The neuroprotective activity of GPE tripeptide analogues does not correlate with glutamate receptor binding affinity

Article abstract of DOI:10.1016/j.bmcl.2006.04.033

The influence of several modifications on the GPE tripeptide structure upon the binding to GluRs and on their neuroprotective effects has been studied. The results indicated that the prevention of neuronal death showed by GPE and some analogues is not dir

Full text of DOI:10.1016/j.bmcl.2006.04.033

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3396–3400
The neuroprotective activity of GPE tripeptide analogues
does not correlate with glutamate receptor binding affinity
a
a
´
˜
´
Sergio A. Alonso De Diego, Marta Gutierrez-Rodrıguez,
a
a
a
´
´
´
M. Jesus Perez de Vega, Rosario Gonzalez-Muniz, Rosario Herranz,
Mercedes Martın-Martınez, Edurne Cenarruzabeitia, Diana Frechilla,
a
d
c,d
*
´
´
c,d
b
a,
´
´
´
´
Joaquın Del Rıo, M. Luisa Jimeno and M. Teresa Garcıa-Lopez
aInstituto de Quı´mica Me´dica Juan de la Cierva, 3, 28006 Madrid, Spain
bCentro de Quı´mica Orga´nica Manuel-Lora Tamayo (CSIC), Juan de la Cierva, 3, 28006 Madrid, Spain
cDivisio´n de Neurociencias, CIMA, Universidad de Navarra, Irunlarrea, 1, 31080 Pamplona, Spain
dDepartamento de Farmacologı´a-Unidad Asociada al CSIC, Universidad de Navarra, Irunlarrea, 1, 31080 Pamplona, Spain
Received 27 February 2006; revised 5 April 2006; accepted 5 April 2006
Available online 2 May 2006
Abstract—The influence of several modifications on the GPE tripeptide structure upon the binding to GluRs and on their neuro-
protective effects has been studied. The results indicated that the prevention of neuronal death showed by GPE and some analogues
is not directly related to their affinity at glutamate receptors.
Ó 2006 Elsevier Ltd. All rights reserved.
The tripeptide Gly-Pro-Glu (GPE, 1) is endogenously
formed from the acid protease-mediated metabolism of
IGF-1 (insulin-like growth factor type 1).1–3 Unlike
IGF-1 and des(1–3)IGF-1, GPE neither binds to IGF-1
receptors nor has any neurotrophic effect, but there is
evidence that it displays remarkable neuromodulatory
activities in the CNS. Thus, GPE stimulates acetylcho-
line and dopamine release in the rat cortex and stria-
tum,4,5 and protects neurons of different brain regions
from diverse induced injuries (hypoxia-ischemia, gluta-
mate, quinolinic acid, etc.).5–8 Furthermore, GPE shows
neuroprotective effects in animal models of neurodegen-
erative processes, such as Huntington’s, Parkinson’s, and
Alzheimer’s diseases.5,9,10 Although the mode of action
of this tripeptide in neuroprotection remains unclear, ini-
tial structural considerations suggested that GPE could
interact with one or more glutamate receptors (GluRs)
and, in fact, it was demonstrated that it binds to the N-
methyl-D-aspartate (NMDA) receptors, but not to the
(2S)-2-amino-3-hydroxy-5-methyl-4-isoxazole propionic
acid (AMPA) or kainate (KA) receptors.1 In this sense,
both agonistic and antagonistic effects of GPE on
NMDA receptors have been reported.1,11 It has also
been shown that GPE stimulates the potassium-evoked
release of acetylcholine and dopamine through interac-
tion with NMDA receptors, along with other unknown
non-NMDA receptor-mediated pathways.1,6,12 Studies
on metabolic stability have demonstrated that GPE has
very short half-life in plasma, which hampers its pre-clin-
ical and clinical trials.13,14
The structural simplicity of GPE converts this tripeptide
into a promising starting point for the search of non-
peptide mimics with improved bioavailability. In the
process to achieve this purpose, through the rational de-
sign of peptidomimetics, the study of the bioactive con-
formation is a key step. In a previous communication,15
we have addressed this issue by exploring the influence
of both the amino acid side-chain orientation and the
cis/trans isomerism at the Gly-Pro peptide bond on the
binding of GPE to glutamate receptors. The significance
of the amino acid side-chain orientation was investigat-
ed by the sequential replacement of both the L-Pro and
L-Glu residues by their enantiomeric forms D-Pro and
D-Glu in 2–4, while the importance of the cis/trans isom-
erism was studied by the replacement of the Pro residue
by pseudoprolines16,17 [(R)-thiazolidine-4-carboxylic
Keywords: Neuroprotection; Glutamate receptors binding; Tripeptides;
GPE; Peptidomimetics.
*
Corresponding author. Tel.: +34 915622900; fax: +34 915644853;
e-mail: iqmgl37@iqm.csic.es
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.04.033

S. A. Alonso De Diego et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3396–3400
3397
acid (Cys[WH,Hpro]) and (R)-2,2-dimethylthiazolidine-4-
H
a
b
carboxylic acid (Cys[WMe,Mepro])18] in analogues 5 and
N
CO Bn
(R)
2
CO2H
CO2H
N
H
N
N
6, and by alkylprolines [(S)-2-methylproline (PMe
)
O
19
Boc
Boc
and (S)-5,5-dimethylproline (dmP)20] in analogues 7
and 8, respectively. The binding affinity of analogues
2–8 for GluRs was evaluated by the measure of the dis-
placement of L-[3H]Glu from rat brain synaptic mem-
branes. In this evaluation, only the GPE analogues 7
and 8, which incorporate alkylprolines inducing prefer-
entially the trans and cis rotamer, respectively, at the
Gly-Pro bond, showed noticeable affinities, which were
higher than that displayed by GPE, and comparable to
that of D-AP5, one of the most potent and selective com-
petitive NMDA antagonists.21 Taking into account
these results, GPE analogues 7 and 8 were also evaluat-
ed as neuroprotective agents in cultured rat hippocam-
pal neurons. These compounds reduced the neuronal
death induced by NMDA administration with a similar
potency to that of GPE, and, in particular, the PMe
derivative 7 increased significantly the neuronal survival
after oxygen–glucose deprivation (OGD) injure.
12
13 (78%)
14 (67%)
c,d
CO2Bn
H
H
(S)
(R)
+
N
CO Bn
N
CO Bn
(S)
2
(S)
2
N
N
O
O
Z-Gly
Z-Gly
15 (42%)
16 (42%)
CO2Bn
CO2Bn
e
e
H
H
(R)
(S)
N
CO H
N
CO H
(S)
(S)
2
2
N
N
O
O
H-Gly
H-Gly
9 (98%)
8 (98%)
CO2H
CO2H
Scheme 1. Reagents: (a) Boc2O, Et3N, DMAP, H2O/dioxane; (b) H-L-
Glu(OBn)-OBn, BOP, DIEA, CH2Cl2; (c) 3.2 M HCl/AcOEt; (d)
Z-Gly-OH, HOAt, HATU, DIEA, CH2Cl2; (e) H2, Pd(C), MeOH.
chromatographically resolved. Finally, the hydrogenoly-
sis-mediated N- and C-deprotection of separated 15 and
16 provided the GPE analogues 8 and 9, respectively.
Our previously reported synthesis of 815 involved the
resolution of racemic dmP-OH (12), by crystallization
of the L-enantiomer with D-tartaric acid, according to
the reported procedure.20 The NMR analysis of tripep-
tides 8 and 9 in (90:10) H2O/D2O solution showed cis/
trans rotamer ratios at the Gly-dmP peptide bond of
(69:31) and (82:18), respectively. The cis form of the ma-
jor preferred conformation was assigned based on the
NOE effects observed between the Gly a-H and dmP
In a recent communication on other series of GPE ana-
logues, we have shown that their neuroprotective activ-
ity does not seem to be directly related to the binding
affinity at GluRs.22 On account of these results, now
we have determined and reported herein the neuropro-
tective activity of GPE analogues 2–6. In addition, to
further assess the significance of the configuration of
the dmP residue of 8, and that of the volume of the
Me substituent in the PMe residue of 7, their analogues
9 and 10, incorporating D-dmP and 2-propyl-L-proline
(PPr), respectively, have been prepared and evaluated.
Moreover, taking into account the high propensity of
the Pro residue to induce folded conformations,23 and
specially reverse turns,24 we have also prepared and
evaluated the analogue 11, where the Pro-Glu peptide
bond has been restricted into a [4.4]-spirolactam b-turn
mimetic25 (Fig. 1).
1
a-H protons in the respective H NMR 1D NOESY
spectra.
The PPr-containing GPE analogue 10 was synthesized
from N-Boc-2-allyl-L-Pro-OH (17), which was previous-
ly prepared by applying the Khalil et al.27 procedure of
N-Boc-protection of sterically hindered amino acid to 2-
allyl-L-Pro-OH.28 As shown in Scheme 2, the coupling
of 17 with H-L-Glu(OBn)-OBn, using DCC and HOAt
as activating reagents, led to the corresponding dipep-
tide 18. N-Boc removal in this dipeptide, followed by
coupling with Z-Gly-OH, using HATU and HOAt as
activating reagents, gave the tripeptide 19, whose
hydrogenolysis-mediated N- and C-deprotection provid-
ed the proposed GPE analogue 10 in a 55% overall yield.
The NMR analysis of this tripeptide in D2O solution
showed the presence of only one preferred conforma-
tion. The NOE effect observed in the 1H NMR spectrum
of 10 between the Gly a-H and the ProPr 5-H protons
indicated the presence of a trans conformation at the
Gly-ProPr peptide bond. It is interesting to note that,
while this work was in progress, Harris et al.29 published
an alternative synthesis of 10 in a 32% overall yield from
2-allyl-L-Pro-OMe, although they did not report any
data of its biological evaluation.
The D-dmP-containing tripeptide 9 was synthesized
from racemic dmP-OH (12),26 as shown in Scheme 1.
After N-Boc protection, the coupling of N-Boc-dmP-
OH (13) with H-L-Glu(OBn)-OBn, using BOP as acti-
vating reagent, led to the dipeptide diastereoisomeric
mixture 14. Then, this mixture was N-deprotected and
coupled with Z-Gly-OH, using HATU and HOAt as
activating reagents, to obtain the corresponding (1:1)
mixture of protected tripeptides 15 and 16, which were
H-Gly-Pro-Glu-OH (GPE)
H-Gly-Pro-D-Glu-OH
H-Gly-Cys[ψH,HPro]-Glu-OH
H-Gly-PMe-Glu-OH
1
3
5
7
9
H-Gly-D-Pro-Glu-OH
H-Gly-D-Pro-D-Glu-OH
H-Gly- Cys[ψMe,MePro]-Glu-OH
H-Gly-dmP-Glu-OH
2
4
6
8
H-Gly-D-dmP-Glu-OH
H-Gly-PPr-Glu-OH
10
N
CO2H
N
The spirolactam GPE analogue 11 was prepared by
applying our previously developed synthesis of [4.4]-
spirolactam b-turn mimetics from N-Boc-2-allyl-L-
Pro-OH.30 As shown in Scheme 2, this methodology
O
H-Gly
11
CO2H
Figure 1. GPE analogues studied.

3398
S. A. Alonso De Diego et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3396–3400
determined, and the results were compared with those
previously reported for compounds 1–8.15 No displace-
ment was found using concentrations up to 100 lM of
compounds 10 and 11. However, the D-dmP-containing
GPE analogue 9 displayed one and two orders of mag-
nitude higher affinity for glutamate receptors than its
epimer at the dmP residue 8 and GPE (1), respectively
(see Table 1). This significant increase in the binding
affinity was unexpected, taking into account the detri-
mental effect on the binding affinity at GluRs observed
when the Pro residue of GPE was replaced by D-Pro
in 2.15
H
N
CO2H
CO2H
CO2Bn
CO2Bn
N
CO2H
N
O
H-Gly
Boc
17
10 (100%)
a
d
H
H
N
b,c
N
CO2Bn
CO2Bn
N
Z-Gly
N
O
O
Boc
19 (56%)
18 (98%)
e
OH
The neuroprotective effects of compounds 2–6 and 9–
11, regardless of their binding affinity at GluRs, were
evaluated in cultured hippocampal neurons exposed to
NMDA (100 lM) or oxygen–glucose deprivation
(OGD), following described procedures.31,32 The re-
sults are shown in Figure 2 compared with those of
GPE and its analogues 7 and 8 previously studied.
Surprisingly, the stereochemistry requirements were
contrary to those for the GluRs binding affinity. Thus,
the GPE diastereoisomers 2 and 3, which did not bind
at all at the higher assayed concentration (100 lM),
displayed the best neuroprotection percentage, higher
than GPE, in both NMDA excitotoxicity (Fig. 2A)
and OGD (Fig. 2B) assays, while, the D-dmP-contain-
ing GPE analogue 9, which, as above-mentioned,
showed the higher GluRs binding potency, did not
significantly prevent the neuronal death in both types
of assays. Similarly, the influence of the ring pucker-
ing and hydrophobicity of the Pro residue on the
GluRs binding affinity and on the neuroprotective
activity was different. Thus, the pseudoproline-con-
taining compounds 5 and 6, which did not displace
L-[3H]glutamate, displayed neuroprotection percentag-
es slightly lower than GPE in the NMDA excitotoxic-
ity assay, but their effect was higher in the oxygen–
glucose deprivation model. As previously suggested,15
the Gly-Pro peptide bond conformation does not seem
to have a significant influence on the neuroprotective
activity. However, the conformational restriction at
the Pro-Glu peptide bond in compounds 10 and 11
was detrimental both for the binding at GluRs and
for the neuroprotection effects.
f
N
O
CO2Bn
N
CO2Bn
N
N
H
O
Boc
TFA
20 (72%)
21
g
CO2Bn
CO2Bn
CO2Bn
CO2Bn
N
b,c
N
N
N
O
O
Z-Gly
Boc
22(45%)
23 (42%)
CO2Bn
CO2Bn
d
N
CO2H
CO2H
N
O
H-Gly
11 (100%)
Scheme 2. Reagents: (a) H-L-Glu(OBn)-OBn, HOAt, DCC, NMM,
CH2Cl2; (b) 3.2 M HCl/AcOEt; (c) Z-Gly-OH, HOAt, HATU, DIEA,
CH2Cl2; (d) H2, Pd(C), MeOH; (e) OsO4, NaIO4, MeOH/H2O; (f)
NaBH4, TFA; (g) Boc2O, TEA, DMAP, CH2Cl2.
involved the oxidation of the dipeptide 18, by treat-
ment with OsO4 and NaIO4 in 2:1 MeOH/H2O mix-
ture for 24 h, to give the hydroxylactam 20, whose
one-step reduction and N-deprotection by reaction
with NaBH4 in neat TFA led to the trifluoroacetate
21, along with traces of decomposition products. To
facilitate the purification of the [4.4]-spirolactam
derivative 21, the Boc-protecting group was reintro-
duced in 22, by the treatment of 21 with Boc2O in
the presence of TEA and a catalytic amount of
4-(dimethylamino)pyridine (DMAP). N-Boc removal
in pure 22, followed by coupling with Z-Gly-OH
and hydrogenolysis-mediated final deprotection as
above-mentioned, provided the conformationally con-
strained tripeptide analogue 11 in a 14% overall yield.
Harris et al.29 have also published an alternative syn-
thesis of 11 in a 10% overall yield from 2-allyl-L-Pro-
OMe, but they neither gave any data of its biological
evaluation. Similarly to the case of the PPr-derived tri-
peptide 10, the NMR analysis of the spirolactam
derivative 11 in D2O showed the presence of only
one preferred conformation, which was assigned as
trans on the basis of the NOE effects observed be-
tween the Gly a-H and the spirolactam 2-H protons.
In conclusion, the results herein reported indicate that
the tridimensional structural requirements of GPE for
binding to GluRs are different to those required for neu-
roprotective activity. Therefore, it seems to confirm that
the prevention of neuronal death showed by GPE and
some analogues is not directly related to their affinity
at glutamate receptors.
Table 1. Displacement of L-[3H]glutamate from rat brain synaptic
membranes
Compound
Ki (lM)
GPE (H-Gly-Pro-Glu-OH) (1)
H-Gly-PMe-Glu-OH (7)
31.24 15.65
7.96 1.83
3.79 0.53
0.33 0.07
Displacement of L-[3H]glutamate from rat brain synap-
tic membranes by the new GPE analogues 9–11 was
H-Gly-dmP-Glu-OH (8)
H-Gly-D-dmP-Glu-OH (9)

S. A. Alonso De Diego et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3396–3400
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This work has been supported by the Spanish Ministry
of Education and Science (SAF 2003-07207-C02) and
Comunidad de Madrid (GR/SAL/0850/2004), and
UTE project FIMA. S.A.A.D.D. holds a predoctoral
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´
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