Selective Inhibition of Fibroblast Activation Protein Protease Based on Dipeptide Substrate Specificity

• Published in: The Journal of biological chemistry 2006-03, Vol.281 (11), p.7437-7444
• Main Authors: Edosada, Conrad Yap, Quan, Clifford, Wiesmann, Christian, Tran, Thuy, Sutherlin, Dan, Reynolds, Mark, Elliott, J. Michael, Raab, Helga, Fairbrother, Wayne, Wolf, Beni B.
• Format: Article
• Language: English
• Subjects: Acetylcysteine - analogs & derivatives; Acetylcysteine - chemistry; Adenosine Deaminase - chemistry; Amino Acid Motifs; Antigens, Neoplasm - chemistry; Biomarkers, Tumor - antagonists & inhibitors; Biomarkers, Tumor - chemistry; Biotin - chemistry; Cell Line; Chromatography, Gel; Cloning, Molecular; Dimerization; Dipeptidyl Peptidase 4 - chemistry; DNA, Complementary - metabolism; Dose-Response Relationship, Drug; Fibroblasts - metabolism; Gelatinases; Glycoproteins - chemistry; Humans; Hydrolysis; Kinetics; Light; Membrane Proteins; Models, Chemical; Models, Molecular; Peptide Hydrolases - chemistry; Peptides - chemistry; Protein Binding; Scattering, Radiation; Serine Endopeptidases - chemistry; Substrate Specificity; Time Factors
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M511112200

Fibroblast activation protein (FAP) is a transmembrane serine peptidase that belongs to the prolyl peptidase family. FAP has been implicated in cancer; however, its specific role remains elusive because inhibitors that distinguish FAP from other prolyl peptidases like dipeptidyl peptidase-4 (DPP-4) have not been developed. To identify peptide motifs for FAP-selective inhibitor design, we used P2-Pro1 and acetyl (Ac)-P2-Pro1 dipeptide substrate libraries, where P2 was varied and substrate hydrolysis occurs between Pro1 and a fluorescent leaving group. With the P2-Pro1 library, FAP preferred Ile, Pro, or Arg at the P2 residue; however, DPP-4 showed broad reactivity against this library, precluding selectivity. By contrast, with the Ac-P2-Pro1 library, FAP cleaved only Ac-Gly-Pro, whereas DPP-4 showed little reactivity with all substrates. FAP also cleaved formyl-, benzyloxycarbonyl-, biotinyl-, and peptidyl-Gly-Pro substrates, which DPP-4 cleaved poorly, suggesting an N-acyl-Gly-Pro motif for inhibitor design. Therefore, we synthesized and tested the compound Ac-Gly-prolineboronic acid, which inhibited FAP with a Ki of 23 ± 3 nm. This was ∼9- to ∼5400-fold lower than the Ki values for other prolyl peptidases, including DPP-4, DPP-7, DPP-8, DPP-9, prolyl oligopeptidase, and acylpeptide hydrolase. These results identify Ac-Gly-BoroPro as a FAP-selective inhibitor and suggest that N-acyl-Gly-Pro-based inhibitors will allow testing of FAP as a therapeutic target.