Biostructural and pharmacological studies of bicyclic analogues of the 3-isoxazolol glutamate receptor agonist ibotenic acid

Article abstract of DOI:10.1021/jm101218a

We describe an improved synthesis and detailed pharmacological characterization of the conformationally restricted analogue of the naturally occurring nonselective glutamate receptor agonist ibotenic acid (RS)-3-hydroxy-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-7-carboxylic acid (7-HPCA, 5) at AMPA receptor subtypes. Compound 5 was shown to be a subtype-discriminating agonist at AMPA receptors with higher binding affinity and functional potency at GluA1/2 compared to GluA3/4, unlike the isomeric analogue (RS)-3-hydroxy-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-5-carboxylic acid (5-HPCA, 4) that binds to all AMPA receptor subtypes with comparable potency. Biostructural X-ray crystallographic studies of 4 and 5 reveal different binding modes of (R)-4 and (S)-5 in the GluA2 agonist binding domain. WaterMap analysis of the GluA2 and GluA4 binding pockets with (R)-4 and (S)-5 suggests that the energy of hydration sites is ligand dependent, which may explain the observed selectivity.

Full text of DOI:10.1021/jm101218a

8354 J. Med. Chem. 2010, 53, 8354–8361
DOI: 10.1021/jm101218a
Biostructural and Pharmacological Studies of Bicyclic Analogues of the 3-Isoxazolol Glutamate
Receptor Agonist Ibotenic Acid†
Karla Frydenvang,‡ Darryl S. Pickering,§ Jeremy R. Greenwood, Niels Krogsgaard-Larsen,‡ Lotte Brehm,‡ Birgitte Nielsen,‡
Stine B. Vogensen,‡ Helle Hald,‡ Jette S. Kastrup,‡ Povl Krogsgaard-Larsen,‡ and Rasmus P. Clausen*,‡
‡Department of Medicinal Chemistry, and §Department of Pharmacology and Pharmacotherapy, Faculty of Pharmaceutical Sciences,
University of Copenhagen, 2 Universitetsparken, DK-2100 Copenhagen, Denmark, and Schro€dinger Inc., 120 West 45th Street, New York,
New York 10036, United States
Received September 18, 2010
We describe an improved synthesis and detailed pharmacological characterization of the conformationally
restricted analogue of the naturally occurring nonselective glutamate receptor agonist ibotenic acid (RS)-
3-hydroxy-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-7-carboxylic acid (7-HPCA, 5) at AMPA receptor
subtypes. Compound 5 was shown to be a subtype-discriminating agonist at AMPA receptors with higher
binding affinity and functional potency at GluA1/2 compared to GluA3/4, unlike the isomeric analogue
(RS)-3-hydroxy-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-5-carboxylic acid (5-HPCA, 4) that binds to all
AMPA receptor subtypes with comparable potency. Biostructural X-ray crystallographic studies of 4 and 5
reveal different binding modes of (R)-4 and (S)-5 in the GluA2 agonist binding domain. WaterMap analysis
of the GluA2 and GluA4 binding pockets with (R)-4 and (S)-5 suggests that the energy of hydration sites is
ligand dependent, which may explain the observed selectivity.
Introduction
AMPA receptors being made up of GluA1-4 subunits and KA
receptors of GluK1-5 subunits.2,7-9 During the past decade,
X-ray crystallographic studies of a soluble construct (GluA2-
S1S2) of the agonist binding domain (ABD) of GluA2 contain-
ing various AMPA receptor ligands have provided structural
information about ligand recognition as well as activation and
desensitization mechanisms.10-12 Furthermore, ABD crystal
structures of the AMPA receptor subunits GluA3 and GluA4,
NMDA receptor subunits GluN1, GluN2A, GluN3A, and
GluN3B, KA receptor subunits GluK1 and GluK2, and the
GluD2 receptor subunit have been reported.13-21 The ABD has
a clamshell-like structure that closes around the ligand, and this
motion probably leads to opening of the ion channel. It has been
observed that the degree of domain closure induced by the
ligand bound to GluA2 correlates with the relative agonist
efficacy of ligands measured by the magnitude of the induced
ion current.22,23 Recently, the structure of a full length homo-
meric GluA2 receptor disclosed that the receptor is tetrameric
with 4-fold symmetry in the ion-channel part and the agonist
binding domain is organized as a dimer of dimers.24 Several
agonists have been developed that are able to selectively activate
one of the three major ionotropic receptor classes. However,
agonists discriminating subtypes within a class have been diffi-
cult to develop. Nevertheless, some compounds such as (RS)-2-
amino-3-(4-chloro-3-hydroxyisoxazol-5-yl)propionic acid
(Cl-HIBO, 3) are able to selectively activate GluA1/2.25 We have
previously reported the agonists (RS)-3-hydroxy-4,5,6,7-tetra-
hydroisoxazolo[5,4-c]pyridine-5-carboxylic acid (5-HPCA, 4)
and (RS)-3-hydroxy-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-
7-carboxylic acid (7-HPCA, 5) that may be considered confor-
mationally restricted analogues of 2 and 1, respectively.26 Re-
cently, we showed that 4 binds to GluA1-4 with comparable
potency and, unlike most other AMPA receptor agonists, it is the
(S)-Glutamate (Glua) is the primary excitatory neurotrans-
mitter in the brain and plays multiple roles in normal functions
of the central nervous system (CNS) but also in a number of
disorders related to the CNS.1-6 Upon synaptic release Glu
activates a highly heterogeneous group of receptors comprising
both the G-protein-coupled metabotropic Glu receptors
(mGluRs) and the ionotropic Glu receptors (iGluRs) that are
ligand-gated ion channels.2,6 The intriguing roles of Glu recep-
tors in several CNS disorders has spurred considerable interest
from both academia and industry in the development of ligands
that interact selectively with subtypes of this group of receptors
in a specific manner. A number of such ligands have been
developed from the naturally occurring amino acid, ibotenic
acid (1, Chart 1) notably (S)-2-amino-3-(3-hydroxy-5-methyl-
isoxazol-4-yl)propionic acid [(S)-AMPA, (S)-2]. On the basis of
structural, functional, and pharmacological characteristics,
iGluRs are categorized into three classes according to selective
activation by the agonists N-methyl-D-aspartic acid (NMDA),
(S)-AMPA, and kainic acid (KA). The iGluRs are homo- or
heteromeric assemblies of subunits forming the ion channels,
†The following coordinates have been deposited in the Protein Data
Bank: (R)-4 in GluA2-S1S2, PDB code 3PD9; (S)-5 in GluA2-S1S2,
PDB code 3PD8.
*To whom correspondence should be addressed. Phone: þ45 35 33 65
66. Fax: þ45 35 33 60 40. E-mail: rac@farma.ku.dk.
a Abbreviations: iGluRs, ionotropic glutamate receptors; KA, kainic
acid; ABD, agonist binding domain; Glu, (S)-glutamate; NMDA, N-methyl-
D-aspartic acid; AMPA, (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-
4-yl)propionic acid; CNS, central nervous system; mGluRs, metabotropic
glutamate receptors; GluA2-S1S2, soluble construct of the agonist
binding domain of GluA2; GluK1-S1S2, soluble construct of the agonist
binding domain of GluK1; DFT, density functional theory; TEVC, two-
electrode voltage clamp.
r
pubs.acs.org/jmc
Published on Web 11/10/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8355
Chart 1. Chemical Structures of Glu and Some Selective iGluR
Agonists 1-5
Table 1. Receptor Binding Affinities of Compounds 1 and 3, (R)-4, and
5 at Three Major Groups of iGluRs in Rat Cortical Synaptosome Assaya
IC50 (μM)
[3H]AMPA
>100
Ki (μM)
[3H]CGP 39653
compd
1b
[3H]KA
22
5.3
18
3c
0.22
0.47
>100
>100
>100
(R)-4d
>100
>100
5
0.37 [0.36, 0.39]
a Values are expressed as the antilog of the log of the mean of three
individual experiments. The numbers in brackets [min, max] indicates
(SEM according to a logarithmic distribution. b Reference 36. c Refer-
ence 25. d Reference 27.
(R)-4 that has similar affinity at all AMPA receptor subtypes.
Since 5 is structurally very similar to 1, we decided to probe the
affinity of 1 toward cloned AMPA receptor subtypes as well,
because activity at individual subtypes may not have been
detected in the initial native receptor binding assay. However,
1 displayed no detectable AMPA receptor affinity but showed
some affinity toward GluK1-3 subtypes as expected from the
initial native receptor binding study. The selectivity of 5
prompted a functional characterization using two-electrode
voltage clamp (TEVC) electrophysiological measurements in
oocytes expressing recombinant rat AMPA receptor subtypes
(Table 3, Figure 1). Also in this assay compound 5 displayed
selectivity toward GluA1/2, albeit with lower potency than 3 but
with similar or improved selectivity. Interestingly, (R)-4 appears
to be a partial agonist at GluA2(Q)i with an efficacy (relative to
Glu) of 0.807 ( 0.024 (n = 18) while 5 is a superagonist with an
efficacy of 1.179 ( 0.026 (n=8).
Scheme 1a
Competitive displacement studies of [3H]AMPA by 4 and
5 were performed in order to compare the binding affinity at
GluA2-S1S2 with the affinity at the full-length receptor. The
Ki of 4 at GluA2-S1S2 is 494 ( 71 nM and that of 5 at GluA2-
S1S2 is 108 ( 9 nM, which is comparable to those at the
wildtype full-length GluA2 receptor.
a Reagents and conditions: (a) sec-BuLi, -78 ꢀC, CO2; (b) HCl (aq),
room temp.
(R)-form of 4 that is the active enantiomer.27 We now report an
improved synthesis and a detailed pharmacological characteriza-
tion of 5 at Glu receptor subtypes and X-ray structures of 4 and 5
in complex with the agonist binding domain of GluA2. Finally,
we have performed a computational WaterMap analysis of the
binding pocket suggesting that the observed subtype selectivity is
closely linked to changes in the free energy of receptor water sites
in response to different ligands.
X-ray Structure. To study the binding modes of (R)-4 and
5 at the agonist binding domain of GluA2, we determined the
two X-ray structures. (R)-4 in complex with GluA2 crystal-
lized with two molecules (molA and molB) in the asymmetric
unit of the crystals, and diffraction data were collected to
˚
2.1 A resolution, whereas three molecules (molA-molC) were
seen in the asymmetric unit of crystals of (S)-5 in complex
˚
with GluA2 and diffraction data were collected to 2.5 A
resolution. For statistics on data collections and structure
refinements, see Table 4. (R)-4 and (S)-5 were unambigu-
ously defined in the electron density maps (parts A and B of
Figure 2). (S)-5 induces full domain closure in the ABD of
GluA2 as previously observed for full agonists: 21.0ꢀ (molA),
19.8ꢀ (molB), and 20.8ꢀ (molC), whereas (R)-4 induces full
domain closure in one molecule (20.1ꢀ, molA) and partial
domain closure in the other molecule (18.5ꢀ, molB).
The amino acid moieties of both (R)-4 and (S)-5 are located
at similar positions in the binding site of GluA2 and form the
same contacts to GluA2 binding site residues as seen in several
other structures of agonists in complex with GluA211,12,22,23
(Figure 2). The ammonium group of (R)-4 forms contacts to the
oxygen atom of Pro478 and the side chain OE2 oxygen atom of
Glu705 (OE1 in (S)-5). In addition, the ammonium group of
(S)-5 makes a hydrogen bond to the side chain hydroxyl group
of Thr480. The carboxylate groups of (R)-4 and (S)-5 are
engaged in salt bridge formation to the NH1 and NH2 side
chain atoms of Arg485. Furthermore, potential hydrogen bonds
are formed to the nitrogen atoms of Thr480 and Ser654.
Results
Chemistry. A new synthetic route (Scheme 1) was employed
for the resynthesis of 5, starting from tert-butyl 3-hydroxy-4,5,
6,7-tetrahydroisoxazolo[5,4-c]pyridine-6-carboxylate (6). Com-
pound 6 was dilithiated using sec-BuLi and reacted with CO2
providing 7. Acidic deprotection of 7 yielded the desired com-
pound 5.
Pharmacology. The affinity of compound 5 for native
AMPA, KA, and NMDA receptors was initially determined
in [3H]AMPA, [3H]KA, and [3H]CGP 39653 competition radio-
ligand displacement assays, respectively, using membranes
prepared from rat cortical brain tissue (Table 1). In this assay,
compound 5 displays selectivity toward AMPA sites with
affinities similar to those of 3 and (R)-4. The affinities of com-
pound 5 were further evaluated at recombinant GluA1-4 and
GluK1-3 expressed as homomeric receptors in Sf9 cell mem-
branes (Table 2). In this assay, compound 5displayed preference
for GluA1/2 over GluA3/4 and weak affinity for GluK1. This
profile is similar to that of 3 but different from the profile of

8356 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Frydenvang et al.
Table 2. Receptor Binding Affinity at Cloned rat iGluR Subtypes Expressed in Sf9 Cellsa
Ki (nM)
(R)-4c
2b
3b
1d
5d
GluA1o
22
17
130
370
360
600
840
640
>100000
>100000
>100000
>100000
2430 ( 545
3230 ( 245
1740 ( 170
180 ( 2
348 ( 31
4520 ( 600
13530 ( 2350
4480 ( 480
>100000
GluA2(R)o
GluA3o
GluA4o
21
40
27500
11900
GluK1(Q)1b
GluK2(VCR)a
GluK3a
1150
>1000000
6800
>100000
10100
>100000
54000 ( 9900e
42700 ( 12500
90500 ( 7400
29800 ( 3500
a Mean ( SEM values are shown for at least three experiments conducted in triplicate at 12-16 ligand concentrations. b Reference 25. c Reference 27
d Hill coefficients were not different from unity. e (RS)-4.
Table 3. TEVC Functional Characterization of 3, (R)-4, and 5 in
Xenopus lævis Oocytes Expressing Recombinant Rat GluA Subtypesa
Table 4. Crystallographic and Structure Refinement Data of (R)-4 and
(S)-5 in Complex with the Agonist Binding Domain of GluA2a
EC50 (μM)
parameter
(R)-4
(S)-5
3b
(R)-4
5
space group
unit cell
P21212
P21212
GluA1i
4.7
1.7
n.d.
82.8 ( 13.4 (5)
34.6 ( 1.5 (5)
637 ( 81 (3)
>1000 (4)
˚
a (A)
99.4
121.3
47.5
2
114.6
164.0
47.6
3
GluA2(Q)i
GluA3i
GluA4i
22.3 ( 1.1 (11)
nd
nd
˚
b (A)
2700
1300
˚
c (A)
molecules/aub
a Mean ( SEM values given with number of experiments in parenthe-
ses. nd, not determined. b Reference 25.
Data Collection
29.4-2.1
137887
˚
resolution range (A)
no. of reflections
unique reflections
average redundancy
completeness (%)
29.3-2.5
109603
32227
34007
4.1
3.4
99.9 (99.9)
10.4 (41.1)
13.7 (4.1)
23.0
98.3 (97.8)
11.4 (39.8)
11.0 (3.0)
39.5
R
sym (%)c
I/σ(I)
Wilson B (A )
2
˚
Refinement
non-hydrogen atoms
amino acid residues
(R)-4/(S)-5
4540
518
2
6551
774
3
396/0/5/1/0/1/1
water/sulfate/zinc/glycerol/
chloride/cacodylate/acetate
Rwork d (%)
Rfree e (%)
rmsd bond length
361/4/0/5/4/0/0
17.7
23.3
17.5
26.0
0.007/1.0
0.006/1.0
˚
(A)/angle (deg)
no. residues in allowed regions
of Ramachandran plotf (%)
98
98
Figure 1. Concentration-response curves of 5 at recombinant rat
homomeric GluA receptors (flip isoforms) expressed in Xenopus
lævis oocytes and measured by TEVC electrophysiology. Data are
pooled from three to five experiments per receptor conducted in
duplicate and expressed as the mean ( SEM of normalized max-
imum responses in each oocyte. Because of limitations in compound
availability, 3 mM was the highest concentration tested. The EC50
for GluA4i is estimated to be >1 mM (Table 3).
2
average B (A ) for protein
˚
25/23
38/34/30
atoms (molA/molB/molC)
(R)-4/(S)-5
19/18
31/63/-/41/
56/-/-
28/25/26
32/-/40/64/
-/42/42
˚
water/sulfate/zinc/glycerol/
chloride/cacodylate/acetate
a
Numbers in parentheses are for the outermost bin: 2.18-2.10 A [(R)-
4], 2.55-2.48 A [(S)-5]. b au: asymmetric unit of the crystal. c Rsym(I) =
˚
P
P
P
P
Whereas the tetrahydropyridine carboxylic acid ring sys-
tems of (R)-4 and (S)-5 bind in approximately the same
region of the receptor, the 3-hydroxyisoxazole ring system is
located in completely different areas of the binding site
(Figure 2C). The binding mode of (R)-4 most closely resem-
bles that of (S)-AMPA and the binding mode of (S)-5 mostly
that of (S)-glutamate.11 This leads to differences in receptor
contact residues of the two isoxazoles. In (R)-4, the 3-hydroxy
group forms in total three potential hydrogen bonds to the side
chain hydroxyl group of Thr655 and two water molecules
(W1 and W2, Figure 2A). These two water molecules are key
elements in the formation of water-mediated networks between
the agonist and receptor. W1 is further connected to the nitrogen
atom of Thr655 and the backbone oxygen atom of Ser652, and
W2 forms additional hydrogen bonds to the nitrogen atom of
Leu650 and the backbone oxygen atom of Leu703. In (S)-5, the
hkl|Ihkl - ÆIhklæ|/ hklIhkl
.
d R = hkl||Fo| - |Fc||/ |Fo|, where |Fo| and
|Fc| are observed and calculated structure factor amplitudes, respec-
tively, for reflection hkl. e 5% of the reflections were set aside for
calculation of the Rfree value. f The Ramachandran plot was calculated
according to Kleywegt and Jones.55
3-hydroxy group does not form a hydrogen bond to Thr655
(Figure 2B), but two water molecules (W1 and W2) are found in
similar positions as water molecules W1 and W2 in the structure
of GluA2 with (R)-4. Because of the different location of the
isoxazole ring system, the 3-hydroxy group only forms a
hydrogen bond to W2. However, two additional water mole-
cules (W3 and W4) are present, making water-mediated hydro-
gen bonds between the 3-hydroxy group of (S)-5 and the
nitrogen atom of Glu705 (W4) and the hydroxyl groups of
Thr686 and Tyr702 (W3). A water molecule corresponding to

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8357
W3 is also observed in the structure of GluA2 with (R)-4, but
here the water molecule (W3) forms a water-mediated hydrogen
bond from the isoxazole nitrogen atom to the side chain hydro-
xyl groups of Thr686 and Tyr702 (Figure 2A). In (S)-5, the
isoxazole nitrogen atom forms a hydrogen bond to the side chain
hydroxyl group of Thr655 and water-mediated hydrogen bonds
through W1 to the nitrogen atom of Lys656 and the oxygen
atom of Leu650. Of note, a water molecule corresponding to W4
is not present in the structure of GluA2 with (R)-4, as this would
lead to too close contacts between the water molecule and the
isoxazole ring system of (R)-4. Finally, the isozaxole oxygen
atom of (S)-5 forms a potential hydrogen bond to the backbone
nitrogen atom of Thr655 and also to the backbone nitrogen
atom and side chain hydroxyl group of Ser654 (Figure 2B). No
hydrogen bonds are formed to the corresponding oxygen atom
in (R)-4, which is located in close contact with Met708.
Molecular Modeling. We recently described the use of the
WaterMap method28 to highlight how comparing differences
in the free energy of ABD hydration sites in the absence of the
ligand can be used to understand selectivity.29 For the ligand
4-AHCP, it was found that it was not the energies of water sites
displaced by the ligand but rather those surrounding it that are of
most importance for GluK1/GluA2 selectivity.
Thus, the energies of hydration sites adjacent to the ligand
depend on the ligand structure, and the dependence may vary
from one receptor subtype to another. It follows that a more
complete understanding of selectivity could be obtained by
comparing a pair of simulations for each complex rather than
just one: both without and with the ligand present. Therefore,
for the GluA1/2 vs GluA3/4-selective ligand (S)-5 and the
nonselective ligand (R)-4, we have examined changes in the
enthalpy, entropy, and free energy of waters surrounding the
ligands’ position in the receptor, between the ligand-free sol-
vated state (“apo”) and that induced by each ligand’s presence
(“holo”). Eight 2 ns explicit solvent molecular dynamics simu-
lations were performed in Desmond/WaterMap30 and ana-
lyzed: GluA2 vs GluA4, with and without the presence of
(S)-5 vs (R)-4 (Table 5, Figure 3).
In the absence of experimental structures of GluA4 in
complex with these ligands, models were constructed by aligning
the high resolution structure of Glu in GluA4 (PDB code 3FAS,
chain B) and copying the ligands from the present GluA2
structures to replace Glu in GluA4 and then performing a light
minimization with the impref protocol using the OPLS2005
force field.30 In theory, when the source of selectivity is unknown
or unclear, it would be most rigorous to integrate over all
affected water sites. However, in particular for GluA1/2 vs
GluA3/4 selectivity, it is known from previous studies that the
primary determinant is the ligand’s relationship to a particular
site, specifically the site occupied by the experimentally observed
water molecule in GluA2 that links the ligand to Tyr702 and
Thr686 via hydrogen bonding (W3 in Figure 2). Indeed, a
quantitative correlation has previously been observed between
the strength of hydrogen bonding to this water molecule and the
magnitude of the GluA1/2 vs GluA3/4 selectivity over several
orders of magnitude.31
With the WaterMap method, the free energy of the W3 site
can be broken down into its enthalpic and entropic compo-
nents (Table 5). Note that for (S)-5 in GluA4, the region
where W3 is normally found is so high in energy (6.4 kcal/
mol) that the site is on the cusp of being depleted/dewetted
(the upper limit for theoretical observation being around
7-8 kcal/mol and in practice somewhat lower for experi-
mental visibility). This accords with experimental structures
Figure 2. Binding modes of the agonists (R)-4 and (S)-5 in the
ligand-binding site of GluA2-S1S2. (A) The (R)-4 complex
(molecule A). The omit Fo - Fc electron-density map contoured
at 3σ level of (R)-4 is shown. (R)-4 (green) and potential hydrogen-
bonding residues of GluA2-S1S2 (green) are represented as sticks,
˚
and dashed lines indicate hydrogen bonds (within 3.2 A). The three
water molecules forming hydrogen bonds to the ligand are shown
as red spheres. (B) The (S)-5 complex (molA). The omit Fo - Fc
electron-density map contoured at 3σ level of (S)-5 is shown. (S)-5
(light blue) and potential hydrogen-bonding residues of GluA2-
S1S2 (light blue) are represented as sticks, and dashed lines indicate
˚
hydrogen bonds (within 3.2 A). The four water molecules forming
hydrogen bonds to the ligand are shown as red spheres. (C) Super-
imposition of the (R)-4 and (S)-5 complexes on domain D1 residues.
Ligands and potential hydrogen-bonding residues of GluA2-S1S2
are represented as sticks. Color coding is as in parts A and B. Water
molecules have been displayed in colors according to the ligands.

8358 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Frydenvang et al.
Table 5. Enthalpic and Entropic Contributions to the Free Energy of Hydration of the W3 Site, with and without Ligand, for (R)-4 and (S)-5 in GluA2
and GluA4
receptor
GluA2
ligand
state
apo
ΔH, kcal/mol
-TΔS, kcal/mol
ΔG, kcal/mol
(R)-4
-0.86
-5.73
-4.87
2
0.83
1.32
-0.03
-4.41
-4.38
0.74
liganded
Δ
apo
0.49
GluA4
(3FAS:B)
(R)-4
(S)-5
(S)-5
-1.26
1.25
2.51
liganded
Δ
apo
-2.33
-4.33
-2.27
-1.35
0.92
-1.08
-1.82
-1.24
0.07
GluA2
1.03
1.42
liganded
Δ
apo
0.39
1.31
0.7
GluA4
(3FAS:B)
1.98
3.9
-1.38
3.17
4.55
liganded
Δ
7.07
6.37
1.92
ΔΔG, kcal/mol
2.5
selectivity ratio
69
GluA4 vs GluA2
Figure 3. Hydration site W3 in holo vs apo receptors color coded according to WaterMap (more green is more favorable; more red is more
unstable): ligand carbons in gray, protein carbons in green. Dotted black lines represent hydrogen bonding to the average position of a water
molecule in the holo simulation. (A) (R)-4 in GluA2. (B) (R)-4 in GluA4. (C) (S)-5 in GluA2. (D) (S)-5 in GluA4. The ligands have opposite
effects on the hydration site with respect to receptor: whereas (R)-4 stabilizes W3 somewhat more in GluA2 than GluA4, (S )-5 destabilizes this
water to some extent in GluA2 and greatly in GluA4.
of GluA1/2-selective ligands, such as (S)-2-amino-3-(4-bro-
mo-3-hydroxyisoxazol-5-yl)propionic acid [(S)-Br-HIBO,
(S)-3a, Chart 1]. When (S)-3a is crystallized in a mutated
GluA2 ABD, where Tyr702 from GluA2 is mutated to the
Phe found in GluA3/4, density depletion occurs and no
explicit water molecule is experimentally observable at this
site (PDB codes 1M5C and 1M5D).12
This observation was previously difficult to understand in
terms of simple interaction energies, since a buried anion was
expected to remain solvated if there was sufficient space for a
water molecule to be present. However, this water turns out to be
both enthalpically and entropically unfavorable in GluA4 in the
presence of (S)-5, sometimes referred to as being “trapped” by
the ligand. By contrast, (R)-4 stabilizes this water site, and a
water molecule at this position is also experimentally observed in
GluA4:Glu (PDB code 3FAS).
Overall, WaterMap predicts a 2.5 kcal/mol (70-fold) differ-
ence for the destabilization of W3 at GluA4 vs GluA2 by (S)-5vs
(R)-4, which is in good agreement with the observed selectivity
difference (40-fold) for the two ligands. Thus, the holo vs apo

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8359
WaterMap analysis provides a new technique for quantitatively
probing the basis of water-mediated ligand subtype selectivity.
solvent was removed in vacuo. After 24 h in a vacuum oven at 45 ꢀC,
1.68 g (7.62 mmol) of 5 was obtained as hygroscopic white crys-
tals. A small sample of 5 was recrystallized as the zwitterion from
2-PrOH and H2O (5:1) in a 75% yield. Mp >200 ꢀC(dec).1HNMR
(D2O): 2.68-2.76 (t, 2H), 3.45-3.65 (m, 2H), 5.07 (s, H exchanges
slowly with D). Rf = 0.17 (n-BuOH/AcOH/H2O) (4:1:1). Anal.
(C7H8N2O4) C, H, N.
Discussion
The compounds 4 and 5 were originally designed as con-
formationally restricted analogues of 2 and 1, respectively, to
obtain information on the active conformation of 1 and 2.
Whereas (R)-4 is AMPA receptor selective (like AMPA (2)
itself), 5 has a different pharmacological profile compared
to 1. Thus, 5 is group-selective for AMPA receptor sites and 1
is group-selective for NMDA receptor sites. In the present study
we demonstrate that 5 also is subtype-selective for GluA1/2
compared to GluA3/4. This is in contrast to (R)-4 that has
similar affinity at all AMPA receptor subtypes. These pharma-
cological results are quite surprising given the close structural
relationship of these compounds. X-ray crystallographic studies
of (S)-5 and (R)-4 bound to the receptor ABD illustrate that the
ligands bind very differently. Although the conformations of
(S)-5and (R)-4do not exactly match the active conformations of
Glu and AMPA (2), respectively, the binding modes are com-
parable. For compound 5,the(S)-enantiomer binds to the ABD,
whereas it is the (R)-enantiomer of 4 that is the active form
(Table 3). Since the functional groups in 4 and 5 are essentially
the same, the pharmacological difference likely arises from the
different binding modes. A WaterMap analysis of the binding
site suggests that the selectivity difference for the two compounds
is related to their different effects on binding pocket solvation.
Thus, (S)-5 destabilizes a water molecule in GluA3/4 compared
to GluA1/2, whereas (R)-4 stabilizes this water molecule. This
difference arises from different binding modes of the two
compounds, since there is no difference in hydrogen bonding
ability of the respective 3-isoxazolol groups as has been observed
for halogen substituted homoibotenic acid analogues, such as
3 and 3a (Chart 1).25 Thus, selectivity of Glu receptor ligands
arises from their dissimilar effectsonthewaterarchitectureinthe
binding pocket as also demonstrated in previous studies.29
In Vitro Pharmacology. Native Receptor Binding Assays.
Affinities for native AMPA, KA, and NMDA receptors in rat
cortical synaptosomes were determined using 5 nM [3H]AMPA
(55.5 Ci/mmol),33 5 nM [3H]KA (58.0 Ci/mmol),34 and 2 nM
[3H]CGP 39653 (Kd = 6 nM, 50.0 Ci/mmol),35 respectively, with
minor modifications as previously described.36 Rat brain mem-
brane preparations used in these receptor binding experiments
were prepared according to a method previously described.37
Recombinant Receptor Binding Assays. Sf9 cells were cultured and
infected with recombinant baculovirus of rat AMPA receptors
(GluA1o-4o) or rat KA receptors (GluK1-3) and membranes pre-
pared and used for binding as previously detailed.38,39 The affinities
of compound 5 for GluA1o, GluA2(R)o, GluA3o, and GluA4o were
determined from competition experiments with 2-5 nM (RS)-
[3H]AMPA and for GluK1(Q)1b, GluK2(V,C,R)a, and GluK3a
using 1-5 nM [3H]SYM2081 (47.9 Ci/mmol). Italic letters in
parentheses indicate the RNA-edited isoforms of the subunits used.
The affinities of compounds 4 and 5 at the soluble GluA2-
S1S2 were assayed as formerly described.13
Electrophysiology. Xenopus lævis oocytes were collected, pre-
pared, injected, and maintained as previously described.31 Two-
electrode voltage clamp electrophysiology was carried out as pre-
viously described.31 The concentration-response relationship and
drug EC50 were determined by curve-fitting of the data to the logistic
equation I = Imax/(1 þ (10logEC /10log[A])nH), where I is the agonist-
50
evoked current, [A] is the agonist concentration, Imax is the calcu-
lated maximum response at saturating agonist concentration, and
nH is the Hill coefficient.
X-ray Structure Determination. X-ray Structure Determina-
tion of ABD Bound (R)-4. The GluA2-S1S2J construct11 was
used, and the protein was expressed, refolded, and purified essen-
tially as reported.40,41 GluA2-S1S2 in complex with (R)-4 was
crystallized by the hanging drop vapor diffusion method at 7 ꢀC.
The protein complex solution contained 7.5 mg/mL GluA2-S1S2
and 15 mM (R)-4in 10 mM Hepes, pH 7.0, 20 mM sodium chloride,
and 1 mM EDTA. Crystals were obtained in drops consisting of
1 μL of complex solution and 1 μL of reservoir solution of 20%
PEG4000, 0.2 M ammonium sulfate, and 0.1 M sodium acetate,
pH 5.5. The reservoir volume was 0.5 mL. The crystals grew within
1 week to a maximum dimension of 0.1 mm.
Experimental Section
Chemistry. General Procedures. Analytical thin-layer chro-
matography (TLC) was performed on silica gel F254 plates
(Merck). All compounds were detected using UV light and/or
by spraying with diluted solutions of KMnO4 or H2SO4/Ce(SO4)2/
ammonium molybdate. 3-Isoxazolols and amino groups were
visualized using standard spraying solutions of FeCl3 and ninhydrin,
respectively. Melting point was determined in a capillary tube and is
uncorrected. The 1H (300 MHz) NMR spectrum was recorded on a
Varian Gemini-2000 BB spectrometer in D2O using dioxane as
internal standard. Chemical shifts are given in ppm (δ). Elemental
analysis was performed by J. Theiner, Microanalytical Laboratory,
Department of Physical Chemistry, University of Vienna, Austria,
and is within (0.4% of the theoretical values. Compounds had
g95% purity according to elemental analysis.
3-Hydroxy-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-7-carboxyl-
ic Acid (7-HPCA, 5). An amount of 2.5 g (10.4 mmol) of tert-butyl
3-hydroxy-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-6-carbox-
ylate 632 was dissolved in 100 mL of dry THF under argon. The
mixture was cooled to -78 ꢀC,andanamountof20mLof1.2M(24
mmol) sec-BuLi was carefully added. The mixture was allowed to
stir for 1.5 h before dry ice was added continuously for 1.5 h. The
mixture was transferred to a separation funnel containing 0.5 M HCl
and EtOAc. The organic phase was dried (MgSO4) and concen-
trated in vacuo to give 7. Without further purification, compound 7
was dissolved in 50 mL of THF to which HCl in ether
(approximately 5 M) was added in excess. The mixture was stirred
for 72 h. Most of the solvent was decanted, while the remaining
Crystals of GluA2-S1S2 in complex with (R)-4 were flash-cooled
to 100 K using ∼20% glycerol added to the reservoir solutions as a
cryoprotectant. Synchrotron data were collected at the X11 beam-
line, DESY Hamburg, Germany, equipped with a MARCCD
˚
detector and at a wavelength of 0.8126 A. A complete data set
˚
was collected to 2.1 A resolution. Diffraction data were processed
with the programs DENZO and SCALEPACK.42 For crystal data
and data collection statistics, see Table 4.
The structure was solved by molecular replacement, using the
program AMoRe43 from CCP4i.44 The structure of GluA2-
S1S2 in complex with (S)-ATPA45 (PDB code 1NNP) was used
as a search model for phasing the data, including protein atoms
only. A clear solution comprising two molecules (molA and
molB) was obtained. Subsequently, the amino acid residues were
traced using ARP/wARP46 except for a few amino acids, which
were manually built using the program O.47 Afterward, (R)-4
was unambiguously fitted into the electron densities within the
ligand binding site of molA and molB, respectively. The struc-
ture was further subjected to refinements in REFMAC548 within
CCP4i, in CNS,49 and in Phenix.50 Between each refinement
step, the structure was inspected and corrected using the pro-
grams O and COOT.51 Gradually, water molecules, glycerol,
and sulfate ions were added to the structure. The GluA2-S1S2

8360 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Frydenvang et al.
construct comprises a Gly-Ala cloning remnant, amino acid
residues 391-506 from segment S1 of the membrane bound
receptor, a two amino acid linker Gly-Thr, and residues
632-775 from segment S2 (numbering without signal peptide).
In molA and molB all residues could be modeled (except for
Asp456), as well as residue Ala from the cloning remnant in the
N-terminus of molB. A summary of structure refinements is
presented in Table 4. Coordinates have been deposited in the
Protein Data Bank (PDB code 3PD9).
X-ray Structure Determination of ABD Bound (S)-5. GluA2-
S1S2 in complex with 5 was crystallized by the hanging drop
vapor diffusion method at 7 ꢀC. The protein complex solution
contained 7.0 mg/mL GluA2-S1S2 and 8.0 mM 5 in 10 mM
Hepes, pH 7.0, 20 mM sodium chloride, and 1 mM EDTA.
Crystals were obtained in drops consisting of 1 μL of complex
solution and 1 μL of reservoir solution of 0.1 M zinc acetate,
0.1 M sodium cacodylate buffer, pH 6.5, and 17.5% PEG 4000.
The reservoir volume was 0.5 mL. The crystals grew within
1 week to a maximum dimension of 0.1 mm.
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was used as search model for phasing the data, that a clear
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refinements is presented in Table 4. Coordinates have been
deposited in the Protein Data Bank (PDB code 3PD8).
The program DynDom52 was employed for analysis of
ligand-induced domain closure relative to the apo structure of
GluA2-S1S2 (PDB code 1FTO, molA). Figures were prepared
with PyMOL.53 Library files for refinement of ligands were
obtained using the PRODRG server,54 the geometry compara-
ble to the geometry obtained from quantum mechanical calculations
(B3LYP, basis set 6-311þG**).
Molecular Modeling. All calculations described were per-
30
€
formed in Schrodinger’s Suite 2010 (release v19108).
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Acknowledgment. Lise Baadsgaard Sørensen is gratefully
thanked for crystallization of the ABD complexes and Desiree
Sprogøe for mounting the crystals for data collection. We also
acknowledge financial support from The Lundbeck Founda-
tion, The Danish Medical Research Council, DANSYNC
(Danish Centre for Synchrotron Based Research), and The
European Community;Access to Research Infrastructure
Action of the Improving Human Potential Programme to the
EMBL Hamburg Outstation.
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