title: Selective inhibition of fibroblast activation protein protease based on dipeptide substrate specificity.
abstract: 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 P(2)-Pro(1) and acetyl (Ac)-P(2)-Pro(1) dipeptide substrate libraries, where P(2) was varied and substrate hydrolysis occurs between Pro(1) and a fluorescent leaving group. With the P(2)-Pro(1) library, FAP preferred Ile, Pro, or Arg at the P(2) residue; however, DPP-4 showed broad reactivity against this library, precluding selectivity. By contrast, with the Ac-P(2)-Pro(1) 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 K(i) of 23 +/- 3 nm. This was approximately 9- to approximately 5400-fold lower than the K(i) 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.
authors.affiliation: Department of Molecular Oncology, Genentech Inc., 1 DNA Way-MS42, South San Francisco, CA 94080, USA.
authors.forename: Conrad Yap
authors.initials: CY
authors.lastname: Edosada
authors.forename: Clifford
authors.initials: C
authors.lastname: Quan
authors.forename: Christian
authors.initials: C
authors.lastname: Wiesmann
authors.forename: Thuy
authors.initials: T
authors.lastname: Tran
authors.forename: Dan
authors.initials: D
authors.lastname: Sutherlin
authors.forename: Mark
authors.initials: M
authors.lastname: Reynolds
authors.forename: J Michael
authors.initials: JM
authors.lastname: Elliott
authors.forename: Helga
authors.initials: H
authors.lastname: Raab
authors.forename: Wayne
authors.initials: W
authors.lastname: Fairbrother
authors.forename: Beni B
authors.initials: BB
authors.lastname: Wolf
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000737:chemistry; D000243:Adenosine Deaminase / Q000737:chemistry*; D020816:Amino Acid Motifs; D000951:Antigens, Neoplasm / Q000737:chemistry; D014408:Biomarkers, Tumor / Q000037:antagonists & inhibitors* / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D002460:Cell Line; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D018819:Dipeptidyl Peptidase 4 / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D010450:Endopeptidases; D005347:Fibroblasts / Q000378:metabolism*; D018093:Gelatinases; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008027:Light; D008565:Membrane Proteins; D008956:Models, Chemical; D008958:Models, Molecular; D010447:Peptide Hydrolases / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D012542:Scattering, Radiation; D012697:Serine Endopeptidases / Q000737:chemistry; D013379:Substrate Specificity; D013997:Time Factors; D000099167:Fibroblast Activation Protein Alpha
chemical_list: D000111:Acetylcysteine; D000243:Adenosine Deaminase; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D001710:Biotin; D018076:DNA, Complementary; D018819:Dipeptidyl Peptidase 4; D010450:Endopeptidases; D018093:Gelatinases; D006023:Glycoproteins; D008565:Membrane Proteins; D010447:Peptide Hydrolases; D010455:Peptides; D012697:Serine Endopeptidases; D000099167:Fibroblast Activation Protein Alpha; C052469:N-acetyl-S-(N-methylcarbamoyl)cysteine; C042807:DPP4 protein, human