Interactions between human glutamate carboxypeptidase II and urea-based inhibitors: Structural characterization

Article abstract of DOI:10.1021/jm800765e

Urea-based, low molecular weight ligands of glutamate carboxypeptidase II (GCPII) have demonstrated efficacy in various models of neurological disorders and can serve as imaging agents for prostate cancer. To enhance further development of such compounds, we determined X-ray structures of four complexes between human GCPII and urea-based inhibitors at high resolution. All ligands demonstrate an invariant glutarate moiety within the S1′ pocket of the enzyme. The ureido linkage between P1 and P1′ inhibitor sites interacts with the active-site Zn₁²⁺ ion and the side chains of Tyr552 and His553. Interactions within the S1 pocket are defined primarily by a network of hydrogen bonds between the P1 carboxylate group of the inhibitors and the side chains of Arg534, Arg536, and Asn519. Importantly, we have identified a hydrophobic pocket accessory to the S1 site that can be exploited for structure-based design of novel GCPII inhibitors with increased lipophilicity.

Full text of DOI:10.1021/jm800765e

J. Med. Chem. 2008, 51, 7737–7743
7737
Interactions between Human Glutamate Carboxypeptidase II and Urea-Based Inhibitors:
Structural Characterization†
Cyril Barinka,‡ Youngjoo Byun,§ Crystal L. Dusich,§,# Sangeeta R. Banerjee,§,# Ying Chen,§,# Mark Castanares,|
Alan P. Kozikowski, Ronnie C. Mease,§ Martin G. Pomper,*,§,| and Jacek Lubkowski*,‡
Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland 21702, Russell H. Morgan Department of Radiology and
Radiological Sciences, Department of Pharmacology & Molecular Sciences, Johns Hopkins Medical Institutions,
Baltimore, Maryland 21231, Department of Medicinal Chemistry and Pharmacognosy, UniVersity of Illinois at Chicago, Chicago, Illinois 60612
ReceiVed June 23, 2008
Urea-based, low molecular weight ligands of glutamate carboxypeptidase II (GCPII) have demonstrated
efficacy in various models of neurological disorders and can serve as imaging agents for prostate cancer. To
enhance further development of such compounds, we determined X-ray structures of four complexes between
human GCPII and urea-based inhibitors at high resolution. All ligands demonstrate an invariant glutarate
moiety within the S1′ pocket of the enzyme. The ureido linkage between P1 and P1′ inhibitor sites interacts
with the active-site Zn12+ ion and the side chains of Tyr552 and His553. Interactions within the S1 pocket
are defined primarily by a network of hydrogen bonds between the P1 carboxylate group of the inhibitors
and the side chains of Arg534, Arg536, and Asn519. Importantly, we have identified a hydrophobic pocket
accessory to the S1 site that can be exploited for structure-based design of novel GCPII inhibitors with
increased lipophilicity.
Introduction
the cell surface, and the enzyme is not extensively shed into
the circulation. Therefore, GCPII represents an attractive target
for the imaging and therapy of a variety of cancers.6-8
Glutamate carboxypeptidase II (GCPII, E.C. 3.4.17.21) is a
type II transmembrane exopeptidase expressed in the nervous
system1 where it hydrolyzes endogenous N-acetylaspartyl
glutamate (NAAGa) to N-acetylaspartate and glutamate, the
latter being the most abundant excitatory neurotransmitter.2
Under normal conditions, glutamate is indispensable for physi-
ological processes such as learning, memory, and developmental
plasticity.3 However, excessive glutamate production and release
can result in neuronal cell death and is implicated in a variety
of neurological conditions including neuropathic/diabetic pain,
stroke, trauma, amyotrophic lateral sclerosis, and schizophrenia.4,5
The upregulation of GCPII expression is also observed in
prostate carcinoma and within the neovasculature of solid
tumors. The expression of GCPII is confined predominantly to
Given the GCPII involvement in a variety of pathologies,
discovery and development of GCPII inhibitors as diagnostic
or therapeutic agents have been extensively pursued for the last
decade (refs 9 and 10 and references therein). GCPII inhibitors
reported to date occupy either solely the S1′ (pharmacophore)
pocket, or the enzyme-inhibitor interactions can extend over
both S1 and S1′ sites. The former type of inhibitor is usually a
derivative or mimetic of glutamic acid linked to a zinc-binding
group such as phospho(i)nate or thiol, and such inhibitors
preferentially bind to the S1′ site of GCPII.11-13 The latter group
encompasses analogues of dipeptides (such as NAAG, the
natural GCPII substrate) with the peptide bond substituted by a
hydrolysis-resistant surrogate.14-17 Among others, this category
includes dipeptide analogues connected by a urea linkage,
previously developed by us.18,19 The relative ease of synthesis
of the urea-based inhibitors of GCPII facilitates SAR studies.
Furthermore, compared to the similar phosphinates, the urea
derivatives are less polar and thus better suited for applications
requiring increased lipophilicity of inhibitors such as stroke
therapy. The efficacy of the urea-based compounds has been
demonstrated in various animal models of neurological
disorders.19-23 Additionally, radiolabeled derivatives were suc-
cessfully applied for in vivo imaging in experimental models
of prostate cancer as well as for in vitro identification of GCPII
in rodent and human brains.24-26
†
PDB accession numbers. Atomic coordinates of the present structures
together with the experimental diffraction amplitudes have been deposited
at the RCSB Protein Data Bank with accession numbers 3D7G (the complex
with 1, (DCMC)), 3D7F (the complex with 2, (DCIT)), 3D7D (the complex
with 3, (DCFBC)), and 3D7H (the complex with 4, (DCIBzL)).
* To whom correspondence should be addressed. for J.L.: phone, 301-
846-5494; fax, 301-846-7517; E-mail, jacek@ncifcrf.gov; address: Mac-
romolecular Crystallography Laboratory, 539 Boyles Street, National Cancer
Institute at Frederick, Frederick, MD 21702. For M.G.P.: phone, 410-955-
2789; fax, 443-956-5055; E-mail, mpomper@jhmi.edu; address: 1550
Orleans Street, 492 CRB II, Johns Hopkins Medical Institutions Baltimore,
MD 21213.
‡
Center for Cancer Research, National Cancer Institute at Frederick.
Russell H. Morgan Department of Radiology and Radiological Sciences,
§
Johns Hopkins Medical Institutions.
The extracellular catalytic portion of GCPII (amino acids
44-750) consists of three structurally distinct domains, each
of them contributing residues implicated in substrate binding.27
The S1′ (pharmacophore) pocket accepts the C-terminal part
of a substrate/inhibitor and preferentially binds glutamate or
glutamate-like moieties. The most prominent feature of the S1
pocket in GCPII is the “arginine patch”, comprising arginines
463, 534, and 536, which is mainly associated with the enzyme’s
preferences for negatively charged P1 residues of the li-
gand.11,27-30 Compared to the S1′ site, the S1 pocket is more
|
Department of Pharmacology & Molecular Sciences, Johns Hopkins
Medical Institutions.
Department of Medicinal Chemistry and Pharmacognosy, University
of Illinois at Chicago.
#
These authors contributed equally.
a Abbreviations: rhGCPII, recombinant human glutamate carboxypep-
tidase II; DCIT, (S)-2-(3-((S)-1-carboxy-2-(4-hydroxy-3-iodophenyl)ethy-
l)ureido)pentanedioic acid; DCMC, (S)-2-(3-((R)-1-carboxy-2-methylthio-
)ethyl)ureido)pentanedioic acid; DCFBC, (S)-2-(3-((R)-1-carboxy-2-(4-
fluorobenzylthio)ethyl)ureido)pentanedioic acid; DCIBzL, (S)-2-(3-((S)-1-
carboxy-(4-iodobenzamido)pentyl)ureido)pentanedioic acid; NAAG,
N-acetylaspartylglutamate; SAR, structure-activity relationship.
10.1021/jm800765e CCC: $40.75
2008 American Chemical Society
Published on Web 11/19/2008

7738 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Barinka et al.
Figure 1. Chemical structures of NAAG and urea-based GCPII inhibitors.
flexible in terms of structural modifications of the GCPII
inhibitors.14,16,19
groups instead of benzyl esters. Preparation of 2 is different
from that used to prepare [125I]2,24 where the radioiodine is
added in the last step of the radiosynthesis. Experimental details
and analytical data of 1 and 2 are described in the Supporting
Information. Compounds 3 and 4 were prepared as described
previously.31,32 Compounds 1, 2, and 3 are potent inhibitors of
GCPII, with IC50 values of 17, 0.5, and 14 nM, respectively.24,32,33
Compound 4 is even more potent with an IC50 value of 0.06
nM.
Recently, we have reported several high-resolution structures
of the GCPII ectodomain in complex with small-molecule
ligands such as mimetics and derivatives of glutamic acid as
well as phosphinate-based analogues of acidic dipeptides.11,29
These structures advanced our understanding of the architecture
of the GCPII active site but did not reveal the complete picture
of GCPII-inhibitor interactions. First, out of a variety of zinc-
binding groups utilized for the design of GCPII inhibitors, only
phospho(i)nates have so far been successfully co-crystallized
with GCPII.11,27,29 Furthermore, lack or limited variability of
the P1 side chains in inhibitors studied previously hampers
detailed analysis of the S1 site. Also, this lack of variability
limits a structure-based design of new GCPII inhibitors by
overlooking the advantage of modifications in the P1 position
that might provide higher affinity interaction with the enzyme.
This manuscript aims to extend our understanding of interac-
tions between human GCPII and low molecular weight ligands.
We describe the first detailed structures of complexes between
human GCPII and urea-based inhibitors. Our data provide an
explanation for earlier SAR studies, offer a deeper insight into
both the architecture of the S1 pocket and enzyme-inhibitor
interactions, and form the basis for the structure-aided design
and development of the next generation of dipeptide-based
GCPII inhibitors and substrates.
Overall Structural Comparison. Structures of rhGCPII/2,
rhGCPII/1, rhGCPII/3, and rhGCPII/4 complexes were deter-
mined by difference Fourier methods and refined at the
resolutions of 1.54, 1.75, 1.69, and 1.55 Å, respectively (Table
1). The orientation of the individual inhibitors in the rhGCPII
active site was unequivocally identified from the positive density
peaks in the Fo-Fc omit maps (Supporting Information, Figure
S1) with the invariant C-terminal glutamate facing the S1′ pocket
of the enzyme and variable positioning of the P1 moieties. All
four complexes of rhGCPII share common fold, with the root-
mean-square deviations between 0.32 and 0.52 Å for ∼695
equivalent CR-atoms in any two complexes compared.
The S1′ Pocket. The S1′ site of human GCPII has strong
preference for glutamate or glutamate-like residues.28,34 Con-
sistent with this observation, the S1′ pocket is occupied by the
invariant glutamate of the urea inhibitors. The R-carboxylate
group of glutamate of the inhibitor forms strong H-bonds with
the guanidinium group of Arg210 (2.8 Å; distances present here
are from rhGCPII/2, the complex refined at the highest resolu-
tion) and the hydroxyl group of Tyr552 (3.1 Å), and the
γ-carboxylate is engaged by the side chains of Asn257 (Nδ2,
2.9 Å) and Lys699 (Nꢀ, 2.7 Å). The shape of the S1′ pocket is
defined by Gly518 and the side chains of Phe209 and Leu428,
which also contribute nonpolar interactions to the inhibitor
binding. Additional stabilization of inhibitor is contributed by
the water interactions of R- and γ-carboxylate groups. The
overall architecture of the S1′ pocket, position of the C-terminal
glutamate, and the enzyme-inhibitor interactions are virtually
identical to those observed for complexes of rhGCPII with free
glutamateandphospho(i)nateanaloguesofglutamate/NAAG11,27,29
(Figure 2). Evidently, the S1′ pocket is “optimized” for
glutamate binding and glutamate-like residues are the best choice
as to be positioned at the C-terminus of GCPII inhibitors. Our
structural observations are fully supported by available SAR
studies showing low tolerance for substitutions at the P1′
position of the inhibitors.14,16,19
Results and Discussion
Synthesis of Inhibitors. Urea-based inhibitors with which
GCPII crystal complexes were determined in this study include
(S)-2-(3-((R)-1-carboxy-2-methylthio)ethyl)ureido)pen-
tanedioic acid (1, DCMC in Figure 1), (S)-2-(3-((S)-1-carboxy-
2-(4-hydroxy-3-iodophenyl)ethyl)ureido)pentanedioic acid (2,
DCIT in Figure 1), (S)-2-(3-((R)-1-carboxy-2-(4-fluorobenzylth-
io)ethyl)ureido)pentanedioic acid (3, DCFBC in Figure 1), and
(S)-2-(3-((S)-1-carboxy-(4-iodobenzamido)pentyl)ureido)pen-
tanedioic acid (4, DCIBzL in Figure 1). All four inhibitors were
stereochemically pure and can be viewed as nonhydrolyzable
analogues of NAAG, with the N-acetylaspartate moiety replaced
by four different side chains. Common features of these
compounds are (a) the ureido linkage substituting for the peptide
bond of a parental dipeptide, and (b) the C-terminal glutarate
moiety (Figure 1). The synthesis of 1 has been reported
previously.18 Our synthesis of 1 differs from the previous
protocol by the use of p-methoxybenzyl esters as protecting

Human GCPII in Complex with Urea-Based Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7739
Table 1. Data Collection and Refinement Statistics
rhGCPII/1
rhGCPII/2
Data Collection Statistics
rhGCPII/3
rhGCPII/4
wavelength (Å)
temperature (K)
space group
1.000
100
I222
unit cell parameters a, b, c (Å)
resolution limits (Å)
101.8, 129.7, 159.3
101.4, 130.0, 158.5
30-1.54
(1.60-1.54)
144557 (9835)
6.1 (4.0)
95.0 (65.3)
17.6 (2.2)
0.071 (0.43)
101.9, 130.5, 159.2
30-1.69
(1.75-1.69)
116666 (11057)
6.5 (4.5)
99.5 (95.3)
14.8 (2.3)
0.094 (0.51)
101.3, 130.5, 158.7
30-1.55
(1.61-1.55)
143433 (9756)
7.1 (4.5)
94.6 (64.9)
21.4 (2.2)
0.068 (0.43)
30-1.75
(1.81-1.75)
a
number of unique reflections
105078 (10129)
6.8 (5.2)
99.6 (96.9)
21.5 (2.4)
redundancy
completeness (%)
I/σI
Rmerge
0.070 (0.49)
Refinement Statistics
resolution limits (Å)
15.0-1.75
15.0-1.54
(1.58-1.54)
142946 (6620)
1446 (66)
0.177 (0.279)
0.195 (0.276)
[0.155 (0.226)]
[0.179 (0.254)]
6603
15.0-1.69
15.0-1.55
(1.59-1.55)
141846 (6804)
1445 (76)
0.183 (0.270)
0.208 (0.290)
[0.161 (0.213)]
[0.190 (0.257)]
6529
(1.79-1.75)
103375 (6801)
1550 (111)
(1.74-1.69)
114765 (7473)
1730 (97)
no. of reflections
no. of reflections in test set
R
Rfree
0.170 (0.252)
0.204 (0.272)
0.179 (0.255)
0.199 (0.293)
b
total number of non-H atoms
number of ligand atoms
number of ions
6552
21
4
6580
28
4
26
4
31
4
number of water molecules
617
657
626
592
average B-factor (Å2)
protein atoms
waters
26.4
38.7
24.5
25.7
38.6
25.7
26.8
38.4
26.8
27.9
39.6
25.4
inhibitor
rms deviations
bond lengths (Å)
bond angles (deg)
0.021
1.92
0.018 [0.015]
1.78 [1.60]
0.020
1.79
0.018 [0.015]
1.74 [1.57]
Ramachandran plot (%)
most favored
90.3
89.2
89.7
89.3
additionally allowed
generously allowed
disallowed
9.2
0.3
10.2
0.3
9.8
0.3
10.0
0.5
0.2 (Lys207)
44--54, 541-543, 654-655
0.2 (Lys207)
44-54, 545--547, 654-655
0.2 (Lys207)
44-54, 654-655
0.2 (Lys207)
44-54, 654-655
missing residues
a Values in parentheses correspond to the highest resolution shells. b Values in brackets correspond to refinement utilizing the anisotropic model for
B-factors.
The Ureido Linkage. The ureido group of the inhibitors
lesser extent Arg463, are quite flexible. The Arg536 side chain
can adopt two distinct conformations referred to as “stacking”
and “binding,” and transition between these two states is
associated with shift of the Arg463 side chain between “up”
and “down” positions, respectively (Supporting Information,
Figure S2). Such flexibility likely contributes toward less
stringent substrate specificity within the S1 site of the enzyme
(in comparison to the S1′ site) and also modulates affinity of
P1 diversified GCPII inhibitors.28,29,36
mimics a planar peptide bond of a GCPII substrate, such as
NAAG, but acts as an amide-bioisostere due to its resistance to
hydrolysis by the enzyme.35 In the structures reported here, the
ureido carbonyl oxygen is engaged by the side chains of Tyr552
(OH, 2.7 Å) and His553 (Nε2, 3.2 Å), and further interacts with
2+
the activated water molecule (2.8 Å) and the Zn1 ion (2.6
Å). The N2 is H-bonded to the Glu424 γ-carboxylate (3.0 Å),
Gly518 carbonyl oxygen (2.9 Å), and the activated water
molecule (3.1 Å), while the second ureido nitrogen donates only
one H-bond to the Gly518 main-chain carbonyl (3.0 Å).
It is interesting to note that the active-site arrangement of
the rhGCPII-ureido complexes mirrors the situation in the
rhGCPII(E424A)/NAAG complex (Barinka, unpublished) with
the distance between the two active-site zinc ions ∼3.3 Å and
the activated water molecule positioned symmetrically in
between them (2.0 Å). This finding validates the prediction of
the urea biostere as a true surrogate of the peptide bond.
While the C-terminal glutamate and the ureido group of all
four inhibitors structurally overlap, there are considerable
positional differences within the S1 pocket of the enzyme
(Figure 3A). The only common denominator of the S1 sites in
four complexes is the engagement of the P1 carboxylate in four
direct contacts with the side chains of Asn519 (Nδ, 2.9 Å),
Arg534 (Nη1, 2.9 Å) and Arg536 (Nη1, 2.9 Å; Nη2, 3.0 Å)
and two water mediated polar interactions (2.8 and 2.7 Å; Figure
3B). Hydrogen bonds between the P1 carboxylate and the
guanidinium group of Arg536 stabilize the latter in its “binding”
conformation in a manner similar to the binding of NAAG.
Because the P1 carboxylate of the studied urea-based com-
pounds contributes prominently to the GCPII-inhibitor interac-
tions in the S1 pocket, it is reasonable to suggest that this
The S1 Site and an “Accessory Hydrophobic Pocket.” The
major structural signature of the S1 pocket of the enzyme is
the “arginine patch” comprising arginines 463, 534, and 536.29,30
The Arg534 is kept in an invariant position via its interaction
with the S1-bound chloride anion, but both Arg536 and, to a

7740 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Barinka et al.
Figure 4. The hydrophobic pocket accessory to the S1 site. The
dissected substrate-binding cavity of GCPII is shown in semitransparent
surface representation (gray). The side chains of amino acids delineating
the “accessory hydrophobic pocket” are shown in stick representation
and colored cyan. The active-site Zn2+ and S1-bound Cl- are colored
blue and yellow, respectively, and water molecules are represented by
red spheres. Compound 4 bound to the active site is in stick
representation.
Figure 2. Structural similarity of the glutamate binding in the S1′
pocket. The X-ray structures of rhGCPII/EPE (PDB code 3bi0),
rhGCPII/glutamate complex (PDB code 2c6g), and the rhGCPII/2
complexes are superimposed using corresponding CR-atoms. The active
site ligands are in stick representation, residues shaping the S1′ pocket
are shown as lines, Zn2+ ions as blue spheres, and conserved water
molecules as red spheres. The H-bonds are indicated by dashed lines
with distances shown in Ångstroms (from the rhGCPII/2 complex).
of 4, have more positional freedom and could extend further
into the S1 pocket.
structural feature should be preserved in future generations of
such inhibitors to retain high-affinity binding for GCPII.
The P1 methylcysteine side chain of 1 is placed into a
“hydrophobic section” of the S1 pocket, defined by the side-
chains of Tyr549, Tyr552, and Tyr700, but its fit is rather loose
with no signature interactions observed. A similar conclusion
can be drawn for the P1 side chain of 2, with the exception of
H-bonds between the P1 hydroxyl group and side-chains of
Glu457 (Oε1, 2.6 Å) and Arg463 (Nη2, 3.2 Å). Each of 2, 3,
and 4 feature a phenyl ring as a terminal part of their P1 side
chain but they differ in the length of a linker connecting the
ring to the ureido group (Figure 1). The linker in 2 consists of
only one methylene group, while those in 3 and 4 feature
equivalents of three and six methylene groups, respectively
(Figure 1). Consequently, the phenyl groups of 3, and especially
Given the positional freedom of the terminal phenyl ring of
3, it is interesting to find it partially inserted into a pocket
accessory to the S1 site with approximate dimensions of 8.5 Å
× 7 Å × 9 Å. The bottom of this hydrophobic “accessory
pocket” is defined by portions of ꢁ-sheets ꢁ13 (Arg463-Asp465)
and ꢁ14 (Arg534-Arg536), while the walls are shaped by the
side chains of Glu457, Arg463, Asp465, Arg534, and Arg536
(Figure 4). The formation of such a pocket is only possible by
simultaneous “atypical” positioning of Arg536 into the “binding”
configuration and Arg463 into the “up” position. On the
contrary, when NAAG, the natural GCPII substrate, is bound
in the active site of the enzyme, the Arg536 side chain is
stabilized in the “binding” conformation, but Arg463 relocates
Figure 3. (A) Superposition of urea-based inhibitors in the active-site of rhGCPII. The rhGCPII-inhibitor complexes were superimposed on
corresponding CR-atoms. The inhibitors are shown in stick representation and protein residues are shown as lines. Note invariant positioning of the
P1′ glutamate contrasting with inhibitor conformational variability in the S1 pocket. (B) Hydrogen-bonding network in the S1 site of GCPII.
Hydrogen bonding interactions (indicated with dashed lines) and distances (in Ångstroms) between 1 bound in the active site and S1 residues of
GCPII. The zinc ions, chloride anion, and water molecules located in the active site are shown as blue, yellow and red spheres, respectively. The
protein and inhibitor atoms are colored red (oxygen), blue (nitrogen), yellow (sulfur), violet (iodine), cyan (fluorine), gray (carbons).

Human GCPII in Complex with Urea-Based Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7741
Figure 5. The flexibility of S1 arginines 463 and 536 defines size of the “accessory pocket”. Dissected view into the active-site of GCPII. The
protein moiety is shown in combination of cartoon and line representation. The residues shaping walls of the accessory pocket are shown as spheres
and inhibitor (substrate) residues are in stick representation. The active site Zn2+ and S1 bound Cl- are colored blue and yellow, respectively. The
R463u and R463d denotes the side chain of Arg463 in the “up” and “down” position, respectively, while R536b and R536s denotes the side chain
of Arg536 in the “binding” and “stacking” configuration, respectively. Notice the closure of the accessory pocket in the unliganded GCPII structure
(A). Shown are complexes of rhGCPII with 2 (C), 1 (D), 3 (E), and 4 (F). The complex between the E424A active-site mutant of GCPII and
NAAG, the GCPII natural substrate, is included for comparison (PDB code 3bxm, (B)).
by 2 Å into the “down” position at the same time, effectively
closing the “accessory pocket.” Similarly, under conditions when
the S1 site is unoccupied or is occupied by a moiety that fails
to enforce the Arg536 “binding” conformation, the Arg536 side
chain is found in both “binding” and “stacking” alternate
positions (accompanied by the “down” and “up” position of
Arg463, respectively) resulting in closure (disappearance) of
the “accessory pocket” (Figure 5). In the case of 4, the phenyl
ring is fully inserted into the pocket (due to presence of longer
P1 spacer), and this fact likely contributes to the tighter inhibitor
binding that is translated into an IC50 value of ∼10-fold lower
as compared to 2 and 3.
The existence of such “remote hydrophobic binding register”
has been predicted in the study by Berkman’s group, where the
authors showed that lengthening of the methylene linker between
the phosphoamidate surrogate and the terminal phenyl group
results in stronger inhibitor binding to GCPII.15 Clearly, the
incorporation of a longer spacer into the P1 side chain allows
for the full insertion of the phenyl ring into the accessory
hydrophobic pocket with concomitant decrease in inhibition
constants. Our observations thus provide a structural rationale
for Berkman’s inhibition data and further underscore the
importance of Arg463 and Arg536 flexibility in influencing the
affinity of various inhibitors to GCPII.29
to the corresponding C-termini (Supporting Information, Figure
S3). This observation is consistent with site-directed mutagenesis
studies, SAR data, and findings reported for the X-ray structures
of GCPII and phosphinate-based inhibitors.14-17,19,29,37 Clearly,
the C-terminal glutamate or glutamate-like residue contributes
prominently to the inhibitor affinity towards GCPII, while the P1
moiety is less important in this respect. Concurrently, however,
the hydrophobic pocket accessory to the S1 site identified here
could be exploited for further optimization of GCPII inhibitors by
modification of the P1 moiety. The appendage of a hydrophobic
functionality (with the linker of the appropriate length) at the P1
position can both enhance inhibitor affinity towards GCPII and
increase liphophilicity of such compounds as a first step toward
facilitating blood-brain barrier penetration, if desired.
Conclusions
We determined high-resolution structures of human GCPII and
four urea-based inhibitors and identified a hydrophobic pocket
accessory to the S1 specificity site. Our data provide a mechanistic
explanation for prior SAR studies on GCPII inhibitors and can
serve as a useful platform for the design of novel GCPII inhibitors
with improved pharmacokinetic properties.
Experimental Section
Expression and Purification of Recombinant Human
GCPII (rhGCPII). Human GCPII (the extracellular part, amino
acids 44-750) was heterologously overexpressed in Drosophila
Thermal displacement parameters for the P1 moieties of the
inhibitors are considerably higher (6.3-15.0 Å2) when compared

7742 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Barinka et al.
Schneider’s S2 cells and purified to homogeneity as described
previously.28 This construct is designated rhGCPII (recombinant
human GCPII). The final protein preparation in 20 mM Tris-HCl,
100 mM NaCl, pH 8.0 was concentrated to 8 mg/mL and stored at
-80°C until further use.
EB005423, and MH080580 and Department of Defense grant
PC050825 (M.G.P.).
Supporting Information Available: Density maps of the
substrate binding cavity of human GCPII in complex with 3, 2, 1,
and 4; flexibility of S1 Arg463 and Arg536; thermal displacement
parameters of urea-based inhibitors; experimental procedures and
analytical data for all intermediates of 1 and 2. This material is
available free of charge via the Internet at http://pubs.acs.org.
Crystallization and X-ray Data Collection. The inhibitors were
dissolved in distilled water to a final concentration of 40 mM, and
the pH of the solution was adjusted to 8.0 with 1 M NaOH. The
rhGCPII stock solution (8 mg/mL) was mixed with 1/10 v/v of
the individual inhibitor, and the complexes were crystallized using
the hanging drop vapor diffusion setup at 293 K. Crystallization
droplets were made by combining 1 µL of the rhGCPII-inhibitor
mixture and 1 µL of the reservoir solution containing 33%
pentaerythriol propoxylate (PO/OH 5/4; Hampton Research), 1%
PEG3350, and 100 mM Tris-HCl, pH 8.0. Crystals belonging to
the space group I222 typically appeared within two days and
reached their final size with approximate dimensions of 0.3 mm ×
0.4 mm × 0.1 mm within a week. For data collection crystals were
flash-frozen in liquid nitrogen directly from the reservoir solution,
and diffraction intensities were collected at 100 K using synchrotron
radiation at the SER-CAT sector 22 beamlines of the Advanced
Photon Source (Argonne, IL) at the X-ray wavelength of 1.0 Å. In
all cases, the diffraction data were collected from a single crystal,
recorded on a CCD detector, and processed using the HKL2000
software package.38
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