Sensitive quantification of fibroblast activation protein and high-throughput screening for inhibition by FDA-approved compounds

Fibroblast activation protein (FAP) has been extensively studied as a cancer biomarker for decades. Recently, small-molecule FAP inhibitors have been widely adopted as a targeting moiety of experimental theranostic radiotracers. Here we present a fast qPCR-based analytical method allowing FAP inhibition screening in a high-throughput regime. To identify clinically relevant compounds that might interfere with FAP-targeted approaches, we focused on a library of FDA-approved drugs. Using the D NA-linked I nhibitor An tibody A ssay (DIANA), we tested a library of 2,667 compounds within just a few hours and identified numerous FDA-approved drugs as novel FAP inhibitors. Among these, prodrugs of cephalosporin antibiotics and reverse transcriptase inhibitors, along with one elastase inhibitor, were the most potent FAP inhibitors in our dataset. In addition, by employing FAP DIANA in the quantification mode, we were able to determine FAP concentrations in human plasma samples. Together, our work expands the repertoire of FAP inhibitors, analyzes the potential interference of co-administered drugs with FAP-targeting strategies, and presents a sensitive and low-consumption ELISA alternative for FAP quantification with a detection limit of 50 pg/ml. • A dual method for (1) sensitive FAP quantification and (2) high-throughput FAP inhibitor screening was developed. • A library of 2,667 FDA-approved compounds was screened within hours. • Prodrugs of antimicrobial (cefetamet, cefcapene, cefditoren, tebipenem) and antiviral (adefovir, tenofovir) compounds were identified as novel FAP inhibitors. • Soluble FAP was quantified in patient plasma samples with a detection limit of 50 pg/ml.