Novel substrate-based inhibitors of human glutamate carboxypeptidase II with enhanced lipophilicity

Article abstract of DOI:10.1021/jm200807m

Virtually all low molecular weight inhibitors of human glutamate carboxypeptidase II (GCPII) are highly polar compounds that have limited use in settings where more lipophilic molecules are desired. Here we report the identification and characterization of GCPII inhibitors with enhanced liphophilicity that are derived from a series of newly identified dipeptidic GCPII substrates featuring nonpolar aliphatic side chains at the C-terminus. To analyze the interactions governing the substrate recognition by GCPII, we determined crystal structures of the inactive GCPII(E424A) mutant in complex with selected dipeptides and complemented the structural data with quantum mechanics/molecular mechanics calculations. Results reveal the importance of nonpolar interactions governing GCPII affinity toward novel substrates as well as formerly unnoticed plasticity of the S1′ specificity pocket. On the basis of those data, we designed, synthesized, and evaluated a series of novel GCPII inhibitors with enhanced lipophilicity, with the best candidates having low nanomolar inhibition constants and clogD > -0.3. Our findings offer new insights into the design of more lipophilic inhibitors targeting GCPII.

Full text of DOI:10.1021/jm200807m

ARTICLE
pubs.acs.org/jmc
Novel Substrate-Based Inhibitors of Human Glutamate
Carboxypeptidase II with Enhanced Lipophilicity†
Anna Plechanovovꢀa,‡,§,b Youngjoo Byun,||,^,b Glenda Alquicer, L'ubica Skultꢀetyovꢀa, Petra Mlꢁcochovꢀa,
Adriana Nꢁemcovꢀa,‡ Hyung-Joon Kim,|| Michal Navrꢀatil,‡,§ Ronnie Mease,|| Jacek Lubkowski,∞
Martin Pomper,|| Jan Konvalinka,‡,§ Lubomír Rulíꢁsek,‡ and Cyril Baꢁrinka*,#
#
#
‡,§
ꢁ
‡Institute of Organic Chemistry and Biochemistry, Gilead Sciences Research Center at IOCB, Academy of Sciences of the Czech
Republic, Flemingovo nꢀamꢁestí 2, 166 10 Praha 6, Czech Republic
§Department of Biochemistry, Faculty of Natural Science, Charles University in Prague, Hlavova 2030, Prague, Czech Republic
Russell H. Morgan Department of Radiology and Radiological Sciences, Johns Hopkins Medical Institutions, 1550 Orleans Street,
Baltimore, Maryland 21231, United States
^College of Pharmacy, Korea University, Sejong-ro, Jochiwon-eup, Yeongi-gun, Chungnam 339-700, South Korea
#Institute of Biotechnology, Academy of Sciences of the Czech Republic, Videnska 1083, 14200 Praha 4, Czech Republic
∞Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland 21702, United States
S Supporting Information
b
ABSTRACT: Virtually all low molecular weight inhibitors of
human glutamate carboxypeptidase II (GCPII) are highly polar
compounds that have limited use in settings where more lipophilic
molecules are desired. Here we report the identification and
characterization of GCPII inhibitors with enhanced liphophilicity
that are derived from a series of newly identified dipeptidic GCPII
substrates featuring nonpolar aliphatic side chains at the C-termi-
nus. To analyze the interactions governing the substrate recogni-
tion by GCPII, we determined crystal structures of the inactive
GCPII(E424A) mutant in complex with selected dipeptides and
complemented the structural data with quantum mechanics/mo-
lecular mechanics calculations. Results reveal the importance of
nonpolar interactions governing GCPII affinity toward novel
substrates as well as formerly unnoticed plasticity of the S10 specificity pocket. On the basis of those data, we designed, synthesized,
and evaluated a series of novel GCPII inhibitors with enhanced lipophilicity, with the best candidates having low nanomolar
inhibition constants and clogD > ꢀ0.3. Our findings offer new insights into the design of more lipophilic inhibitors targeting GCPII.
’ INTRODUCTION
by small-molecule ligands is considered a viable option for the
treatment and diagnosis of a variety of pathologies that involve
glutamatergic transmission. Indeed, GCPII inhibitors have de-
monstrated efficacy in experimental models of stroke,10 diabetic
neuropathy,11 amyotrophic lateral sclerosis,12 neuropathic and
inflammatory pain,13ꢀ15 schizophrenia,16 and drug addiction.17,18
Additionally, radiolabeled GCPII-specific ligands were used to
quantitatively visualize GCPII in rodent and human tissue
ex vivo,19,20 thus expanding their use into the diagnostic area.
Effective targeting of GCPII residing in nervous tissue requires
inhibitors that can penetrate the neuronal compartment. That
can be achieved by designing inhibitors that can leverage active
transport mechanisms or by using liphophilic compounds with
enhanced penetration across the bloodꢀbrain barrier. As the
Human glutamate carboxypeptidase II (GCPII) is a trans-
membrane metallopeptidase with the expression pattern re-
stricted primarily to nervous and prostatic tissues.1ꢀ3 As the
expression levels of the prostate form of the enzyme are highly
elevated in prostate carcinoma and its metastases, GCPII serves
as a membrane-bound marker for prostate cancer imaging and
experimental therapy.4ꢀ8
Within the nervous system, GCPII catabolizes N-Ac-Asp-Glu
(NAAG), the dipeptidic neuropeptide, liberating glutamate into
the extrasynaptic space.1 Both NAAG and glutamate act as
potent neurotransmitters, and their extracellular concentrations
must be tightly regulated to secure normal brain functioning. Not
surprisingly, dysregulated NAAG (and glutamate) metabolism
and signaling are associated with a number of neurological
disorders.9 Given the intimate involvement of GCPII in NAAG
(glutamate) metabolism, the modulation of its enzymatic activity
Received: June 22, 2011
Published: September 19, 2011
r
2011 American Chemical Society
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Table 1. Formulas and Kinetic Parameters of Novel GCPII Dipeptidic Substratesa
compd
KM (μM)
kcat (sꢀ1
NAb
)
kcat/KM (L mmolꢀ1 sꢀ1
)
3
3
1S
NAb
NAb
2S
85 ( 21
29 ( 8
31 ( 12
19 ( 4
12 ( 1
8 ( 2
0.23 ( 0.05
0.25 ( 0.03
0.42 ( 0.05
0.24 ( 0.01
0.44 ( 0.04
0.60 ( 0.03
0.62 ( 0.10
0.07 ( 0.002
1.1 ( 0.2d
2.7
3S
8.6
4S
13.5
12.6
36.7
75.0
62.0
2.9
5S
6S
7S
8S
10 ( 2
24.8 ( 2.9c
1.2 ( 0.5d
NAAM
NAAG
930d
a Ac-Asp-Glu (NAAG), natural GCPII substrate in mammalian nervous system; Ac-Asp-Met (NAAM), non-natural GCPII substrate from the dipeptidic
library screen; 1S...8S, novel GCPII dipeptidic substrates featuring nonpolar aliphatic side chain at the P10 position. The kinetic parameters were
determined by saturation kinetics employing precolumn derivatization of the reaction products (i.e., released C-terminal amino acids) with AccQ-Fluor,
followed by HPLC separation on a C18(2) Luna column and fluorimetric detection. b Not analyzed, substrate hydrolysis below the detection limit.
c From ref 21. d From ref 29.
early efforts in identifying low molecular weight GCPII inhibitors
relied mainly on the knowledge of GCPII substrate specificity,
the two major categories of GCPII-specific inhibitors that exist at
present are either analogues of NAAG (the GCPII substrate) or
derivatives of glutamic acid (the reaction product). Conse-
quently, nearly all currently used GCPII inhibitors are highly
polar with their total molecular charges under physiological pH
conditions ranging typically between ꢀ2 and ꢀ3 and with lower
likelihood of CNS penetration. The design of GCPII-specific
inhibitors with increased liphophilicity/hydrophobicity is there-
fore highly desirable, with the ultimate goal of improving
bioavailability and tissue penetration while maintaining potency
and specificity for the use in clinical practice.
The aim of this work is to explore new directions in the
rational design of GCPII inhibitors by increasing their lipophi-
licity and to explain in detail the observed nonpolar interaction
patterns. To achieve this goal, we use a combination of experi-
mental and theoretical approaches including X-ray crystallography,
site-directed mutagenesis, and hybrid quantum mechanical and
molecular mechanical (QM/MM) calculations. Starting from the
detailed characterization of GCPIIꢀmethionine interactions
within the S10 pocket, we designed and characterized novel
GCPII substrates featuring aliphatic side chains at the C-terminal
amino acid. Finally, on the basis of our kinetic data, we synthesized
and evaluated a series of novel substrate-based GCPII inhibitors
with enhanced lipophilicity.
In our previous work, we reported the analysis of the substrate
specificity of human GCPII, and in addition to the known GCPII
substrates featuring C-terminal glutamate, we identified P10
methionine as a residue effectively recognized by the enzyme.21
Taking into the account the substitution of the glutamate negatively
charged side chain by the more hydrophobic side chain of methio-
nine, we hypothesized that in addition to salt bridges and/or
hydrogen bonds, nonpolar interactions at the S10 site of GCPII
are likely to play an important role in the positioning of the
C-terminal amino acid residue of GCPII substrates. These argu-
ments are also consistent with our other findings concerning the
importance of specific amino acid residues in the GCPII S1 and S10
sites,22 the detailed study of GCPII reaction mechanism,23 and
previously reported series of GCPII-specific inhibitors.7,24ꢀ28
’ RESULTS
Novel GCPII Substrates: Ac-Asp-Ala...Ac-Asp-Ano Series.
Since dipeptides with the C-terminal methionine, but not with
branched hydrophobic amino acids such as valine or leucine, are
efficiently hydrolyzed by human GCPII,21,29 we hypothesized
that the enzyme might be able to process dipeptides bearing an
unbranched, aliphatic side chain at the C-terminal moiety. To
test this prediction, we analyzed the hydrolysis of a series of eight
dipeptides by recombinant human GCPII (rhGCPII, Table 1).
The series was chosen to examine systematically the effect of P10
aliphatic side chain length on the hydrolysis by rhGCPII. The
substrate cleavage was followed by high-performance liquid
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Figure 1. (A) Superposition of NAAM, 7S, and 8S substrates in the substrate binding pocket of GCPII(E424A). The substrates are shown in
stick representation with carbon atoms colored green (NAAM), gray (7S), and magenta (8S). Selected GCPII residues surrounding the binding
pocket are shown in line representation. The zinc ions are shown as orange spheres. (B) Superposition of NAAG (PDB code 3BXM) 8S substrate
binding sites of GCPII. Substrates and the amino acids Lys699 are shown in stick representation, zinc ions and waters as orange and red spheres,
respectively. Selected GCPII residues surrounding the binding pocket are shown in line representation. Atoms are colored as follows: cyan
(NAAG carbon), green (8S carbon), blue (nitrogen), and red (oxygen). Note the displacement of the Lys699 side chain by 3.9 Å in the 8S complex
(green) due to steric clash with the 8S side chain. (C, D) S10 pocket plasticity defined by the “Lys699 swing”. (C) Composite figure showing GCPII
substrate binding site (surface representation) derived from the NAAG (PDB code 3BXM) complex and 8S (stick representation). Note the steric clash
between the side chain of Lys699 (green) and the side chain of the C-terminal residue in 8S. (D) GCPII substrate binding site (surface representation)
from the complex with 8S (stick representation). The swing-out of the Lys699 side chain (green) enlarges the S10 pocket to accommodate longer (or
more bulky) side chains of P10 part of substrates/inhibitors. The figure was generated using The PyMOL Molecular Graphics System, version 0.99,
Schr€odinger, LLC.
chromatography (HPLC), and the results are summarized in
Table 1. Except for 1S (Ac-Asp-Gly), a substrate lacking a side
chain at the C-terminal amino acid, all other dipeptidic substrates
tested were hydrolyzed by rhGCPII. The least efficient substrate
in this series was 2S (Ac-Asp-Ala), i.e., the substrate with the
shortest amino acid side chain, and gradual extension of the
hydrocarbon side chain of the C-terminal amino acid resulted in
the monotonic improvement of the overall catalytic efficiency.
This trend is documented by the fact that compared to Ac-Asp-
Ala, the rhGCPII hydrolysis of 8S (Ac-Asp-Ano), the dipeptide
with the longest (heptyl) C-terminal side chain, is approximately
20-fold more efficient (Table 1).
rhGCPII(E424A)/Substrate Complexes. To elucidate struc-
tural features that govern interactions between GCPII and
nonpolar side chains of P10 residues, we determined X-ray
structures of the inactive rhGCPII(E424A) mutant23 in complex
with three of the biochemically characterized substrates Ac-Asp-
Met (NAAM), 7S (Ac-Asp-Aoc), and 8S (Ac-Asp-Ano) at a
resolution of 1.66, 1.65, and 1.70 Å, respectively. (Note: Glu424
acts as a proton shuttle during substrate hydrolysis by GCPII, and
as such, it is indispensable for the enzymatic activity of the
enzyme. By mutating Glu424 to alanine, we constructed the
inactive GCPII(E424A) mutant that cannot hydrolyze cognate
substrates and thus serves as an excellent tool for elucidating/
approximating enzymeꢀsubstrate interactions.) All three struc-
tures were determined using difference Fourier methods, and the
refinement statistics of the final models are summarized in the
Supporting Information Table S1. The overall fold of the
rhGCPII(E424A) protein in individual complexes is nearly
identical to the arrangement observed for the rhGCPII(E424A)
complex with NAAG, a natural GCPII substrate reported earlier
(PDB code 3BXM).23 The only major structural deviations in the
substrate binding cavity are observed for the Lys699 side chain
that comes into contact with the side chains of C-terminal amino
acids of novel substrates. The superposition of the active-site-
bound substrates in the S10 pocket and their comparison to the
rhGCPII(E424A)/NAAG complex are depicted in Figure 1.
Substrate Orientation in the GCPII Binding Pocket. Posi-
tioning of all three dipeptides within the GCPII binding pocket
can be unambiguously assigned from the electron density map
and conforms to a canonical model, where the S1 pocket of
GCPII is occupied by the acetyl-aspartyl moiety and the C-term-
inal part of a substrate extends into the S10 site. Even though an
equimolar mixture of (10-R,S)-diastereomers was used in the
cases of 7S and 8S dipeptides, only the 10-S-stereoisomers are
observed in the GCPII complexes (Figure 1). That observation is
consistent with known preferences of GCPII toward L-amino
acids in the P10 position of substrates and (S)-stereoisomers of
inhibitors.1,30
S10 Pocket Interactions. The enzymeꢀsubstrate interactions
within the S10 site include both polar and nonpolar contacts. The
arrangement of polar interactions is analogous to the polar
contacts reported earlier for the rhGCPII(E424A) complex with
NAAG23 and includes direct hydrogen bonding/ionic interac-
tions between the C-terminal α-carboxylate and side chains of
Arg210, Tyr700, and Tyr552 as well as several water mediated
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Table 2. Effects of the K699S Mutation on NAAG, NAAM, and 8S Hydrolysisa
KM (μM)
kcat (sꢀ1
)
kcat/KM (L mmolꢀ1 sꢀ1
)
3
3
substrate
WT
K699S
WT
K699S
WT
K699S
NAAG
NAAM
8S
1.2 ( 0.5b
7.5 ( 1.6
10.4 ( 2.5
40.5 ( 22.7
2.6 ( 1.1
2.9 ( 0.7
1.1 ( 0.2b
0.34 ( 0.08
0.25 ( 0.04
0.37 ( 0.05
930b
42.7
59.6
8.4
96.2
0.32 ( 0.07
0.62 ( 0.10
127.6
a The kinetic parameters of wild-type (WT) and mutated (K699S) GCPII toward individual substrates were determined by saturation kinetics. The
reaction products were derivatized with AccQ-Fluor and separated by HPLC on a C18(2) Luna column with fluorimetric detection. b The kinetic
parameters of the NAAG hydrolysis by rhGCPII were taken from ref 29 and are included here for the purpose of comparison. These values were
determined by the radioenzymatic assay using 3H-NAAG as a substrate.
Table 3. Inhibition of GCPII by Novel Substrate-Based Inhibitorsa
inhibitor
ZJ-43
X moiety
Ki (nM)
CI (95% confidence)
MW
ClogD
1.08 (0.75b)
4390 (1320b)
0.85ꢀ1.36
3344ꢀ5762
231ꢀ458
86ꢀ165
96ꢀ219
34ꢀ92
304.13
477.04
491.06
505.07
519.09
533.10
547.12
561.13
575.15
551.40
549.06
ꢀ6.13
ꢀ3.76
ꢀ3.41
ꢀ2.88
ꢀ2.35
ꢀ1.82
ꢀ1.29
ꢀ0.76
ꢀ0.23
ꢀ2.37
ꢀ5.16
1I
ꢀH
ꢀCH3
2I
325
3I
ꢀCH2CH3
ꢀ(CH2)2CH3
ꢀ(CH2)3CH3
ꢀ(CH2)4CH3
ꢀ(CH2)5CH3
ꢀ(CH2)6CH3
ꢀMet
121
145 (52.3b)
4I
5I
56
6I
20
10ꢀ40
7I
72
43ꢀ123
21ꢀ38
8I
29
9I
23
12ꢀ44
10I (DCIBzL)
ꢀGlu
0.01b
0.007ꢀ0.013
a Inhibitory properties of the novel compounds were determined using the Amplex Red assay, and the results are summarized in the table. b Values from
ref 32.
contacts. On the other hand, two hydrogen-bonding interactions
between the glutamate γ-carboxylate and the Asn257 and Lys699
side chains are lost in the case of dipeptides with P10 nonpolar
side chains. Instead, the positioning of the C-terminal aliphatic
side chain relies mainly on nonpolar contacts with the side chains
of Phe209, Asn257, Leu428, Lys699 and main chains of Gly427
and Gly517.
presence of a bulkier substrate/inhibitor moiety, thus contribut-
ing prominently to the plasticity of the S10 pocket.
To verify the “steric crowding” hypothesis, we constructed a
GCPII(K699S) mutant, where Lys699 is mutated to serine.
Compared to wild-type GCPII, the K699S mutation results in
approximately 3-fold stronger binding of 8S as determined by the
kinetic assay (Table 2). These data suggest that the short side
chain of Ser699 is more accommodating toward the bulkier
C-terminal side chains of a substrate than the long Lys699 side
chain. On the other hand, a much lower affinity of NAAG toward
the K699S mutant (∼30-fold increase in the Michaelis constant
compared to the wild-type enzyme) confirms the importance of
the Lys699-Glu γ-carboxylate salt bridge for binding of com-
pounds featuring C-terminal glutamate.
Substrate-Based Inhibitors. On the basis of our kinetic and
structural data, we designed and evaluated a series of novel
inhibitors, where the N-Ac-Asp moiety and the peptide bond of a
substrate are replaced by the (4-iodobenzoylamino)hexanoyl func-
tionality and the nonhydrolyzable urea surrogate, respectively. The
rationale for the N-Ac-Asp to (4-iodobenzoylamino)hexanoyl
Flexibility of the Lys699 Side Chain Shapes the S10 Pocket.
Extended C-terminal side chains of several novel substrates
(most prominently 7S and 8S) are too long (∼8.5 Å from Cα
to the terminal aminononanoic side chain carbon atom in the
extended conformation) to fit conveniently into the standard-
size S10 pocket observed repeatedly for glutamate-like moieties.
Instead, the S10 pocket has to be reshaped to accommodate such
residues. Our structural data show that the change in size and
shape is realized by the out-swing of the Lys699 side chain with
the positional shift of the Nζ by 3.9 Å. Simultaneously, the
C-terminal side chains adopt a bow-shaped conformation to fill
in the S10 pocket (Figure 1). Clearly, the flexibility of the Lys699
side chain is crucial for alleviating steric crowding imposed by the
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Figure 2. Stereoview of the electron density map of the GCPII/9I complex. The 2Fo ꢀ Fc map is contoured at 1σ (blue), and the Fo ꢀ Fc electron
density maps are contoured at ꢀ3σ (red) and +3σ (green). Carbon atoms of the inhibitor and GCPII are colored brown and gray, respectively.
The following coloring scheme was used for individual atoms: oxygen (red), nitrogen (blue), iodine (green), sulfur (yellow), zinc (pink).
Figure 3. Stereoview of superimposed active site regions of GCPII/9I and GCPII(E424A)/NAAM complexes (inhibitor and its parent substrate).
A fragment of the GCPII/9I (wild-type GCPII and 9I inhibitor) complex is shown in light-brown, and an equivalent fragment of the GCPII(E424A)/
NAAM (the inactive mutant and NAAM substrate) complex is shown in green. The following coloring scheme was used for individual atoms: oxygen
(red), nitrogen (blue), iodine (green), sulfur (yellow). Two active site zinc atoms are shown as pink spheres, and the substrate/inhibitor is shown in a
thick ball-and-stick representation.
substitution was driven by our prior observation that the
incorporation of this functionality into the glutamateꢀurea
scaffold increases the affinity of resulting compounds by several
orders of magnitude.24 Inhibitory properties of the novel com-
pounds were determined using the Amplex Red assay, and the
results are summarized in Table 3. The Ki values in the series
follow the general trend observed for the parent substrates, with
the inhibitor potency increasing with the elongation of the P10
side chain. In this series, compound 1I has the lowest affinity
toward GCPII (Ki = 4390 nM), while the inhibition constants
monotonically decrease from 1I through 6I and plateau for
compounds 6Iꢀ8I, reaching low nanomolar affinity (∼20 nM).
The “plateau effect” observed for the inhibitor series mirrors
results from the kinetic measurements, pointing toward identi-
cal/similar positioning of P10 moieties of substrates/inhibitors.
As a result, structural/biochemical observations for one type of
ligand, substrate or inhibitor, can likely be extrapolated to the
corresponding counterpart and exploited for the design of
substrate-based inhibitors in general.
GCPII/9I Complex: X-ray Structure. To confirm the assump-
tion that binding modes to an inhibitor and its parent substrate
by GCPII are similar, we determined an X-ray structure of GCPII
in complex with 9I, a urea-based inhibitor derived from NAAM,
at 1.65 Å resolution. The binding mode of 9I in the GCPII
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Table 4. Calculated Interaction Energies of the Model
Ligands Representing S10 Residues with the P10 Side Chains
of the Selected Substrates/Inhibitors Using B-LYP(+D)/
TZVPP Method and QM/MM Optimized Geometries of
GCPII/Ligand Complexesa
Met
AOC
ANO
Phe209
ꢀ1.4
ꢀ4.8
ꢀ1.3
ꢀ1.3
ꢀ2.1
ꢀ0.3
ꢀ11.2
ꢀ2.4
ꢀ1.7
ꢀ1.9
ꢀ2
ꢀ2.2
ꢀ2.4
ꢀ2.1
ꢀ2.1
ꢀ1.4
ꢀ1.6
ꢀ11.5
Asn257
Gly427(bb)
Leu428
Gly518(bb)
Lys699(n-p)
∑Eij
a All values are in kcal molꢀ1
ꢀ2
ꢀ1.4
ꢀ11.4
.
3
Figure 4. Model system used for the calculations of interaction energies,
the S10 residues (side chains of Phe209, Asn257, Leu428, backbone of
Gly427 and Gly518, and nonpolar part of the Lys699 side chain)
interacting with the nonpolar side chains of the substrates/inhibitors.
The model system truncated from the GCPII(E424A)/NAAM complex
(Met side chain and model functional groups representing the key
interacting residues from the enzyme) is used in the figure.
Figure S1aꢀc (Supporting Information), whereas the newly
predicted structures are depicted in Figure S2.
The most notable structural features of the QM/MM opti-
mized structures not attainable from the X-ray crystallography
can be summarized as follows:
(1) In wild-type complexes, a hydroxide anion, not a water
molecule, bridges the two zinc ions in the active site, since the
water molecule spontaneously deprotonates upon binding to the
(Zn)2 site. Most likely, the proton resides on the Glu424 moiety
and is ready for the second step of the peptide bond cleavage as
shown in the previous study.23
(2) In the complexes with the nonpolar side chains at the
C-terminus, the charge of the Lys699 residue which is otherwise
the key residue for the interaction with (and the recognition of)
the C-terminal (P10) glutamate in GCPII natural substrates is
attenuated by a chain of three water molecules that are strongly
polarized by the interaction with the charged NH3+ group with
the OꢀH bonds elongated by 0.1ꢀ0.2 Å in comparison with
their standard values.
specificity pockets is unambiguously defined by the Fo ꢀ Fc omit
map (Figure 2) and mirrors the orientation and positioning of
10I (DCIBzL, a urea-based compound featuring C-terminal
glutamate). More importantly, though, the C-terminal methio-
nine in the GCPII/9I complex (together with surrounding
GCPII side chains) spatially overlaps with the corresponding
part of NAAM, its parent substrate (Figure 3). Taken together,
these data suggest transferability of kinetic/enzymatic data into
the inhibitory profiles of daughter compounds.
GCPII/Ligand Complexes: QM/MM Modeling. On the basis
of the above-described crystallographic data, we carried out the
QM/MM calculations to extend the experimental structural
information over additional aspects of the ligandꢀGCPII bind-
ing such as the protonation equilibria between protein and ligand
(3) In QM/MM optimized structures, we have not observed
any pronounced structural changes related to the protonation of
the Glu424 residue, as the extra “catalytic” proton23 is not directly
involved in any hydrogen bond. Neither have we noticed any
significant effect of the E424A mutation on the overall constitu-
tion of the GCPII active site and positioning of the substrate. The
superpositions of the wild-type GCPII, GCPII(E424A), and
protonated wild-type GCPII structures are given in the Support-
ing Information (Figures S3ꢀS5).
or energies associated with the enzyme ligand interaction. In
3 3 3
total, seven structures were modeled and examined, including
complexes of NAAM, 7S, and 8S with both wild-type GCPII and
the GCPII(E424A) mutant, as well as the complex of 8I and 10I
with wild-type GCPII. Moreover, we have considered the
protonation of the Glu424 residue (that acts as a proton shuttle
in the GCPII catalytic cycle), which resulted in a total of 13
systems that were studied. A fairly large quantum region (∼350
atoms) and a neighboring part of the protein optimized at the
MM level (additional 110 amino acid residues) should allow for a
sufficient degree of flexibility in the binding site.
Interaction Energies of Nonpolar Side Chains with the S10
Site Residues. We utilized the results of the QM/MM calcula-
tions to provide semiquantitative arguments to the discussions
concerning the origin of the nonpolar interactions between
ligands and the S10 site of GCPII. We have calculated interaction
energies of the model ligands representing the side chains of Met,
2-aminooctanoic (AOC), and 2-aminononanoic (ANO) acids
with the small models of interacting residues. The system is
depicted in Figure 4, and the results are summarized in Table 4. It
is emphasized that the results are only semiquantitative because
of the simplicity of the model functional groups representing the
residues, but a few observations can be made.
The resulting optimized structures can be found in the
Supporting Information (including partial charges on all atoms).
The excellent structural agreement between the equilibrium
QM/MM structures and their crystallographic counterparts
allowed more detailed energy analysis of the interactions be-
tween substrates/inhibitors and GCPII (vide infra). Moreover,
the QM/MM calculations yielded the equilibrium structures of
the wild-type GCPII with NAAM, 7S, 8S and the model structure
of the GCPII complex with 8I (i.e., structures that have not been
determined experimentally), which add to the already extensive
structural information obtained in this study. The alignments
of the calculated and experimental structures are depicted in
Concerning the overall value of the interaction energy, one
may observe that for AOC and ANO side chains it is marginally
higher (0.2ꢀ0.4 kcal molꢀ1) than for the Met side chain. It
3
qualitatively correlates with the observed decrease in KM values
(NAAM vs 7S and 8S). However, these differences are very
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small, and admittedly, the same correlation does not extend to
the 7S vs 8S comparison.
connected via a urea linker (Table 3). The most potent molecule
(compound 8I) has Ki = 29 nM and ClogD = ꢀ0.23. Although
the binding affinity of 8I is markedly lower compared to the
parent glutamate-based compound (29 nM vs 10 pM, re-
spectively), its affinity is sufficient for imaging PCa.32 Further-
more, substantially increased lipophilicity (ꢀ0.23 vs ꢀ5.16) can
be translated into a better pharmacokinetic profile in the
periphery, with increased likelihood of the penetration into the
CNS. Last but not least, in the phase I human clinical trial using
N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-
In the case of methionine, more than 40% of the overall
interaction energy (ꢀ11.1 kcal molꢀ1) with the neighboring
3
residues comes from the interaction with Asn257 side chain
(ꢀ4.8 kcal molꢀ1). Other nonpolar residues contribute by
3
3
∼ ꢀ1.5 kcal molꢀ1 per residue with the exception of the nonpolar
part of the Lys699 side chain (modeled as CH3(CH2)2CH3) that
contributes negligibly. For the C-terminal AOC and ANO, there
is a notable increase in the interaction of Phe209 and the
nonpolar part of Lys699 (by ∼1 kcal/molꢀ1) and a slight
increase in the interaction energies of other nonpolar residues
that more than compensates the energetic loss in the interaction
of AOC/ANO with the Asn257. The same stabilizing role can be
also postulated for several of our inhibitors previously published
and highlights the importance of nonpolar and πꢀπ stacking
interactions in biological systems.
4-[18F]fluorobenzyl-L-cysteine ([18F]DCFBC),35 one of the
glutamate-based PET agents targeting GCPII that was developed
for prostate cancer imaging, we observed somewhat increased
signal from the blood pool in human subjects, suggesting
potential binding of the compound to an unidentified plasma
protein. The prime suspect in the case is plasma glutamate
carboxypeptidase, a circulating plasma protein with 27% overall
sequence identity and overlapping substrate specificity to
GCPII.36,37 Given the substitution of glutamate by non-natural
amino acids in novel inhibitors presented here, the likelihood of
off-target interactions with endogenous proteins might be less
pronounced in the latter. To prove these assumptions, however,
additional in vivo studies are needed.
Finally, we observed that the interaction energies between the
P10 side chain of the substrate/inhibitor and the S10 residues are
almost perfectly pairwise additive; i.e., the total interaction
energy almost equals the sum of pair interaction energies. In
summary, these calculations provide semiquantitative insight
into the arguments about the origin of the hydrophobicity of
the S10 site, given in this study.
For both substrate and inhibitor synthesis, 2-amino acids with
pentyl to heptyl side chains were used as a racemic mixture for
both substrate and inhibitor synthesis. The corresponding pro-
ducts (substrates and inhibitors) are therefore equimolar mixtures
of two diastereomers. In the case of inhibitors, the individual
diastereomers were separated by HPLC and their inhibition
potency was assayed. As expected, only compounds with the (S)-
stereochemistry at the C-terminus were inhibitory, while their
(R)-counterparts turned out to be inactive (data not shown). In
the case of substrates, a mixture of diastereomers was used for
kinetic studies. Following the substrate incubation with rhGCPII
for 24 h at 37 ꢀC, we analyzed the reaction mixture using HPLC
with UV detection after precolumn derivatization of released
C-terminal amino acids with Marfey’s reagent (1-fluoro-2,4-
dinitrophenyl-5-L-alanineamide), a chiral reagent used for dis-
tinguishing (S)- and (R)-amino acids. In all cases, we observed
peaks corresponding to only a single, presumably (S), enantio-
mer (data not shown). Since previously reported data suggested
that GCPII is inactive toward (R)-amino acids at the P10 position,
we concluded that only (S)-amino acid containing dipeptides
serve as efficient substrates of rhGCPII.
The SAR studies suggest that the nonprime GCPII specificity
pocket(s) are rather insensitive to structural changes of GCPII
inhibitors and can accommodate (or at least tolerate) surprising
diversity of functional groups of inhibitors.4,7,24,26,28,34 On the
contrary, the S10 (or pharmacophore) pocket in GCPII is highly
selective for glutamate and glutamate-like moieties. The selec-
tivity is achieved via an intricate network of mostly polar
interactions between GCPII and an inhibitor, with the most
prominent being the Arg210-α-carboxylate and Lys699-γ-
carboxylate ion pairing.22,31 Structural data that characterize
the S10 pocket as fairly compact, small-sized, and unyielding, in
contrast to the much larger and quite flexible nonprime site (the
funnel emanating from the active site zinc to the surface of the
protein), are in agreement with these observations.
’ DISCUSSION
Glutamate-based functionalities are instrumental for selective
targeting of human GCPII in applications ranging from prostate
cancer (PCa) imaging to the experimental treatment of neuro-
degenerative conditions.8 Since the GCPII pharmacophore (S10)
pocket is “optimized” for glutamate-like scaffolds, the presence of
these functionalities ensures both high affinity and specificity of
corresponding inhibitors.31,32 Several groups reported structureꢀ
activity relationship (SAR) studies focusing on substituting the
P10 glutamate in GCPII inhibitors. Majer et al.33 designed and
tested a series of thiol-based inhibitors containing a benzyl
moiety at the P10 position to increase lipophilicity of 2-(3-
mercaptopropyl)pentanedioic acid (2-MPPA), the first orally
available GCPII inhibitor. In addition to higher lipophilicity, the
best candidates were found to be more potent than the parent
molecule and showed effectiveness in a rat chronic constriction
injury model of neuropathic pain. The Kozikowski group34
applied the SAR approach using N-[[[(1S)-1-carboxy-3-methyl-
butyl]amino]carbonyl]-L-glutamic acid (ZJ-43, a urea-based
NAAG analogue as a lead compound) to decrease polarity for
more efficient targeting of GCPII in the nervous system,
especially in the PNS. We evaluated a series of DCIBzL-based
isosteres and identified several non-glutamate inhibitors with Ki
values below 20 nM that exhibited selective binding to GCPII-
expressing tumors by single photon emission computed tomo-
graphy (SPECT-CT) imaging in mice.32
This report uses the rational design to extend and complement
the above-mentioned SAR studies with the objective of preparing
potent GCPII inhibitors with enhanced lipophilicity. On the
basis of our earlier kinetic data, we first designed and character-
ized a set of novel dipeptidic GCPII substrates and provided the
structural evidence for recognition of such dipeptides by GCPII.
Next, we designed a series of inhibitors, where the P10 moiety is
derived from the dipeptidic substrates and the P1 part features
4-iodobenzoyl-ε-lysine, the functionality shown by us previously
to augment interactions with GCPII;24 the P1 and P10 parts are
This report expands the above concept in two ways: (i) it
documents for the first time substantial plasticity of the GCPII
pharmacophore pocket achieved by the relocation of the Lys699
side chain leading to the considerable enlargement (by 3.9 Å) of
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the S10 site. Furthermore, this work directly to S1' site; (ii) it
directly demonstrates the importance of nonpolar interactions,
mediated by the side chains of Phe209, Asn257, Leu428, and
Lys699 for GCPII affinity toward small-molecule compounds
featuring hydrophobic moieties in the P10 position. Although
ionic interactions between the Arg210 guanidinum group and the
α-carboxylate group of the C-terminal (P10 position) residue are
common to all dipeptidic substrates tested in this study, these are
obviously not sufficient with respect to efficient substrate position-
ing and subsequent hydrolysis, as the dipeptide with glycine in the
P10 position is not cleaved. Additionally, the dipeptide with a
C-terminal alanine, the amino acid with the shortest side chain, in
the P10 position is the least efficient GCPII substrate (see Table 1).
In summary, the new findings presented here expand the
chemical space that can be explored during the rational design of
GCPII inhibitors with increased lipophilicity. By linking a
lipophilic “non-glutamate” C-terminal moiety to a nonpolar P1
functionality, one can design inhibitors with increased lipophi-
licity that are more likely to penetrate the bloodꢀbrain barrier.
However, lipophilicity is only one of physicochemical parameters
related to the druglike molecular properties (others being
molecular weight, polar surface area, number of hydrogen bond
donors/acceptors, number of rotatable bonds). In this regard,
compounds presented here can be viewed as a precedent for the
development of GCPII-specific inhibitor analogues, with the
ultimate goal of designing the truly BBB-permeable compounds.
Crystallization and Data Collection. The rhGCPII(E424A)
stock solution was mixed with 1/10 volume of 100 mM aqueous solutions
of NAAM, 7S, and 8S. For the GCPII/9I complex, rhGCPII stock
1
solution (8 mg/mL) was mixed with /10 volume of 9I (3 mM final
concentration). The protein/dipeptide or protein/inhibitor mixtures
were combined with an equal volume of the reservoir solution contain-
ing 33% (v/v) pentaerythritol propoxylate PO/OH 5/4 (Hampton
Research), 0.5% (w/v) PEG 3350, and 100 mM Tris-HCl, pH 8.0.
Crystals were grown from 2 μL droplets by the hanging-drop vapor-
diffusion method at 293 K. For each complex, the diffraction intensities
were collected from a single crystal at 100 K using synchrotron radiation
(λ = 1.00 Å) at the SER-CAT beamline 22-ID at the Advanced Photon
Source (Argonne, IL, U.S.) equipped with a MAR225 CCD detector.
The data sets were indexed, integrated, and scaled using the HKL
software package (Table S1).38
Structure Determination and Refinement. Structure deter-
mination of GCPII complexes presented here were carried out using
difference Fourier methods with the ligand-free rhGCPII (PDB code
2OOT)39 as a starting model. Calculations were performed with the
program Refmac 5.5,40 and the refinement protocol was interspersed
with manual corrections to the model employing the program Coot.41
The restrains library and the coordinate files for individual inhibitors
were prepared using the PRODRG server,42 and the inhibitors/sub-
strates were fitted into the positive electron density map in the final
stages of the refinement. Approximately 1% of the randomly selected
reflections were kept aside for cross-validation (Rfree) during the
refinement process. The quality of the final models was evaluated using
MolProbity.43 The data collection and refinement statistics are summar-
ized in Table S1. Atomic coordinates of the present structures together
with the experimental structure factor amplitudes were deposited at the
RCSB Protein Data Bank under accession numbers 3SJX [GCPII-
(E424A)/NAAM], 3SJG [GCPII(E424A)/7S], 3SJE [GCPII(E424A)/
8S], and 3SJF (GCPII/9I).
’ CONCLUSIONS
In this study we (i) report the design and characterization of a
novel set of dipeptidic GCPII substrates, (ii) provide structural
and computational evidence for recognition of such dipeptidic
substrates by GCPII, and (iii) report the design and evaluation of
novel substrate-based GCPII inhibitors with nanomolar affinity
and increased lipophilicity. Besides contributing to the under-
standing of GCPII function, these data also serve as a starting
point for the design of “non-glutamate” small molecule GCPII
ligands with the increased lipophilicity that may represent a novel
and important class of inhibitors of GCPII.
Substrate Synthesis. Glycine, (S)-alanine, (S)-norleucine, (S)-
methionine, (S)-aminobutyric acid, (S)-glutamic acid, and (S)-norvaline
were purchased from Sigma. (S,R)-forms of 2-aminoheptanoic, octanoic,
and nonanoic acids were purchased from Fluka. N-Acetylated dipeptides
were synthesized using standard Fmoc-based approach on a Wang
hydroxymethylphenyl-functionalized resin.44 The identity and purity
of the peptides were checked by mass spectrometry, reversed-phase
HPLC, and amino acid analysis, and the purity was determined to be
>95% (data not shown).
’ EXPERIMENTAL SECTION
Determination of Kinetic Constants. Kinetic constants of
N-terminally acetylated dipeptidic substrates were determined by
high-performance liquid chromatography with fluorimetric detection
(excitation 250 nm, emission 395 nm) of precolumn derivatized amino
acids. Typically, 1ꢀ2 μg/mL rhGCPII in 20 mM MOPS, 20 mM NaCl,
pH 7.4, was reacted with 5ꢀ300 μM relevant substrate for 30 min at
37 ꢀC in a final volume of 120 μL. The reaction was stopped by the
addition of 20 μL of 100 mM EDTA, pH 9.2, and the pH of the reaction
mixture was adjusted by the addition of 40 μL of 100 mM borate buffer,
pH 9.0. The released amino acids were derivatized using 20 μL of
2.5 mM AccQ-Fluor reagent (Waters, Milford, MA, U.S.) dissolved in
acetonitrile. Then 30 μL of the resulting mixture was applied to a Luna
C18(2) column (250 mm ꢁ 4.6 mm, 5 μm particle size, Phenomenex)
mounted to a Waters Alliance 2795 system equipped with a Waters 2475
fluorescence detector.
Wild-Type rhGCPII Expression and Purification. The cloning,
expression, and purification of recombinant human GCPII (rhGCPII)
have been described previously.21 Briefly, the extracellular part of human
glutamate carboxypeptidase II, which spans amino acid residues 44ꢀ750,
was cloned into the pMT/BiP/V5-His A plasmid (Invitrogen, Carlsbad,
U.S.), and the recombinant protein (designated rhGCPII) was expressed
in Drosophila Schneider’s S2 cells and purified to homogeneity.
Expression and Purification of rhGCPII(E424A) and
rhGCPII(K699S) Mutants. Cloning, expression, and purification of
the rhGCPII(E424A) and rhGCPII(K699S) mutants have been de-
scribed elsewhere.22,23 In short, desired mutations were introduced into
a GCPII coding sequence using QuikChange site-directed mutagenesis
kit (Stratagene, La Jolla, CA, U.S.) and the mutated protein expressed in
S2 cells. Purification protocols included ion-exchange chromatography
steps (QAE-Sephadex A50, source S15) followed by affinity chroma-
tography on lentil lectin and size-exclusion chromatography on a
Superdex 200 column. The final protein preparations were >95% pure
as determined by silver-stained SDSꢀPAGE (data not shown). For
crystallization experiments, purified rhGCPII(E424A) was dialyzed
against 20 mM MOPS, 20 mM NaCl, pH 7.4, and concentrated to
8.7 mg/mL.
Synthesis of GCPII Inhibitors. Some of the tested inhibitors
(1I, 4I, DCIBzL, and 9I) were already reported previously by our
laboratory.32 The other GCPII inhibitors (2I, 3I, 5Iꢀ8I) were prepared
by a general procedure as follows.
To Nε-Boc-L-lysine tert-butyl ester hydrochloric acid (339 mg,
1 mmol) in 20 mL of CH2Cl2 at ꢀ78 ꢀC was added triphosgene (98 mg,
0.33 mmol) followed by triethylamine (1 mL). The reaction mixture was
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stirred at ꢀ78 ꢀC for 1 h, and the dry ice/acetone bath was removed. The
stirring was continued for another 30 min at room temperature and the
reaction mixture cooled back to ꢀ78 ꢀC. To the reaction mixture were
added the individual amino acid (1 mmol) in anhydrous DMF (10 mL)
and triethylamine (1 mL). After the mixture was stirred overnight, the
excess solvent was removed under reduced pressure and the residue was
purified by reverse phase HPLC to give urea compounds (20ꢀ50%
yield). The urea compounds (0.1 mmol) were dissolved in 1 mL of TFA
and stirred at room temperature for 60 min. The completion of the
deprotection reaction was monitored by ESI-MS. Excess TFA was
removed by reduced pressure. The residue was dissolved in DMF
(5 mL) and triethylamime (0.5 mL), followed by the addition of
4-iodosuccinimide benzoate (0.12 mmol). The reaction mixture was
stirred at room temperature for 3 h. The excess solvent was removed and
the residue was purified by reverse phase HPLC to give the target
compounds (40ꢀ60% yield). The final purity of inhibitors was >95% as
determined by analytical HPLC.
(S)-2-(3-((S)-1-Carboxyethyl)ureido)-6-(4-iodobenzamido)-
hexanoic Acid (2I). HPLC conditions: H2O/CH3CN (0 min, 90/
10 f 30 min, 50/50, 0.1% TFA), flow rate 3 mL/min, retention time 23
min. 1H NMR (400 MHz, H2O-d2) δ: 8.17 (d, J = 8.0 Hz, 2H), 7.80 (d,
J = 7.2 Hz, 2H), 4.40ꢀ4.44 (m, 2H), 3.41ꢀ3.44 (m, 2H), 1.95ꢀ2.05 (m,
2H), 1.86ꢀ1.89 (m, 2H), 1.67ꢀ1.72 (m, 2H). [M + H] calculated for
C17H22IN3O6 492.1, found 491.9. Yield: 21%.
(S)-2-(3-((S)-1-Carboxypropyl)ureido)-6-(4-iodobenzami-
do)hexanoic Acid (3I). HPLC conditions: H2O/CH3CN (0 min,
75/25 f 30 min, 40/60, 0.1% TFA), flow rate 3 mL/min, retention time
13 min. 1H NMR (400 MHz, CD3CN) δ: 7.81 (d, J = 6.8 Hz, 2H), 7.46
(d, J = 6.8 Hz, 2H) 4.08 (m, 1H), 4.01 (m, 1H), 3.26 (d, J = 6.8 Hz, 2H),
1.70ꢀ1.75 (m, 2H), 1.63ꢀ1.68 (m, 2H), 1.60ꢀ1.64 (m, 2H),
1.32ꢀ1.38 (m, 2H), 0.84 (t, J = 7.2 Hz, 3H). [M + H] calculated for
C18H24IN3O6 506.1, found 505.8. Yield: 19%.
(S)-2-(3-((S)-1-Carboxypentyl)ureido)-6-(4-iodobenzami-
do)hexanoic Acid (5I). HPLC conditions: H2O/CH3CN (0 min,
75/25 f 30 min, 40/60, 0.1% TFA), flow rate 3 mL/min, retention time
15 min. 1H NMR (400 MHz, MeOH-d4) δ: 7.82 (d, J = 7.2 Hz, 2H),
7.56 (d, J = 7.2 Hz, 2H), 4.25 (m, 1H), 4.22 (m, 1H), 3.31ꢀ3.39 (m,
2H), 1.83ꢀ1.89 (m, 2H), 1.62ꢀ1.67 (m, 4H), 1.45ꢀ1.49 (m, 2H),
1.32ꢀ1.39 (m, 4H), 0.89 (t, 3H). [M + H] calculated for C20H28IN3O6
534.1, found 533.8. Yield: 17%.
In Vitro GCPII Inhibitory Constants. Inhibition constants were
determined using a fluorescence-based assay according to a previously
reported procedure.45 Briefly, lysates of LNCaP cell extracts (25 μL)
were incubated with the inhibitor (12.5 μL) in the presence of 4 μM
NAAG (12.5 μL) for 120 min. The amount of the released glutamate
was determined by incubating with a working solution (50 μL) of the
Amplex Red glutamic acid kit (Invitrogen Corp., CA, U.S.) for 60 min.
The fluorescence was measured using a VICTOR3V multilabel plate
reader (Perkin-Elmer Inc., Waltham, MA, U.S.) with excitation at 490 nm
and emission at 640 nm. Inhibition curves were determined using semilog
plots, and IC50 values were calculated. Assayswere performed in triplicate.
Enzyme inhibitory constants (Ki) were generated using the Chengꢀ
Prusoff conversion. Data analysis was performed using GraphPad Prism,
version 4.00, for Windows (GraphPad Software, San Diego, CA).
Quantum Chemical Calculations and QM/MM Modeling.
All QM/MM calculations were carried out with the COMQUM
program.46 In the current version, the program uses Turbomole 5.747
for the QM part and AMBER 8 (University of California, San Francisco,
U.S.) with the Cornell force field48 for the MM part. The details of the
QM/MM procedure that are essentially identical to the ones described
in ref 23 can be found in the Supporting Information.
Protein (GCPII) Setup. All structural models used in QM/MM
calculations were based on the reported crystal structures and on our
previous work23 where we carefully described the strategy to include
missing loops, addition of hydrogens, protonation states of His residues,
and construction of solvation sphere, including initial simulated anneal-
ing protocol leading to the optimization of positions of all the atoms
missing in the crystal structures. All these structural details can be found
in the PDB files deposited. Therefore, starting from the QM/MM
optimized structure of the NAAG (GCPII substrate) complexed with
the enzyme,23 we have replaced NAAG with the studied substrates/
inhibitors (NAAM, 7S, 8S, S8, 8I, 10I) using their experimental crystal
structures as templates. In case of 8I, for which the crystal structure has
not been yet determined, we used GCPII/DCIBzL and GCPII-
(E424A)/7S structures as templates for positioning their P1 and P10
parts, respectively. Because of the high similarity of the structures, we
considered this approach as physically more sound, since the (highly
optimized) QM/MM structure already contained all hydrogen atoms
and missing parts of the protein. This approach is also justified a
posteriori by an excellent agreement of the equilibrium QM/MM and
crystal structures for a given enzyme/inhibitor complex. The only
significant structural change involved the Lys699 residue which swings
by ∼3.5 Å upon binding of the inhibitors with longer alkyl chains as
described above. Also, we have tried to carefully adapt the positions of two
to three water molecules in the vicinity of the active site to match their
positions in the crystal structures. The quantum region in the calcula-
tions consisted of the following residues: two Zn2+ ions, bringing hydro-
xide moiety, inhibitor, side chains of Phe209, Arg210, Asn257, His377,
Asp387, Glu424, Glu425, Asp453, Arg463, Asp465, Arg534, Arg536,
Tyr552, His553, Lys699, Tyr700, the Gly427-Leu428 chain (capped by
CHO moiety of the Phe426 at the N-terminus and NHꢀCH3 moiety of
the Leu429 residue at the C-terminus), the Ser517-Asp520 chain (capped
by CHO moiety of the Gly516 at the N-terminus and NHꢀCH3 moiety
ofthe Phe521), and six to eightwater moleculesinthevicinity of the active
site. It resulted in fairly large QM system of ∼350 atoms.
(S)-2-(3-((S)-1-Carboxyhexyl)ureido)-6-(4-iodobenzamido)-
hexanoic Acid (6I). HPLC conditions: H2O/CH3CN (58/42, 0.1%
1
TFA), flow rate 3 mL/min, retention time 13.5 min. H NMR (400
MHz, CD3CN) δ: 7.80 (d, J = 7.2 Hz, 2H), 7.49 (d, J = 7.2 Hz, 2H),
4.13ꢀ4.18 (m, 1H), 4.08ꢀ4.12 (m, 1H), 3.27 (t, J = 6.8 Hz, 2H),
1.86ꢀ1.89 (m, 1H), 1.74ꢀ1.78 (m, 2H), 1.62ꢀ1.66 (m, 3H),
1.52ꢀ1.55 (m, 2H), 1.29ꢀ1.37 (m, 6H), 0.87 (t, J = 6.2 Hz, 3H).
[M + H] calculated for C21H30IN3O6 548.2, found 547.8. Yield: 18%.
(S)-2-(3-((S)-1-Carboxyheptyl)ureido)-6-(4-iodobenzami-
do)hexanoic Acid (7I). HPLC conditions: H2O/CH3CN (58/42,
0.1% TFA), flow rate 3 mL/min, retention time 16.5 min. 1H NMR (400
MHz, CD3CN) δ: 7.82 (d, J = 7.2 Hz, 2H), 7.50 (d, J = 7.2 Hz, 2H),
4.15ꢀ4.18 (m, 1H), 4.09ꢀ4.12 (m, 1H), 3.30 (t, J = 6.8 Hz, 2H), 1.83ꢀ
1.89 (m, 1H), 1.62ꢀ1.67 (m, 3H), 1.45ꢀ1.49 (m, 2H), 1.26ꢀ1.38 (m,
10H), 0.89 (t, J = 6.2 Hz, 3H). [M + H] calculated for C22H32IN3O6
562.1, found 561.9. Yield: 16%.
(S)-2-(3-((S)-1-Carboxyoctyl)ureido)-6-(4-iodobenzami-
do)hexanoic Acid (8I). HPLC conditions: H2O/CH3CN (58/42,
0.1% TFA), flow rate 3 mL/min, retention time 19.5 min. 1H NMR (400
MHz, CD3CN) δ: 7.79 (d, J = 8.0 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H),
4.11ꢀ4.16 (m, 1H), 4.04ꢀ4.08 (m, 1H), 3.26 (t, J = 6.8 Hz, 2H), 1.73ꢀ
1.81 (m, 1H), 1.52ꢀ1.63 (m, 3H), 1.32ꢀ1.38 (m, 2H), 1.17ꢀ1.30 (m,
12H), 0.88 (t, J = 6.2 Hz, 3H). [M + H] calculated for C23H34IN3O6
576.1, found 575.8. Yield: 14%.
Quantum Chemical Calculations. All quantum chemical calcu-
lations were performed at the density functional theory (DFT) level.
Geometry optimizations were carried out at the PerdewꢀBurkeꢀ
Ernzerhof (PBE) level.49 The DFT/PBE calculations were expedited
by expanding the Coulomb integrals in an auxiliary basis set, the
resolution-of-identity (RI-J) approximation.50 In QM/MM optimiza-
tions, the def2-SVP basis set was employed for all atoms.51 The
calculations of the interaction energy of the nonpolar side chains of
the inhibitors with the S10 residues and its decomposition into the pair
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contributions were carried out using the recent DFT-D3 computational
protocol (DFT with the empirical dispersion) of Grimme52 that was
shown to yield, in combination with the B-LYP functional53 and TZVPP
basis set,51 excellent values of the interaction energies for noncovalently
bound complexes.
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S
Supporting Information. Details of data collection and
b
refinement statistics, spectroscopic data for inhibitors, and QM/
MM calculations together with resulting PDB structures. This
material is available free of charge via the Internet at http://pubs.
acs.org.
Accession Codes
†Atomic coordinates of the present structures together with the
experimental structure factor amplitudes were deposited at the
RCSB Protein Data Bank under accession numbers 3SJX [GCPII-
(E424A)/NAAM], 3SJG [GCPII(E424A)/7S], 3SJE [GCPII-
(E424A)/8S], and 3SJF (GCPII/9I).
’ AUTHOR INFORMATION
Corresponding Author
*Phone: +420-296-443-615. Fax: +420-296-443-610. E-mail:
cyril.barinka@img.cas.cz.
(9) Neale, J. H.; Olszewski, R. T.; Gehl, L. M.; Wroblewska, B.;
Bzdega, T. The neurotransmitter N-acetylaspartylglutamate in models
of pain, ALS, diabetic neuropathy, CNS injury and schizophrenia. Trends
Pharmacol. Sci. 2005, 26, 477–484.
Author Contributions
bThese author contributed equally.
(10) Slusher, B. S.; Vornov, J. J.; Thomas, A. G.; Hurn, P. D.;
Harukuni, I.; Bhardwaj, A.; Traystman, R. J.; Robinson, M. B.; Britton,
P.; Lu, X. C.; Tortella, F. C.; Wozniak, K. M.; Yudkoff, M.; Potter, B. M.;
Jackson, P. F. Selective inhibition of NAALADase, which converts NAAG
to glutamate, reduces ischemic brain injury. Nat. Med. 1999, 5, 1396–1402.
(11) Zhang, W.; Slusher, B.; Murakawa, Y.; Wozniak, K. M.; Tsukamoto,
T.; Jackson, P. F.; Sima, A. A. GCPII (NAALADase) inhibition prevents long-
term diabetic neuropathy in type 1 diabetic BB/Wor rats. J. Neurol. Sci. 2002,
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(12) Ghadge, G. D.; Slusher, B. S.; Bodner, A.; Canto, M. D.;
Wozniak, K.; Thomas, A. G.; Rojas, C.; Tsukamoto, T.; Majer, P.;
Miller, R. J.; Monti, A. L.; Roos, R. P. Glutamate carboxypeptidase II
inhibition protects motor neurons from death in familial amyotrophic
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’ ACKNOWLEDGMENT
The authors thank Jana Starkova for excellent technical
assistance. Financial support from EMBO (Installation Grant
No. 1978), Ministry of Education, Youth and Sports of the Czech
Republic (Projects ME10031, LC 512), IBT (Grant AV0Z-
50520701), and IOCB (Grant AV0Z40550506) is gratefully
acknowledged. This work was also supported in part by the
Intramural Research Program of the NIH, National Cancer
Institute, Center for Cancer Research (J.L. and C.B.) and by
Grant R21/R33 MH080580 (M.P.). Use of the Advanced
Photon Source was supported by the U.S. Department of Energy,
Office of Science, Office of Basic Energy Sciences, under Con-
tract No. W-31-109-Eng38.
(13) Carpenter, K. J.; Sen, S.; Matthews, E. A.; Flatters, S. L.;
Wozniak, K. M.; Slusher, B. S.; Dickenson, A. H. Effects of GCP-II
inhibition on responses of dorsal horn neurones after inflammation and
neuropathy: an electrophysiological study in the rat. Neuropeptides 2003,
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’ ABBREVIATIONS USED
rhGCPII, recombinant human glutamate carboxypeptidase
II; QM/MM, quantum mechanics/molecular mechanics;NAAM,
N-acetyl-aspartyl-methionine; NAAG, N-acetyl-aspartyl-glutamate;
ANO, 2-aminononanoic acid; AOC, 2-aminooctanoic acid;
SAR, structureꢀactivity relationship; ESI-MS, electrospray
ionization mass spectrometry; PET, positron emission tomography;
Ac, acetyl group; GCPII, glutamate carboxypeptidase II;
SPECT-CT, single photon emission computed tomography; HPLC,
high performance liquid chromatography; DFT, density functional
theory; PBE, PerdewꢀBurkeꢀErnzerhof; PCa, prostate cancer
(14) Chen, S. R.; Wozniak, K. M.; Slusher, B. S.; Pan, H. L. Effect of
2-(phosphono-methyl)-pentanedioic acid on allodynia and afferent
ectopic discharges in a rat model of neuropathic pain. J. Pharmacol.
Exp. Ther. 2002, 300, 662–667.
(15) Yamamoto, T.; Nozaki-Taguchi, N.; Sakashita, Y.; Inagaki, T.
Inhibition of spinal N-acetylated-alpha-linked acidic dipeptidase pro-
duces an antinociceptive effect in the rat formalin test. Neuroscience
2001, 102, 473–479.
(16) Olszewski, R. T.; Bukhari, N.; Zhou, J.; Kozikowski, A. P.;
Wroblewski, J. T.; Shamimi-Noori, S.; Wroblewska, B.; Bzdega, T.;
Vicini, S.; Barton, F. B.; Neale, J. H. NAAG peptidase inhibition reduces
locomotor activity and some stereotypes in the PCP model of schizo-
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dependence in C57/Bl mice. Psychopharmacology (Berlin, Ger.) 2005,
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