An enzyme-coupled continuous fluorescence assay for farnesyl diphosphate synthases

Area of Science:

• Biochemistry
• Enzymology
• Drug Discovery

Background:

• Farnesyl diphosphate synthase (FDPS) is a key enzyme in metabolic pathways, including cholesterol synthesis.
• Existing assays for FDPS activity are often discontinuous, use radiolabeled substrates, or indirectly measure pyrophosphate release.
• There is a need for sensitive, continuous, and high-throughput assays to facilitate the development of FDPS inhibitors.

Purpose of the Study:

• To develop a novel, continuous coupled enzyme assay for measuring farnesyl diphosphate synthase (FDPS) activity.
• To improve upon the sensitivity and convenience of existing FDPS assays.
• To facilitate the screening of potential FDPS inhibitors.

Main Methods:

• Developed a continuous coupled enzyme assay utilizing protein farnesyltransferase (PFTase) to incorporate the farnesyl diphosphate (FPP) product into a peptide.
• Employed a dansylated peptide whose fluorescence increases upon farnesylation, allowing for real-time monitoring of FPP production.
• Validated the assay's performance in a 96-well plate format and compared its sensitivity and inhibitor IC(50) values to existing methods.

Main Results:

• The new assay demonstrates higher sensitivity compared to existing coupled enzyme assays for FDPS.
• The assay is suitable for high-throughput screening in a 96-well plate format.
• The assay accurately reproduced IC(50) values for known FDPS inhibitors, validating its reliability.

Conclusions:

• A simple, safe, and continuous assay for FDPS activity has been successfully developed.
• This assay significantly enhances the ability to screen for and identify inhibitors of FDPS.
• The method is expected to accelerate research and development targeting FDPS as a therapeutic target.