Identification of selective and potent inhibitors of fibroblast activation protein and prolyl oligopeptidase

Abstract

Fibroblast activation protein (FAP) is a serine protease selectively expressed on reactive stromal fibroblasts of epithelial carcinomas. It is widely believed to play a role in tumor invasion and metastasis and therefore to represent a potential new drug target for cancer. Investigation into its biological function, however, has been hampered by the current unavailability of selective inhibitors. The challenge has been in identifying inhibitors that are selective for FAP over both the dipeptidyl peptidases (DPPs), with which it shares exopeptidase specificity, and prolyl oligopeptidase (PREP), with which it shares endopeptidase specificity. Here, we report the first potent FAP inhibitor with selectivity over both the DPPs and PREP, N-(pyridine-4-carbonyl)-d-Ala-boroPro (ARI-3099, 6). We also report a similarly potent and selective PREP inhibitor, N-(pyridine-3-carbonyl)-Val-boroPro (ARI-3531, 22). Both are boronic acid based inhibitors, demonstrating that high selectivity can be achieved using this electrophile. The inhibitors are stable, easy to synthesize, and should prove to be useful in helping to elucidate the biological functions of these two unique and interesting enzymes, as well as their potential as drug targets.

Figure 1

P₁ and P₂ specificity of FAP. Percentage of intact peptide remaining following a 24 h incubation at 37 °C of FAP with either the GXYSWS-NH₂ or Ac-GXYSWS-NH₂ peptide library for P₁ analysis or the XPYSWS-NH₂ or Ac-XPYSWS-NH₂ peptide library for P₂ analysis. The dark columns represent the peptide libraries with a free N-terminus, while the light columns represent the acetyl blocked libraries. Error bars show the mean ± SEM.

Figure 2

Lineweaver–Burk plots for inhibition of FAP and PREP by 6. FAP (A) and PREP (B) either alone (closed squares) or with inhibitor concentrations equal to 0.5 IC₅₀ (open squares), IC₅₀ (closed squares), or 2 IC₅₀ (open squares) were incubated with varying concentrations of Suc-GP-AMC ((0.1–5)K_m) at 37 °C.

Figure 3

Inhibition of cellular prolyl endopeptidase activity in mock and murine FAP transfected HEK293 cells. Mock (dark columns) and mFAP transfected (light columns) HEK293 cells were incubated with cell culture medium alone or 1 μM 2, 6, or 22 for 15 min at 37 °C. The endopeptidase activity was assessed via cleavage of a Z-GP-AMC substrate: ***, P < 0.0001 vs no inhibitor control. Error bars show the mean ± SEM.

Figure 4

Effect of the presence of PREP on FAP inhibition by 6. (A) Recombinant FAP (1 nM) was mixed with varying concentrations of recombinant PREP and assayed for FAP inhibition by 36 nM (IC₅₀) or 1 μM 6. (B) Standard curves were prepared for recombinant FAP (closed circles) and recombinant PREP (open circles) activity using 25 μM FAP specific substrate or Z-GP-AMC, respectively. FAP activity associated with 25 000 HEK-mFAP cells (red star) was measured using an FAP specific substrate and determined to be 2 nM. PREP activity associated with mock transfected HEK293 cells (blue star) was measured using Z-GP-AMC and determined to be 2.9 nM. This yields a cellular PREP to FAP ratio of 1:1.5. Error bars show the mean ± SEM.

Figure 5

Cytotoxicity of 6 in mFAP transfected HEK293 cells. Cells were treated with varying concentrations of 6 (light columns), 20 (dark columns), or bortezomib as a positive control (striped columns) for 48 h. Cytotoxicity was assessed using a CellTiter-Blue cell viability assay with calculation of percent cell viability based on vehicle treated control cells. Error bars show the mean ± SEM.