Diaphorase Coupling Protocols for Red-Shifting Dehydrogenase Assays

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.