implementing kinetic and endpoint modes for different dehydrogenase activities.
- Developing counterassays to detect compound interference.
Main Results:
- Established robust and miniaturized 1,536-well assays for IDH1 and IDH1 R132H.
- Demonstrated reduced spectral interference from compound libraries.
- Validated the coupling technique for both NAD(P)H-producing and consuming dehydrogenases.
- Developed counterassays for reliable interference detection.
Conclusions:
- The diaphorase coupling method provides a reliable strategy for high-throughput screening of dehydrogenases.
- This approach minimizes compound interference, enhancing assay accuracy.
- The developed protocols serve as a foundation for screening diverse dehydrogenases as cancer drug targets.
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