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

Article abstract of DOI:10.1021/jm400351a

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.

Full text of DOI:10.1021/jm400351a

Article
pubs.acs.org/jmc
Identification of Selective and Potent Inhibitors of Fibroblast
Activation Protein and Prolyl Oligopeptidase
Sarah E. Poplawski,†,‡ Jack H. Lai,†,‡ Youhua Li,† Zhiping Jin,† Yuxin Liu,† Wengen Wu,† Yong Wu,†
Yuhong Zhou,† James L. Sudmeier,† David G. Sanford,†,‡ and William W. Bachovchin*,†,‡
†Department of Biochemistry, Tufts University Sackler School of Biomedical Sciences, 136 Harrison Avenue, Boston, Massachusetts
02111, United States
‡Arisaph Pharmaceuticals, Inc., 100 High Street, Suite 1202, Boston, Massachusetts 02110, United States
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.
The unique expression profile of FAP, coupled with studies
indicating that it plays an important role in supporting tumor
growth and metastasis, has generated interest in FAP as a
therapeutic target.13−15 Tumors have been reported to grow
more slowly in FAP knockout mice and more rapidly when
transfected with FAP.16,17 Kramen et al. have reported that
genetic depletion of FAP-positive fibroblasts in a mouse lung
cancer model results in hypoxic necrosis of tumor and stromal
cells via elicitation of an immune response mediated by TNF-α
and IFN-γ.18 This is especially exciting, as it indicates that FAP
may promote tumor growth, at least in part, by helping to hold
the host’s antitumor immune response at bay and therefore
suggests that FAP targeted therapies may help to turn on
antitumor immunity. The mechanisms through which FAP
mediates the above effects, however, are essentially unknown. It
is not even clear whether or not its protease activity is involved,
although inhibitors have been reported to have antitumor
effects, including those mediated via enhancement of antitumor
immunity.19,20 The inhibitors used in these experiments were
not entirely specific for FAP, however, usually cross-reacting
with the DPPs, PREP, or both, rendering the connection
between the protease activity and tumor growth promoting
activities of FAP less than certain.
INTRODUCTION
■
Fibroblast activation protein (FAP) is a type II, membrane-
bound serine protease highly expressed on activated stromal
fibroblasts of essentially all epithelial cell carcinomas but not on
the cancer cells themselves, normal fibroblasts, or normal
tissues.1−4 FAP is most closely related to dipeptidyl peptidase
IV (DPPIV, EC 3.4.14.5), with which it shares 54% sequence
homology, membership in protease clan SC subfamily S9B, and
the relatively rare ability to cleave peptides after proline
residues. Other human enzymes with post-proline cleaving
activity include dipeptidyl peptidase 8 (DPP8), dipeptidyl
peptidase 9 (DPP9), dipeptidyl peptidase II (DPPII, EC
3.4.14.2), and prolyl oligopeptidase (PREP, EC 3.4.21.26).5−7
The dipeptidyl peptidases (DPPs) are exclusively exopepti-
dases, whereas PREP is exclusively an endopeptidase. FAP is
unique in having both exo- and endopeptidase activities.4,8
Post-proline cleaving enzymes are expected to have
important biological functions in part because of the unique
structure of proline but also because many bioactive peptides
contain conserved proline residues at sites that appear to serve
as key recognition elements for proteolytic processing.9 Of the
human post-proline cleaving enzymes, only the biological
function of DPPIV has thus far been discovered and described.
DPPIV regulates the incretin hormones glucagon-like peptide 1
(GLP-1) and glucose-dependent insulinotropic peptide (GIP)
and, as a result, is now a validated target for the treatment of
type 2 diabetes.10−12
Received: October 24, 2012
© XXXX American Chemical Society
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The current lack of specific FAP inhibitors is therefore an
important factor hampering investigation of FAP’s biological
functions and its potential as a new target for anticancer drugs.
The problem stems from FAP’s dual function as both an exo-
and endopeptidase. Identifying inhibitors that are specific for
FAP over the DPPs, or for FAP over PREP, is relatively simple,
and such inhibitors exist.21,22 Identifying inhibitors that are
specific for FAP over both the DPPs and PREP at the same
time is more difficult.
Tsai et al. have reported that several substituted 4-
carboxymethylpyroglutamic acid diamides are selective inhib-
itors of FAP.23 While they screened these compounds against
several of the DPPs, they did not screen against PREP, so it is
not clear whether or not these inhibitors are truly specific and
to what degree. Ryabtsova et al. described the design of a series
of acylated Gly-(2-cyano)pyrrolidines to specifically address
FAP selectivity versus PREP.24 The bulk of the inhibitors
described were not particularly potent, however, and they
ultimately concluded that “selectivity toward the endopeptidase
PREP was found to be less evident” in comparison to the other
dipeptidyl peptidases. Thus, no FAP inhibitors thus far claimed
to be specific for FAP have been demonstrated to be selective
over PREP. PREP, like DPPIV and FAP, belongs to protease
clan SC but to a different subfamily, S9A. Also similar to FAP, it
has both soluble and membrane-bound forms, although the
soluble form of PREP is generally localized the cytosol, whereas
the soluble form of FAP is in the plasma.25,26 PREP is highly
conserved in mammals and widely distributed in cells and
tissues, with especially high concentrations found in the
brain.27,28
3531, 22) displays even greater selectivity for PREP over FAP
(77 000-fold) while also not inhibiting the DPPs.
The fact that boronic acid-based compounds can discrim-
inate between enzymes with specificities as closely overlapping
as the post-proline cleaving enzymes demonstrates the high
selectivity that can be achieved with this electrophile.
RESULTS AND DISCUSSION
■
P1 and P2 Substrate Specificity of FAP. Specificity
profiling demonstrates that FAP, like all members of this family,
strongly prefers cleaving after proline residues. This applies to
both its exo- and endopeptidase activities (Figure 1, top). The
Small molecule inhibitors of serine proteases are often
divided into two categories, covalent and noncovalent.
Covalent inhibitors are those that incorporate functional
groups designed to be attacked by, and to form a covalent
bond with, the active site serine. Such functional groups have
been called transition-state analogues, serine traps, or electro-
philes. Covalent inhibitors can be reversible or irreversible.
Examples of reversible covalent inhibitors include those
incorporating aldehyde, nitrile, boronic acid, or α-ketoamide
functional groups. Examples of irreversible covalent inhibitors
include those incorporating chloromethyl ketone, phosphoro-
fluoridate, or phosphonate ester groups. At the present time,
conventional wisdom still appears to strongly prefer non-
covalent over covalent inhibitors, whether reversible or not, for
use as drugs.29 There are several reasons for this, but the most
important is a belief that covalent inhibitors are too reactive to
yield inhibitors specific enough to be safe for use in
humans.30,31 Thus, statements can be found in the literature
prior to 2000 to the effect that covalent inhibitors, including
those incorporating any of the electrophiles mentioned above
would be too reactive to be sufficiently specific to ever yield a
marketed drug.32 As of 2011, however, there are at least 39
examples of covalent inhibitors approved by the FDA. Of the
15 drugs on the market that target serine or threonine
proteases, seven, or nearly half, contain an electrophile. These
include one boronic acid, two α-ketoamides, and two nitriles.33
Here we report the design and characterization of boronic
acid based inhibitors for FAP and PREP that combine low
nanomolar potency with high selectivity. N-(Pyridine-4-carbon-
yl)-D-Ala-boroPro (ARI-3099, 6) is more than 350-fold
selective for FAP over PREP, with negligible potency against
the DPPs, while N-(pyridine-3-carbonyl)-Val-boroPro (ARI-
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.
P2 specificity, however, is quite different for the exo- and
endopeptidase activities (Figure 1, bottom). As an exopepti-
dase, FAP accepts essentially all residues at P2 with proline,
large, hydrophobic, aromatic, and positively charged residues
preferred most and negatively charged side chains preferred
least. As an endopeptidase, FAP exhibits an absolute require-
ment for glycine at P2; no other naturally occurring amino acid
is accepted. This agrees with previous FAP specificity studies
and explains why many FAP inhibitors are based upon an X-
Gly-Pro sequence.21,34,35 While this structural motif confers
specificity against DPPs, it generally does not prevent cross-
reactivity with PREP.
Selective Inhibitors of FAP. A recent review cited Tran et
al. as having reported that a nonpeptidic boroproline inhibitor,
1-(2-(1-oxoisoindolin-2-acetyl)-boroPro (4), is approximately
400-fold selective for FAP over PREP.36 Tran et al. actually
reported that 4 is only 3-fold selective for PREP over FAP,22
a
finding with which our results essentially agree (Table 1).
Furthermore, Tran et al. reported that a P2 D-alanine-containing
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a
Table 1. FAP Selectivity of Compounds 1−6
a
The FAP selectivity is equal to IC50(PREP)/IC50(FAP). IC50 values are expressed with SEM.
inhibitor, N-acetyl-D-Ala-boroPro (3), exhibits modest potency
(Ki of 350 nM) and specificity (7.7-fold) for FAP over PREP.
Our results agree that 3 inhibits FAP with modest potency but
disagree that it is selective for FAP over PREP; we find instead
that 3 is modestly selective for PREP over FAP. Nevertheless,
both results suggest that FAP is capable of tolerating a P2 D-
alanine in the context of an X-boroPro-based inhibitor.
Of the compounds reported here, Gly-boroPro (1)
demonstrates the greatest degree of selectivity for FAP over
PREP (900-fold). Because of the presence of the free N-
terminal amino group, however, 1 exhibits essentially no
selectivity for FAP over the DPPs. Addition of various N-
terminal blocking groups to 1, as shown with 2 and 5, provides
increased potency against FAP, elimination of cross-reactivity
with the DPPs, but little to no selectivity for FAP over PREP.
Replacement of the P2 glycine of 5 with a D-alanine (6)
provides 360-fold selectivity for FAP over PREP while retaining
FAP potency in the low nanomolar range.
Optimization of the N-Terminal Blocking Group. A
number of variations of the X-D-Ala-boroPro scaffold were
assessed, with 6 possessing the most optimal combination of
potency and selectivity. Both the presence of the nitrogen and
its position in the pyridine ring proved to be important. The
former provides selectivity for FAP, while the latter provides
additional potency, as is evident in comparing 6 with 7 and 8
(Table 2). The nitrogen atom at the 4- or para position appears
optimal. Without a solved structure of FAP complexed to 6, we
cannot be certain why this is the case. However, through
molecular modeling beginning with the existing homologous X-
ray crystal structure of the DPPIV tetrahedral intermediate
complexed with diprotinA, as performed by previous
researchers,21,34 we discovered a reasonable hypothesis for
the role of the pyridine group. Positioning the -boroPro moiety
into the hydrophobic S1 pocket of FAP in a manner consistent
with the diprotinA structure, the P2 D-alanyl and 4-
pyridylcarbonyl groups are oriented for an optimal fit. The
resulting model strongly suggests that a protonated pyridine
nitrogen atom in the 4-position could form a hydrogen bond
with the backbone carbonyl oxygen of Glu204. The pyridyl
nitrogen should have a pKa of approximately 5.5, so it would
not be unreasonable for it to be protonated in the complex.
PREP does not have a corresponding carbonyl oxygen or any
other functional group that could act as a hydrogen bond
acceptor in this position.
Addition of halo substituents (F and Cl) to the pyridine ring
also appears to further improve potency against FAP but with a
2- to 3-fold loss in selectivity over PREP (9−12). There are
several possible explanations for this. The electron withdrawing
effect of fluorine and chlorine would be expected to reduce the
pKa of the pyridyl nitrogen, making it a stronger acid. Although
this would reduce the fraction of protonated inhibitor in
solution, it would make the pyridyl N−H a stronger hydrogen
bond donor in the complex. Fluorine at this position enhances
binding considerably less than chlorine, however, suggesting
that the effect is more likely mediated by a steric or torsion
angle change rather than an electronic effect. As torsion angle
changes could have an effect on PREP binding as well, this may
explain why these inhibitors are less selective for FAP over
PREP in comparison to 6.
Effects of P2 D-Amino Acid Variation on FAP and PREP
Inhibition. The finding that a D-alanine in P2 provides
significant selectivity for FAP over PREP prompted us to
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a
Table 2. Comparison of FAP vs PREP against a Panel of N-Substituted D-Ala-boroPro-Based Inhibitors
a
The FAP selectivity is equal to IC50(PREP)/IC50(FAP). IC50 values are expressed with SEM.
examine other D-amino acids in this position (Table 3). For this
purpose we employed a dipeptide-boroPro scaffold with an N-
(4-quinolinoyl) group substituted at the N-terminus. As before,
placement of D-alanine at the P2 position in this scaffold yields a
potent and highly selective FAP inhibitor (13). Increasing the
size of the D-amino acid at P2 quickly leads to losses of both
potency and FAP selectivity. In fact, the larger P2 substitutions
lead to such large and selective losses in potency toward FAP
that the inhibitors become selective for PREP over FAP (15,
17, 18, and 19).
Selective Inhibitors of PREP. Identifying potent and
selective inhibitors for PREP is a far more simple task than for
FAP. N-Terminal substitution provides, at once, selectivity for
PREP over all of the DPPs and over the exopeptidase activity of
FAP. The problem then reduces to discriminating between the
endopeptidase activities of PREP and FAP, which is relatively
straightforward, as FAP’s endopeptidase activity has rather
stringent requirements at P2, accepting so far only glycine or D-
alanine, whereas PREP is more promiscuous and will accept a
variety of amino acids at this position.37 Thus, blocking the N-
terminus of Val-boroPro (20), which exhibits little to no
selectivity against any of the post-proline cleaving enzymes,
produces 22, which combines 1 nM potency against PREP with
greater than 77 000-fold selectivity over FAP, as well as the
DPPs (Table 4). Substituting glycine for valine at P2 in 22
produces a potent inhibitor (21) of both FAP and PREP,
demonstrating that the P2 valine provides selectivity for PREP
over FAP.
Determination of Competitive or Noncompetitive
Inhibition. Given the structural similarities of 6 and 22 to
the substrates used for IC50 determinations, it was anticipated
that they would be competitive inhibitors of FAP and PREP.
This is in fact the case, as shown by the Lineweaver−Burk plots
for 6 in Figure 2. This kinetic analysis also provided Ki values
for these two inhibitors (Table 5). The Ki values of 6 against
FAP and PREP are lower than the IC50 values, but the ratio of
selectivity remains the same, as expected. As before, the
selectivity of 22 for PREP over FAP could only be quantified as
greater than 140 000-fold, as 22 does not inhibit FAP at the
highest concentrations tested.
Inhibitor Selectivity in Vitro in Mock and mFAP
Transfected Cells. Although 6 and 22 are here shown to be
potent and selective inhibitors of FAP and PREP, one may
question whether the selectivity can be demonstrated in a more
biologically relevant milieu, such as in cell cultures. Therefore,
HEK293 cells transfected with either murine FAP (mFAP) or a
mock vector were incubated with 1 μM 2, 6, or 22. Compound
2, which is a relatively poor inhibitor of DPPIV, DPP8, and
DPP9, but a potent inhibitor of both FAP and PREP, inhibited
virtually all of the cellular prolyl endopeptidase activity in both
cell lines (Figure 3). The PREP specific inhibitor 22 inhibited
all activity in the mock-transfected cells but only approximately
60% in the mFAP transfected cells. This demonstrates that all
of the endogenous prolyl endopeptidase activity in the HEK293
cells can be attributed to PREP, in agreement with previously
published results that showed that HEK293 cells do not express
D
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Table 3. Comparison of FAP vs PREP against a Panel of P2
a
D-Amino Acid Substituted Inhibitors
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)Km) at 37 °C.
a
Table 5. Ki Values for 6 and 22 against FAP and PREP
Ki (nM)
inhibitor
FAP
PREP
FAP selectivity
340
6
9.0 0.9
3100 260
0.73 0.15
22
a
Values are expressed with SEM.
a
The FAP selectivity is equal to IC50(PREP)/IC50(FAP). IC50 values
are expressed with SEM.
Figure 3, as well as any future in vivo assays, involves a mixture
of enzymes rather than a single purified enzyme. The chief
competitor for a FAP inhibitor in vivo will likely be PREP.
Therefore, the effect of varying concentrations of PREP on FAP
inhibition by 6 was assessed (Figure 4A).
Focusing on 36 nM and 1 μM 6, concentrations equal to the
reported IC50 and the amount used to assess cell-associated
enzyme activity, it was shown that concentrations of PREP
equal to 0.01- to 100-fold the concentration of FAP had no
significant effect on the observed inhibition by 6. In the case of
endogenous FAP.38 Finally, the FAP specific inhibitor 6
reduced activity only in the mFAP transfected cells but not
in mock transfected cells. Not only do these findings
demonstrate specificity in a cell-culture-based setting, they
also indicate that 6 inhibits mouse and human FAP.
Effect of the Presence of PREP on the Selectivity of 6
toward FAP. Unlike the in vitro assays used to determine the
previously reported IC50 values, the cellular-based assay in
a
Table 4. PREP Selectivity of Compounds 20−22
a
The PREP selectivity is equal to IC50(FAP)/IC50(PREP). IC50 values are expressed with SEM.
E
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FAP to PREP ratio of 1:1.5, well within the range tested in
Figure 4A, further validating the cell-based results.
Cytotoxicity of 6 in mFAP Transfected Cells. Previously,
it was discussed that inhibition of FAP activity in vivo has been
associated with antitumor activity in a number of mouse tumor
models.16,17,20 Consequently, the cytotoxicity of 6 in mFAP
transfected HEK293 cells was assessed, as these cells are known
to express FAP and to represent a cell line that has been shown
to rely on FAP activity for maximal tumor growth in vivo17
(Figure 5). No cytotoxicity was observed in this assay. This
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 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.
does not necessarily prove that inhibition of FAP by 6 would
not have an effect in vivo, however, as 20 has been shown to
lack cytotoxicity against several tumor cell lines while still
attenuating tumor growth in vivo in mice inoculated with those
same cells.20 In partial agreement with this, 20 was also shown
here to lack cytotoxicity. Conversely, bortezomib, which
represents a class of boronic acid inhibitors that target the
proteasome, has been shown to be cytotoxic to cells in vitro
and in vivo and was thus used here as a positive control.39
These results suggest that any reduction in tumor volume
resulting from FAP inhibition in vivo must be mediated by
more complex pathways than those that can be mimicked using
a cell-based assay alone.
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.
CONCLUSIONS
■
Here we report the first FAP inhibitor, 6, that combines low
nanomolar potency with selectivity against both the DPPs and
PREP, as well as 22, a corresponding potent and specific
inhibitor for PREP. Both are boronic acid based inhibitors,
demonstrating that high selectivity can be achieved with the use
of this electrophile, notwithstanding beliefs to the contrary.
These inhibitors should prove to be useful in helping to
elucidate the biological function of these important enzymes
and to evaluate their potential as drug targets.
PREP specific inhibition by 22, similar results would be
anticipated given that 22 is much more selective for PREP over
FAP than 6 is for FAP over PREP.
In order to determine if this range covers the enzyme
activities associated with the cells used for the experiment in
Figure 3, their FAP and PREP activities were measured and the
relative concentrations of each calculated using a standard curve
prepared from purified enzyme (Figure 4B). This yielded an
EXPERIMENTAL SECTION
Inhibitor Synthesis. Compound identities were confirmed via H
■
1
NMR and LC/MS, using the HPLC aspect of the latter to assess a
level of relative purity of ≥95% for each inhibitor. Specifically, NMR
spectra of the compounds in D2O were recorded on a Bruker Avance
300 MHz spectrometer with a 5 mm dual 1H/X probe. Chemical
shifts were reported relative to DSS. Mass spectra and HPLC retention
times were recorded on a Hewlett-Packard HP LC/MSD system with
F
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Table 6. Chemical Building Blocks Utilized for the Synthesis of the N-Terminally Blocked X-boroPro Dipeptides
1
UV detector (monitoring at 215 and 254 nm), using a Discovery C18
569232-U RP-HPLC column (12.5 cm, 4.6 mm, 5 μm) with solvent
gradient A (water, 0.1% TFA) and B (acetonitrile, 0.08% TFA)
flowing at 0.5 mL/min. Unless otherwise noted, all HPLC retention
times are given for an eluent gradient of 2% B for the first 5 min, 2−
98% B over 10 min, and 98% B for the final 5 min.
Dipeptide Boronic Acid Synthesis. Synthesis of the X-boroPro
dipeptides (1 and 20) was performed essentially as described
previously40 except that the initial coupling step was performed with
HATU/DIPEA rather than EDC/HOBt, and the N- and C-terminal
protection groups of the N-Boc-(S)-X-(R)-boroPro-(1S,2S,3R,5S)-
pinanediol ester products were removed simultaneously. For the latter,
to a stirred solution of the protected product in CH2Cl2 cooled to −78
°C, an equimolar amount of a 1.0 M solution of BCl3 in CH2Cl2 was
added. After 1 h, the product was extracted into water and purified on
a Supelco Discovery-C18 column, eluting with water (0.1% TFA) and
acetonitrile (0.08% TFA). Lyophilization yielded the free dipeptide
boronic acids.
Glycinyl-(R)-boroproline (1). H NMR (D2O) δ 1.69−2.07 (m,
4H, BCHCH2CH2), 3.03−3.05 (m, 1H, CH2CHB), 3.36−3.50 (m,
2H, CH2CH2N), 3.88 (s, 2H, H2NCH2CO). 13C NMR (D2O) δ
29.13, 29.53, 42.89, 49.11, 50.12, 166.89. 11B NMR (D2O) δ 11.03.
LC−MS (ESI+) m/z (rel intensity): 309.2, [2 × (M − H2O) + H+];
155.2, [M − H2O + H+]. tR = 5.2 min.
1
(S)-Valinyl-(R)-boroproline (20). H NMR (D2O) δ 0.98 (d, J =
6.9 Hz, 3H, CH3CHCH3), 1.08 (d, J = 6.9 Hz, 3H, CH3CHCH3),
1.60−2.34 (m, 5H, CH3CHCH3 and BCHCH2CH2), 3.03−3.09 (m,
1H, CH2CHB), 3.43−3.75 (m, 2H, CH2CH2N), 4.12 (d, J = 6.3 Hz,
1H, H2NCHCO). LC−MS (ESI+) m/z (rel intensity): 393.3, [2 × (M
− H2O) + H+]; 197.1, [M − H2O + H+], tR = 11.1 min.
N-Terminally Blocked Dipeptide Boronic Acids. Synthesis of
the N-terminally blocked X-boroPro dipeptides was similar to that of
the dipeptide boronic acids but fell under two synthetic schemes,
specifically, those that coupled the N-terminal blocking group to the P2
residue first (2, 16, and 19) and those that coupled the P1 and P2
residues in the initial step, followed by the addition of the N-terminal
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blocking group (5−15, 17, 18, and 22). The coupling methods for the
blocking group additions, as well as descriptions of the specific
chemical building blocks, are highlighted in Table 6. In the cases of 3,
4, and 21, the P2 residues with the desired N-terminal blocking group
were commercially available, and these were coupled directly to the
pinanediol protected P1 boroproline residue.
N-(Pyridine-2-fluoro-4-carbonyl)-(R)-alaninyl-(R)-boropro-
1
line (11). H NMR (D2O) δ 1.37−1.49 (d, 3H, HNCHCH3), 1.61−
2.21 (m, 4H, BCHCH2CH2), 2.93−3.03 (m, 1H, CH2CHB), 3.62−
3.69 (m, 2H, CH2CH2N), 4.61−4.80 (m, 1H, HNCHCO), 7.31−7.82
(m, 2H, aromatic H), 8.26−8.33 (d, 1H, aromatic H), 8.81−8.85 (s,
1H, aromatic H). The HPLC retention time is for an eluent gradient
of 2% B for the first 3 min, 2−98% B over 3 min, and 98% B for the
final 5 min. LC−MS (ESI+) m/z (rel intensity): 583.3, [2 × (M −
H2O) + H+]; 292.2, [M − H2O + H+], tR = 7.1 min.
N-(1-Naphthalenecarbonyl)glycinyl-(R)-boroproline (2). 1H
NMR (D2O) δ 1.73−2.13 (m, 4H, BCHCH2CH2), 3.08−3.14 (m,
1H, CH2CHB), 3.55−3.74 (m, 2H, CH2CH2N), 4.25−4.39 (d, J =
15.2 Hz, 2H, HNCH2CO), 7.59−7.76 (m, 4H, aromatic H), 8.00−
8.18 (m, 3H, aromatic H). LC−MS (ESI+) m/z (rel intensity): 617.3,
[2 × (M − H2O) + H+]; 309.2, [M − H2O + H+], tR = 14.4 min.
N-Acetyl-(R)-alaninyl-(R)-boroproline (3). 1H NMR (D2O) δ
1.30−1.32 (d, J = 7.0 Hz, 3H, HNCHCH3), 1.56−2.09 (m, 4H,
BCHCH2CH2), 2.96−2.97 (m, 1H, CH2CHB), 3.58−3.63 (m, 2H,
CH2CH2N), 4.25−4.39 (q, J = 7.0 Hz, 3H, H3CO). LC−MS (ESI+)
m/z (rel intensity): 421.2, [2 × (M − H2O) + H+]; 251.3, [M + Na+];
211.3, [M − H2O + H+], tR = 12.2 min.
N-(Pyridine-2-chloro-4-carbonyl)-(R)-alaninyl-(R)-boropro-
1
line (12). H NMR (D2O) δ 1.36−1.47 (d, 3H, HNCHCH3), 1.59−
2.11 (m, 4H, BCHCH2CH2), 2.88−3.01 (m, 1H, CH2CHB), 3.62−
3.71 (m, 2H, CH2CH2N), 4.66−4.79 (m, 1H, HNCHCO), 7.66−7.71
(d, 1H, aromatic H), 7.76−7.80 (s, 1H, aromatic H), 8.44−8.49 (d,
1H, aromatic H). The HPLC retention time is for an eluent gradient
of 2% B for the first 3 min, 2−98% B over 3 min, and 98% B for the
final 5 min. LC−MS (ESI+) m/z (rel intensity): 615.1, [2 × (M −
H2O) + H+]; 308.0, [M − H2O + H+], tR = 7.7 min.
1
1-(2-(1-Oxoisoindolin-2-acetyl)-(R)-boroproline (4). H NMR
N-(Quinoline-4-carbonyl)-(R)-alaninyl-(R)-boroproline (13).
1H NMR (D2O) δ 1.45−1.48 (d, J = 6.9 Hz, 3H, HNCHCH3),
1.70 − 2.13 (m, 4H, BCHCH2CH2), 3.02−3.07 (m, 1H, CH2CHB),
3.72−3.77 (m, 2H, CH2CH2N), 4.88−4.97 (m, 1H, HNCHCO),
7.97−8.33 (m, 5H, aromatic H), 9.18−9.20 (d, 1H, NCHCH,
aromatic H). LC−MS (ESI+) m/z (rel intensity): 647.3, [2 × (M −
H2O) + H+]; 324.3, [M − H2O + H+], tR = 12.8 min.
(D2O) δ 1.67−2.07 (m, 4H, BCHCH2CH2), 2.99−3.04 (m, 1H,
CH2CHB), 3.47−3.62 (m, 2H, CH2CH2N), 4.41 (s, 2H, CCH2N),
4.51 (s, 2H, HNCH2CO), 7.49−7.76 (m, 4H, aromatic H). LC−MS
(ESI+) m/z (rel intensity): 541.4, [2 × (M − H2O) + H+]; 271.1, [M
− H2O + H+], tR = 8.4 min.
N-(Pyridine-4-carbonyl)glycinyl-(R)-boroproline (5). 1H NMR
(D2O) δ 1.52−2.13 (m, 4H, BCHCH2CH2), 2.91−3.02 (m, 1H,
CH2CHB), 3.36−3.64 (m, 2H, CH2CH2N), 4.23 (s, 2H, HNCH2CO),
8.33−8.35 (d, J = 5.4 Hz, 2H, aromatic H), 8.91−8.93 (d, J = 5.4 Hz,
2H, aromatic H). LC−MS (ESI+) m/z (rel intensity): 519.2, [2 × (M
− H2O) + H+]; 260.1, [M − H2O + H+], tR = 11.9 min.
N-(Pyridine-4-carbonyl)-(R)-alaninyl-(R)-boroproline (6). 1H
NMR (D2O) δ 1.45−1.47 (d, 3H, HNCHCH3), 1.68−2.16 (m, 4H,
BCHCH2CH2), 2.98−3.04 (m, 1H, CH2CHB), 3.66−3.71 (m, 2H,
CH2CH2N), 4.83−4.86 (m, 1H, HNCHCO), 8.36−8.38 (d, 2H,
aromatic H), 8.94−8.96 (d, 2H, aromatic H). LC−MS (ESI+) m/z (rel
intensity): 547.2, [2 × (M − H2O) + H+]; 274.1, [M − H2O + H+], tR
= 12.0 min.
N-(Quinoline-4-carbonyl)-(R)-serinyl-(R)-boroproline (14). 1H
NMR (D2O) δ 1.75−2.20 (m, 4H, BCHCH2CH2), 3.05−3.11 (m, 1H,
CH2CHB), 3.78−3.82 (m, 2H, CH2CH2N), 3.87−4.03 (m, 2H,
CHCH2OH), 5.13−5.17 (m, 1H, HNCHCO), 8.01−8.36 (m, 5H,
aromatic H), 9.21−9.23 (d, 1H, NCHCH, aromatic H). LC−MS
(ESI+) m/z (rel intensity): 679.3, [2 × (M − H2O) + H+]; 340.2, [M
− H2O + H+], tR = 12.4 min.
N-(Quinoline-4-carbonyl)-(R)-methioninyl-(R)-boroproline
1
(15). H NMR (D2O) δ 1.74−2.22 (m, 4H, BCHCH2CH2), 2.04−
2.22 (m, 5H, CHCH2CH2SCH3), 2.67−2.75 (m, 1H, CH2CHB),
2.67−3.10 (m, 2H, CH2CH2N), 3.79−3.83 (m, 2H, CH2CH2S), 5.14−
5.19 (m, 1H, HNCHCO), 8.01−8.32 (m, 5H, aromatic H), 9.21−9.23
(d, 1H, NCHCH, aromatic H). LC−MS (ESI+) m/z (rel intensity):
767.3, [2 × (M − H2O) + H+]; 384.5, [M − H2O + H+], tR = 12.9
min.
1
N-(Benzoyl)-(R)-alaninyl-(R)-boroproline (7). H NMR (D2O)
δ 1.38−1.51 (d, 3H, HNCHCH3), 1.60−2.24 (m, 4H, BCHCH2CH2),
2.93−3.08 (m, 1H, CH2CHB), 3.62−3.77 (m, 2H, CH2CH2N), 4.70−
4.85 (m, 1H, HNCHCO), 7.49−7.82 (m, 5H, aromatic H). LC−MS
(ESI+) m/z (rel intensity): 545.3, [2 × (M − H2O) + H+]; 273.1, [M
− H2O + H+], tR = 13.9 min.
N-(Quinoline-4-carbonyl)-(R)-ethyl(glycinyl)-(R)-boroproline
(16). 1H NMR (D2O) δ 1.02−1.07 (m, 3H, CHCH2CH3), 1.72−1.91
(m, 2H, CHCH2CH3), 1.72−2.16 (m, 4H, BCHCH2CH2), 3.03−3.08
(m, 1H, CH2CHB), 3.76−3.81 (m, 2H, CH2CH2N), 4.84−4.89 (m,
1H, HNCHCO), 7.99−8.32 (m, 5H, aromatic H), 9.20−9.21 (d, 1H,
NCHCH, aromatic H). LC−MS (ESI+) m/z (rel intensity): 675.3, [2
× (M − H2O) + H+]; 338.4, [M − H2O + H+], tR = 13.2 min.
N-(Quinoline-4-carbonyl)-(R)-propyl(glycinyl)-(R)-boropro-
line (17). 1H NMR (D2O) δ 0.93−0.98 (m, 3H, CH2CH2CH3),
1.45−1.52 (m, 2H, CH2CH2CH3), 1.73−1.84 (m, 2H, CHCH2CH2),
1.73−2.14 (m, 4H, BCHCH2CH2), 3.01−3.07 (m, 1H, CH2CHB),
3.75−3.80 (m, 2H, CH2CH2N), 4.91−4.96 (m, 1H, HNCHCO),
7.99−8.30 (m, 5H, aromatic H), 9.20−9.21 (d, 1H, NCHCH,
aromatic H). LC−MS (ESI+) m/z (rel intensity): 703.5, [2 × (M −
H2O) + H+]; 352.3, [M − H2O + H+], tR = 13.7 min.
N-(Quinoline-4-carbonyl)-(R)-butyl(glycinyl)-(R)-boroproline
(18). 1H NMR (D2O) δ 0.86−0.91 (m, 3H, CH2CH2CH3), 1.34−1.45
(m, 4H, CH2CH2CH2CH3), 1.81−1.85, (m, 2H, CHCH2CH2), 1.81−
2.14 (m, 4H, BCHCH2CH2), 3.01−3.07 (m, 1H, CH2CHB), 3.74−
3.81 (m, 2H, CH2CH2N), 4.89−4.94 (m, 1H, HNCHCO), 7.99−8.30
(m, 5H, aromatic H), 9.19−9.21 (d, 1H, NCHCH, aromatic H). The
HPLC retention time is for an eluent gradient 2% B for the first 3 min,
2−98% B over 6 min and 98%B for the final 4 min. LC−MS (ESI+)
m/z (rel intensity): 365.7, [M − H2O + H+], tR = 8.5 min.
N-(Pyridine-3-carbonyl)-(R)-alaninyl-(R)-boroproline (8). 1H
NMR (D2O) δ 1.38−1.51 (d, 3H, HNCHCH3), 1.58−2.20 (m, 4H,
BCHCH2CH2), 2.79−3.05 (m, 1H, CH2CHB), 3.59−3.74 (m, 2H,
CH2CH2N), 4.72−4.87 (m, 1H, HNCHCO), 8.12−8.23 (m, 1H,
aromatic H), 8.94−9.03 (m, 2H, aromatic H), 9.18−9.21 (s, 1H,
aromatic H). The HPLC retention time is for an eluent gradient of 2%
B for the first 3 min, 2−98% B over 6 min, and 98% B for the final 4
min. LC−MS (ESI+) m/z (rel intensity): 547.2, [2 × (M − H2O) +
H+]; 274.1, [M − H2O + H+], tR = 7.3 min.
N-(Pyridine-3-fluoro-4-carbonyl)-(R)-alaninyl-(R)-boropro-
1
line (9). H NMR (D2O) δ 1.41−1.50 (d, 3H, HNCHCH3), 1.61−
2.22 (m, 4H, BCHCH2CH2), 2.89−3.04 (m, 1H, CH2CHB), 3.59−
3.74 (m, 2H, CH2CH2N), 4.79−4.84 (m, 1H, HNCHCO), 8.02−8.09
(m, 1H, aromatic H), 8.69−8.74 (d, 1H, aromatic H), 8.80−8.84 (d,
1H, aromatic H). The HPLC retention time is for an eluent gradient
of 2% B for the first 3 min, 2−98% B over 3 min, and 98% B for the
final 5 min. LC−MS (ESI+) m/z (rel intensity): 583.2, [2 × (M −
H2O) + H+]; 292.0, [M − H2O + H+], tR = 7.2 min.
N-(Pyridine-3-chloro-4-carbonyl)-(R)-alaninyl-(R)-boropro-
1
line (10). H NMR (D2O) δ 1.34−1.46 (d, 3H, HNCHCH3), 1.62−
2.21 (m, 4H, BCHCH2CH2), 2.91−3.04 (m, 1H, CH2CHB), 3.62−
3.74 (m, 2H, CH2CH2N), 4.77−4.82 (m, 1H, HNCHCO), 7.89−7.93
(d, 1H, aromatic H), 8.71−8.76 (d, 1H, aromatic H), 8.81−8.85 (s,
1H, aromatic H). The HPLC retention time is for an eluent gradient
of 2% B for the first 3 min, 2−98% B over 3 min, and 98% B for the
final 5 min. LC−MS (ESI+) m/z (rel intensity): 615.2, [2 × (M −
H2O) + H+]; 308.0, [M − H2O + H+], tR = 7.6 min.
N-(Quinoline-4-carbonyl)-(R)-phenyl(glycinyl)-(R)-boropro-
line (19). 1H NMR (D2O) δ 1.70−1.72 (m, 2H, BCHCH2CH2),
1.73−2.00 (m, 2H, BCHCH2CH2), 2.99−3.06 (m, 1H, CH2CHB),
3.26−3.68 (m, 2H, CH2CH2N), 6.00 (s, 1H, HNCHCO), 7.42−7.46
(m, 5H, phenyl H), 7.93−8.31 (m, 5H, aromatic H), 9.13−9.15 (d,
H
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1H, NCHCH, aromatic H). LC−MS (ESI+) m/z (rel intensity): 386.1,
[M − H2O + H+], tR = 14.1 min.
crystallographic structure of the DPPIV tetrahedral intermediate
complexed with diprotinA (PDB accession code 1NU8), the -boroPro
moiety of 6 was positioned in the similarly shaped hydrophobic S1
pocket of FAP (PDB accession code 1Z68). The boron was placed at
N-(Pyridine-3-carbonyl)glycinyl-(R)-boroproline (21). 1H
NMR (D2O) δ 1.68−2.10 (m, 4H, BCHCH2CH2), 3.02−3.08 (m,
1H, CH2CHB), 3.47−3.69 (m, 2H, CH2CH2N), 4.29 (s, 2H,
HNCH2CO), 8.18−8.22 (m, 1H, NCHCHCH, aromatic H), 8.94−
8.96 (m, 2H, NCHCHCH, aromatic H), 9.22 (s, 1H, NCHCCO,
aromatic H). LC−MS (ESI+) m/z (rel intensity): 519.2, [2 × (M −
H2O) + H+]; 260.1, [M − H2O + H+], tR = 11.5 min.
624
́
nearly the same distance, 1.6 Å, from the Ser oxygen as previously
reported B−O bonds. Tetrahedral angles for boron were maintained as
closely as possible, utilizing the free rotation of ligand Cα−B and
enzyme Ser624 Cα−Cβ single bonds. The ligand 6 dihedral angles were
varied sequentially from ψ2 to ϕ2 of the D-alanyl group to ψ3 of the 4-
pyridylcarbonyl group to optimize possible hydrogen bond receptors
for the pyridinium N−H+ group (ψ3 = −160°, ω3 = −177°, ϕ2 = 94°,
ψ2 = 65°, ω2 = −177°, ϕ1 = −73°). This placed the pyridinium N−H+
proton in position for hydrogen bonding to the backbone carbonyl
N-(Pyridine-3-carbonyl)-(R)-valinyl-(R)-boroproline (22). 1H
NMR (D2O) δ 0.96−1.03 (m, 6H, CHCH(CH3)2), 1.69−2.15 (m,
4H, BCHCH2CH2), 2.15−2.22 (m, 1H, CHCH(CH3)2), 2.95−2.99
(m, 1H, CH2CHB), 3.57−4.01 (m, 2H, CH2CH2N), 4.61−4.63 (m,
1H, HNCHCO), 8.16−8.21 (m, 1H, NCHCHCH, aromatic H),
8.92−8.95 (m, 2H, NCHCHCH, aromatic H), 9.16 (s, 1H,
NCHCCO, aromatic H). The HPLC retention time is for an eluent
gradient 2% B for the first 3 min, 2−98% B over 6 min, and 98% B for
the final 4 min. LC−MS (ESI+) m/z (rel intensity): 602.4, [2 × (M −
H2O)]; 302.2, [M − H2O + H+], tR = 6.7 min.
oxygen atom of Glu204, with an H−O distance of 1.87 Å and an N−
́
H...O angle of 121°.
Characterization of Competitive/Noncompetitive Inhibition.
The enzyme kinetics for FAP or PREP with or without varying
concentrations ((0.5−2)IC50) of 6 or 22 were measured in the
appropriate assay buffer at 37 °C, using Suc-GP-AMC at
concentrations equivalent to (0.1−5)× its respective Km values for
these enzymes. The enzyme concentration was 5 nM. 6 was added
concurrent with the substrate and enzyme, while 22 was allowed to
preincubate with the enzyme for 10 min prior to the addition of the
substrate due to observed slow binding kinetics. The fluorescence was
continuously monitored at excitation and emission wavelengths of 380
and 460 nm, respectively. Standard curves for 7-amino-4 methyl-
coumarin (Aldrich) were used to convert the fluorescence units into
the concentration of the released fluorophore. From this, the slope of
[AMC] versus time yielded the reaction rate for each condition. The
resulting Lineweaver−Burk plots were prepared by plotting 1/V,
where V was equal to the reaction rate calculated above, versus 1/[S]
and were analyzed with a linear regression analysis, using GraphPad
software. Ki values were also determined using GraphPad software,
fitting the V vs [S] results to a competitive inhibition analysis.
Inhibition of Cellular Enzyme Activity in HEK293 Cells. Mock
or murine FAP (mFAP) transfected HEK293 cells were kindly
provided by Jonathan Cheng from the Fox Chase Cancer Center. The
cells were equilibrated in RPMI 1640 at a density of 25 000 cells/well,
treated with 1 μM 2, 6, 22, or RPMI 1640 alone, and allowed to
incubate for 15 min at 37 °C. The substrate was 25 μM Z-Gly-Pro-
AMC. The fluorescence was measured at 37 °C, monitoring an
excitation wavelength of 380 nm and an emission wavelength of 460
nm. The percent activity was determined versus the RPMI 1640
control.
In Vitro Mixed FAP and PREP Inhibition Assay. Enzymatic
activity of 1 nM FAP combined with 0.01−100 nM PREP was
measured at 25 °C, monitoring the fluorescence at an excitation
wavelength of 380 nm and an emission wavelength of 460 nm. The
substrate was the N-terminally blocked FAP specific substrate, so as to
be able to differentiate FAP activity in the presence of PREP. The final
reaction mixture contained 25 μM substrate, enzyme, a 1:1 mixture of
buffer C and buffer D, and either 36 nM or 1 μM 6 in a total volume of
210 μL. The enzymes were allowed to preincubate with the inhibitor
for 10 min at room temperature prior to addition of the substrate. The
percent of FAP activity was determined based on a simultaneously run
control for each condition, which lacked treatment with the inhibitor.
Measurement of FAP and PREP Activity in HEK293 Cells.
Both mock and mFAP transfected HEK293 cells were equilibrated in
RPMI 1640 at a density of 25 000 cells/well. The mock transfected
cells were treated with 25 μM Z-GP-AMC, and the mFAP transfected
cells were treated with 50 μM N-terminally blocked FAP specific
inhibitor in a total volume of 200 μL/well. The fluorescence was
measured at 37 °C, monitoring an excitation wavelength of 380 nm
and an emission wavelength of 460 nm. To calculate the relative
cellular PREP and FAP concentrations, standard activity curves were
prepared for each using varying concentrations of purified enzyme and
Z-GP-AMC for PREP and the N-terminally blocked FAP specific
substrate for FAP. The relative concentration of active enzyme
associated with the cells was calculated via interpolation from the
standard curves and the measured cellular activity.
Synthesis of Bortezomib. Bortezomib was synthesized as
described previously.41
Biological Materials. The peptide libraries XPYSWS-NH2, Ac-
XPYSWS-NH2, GXYSWS-NH2, and Ac-GXYSWS-NH2, where X
represents all natural amino acids except cysteine were synthesized
by the Tufts University Core Facility. The DPPIV enzyme used for the
specificity studies was previously purified by our laboratory at Tufts
University from human placenta, and the purified human FAP enzyme
used for these studies was kindly provided by Syrxx, Inc. For the in
vitro IC50 determination assays, recombinant human DPPIV, DPP9,
FAP, and PREP were purchased from R&D Systems, and DPP8 was
from Biomol International. Buffer systems used were A (25 mM Tris,
pH 8.0), B (50 mM Tris, pH 7.5), C (50 mM Tris, 140 mM NaCl, pH
7.5), D (25 mM Tris, 250 mM NaCl, pH 7.5), and E (20 mM Tris, 20
mM KCl, pH 7.4). Fluorogenic substrates were Gly-Pro-AMC, Z-Gly-
Pro-AMC, or Suc-Gly-Pro-AMC purchased from Bachem or an N-
terminally blocked FAP specific substrate.42 The cell culture medium
was RPMI 1640 without phenol red and supplemented with 2 mM L-
glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L
glucose, 100 IU/mL penicillin, and 100 μg/mL streptomycin.
Substrate Specificity Assay. Peptide libraries (0.21 mM) were
incubated for 24 h with 1 nM FAP in buffer E at 37 °C. The reaction
was quenched by the addition of 1.2 N HCl. The samples were
analyzed by reverse-phase HPLC−MS on a Thermo Finnigan LCQ
Duo, quantifying the peaks in the resulting base peak chromatograms.
Relative cleavage values were determined by comparing the
postquench abundance of intact peptides to those in the initial library.
In Vitro Enzyme IC50 Assays. Enzymatic activity of DPPIV,
DPP8, DPP9, FAP, and PREP was measured at 25 °C on a Molecular
Devices M2e multidetection microtiter plate reader, monitoring the
fluorescence at an excitation wavelength of 380 nm and an emission
wavelength of 460 nm. The substrate was either H-Gly-Pro-AMC for
the DPPIV, DPP8, and DPP9 assays or Z-Gly-Pro-AMC for the FAP
and PREP assays. The reaction mixture contained 25 μM substrate,
enzyme, buffer A (DPPIV and DPP9), buffer B (DPP8), buffer C
(FAP), or buffer D (PREP) and a suitable amount of inhibitor
(ranging between 10−4 and 10−11 M) in a total volume of 210 μL. The
final enzyme concentrations were 0.1, 0.8, 0.4, 1.2, and 0.6 nM for
DPPIV, DPP8, DPP9, FAP, and PREP, respectively. The IC50 value is
defined as the concentration of inhibitor required to reduce the
enzyme activity by 50% after a 10 min preincubation with the enzyme
at 25 °C prior to addition of the substrate.
Inhibitor stock solutions (100 mM) were prepared in either a pH
2.0 HCl solution for compounds 1 and 20 or DMSO. Those prepared
in pH 2.0 solution were preincubated at 25 °C for 4 h prior to dilution.
Immediately prior to the commencement of the experiment, the 100
mM stocks were further diluted to 10−3 M in the appropriate assay
buffer, from which 1:10 serial dilutions were prepared. All inhibitors
were tested in triplicate.
Molecular Modeling of 6 Bound to FAP. Using Insight II
(version 2005.03) and beginning with the homologous X-ray
I
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Journal of Medicinal Chemistry
Article
Cell Viability Assay. mFAP transfected HEK293 cells were
equilibrated in RPMI 1640 at a density of 25 000 cells/well. Cells were
treated with RPMI 1640 alone or varying concentrations of 6, 20, or
bortezomib (ranging between 10−3 and 10−11 M) in a final assay
volume of 100 μL. Following a 48 h incubation at 37 °C, all wells were
treated with CellTiter-Blue reagent from Promega and incubated an
additional 2 h at 37 °C and the fluorescence was measured, monitoring
an excitation wavelength of 560 nm and an emission wavelength of
590 nm. Percent cell viability relating to the inhibitors was determined
versus the cells treated with RPMI 1640 alone.
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́
AUTHOR INFORMATION
Corresponding Author
*Phone: 617-636-6881. E-mail: william.bachovchin@tufts.edu.
■
(13) Cheng, J. D.; Dunbrack, R. L., Jr.; Valianou, M.; Rogatko, A.;
Alpaugh, R. K.; Weiner, L. M. Promotion of tumor growth by murine
fibroblast activation protein, a serine protease, in an animal model.
Cancer Res. 2002, 62, 4767−4772.
Notes
The authors declare no competing financial interest.
(14) Scott, A. M.; Wiseman, G.; Welt, S.; Adjei, A.; Lee, F. T.;
Hopkins, W.; Divgi, C. R.; Hanson, L. H.; Mitchell, P.; Gansen, D. N.;
Larson, S. M.; Ingle, J. N.; Hoffman, E. W.; Tanswell, P.; Ritter, G.;
Cohen, L. S.; Bette, P.; Arvay, L.; Amelsberg, A.; Vlock, D.; Rettig, W.
J.; Old, L. J. A phase I dose-escalation study of sibrotuzumab in
patients with advanced or metastatic fibroblast activation protein-
positive cancer. Clin. Cancer Res. 2003, 9, 1639−1647.
ACKNOWLEDGMENTS
■
This work was supported by Arisaph Pharmaceuticals, Inc.
ABBREVIATIONS USED
■
FAP, fibroblast activation protein; PREP, prolyl oligopeptidase;
DPPIV, dipeptidyl peptidase IV; DPPII, dipeptidyl peptidase II;
DPP8, dipeptidyl peptidase 8; DPP9, dipeptidyl peptidase 9;
GLP-1, glucagon-like peptide 1; GIP, glucose-dependent
insulinotropic peptide; Ki, inhibition constant; IC50, median
inhibition concentration
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