Peptide substrate profiling defines fibroblast activation protein as an endopeptidase of strict Gly₂‐Pro₁‐cleaving specificity

Abstract

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.

Phase II trial of single agent Val-boroPro (talabostat) inhibiting fibroblast activation protein in patients with metastatic colorectal cancer

“…9 Although no objective responses were seen, this single agent study allowed investigation of the pharmacodynamic effects of FAP inhibition, thus providing valuable information about the effects of Val-boroPro on FAP enzymatic activity in vivo. FAP enzymatic activity was analyzed in patient plasma samples before and during Val-boroPro treatment using an endopeptidase substrate 48,49 that cannot be cleaved by exopeptidases like DPP-IV (data not shown). FAP selective enzymatic activity was reduced during treatment compared to pre-treatment levels, indicating that Val-boroPro is able to inhibit the enzymatic activity in vivo.…”

Targeting fibroblast activation protein inhibits tumor stromagenesis and growth in mice

“…Also, the widespread expression of many of these enzymes is likely to limit their potential as therapeutic targets. In contrast, FAP (also called FAPα or seprase) has recently gained attention as a potential target, due to its tightly regulated expression in the tumor stroma and structurally defined proteolytic activity (7)(8)(9)(10)(11); however, its function in tumors is largely unknown.…”

Selective Fluorescence Probes for Dipeptidyl Peptidase ActivityFibroblast Activation Protein and Dipeptidyl Peptidase IV

“…It has been reported that FAP, but not DPP-IV, possesses endopeptidase activity (3,5,6). To confirm the endopeptidase specificity of the protease, we assayed FAP and DPP-IV against TSGP-2SPO, Ac-GP-2SBPO, and Ac-GPGP-2SBPO (Fig.2).…”

“…Although FAP and DPP-IV share a high sequence identity and may have arisen by gene duplication, it was reported that each has distinct enzyme activity profile. Apart from having prolyl exopeptidase activity like its DPP-IV homologue, FAP also functions as a prolyl endopeptidase (3,4) and has a collagenolytic activity capable of degrading gelatin and type I collagen (5,6).…”

“…Protease-targeted fluorogenic substrates using 7-amino-4-methylcoumarin (AMC) or 7-amino-4-trifluoromethyl coumarin (AFC) as a reporter have commonly been used for detecting protease activity in vitro (4)(5)(6). The fluorescence property of the coumarin reporters is masked by the peptide amide linkage.…”