A high throughput assay for phosphoribosylformylglycinamidine synthase

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_M 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 LOPAC¹²⁸⁰ 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.