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-GlyPro substrates, which DPP-4 cleaved poorly, suggesting an N-acylGly-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 ϳ9-to ϳ5400-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.Tumor-associated stromal cells can promote epithelial tumorigenesis (1, 2), suggesting that stromal proteins may represent novel therapeutic targets. Fibroblast activation protein (FAP), 2 a transmembrane serine peptidase, is one potential target because it is highly expressed by stromal fibroblasts in most epithelial cancers (3-7). Increased FAP expression in tumors correlates with increased FAP activity relative to normal tissues (8, 9), and FAP overexpression promotes tumorigenesis in xenograft models (10 -12). This effect requires catalytically active FAP (12), suggesting that FAP activity promotes tumor growth and that FAP inhibition may have therapeutic value.FAP belongs to the prolyl peptidase family, which comprises serine proteases that typically cleave peptide substrates after a proline residue. This family has been implicated in several diseases, including diabetes, cancer, and mood disorders (13,14), and includes dipeptidyl peptidase-4 (DPP-4), DPP-7, DPP-8, DPP-9, prolyl oligopeptidase, acylpeptide hydrolase, and prolyl carboxypeptidase. These proteases differ in structure at the N terminus, but each has a C-terminal ␣␤-hydrolase domain that contains the catalytic Ser, Asp, and His residues. FAP, like its most closely related family member, DPP-4, is a type II transmembrane protein; both have a short cytoplasmic tail, a transmembrane domain, and a ␤-propeller domain containing several sites of N-linked glycosylation (5,(15)(16)(17)(18)(19)(20). Crystallographic data for FAP and DPP-4 show that the ␤-propeller has important substrate ...

Fibroblast activation protein (FAP) is a serine protease of undefined endopeptidase specificity implicated in tumorigenesis. To characterize FAP's P 4 -P 0 2 specificity, we synthesized intramolecularly quenched fluorescent substrate sets based on the FAP cleavage site in a 2 -antiplasmin (TSGP-NQ). FAP required substrates with Pro at P 1 and Gly or D D-amino acids at P 2 and preferred small, uncharged amino acids at P 3 , but tolerated most amino acids at P 4 , P 0 1 and P 0 2 . These substrate preferences allowed design of peptidyl-chloromethyl ketones that inhibited FAP, but not the related protease, dipeptidyl peptidase-4. Thus, FAP is a narrow specificity endopeptidase and this can be exploited for inhibitor design.

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Numerous studies implicate the prolyl peptidase, fibroblast activation protein (FAP) in tumorigenesis; however, FAP-selective inhibitors have not yet been developed to fully validate FAP as a therapeutic target. Herein, we review recent efforts aimed at validating and inhibiting FAP for cancer therapy and highlight future directions for successful targeting of this prolyl peptidase.

Fibroblast activation protein (FAP) and dipeptidyl peptidase-4 (DPP-4) are highly homologous serine proteases of the prolyl peptidase family and therapeutic targets for cancer and diabetes, respectively. Both proteases display dipeptidyl peptidase activity, but FAP alone has endopeptidase activity. FAP Ala657, which corresponds to DPP-4 Asp663, is important for endopeptidase activity; however, its specific role remains unclear, and it is unknown whether conserved DPP-4 substrate binding residues support FAP endopeptidase activity. Using site-directed mutagenesis and kinetic analyses, we show here that Ala657 and five conserved active site residues (Arg123, Glu203, Glu204, Tyr656, and Asn704) promote FAP endopeptidase activity via distinct mechanisms of transition state stabilization (TSS). The conserved residues provide marked TSS energy for both endopeptidase and dipeptidyl peptidase substrates, and structural modeling supports their function in binding both substrates. Ala657 also stabilizes endopeptidase substrate binding and additionally dictates FAP reactivity with transition state inhibitors, allowing tight interaction with tetrahedral intermediate analogues but not acyl-enzyme analogues. Conversely, DPP-4 Asp663 stabilizes dipeptidyl peptidase substrate binding and permits tight interaction with both transition state analogues. Structural modeling suggests that FAP Ala657 and DPP-4 Asp663 confer their contrasting effects on TSS by modulating the conformation of conserved residues FAP Glu204 and DPP-4 Glu206. FAP therefore requires the combined function of Ala657 and the conserved residues for endopeptidase activity.