Structure-activity relationships of a small-molecule inhibitor of the PDZ domain of PICK1

Article abstract of DOI:10.1039/c0ob00025f

Recently, we described the first small-molecule inhibitor, (E)-ethyl 2-cyano-3-(3,4-dichlorophenyl)acryloylcarbamate (1), of the PDZ domain of protein interacting with Cα-kinase 1 (PICK1), a potential drug target against brain ischemia, pain and cocaine addiction. Herein, we explore structure-activity relationships of 1 by introducing subtle modifications of the acryloylcarbamate scaffold and variations of the substituents on this scaffold. The configuration around the double bond of 1 and analogues was settled by a combination of X-ray crystallography, NMR and density functional theory calculations. Thereby, docking studies were used to correlate biological affinities with structural considerations for ligand-protein interactions. The most potent analogue obtained in this study showed an improvement in affinity compared to 1 and is currently a lead in further studies of PICK1 inhibition.

Full text of DOI:10.1039/c0ob00025f

Organic &
Biomolecular
Chemistry
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Volume 8 | Number 19 | 7 October 2010 | Pages 4185–4484
ISSN 1477-0520
FULL PAPER
Anders Bach et al.
PERSPECTIVE
Georgiy B. Shul’pin
Structure–activity relationships
of a small-molecule inhibitor
of the PDZ domain of PICK1
Selectivity enhancement
in functionalization of C–H bonds:
A review

PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Structure–activity relationships of a small-molecule inhibitor of the PDZ
domain of PICK1†
Anders Bach,a Nicolai Stuhr-Hansen,a Thor S. Thorsen,b Nicolai Bork,c Irina S. Moreira,d Karla Frydenvang,a
Shahrokh Padrah,a S. Brøgger Christensen,a Kenneth L. Madsen,b Harel Weinstein,d Ulrik Gether*b and
Kristian Strømgaard*a
Received 21st April 2010, Accepted 17th June 2010
DOI: 10.1039/c0ob00025f
Recently, we described the first small-molecule inhibitor, (E)-ethyl
2-cyano-3-(3,4-dichlorophenyl)acryloylcarbamate (1), of the PDZ domain of protein interacting with
Ca-kinase 1 (PICK1), a potential drug target against brain ischemia, pain and cocaine addiction.
Herein, we explore structure–activity relationships of 1 by introducing subtle modifications of the
acryloylcarbamate scaffold and variations of the substituents on this scaffold. The configuration
around the double bond of 1 and analogues was settled by a combination of X-ray crystallography,
NMR and density functional theory calculations. Thereby, docking studies were used to correlate
biological affinities with structural considerations for ligand–protein interactions. The most potent
analogue obtained in this study showed an improvement in affinity compared to 1 and is currently a
lead in further studies of PICK1 inhibition.
transporters and receptors have been found to interact with the
Introduction
PDZ domain of PICK1, including the monoaminergic (dopamine,
The inhibition of specific protein–protein interactions involved
serotonin, norepinephrine) transporters, and several subunits of
in signal transduction, cell–cell communication, apoptosis, and
many other vital cellular processes with small organic molecules
the ionotropic glutamate receptors including the a-amino-3-
hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), kainate,
and the metabotropic glutamate receptors.4,23,24 PICK1 was found
to be important for various forms of synaptic plasticity,20,25–27
including long-term depression (LTD) and long-term potentiation
(LTP) presumably via its interaction with the AMPA receptors,
and has been suggested as a therapeutic target for treating brain
ischemia,28,29 pain,4,30,31 and cocaine addiction.32 Hence, specific
small-molecule inhibitors of the PDZ domain of PICK1 could
support the study of a variety of physiological processes and
provide leads for potential therapeutic interventions.
PDZ domains are considered difficult to target with small-
molecules because of their shallow and elongated binding
pocket,33,34 and only relatively few examples of potent (Ki < 50 mM)
PDZ inhibitors are found in the literature,35–47 most of which are
peptide-derived.40–44,48 However, by screening ~43 000 compounds
we have recently identified a small-molecule inhibitor, (E)-ethyl 2-
cyano-3-(3,4-dichlorophenyl)acryloylcarbamate (1, Fig. 1), of the
PICK1 PDZ domain with Ki = 9.6 mM. This affinity is in the
same range as observed for the endogenous C-terminal peptide
ligands.49 Biochemical studies demonstrated that 1 binds reversibly
to PICK1 and exhibits selectivity with respect to cognate PDZ
domains such as the three PDZ domains of postsynaptic density
protein-95 (PSD-95), or PDZ4-5 of glutamate receptor interacting
protein-1 (GRIP1). Additionally, 1 is membrane permeable and
binds the PICK1 PDZ domain in living cells, as demonstrated
by intracellular fluorescence resonance energy transfer (FRET).
Furthermore, 1 affected AMPA receptor trafficking and inhibited
induction of LTD and LTP in hippocampal CA1 neurons.49
Structurally, 1 is promising for further studies because it does not
contain a carboxylic acid group as found in most PDZ ligands,36–47
which could partially explain its ability to cross membranes.
Here, we explore structure–activity relationships (SARs) of 1, in
has great potential in probing biological questions and pursuing
therapeutic modalities.1–3 PSD-95/Discs-large/ZO-1 (PDZ)
domains are involved in several potentially therapeutic relevant
protein–protein interactions,4–6 and they often function as scaf-
folding modules in proteins that are involved in trafficking and
assembling of large protein complexes in the cell. PDZ domains
generally recognize the C-terminal part of the interacting protein
and they are highly abundant in eukaryotic organisms.7–10
The PDZ domain of protein interacting with Ca-kinase
(PICK1) was first shown to interact with protein kinase Ca
(PKCa) to mediate contacts between PKCa and integral mem-
brane proteins in the central nervous system,11 whereby phospho-
rylation of these proteins is regulated.12–15 By comprising both the
PDZ domain and the Bin/amphiphysin/Rvs (BAR) domain,16
a membrane-binding and curvature-sensing module,17,18 PICK1
can generate diverse protein complexes and regulate their mem-
brane clustering and trafficking.19–22 Several membrane bound
aDepartment of Medicinal Chemistry, University of Copenhagen, Uni-
versitetsparken 2, DK-2100, Copenhagen, Denmark. E-mail: krst@
farma.ku.dk; Fax: (+45) 3533 6041; Tel: (+45) 3533 6114
bDepartment of Neuroscience and Pharmacology, Panum Institute, Univer-
sity of Copenhagen, Blegdamsvej 3B, DK-2200, Copenhagen, Denmark.
E-mail: gether@sund.ku.dk; Fax: (+45) 3532 7610; Tel: (+45) 3532 7548
cRisø National Laboratory for Sustainable Energy, Technical University of
Denmark, Frederiksborgvej 399, DK-4000, Roskilde, Denmark
dDepartment of Physiology and Biophysics, Weill Medical College of Cornell
University, New York, NY 10021, USA
† Electronic supplementary information (ESI) available: General chem-
istry procedures. Procedures and data for purified compounds. Experimen-
tal for biochemical assays and computational docking studies. Analysis of
X-ray crystallographic data, NMR data and density functional theory
(DFT) calculations for determining the double bond configuration. See
DOI: 10.1039/c0ob00025f
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Fig. 1 Structure of lead compound 1 and the concordant generic structure together with its retrosynthetic analysis. Substituents explored in this SAR
study are indicated R1–3
.
order to elucidate the structural requirements of 1 and analogues
for PICK1 affinity (Fig. 1). A fluorescence polarization assay,
which quantifies the ability to displace a fluorescently labelled
undecapeptide representing the extreme C-terminal of the human
dopamine transporter (hDAT), was used to determine affinities
between compounds and PICK1. The results from key compounds
was confirmed in a glutathione S-transferase (GST) fusion protein
pull-down assay.49 The compounds were also tested against the
three PDZ domains of PSD-95 to examine selectivity,42 and finally,
we analyzed the SAR in a structural context with computational
docking studies.
3-(3,4-dichlorophenyl)propanoylcarbamate (3) was obtained by
hydrogenation of the double bond of 1, which was a highly
selective reaction as neither the cyano group or halogens were
affected (Scheme 2a). The des-cyano analogue of 1, (E)-ethyl
3-(3,4-dichlorophenyl)acryloylcarbamate (5), was synthesized by
amidolysis of 4 with the anion of urethane (Scheme 2b). However,
presumably a self-condensation of urethane anions leading to
ethoxide, which reacted with 4, was responsible for the low yield
of the reaction as the undesired ethyl (E)-3,4-dichlorocinnamate
(6; electronic supplementary information†) was observed as the
major product. The ester functionality was replaced with an alkyl
moiety by aminolysis of 2 with butylamine (expelling urethane),
providing 7 in high yield. A Knoevenagel condensation of 7
with 3,4-dichlorobenzaldehyde resulted in (E)-N-butyl-2-cyano-
3-(3,4-dichlorophenyl)acrylamide (8) (Scheme 2c). Finally, in
order to investigate the significance of the carbonyl moiety we
prepared (Z)-ethyl 2-cyano-3-(3,4-dichlorophenyl)allylcarbamate
(14) (Scheme 2d). The precursor 9 was produced by a Baylis–
Hillman reaction, followed by treatment with phosphorus tribro-
mide to provide 10 and 11 in a 1 : 1 mixture (GC-MS). As these
compounds were inseparable for all practical purposes, we treated
the mixture with methanolic ammonia to furnish 12 and 13, which
were isolated by standard flash chromatography. Compound 12
was readily acylated with ethyl chloroformate affording the desired
14. The Z geometry in 14 (as depicted in Scheme 2d) was verified by
NOESY experiments and supported by density functional theory
(DFT) calculations (electronic supplementary information†).
In order to probe the importance of the terminal alkyl tail
of 1 and to study the possibilities of introducing substituents
on the central nitrogen atom, a series of cyanoacetylcarbamate
intermediates were prepared (Scheme 3). Compounds 15–18 were
synthesized from commercially available building blocks using
the same protocol as for the synthesis of 2 (Scheme 3a). For
the preparation of intermediates 20, 23 and 24, the starting
carbamate had to be prepared. Compound 20 was synthesized
by converting adamantan-1-ol into carbamate 19 by reaction
with trichloroacetyl isocyanate and basic hydrolysis,54,55 followed
by condensation with 2-cyanoacetic acid (Scheme 3b). Ethyl
butylcarbamate (21) and ethyl benzylcarbamate (22) were pre-
pared by aminolysis of ethyl chloroformate with butylamine
or benzylamine, respectively, and 21 and 22 were subsequently
reacted with 2-cyanoacetic acid to furnish 23 and 24 (Scheme 3c).
Results and discussion
Chemistry
The synthetic route for compound 1 and its analogues was identi-
fied by a retrosynthetic analysis of the generic acryloylcarbamate
structure (Fig. 1). A condensation reaction with 2-cyanoacetic
acid and carbamates was chosen to obtain the intermediate
cyanoacetylcarbamates, which could subsequently be reacted
with aromatic aldehydes in a Knoevenagel condensation reaction
to furnish the target compounds. Besides being efficient, this
route has the advantage of being versatile and allows convenient
synthesis of analogues, as several building blocks are commercially
available.
To prepare 1, the cyanoacetylcarbamic acid ethyl ester (2)
intermediate was first synthesized from 2-cyanoacetic acid and
ethyl carbamate (urethane) using phosphorus chloride oxide
(POCl3) as a condensation agent (Scheme 1). By using POCl3
and small amounts of DMF to catalyze the reaction, in aprotic
solvents, side reactions are limited and yields increased compared
to using acetic acid anhydride as condensation agent and/or
protic solvents.50 The Knoevenagel reaction between 2 and 3,4-
dichlorobenzaldehyde to provide 1 was initially carried out at room
temperature using piperidine as a catalyst.51 However, we found
that adding small amounts of acetic acid improved both yield and
purity, as previously shown for other Knoevenagel reactions,52,53
while refluxing did not improve the reaction.
To investigate the biological importance of the acryloylcarba-
mate scaffold we introduced subtle modifications by individually
modifying the alkene, the cyano, the ester, and the carbonyl group
as in compounds 3, 5, 8 and 14 (Scheme 2). Ethyl 2-cyano-
Scheme 1 (a) POCl3, DMF, toluene, 70 ◦C, 1.5 h (82%); (b) 3,4-dichlorobenzaldehyde, piperidine, AcOH, DMF, 2 h (43%).
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Scheme 2 (a) H2 (1 atm.), Pd/C (3.5 mol%), EtOAc, rt, 24 h (76%); (b) SOCl2, reflux, 2 h (73%); (c) sodium (ethoxycarbonyl)amide, DMF, rt, 1 h
(14%); (d) BuNH2, MeOH, rt, 1 h (91%); (e) 3,4-dichlorobenzaldehyde, piperidinium acetate (cat.), DMF, rt, 48 h (47%); (f) acrylonitrile, DABCO,
dioxane–water (1 : 1), rt, 24 h (68%); (g) PBr3, Et2O, rt, 2 h (69%); (h) NH3, MeOH, rt, 2 h (12: 19%; 13: 65%); (i) EtOCOCl, Et3N, THF, rt, 1 h (64%).
Analogues with a range of acryloylcarbamate substituents, R1–3
(Fig. 1), were prepared applying the same synthetic method as
described for 1, where cyanoacetylcarbamate intermediates and
commercially available aromatic aldehydes were reacted generally
in good yields and purities after recrystallization. In the first class
of analogues, 25–35 (Table 1) and 36 (Table 2), the aromatic and the
ethyl moieties were varied. Subsequently, N-alkylated carbamates,
compounds 37–40 (Table 2), were prepared and finally subtle
changes of the chloro substituents of the phenyl group were made
as in compounds 41–49 (Table 3).
demonstrate a reversible binding mode,49 and the close analogue
8, which also possesses a Michael acceptor site, is inactive.
Finally, as 1 shows no affinity for PDZ domains from PSD-95
or GRIP1,49 1 is considered a specific and reversible inhibitor of
PICK1.
Since SAR studies of 1 had not been carried out thus far,
we explored the steric requirements of the aromatic and the
ethyl moieties (R1 and R2, respectively, Fig. 1), and introduced
relatively large and hydrophobic modifications in these two
regions providing a matrix of 12 analogues (1, 25–35, Table 1).
The analogues yielded unambiguous information regarding the
substitutions allowed in these regions, as an anthracene in the R1
position (analogues 26, 30 and 34), and adamantane in the R2
position (analogues 32–35), abolished affinity. On the other hand,
introducing a naphthalene (25 and 29) or a biphenyl group (27 and
31), instead of dichlorophenyl, did not affect Ki values significantly
compared to 1. Similarly, an isopropyl group instead of ethyl in the
R2 position, as in compounds 28–31, did not influence the affinity
either (Table 1). We therefore probed the possibility of increasing
the length of the alkyl group (as in 36), but the affinity remained
equal to that of 1 (Table 2). Taken together, these results suggest
Structure–activity relationship studies
All final compounds were investigated for their ability to bind
the PICK1 PDZ domain in a fluorescence polarization assay.23,49
Compounds 3, 5, 8 and 14 were all devoid of affinity at up
to 500 mM concentrations (data not shown) thus demonstrat-
ing the importance of an intact acryloylcarbamate scaffold. A
general concern of 1 is that it binds PICK1 irreversibly as a
Michael acceptor allowing nucleophilic attack from PDZ amino
acid side chains on the alkene bond.56 But biochemical studies
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Scheme 3 (a) POCl3, DMF, toluene, 70 ◦C, 1-2 h; (b) Cl3CCONCO, CH2Cl2, 0 ◦C → 23 ◦C, 2 h; (c) K2CO3, MeOH, 50 ◦C, 16 h (87%); (d) 2-cyanoacetic
acid, POCl3, DMF, toluene, 70 ◦C, 1.5 h; (e) Et3N, THF, rt, 2 h
Table 1 Structures and affinities of compound 1 and analogues 25–35a,b
Table 2 Structures and affinities of compound 1 and analogues 36–40
R2
Compound
R2
R3
Ki/mMa
1
Ethyl
Butyl
Ethyl
Ethyl
Ethyl
Ethyl
H
H
Methyl
Ethyl
Butyl
Benzyl
9.6 0.22
12 2.8
55 13
40 3.1
19 4.7
14 1.7
36
37
38
39
40
R1
1
28
11 1.7
32
NA
9.6 0.22
a Ki values are shown as mean SEM based on at least three individual
measurements.
25
29
33
12 2.5
15 4.2
NA
Table 3 Structures and affinities of compound 1 and analogues 41–49
26
30
34
NA
NA
NA
Compound
X
Y
Ki/mMa
1
Cl
H
H
CH3
H
F
Br
NO2
NO2
CF3
Cl
H
Cl
CH3
CH3O
CF3
NO2
Cl
Br
Cl
9.6 0.22
84 2.2
23 0.7
23 3.0
89 20
17 2.7
26 2.3
22 6.1
11 1.2
7.2 0.7
27
12 2.3
31
14 2.9
35
NA
41
42
43
44
45
46
47
48
49
a Ki values are shown as mean SEM in mM based on at least three
individual measurements. b NA: no affinity.
a Ki values are shown as mean SEM based on at least three individual
measurements.
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that modifying the ethyl moiety by increasing length and/or bulk
do not affect affinity.
A recurring problem in studies of protein–protein interac-
tions is the possibility of false positives and specifically in
fluorescence-based assays where artefacts due to autofluorescence
or absorbance are common phenomena.1,61–63 The results of the
fluorescence polarization assay were therefore verified for selected
analogues in a semi-quantitative and non-fluorescence based,
pull-down assay. As seen in the fluorescence polarization assay,
compound 26 had no activity towards the PICK1 PDZ domain
and 37 showed impaired affinity compared to 47 (Fig. 2). For the
remaining compounds tested, the results showed no statistically
significant differences compared to 1, but the pull-down assay
was generally in agreement with results from the fluorescence
polarization assay. In order to verify that modifying compound 1
did not alter the selectivity profile for PICK1 compared to the three
individual PDZ domains of PSD-95,49 analogues 3, 5, 8, 14 and 25–
49 were tested in a concentration of 500 mM towards PDZ1, PDZ2
and PDZ3 of PSD-95, respectively, in a fluorescence polarization
assay as previously described.42 The results demonstrated that
none of the compounds showed affinity towards any of the PDZ
domains of PSD-95 (data not shown).
In order to evaluate the effect of increasing the bulk in the
central part of 1, substitutions were introduced on the nitrogen in
the acryloylcarbamate moiety (R3). Specifically the nitrogen was
alkylated with methyl, ethyl, butyl, and benzyl groups, leading to
analogues 37–40. Compounds 37 and 38, with methyl and ethyl
substitutions, respectively, had reduced affinity, but affinity was
regained by increasing size and bulkiness of the N-substitution,
as in compounds 39 and 40 (Table 2). Accordingly, affinity is not
improved by adding any hydrophobic functionality in this region,
but larger alkyl or aryl groups lead to compounds that are almost
equipotent to 1, which indicates that these groups occupy an area
in the PDZ domain where variations are tolerated.
Finally, we focused on the chloro substituents on the aromatic
ring in relation to affinity towards PICK1. In the screening
that had led to the discovery of 1,49 a related structure without
chloro substituents on the phenyl ring (41) was identified as a
weak inhibitor. To confirm this, compound 41 was synthesized
together with the analogue comprising a 4-chloro substituent (42)
and tested in the fluorescence polarization assay (Table 3). This
revealed a stepwise reduction of affinity when removing one or
both chlorine atoms, since 42 and 41 demonstrated a 2.4- and 9-
fold reduction in affinity compared to 1, respectively. Also, it was
seen that methyl or methoxy groups could not substitute for the
chlorines, as the 3,4-dimethyl substituted analogue (43) showed~2-
fold higher Ki value compared to 1, and the 4-methoxy substituted
analogue (44) demonstrated a 4-fold higher Ki compared to 42.
Thus, chloro substituents are favoured, but whether this was due
to the electronic properties (electronegativity) of the chlorines,
their lipophilicity, or a combination of these properties was not
obvious. Therefore, we explored more subtle modifications in
the 3- and 4-positions inspired by the Topliss ‘decision tree’.57–60
This approach explicitly addresses the situation where a 3,4-
dichloro analogue is more potent than a 4-chloro analogue, which
is more potent than the non-substituted analogue. Hence, we
synthesized compound 45, where the size and lipophilicity of the
3-substituent were reduced while the electronic properties were
preserved with fluorine, concurrent with increasing the inductive
effect and lipophilic properties of the substituent in the 4-position
by introducing a trifluoromethyl substituent. Introducing a 4-nitro
group similarly increased the negative polarity of the region while
a simultaneous substitution with 3-bromo increased lipophilicity,
as in 46. However, none of these combinations of substitutions
provided increased affinity compared to 1 (Table 3). When the
3-position was substituted with a nitro group, while the 4-chloro
group was preserved, as in 47, a 2.3 fold affinity decrease was seen
compared to 1. Substitution of the 4-chloro in compound 47 with
the larger and more lipophilic bromo, leading to 48, increased
affinity relatively to 47, thus yielding an affinity similar to 1. This
indicates that a large lipophilic substituent in the 4-position is
favourable. Finally, a small but statistical significant increase in
affinity compared to 1 was observed, when the negative polarity
and lipophilicity in the 3-position were increased by introducing
a trifluoromethyl group as in compound 49. This substitution
was specifically suggested in the Topliss ‘decision tree’ to possess
increased affinity compared to the 3,4-dichloro analogue, and
accordingly 49 is observed to be the most potent compound in
this series with Ki = 7.2 mM (Table 3).
Fig. 2 Semi-quantitative pull-down assay of selected compounds towards
PICK1. DMSO indicates the amount of protein pulled down when no
compound is added but only the equivalent amount of DMSO. The
columns represent the average values and the error bars represent standard
deviations based on three individual experiments.
Molecular modelling and docking studies
In the absence of NMR or X-ray crystal structures of 1 in complex
with PICK1, docking studies were used to gain detailed insights
into the molecular recognition responsible for PICK1 binding.
However, the regiochemistry of 1 needed to be settled in order
to carry out docking studies. Commercial vendors suggest a
Z-configuration around the double bond, but whether this applies
to our synthesis procedure of 1 was not certain. NMR clearly
showed that only one regioisomer was formed in the Knoevenagel
reactions independently of the character of the aromatic moiety,
but because there is only one hydrogen atom on the double
bond, NMR techniques cannot provide sufficient evidence for
either the E- or Z-isomer. Several attempts to prepare crystals
of 1 for single crystal X-ray experiments failed, but instead we
obtained suitable crystals of the des-chloro analogue, 41, and
the structural determination unequivocally demonstrated that
compound 41 possesses the E-configuration (Fig. 3). In order
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affinity and selectivity within PDZ domains,9,23,42,65 and mediates
hydrophobic interactions with Lys83 and Val84 (Fig. 4d). For
the des-cyano analogue 5, the pose of 1 correlates with the
observed inactivity of 5, since the important contribution to
affinity from the cyano group is missing. The modified regions
of compounds 3, 8 and 14 do not mediate direct contacts with the
PDZ domain, however, the lack of affinity of these compounds
can be explained by introduction of increased flexibility that
apparently is unfavourable for binding.
Next, we evaluated the effects of variations in the aromatic and
the ethyl groups, compounds 25–36. The anthracene moiety in 26,
30 and 34 is too large to fit the P0 binding pocket and leads to
a change in orientation in the binding pocket that is associated
with the loss of interactions and hence affinity. In contrast,
compounds 25 and 27 superimpose well with 1 in the binding
pocket (Fig. 4b), and their binding affinities are indeed very similar
(Table 1). Modification of the alkyl tail, which interacts with the
P-2 region, to isopropyl and butyl (compounds 28–31 and 36)
does not alter affinity significantly, probably because this part
points away from the PDZ domain and further extensions do not
introduce additional interactions (Fig. 4d). However, introduction
of adamantyl (32–35) at this place abolished affinity, due to its large
size and bulkiness, which is not favourable in this solvent-exposed
part of the PDZ domain.
Fig. 3 Perspective drawing (ORTEP-3)68 of compound 41. Displacement
ellipsoids of the non-hydrogen atoms are shown at the 50% probability
level. Hydrogen atoms are represented by spheres of arbitrary size.
to exclude discrepancies due to the difference in the aromatic
moieties between 1 and 41, we performed quantum mechanical
modelling using density functional theory (DFT), demonstrating
that for both compounds the E-configuration is stabilized by 23–
25 kJ mol-1 compared to the Z-configuration.
The docking pose of (E)-1 using the crystallographic structure
of the human PDZ domain of PICK164 suggested three structural
areas as the primary contributors to binding (Fig. 4): (i) The
dichloro- and aromatic moieties establish hydrophobic interac-
tions with the P0 hydrophobic pocket created by four isoleucines
from the PICK1 PDZ domain. This area is normally occupied by
the first (P0) amino acid from peptide ligands (Fig. 4a and 4b); (ii)
the cyano group facilitates ligand–protein shape-complementarity,
as it is found buried in a small cavity created by the aB helix and bB
beta-sheet of the PDZ domain (Fig. 4c); (iii) the alkyl tail binds
near the P-2 region, which for peptide ligands is important for
Upon alkylation of the carbamate nitrogen, the conformation
of the compound changes thereby preventing steric clashing
between the N-substituent and the cyano group, as established
from conformational analysis of compounds 37–40 calculated
in the absence of protein. Docking studies of 37 suggested that
Fig. 4 The binding of compound 1 in the PDZ domain of PICK1. The protein is rendered in green and the ligand in stick representation with the chlorine
atoms shown in deep blue, oxygens in red, carbons in purple, aromatic carbons in white and nitrogen atoms in blue. Panels (a)–(d) show magnifications of the
corresponding regions indicated by the labelled frames in the main figure. These (a)–(d) framed regions contain the ligand moieties that contribute most to
binding, i.e., the chloro substituents, the phenyl moiety, the cyano group and the alkyl tail, respectively. In panel (a) the positions of the 3-trifluoromethyl
and the 4-methoxy moieties of compound 49 and 44, are shown in orange and cyan, respectively; in (b) the naphthyl and biphenyl analogues, compounds
25 and 27 are shown in yellow and wheat; in (c) the ethyl and benzyl analogues, compounds 38 and 40 are shown in white and orange; in panel (d)
compounds 28 and 29 with the isopropyl substituents are shown in blue and pink.
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the aromatic region was positioned at P0 similar to 1, and
that the N-methyl group pointed towards the exterior, but the
changed conformation of 37 prevented favourable interactions
of the cyano and ethyl groups. A similar observation was made
for 38, whereas the increased length of the alkyl group in 39
was found to compensate for the lost interactions by making
hydrophobic contacts with Val84 and Ala87 from the aB helix
of PDZ. The same hydrophobic interactions were observed for the
N-benzyl group of 40. This computational evaluation correlates
well with the biological data showing that N-methylation, as in
37, impaired binding affinity, while larger and more hydropho-
bic N-substituents gradually lead to increased potency (38–40,
Fig. 4c).
Compounds 41–49 probe the role of the 3- and 4 substitutions on
the aromatic ring (Table 3). The compound with the unsubstituted
phenyl, 41, appears to be buried deeper in the P0 pocket, and this
affects the position of the cyano and ethyl groups that can no
longer interact with PDZ in the same favourable way as for 1. The
4-chloro analogue (42) presents the same orientation as 1, but the
lack of the 3-chloro substituent decreases the number of favourable
interactions with the P0 pocket. In the fluorescence polarization
assay, compound 43 with the 3,4-dimethyl substituents demon-
strated lower affinity than 1. This correlates with the docking
studies, where it is seen that 43 penetrates deeper into the P0
pocket, hence affecting the position of the cyano and ethyl groups
as also seen for 41. For compound 44, the model indicates that the
methoxy group induces a different orientation of the ligand that
prevents the interaction between the cyano group and the PDZ
domain (Fig. 4a).
Compounds 46–48 bind in similar orientations, with the nitro
group interacting with backbone amides from a conserved Gly-
Leu-Gly-Phe (GLGF) motif in the PDZ domain, which coor-
dinates the C-terminal carboxylate group from peptide ligands
via hydrogen bonds.66,67 However, the halogens either fit into
this pocket (47, 48), or do not fit (46). Although the overall
conformation of these compounds is very similar to compound
1, the cyano group presents a different orientation and cannot
establish interactions with the PDZ domain, which results in a
lower affinity for compound 46 and 47. For compound 48, the
bromine fills out the P0 cavity more efficiently than chlorine,
explaining the higher affinity compared to 47. For compound 45,
the interaction with the GLGF loop involves the 4-trifluoromethyl
group, and 45 is found in a conformation that prevents interaction
between the cyano group and the PDZ domain. Compound 49,
with a 3-trifluoromethyl and 4-chloro substituent, completely
superimposes with 1 (Fig. 4a), and hydrogen bond interaction
between the fluorines of the 3-trifluoromethyl group and the
GLGF loop, is in agreement with the observed higher affinity
of 49 towards PICK1 relative to 1.
3-(3,4-dichlorophenyl)acryloylcarbamate (1). To the best of our
knowledge, this work constitutes the first medicinal chemistry
assessment of any small-molecule PDZ domain inhibitor – a class
of potentially therapeutic relevant protein domains well-known
for their abundance and central roles in cell biology, but also for
being difficult to target with small-molecules.
Analogues of 1 were prepared by a condensation reac-
tion between 2-cyanoacetic acids and carbamates to generate
cyanoacetylcarbamates, which were subsequently reacted with
aromatic aldehydes in a Knoevenagel condensation reaction to
provide the final compounds in good yields. Subtle modifications
of the acryloylcarbamate backbone as in 3, 5, 8 and 14 revealed that
preservation of this scaffold is an essential prerequisite for affinity.
The aromatic and ethyl moieties were explored, in compounds
25–36, providing basic information on the steric requirements for
biological activity. N-Alkylated carbamates, compounds 37–40,
demonstrated that the size of potential alkyl substituents on the
carbamate nitrogen is critical for maintaining affinity to PICK1.
The analogues with subtle changes of the phenyl substituents,
compounds 41–49 led to the conclusion that electronegative and
lipophilic substituents are favoured in both the 3- and 4-positions
of the phenyl moiety. Strengthening both of these properties in the
3-position by substituting chlorine with trifluoromethyl resulted
in the most potent compound (49) with a small but significant
improvement in affinity compared to 1.
X-ray crystallography, NMR and DFT calculations settled the
configuration around the double bond, a premise for conducting
docking studies, and revealed that the aromatic and the cyano
groups are found on the same side of the double bond in 1
and analogues. Docking of all the compounds in the PICK1-
PDZ binding site provided a structural context for the results
of the experimental SAR study, and in general, there was a good
correlation between the suggested poses and the affinity trends.
Based on these investigations, it is concluded that modifications of
the aromatic moiety is the most promising strategy for improving
affinity, whereas other regions such as the carbamate nitrogen
are more sensitive to structural changes. In addition, this work
demonstrated that specificity is a robust feature for the lead
compound and not easily affected by modifications. Therefore, this
family of compounds should be useful in further studies probing
the biological importance of PICK1.
Acknowledgements
This work was supported by the Lundbeck Foundation (K.S.) and
the Drug Research Academy, Faculty of Pharmaceutical Sciences,
University of Copenhagen, Denmark (Ph.D. scholarship to A.B),
by University of Copenhagen Program of Excellence (BioScaRT)
(U.G., A.B., K.S., K.L.M) and by the National Institutes of
Health grant P01 DA012408 (to H.W., U.G.). The technical
assistance of Mr. Flemming Hansen with the X-ray data collection
is gratefully acknowledged. We thank Dr. Tejs Vegge and the
Danish Center for Scientific Computing and the Cofrin Center for
Biomedical Information in the HRH Prince Alwaleed Bin Talal
Bin Abdulaziz Alsaud Institute for Computational Biomedicine
at Weill Cornell Medical College for computational time and
resources.
Conclusion
PICK1 is suggested as a drug target against severe neurological
disorders and possesses intriguing roles in neurobiology. Thus,
pursuing inhibitors of PICK1 is important to aid biological studies
and possibly develop new therapeutic modalities. In order to
understand the molecular requirements for PICK1 activity and
improve affinity, the SAR study presented here characterizes the
first small-molecule PICK1 PDZ inhibitor, (E)-ethyl 2-cyano-
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 4281–4288 | 4287
©

37 N. Fujii, J. J. Haresco, K. A. Novak, R. M. Gage, N. Pedemonte, D.
Stokoe, I. D. Kuntz and R. K. Guy, Bioorg. Med. Chem. Lett., 2007,
17, 549–552.
38 A. Mayasundari, A. M. Ferreira, L. He, N. Mahindroo, D. Bashford
and N. Fujii, Bioorg. Med. Chem. Lett., 2008, 18, 942–945.
39 J. Shan and J. J. Zheng, J. Comput.-Aided Mol. Des., 2009, 23, 37–47.
40 M. C. Hammond, B. Z. Harris, W. A. Lim and P. A. Bartlett, Chem.
Biol., 2006, 13, 1247–1251.
41 M. Aarts, Y. Liu, L. Liu, S. Besshoh, M. Arundine, J. W. Gurd, Y. T.
Wang, M. W. Salter and M. Tymianski, Science, 2002, 298, 846–850.
42 A. Bach, C. N. Chi, T. B. Olsen, S. W. Pedersen, M. U. Røder, G. F.
Pang, R. P. Clausen, P. Jemth and K. Strømgaard, J. Med. Chem., 2008,
51, 6450–6459.
43 A. Piserchio, G. D. Salinas, T. Li, J. Marshall, M. R. Spaller and D. F.
Mierke, Chem. Biol., 2004, 11, 469–473.
References
1 M. R. Arkin and J. A. Wells, Nat. Rev. Drug Discovery, 2004, 3, 301–
317.
2 J. A. Wells and C. L. McClendon, Nature, 2007, 450, 1001–1009.
3 T. Berg, Curr. Opin. Drug Discovery Dev., 2008, 11, 666–674.
4 K. K. Dev, Nat. Rev. Drug Discovery, 2004, 3, 1047–1056.
5 L. L. Blazer and R. R. Neubig, Neuropsychopharmacology, 2009, 34,
126–141.
6 M. D. Houslay, Br. J. Pharmacol., 2009, 158, 483–485.
7 H. C. Kornau, L. T. Schenker, M. B. Kennedy and P. H. Seeburg,
Science, 1995, 269, 1737–1740.
8 E. Kim, M. Niethammer, A. Rothschild, Y. N. Jan and M. Sheng,
Nature, 1995, 378, 85–88.
9 Z. Songyang, A. S. Fanning, C. Fu, J. Xu, S. M. Marfatia, A. H. Chishti,
A. Crompton, A. C. Chan, J. M. Anderson and L. C. Cantley, Science,
1997, 275, 73–77.
44 D. G. Udugamasooriya, S. C. Sharma and M. R. Spaller, Chem-
BioChem, 2008, 9, 1587–1589.
10 E. Kim and M. Sheng, Nat. Rev. Neurosci., 2004, 5, 771–781.
11 J. Staudinger, J. Zhou, R. Burgess, S. J. Elledge and E. N. Olson, J. Cell
Biol., 1995, 128, 263–271.
12 J. L. Perez, L. Khatri, C. Chang, S. Srivastava, P. Osten and E. B. Ziff,
J. Neurosci., 2001, 21, 5417–5428.
13 W. L. Wang, S. F. Yeh, Y. I. Chang, S. F. Hsiao, W. N. Lian, C. H. Lin,
C. Y. Huang and W. J. Lin, J. Biol. Chem., 2003, 278, 37705–37712.
14 K. K. Dev, Y. Nakajima, J. Kitano, S. P. Braithwaite, J. M. Henley and
S. Nakanishi, J. Neurosci., 2000, 20, 7252–7257.
15 A. Baron, E. Deval, M. Salinas, E. Lingueglia, N. Voilley and M.
Lazdunski, J. Biol. Chem., 2002, 277, 50463–50468.
16 B. J. Peter, H. M. Kent, I. G. Mills, Y. Vallis, P. J. Butler, P. R. Evans
and H. T. McMahon, Science, 2004, 303, 495–499.
17 H. T. McMahon and J. L. Gallop, Nature, 2005, 438, 590–596.
18 G. Khelashvili, D. Harries and H. Weinstein, Biophys. J., 2009, 97,
1626–1635.
45 M. P. Belmares, L. U. Peter, K. A. Mendoza, PCT 079406 A1, 2007.
46 H.-J Lee, N. X. Wange, D.-L. Shi and J. J. Zheng, Angew. Chem., Int.
Ed., 2009, 48, 6448–6452.
47 D. Grandy, J. Shan, X. Zhang, S. Rao, S. Akunuru, H. Li, Y. Zhang, I.
Alpatov, X. A. Zhang, R. A. Lang, D. L. Shi and J. J. Zheng, J. Biol.
Chem., 2009, 284, 16256–16263.
48 A. Bach, C. N. Chi, G. F. Pang, L. Olsen, A. S. Kristensen, P. Jemth
and K. Strømgaard, Angew. Chem., Int. Ed., 2009, 48, 9685–9689.
49 T. S. Thorsen, K. L. Madsen, N. Rebola, M. Rathje, V. Anggono,
A. Bach, I. S. Moreira, N. Stuhr-Hansen, T. Dyhring, D. Peters, T.
Beuming, R. Huganir, H. Weinstein, C. Mulle, K. Strømgaard, L. C. B.
Rønn and U. Gether, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 413–
418.
50 C. O’Murchu, US Patent 3810934, 1974.
51 G. B. Kharas, A. M. Fuerst, E. L. Feitl, M. E. Pepper, F. C. Prillaman,
J. R. Pyo, G. M. Rogers, A. Z. Tadros, L. G. Umek and K. Watson,
J. Macromol. Sci., Part A: Pure Appl. Chem., 2004, 41, 629–635.
52 B. C. Cantello, M. A. Cawthorne, G. P. Cottam, P. T. Duff, D. Haigh,
R. M. Hindley, C. A. Lister, S. A. Smith and P. L. Thurlby, J. Med.
Chem., 1994, 37, 3977–3985.
19 J. Xia, X. Zhang, J. Staudinger and R. L. Huganir, Neuron, 1999, 22,
179–187.
20 S. M. Gardner, K. Takamiya, J. Xia, J. G. Suh, R. Johnson, S. Yu and
R. L. Huganir, Neuron, 2005, 45, 903–915.
21 J. G. Hanley and J. M. Henley, EMBO J., 2005, 24, 3266–3278.
22 K. L. Madsen, J. Eriksen, L. Milan-Lobo, D. S. Han, M. Y. Niv, I.
Ammendrup-Johnsen, U. Henriksen, V. K. Bhatia, D. Stamou, H. H.
Sitte, H. T. McMahon, H. Weinstein and U. Gether, Traffic, 2008, 9,
1327–1343.
23 K. L. Madsen, T. Beuming, M. Y. Niv, C. W. Chang, K. K. Dev, H.
Weinstein and U. Gether, J. Biol. Chem., 2005, 280, 20539–20548.
24 G. E. Torres, W.-D. Yao, A. R. Mohn, H. Quan, K.-M. Kim, A. I.
Levey, J. Staudinger and M. G. Caron, Neuron, 2001, 30, 121–134.
25 M. I. Daw, R. Chittajallu, Z. A. Bortolotto, K. K. Dev, F. Duprat, J. M.
Henley, G. L. Collingridge and J. T. Isaac, Neuron, 2000, 28, 873–886.
26 J. Xia, H. J. Chung, C. Wihler, R. L. Huganir and D. J. Linden, Neuron,
2000, 28, 499–510.
53 R. G. Giles, N. J. Lewis, J. K. Quick, M. J. Sasse, M. W. J. Urquhart
and L. Youssef, Tetrahedron, 2000, 56, 4531–4537.
54 J. J. Rohde, M. A. Pliushchev, B. K. Sorensen, D. Wodka, Q. Shuai, J.
Wang, S. Fung, K. M. Monzon, W. J. Chiou, L. Pan, X. Deng, L. E.
Chovan, A. Ramaiya, M. Mullally, R. F. Henry, D. F. Stolarik, H. M.
Imade, K. C. Marsh, D. W. Beno, T. A. Fey, B. A. Droz, M. E. Brune,
H. S. Camp, H. L. Sham, E. U. Frevert, P. B. Jacobson and J. T. Link,
J. Med. Chem., 2007, 50, 149–164.
55 P. Kocovsky´, Tetrahedron Lett., 1986, 27, 5521–5524.
56 M. H. Potashman and M. E. Duggan, J. Med. Chem., 2009, 52, 1231–
1246.
57 J. G. Topliss, J. Med. Chem., 1972, 15, 1006–1011.
58 Y. C. Martin and W. J. Dunn, 3rd, J. Med. Chem., 1973, 16, 578–579.
59 W. C. Patt, J. J. Edmunds, J. T. Repine, K. A. Berryman, B. R. Reisdorph,
C. Lee, M. S. Plummer, A. Shahripour, S. J. Haleen, J. A. Keiser, M. A.
Flynn, K. M. Welch, E. E. Reynolds, R. Rubin, B. Tobias, H. Hallak
and A. M. Doherty, J. Med. Chem., 1997, 40, 1063–1074.
60 J. G. Topliss, J. Med. Chem., 1977, 20, 463–469.
27 A. Terashima, K. A. Pelkey, J. C. Rah, Y. H. Suh, K. W. Roche, G. L.
Collingridge, C. J. McBain and J. T. Isaac, Neuron, 2008, 57, 872–
882.
28 J. D. Bell, E. Park, J. Ai and A. J. Baker, Cell Death Differ., 2009, 16,
1665–1680.
29 R. M. Dixon, J. R. Mellor and J. G. Hanley, J. Biol. Chem., 2009, 284,
14230–14235.
61 S. Turconi, K. Shea, S. Ashman, K. Fantom, D. L. Earnshaw, R. P.
Bingham, U. M. Haupts, M. J. Brown and A. J. Pope, J. Biomol.
Screening, 2001, 6, 275–290.
62 A. J. Pope, U. M. Haupts and K. J. Moore, Drug Discov. Today, 1999,
4, 350–362.
63 J. C. Owicki, J. Biomol. Screening, 2000, 5, 297–306.
64 J. M. Elkins, E. Papagrigoriou, G. Berridge, X. Yang, C. Phillips, C.
Gileadi, P. Savitsky and D. A. Doyle, Protein Sci., 2007, 16, 683–694.
65 C. N. Chi, A. Engstro¨m, S. Gianni, M. Larsson and P. Jemth, J. Biol.
Chem., 2006, 281, 36811–36818.
66 D. A. Doyle, A. Lee, J. Lewis, E. Kim, M. Sheng and R. MacKinnon,
Cell, 1996, 85, 1067–1076.
30 K. K. Dev, Curr. Top. Med. Chem., 2007, 7, 3–20.
31 E. M. Garry, A. Moss, R. Rosie, A. Delaney, R. Mitchell and S. M.
Fleetwood-Walker, Mol. Cell. Neurosci., 2003, 24, 10–22.
32 C. Bellone and C. Luscher, Nat. Neurosci., 2006, 9, 636–641.
33 P. J. Hajduk, J. R. Huth and S. W. Fesik, J. Med. Chem., 2005, 48,
2518–2525.
34 D. C. Fry and L. T. Vassilev, J. Mol. Med., 2005, 83, 955–963.
35 M. Joshi, C. Vargas, P. Boisguerin, A. Diehl, G. Krause, P. Schmieder,
K. Moelling, V. Hagen, M. Schade and H. Oschkinat, Angew. Chem.,
Int. Ed., 2006, 45, 3790–3795.
36 N. Fujii, J. J. Haresco, K. A. Novak, D. Stokoe, I. D. Kuntz and R. K.
Guy, J. Am. Chem. Soc., 2003, 125, 12074–12075.
67 A. Y. Hung and M. Sheng, J. Biol. Chem., 2002, 277, 5699–5702.
68 L. J. Farrugia, J. Appl. Crystallogr., 1997, 30, 565–565.
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