A cell-based fluorescent assay for FAP inhibitor discovery

Area of Science:

• Biochemistry
• Assay Development
• Drug Discovery

Background:

• Fibroblast activation protein (FAP) is a target for cancer therapy.
• Existing methods for FAP inhibitor discovery can be time-consuming.
• A need exists for a rapid and efficient assay to screen FAP inhibitors.

Purpose of the Study:

• To develop a convenient and fast cell-based fluorescent assay for discovering FAP inhibitors.
• To optimize assay conditions for reliable IC50 determination.
• To validate the assay's utility with known FAP inhibitors.

Main Methods:

• Utilized U87MG cell line and FAP-specific substrate Suc-Gly-Pro-AMC.
• Developed a 96-well plate assay with phosphate-buffered saline incubation.
• Optimized substrate specificity, cross-reaction, and assay conditions.
• Determined IC50 values for seven known FAP inhibitors.

Main Results:

• The assay provides fast determination of multiple IC50 values within 30 minutes.
• Optimized conditions ensured assay reliability and specificity.
• Results for known inhibitors were consistent with reported trends.
• The assay demonstrated practical utility for FAP inhibitor screening.

Conclusions:

• A practical and efficient cell-based fluorescent assay for FAP inhibitor discovery has been established.
• This assay accelerates the identification and characterization of novel FAP inhibitors.
• The developed method is valuable for advancing FAP-targeted therapeutics.