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A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors
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Evaluation of binding, affinity and metabolic regulatory activity of a transketolase inhibitor

Dan Jia1, Chunliang Liu2, Wenyu Liu3

  • 1School of Pharmacy, Second Military Medical University/Naval Medical University, Shanghai, P.R. China; International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University/Naval Medical University, Shanghai, P.R. China; National Center for Liver Cancer, Second Military Medical University/Naval Medical University, Shanghai, P.R. China.

Methods in Enzymology
|November 7, 2025
PubMed

Insights

Transketolase inhibitors show potential for cancer therapy by modulating metabolic pathways. This study systematically evaluated a transketolase inhibitor's binding affinity and metabolic regulatory activity, confirming its anti-tumor potential.

Area of Science:

  • Biochemistry
  • Enzymology
  • Metabolic Pathways

Background:

  • Transketolase (TKT) is a key enzyme in the pentose phosphate pathway, crucial for nucleotide synthesis and tumorigenesis.
  • TKT inhibitors offer therapeutic potential for cancer and infectious diseases by modulating enzyme activity and metabolic flux.

Purpose of the Study:

  • To systematically evaluate the binding affinity and metabolic regulatory activity of a transketolase inhibitor.
  • To assess the anti-tumor activity of the TKT inhibitor using patient-derived organoids.

Main Methods:

  • Binding affinity assessed via 2D TKT protein biological chromatography, DARTS, CETSA, SPR, competitive binding, and molecular docking.
  • Metabolic regulatory activity determined using spectrophotometric assays and targeted metabolite analysis.
  • Anti-tumor activity evaluated in patient-derived organoids.

Main Results:

  • Comprehensive characterization of a transketolase inhibitor's binding and metabolic effects.
  • Demonstrated anti-tumor activity of the inhibitor in patient-derived organoids.
  • Established the inhibitor's potential for modulating metabolic flux.

Conclusions:

  • The evaluated transketolase inhibitor exhibits significant potential for therapeutic applications, particularly in oncology.
  • This systematic evaluation provides a foundation for further drug development targeting transketolase.
  • The inhibitor effectively modulates metabolic pathways and demonstrates anti-tumor efficacy.
Keywords:
Binding affinityInhibitorsMetabolic regulatory activityTransketolase

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