Design, synthesis, and characterization of fatty acid derivatives of a dimeric peptide-based postsynaptic density-95 (PSD-95) inhibitor

Article abstract of DOI:10.1021/jm501755d

Dimeric peptide-based inhibitors of postsynaptic density-95 (PSD-95) can reduce ischemic brain damage and inflammatory pain in rodents. To modify the pharmacokinetic profile, we designed a series of fatty acid linked dimeric ligands, which potently inhibits PSD-95 and shows improved in vitro blood plasma stability. Subcutaneous administration in rats showed extended stability and sustained release of these ligands. This can facilitate new pharmacological uses of PSD-95 inhibitors and further exploration of PSD-95 as a drug target.

Full text of DOI:10.1021/jm501755d

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Brief Article
Design, Synthesis, and Characterization of Fatty Acid Derivatives of
a Dimeric Peptide-Based Postsynaptic Density-95 (PSD-95) Inhibitor
Klaus B. Nissen, Julie J. Andersen, Linda M. Haugaard-Kedström, Anders Bach, and Kristian Strømgaard
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501755d • Publication Date (Web): 15 Jan 2015
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Journal of Medicinal Chemistry
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Design, Synthesis, and Characterization of Fatty Acid Deriva-
tives of a Dimeric Peptide-Based Postsynaptic Density-95
(PSD-95) Inhibitor
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Klaus B. Nissen, Julie J. Andersen, Linda M. Haugaard-Kedström, Anders Bach, and Kristian
Strømgaard*
Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copen-
hagen, Denmark.
ABSTRACT: Dimeric peptide-based inhibitors of postsynaptic density-95 (PSD-95) can reduce ischemic brain damage
and inflammatory pain in rodents. To modify the pharmacokinetic profile we designed a series of fatty acid linked dimeric
ligands, which potently inhibits PSD-95 and shows improved in vitro blood plasma stability. Subcutaneous administration
in rats showed extended stability and sustained release of these ligands. This can facilitate new pharmacological uses of
PSD-95 inhibitors and further exploration of PSD-95 as a drug target.
The family of ionotropic glutamate (iGlu) receptors
are ligand-gated ion channels that mediate the majority
of excitatory synaptic transmission in the vertebrate brain
and are crucial for normal brain function.1-3 Dysfunction
of iGlu receptors is involved in a range of neurological
and psychiatric diseases and inhibition of iGlu receptors
is considered a promising strategy for treating brain dis-
eases, such as Alzheimer’s disease, ischemic brain damage
and chronic pain.1, 4 Particularly, overstimulation of the N-
The clinically most advanced PSD-95 inhibitor is Tat-
NR2B9C (NA-1), which is being developed for the treat-
ment of ischemic brain damage after stroke and has re-
cently successfully passed phase II clinical trials.22-23 How-
ever, NA-1 suffers from low affinity to PSD-95 (Ki = 4.6
ꢀM),16 and in an attempt to address this, we have previ-
ously developed plasma stable, high-affinity dimeric lig-
ands 1 (UCCB01-125) and 2 (UCCB01-144) (Figure 1) that
bind both PDZ1 and PDZ2 of PSD-95 simultaneously.
These compound have up to 1000-fold increased affinity
for PSD-95 compared to NA-1.16, 24 1 was recently shown to
reduce the mechanical hypersensitivity associated with
chronic inflammatory pain in mice without affecting at-
tention, long-term memory or motor performance, in
contrast to conventional inhibitors of NMDA receptors.25
2, containing a cell penetrating peptide (CPP) moiety, was
shown to reduce brain damage and improve motor func-
tion in mice subjected to focal cerebral ischemia.16 Both 1
methyl-D-aspartate (NMDA) subtype of iGlu receptors is
involved in the pathology of ischemia and neuropathic
pain and several clinical candidates targeting the NMDA
receptor have been in development, but most compounds
5-6
have failed to reach the market.1,
This has generally
been attributed to a narrow therapeutic window of
NMDA inhibition, leading to severe side-effects or low
efficacy of the drugs in clinical trials.1, 6
The scaffolding protein postsynaptic density-95 (PSD-
95) has been shown to simultaneously bind the NMDA
receptor and the enzyme neuronal nitric oxide synthase
(nNOS)7-9 via its PSD-95/discs large/zonula occludens
(PDZ) domains. Upon activation of the NMDA receptor,
Ca2+ influx into the neuron stimulates nNOS to generate
nitric oxide (NO), which is neurotoxic in high concentra-
tions.10-12 Ligands that bind the first two PDZ domains
(PDZ1 and PDZ2) of PSD-95 inhibit formation of the
NMDA/PSD-95/nNOS ternary complex, thus uncoupling
nNOS and reducing NO formation.13 These ligands have
been shown to reduce ischemic brain damage11-16 and
reduce the symptoms of chronic pain in rodents.17-20 Im-
portantly, inhibition of PSD-95 does not block synaptic
and 2 demonstrated improved in vitro plasma stability
24
compared to NA-1,16,
and here we were interested in
extending this stability further, and create compounds
with modified in vivo pharmacokinetic profiles, without
compromising biological activity.
Figure 1. Structures of plasma stable, high affinity dimeric ligands
binding simultaneously to PDZ1 and PDZ2 of PSD-95. 1 consists of
two PSD-95 binding peptides linked by a PEG(4) moiety. 2 further-
more contains the cell penetrating Tat sequence (YGRKKRRQRRR),
attached via the nitrogen atom on the PEG-based ‘NPEG’ linker, to
facilitate brain delivery. Letters indicate amino acids except for N
(nitrogen) and O (oxygen).
21
activity or other Ca2+-mediated signal events.13, There-
fore, inhibition of PSD-95 is a promising strategy for the
treatment of ischemic brain damage and neuropathic
pain.
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Scheme 1. Synthesis of FA-Linked Dimeric Ligands 7-18 and 22-24.a
O
O
O
IETXV
IETXV
IETXV
IETXV
1
2
3
4
5
6
7
8
O
O
O
O
R1
O
n
O
m
R1
O
n
O
O
O
O
O
O
b,c
R2
a
H
N
H2
N
N
N
N
H
R1
O
n
O
O
O
Fmoc
R2
N
H
OH
IETXV
OH
IETXV
m
O
O
4: X=A n=2, R1=H
O
15: X=A, n=3, m=10, R1=H, R2=CH3
16
m = 10/16
R2 = CH3 or COOCH3
n = 2-3
1 = H or COOtBu
7: X=A,n=2, m=10, R1=H, R2=CH3
8
: X=A, n=2, m=10, R1=H, R2=COOH
9: X=A, n=2, m=16, R1=H, R2=CH3
3
: X=A
19: X=D
R
5
: X=A n=2, R1=COOtBu
: X=A, n=3, m=10, R1=H, R2=COOH
6: X=A n=3, R1=H
20: X=D n=2, R1=H
17: X=A, n=3, m=16 R1=H, R2=CH3
18: X=A, n=3, m=16, R1=H, R2=COOH
10: X=A, n=2, m=16, R1=H, R2=COOH
21
: X=D n=2, R1=COOtBu
11
22
: X=D, n=2, m=16, R1=H, R2=CH3
: X=A, n=2, m=10, R1=COOH, R2=CH3
12: X=A, n=2, m=10, R1=COOH, R2=COOH 23: X=D, n=2, m=16, R1=H, R2=COOH
13 24
: X=A, n=2, m=16, R1=COOH, R2=CH3
14: X=A, n=2, m=16, R1=COOH, R2=COOH
: X=D, n=2, m=16, R1=COOH, R2=CH3
9
10
11
12
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aReagents and conditions: (a) HATU, collidine, DMF (1 h), repeat coupling with HBTU, DIEA, DMF, then 20 % piperidine in DMF. (b) HBTU,
DIPEA, DMF/DCM, 45 min, then TFA/TIPS/H2O (90/5/5); (c) 0.5 M LiOH, H2O/ACN (75/25), 30 min, then TFA to pH<2.
In general, the pharmacokinetic profile of peptides is
influenced in particular by enzymatic degradation, renal
clearance and hepatic metabolism.26 The former has been
addressed in our in vitro stability assay, suggesting that
dimerization per se significantly improves enzymatic
stability.16, 24 An attractive strategy for further increasing
the plasma half-life is to facilitate association with human
serum albumin (HSA).27 Specifically, conjugation of fatty
acids (FA) has been successfully exploited to extend the
plasma half-lives of therapeutic proteins and peptides
such as insulin, glucagon-like peptide 1 (GLP−1), and pan-
creatic polypeptide peptide.28-33 It was demonstrated that
the type of FA as well as a linker unit between the peptide
and the FA are crucial determinants for appropriate in
vivo profile. In addition, conjugation of peptides with FAs
can also improve cell permeability,34 as shown by
myristoylating a potential anti-cancer peptide.35
four different FAs, dodecanoic acid (C12 acid), dodecane-
dioic acid monomethyl ester (C12 diacid methyl ester),
octadecanoic acid (C18 acid) or octadecanedioic acid
monomethyl ester (C18 diacid methyl ester, Scheme S1),
respectively were readily coupled to the primary amino
group of 4−6. The resin-bound derivatives were then
cleaved from the resin with concomitant deprotection of
the side-chain protecting groups, and finally, the mono
methyl protected FAs were deprotected by saponification
of the cleaved product followed by acidification (Scheme
1). The crude FA linked dimeric ligands (7−18) were lyoph-
ilized and purified by preparative HPLC providing the
target compounds in low-to-medium yields (2-40%) and
high purity (>95%, Table S1).
Table 1. Affinity of Dimeric Ligands 1−2 and FA-
Linked Dimeric Ligands 7−18 for PSD-95 PDZ1-2 as
determined by FP.
Here, we have designed and synthesized a series of
systematically modified analogues of dimeric ligand 1,
where different FAs and linkers were examined. We em-
ployed three different linkers, γ−glutamic acid (γ−Glu), γ-
butyric acid (GABA) and 5-amino valeric acid (5-Ava),
similarly to what has been used previously,28-30, 36 and four
different saturated FAs, with either 12 or 18 methylene
groups, and with or without a ϖ-terminal carboxylate.
These FA derivatives of 1 were subsequently examined for
their affinity to PSD-95, as well as their in vitro and in vivo
plasma half-lives.
Linker
FA
Ki,PSD-
Ki,PSD-95
Cmpd
95 (nM)a
+HSA (nM)a
n
-
R1
-
m
-
R2
-
14.3 ± 1.1
4.3 ± 0.1
9.7 ± 1.0
1
-
10.5 ± 0.9
2
-
-
-
In the design of FA derivatives of 1, we took advantage
of our previously developed NPEG linker, which connects
the peptide ligands in 2 (Figure 1).16 The secondary amino
group in the NPEG linker can be selectively derivatized
with functional moieties, as demonstrated with the CPP
sequence in 2 (Figure 1).16 Moreover, this amino group is
introduced in the dimeric ligands at a position that is
presumed to be spatially secluded from the PDZ domain
binding peptide moieties,16 suggesting that derivatization
at this point will not affect the affinity for PSD-95. We
therefore substituted the conventional PEG linker in 1
with the customized NPEG linker, and prepared interme-
diate 3, as described previously (Scheme 1). Next, the
three different linker moieties were attached, by coupling
of 3 with either Fmoc-GABA-OH, Fmoc-γ-Glu-OtBu or
Fmoc-5-Ava, followed by deprotection to provide resin-
bound compounds 4−6 (Scheme 1). Subsequently, the
7
10
10
16
16
10
10
16
16
10
10
16
16
CH3
13.6 ± 0.6
15.4 ± 1.2
11.1 ± 0.6
11.4 ± 0.6
30.2 ± 4.2
33.7 ± 1.6
17.0 ± 0.6
9.2 ± 1.6
20.5 ± 1.1
13.9 ± 2.1
26.5 ± 0.2
11.5 ± 1.2
27.2 ± 2.9
27.9 ± 2.7
1889 ± 155
5717 ± 298
24.0 ± 0.6
26.4 ± 2.6
1391 ± 184
3594 ± 132
13.4 ± 1.4
8
COOH
CH3
2
2
3
H
9
10
11
COOH
CH3
12
13
14
15
16
17
18
COOH
CH3
COOH
COOH
CH3
COOH
CH3
18.5 ± 0.6
1889 ± 100
2926 ± 335
H
COOH
aData shown as mean ± SEM, n≥3.
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Journal of Medicinal Chemistry
with previously published results,16, 24 with Ki values of 14.3
1
2
3
4
5
6
7
8
± 1.1 and 4.3 ± 0.1 nM, respectively. Noticeable, all FA
derived dimeric ligands showed affinities in the same
range with Ki values between 9–34 nM (Table 1 and Figure
2A). Thus, clearly the strategy of introducing FAs in the
NPEG linker did not compromise PSD-95 affinity regard-
less of the type of linker or FA that was used. Next, we
investigated whether HSA influenced the ability of the FA
linked dimers to bind PSD-95. The FP assay was therefore
performed with buffer containing 1% HSA (~150 µM),
which is slightly lower than physiological blood concen-
trations. At higher HSA concentrations binding of the
fluorescent dimeric probe to HSA compromised the FP
assay. Here, the affinity of the FA derivatives for PSD-95
PDZ1-2 showed a significant and systematic change: De-
rivatives with shorter FAs, such as C12 acid (7, 11 and 15) or
C12 diacid (8, 12 and 16) were largely unaffected by the
presence of HSA, while the affinities of the FA derivatives
comprising the longer C18 acid (9, 13 and 17) or C18 diacid
(10, 14 and 18) were severely reduced (Figure 2B). In par-
ticular, C18 diacid containing analogues showed 250-520
fold increases in Ki values, while the C18 acid derivatives
had 70- to 170-fold reduced affinities. FA derivatives 10
and 14 were the most potent derivatives when HSA was
not present with Ki values of 11.4 ± 0.6 and 9.2 ± 1.6 nM,
respectively, but also the least potent in the modified FP
assay with HSA, where Ki values were measured to 5717 ±
298 and 3594 ± 132 nM, respectively. For the three differ-
ent libkers, we did not observe any systematic difference
when HSA was present in the FP assay (Table 1).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Figure 2. Affinity for PSD-95 PDZ1-2 of dimeric ligands 1−2 and 7−18
30
31
32
33
34
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42
43
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46
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as determined by FP. A: FP assay without HSA. B: FP assay with 1 %
HSA. Data shown as mean in nM ± SEM, n ≥ 3.
Compounds 9 and 13 were among the most hydropho-
bic compounds, as assessed by the retention time (Rt) in
analytical HPLC (Table S2). Also, their affinities were
greatly affected by HSA and the water solubility of these
ligands was reduced. In an attempt to increase the hydro-
philicity, we synthesized compounds 22−24 (Scheme 1)
corresponding to 9, 10 and 13, but where the peptide se-
quence IETAV is replaced with IETDV, which should
increase hydrophilicity, but not affect the affinity towards
PDZ1-2 of PSD-95.16, 24 However, when incorporated into
the dimeric structure and attaching the FA, the overall
hydrophobicity of 22−24 was not affected according to the
HPLC retention times (Table S2). When tested in the FP
assay compounds 22 and 24 showed similar affinities to
the corresponding compounds, 9 and 13, respectively,
both with and without HAS in the FP assay, while 23 were
less affected by HSA relative to 10 (Tables 2 and 3).
In addition, three FA linked dimeric ligands containing
the peptide sequence IETDV instead of IETAV were syn-
thesized, similarly to the procedure described above:
Resin-bound, protected IETDV (19) was coupled with
Fmoc-GABA-OH or Fmoc-γ-Glu-OtBu to give 20 and 21,
which were coupled with C18 acid and C18 diacid methyl
ester to give 22−24 (Scheme 1).
The affinity of the FA linked dimeric ligands for the
tandem PDZ1-2 domain of PSD-95 was evaluated in a
fluorescence polarization (FP) assay, as previously de-
scribed.16 In addition, we examined the influence of HSA
on affinity to PDZ1-2 as an indirect measurement of HSA
affinity. Without the presence of HSA, the measured
affinities of 1 and 2 were comparable
Next, we wanted to determine if FA derivatization of the
dimeric ligands influenced the in vitro plasma stability,
and four analogues, 7, 10, 13 and 22, were selected for
further analysis. However, when subjected to a standard
in vitro plasma stability assay24 sample recoveries were
very low (<5%), due to co-precipitation of FA derivatized
ligands and plasma proteins during trichloroacetic acid
Table 2. FA-linked Dimeric Ligands with an IETDV
Peptide Ligand.
Pep-
tide
Ki,PSD-
Ki,PSD-95
Cmpd
Linker
FA
95 (nM)a
+HSA (nM)a
8.0 ± 0.8
29.3 ± 0.7
6.7 ± 0.6
2514 ± 149
770 ± 27
22
23
24
GABA
GABA
γ-Glu
C18:COOH
C18(COOH)2
C18:COOH
IETDV
(TCA)
precipitation.
A
modified
version
of
3806 ± 287
aData shown as mean ± SEM, n≥3.
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Table 3. Pharmacokinetic Parameters of FA-Linked
Dimeric Ligands after s.c. Adminstration in Rats.
1
2
3
4
5
6
7
8
9
Dose
3
T
(h)a
½
Tmax (h)a
0.5 ± 0.0
0.5 ± 0.0
0.8 ± 0.2
4.7 ± 0.7
Cmpd
1
Linker
FA
-
Peptide
0.6 ± 0.1
0.5 ± 0.1
0.8 ± 0.05
8.1 ± 0.5
-
IETAV
30
30
7
GABA
GABA
C12
IETAV
IETAV
C17:0-
COOH
15
10
15
10
10.7 ± 0.6
16.3 ± 2.8
6.0 ± 1.2
8.0 ± 0.0
13
C18:0
IETAV
IETDV
γ-Glu
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
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38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. In vitro plasma stability of dimeric ligands 1, 7, 10, 13 and
22. Half-lives (first order decay) are indicated in parenthesis.
22
GABA
C18:0
aData shown as mean ± SEM, n≥3.
this assay was therefore implemented, where solid guani-
dine hydrochloride (GnHCl) was added to the samples
prior to TCA precipitation, providing sample recoveries of
>80%. Thus, the dimeric ligands were incubated in hu-
man plasma, analyzed at different time points by HPLC
and peak areas were normalized to the amount at T0 and
fitted to a first order decay model to determine the half-
(Figure 4). The effect was smallest for 7, but noticeable for
10, 13, and 22, which showed T1/2 between 8-16 hours,
corresponding to a 16-to 32-fold increase relative to 1
without FA (Table 3). The increased Tmax is likely due to a
prolonged absorption from the injection site, illustrating
that FA linked dimeric PSD-95 inhibitors, especially 10, 13,
and 22, can be administered by a s.c. depot injection
where the compound is released slowly and consistently
into the blood. Thereby relevant blood concentrations
may be achieved with fewer administrations.
life (T ). The half-life of the original dimeric ligand 1 was
½
determined to 1.7 h, which was in the same range as pre-
viously determined,24 whereas all the FA linked dimeric
ligands showed significantly improved half-lives (Figure
3): The dimeric ligand linked to the shorter C12 acid FA, 7,
In conclusion, we have designed and synthesized a
range of dimeric ligands conjugated to four different FAs
via three different linkers. The FA linked dimeric ligands
were prepared from a common NPEG dimeric derivative
providing a systematic investigation of PSD-95 affinity
with and without the presence of HSA. PSD-95 affinities
of FA-derived dimeric ligands were not compromised, as
all ligands showed affinities in the low nanomolar range.
However, when adding HSA to the assay affinities were
reduced significantly for ligands comprising a long C18 FA,
whereas C12 FAs did not affect affinity. Compounds 1 and 2
(without FA) were not influenced by HSA, corroborating
that indeed FA leads to interaction with HSA. Selected
FA-linked dimeric ligands were investigated in an in vitro
plasma stability assay, and especially the C18 FA-
containing ligands showed a dramatic increase in stability
compared to 1. Importantly, this property translates into
greatly increased in vivo half-lives for up to 16 hours and
sustained release into the blood in rats given a s.c. depot
injection. Overall, dimeric PSD-95 inhibitors modified
with C18 FAs bind strongly to both PSD-95 and HSA, are
highly stable in blood plasma, and demonstrate very long
in vivo half-lives. Thus these compounds comprise a novel
class of PSD-95 inhibitors with unique in vivo pharmaco-
kinetic properties. Future studies are required to examine
if this profile translates into improved efficacy in animal
disease models, and if these compounds can facilitate the
development of new pharmacological strategies and ex-
aminations of PSD-95 as a drug target, where a long-term
and consistent PSD-95 inhibition is desirable.
had a T = 23.6 h, whereas the dimeric ligands linked to
½
the longer C18 FAs, 10, 13 and 22, all showed T > 24 h. In
½
this assay, using human plasma, it is expected that the
HSA concentration is in the range of 0.5-0.8 mM, as in
normal blood samples.37-39 Thus, the dramatically in-
creased half-lives observed is most likely attributed to the
increased HSA affinity of the FA linked dimeric ligands,
which lowers the free concentration of ligand available for
enzymatic digestion. Additionally, steric hindrance medi-
ated by FA could also prevent enzymatic degradation.
To explore if the increased in vitro plasma stability of
lipidated compounds translates to in vivo conditions, we
established the plasma pharmacokinetic profiles of 1 and
analogue 7, 10, 13 and 22 in rats. We measured the con-
centration of compound in blood following a single sub-
cutaneous (s.c.) bolus injection of male Wistar rats. Clear-
ly, FA linked dimeric ligands have higher T and it takes
½
longer before maximal blood concentration is reached
(Tmax) compared to dimeric ligand without FA (1)
EXPERIMENTAL SECTION
General synthetic procedure. See Supporting Infor-
mation.
Figure 4. Plasma profiles of dimeric ligands 1 (two doses) and FA-
derived dimeric ligands 7, 10, 13 and 22 after s.c. administration in
rats.
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kristian.stromgaard@sund.ku.dk, +45 35336114
Synthesis of 4-6, 20-21. The protected linkers (Fmoc-
1
2
3
4
Glu-OtBu, Fmoc-GABA-OH and Fmoc-5-Ava-OH were
coupled to the nitrogen of the NPEG linker of 3 and 19 by
two consecutive couplings. For the first coupling, Fmoc-
Glu-OtBu, Fmoc-GABA or Fmoc-5-Ava-OH (2 eq.) was
ACKNOWLEDGMENTS
We are grateful for financial support from the Lundbeck
Foundation (K.S.).
5
6
7
8
activated
by
O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-
ABBREVIATIONS USED
tetramethyluronium hexafluorophosphate (HATU, 3 eq,
0.5M) and collidine (4 eq) in DMF) before addition to the
drained resin. After 45 min of shaking and a DMF flow-
wash, the coupling was repeated with HBTU/DIEA as
coupling reagent and the reaction was continued over-
night. The Fmoc group was then removed with 2 x 20%
piperidine in DMF (2 x 5 min shaking, DMF wash in be-
tween).
4: MS (ESI+) calcd. for C65H112N12O27 [M + H]+, 1493.8;
found m/z 1493.9, calcd. For [M + 2H]+, 747.4; found m/z
747.5. 5: MS (ESI+) calcd. for C64H112N12O25 [M + H]+,
1449.8; found m/z 1449.8, calcd. For [M + 2H]+ 725.4;
found m/z 725.6. 6: MS (ESI+) calcd. for C65H114N12O25 [M
+ H]+, 1463.8; found m/z 1464.1, calcd. For [M + 2H]+, 731.9
found m/z 732.3. 20: MS (ESI+) calcd. for C67H112N12O31 [M
+ H]+, 1581.8; found m/z 1581.8, calcd. For [M + 2H]+, 791.4;
found m/z 791.5. 21: MS (ESI+) calcd. for C66H112N12O29 [M
+ H]+, 1537.8; found m/z 1537.8, calcd. For [M + 2H]+
769.4; found m/z 769.6.
5-Ava, 5-amino valeric acid; CPP, cell penetrating peptide;
FA, fatty acid; FP, fluorescence polarization; GABA, γ-butyric
acid; γ−Glu, γ−glutamic acid; HSA, human serum albumin;
iGlu; ionotropic glutamate; nNOS, neuronal nitric oxide
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synthase; NO, nitric oxide; NMDA, N-methyl-D-aspartate;
PDZ, PSD-95, discs large, zonula occludens-1; PEG, polyeth-
ylene glycol; PPIs, protein-protein interactions; PSD-95,
postsynaptic density-95. SAR, structure-activity relationship.
REFERENCES
(1) Traynelis, S. F.; Wollmuth, L. P.; McBain, C. J.; Menniti, F.
S.; Vance, K. M.; Ogden, K. K.; Hansen, K. B.; Yuan, H.; Myers, S.
J.; Dingledine, R. Glutamate receptor ion channels: structure,
regulation, and function. Pharmacol. Rev. 2010, 62, 405–496.
(2) Mayer, M. L. Glutamate receptors at atomic resolution.
Nature 2006, 440, 456-462.
(3) Mayer, M. L. Structure and mechanism of glutamate recep-
tor ion channel assembly, activation and modulation. Curr. Opin.
Neurobiol. 2011, 21, 283-290.
(4) Lemoine, D.; Jiang, R.; Taly, A.; Chataigneau, T.; Specht,
A.; Grutter, T. Ligand-gated ion channels: new insights into
neurological disorders and ligand recognition. Chem. Rev. 2012,
112, 6285-6318.
(5) Ogden, K. K.; Traynelis, S. F. New advances in NMDA re-
ceptor pharmacology. Trends Pharmacol. Sci. 2011, 32, 726-733.
(6) Muir, K. W., Glutamate-based therapeutic approaches:
clinical trials with NMDA antagonists. Curr. Opin. Pharmacol.
2006, 6, 53-60.
(7) Niethammer, M.; Kim, E.; Sheng, M. Interaction between
the C terminus of NMDA receptor subunits and multiple mem-
bers of the PSD-95 family of membrane-associated guanylate
kinases. J. Neurosci. 1996, 16, 2157-2163.
(8) Kornau, H. C.; Schenker, L. T.; Kennedy, M. B.; Seeburg, P.
H. Domain interaction between NMDA receptor subunits and
the postsynaptic density protein PSD-95. Science 1995, 269, 1737-
1740.
(9) Sattler, R.; Xiong, Z.; Lu, W. Y.; Hafner, M.; MacDonald, J.
F.; Tymianski, M. Specific coupling of NMDA receptor activation
to nitric oxide neurotoxicity by PSD-95 protein. Science 1999,
284, 1845-1848.
(10) Sattler, R.; Charlton, M. P.; Hafner, M.; Tymianski, M.
Distinct influx pathways, not calcium load, determine neuronal
vulnerability to calcium neurotoxicity. J. Neurochem. 1998, 71,
2349-2364.
(11) Tymianski, M. Emerging mechanisms of disrupted cellular
signaling in brain ischemia. Nat. Neurosci. 2011, 14, 1369-1373.
(12) Aarts, M. M.; Tymianski, M. Novel treatment of excitotox-
icity: targeted disruption of intracellular signalling from gluta-
mate receptors. Biochem. Pharmacol. 2003, 66, 877-886.
(13) Aarts, M.; Liu, Y.; Liu, L.; Besshoh, S.; Arundine, M.; Gurd,
J. W.; Wang, Y.-T.; Salter, M. W.; Tymianski, M. Treatment of
ischemic brain damage by perturbing NMDA receptor-PSD-95
protein interactions. Science 2002, 298, 846-850.
(14) Cook, D. J.; Teves, L.; Tymianski, M. Treatment of stroke
with a PSD-95 inhibitor in the gyrencephalic primate brain.
Nature 2012, 483, 213-217.
(15) Sun, H. S.; Doucette, T. A.; Liu, Y.; Fang, Y.; Teves, L.;
Aarts, M.; Ryan, C. L.; Bernard, P. B.; Lau, A.; Forder, J. P.; Salter,
Synthesis of 7‒18 and 22‒24. The fatty acids (C12, C12
diacid monoester, C18 and C18 monoester, 4 eq.) were cou-
pled to the linker-dimer conjugates (4‒6 and 20‒21) using
HBTU (4 eq., 0.5M) and DIEA (8 eq.) in dry DMF for the
shorter-chain acids and dry DCM:DMF (75:25) for the
longer-chain acids. After the coupling was complete, the
FA-linked dimeric ligands were cleaved from the resin
with concomitant side-chain deprotection using
TFA/TiPS/H2O (90:5:5). The methyl protection group of
the mono-protected FAs was then removed by stirring the
cleaved products in 0.5M LiOH in H20/ACN 75/25 (100
mL) for 30 minutes followed by acidification with TFA.
The resulting mixture was lyophilized. Purification of the
crude FA-linked dimeric ligands was conducted by dis-
solving the ligand in the smallest possible volume of 100%
DMSO (<10 mL) before purification by large-scale C18 RP-
HPLC. The semi-pure fractions (50-90% purity) were
subsequently lyophilized, redissolved in a small volume of
DMSO, diluted to 10 mL with ACN/H2O/TFA (70/30/0.1)
and purified by C4 RP-HPLC to give the pure compounds
in >95% purity (Scheme 1). Characterization of 7‒18 and
22‒24 is shown in table S1.
ASSOCIATED CONTENT
Supporting Information. General information on chemis-
try and peptide synthesis, synthetic procedure for C18 diacid
methyl ester, procedures for protein expression, fluorescence
polarization assay, plasma stability assay, and in vivo phar-
macokinetic studies; and Table S1-S2 and Scheme S1. This
material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
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M. W.; Wang, Y. T.; Tasker, R. A.; Tymianski, M. Effectiveness of
(26) Tang, L.; Persky, A. M.; Hochhaus, G.; Meibohm, B.
1
2
PSD95 inhibitors in permanent and transient focal ischemia in
the rat. Stroke 2008, 39, 2544-2553.
Pharmacokinetic aspects of biotechnology products. J. Pharm.
Sci. 2004, 93, 2184-2204.
(16) Bach, A.; Clausen, B. H.; Møller, M.; Vestergaard, B.; Chi,
C. N.; Round, A.; Sørensen, P. L.; Nissen, K. B.; Kastrup, J. S.;
Gajhede, M.; Jemth, P.; Kristensen, A. S.; Lundström, P.; Lam-
bertsen, K. L.; Strømgaard, K. A high-affinity, dimeric inhibitor
of PSD-95 bivalently interacts with PDZ1-2 and protects against
ischemic brain damage. Proc. Natl. Acad. Sci. U. S. A. 2012, 109,
3317-3322.
(17) Florio, S. K.; Loh, C.; Huang, S. M.; Iwamaye, A. E.; Kitto,
K. F.; Fowler, K. W.; Treiberg, J. A.; Hayflick, J. S.; Walker, J. M.;
Fairbanks, C. A.; Lai, Y. Disruption of nNOS-PSD95 protein-
protein interaction inhibits acute thermal hyperalgesia and
chronic mechanical allodynia in rodents. Br. J. Pharmacol. 2009,
158, 494-506.
(18) Tao, F.; Su, Q.; Johns, R. A. Cell-permeable peptide Tat-
PSD-95 PDZ2 inhibits chronic inflammatory pain behaviors in
mice. Mol. Ther. 2008, 16, 1776-1782.
(19) LeBlanc, B. W.; Iwata, M.; Mallon, A. P.; Rupasinghe, C.
N.; Goebel, D. J.; Marshall, J.; Spaller, M. R.; Saab, C. Y. A cyclic
peptide targeted against PSD-95 blocks central sensitization and
attenuates thermal hyperalgesia. Neuroscience 2010, 167, 490-
500.
(20) D'Mello, R.; Marchand, F.; Pezet, S.; McMahon, S. B.;
Dickenson, A. H. Perturbing PSD-95 interactions with NR2B-
subtype receptors attenuates spinal nociceptive plasticity and
neuropathic pain. Mol. Ther. 2011, 19, 1780-1792.
(21) Soriano, F. X.; Martel, M. A.; Papadia, S.; Vaslin, A.; Bax-
ter, P.; Rickman, C.; Forder, J.; Tymianski, M.; Duncan, R.; Aarts,
M.; Clarke, P.; Wyllie, D. J.; Hardingham, G. E. Specific targeting
of pro-death NMDA receptor signals with differing reliance on
the NR2B PDZ ligand. J. Neurosci. 2008, 28, 10696-10710.
(22) Cook, D. J.; Teves, L.; Tymianski, M. A translational para-
digm for the preclinical evaluation of the stroke neuroprotectant
Tat-NR2B9c in gyrencephalic nonhuman primates. Sci. Transl.
Med. 2012, 4, 154ra133.
(23) Hill, M. D.; Martin, R. H.; Mikulis, D.; Wong, J. H.; Silver,
F. L.; Terbrugge, K. G.; Milot, G.; Clark, W. M.; Macdonald, R. L.;
Kelly, M. E.; Boulton, M.; Fleetwood, I.; McDougall, C.; Gunnars-
son, T.; Chow, M.; Lum, C.; Dodd, R.; Poublanc, J.; Krings, T.;
Demchuk, A. M.; Goyal, M.; Anderson, R.; Bishop, J.; Garman, D.;
Tymianski, M.; ENACT trial investigators. Safety and efficacy of
NA-1 in patients with iatrogenic stroke after endovascular aneu-
rysm repair (ENACT): a phase 2, randomised, double-blind,
placebo-controlled trial. Lancet Neurol. 2012, 11, 942-950.
(24) Bach, A.; Chi, C. N.; Pang, G. F.; Olsen, L.; Kristensen, A.
S.; Jemth, P.; Strømgaard, K. Design and synthesis of highly
potent and plasma-stable dimeric inhibitors of the PSD-95-
NMDA receptor interaction. Angew. Chem. Int. Ed. 2009, 48,
9685-9689.
(25) Andreasen, J. T.; Bach, A.; Gynther, M.; Nasser, A.; Mo-
gensen, J.; Strømgaard, K.; Pickering, D. S. UCCB01-125, a dimeric
inhibitor of PSD-95, reduces inflammatory pain without disrupt-
ing cognitive or motor performance: comparison with the
NMDA receptor antagonist MK-801. Neuropharmacology 2013,
67, 193-200.
(27) Kontermann, R. E. Strategies for extended serum half-life
of protein therapeutics. Curr. Opin. Biotechnol. 2011, 22, 868-876.
(28) Jonassen, I.; Havelund, S.; Høeg-Jensen, T.; Steensgaard,
D. B.; Wahlund, P. O.; Ribel, U. Design of the novel protraction
mechanism of insulin degludec, an ultra-long-acting basal insu-
lin. Pharm. Res. 2012, 29, 2104-2114.
(29) Madsen, K.; Knudsen, L. B.; Agersoe, H.; Nielsen, P. F.;
Thøgersen, H.; Wilken, M.; Johansen, N. L. Structure-activity
and protraction relationship of long-acting glucagon-like pep-
tide-1 derivatives: importance of fatty acid length, polarity, and
bulkiness. J. Med. Chem. 2007, 50, 6126–6132.
(30) Havelund, S.; Plum, A.; Ribel, U.; Jonassen, I.; Vølund, A.;
Markussen, J.; Kurtzhals, P. The mechanism of protraction of
insulin detemir, a long-acting, acylated analog of human insulin.
Pharm. Res. 2004, 21, 1498–1504.
(31) Bellmann-Sickert, K.; Elling, C. E.; Madsen, A. N.; Little, P.
B.; Lundgren, K.; Gerlach, L.-O.; Bergmann, R.; Holst, B.;
Schwartz, T. W.; Beck-Sickinger, A. G. Long-acting lipidated
analogue of human pancreatic polypeptide is slowly released
into circulation, J. Med. Chem. 2011, 54, 2658–2667.
(32) Mäde, V.; Bellmann-Sickert, K.; Kaiser, A.; Meiler, J.;
Beck-Sickinger, A. G. Position and length of fatty acids strongly
affect receptor selectivity pattern of human pancreatic polypep-
tide analogues. ChemMedChem, 2014, 9, 2463–2474.
(33) Mäde, V.; Babilon, S.; Jolly, N.; Wanka, L.; Bellmann-
Sickert, K.; Diaz Gimenez, L. E.; Mörl, K.; Cox, H. M.; Gurevich,
V. V.; Beck-Sickinger, A. G. Peptide modifications differentially
alter G protein-coupled receptor internalization and signaling
bias. Angew. Chem. Int. Ed. 2014, 53, 10067-10071.
(34) Toth, I. A novel chemical approach to drug delivery: lipid-
ic amino acid conjugates. J. Drug Targeting 1994, 2, 217-239.
(35) Muders, M. H.; Vohra, P. K.; Dutta, S. K.; Wang, E.; Ikeda,
Y.; Wang, L.; Udugamasooriya, D. G.; Memic, A.; Rupasinghe, C.
N.; Baretton, G. B.; Aust, D. E.; Langer, S.; Datta, K.; Simons, M.;
Spaller, M. R.; Mukhopadhyay, D. Targeting GIPC/synectin in
pancreatic cancer inhibits tumor growth. Clin. Cancer Res. 2009,
15, 4095-4103.
(36) Knudsen, L. B.; Nielsen, P. F.; Huusfeldt, P. O.; Johansen,
N. L.; Madsen, K.; Pedersen, F. Z.; Thøgersen, H.; Wilken, M.;
Agersø, H. Potent derivatives of glucagon-like peptide-1 with
pharmacokinetic properties suitable for once daily administra-
tion. J. Med. Chem. 2000, 43, 1664-1669.
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4
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35
36
37
38
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(37) Hackett, M. J.; Zaro, J. L.; Shen, W. C.; Guley, P. C.; Cho,
M. J. Fatty acids as therapeutic auxiliaries for oral and parenteral
formulations. Adv. Drug Deliv. Rev. 2013, 65, 1331-1339.
(38) Kragh-Hansen, U.; Chuang, V. T.; Otagiri, M. Practical
aspects of the ligand-binding and enzymatic properties of hu-
man serum albumin. Biol. Pharm. Bull. 2002, 25, 695–704.
(39) Sleep, D.; Cameron, J.; Evans, L. R. Albumin as a versatile
platform for drug half-life extension. Biochim. Biophys. Acta
2013, 1830, 5526–5534.
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