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Originally published In Press as doi:10.1074/jbc.M511112200 on January 12, 2006

J. Biol. Chem., Vol. 281, Issue 11, 7437-7444, March 17, 2006
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Selective Inhibition of Fibroblast Activation Protein Protease Based on Dipeptide Substrate Specificity*Formula

Conrad Yap Edosada{ddagger}, Clifford Quan§, Christian Wiesmann, Thuy Tran§, Dan Sutherlin§, Mark Reynolds§, J. Michael Elliott||, Helga Raab||, Wayne Fairbrother, and Beni B. Wolf{ddagger}1

From the Departments of {ddagger}Molecular Oncology, §Medicinal Chemistry, Protein Engineering, and ||Protein Chemistry, Genentech, Inc., South San Francisco, California 94080

Received for publication, October 12, 2005 , and in revised form, December 15, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {alpha}beta-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 beta-propeller domain containing several sites of N-linked glycosylation (5, 15–20). Crystallographic data for FAP and DPP-4 show that the beta-propeller has important substrate binding sites and suggest that this domain precludes access of large substrates to the {alpha}beta-hydrolase domain (15–21).

DPP-4 regulates biological processes by cleaving regulatory peptides of < ~10 kDa, including glucagon-like peptide-1, glucose-dependent insulinotropic peptide, and stromal-derived factor-1 (22–25). DPP-4 cleaves these peptides via a well characterized dipeptidyl peptidase activity that removes P2-Pro1 or P2-Ala1 dipeptides, (P2 represents any amino acid) from the N terminus of the substrate. DPP-4 similarly cleaves P2-Pro1-based synthetic peptides and does so with high catalytic efficiency and broad specificity (25, 26). In contrast, endogenous peptide substrates of FAP are not known, and the activity of the protease against synthetic substrates remains poorly characterized. FAP also cleaves proteins such as gelatin (8, 27) and {alpha}2-antiplasmin (28), suggesting that the two proteases cleave distinct substrates.

The dipeptidase inhibitor Val-BoroPro shows efficacy in tumor models (12, 29) that correlates with FAP inhibition (12). However, because Val-BoroPro also inhibits DPP-4, -7, -8, and -9 (30–32), its mechanism of action remains unclear. To overcome this lack of selectivity, we sought here to define the substrate specificity of FAP with the goal of identifying a FAP-selective motif for inhibitor design. We identified N-acyl-Gly-Pro dipeptides as FAP-selective substrate motifs and synthesized a representative boronic acid inhibitor, Ac-Gly-BoroPro. This inhibitor showed ~9- to ~5400-fold selectivity for FAP inhibition relative to other prolyl peptidases, suggesting that N-acyl-Gly-Pro-based inhibitors will aid in testing whether FAP is a valid therapeutic target.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Ala-Pro-7-amino-4-trifluoromethylcoumarin (AFC), Phe-Pro-AFC, Gly-Pro-AFC, Ile-Pro-AFC, acetyl (Ac)-Gly-Pro-AFC, and Lys-Ala-AFC were from Enzyme Systems Products. Benzyloxycarbonyl (Z)-Gly-Pro-7-amino-4-methylcoumarin (AMC), Gly-Ala-AMC, Ac-Ala-AMC, and amino acid derivatives were from Bachem. An Ac-P2-Pro1-AFC substrate library, where P2 was varied with all amino acids (except Cys and Trp), was custom synthesized by Enzyme Systems Products. N-substituted-Gly-Pro-7-amino-4-methyl-3-carbamoylcoumarin (AMCC) substrates and a P2-Pro1-AMCC substrate library were prepared essentially as described by Maly et al. (33) with the exception that 7-amino-4-methyl-3-coumarinylacetic acid was used as the fluorophore. Acetylated substrates were prepared by treating peptides on resin with acetic anhydride in 10% triethylamine/dichloromethane until the resin was negative to the Kaiser ninhydrin test (34). Formylated substrates were prepared as described (35). Ac-Gly-Proline boronic acid (BoroPro) was synthesized as described by Gibson et al. (36) except that Ac-Gly was substituted for Val, making deprotection unnecessary. Substrates and inhibitors were purified by reverse phase chromatography and verified by matrix-assisted laser-desorption ionization mass spectrometry. N-glycanase was from Sigma.

Protease Cloning and Expression—cDNAs encoding the extracellular domains of FAP (amino acids 38–760) and DPP-4 (amino acids 39–766) were generated by polymerase chain reaction (PCR) using Quick Clone cDNA library (Stratagene) as a template. PCR products were TA-cloned into pGemT (Promega) and confirmed by DNA sequencing. Confirmed cDNAs were then subcloned into pFLAG-CMV1 (Sigma) for expression as N-terminal FLAG-tagged proteins. Plasmids containing full-length DPP-7, DPP-8, DPP-9, prolyl oligopeptidase, and acylpeptide hydrolase were obtained from Origene and used as templates to generate pFLAG-CMV1 expression constructs encoding each protease as above. These constructs encoded amino acids 26–492 of DPP-7, 2–883 of DPP-8, 2–864 of DPP-9, 2–710 of prolyl oligopeptidase, and 2–732 of acylpeptide hydrolase.

For protein production, we transfected 293 cells with plasmids encoding proteases using calcium phosphate and purified proteins from serum-free conditioned medium by affinity chromatography with M2-anti-FLAG resin (Sigma). Proteins were >95% pure as determined by SDS-PAGE with Coomassie Blue staining, with the exception of DPP8, which had a purity of ~70%. Protein concentrations were determined by the bichinconic acid method (Bio-Rad).

Gel Filtration Chromatography and Light Scattering Analysis—To calculate the stoichiometry of purified FAP and DPP-4, we measured the molecular mass of each protease in solution using multiangle light scattering in combination with gel filtration chromatography and interferometric refractometry. This method allows accurate determination of the molecular mass of a protein based on protein concentration, refractive index, and the degree of light scattering (37). Proteases (50 µg) in 50 mM Tris (pH 7.4), and 100 mM NaCl were loaded onto a Shodex KW-803 gel filtration column (flow rate 0.5 ml/min). The column was developed using an Agilent 1100 HPLC system with a MiniDawn 3-angle light scattering detector (Wyatt Technology) and an OPTILAB DSP interferometric refractometer (Wyatt Technology) in line. ASTRA software was used for molecular mass calculations.

Protease Assays—Protease activity was monitored continuously using a SpectraMax M2 microplate reader (Molecular Devices) in the kinetic mode. Assays were conducted at 23 °C in 50 mM Tris (pH 7.4), 100 mM NaCl, 1 mM EDTA. The excitation/emission wavelengths were 360/460 nm for AMC/AMCC substrates and 400/505 nm for AFC substrates. Standard curves of the appropriate fluorescent product versus concentration were used to convert relative fluorescence units to moles of product produced. Generally, kinetic constants (kcat, Km) were determined with initial rate (v0) measurements, using substrate concentrations in the range of 0.1–5 Km and protease concentrations as indicated in the figure legends. Kinetic parameters were calculated from Michaelis-Menten plots (v0 versus [S]) with nonlinear regression analysis using GraphPad software. kcat values were calculated assuming that each protease was 100% active.

When saturating amounts of substrate could not be achieved, catalytic efficiencies (kcat/Km) were determined under pseudo-first order conditions ([S] <<the estimated Km) and fit to the following equation: Ln[St/S0] =-kobst, where St is the concentration of substrate remaining at time t, S0 is the initial substrate concentration, and kobs is the apparent first order substrate cleavage constant equal to (kcat/Km) x ET, the total enzyme concentration (38). The linear relationship of Ln[St/S0] versus time allows calculation of kcat/Km by dividing the slope of the plot by ET.

Inhibition Kinetics—Ki values for inhibition of proteases by Ac-Gly-BoroPro were determined using the method of progress curves for analysis of tight binding competitive inhibitors (39, 40). Briefly, proteases were added to a reaction mixture containing inhibitor and substrate (Ac-Ala-AMC for acylpeptide hydrolase, Z-Gly-Pro-AMC for prolyl oligopeptidase, and Ala-Pro-AFC for all others) in assay buffer at 23 °C. Protease activity was followed continuously as described above to monitor time-dependent inhibition. Reactions contained inhibitor concentrations at least 20-fold greater than protease concentrations, such that the protease-inhibitor complex does not significantly deplete the free inhibitor. Data were plotted as v0/vi - 1 versus [I], where v0 is the rate of substrate hydrolysis in the absence of inhibitor, vi is the steady state rate of substrate hydrolysis in the presence of inhibitor, and [I] is the concentration of Ac-Gly-BoroPro. Plots of v0/vi - 1 versus [I] were linear, and the apparent inhibition constant, Kapp, was determined from the reciprocal of the slope. Ki, the true equilibrium inhibition constant, was determined according to the following relationship: Ki = Kapp/(1+ [S]/Km), where [S] is the concentration of substrate used in the assay and Km is the Michaelis constant for substrate cleavage.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Protease Expression and Characterization—DPP-4 exists in serum as a soluble glycoprotein beginning at residue 39 (41). We therefore expressed and purified recombinant Ser-39-DPP-4 and an analogous soluble FAP molecule beginning at amino acid Thr-38. When analyzed by SDS-PAGE under reducing conditions, FAP migrated with an apparent molecular mass of 97 kDa (Fig. 1A), whereas DPP-4 migrated with a molecular mass of 105–115 kDa (Fig. 1B). These molecular masses are 15–20 kDa greater than expected based on primary amino acid sequence and decreased upon treatment with N-glycanase (not shown), indicating that each protease is N-glycosylated. To further characterize each protease, we determined molecular mass in solution using multiangle light scattering in combination with gel filtration chromatography and interferometric refractometry. This analysis showed that FAP exists predominantly as a dimer with a molecular mass of 200 ± 15 kDa (Fig. 1A). Small amounts of monomeric (elution volume 9.0 ml) and multimeric (elution volume <8.0 ml) FAP were also observed. The predominant elution peak of DPP-4 had a molecular mass of 220 ± 15 kDa (Fig. 1B), indicating a dimeric composition. The dimeric nature of our soluble protease preparations is consistent with the dimeric composition of FAP and DPP-4 crystal structures (15–19), suggesting that they are structurally intact.

Dipeptide Substrate Specificity—Although FAP cleaves certain proline-containing DPP-4 substrates (8, 15), the full spectrum of dipeptidase specificity of FAP remains undefined. Therefore, to better understand the specificity of FAP, we examined the activity of the protease against a P2-Pro1-AMCC dipeptide substrate library (Fig. 2), where P2 was varied with all amino acids (except Cys and Trp) and amide bond hydrolysis occurs between the P1 Pro and the fluorogenic leaving group, AMCC. Even though DPP-4 can cleave dipeptides with a P1 Ala residue, we limited the P1 position of our library to Pro because FAP showed little activity against P2-Ala dipeptide substrates in preliminary studies. The dipeptide cleavage profile of the library provides information regarding protease S2 subsite specificity. As shown in Fig. 2A, FAP preferred Ile, Pro, and Arg in the P2 position and showed little activity against dipeptides with Asp, His, or Asn in the P2 position. DPP-4 readily cleaved all substrates in the library, indicating broad specificity at the S2 subsite (Fig. 2B).


Figure 1
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  		FIGURE 1.
FAP and DPP-4 are dimers in solution. The extracellular domains of FAP (A) and DPP-4 (B) were expressed and purified as described under "Experimental Procedures." Each protein was then subjected to SDS-PAGE under reducing conditions (left panels) and analyzed by gel filtration chromatography and multiangle light scattering (right panels). The molecular mass value determined across the predominant peak in each chromatogram is indicated (average ± S.D.; n = 3).

 


Figure 2
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  		FIGURE 2.
FAP and DPP-4 have distinct dipeptide substrate specificities. FAP (37 nM) and DPP-4 (6.8 nM) were reacted with P2-Pro1-AMCC (1 µM; top panels) and acetyl-P2-Pro1-AFC (10 µM; bottom panels) dipeptide substrates, where P2 was varied with all amino acids (except Cys and Trp). Initial rates of substrate hydrolysis were determined as described under "Experimental Procedures." Amino acid residues are shown in single-letter code.

 
Kinetic Constants for Cleavage of Dipeptide Substrates—To extend the results obtained with the dipeptide substrate library, we determined kinetic parameters for cleavage of commercially available dipeptide substrates by FAP (Table 1). The catalytic efficiency (kcat/Km) for substrate cleavage was greatest with Ile-Pro-AFC, followed by Ala-Pro-, Gly-Pro-, and Phe-Pro-AFC, consistent with the dipeptide substrate library results (Fig. 2). With the exception of Ile-Pro-AFC, these differences reflect differences in kcat values, as the Km for each substrate was ~250 µM. The greater catalytic efficiency observed for Ile-Pro-AFC hydrolysis was due to both kcat and Km effects as the observed Km was ~2.5-fold lower than the other P2-Pro1-AFC substrates. FAP showed markedly less activity against P2-Ala1-based substrates. GA-AMC was not cleaved, and the catalytic efficiency for Lys-Ala-AFC cleavage was 400–1000-fold less than the catalytic efficiency for cleavage of P2-Pro1-based peptides (Table 1), indicating that FAP prefers Pro in the P1 position.


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  		TABLE 1
Kinetic constants (average ± S.E., n =3) for hydrolysis of dipeptide substrates by FAP and DPP-4 Substrate cleavage was at 23 °C in 50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, pH 7.4.

 


Figure 3
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  		FIGURE 3.
FAP and DPP-4 show differential activity against Gly-Pro-AFC and Ac-Gly-Pro-AFC. Substrate-velocity curves for cleavage of Gly-Pro-AFC and Ac-Gly-Pro-AFC by FAP (37 nM) (A) and DPP-4 (10.5 nM) (B) are shown. Each value represents the average ± S.E. (n = 3).

 
For comparison, we determined kinetic constants for cleavage of Ala-Pro-AFC and Gly-Pro-AFC by DPP-4 (Table 1). The catalytic efficiency for Ala-Pro-AFC hydrolysis was greater than that for Gly-Pro-AFC, consistent with the dipeptide library. Strikingly, the catalytic efficiencies for dipeptide hydrolysis by DPP-4 were consistently ~100-fold greater than observed with FAP, reflecting both an increase in kcat and decrease in Km.

N-acetyl-dipeptide Substrate Specificity—Given reports suggesting that FAP has endopeptidase activity (8, 28), we next examined the endopeptidase specificity of the protease. For this, we assayed FAP against an Ac-P2-Pro1-AFC substrate library in which the acetyl group forms an amide bond with the P2 amino acid, thereby mimicking an endopeptidase substrate. Thus, in contrast with the P2-Pro1 library, which contains a free N terminus, the acetylated library is N-terminal blocked. FAP showed a marked preference for a P2 Gly residue in the acetylated library, having little activity against all other substrates in the library (Fig. 2C). Strikingly, DPP-4 had little activity against Ac-Gly-Pro-AFC and all other substrates in the acetylated library (Fig. 2D). These data suggest that DPP-4 has limited endopeptidase activity and that FAP endopeptidase activity is restricted to Gly-Pro-containing substrates.


Figure 4
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  		FIGURE 4.
FAP and DPP-4 show differential activity against N-substituted-Gly-Pro-AMCC substrates. N-substituted-Gly-Pro-AMCC substrates (A) were synthesized as described under "Experimental Procedures." R represents the varied N substitution. FAP (37 nM) (B) and DPP-4 (6.8 nM) (C) were reacted with each substrate (25 µM) and initial substrate hydrolysis velocities determined. Rates of substrate hydrolysis relative to the rate of Gly-Pro-AMCC are shown. Each value represents the average ± S.E. (n = 3).

 
Kinetic Analysis of Gly-Pro- and Ac-Gly-Pro-AFC Hydrolysis—In light of the Ac-P2-Pro1-AFC library results, we performed kinetic analyses with Ac-Gly-Pro-AFC and Gly-Pro-AFC substrates as their hydrolysis rates may be directly compared. FAP cleaved each substrate with similar efficiency as shown in Fig. 3A. FAP showed a moderate increase in Km (330 ± 30 µM) for Ac-Gly-Pro-AFC relative to Gly-Pro-AFC (Table 1); however, a concomitant increase in kcat (7.7 ± 0.2 s-1) for the acetylated substrate resulted in an equivalent catalytic efficiency for both substrates. By contrast, DPP-4 only showed significant activity against Gly-Pro-AFC (Fig. 3B). The catalytic efficiency for cleavage of Ac-Gly-Pro-AFC by DPP-4 was equivalent to 36 ± 3 M-1 s-1 when determined under pseudo-first order conditions. This value is over four orders of magnitude lower than that for cleavage of Gly-Pro-AFC (Table 1), indicating a marked preference for the substrate with a free N terminus.


Figure 5
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  		FIGURE 5.
Ac-Gly-BoroPro selectively inhibits FAP. Various concentrations of Ac-Gly-BoroPro were reacted with FAP (1.0 nM) (A) and DPP-4 (0.1 nM) (B) in the presence of Ala-Pro-AFC (500 µM for FAP; 100 µM for DPP-4), and time-dependent inhibition of each protease was monitored. The top panels show representative progress curves for inhibition of each protease by Ac-Gly-BoroPro. Apparent inhibition constants (Kapp) were determined for each protease by plotting v0/vi - 1 against [Ac-Gly-BoroPro] (bottom panels). Each value represents the average ± S.E. (n = 3).

 
Cleavage of N-substituted-Gly-Pro-AMCC Substrates—To test whether FAP could cleave other N-substituted-Gly-Pro-based substrates, we synthesized N-blocked dipeptide substrates (N-methyl-, formyl-, succinyl-, benzyloxycarbonyl- (Z-), biotinyl-Gly-Pro-AMCC) and a peptide modeled after the FAP cleavage site in {alpha}2-antiplasmin (Ac-Thr-Ser-Gly-Pro-AMCC) (28). With the exception of succinyl-Gly-Pro-AMCC, FAP cleaved all N-substituted-Gly-Pro-AMCC substrates at 35–165% of the rate for Gly-Pro-AMCC hydrolysis (Fig. 4B), indicating that the protease tolerates other N-terminal blocking groups and a tetrapeptide substrate. Kinetic analysis with commercially available Z-Gly-Pro-AMC showed a catalytic efficiency of 7.4 ± 0.6 x 103 M-1 s-1, which is ~3-fold lower than that for Gly-Pro-AFC (Table 1), consistent with these results. In contrast with FAP, DPP-4 cleaved only N-methyl-Gly-Pro-AMCC at a rate somewhat comparable with Gly-Pro-AMCC (Fig. 4C). A low rate of hydrolysis was obtained with formyl-Gly-Pro-AMCC, but no cleavage of succinyl-, Z-, biotinyl-, or Ac-Thr-Ser-Gly-Pro-AMCC substrates was observed at concentrations up to 1 mM, indicating that DPP-4 does not tolerate N-acyl-Gly-Pro-based substrates. DPP-4 also showed little activity against commercially available Z-Gly-Pro-AMC (kcat/Km = 15 ± 1 M-1 s-1).

Ac-Gly-BoroPro Selectively Inhibits FAP—Having defined N-acyl-Gly-Pro dipeptides as FAP-selective substrates, we next asked whether the Ac-Gly-Pro motif would confer FAP-selective inhibition. For this, we coupled the Ac-Gly-Pro specificity motif to an electrophilic boronic acid moiety capable of reacting with the active site serine of the protease (42). The resulting peptide boronic acid, Ac-Gly-BoroPro, was then tested for FAP and DPP-4 inhibition. FAP reacted readily with submicromolar concentrations of Ac-Gly-BoroPro, reaching steady state inhibition levels rapidly as shown in the progress curves of Fig. 5A. In contrast, DPP-4 required higher Ac-Gly-BoroPro concentrations for inhibition and a longer time to reach steady state inhibition levels (Fig. 5B). The steady states of product formation in the absence (v0) and presence (vi) of inhibitor were used to calculate apparent inhibition constants (Kiapp) by plotting v0/vi - 1 against inhibitor concentration (Fig. 5, C and D). The calculated inhibition constants (Ki) were 23 ± 3 nM for FAP and 377 ± 18 nM for DPP-4 (Table 2 and Structure 1), indicating an ~16-fold selectivity for FAP inhibition.


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  		TABLE 2
Inhibition of prolyl peptidases by Ac-Gly-BoroPro Ki values were determined using the method of progress curves as described under "Experimental Procedures." Ac-Gly-BoroPro is shown in Structure 1.

 
Besides FAP and DPP-4, the prolyl peptidase family includes DPP-7, DPP-8, DPP-9, prolyl oligopeptidase, and acylpeptide hydrolase, and many of these proteases show ubiquitous distribution (14). To establish whether Ac-Gly-BoroPro inhibits these prolyl peptidases we cloned and expressed each protease and established assays to monitor their activity. Km values determined in these assays are given in supplemental Table S1. Next, we determined Ki values for their inhibition by Ac-Gly-BoroPro using the progress curve method described above. Strikingly, Ac-Gly-BoroPro inhibited these prolyl peptidases with Ki values ranging from ~9- to ~5400-fold higher than that for FAP inhibition (Table 2 and Structure 1), indicating that the Ac-Gly-Pro motif confers significant FAP selectivity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
FAP remains invalidated as a therapeutic target because attempts to fully block FAP activity with antibodies have not been successful (43) and FAP-selective small molecule inhibitors have not been developed. In this study, we identified Ac-Gly-Pro as a FAP-selective substrate motif and developed a FAP-selective inhibitor, Ac-Gly-BoroPro, based on this motif. Our results highlight the unique substrate specificity of FAP and suggest that N-acyl-Gly-Pro-based inhibitors will allow testing the potential of FAP as a therapeutic target.

Our data define FAP as a dual activity protease, having both dipeptidase and Gly-Pro-cleaving endopeptidase activity. This substrate specificity distinguishes FAP from other prolyl peptidases that act as single activity proteases, including DPPs-4, -7, -8, and -9, which act solely as dipeptidases (22, 23, 44–46), and prolyl oligopeptidase, which displays only endopeptidase activity (13). Additionally, the dual activity of FAP is distinct from acylpeptide hydrolase (47) and prolyl carboxypeptidase (48), which lack both dipeptidase and endopeptidase activity. Acylpeptide hydrolase acts as an N-acetyl amino acid hydrolase, and prolyl carboxypeptidase acts as a Pro-X carboxypeptidase. Thus, the unique substrate specificity of FAP is distinct from other prolyl peptidases.


Figure 6
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  		STRUCTURE 1
 
Determination of the dipeptide specificity of FAP provides insight into potential biological substrates and is in accord with known macromolecular substrates of the protease. Although endogenous FAP dipeptidase substrates are unknown, the broad specificity observed with the P2-Pro1 substrates suggests that FAP, like DPP-4, may cleave bioactive peptides beginning with a P2-Pro1 motif. Cancer-associated cytokines and chemokines represent such potential dipeptidase substrates. For endopeptidase substrates, the Gly2-Pro1 requirement we observed with acetylated dipeptide substrates is similarly found in the FAP cleavage site in {alpha}2-antiplasmin (TSGP12-N13Q) (28) and probable cleavage sites in gelatin. We have also confirmed the Gly2-Pro1 requirement using intramolecularly quenched fluorescent substrates and demonstrated that FAP prefers small uncharged amino acids at P3, but lacks specificity at P4, P1', and P2.3 Together, the data suggest that the Gly2-Pro1 motif is a critical determinant of FAP endopeptidase activity.

Based on the unique reactivity of FAP with N-acyl-Gly-Pro-based substrates, we developed Ac-Gly-BoroPro, which selectively inhibited FAP relative to other prolyl peptidases. This selectivity profile and the N-acyl linkage in Ac-Gly-BoroPro differentiate it from other boronic acid inhibitors targeting prolyl peptidases, including Val-BoroPro (30–32), N-alkyl-Gly-BoroPro (49) inhibitors, and Boro-norleucine (32) inhibitors. Val-BoroPro and N-alkyl-Gly-BoroPro inhibitors target most prolyl peptidases, whereas Boro-norleucine-based inhibitors selectively target DPP-7. Additionally, these inhibitors contain a free amine at their N terminus, which allows intra-molecular reaction with the electrophilic boron, resulting in cyclization and inhibitor inactivation. In contrast, the N-acyl-linkage in Ac-Gly-BoroPro blocks the N terminus of the inhibitor, making it less nucleophilic and therefore unlikely to cyclize. Importantly, Ac-Gly-BoroPro shows poor reactivity with DPP-8 and DPP-9 as selective inhibition of these proteases causes severe toxicity in animals (50). Specific areas for in vivo testing of FAP-selective inhibitors will include not only cancer but also other biological processes in which FAP may act, such as cirrhosis (51) and hematopoiesis (52).


Figure 7
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  		FIGURE 6.
A model for binding of substrates and inhibitors to FAP and DPP-4. A, the structure of FAP (Protein Data Bank code 1Z68; Ref. 15) superimposed onto the structure of DPP-4 (Protein Data Bank code 1NU8 [PDB] ; Ref. 21) bound to the tripeptide, diprotinA (Ile-Pro-Ile). Selected residues are shown as sticks with the carbon atoms of FAP colored cyan, DPP-4 carbon atoms colored white, and carbon atoms of diprotinA in yellow. Nitrogen atoms are colored blue and oxygen atoms red. Selected potential hydrogen bonds are depicted as blue dashed lines. Amino acids are numbered according to the FAP sequence. The only non-conserved residue, FAP Ala-657, which corresponds to DPP-4 Asp-663, is highlighted in red. Note that the substrate binding residues of FAP and DPP-4 superimpose very well and both proteins can readily accommodate the binding of a tripeptide with a free N terminus. Note that Glu-203, Glu-204, and Tyr-656 are hydrogen bonded to the positively charged N terminus of the substrate and that a P3 residue cannot be accommodated. B, model of the Ac-Gly-BoroPro inhibitor bound to FAP (15) based on the crystal structure of DPP-4 bound to diprotinA (21). Carbon atoms of the inhibitor are colored yellow and the boron in pink. Note that the N terminus of the inhibitor is acetylated and therefore not positively charged like the N terminus of the dipeptidase substrate depicted in panel A. The P2 residue must adopt a positive {phi} dihedral angle for the protease to accommodate the N-terminal acetyl group which is circled. The only amino acid that can readily adopt a positive {phi} dihedral angle is glycine. C, surface presentation of FAP with the Ac-Gly-BoroPro inhibitor docked into the substrate binding site of the protease to illustrate the steric requirements for the P2 residue. The carbon atoms of the inhibitor are indicated in yellow, the boron in pink, and the N-terminal acetyl group is circled. The left panel shows the acetyl group of the inhibitor in the accepted position with the P2 Gly in the positive {phi} conformation (+{Phi}). The right panel depicts the acetyl group of the inhibitor in the non-accepted position with the P2 Gly in the negative {phi} conformation (-{Phi}). Note when the glycine adopts a negative {phi} dihedral angle steric clashes occur between the N-terminal acetyl group of the inhibitor and the protease.

 
Although FAP and DPP-4 share a high sequence identity, our data show that each has distinct activity, with DPP-4 acting as a dipeptidase and FAP functioning as both a dipeptidase and narrow specificity endopeptidase. Recent structural studies with each protease (15, 21) suggest a mechanistic explanation for our results, as illustrated in Fig. 6. In Fig. 6A, the crystal structure of DPP-4 covalently bound to diprotinA (Ile-Pro-Ile) illustrates the interactions between this protease and a low turnover dipeptidase substrate. Superposition of this crystal structure onto the structure of FAP shows that key substrate binding residues in both proteins are in very similar positions and that diprotinA readily fits into the active site of each protease (Fig. 6A). Note that the P2 Ile residue of the substrate contains an aliphatic side chain and a free N terminus that is positively charged. The aliphatic side chain is oriented outward, away from the protease. This orientation can accommodate side chains of various sizes, which explains the broad P2 dipeptidase specificity we observed with each protease. In contrast, the N terminus of the P2 Ile is oriented toward the protease and hydrogen bonds with Glu-203, Glu-204, and Tyr-656. Because of this orientation, a P3 substrate residue cannot be accommodated.

To understand how FAP and DPP-4 interact with an endopeptidase inhibitor, we modeled Ac-Gly-BoroPro into the active site of each protease as shown in Fig. 6B. Compared with the dipeptidase substrate in Fig. 6A, the inhibitor contains an additional peptide bond linking the P2 Gly residue to the acetyl group. The acetyl group therefore mimics a P3 residue, and the N terminus is no longer positively charged. The protease can only accommodate the acetyl group when the P2 residue adopts a conformation with a positive {phi} dihedral angle as illustrated in Fig. 6B and the left panel of 6C. When the P2 residue assumes a negative {phi} conformation (Fig. 6C, right panel), the acetyl group of the inhibitor sterically clashes with the protease. Thus, endopeptidase substrates and inhibitors must contain an amino acid able to adopt a positive {phi} dihedral angle to avoid this clash. Glycine is the only natural amino acid that meets this criterion, explaining the requirement of FAP for endopeptidase substrates and inhibitors with a Gly at P2.

Based on the superposition of FAP and DPP-4 it is more difficult to comprehend why DPP-4 does not share the endopeptidase activity of FAP. Both proteases readily accommodate the endopeptidase inhibitor Ac-Gly-BoroPro (Fig. 6B), and they differ only in a single position near the N terminus of P2: FAP Ala-657, which corresponds to Asp-663 in DPP-4 (Fig. 6, A and B). Aertgeerts et al. (15) recently interchanged the Ala and Asp residues using site-directed mutagenesis and showed that the presence of Asp at position 657/663 markedly favors dipeptidase activity, whereas Ala at this position allows endopeptidase activity. These data suggest that increased acidity near the active site provided by the Asp confers a requirement for substrates with a free N terminus. The mechanism by which Ala-657 allows endopeptidase activity remains unclear and warrants future study.

In summary, we have defined the dipeptide substrate specificity of FAP and developed the FAP-selective inhibitor, Ac-Gly-BoroPro, based on FAP substrate preferences. Our findings distinguish FAP from other prolyl peptidases and suggest that N-acyl-Gly-Pro-based inhibitors will aid in elucidating FAP biology and utility as a potential therapeutic target.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1. Back

1 To whom correspondence should be addressed: Genentech, Inc., 1 DNA Way-MS42, South San Francisco, CA 94080. Tel.: 650-467-1954; Fax: 650-225-6327; E-mail: bbwolf{at}gene.com.

2 The abbreviations used are: FAP, fibroblast activation protein; Ac, acetyl; AFC, 7-amino-4-trifluoromethylcoumarin; AMC, 7-amino-4-methylcoumarin; AMCC, 7-amino-4-methyl-3-carbamoylmethylcoumarin; BoroPro, prolineboronic acid; APH, acylpeptide hydrolase; DPP, dipeptidyl peptidase; Z, benzyloxycarbonyl. Back

3 Edosada, C. Y., Quan, C., Tran, T., Pham, V., Wiessman, C., Fairbrother, W., and Wolf, B. B. (2006) FEBS Lett., in press. Back


   ACKNOWLEDGMENTS
 
We thank Avi Ashkenazi, Bob Lazarus, and Daniel Kirchhofer for critically reading the manuscript and Janie Pena for graphics support.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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