Design of highly potent urea-based, exosite-binding inhibitors selective for glutamate carboxypeptidase II

Article abstract of DOI:10.1021/acs.jmedchem.5b00278

We present here a structure-aided design of inhibitors targeting the active site as well as exosites of glutamate carboxypeptidase II (GCPII), a prostate cancer marker, preparing potent and selective inhibitors that are more than 1000-fold more active tow

Full text of DOI:10.1021/acs.jmedchem.5b00278

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Brief Article
Design of highly potent urea-based, exosite-binding
inhibitors selective for glutamate carboxypeptidase II
Jan Tykvart, Jiri Schimer, Andrej Jancarik, Jitka Barinkova, Vaclav Navratil, Jana
Starkova, Karolina Sramkova, Jan Konvalinka, Pavel Majer, and Pavel Sacha
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00278 • Publication Date (Web): 29 Apr 2015
Downloaded from http://pubs.acs.org on May 6, 2015
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Design of highly potent urea-based, exosite-binding inhibitors selec-
tive for glutamate carboxypeptidase II
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,2‡
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Jan Tykvart , Jiří Schimer , Andrej Jančařík , Jitka Bařinková , Václav Navrátil , Jana Starková ,
1
1,2*
1
1,2*
Karolína Šrámková , Jan Konvalinka , Pavel Majer and Pavel Šácha
1
Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the
Czech Republic, Flemingovo n. 2, Prague 6, Czech Republic
2
Department of Biochemistry, Faculty of Science, Charles University, Albertov 6, Prague 2, Czech Republic
KEYWORDS: PSMA, GCPII, GCPIII, inhibitor selectivity, imaging, structure-aided drug design
ABSTRACT: We present here a structureꢀaided design of inhibitors targeting the active site as well as exosites of glutamate carꢀ
boxypeptidase II (GCPII), a prostate cancer marker, preparing potent and selective inhibitors that are more than 1000ꢀfold more
active toward GCPII than its closest human homolog, glutamate carboxypeptidase III (GCPIII). Additionally, we demonstrate that
the prepared inhibitor conjugate can be used for sensitive and selective imaging of GCPII in mammalian cells.
is essential to facilitate their development as potential
theranostic agents for PCa.
INTRODUCTION
Prostate carcinoma (PCa) was the most commonly diagꢀ
Recently, we reported the structureꢀactivity relationship
nosed cancer and the leading cause of cancer death among
study of GCPII inhibitors modified with linkers and biotin
men in the United States in 2014, with an estimated 233,000
molecules, which enable facile preparation of GCPIIꢀtargeting
1
18
diagnoses and 30,000 deaths. Glutamate carboxypeptidase II
nanoparticles. Nevertheless, the GCPII inhibitors with PEG
linkers showed slightly decreased inhibitory potency. In the
present work, we set out to improve their potency by increasꢀ
ing the linker's rigidity. Additionally, we set out to investigate
the specificity of these inhibitors against GCPIII.
(
(
GCPII), also known as prostate specific membrane antigen
PSMA), is a promising biomarker of this pathological condiꢀ
2ꢀ5
tion. Additionally, GCPII has been investigated as a potenꢀ
tial biomarker of other solid tumors because of its reported
6
ꢀ7
expression in tumor neovasculature.
Lowꢀmolecularꢀweight inhibitors can be used to target
RESULTS AND DISCUSSION
2, 8ꢀ10
GCPII.
Specificity of these inhibitors is an issue that
To decrease the mobility of the linker region, we designed a
rigid arm formed by joined pyridine, piperazine, and benzene
rings. As shown in Figure 1, the addition of this rigid arm led
to a 15ꢀfold increase in potency for inhibitors with terminal
PEG ꢀbiotin [compare K (3) = 1.6 nM to K (6) = 0.11 nM] but
needs to be properly addressed, to exclude any possible offꢀ
target effects. In principle, any close homolog with similar
enzymatic activity is likely to be affected by these targeting
inhibitors. Glutamate carboxypeptidase III (GCPIII) is the
most likely candidate for offꢀtarget activity of GCPII inhibiꢀ
tors. GCPIII shares 67% sequence identity with GCPII and has
12
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i
did not affect the potency of inhibitors with terminal acetyl
groups [compare K (2) = 45 pM to K (5) = 49 pM]. As we
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1ꢀ12
similar enzymatic acitivity.
Its tissue distribution is similar
showed previously, inhibitor conjugates with a terminal
PEG ꢀbiotin moiety can be used to easily modify nanopartiꢀ
to that of GCPII, according to RTꢀPCR analyses, but it is more
12
12ꢀ13
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highly expressed in the testes, ovaries, and placenta.
In
cles via the biotinꢀstreptavidin interaction. The improvement
in inhibitor potency should also affect the binding properties
of the nanoparticles.
addition to its “GCPIIꢀlike activity,” GCPIII was recently also
13
shown to degrade βꢀcitrylglutamate (BCG). Even though the
physiological function of this pseudopeptide remains unꢀ
known, it may play an important role in spermatogenesis and
brain development, based on its elevated expression in testicuꢀ
Additionally, because the length of the rigid arm should be
sufficient to reach outside the GCPII entrance funnel, we preꢀ
pared a smallꢀmolecule agent for visualization of GCPII by
replacing the terminal PEG12ꢀbiotin with a fluorophore (comꢀ
pound 7). Interestingly, the addition of fluorescein moiety
resulted in a further improvement of inhibitor potency, leading
1
4ꢀ15
lar germinal cells and newborn brain tissue.
Additionally,
16ꢀ17
BCG has been proposed to serve as an iron chelator.
Curꢀ
rently, GCPIII is the only known enzyme responsible for BCG
degradation, and no data are available for educated assessment
of the potential disruption of BCG metabolism by inhibition of
GCPIII. To the best of our knowledge, no studies investigating
GCPIIꢀtargeting inhibitors have yet analyzed their activity
towards GCPIII. Analysis of the specificity of these inhibitors
to a picomolar inhibition constant [K (7) = 8.6 pM].
i
To elucidate the binding mechanism of the inhibitors with
the rigid arm, we prepared diffractionꢀquality crystals of
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Figure 1. Structures and inhibition constants of GCPII inhibitors. The compounds were derived from a ureaꢀbased inhibitor scaffold.
The εꢀamino group of lysine was subsequently modified, and the IC50 values for AviꢀGCPII and AviꢀGCPIII were determined using an
HPLCꢀbased enzymatic assay with folylꢀγꢀLꢀglutamate (FolGlu) as substrate. K values were subsequently calculated using ChengꢀPrusoff
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equation for competitive inhibition mode and kinetic parameters of substrate cleavage in reaction buffer (KM (AviꢀGCPII) = 38.6 ± 2.3 nM
and KM (AviꢀGCPIII) = 1020 ± 40 nM) and are shown as means with standard error (SE) from duplicate experiments. The selectivity represents
the fold difference in K values for AviꢀGCPII and AviꢀGCPIII. *K values taken from ref.
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AviꢀGCPII in complex with 7. We solved the structure by
molecular replacement and refined the final model to 1.86 Å
resolution (PDB code 4X3R; data collection and refinement
statistics are shown in the Supporting Information). We identiꢀ
fied a clear electron density map for the portion of 7 comprisꢀ
ing the inhibitor scaffold, bromobenzyl moiety, and first three
rings of the rigid arm. The terminal piperazine ring and the
fluorescein moiety are unstructured, showing no clear electron
density (Figure 2A). We identified two distinct inhibitor bindꢀ
ing modes in our structure.
both modeled with 40% occupancy and differing only in the
position of the bromobenzyl moiety. The rigid arm is posiꢀ
tioned above the arginineꢀpatch, with the pyridine ring formꢀ
ing a πꢀcation interaction with the side chain of Arg463
(3.6 Å). Arg463 is additionally stabilized by waterꢀmediated
hydrogen bonds between its guanidinium group (2.9 Å and
2.8 Å) and the carbonyl oxygen of 7 (2.6 Å). The stability of
this conformation is also increased by a Tꢀshaped stacking
interaction of the piperazine ring and the Trp541 side chain
(3.4 Å) and a πꢀcation interaction of the benzene ring and the
Arg511 side chain (3.6 Å). These two residues form the soꢀ
called areneꢀbinding site (ABS), which is involved in binding
In the first binding mode, which was modeled with 20% ocꢀ
cupancy, the bromobenzyl moiety is located within the argiꢀ
nineꢀpatch formed by Arg534, Arg536, and Arg463. The
Arg463 sideꢀchain forms a πꢀcation interaction with the broꢀ
mobenzyl group (3.5 Å) and simultaneously interacts with the
nitrogen atom of the pyridine ring (3.2 Å; Figure 2A1). This
conformation is identical to that of the previously observed
binding mode of 3 (PDB code 4NGQ), but seems not to be the
most favorable for 7. This observation is supported by the fact
that we detected electron density only for the first pyridine
ring of the rigid arm, which suggests that the rigid arm is not
very wellꢀstructured in this conformation.
19
of the endogenous GCPII substrate folylꢀpolyꢀγꢀLꢀglutamate.
The high similarity of the binding modes of 7 and of the enꢀ
dogenous GCPII substrate folylꢀdiꢀγꢀLꢀglutamate is illustrated
in Figure 2B1 and B2.
The bromobenzyl group does not form any direct interacꢀ
tions with the protein in the second conformation, suggesting
that its removal may lead to conformational stabilization and
higher inhibitory potency. Interestingly, we investigated the
inhibition potencies of the inhibitors with [K (5) = 49 pM] and
i
without [K (4) = 71 pM] the bromobenzyl moiety and obꢀ
i
served the opposite effect. This phenomenon could be exꢀ
plained by a potential second conformational state of 5, with
the bromobenzyl group bound within arginineꢀpatch, which
increases the overall entropy of the system.
In the second binding mode (Figure 2A2), the bromobenzyl
group of 7 is not present within the arginineꢀpatch and is loꢀ
cated within the entrance funnel in two distinct conformations,
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Figure 2. Binding modes of 7 in complex with Avi-GCPII (PDB code 4X3R). The inhibitor molecule and selected AviꢀGCPII amino
acid side chains are depicted in ballꢀandꢀstick representation with oxygen atoms colored red, nitrogen atoms blue, bromine atoms dark red,
and carbon atoms colorꢀcoded. Zinc ions are shown as magenta spheres and the chloride ion as an orange sphere. A: Two different binding
modes of 7 (depicted in cyan) are shown together with the 2F ꢀF electron density map (contoured at 1 σ). Amino acid side chains involved
in binding of the inhibitor linker region are shown: R463 in red, R511 in blue, R534 and R536 in green, and W541 in yellow. The individꢀ
ual inhibitor binding modes are depicted in A1 and A2. The dashed lines (distances shown) indicate hydrogen bonds. The modeled occuꢀ
pancy of the given conformational state is shown. B: Comparison of the binding modes of 7 (B1) and folylꢀγꢀdiꢀLꢀglutamate (FolGlu2;
PDB code 4MCQ; B2). Colorꢀcoding is the same as in panel A with FolGlu2 depicted in orange. Selected parts of protein molecules are
shown as gray surfaces.
c
o
Finally, to analyze the selectivity of our compounds, we
tested their activity toward human GCPIII. As summarized in
Figure 1, the overall inhibitory potency of all tested comꢀ
pounds is lower for GCPIII than GCPII, with the lead comꢀ
we were able to detect GCPII and GCPIII with monoclonal
20
antibody and also confirmed that both proteins were enzyꢀ
matically active. Moreover, we were unable to detect GCPII or
GCPIII expression and activity in the presence of dox, which
demonstrated the high efficiency of our inducible expression
system. For facile validation of our model system, we preꢀ
pared direct fluorophore conjugates with inhibitors containing
the rigid linker. We synthesized conjugates bearing either a
fluorescein moiety (7) or DYꢀ676 moiety (S6). Since S6 disꢀ
played nonꢀspecific binding, we continued further experiments
with 7, which inhibited GCPII 1,900ꢀfold more potently than
GCPIII. Using both confocal microscopy and flow cytometry,
we were able to selectively visualize GCPII but not GCPIII
(Figure 3C and D).
pound 1 showing 48ꢀfold lower potency [K (1GCPII) = 0.17 nM
i
vs. K (1GCPIII) = 8.2 nM]. Further optimization of inhibitors that
i
bind to GCPII exosites—the arginineꢀpatch in the case of the
bromobenzyl moiety (2) or ABS in the case of the rigid arm
(4)—led to additional 7ꢀfold and 29ꢀfold increases in inhibitor
selectivity, respectively. This mechanism seems to be additive
because the combination of both motifs led to an approximateꢀ
ly 100ꢀfold increase in selectivity compared to parent comꢀ
pound, with the most selective compound (6) showing 6,600ꢀ
fold greater potency toward GCPII than GCPIII
[
K (6
) = 110 pM vs. K (6GCPIII) = 730 nM]. The larger imꢀ
i
GCPII i
Furthermore, we analyzed the selectivity of several common
21
pact on inhibitor selectivity for the rigid arm moiety compared
to the bromobenzyl moiety can be explained by different amiꢀ
no acids occupying the ABS (Trp541 for GCPII and Lys531
for GCPIII). Following this reasoning, any compound that
utilizes the GCPII exosites for effective binding will likely
show high selectivity for GCPII over GCPIII.
GCPII inhibitors described in the literature (2ꢀPMPA, ZJꢀ
22
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4
3, DCIBzL ) and one commercially available imaging
24
agent (DKFZꢀPSMAꢀ11 ). The data summarized in Table 1
show that a simple inhibitor such as 2ꢀPMPA, with just a P1’
siteꢀbinding glutamate and zincꢀbinding group, is not highly
selective. On the other hand, the ureaꢀbased inhibitor ZJꢀ43
(GluꢀNHꢀC(O)ꢀNHꢀIle) already shows a significant increase in
selectivity, which may be caused either by replacement of the
zincꢀbinding group (phosphonate vs. urea) or by extension of
the inhibitor to the P1 site.
To demonstrate the general applicability and selectivity of
these inhibitors, we created GCPIIꢀ and GCPIIIꢀtransfected
cell lines using the inducible TetꢀOff gene expression system,
which enables regulation of the target protein expression by
addition of doxycycline (dox). As shown in Figure 3A and B,
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Figure 3. Analysis of the specific binding of 7 to GCPII and GCPIII-expressing cells. HEK293ꢀTetꢀOff cells stably transfected with
pTRETightGCPII or pTRETightGCPIII were grown in the presence [dox(+)] or absence [dox(ꢀ)] of 200 nM doxycycline (dox). A: Westꢀ
ern blot analysis of GCPII and GCPIII expression in HEK293 cells. Lysates were prepared from GCPIIꢀ and GCPIIIꢀtransfectants, loaded
onto a gel (5 µg and 100 µg total protein, respectively), and analyzed by Western blot using GCPꢀ04 monoclonal antibody, which recogꢀ
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nizes both GCPII and GCPIII. B: Enzymatic activity analysis of GCPII and GCPIII expression in HEK293 cells. The same cell lysate
preparation used for Western blot analysis was probed for enzymatic activity toward NAAG, the endogenous substrate of both GCPII and
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3
GCPIII. GCPIIꢀ and GCPIIIꢀtransfectant lysates (1 µg total protein) were incubated for 15 h at 37 °C with 100 nM HꢀNAAG. Released
3
3
HꢀLꢀglutamate was then separated from uncleaved HꢀNAAG on ionꢀexchange resin and detected using liquid scintigraphy. C: Confocal
fluorescence microscopy of GCPIIꢀ and GCPIIIꢀtransfectants using 7. The cell transfectants were grown to approximately 20% confluence.
Compound 7 (100 nM final concentration, green) was added into the growth media for 20 min, and the cell nuclei were subsequently
stained with Hoechst dye (blue). All confocal images were taken using the same microscope settings and processed using ZEN 2011 softꢀ
ware (Carl Zeiss Microscopy). D: Flow cytometry analysis of GCPIIꢀ and GCPIIIꢀtransfectants using 7. The cell transfectants were grown,
harvested, and incubated in the presence of 100 nM 7 for 30 min. Then, the cells were analyzed by flow cytometry (fluorescence detection
ex./em. 495 nm/519 nm). Each experiment was conducted in triplicate; 10,000 living cells were analyzed for each measurement. D1:
Graph illustrating the mean fluorescence intensity (MFI) of analyzed transfectants. Data are shown as mean values with standard deviation
(SD). D2: Histograms showing the population distribution based on MFI. Each histogram was created from a single representative experiꢀ
ment. GCPIIꢀtransfected cells expressing [dox(ꢀ)] and not expressing [dox(+)] GCPII are compared. The number in the top left corner indiꢀ
cates the percentage of the GCPIIꢀexpressing [dox(ꢀ)] population that has a higher MFI signal than the highest 5% of the population not
expressing GCPII [dox(+)]. This number is shown as a mean of triplicate measurements with SD. A comparable histogram is shown for
GCPIIIꢀtransfected cells.
Table 1. Inhibition constants of common GCPII inhibitors
erate larger moieties. Therefore, it can be expected that any
elongation of the GCPII inhibitor may lead to an increase in its
selectivity. Nevertheless, the presented data, as well as data for
AviꢀGCPII
AviꢀGCPIII
selecꢀ
tivity
23
K [nM]
i
K [nM]
i
DCIBzL, an inhibitor that binds to the GCPII arginine patch,
suggest that involvement of an exosite binding moiety further
significantly increases the inhibitor’s selectivity.
2ꢀPMPA
0.28 ± 0.02
0.58 ± 0.03
2.7 ± 0.1
150 ± 10
13 ± 1
10
ZJꢀ43
260
720
830
It should be noted that, despite its high selectivity, DKFZꢀ
PSMAꢀ11 is still able to inhibit GCPIII with a nanomolar inꢀ
DKFZꢀPSMAꢀ11
DCIBzL
0.018 ± 0.001
0.0030 ± 0.0001
hibition constant (K = 13 nM) and thus may disrupt BCG
i
2.5 ± 0.1
metabolism. Generally, we believe that the determination of
the selectivity of imaging agents (i.e., inhibitory activity toꢀ
wards GCPIII) is important for proper assessment of their
working concentrations during imaging experiments to ensure
that these imaging agents will exclusively target GCPII.
K values were determined as described in Figure 1 and are
i
shown as mean values with SE from duplicate experiments.
Additional elongation of the inhibitor towards the S2 site of
the enzyme (DKFZꢀPSMAꢀ11) led to further increase in the
selectivity, suggesting that the entrance funnel of GCPII is
more flexible than that of GCPIII and thus more prone to tolꢀ
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Scheme 1. Synthesis of compounds 5, 6, and 7.
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Boc
S1
Boc
HN
N
N
S2
N
a
b
N
N
I
I
N
NH
Boc
c
Br
N
S4
O
H
H
N
H
N
N
N
S3
0
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0
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7
8
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0
O
O
N
O
O
O
O
N
d
H N
2
N
OH
N
O
CF COO
3
Br
N
S5
N
O
H
N
H
N
N
N
OH
e
O
O
O
OH
O
OH
R
g
O
N
e-g
R =
N
5
Br
O
N
O
N
f
O
H
H
N
O
N
N
N
HN
OH
H
O
O
O
NH
OH
O
OH
OH
S
H
O
O
N
12
O
O
7
OH
H
6
O
a) Boc O, Et N, DCM; b) piperazine, Pd dba Xanthphos, KOt-Bu, Tol, 80 °C; c) 6-chloronicotinic acid, DIEA,
2
3
2
3,
DMAc, 130°C; d) 1) TBTU, DIEA, DMF, 2) TFA; e) Ac-NHS, DIEA, DMF; f) Biotin-PEG12-NHS, DIEA, DMF;
g) 5(6)-carboxyfluorescein TBTU, DIEA, DMF
shown for each compound, including R ), equipped with a
Waters SunFire C18 OBD Prep Column, 5 µm, 19 x 150 mm.
t
CONCLUSION
In this work, we developed potent and highly specific inhibꢀ
itors of GCPII by structureꢀaided drug design. The specificity
was achieved by exploiting exosites identified in the structure
of GCPII that are not present in the structure of its closest
homolog, human GCPIII. We designed a structurally rigid
linker that not only ensures high selectivity but also enables
efficient connection of a GCPII inhibitor with other functional
moieties, such as a fluorophore (7) or biotin (6). Finally, using
GCPII and GCPIIIꢀtransfected mammalian cells, we demonꢀ
strated high efficiency of 7 as a laboratory tool for facile and
specific GCPII visualization.
The purity of compounds was tested on an analytical Jasco
PUꢀ1580 HPLC (flow rate 1 mL/min, invariable gradient 2ꢀ
00% ACN in 30 min, R shown for each compound) equipped
with a Watrex C18 Analytical Column, 5 µm, 250 x 5 mm.
The final inhibitors were all of at least 99% purity. Structure
was further confirmed by HRMS using an LTQ Orbitrap XL
(Thermo Fisher Scientific) and by NMR (Bruker Avance™
500 MHz equipped with a cryoprobe or Bruker Avance™ 400
MHz).
1
t
The synthesis and characterization of 1, 2, and 3 have been
previously described.
18
EXPERIMENTAL SECTION
Compounds 5, 6, and 7 were synthesized as shown in
Scheme 1.
Organic synthesis. All chemicals were purchased from
SigmaꢀAldrich, unless stated otherwise. All inhibitors tested in
biological assays were purified on a preparative scale Waters
Delta 600 HPLC instrument (flow rate 7 mL/min, gradient
22
ZJꢀ43 was prepared as previously described, 2ꢀPMPA was
a kind gift from Barbara S. Slusher, DCIBzL was a kind gift
from Cyril Barinka, and DKFZꢀPSMAꢀ11 was purchased from
ABX Advanced Biochemical Compounds.
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activate the carboxylic acid. Then, S5 (30 mg, 31 µmol, 1.0
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
eq) dissolved in 0.5 mL DMF was added to the mixture. The
reaction was left to proceed overnight. Volatiles were then
evaporated, and the crude product was purified by preparative
scale HPLC (grad: 15ꢀ50% ACN in 50 min, R = 43 min). 12
t
Pure product (12 mg) was isolated upon dry freezing (isolated
yield = 32%). Analytical HPLC R = 19.1 min. HRMS (ESIꢀ)
t
ꢀ
m/z for C H O N Br [MꢀH] calc. 1193.32613, found
60
58 14
8
1
193.32656.
Compound 5: (S)ꢀ2ꢀ(3ꢀ((S)ꢀ5ꢀ(6ꢀ(4ꢀ(4ꢀ(4ꢀacetylpiperazinꢀ1ꢀ
yl)phenyl)piperazinꢀ1ꢀyl)ꢀNꢀ(4ꢀbromobenzyl)nicotinamido)ꢀ1ꢀ
carboxypentyl)ureido)pentanedioic acid. S5 (35 mg, 37 µmol,
1.0 eq) was dissolved in 0.5 mL DMF along with 20 µL DIEA
(114 µmol, 3.0 eq). To this mixture, 11.5 mg of 2,5ꢀ
dioxopyrrolidinꢀ1ꢀyl acetate (73 µmol, 2.0 eq) was added in
one portion, and the reaction was stirred overnight. After 16 h,
volatiles were evaporated, and the crude product was purified
Synthesis and characterization of 4.
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
29
N
28
27
30
31
26
N
25
23
22
24
N
19
21
N
20
15
8
6
1
8
O
H
N
13
11
10
H
N
H
N
3
16
7
1
N
by preparative scale HPLC (grad: 15ꢀ50% ACN in 60 min, R
17
4
OH
t
14
12
2
1
O
O
=
35 min). H NMR (500 MHz, DMSO): δ 8. 22 (bs, 1H, Hꢀ
O
OH
O
5
OH
9
2
1
5), 7.67 (d, J = 6.9 Hz, 1H, Hꢀ22), 7.55 (d, J = 8.4 Hz, 2H, Hꢀ
8), 7.18 (m, 6H, Hꢀ17, Hꢀ29, Hꢀ30), 6.98 (d, J = 8.8 Hz, 1H,
Compound 4: (S)ꢀ2ꢀ(3ꢀ((S)ꢀ5ꢀ(6ꢀ(4ꢀ(4ꢀ(4ꢀacetylpiperazinꢀ1ꢀ
yl)phenyl)piperazinꢀ1ꢀyl)nicotinamido)ꢀ1ꢀ
carboxypentyl)ureido)pentanedioic acid. The synthesis of 4
was nearly identical to that of the bromobenzylated derivative
Hꢀ23), 6.33 (d, J = 8.1 Hz, 1H, Hꢀ6), 6.30 (d, J = 8.1 Hz, 1H,
Hꢀ8), 4.58 (s, 2H, Hꢀ15), 4.10 (dd, J = 13.1, 7.8 Hz, 1H, Hꢀ4),
4
3
.04 (bm, 1H, Hꢀ10), 3.81 (bs, 4H, Hꢀ26), 3.64 (bs, 4H, Hꢀ33),
.37 (bs, 4H, Hꢀ27), 3.20 (bm, 6H,Hꢀ32, Hꢀ14, partial overlap
5
with the difference that a derivative without amine substituꢀ
tion was used in step d. The final product precipitated from a
with residual peak from water), 2.31 – 2.16 (m, 2H, Hꢀ2), 2.05
(s, 3H, Hꢀ35), 1.92 (ddt, J = 12.0, 9.1, 6.0 Hz, 1H, Hꢀ3a), 1.71
(ddt, J = 12.0, 9.1, 6.8 Hz, 1H, Hꢀ3b), 1.65 – 1.38 (m, 4H, Hꢀ
mixture of ACN/H O (2:1). The isolated yield after the 3 steps
2
1
was 15%. H NMR (500 MHz, DMSO) δ 8.60 (d, J = 2.7 Hz,
1
3
1H, Hꢀ21), 8.25 (t, J = 5.6 Hz, 1H, Hꢀ15), 7.96 (dd, J = 9.0,
11, Hꢀ13), 1.20 (d, J = 27.7 Hz, 2H, Hꢀ12). C NMR (126
2
6
4
3
.5 Hz, 1H, Hꢀ18), 6.95 – 6.87 (m, 5H, Hꢀ25, Hꢀ26, Hꢀ19),
.33 (d, J = 8.3 Hz, 1H, Hꢀ6), 6.31 (d, J = 8.3 Hz, 1H, Hꢀ8),
.12 – 4.02 (m, 2H, Hꢀ4, Hꢀ10), 3.74 – 3.70 (m, 4H, Hꢀ22),
.55 (dd, J = 10.4, 5.8 Hz, 4H, Hꢀ23), 3.21 (dd, J = 13.1, 6.7
MHz, DMSO): δ 174.48 (Cꢀ9), 174.19 (Cꢀ5), 173.76 (Cꢀ1),
1
2
69.01 (Cꢀ20), 168.38 (Cꢀ34), 158.35 (q, TFA), 157.91 (Cꢀ
4), 157.26 (Cꢀ7), 150.61 (Cꢀ25), 145.58 (Cꢀ31, Cꢀ28), 137.19
(Cꢀ18,Cꢀ22), 131.48 (Cꢀ17), 121.16 (Cꢀ19), 120.22 (Cꢀ29),
Hz, 2H, Hꢀ14), 3.12 – 3.07 (m, 4H, Hꢀ29), 3.03– 2.92 (m, 4H,
Hꢀ28), 2.31 – 2.17 (m, 2H, Hꢀ2), 2.03 (s, 3H, Hꢀ31), 1.95 –
1
1
4
2
18.27 (Cꢀ30), 117.39 (Cꢀ21), 115.51 (q, J = 290.8 Hz, TFA),
06.77 (Cꢀ23), 52.12 (Cꢀ4), 51.65 (Cꢀ10), 50.80 (Cꢀ32),
4.82ꢀ43.62 (Cꢀ14, Cꢀ15, Cꢀ26, Cꢀ27, Cꢀ33), 31.71 (Cꢀ11),
9.90 (Cꢀ2), 27.54 (Cꢀ13), 27.02(Cꢀ3), 22.36 (Cꢀ12), 21.19
1
.87 (m, 1H, Hꢀ3a), 1.75 – 1.63 (m, 1H, Hꢀ3b), 1.60 – 1.44
13
(m, 3H, Hꢀ11a, Hꢀ13), 1.37 – 1.20 (m, 3H, Hꢀ11b, Hꢀ12). C
NMR (126 MHz, DMSO) δ 174.58 (Cꢀ9), 174.22 (Cꢀ5),
173.76 (Cꢀ1), 168.21 (Cꢀ16), 164.79 (Cꢀ30), 159.71 (Cꢀ20),
157.30 (Cꢀ7), 147.79 (Cꢀ21), 144.98 (Cꢀ24 or Cꢀ27), 144.79
(
Cꢀ35). Analytical HPLC R = 17.4 min. HRMS (ESI+) m/z
t
+
for C H O N Br [M+H] calc. 877.28896, found 877.28658.
41
50
9
8
Compound 6: (S)ꢀ2ꢀ(3ꢀ((S)ꢀ5ꢀ(Nꢀ(4ꢀbromobenzyl)ꢀ6ꢀ(4ꢀ(4ꢀ
(Cꢀ24 or Cꢀ27), 136.50 (Cꢀ18), 118.92 (Cꢀ26), 117.53 (Cꢀ25),
(4ꢀ(41ꢀoxoꢀ45ꢀ((3aS,4S,6aR)ꢀ2ꢀoxohexahydroꢀ1Hꢀthieno[3,4ꢀ
1
5
17.36 (Cꢀ17), 105.67 (Cꢀ19), 52.29 (Cꢀ4), 51.65 (Cꢀ10),
0.01 (Cꢀ28), 49.61, 49.38, 45.66, 44.38, 40.82 (Cꢀ14, Cꢀ15,
d]imidazolꢀ4ꢀyl)ꢀ4,7,10,13,16,19,22,25,28,31,34,37ꢀ
dodecaoxaꢀ40ꢀazapentatetracontanꢀ1ꢀoyl)piperazinꢀ1ꢀ
yl)phenyl)piperazinꢀ1ꢀyl)nicotinamido)ꢀ1ꢀ
Cꢀ26, Cꢀ27, Cꢀ33), 31.83 (Cꢀ11), 29.93 (Cꢀ2), 28.98 (Cꢀ13),
7.54 (Cꢀ3), 22.71 (Cꢀ12), 21.24 (Cꢀ31). Analytical HPLC Rt
2
=
carboxypentyl)ureido)pentanedioic acid. S5 (18 mg, 21 µmol,
ꢀ
14.0 min. HRMS (ESIꢀ) m/z for C H O N [MꢀH] calc.
34 45 9 8
1
.1 eq) was dissolved in 0.5 mL DMF along with DIEA (8 µl,
7
09.33040, found 709.33016.
48 µmol, 2.5 eq). To this mixture, biotinꢀPEG ꢀCOONHS (18
1
2
Description and characterization of the reaction intermediꢀ
mg, 19 µmol, 1.0 eq) was added in one portion, and the reacꢀ
tion was left to proceed overnight. After 12 h, volatiles were
evaporated, and the product was purified by preparative scale
ates (S1-S6) and descriptions of all biochemical and molecular
biology methods used are provided in the Supporting Inforꢀ
mation.
HPLC (grad: 30ꢀ80% ACN in 50 min, R = 22 min). Pure
t
product (8 mg) was isolated upon dry freezing (isolated yield
ASSOCIATED CONTENT
=
25%). Analytical HPLC R = 18.3 min. HRMS (ESIꢀ) m/z
t
ꢀ
Supporting Information. Descriptions of the synthesis and charꢀ
acterization of intermediate compounds, descriptions of biochemꢀ
ical and molecular biology techniques used in this study, and a
table with crystallographic data collection and refinement statisꢀ
tics are shown. This material is available free of charge via the
Internet at http://pubs.acs.org.
for C H O N BrS [MꢀH] calc. 1660.70768, found
76
115 23 11
1
660.70441.
Compound 7: (S)ꢀ2ꢀ(3ꢀ((S)ꢀ5ꢀ(Nꢀ(4ꢀbromobenzyl)ꢀ6ꢀ(4ꢀ(4ꢀ
(4ꢀ(4ꢀcarboxyꢀ3ꢀ(6ꢀhydroxyꢀ3ꢀoxoꢀ3Hꢀxanthenꢀ9ꢀ
yl)benzoyl)piperazinꢀ1ꢀyl)phenyl)piperazinꢀ1ꢀ
yl)nicotinamido)ꢀ1ꢀcarboxypentyl)ureido)pentanedioic acid. A
mixture of 5ꢀ and 6ꢀcarboxy fluorescein (12 mg, 31 µmol, 1.0
eq) and DIEA (19 µL, 110 µmol, 3.5 eq) were dissolved in 0.5
mL DMF, and TBTU (10 mg, 31 µmol, 1.0 eq) was added in
one portion. The reaction mixture was stirred for 30 min to
AUTHOR INFORMATION
Corresponding Authors
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Journal of Medicinal Chemistry
*
JK; Institute of Organic Chemistry and Biochemistry, ASCR,
Labeled PSMAꢀSpecific Small Molecule in TumorꢀBearing Mice.
Prostate Cancer 2014, 2014, 104248.
10) Mease, R. C.; Foss, C. A.; Pomper, M. G. PET imaging in
prostate cancer: focus on prostateꢀspecific membrane antigen. Curr Top
Med Chem 2013, 13, 951ꢀ962.
1
2
3
4
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1
1
1
1
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6
v.v.i.; Flemingovo n. 2, Prague 6, 166 10, Czech Republic; tel:
00420 220 183 218, fax: 00420 220 183 578;
Email: konval@uochb.cas.cz
(
*
PS; Institute of Organic Chemistry and Biochemistry, ASCR,
v.v.i.; Flemingovo n. 2, Prague 6, 166 10, Czech Republic; tel:
0420 220 183 452, fax: 00420 220 183 578;
(11)
Hlouchova, K.; Barinka, C.; Klusak, V.; Sacha, P.; Mlcochova,
P.; Majer, P.; Rulisek, L.; Konvalinka, J. Biochemical characterization of
human glutamate carboxypeptidase III. J Neurochem 2007, 101, 682ꢀ696.
0
Email: pavelsacha@gmail.com
(12)
Pangalos, M. N.; Neefs, J. M.; Somers, M.; Verhasselt, P.;
Bekkers, M.; van der Helm, L.; Fraiponts, E.; Ashton, D.; Gordon, R. D.
Isolation and expression of novel human glutamate carboxypeptidases
with Nꢀacetylated alphaꢀlinked acidic dipeptidase and dipeptidyl peptidase
IV activity. J Biol Chem 1999, 274, 8470ꢀ8483.
Author Contributions
All authors have given approval to the final version of the manuꢀ
script.
‡
0
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(13)
Collard, F.; Vertommen, D.; Constantinescu, S.; Buts, L.; Van
These authors contributed equally.
Schaftingen, E. Molecular identification of betaꢀcitrylglutamate hydrolase
as glutamate carboxypeptidase 3. J Biol Chem 2011, 286, 38220ꢀ38230.
Funding Sources
(14)
Miyake, M.; Kume, S.; Kakimoto, Y. Correlation of the level
This work was supported by grant P208ꢀ12ꢀG016 (Center of Exꢀ
cellence) from the Grant Agency of the Czech Republic and NPU
I project LO 1302 from the Ministry of Education of the Czech
Republic.
of betaꢀcitrylꢀLꢀglutamic acid with spermatogenesis in rat testes. Biochim
Biophys Acta 1982, 719, 495ꢀ500.
(15)
Narahara, M.; Tachibana, K.; Adachi, S.; Iwasa, A.; Yukii, A.;
HamadaꢀKanazawa, M.; Kawai, Y.; Miyake, M. Immunocytochemical
localization of betaꢀcitrylꢀLꢀglutamate in primary neuronal cells and in the
differentiation of P19 mouse embryonal carcinoma cells into neuronal
cells. Biol Pharm Bull 2000, 23, 1287ꢀ1292.
ACKNOWLEDGMENT
We would like to acknowledge Sebastian Zoll, Ph.D., for collectꢀ
ing the xꢀray dataset on synchrotron and Petr Pachl, Ph.D., for
integration and scaling of this dataset.
(16)
HamadaꢀKanazawa, M.; Kouda, M.; Odani, A.; Matsuyama,
K.; Kanazawa, K.; Hasegawa, T.; Narahara, M.; Miyake, M. betaꢀCitrylꢀ
Lꢀglutamate is an endogenous iron chelator that occurs naturally in the
developing brain. Biol Pharm Bull 2010, 33, 729ꢀ737.
(17)
HamadaꢀKanazawa, M.; Narahara, M.; Takano, M.; Min, K. S.;
ABBREVIATIONS
Tanaka, K.; Miyake, M. betaꢀCitrylꢀLꢀglutamate acts as an iron carrier to
activate aconitase activity. Biol Pharm Bull 2011, 34, 1455ꢀ1464.
ABS, areneꢀbinding site; AviꢀGCPII/III, extracellular portions of
GCPII (44ꢀ750) or GCPIII (36ꢀ740) with Nꢀterminal AviTEV tag;
dox, doxycycline; GCPII/III, glutamate carboxypeptidase II/III;
NAAG, NꢀacetylꢀLꢀaspartylꢀLꢀglutamate; PCa, prostate carcinoꢀ
ma; MFI, mean fluorescence intensity; PSMA, prostateꢀspecific
membrane antigen
(18)
Tykvart, J.; Schimer, J.; Barinkova, J.; Pachl, P.; Postovaꢀ
Slavetinska, L.; Majer, P.; Konvalinka, J.; Sacha, P. Rational design of
ureaꢀbased glutamate carboxypeptidase II (GCPII) inhibitors as versatile
tools for specific drug targeting and delivery. Bioorg Med Chem 2014, 22,
4
099ꢀ4108.
(19) Navratil, M.; Ptacek, J.; Sacha, P.; Starkova, J.; Lubkowski, J.;
Barinka, C.; Konvalinka, J. Structural and biochemical characterization of
the folylꢀpolyꢀgammaꢀlꢀglutamate hydrolyzing activity of human
glutamate carboxypeptidase II. FEBS J 2014, 281, 3228ꢀ3242.
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