Validation of high-affinity binding sites for succinic acid through distinguishable binding of gamma-hydroxybutyric acid receptor-specific NCS 382 antipodes

Article abstract of DOI:10.1016/j.bmcl.2008.08.083

Gamma-hydroxybutyric acid (GHB) binding to multiple sites for the tricarboxylic acid cycle intermediate succinic acid (SUC) has been disclosed recently. In order to better characterize these targets, distinguishable binding of GHB receptor-specific NCS 38

Full text of DOI:10.1016/j.bmcl.2008.08.083

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6290–6292
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Validation of high-affinity binding sites for succinic acid through
distinguishable binding of gamma-hydroxybutyric acid
receptor-specific NCS 382 antipodes
b
a
c
c
c
Tünde Molnár a, , Júlia Visy , Ágnes Simon , István Moldvai , Eszter Temesvári-Major , Gábor Dörnyei ,
*
Erzsébet Kútiné Fekete a, Julianna Kardos a
a Department of Neurochemistry, Chemical Research Center, Hungarian Academy of Sciences, Budapest, Hungary
b Department of Molecular Pharmacology, Chemical Research Center, Hungarian Academy of Sciences, Budapest, Hungary
c Department of Natural Organic Compounds, Chemical Research Center, Hungarian Academy of Sciences, Budapest, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
Gamma-hydroxybutyric acid (GHB) binding to multiple sites for the tricarboxylic acid cycle intermediate
succinic acid (SUC) has been disclosed recently. In order to better characterize these targets, distinguish-
able binding of GHB receptor-specific NCS 382 antipodes to [3H]-SUC or [3H]-GHB labelled sites in rat
brain synaptic membranes was explored. Eutomer binding parameters suggest identity of the high-affin-
ity target for SUC with a synaptic GHB receptor subtype.
Received 18 July 2008
Accepted 23 August 2008
Available online 28 August 2008
Keywords:
Succinic acid
Ó 2008 Elsevier Ltd. All rights reserved.
Gamma-hydroxybutyric acid (GHB)
NCS 382
Succinic acid (SUC), the intermediary metabolite of the tricar-
boxylic acid (TCA) cycle has a central role in the control of cellular
energy supply.1 In brain cells, oxidative metabolism of the major
inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is
directly coupled to the TCA cycle through SUC formed from gam-
ma-hydroxybutyric acid (GHB).2–4 Binding interaction between
SUC and [3H]-GHB-labelled sites has been disclosed in synaptic
membranes isolated from rat forebrain and human basal ganglia
areas.5 Moreover, GHB displaced [3H]-SUC specifically bound to
high- and low-affinity synaptic binding sites.6 These findings
indicate that in addition to cellular energy supply, SUC may also
control synaptic activity.6 The remarkable similarities of SUC-sen-
sitive [3H]-GHB and GHB-sensitive [3H]-SUC ligand binding
profiles5,6 suggest that SUC and GHB can compete for the same
synaptic recognition sites. Identity of sites, however, is called into
question by the preferred binding of the GHB receptor-specific li-
gand 6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene-
acetic acid ((rac)-NCS 382) to the high-affinity SUC binding site.6
The aim of the present study was to qualify putative interaction
of SUC with synaptic GHB receptor sites by comparing enantiose-
lectivity profiles of [3H]-SUC or [3H]-GHB binding in rat forebrain
synaptic membranes. To this end, new and known chiral
compounds have been assayed for [3H]-SUC or [3H]-GHB binding.
Design and synthesis of new chiral sets of 4-hydroxypiperidine-
2-carboxylic acid derivatives (Fig. 1A) were based on the close
structural similarity of (2R,4R)-4-hydroxypiperidine-2-carboxylic
acid to the high-affinity GHB analogue (R)-3-hydroxycyclohex-1-
ene-carboxylic acid ((R)-HOCHCA7; Fig. 1B). The (R) and (S) antip-
odes of (rac)-NCS 382 were prepared by a novel chiral HPLC sepa-
ration method.
(R)-HOCHCA has been reported to have much higher binding
affinity to GHB binding sites present in rat cortex preparations
than GHB7, indicating that with its OH and the COOH groups locked
in a ring, its structure closely mimics the bioactive conformation of
the endogenous ligand. In addition to the matching structural pre-
requisites, the novel 4-hydroxypiperidine-2-carboxylic acid deriv-
atives 1–8 (Fig. 1A; c.f. Supplementary Scheme 1) contain two
chiral centres providing chiral sets of possible GHB analogues.
The derivatives have been synthesised by a combined procedure
of Bio-Méga and Ciba groups.8,9 Details of synthesis and purifica-
tion steps are given as Supplementary information. To explore pos-
sible enantioselectivity profiles of GHB and SUC sites present in
synaptic membranes5,6, binding of chiral 4-hydroxypiperidine-2-
carboxylic acid derivatives 1–8 to sites labelled by [3H]-GHB
(49.5 Ci/mM5) or [3H]-SUC (40 Ci/mM; Sigma-Aldrich, Budapest,
Hungary) have been performed.5,6 Surprisingly, among the (4S) iso-
mers, (2R,4S)-4-hydroxypiperidine-2-carboxylic acid (6) indicated
some low binding affinity to SUC and GHB binding sites (26 4%
and 30 14% inhibition at 100 lM, respectively). Other derivatives,
structurally related to (4R) isomers, were all devoid of significant
* Corresponding author. Tel.: +36 1 438 1167; fax: +36 1 438 1143.
displacing activity (p < 0.05) (Supplementary, Table 1). Even
E-mail address: tmolnar@chemres.hu (T. Molnár).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.083

T. Molnár et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6290–6292
6291
Figure 1. Structural analysis of 4-hydroxypiperidine-2-carboxylic acid derivatives.
(A) Chemical structures of 4-hydroxypiperidine-2-carboxylic acid derivatives 1–8.
(B) (2R,4R)-4-hydroxypiperidine-2-carboxylic acid (grey carbon atoms) fitted on
(R)-HOCHCA (cyan carbon atoms) using Surflex-Sim, in which a score value greater
than 0.70 is generally significant in terms of similarity and indicates a functional
relationship of the molecules. (C) Relative orientation of (2R,4R)-4-hydroxypiper-
idine-2-carboxylic acid (grey carbon atoms) and (R)-NCS 382 (cyan carbon atoms),
after simultaneously fitting their COOH and OH groups using PyMOL 0.99 (DeLano
Scientific LLC, http://www.pymol.org). Carbon atoms of the GHB moiety of (R)-NCS
382 are highlighted in light green. Oxygen and nitrogen atoms are coloured
according to atom type.
though, the structure of (2R,4R)-4-hydroxypiperidine-2-carboxylic
acid (8) is strikingly similar to (R)-HOCHCA, as indicated by a high
similarity score (0.92, Surflex-Sim program10; Fig. 1B), these scaf-
folds did not prove to be efficient GHB analogues. The lack of activ-
ity may be attributed to the fact, that GHB adopts a different
conformation in (2R,4S)-4-hydroxypiperidine-2-carboxylic acid
derivatives and in (R)-NCS 382, illustrated by fitting their func-
tional OH and COOH groups simultaneously (Fig. 1C).
Figure 2. Displacement of specifically bound [3H]-SUC (A) and [3H]-GHB (B) by
NCS-382 antipodes in rat forebrain synaptic membrane fractions. Symbols: (R)-NCS
382 (green, ); (S)-NCS 382 (blue, ); ‘‘mixed” (rac)-NCS 382 (red, ). To define
non-specific binding 30 mM succinate or 1 mM GHB was used. Data are presented
as means SD obtained from experiments with 3 different rat forebrain prepara-
tions (N = 3) performed in duplicate measurements. Displacement curves were
fitted by one-site competition approximation (OriginPro 7.5).
In order to compare enantioselectivity profiles of SUC and GHB
recognition sites, binding of HPLC separated enantiomers of (rac)-
NCS 382 (Tocris, Bristol, UK) to synaptic sites labelled by [3H]-
SUC or [3H]-GHB have been performed as described above. The chi-
ral HPLC separation was performed with a system composed of a
ration values for the ‘‘mixed” (rac)-NCS 382 and (R)-NCS 382 were
similar (IC50,RS = 0.5 0.1 lM vs IC50,R = 0.4 0.1 lM; Fig. 2A).
These findings are consistent with our previous results5 obtained
with unresolved (rac)-NCS 382, and suggest that identity of the
high-affinity SUC binding sites with the (R)-NCS 382-selective rec-
ognition site of the synaptic GHB receptor. Furthermore, we may
hypothesize the existence of an NCS 382-insensitive low-affinity
binding site for SUC. The existence of the presumed (R)-NCS 382-
insensitive low-affinity SUC binding site has been challenged in
measurements of [3H]-GHB binding in rat forebrain synaptic mem-
branes. Similarly to [3H]-SUC binding, none of the (R)-NCS 382 or
(S)-NCS 382 antipodes could totally displace [3H]-GHB from its
Jasco PU-980 pump, a Rheodyne 7125 injector (20 ll loop), a Jasco
MD 2010 Plus UV/VIS photodiode-array detector and a ChromPass
chromatographic software. Successful separation of NCS 382 enan-
tiomers was performed on a Chiracel OJ column (250 Â 4.6 mm I.D.
Daicel Chemical Ind. Tokyo, Japan). The mobile phase was 85% n-
hexane, 15% 2-propanol and 0.1% trifluoroacetic acid, flow rate
was 1 mL/min. The two fractions were baseline separated having
retention times of 10.1 and 12.7 min. The products of twenty runs
were combined; enantiomeric purities (% ee P 97.5) were checked
by the same HPLC method.
specific synaptic binding sites (IC50,R = 0.8 0.3
lM
and
IC50,S = 93 18 lM; Fig. 2B). The (R)-NCS 382 antipode showed ap-
prox. 115-fold higher binding affinity to [3H]-GHB labelled sites
than the (S)-NCS 382 antipode (Fig. 2B). The higher binding selec-
tivity obtained with the (R) and (S) enantiomers (this study) com-
(R)- and the (S)-NCS 382 partially displaced [3H]-SUC from its
synaptic binding sites with IC50 values of 0.4 0.1
lM and
74 M, respectively (Fig. 2A) and (R)-NCS 382 showed approx.
9
l
pared to previously reported data11 (c.f. IC50,R = 0.2 0.01
l
M vs
200-fold higher binding affinity to [3H]-SUC labelled synaptic sites
than (S)-NCS 382. Neither the (R)-NCS 382 nor the (S)-NCS 382
antipodes displaced specifically bound [3H]-SUC completely
(Fig. 2A). As a proof of the evidence, ‘‘mixed” (rac)-NCS 382 ob-
tained from the resolved (R)- and (S)-NCS 382 antipodes was as-
sayed for [3H]-SUC binding as well. Maximal displacements
exerted by the ‘‘mixed” (rac)-NCS 382 and the (R)-NCS 382 anti-
pode displayed saturation approximately at the same level
(17 1% and 26 1%, respectively; Fig. 2A). In addition, half-satu-
IC50,S = 12.9 2.3 lM) may be due to the higher homogeneity of
purified synaptic membrane preparation that had been used. In
contrast to the [3H]-SUC binding, however, when we used ‘‘mixed”
(rac)-NCS 382, we got total displacement of specifically bound
[3H]-GHB with an IC50 value (0.9 0.2
previously reported (rac)-NCS 382 data (c.f. Ki = 1.2 0.2
l
M; Fig. 2B) similar to our
l
M4).
It follows, that there may be two synaptic GHB receptor sub-
types, distinguishable by NCS 382 antipodes of high enantiomeric
purity, showing opposite enantioselectivity. Similar enantioselec-

6292
T. Molnár et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6290–6292
tivity profiles, disclosing the (R)-NCS 382 antipode as the high-
affinity eutomer indicate the identity of the high-affinity SUC bind-
ing site with the GHB receptor recognition site. In conclusion, these
results together with our previous findings5,6 suggest that the tar-
get of the high-affinity SUC binding may be a synaptic GHB recep-
tor subtype.
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Acknowledgments
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Edit Simon-Trompler (Group for Radiosyntheses, Institute of
Biomolecular Chemistry, Chemical Research Center) for the synthe-
sis of the radioligand [3H]-GHB and the financial support of grants
MediChem2 (1/A/005/2004 NKFP), OTKA (T049721) and Trans-
porter Explorer (GVOP 3.1.1-2004-05-00) are acknowledged.
Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.08.083.