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 DNA-linked Inhibitor Antibody Assay (DIANA), we tested a library of 2667 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.
- MeSH
- cefalosporiny chemie farmakologie MeSH
- endopeptidasy * metabolismus MeSH
- knihovny malých molekul farmakologie chemie MeSH
- lidé MeSH
- membránové proteiny * antagonisté a inhibitory metabolismus MeSH
- molekulární struktura MeSH
- rychlé screeningové testy * MeSH
- schvalování léčiv MeSH
- serinové endopeptidasy * metabolismus MeSH
- Úřad Spojených států pro potraviny a léky MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- želatinasy * antagonisté a inhibitory metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Spojené státy americké MeSH
Identifying protein targets of bioactive small molecules often requires complex, lengthy development of affinity probes. We present a method for stochastic modification of small molecules of interest with a photoactivatable phenyldiazirine linker. The resulting isomeric mixture is conjugated to a hydrophilic copolymer decorated with biotin and a fluorophore. We validated this approach using known inhibitors of several medicinally relevant enzymes. At least a portion of the stochastic derivatives retained their binding to the target, enabling target visualization, isolation, and identification. Moreover, the mix of stochastic probes could be separated into fractions and tested for binding affinity. The structure of the active probe could be determined and the probe resynthesized to improve binding efficiency. Our approach can thus enable rapid target isolation, identification, and visualization, while providing information required for subsequent synthesis of an optimized probe.
- MeSH
- afinitní značky chemická syntéza chemie účinky záření MeSH
- aspartátové endopeptidasy antagonisté a inhibitory chemie MeSH
- biotin chemie MeSH
- diazomethan analogy a deriváty chemická syntéza účinky záření MeSH
- fluoresceiny chemie MeSH
- fluorescenční barviva chemie MeSH
- glutamátkarboxypeptidasa II antagonisté a inhibitory chemie MeSH
- hmotnostní spektrometrie metody MeSH
- inhibitory enzymů chemická syntéza chemie účinky záření MeSH
- konfokální mikroskopie metody MeSH
- kyseliny polymethakrylové chemie MeSH
- lidé MeSH
- membránové proteiny antagonisté a inhibitory chemie MeSH
- nádorové buněčné linie MeSH
- proteomika metody MeSH
- serinové endopeptidasy chemie MeSH
- ultrafialové záření MeSH
- želatinasy antagonisté a inhibitory chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Fibroblast activation protein (FAP, seprase) is a serine protease with post-proline dipeptidyl peptidase and endopeptidase enzymatic activity. FAP is upregulated in several tumor types, while its expression in healthy adult tissues is scarce. FAP molecule itself and FAP+ stromal cells play an important although probably context-dependent and tumor type-specific pathogenetic role in tumor progression. We provide an overview of FAP expression under both physiological and pathological conditions with focus on human malignancies. We also review and critically analyze the results of studies which used various strategies for the therapeutic targeting of FAP including the use of low molecular weight inhibitors, FAP activated prodrugs, anti-FAP antibodies and their conjugates, FAP-CAR T cells, and FAP vaccines. A unique enzymatic activity and selective expression in tumor microenvironment make FAP a promising therapeutic target. A better understanding of its role in individual tumor types, careful selection of patients, and identification of suitable combinations with currently available anticancer treatments will be critical for a successful translation of preclinically tested approaches of FAP targeting into clinical setting.
- MeSH
- cílená molekulární terapie metody MeSH
- inhibitory enzymů terapeutické užití MeSH
- lidé MeSH
- membránové proteiny antagonisté a inhibitory genetika metabolismus MeSH
- nádorové mikroprostředí účinky léků genetika MeSH
- nádory farmakoterapie genetika metabolismus MeSH
- regulace genové exprese enzymů účinky léků MeSH
- regulace genové exprese u nádorů účinky léků MeSH
- serinové endopeptidasy genetika metabolismus MeSH
- želatinasy antagonisté a inhibitory genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Proteases are directly involved in cancer pathogenesis. Expression of fibroblast activation protein (FAP) is upregulated in stromal fibroblasts in more than 90% of epithelial cancers and is associated with tumor progression. FAP expression is minimal or absent in most normal adult tissues, suggesting its promise as a target for the diagnosis or treatment of various cancers. Here, we report preparation of a polymer conjugate (an iBody) containing a FAP-specific inhibitor as the targeting ligand. The iBody inhibits both human and mouse FAP with low nanomolar inhibition constants but does not inhibit close FAP homologues dipeptidyl peptidase IV, dipeptidyl peptidase 9, and prolyl oligopeptidase. We demonstrate the applicability of this iBody for the isolation of FAP from cell lysates and blood serum as well as for its detection by ELISA, Western blot, flow cytometry, and confocal microscopy. Our results show the iBody is a useful tool for FAP targeting in vitro and potentially also for specific anticancer drug delivery.
- MeSH
- ELISA MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- membránové proteiny antagonisté a inhibitory chemie MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- polymery chemie MeSH
- průtoková cytometrie MeSH
- serinové endopeptidasy chemie MeSH
- western blotting MeSH
- želatinasy antagonisté a inhibitory chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
HYL-20 (GILSSLWKKLKKIIAK-NH2) is an analogue of a natural antimicrobial peptide (AMP) previously isolated from the venom of wild bee. We examined its antimicrobial activity against three strains of Enterococcus faecalis while focusing on its susceptibility to proteolytic degradation by two known proteases-gelatinase (GelE) and serine protease (SprE)-which are secreted by these bacterial strains. We found that HYL-20 was primarily deamidated at its C-terminal which made the peptide susceptible to consecutive intramolecular cleavage by GelE. Further study utilising 1,10-phenanthroline, a specific GelE inhibitor and analogous peptide with D-Lys at its C-terminus (HYL-20k) revealed that the C-terminal deamidation of HYL-20 is attributed to not yet unidentified protease which also cleaves internal peptide bonds of AMPs. In contrast to published data, participation of SprE in the protective mechanism of E. faecalis against AMPs was not proved. The resistance of HYL-20k to C-terminal deamidation and subsequent intramolecular cleavage has resulted in increased antimicrobial activity against E. faecalis grown in planktonic and biofilm form when compared to HYL-20.
- MeSH
- antibakteriální látky chemická syntéza metabolismus farmakologie MeSH
- bakteriální proteiny antagonisté a inhibitory chemie metabolismus MeSH
- biofilmy účinky léků růst a vývoj MeSH
- Enterococcus faecalis účinky léků enzymologie růst a vývoj ultrastruktura MeSH
- fenantroliny farmakologie MeSH
- inhibitory enzymů farmakologie MeSH
- kationické antimikrobiální peptidy chemická syntéza metabolismus farmakologie MeSH
- mikrobiální testy citlivosti MeSH
- plankton účinky léků enzymologie růst a vývoj ultrastruktura MeSH
- proteolýza MeSH
- sekvence aminokyselin MeSH
- serinové endopeptidasy chemie metabolismus MeSH
- substituce aminokyselin MeSH
- včely chemie fyziologie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- želatinasy antagonisté a inhibitory chemie metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
