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A high throughput assay for phosphoribosylformylglycinamidine synthase

  • 0The Hormel Institute, University of Minnesota, Austin, MN 55912, USA.
Slas Discovery : Advancing Life Sciences R & D +

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January 17, 2025

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January 17, 2025

View abstract on PubMed

Summary

This summary is machine-generated.

Researchers developed a new assay to detect inhibitors of phosphoribosylformylglycinamidine synthase (PFAS), an enzyme crucial in cancer metabolism. This high-throughput screening assay is key for discovering new liver cancer drugs.

Area Of Science

  • Biochemistry
  • Enzymology
  • Cancer Metabolism

Background

  • Metabolic reprogramming of purine biosynthesis is a hallmark of cancer.
  • Phosphoribosylformylglycinamidine synthase (PFAS) is critical in de novo purine biosynthesis and prognostic for liver cancer survival.
  • No specific inhibitors of PFAS activity are currently known, highlighting a therapeutic gap.

Purpose Of The Study

  • To develop a novel, continuous, spectrophotometric assay for the synthase domain of PFAS.
  • To establish an assay suitable for high-throughput screening (HTS) to identify PFAS inhibitors.
  • To facilitate the discovery of small molecule inhibitors for PFAS as potential cancer therapeutics.

Main Methods

  • Developed a mechanism-based fluorescent assay utilizing the acid phosphatase substrate 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP).
  • Characterized PFAS enzyme kinetics, determining a K<sub>M</sub> of 108 ± 7 µM for DiFMUP turnover.
  • Optimized and miniaturized the assay for a 1,536-well format and performed a pilot HTS using the LOPAC<sup>1280</sup> library.

Main Results

  • The developed assay demonstrated excellent performance metrics for HTS, including an average Z' of 0.94 ± 0.02 and signal to noise of 5.01 ± 0.06.
  • Pilot HTS yielded a hit rate of 1.18%, indicating the assay's effectiveness in identifying potential inhibitors.
  • The assay showed excellent inter-plate correlation, ensuring reproducibility and reliability.

Conclusions

  • A robust and sensitive continuous spectrophotometric assay for PFAS synthase activity has been established.
  • This assay is amenable to HTS and provides a critical tool for advancing PFAS enzymology studies.
  • The assay will serve as a foundation for discovering functional probes and developing novel small molecule inhibitors for cancer therapy.

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