Epilepsy Research 138 (2017) 124–131

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Epilepsy Research
journal homepage: www.elsevier.com/locate/epilepsyres

Phenylalanine derivatives with modulating effects on human α1-glycine T

receptors and anticonvulsant activity in strychnine-induced seizure model in
male adult rats
⁎
Bassem Sadeka, , Murat Oza,b, Syed M. Nurulaina,c, Petrilla Jayaprakasha, Gniewomir Lataczd,
Katarzyna Kieć-Kononowiczd, Ewa Szymańskad
a
Department of Pharmacology and Therapeutics, College of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, P.O. Box 17666, United Arab
Emirates
b
Department of Basic Medical Sciences, College of Medicine, Qatar University, Doha, Qatar
c
Department of Bioscience, COMSATS Institute of Information Technology, Islamabad 45550, Pakistan
d
Department of Technology and Biotechnology of Drugs Jagiellonian University Medical College, Medyczna 9, PL 30-688 Krakow, Poland

A R T I C L E I N F O A B S T R A C T

Keywords: The critical role of α1-glycine receptor (α1-GLYRs) in pathological conditions such as epilepsy is well known. In
Human α1-glucine receptor the present study, structure-activity relations for a series of phenylalanine derivatives carrying selected hydrogen
Phenyl alanine derivatives bond acceptors were investigated on the functional properties of human α1-GLYR expressed in Xenopus oocytes.
Inhibitor The results indicate that one particular substitution position appeared to be of special importance for control of
Potentiator
ligand activity. Among tested ligands (1-8), the biphenyl derivative (2) provided the most promising
Anticonvulsant
Strychnine
antagonistic effect on α1-GLYRs, while its phenylbenzyl analogue (5) exhibited the highest potentiation effect.
Moreover, ligand 5 with most promising potentiating effect showed in-vivo moderate protection when tested in
strychnine (STR)-induced seizure model in male adult rats, whereas ligand 2 with highest antagonistic effect
failed to provide appreciable anti(pro)convulsant effect. Furthermore, ligands 2 and 5 with the most promising
effects on human α1-GLYRs were examined for their toxicity and potential neuroprotective effect against
neurotoxin 6-hydroxydopamine (6-OHDA). The results show that ligands 2 and 5 possessed neither significant
antiproliferative effects, nor necrotic and mitochondrial toxicity (up to concentration of 50 μM). Moreover,
ligand 2 showed weak neuroprotective effect at the 50 μM against 100 μM toxic dose of 6-OHDA. Our results
indicate that modulatory effects of ligands 2 and 5 on human α1-GLYRs as well as on STR-induced convulsion
can provide further insights for the design of therapeutic agents in treatment of epilepsy and other pathological
conditions requiring enhanced activity of inhibitory glycine receptors.

1. Introduction
In central, as well as peripheral, nervous system, GLY functions as a
neurotransmitter at inhibitory synapses, where it activates strychnine-
sensitive glycine receptors (GLYRs) which are composed of five
subunits (α1-4 and β) (Lynch, 2004) and belong to pentameric Cys-
loop ligand-gated ion channel superfamily (Cys-loop LGICs)(Castro
et al., 2012; Lester et al., 2004).
It has been well established that GLYRs play important roles in
various pathological conditions such as epilepsy, hyperexcitability,
motor-control, and pain sensation (Callister and Graham, 2010; Camp
et al., 2010). In recent studies, the critical role of GLYRs in modulating
temporal lobe epilepsy has been proposed based on clinical findings
that patients suffering from temporal lobe epilepsy disease would
possibly be advantaged most from facilitated glycinergic inhibition,
e.g. through GLYR modulators. Accordingly, mounting evidences have
accentuated the contribution of GLYRs to tonic hippocampal inhibition
in response to glycine, and glycine and taurine as well as glycine uptake
inhibitors were found to be of anticonvulsive role in epilepsy models
(Bedet et al., 2006; Eichler et al., 2008; Meier and Grantyn, 2004; Song

Abbreviations: ANOVA, analysis of variance; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N9,N9-tetraacetic acid; DA, dopamine; DMSO, dimethylsulfoxide; MBS, Barth’s solution;
nAChR, nicotinic acetylcholine receptor; α1-GLYRs, α1-glycine receptor; ROS, radical oxygen species; 6-OHDA, 6-hydroxydopamine; ATP, adenosine triphosphate; HEK-293, human
embryonic kidney cell line; DMEM, Dulbecco’s Modified Eagle’s Medium; STR, strychnine; VPA, valproic acid; AED, antiepileptic drug; AMPA, (RS)-2-amino-3-(3-hydroxy-5-methyl-4-
isoxazolyl)propionic acid; KA, kainite; NMDA, N-methyl-D-aspartic acid; GluK1, homomeric kainate receptor
⁎
Corresponding author.
E-mail address: bassem.sadek@uaeu.ac.ae (B. Sadek).

http://dx.doi.org/10.1016/j.eplepsyres.2017.05.008
Received 22 February 2017; Received in revised form 1 May 2017; Accepted 19 May 2017
Available online 26 May 2017
0920-1211/ © 2017 Elsevier B.V. All rights reserved.
B. Sadek et al.
et al., 2006; Zhang et al., 2008). Also, STR-sensitive glycine receptors
were previously found to depress hyperexcitability in rat dentate gyrus
(Eichler et al., 2008; Kirchner et al., 2003; Mori et al., 2002). Based on
the aforementioned role of GLYRs in central and peripheral nervous
system pathologies, and as a continuation of our research (Szymanska
et al., 2011, 2009), in the present study effects of a series of unnatural
racemic phenylalanine derivatives (1–8) on the functional properties of
the cloned human α1-GLYR expressed in Xenopus oocytes were
investigated. Moreover, selected compounds have been examined for
their anti(pro)convulsant activity in STR-induced seizure model in
adult rats.
In addition, the most interesting phenylalanine derivatives were
tested for their toxicity using antiproliferative EZ4U test as well as for
their potential neuroprotective properties in cell-death in vitro model
using neurotoxin 6-OHDA. Cytotoxicity of 6-OHDA is believed to result
from radical oxygen species (ROS) derived by 6-OHDA intraneuronal
autooxidation as well as a possible direct effect of 6-OHDA on the
mitochondrial respiratory chain (Rodriguez-Pallares et al., 2007), and
recent studies demonstrated the direct association of mitochondrial
dysfunction and oxidative stress with epileptogenesis and acquired
chronic epilepsy (Blum-Degen et al., 1998; Waldbaum and Patel, 2010).
In this context, the new multi-target acting anti-epileptic compounds
which would act not only via epilepsy-relevant receptors but also
possessing neuroprotective activity preventing mitochondria from
ROS may contribute towards development of the therapy of epilepsy.
2. Materials and methods
2.1. Human α1-glycine receptor recordings from oocytes
Mature female Xenopus laevis frogs were purchased from Xenopus
Express (Haute-Loire, France), housed in dechlorinated tap water at
19–21 °C with a 12/12-h light/dark cycle, and fed food pellets supplied
by Xenopus Express. The procedures followed in this study were in
accordance with the Guide for the Care and Use of Laboratory Animals
(8th edition) of the National Institutes of Health (Bethesda, MD) and
approved by the Institutional Animal Care and Use Committee at the
UAEU.
As described previously by Sadek et al., 2014 (Ashoor et al.,
2013a,b; Mahgoub et al., 2013; Sadek et al., 2012, 2014a, 2015a),
clusters of oocytes were removed surgically under benzocaine (Sigma,
St. Louis, MO) local anesthesia (0.15% w/V), and individual oocytes
were dissected manually in a solution containing (in mM): NaCl, 88;
KCl, 1; NaHCO3, 2.4; MgSO4, 0.8; HEPES, 10 (pH 7.5). Dissected
oocytes were then stored 2–7 days in modified Barth’s solution (MBS)
containing (in mM): NaCl, 88; KCl, 1; NaHCO3, 2.4; CaCl2, 2; MgSO4,
0.8; HEPES, 10 (pH 7.5), supplemented with sodium pyruvate, 2 mM,
penicillin, 10,000 IU/L, streptomycin, 10 mg/L, gentamicin, 50 mg/L,
and theophylline, 0.5 mM. Briefly, oocytes were placed in a 0.2 ml
recording chamber and superfused at a rate of 2–3 ml/min. The bathing
solution consisted of (in mM): NaCl, 95; KCl, 2; CaCl2, 2; and HEPES 5
(pH 7.5). The cells were impaled with two glass microelectrodes filled
with a 3 M KCl. The oocytes were routinely voltage clamped at a
holding potential of −70 mV using a GeneClamp-500 amplifier (Axon
Instruments Inc., Burlingame, CA). Glycine-induced currents were
evoked every 5 min by 3–4 s application of 30 μM glycine. During
10 min application time, strychnine or series of phenylalanine deriva-
tives were applied externally by addition to the superfusate. These
compounds (strychnine and series of phenylalanine derivatives) were
also included in glycine containing agonist solution.All chemicals used
in preparing the solutions were from Sigma-Aldrich (St. Louis, MO,
USA) whereas test compounds 1–8 were synthesized as racemates in the
Department of Drug Design and Pharmacology, Faculty of Health and
Medical Sciences, University of Copenhagen/Denmark or in the Depart-
ment of Technology and Biotechnology of Drugs, Jagiellonian Uni-
versity Medical College, Kraków/Poland, according to previously
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Epilepsy Research 138 (2017) 124–131
described procedures (Szymanska et al., 2011, 2009). Stock solutions
of the respective test compound used in this study were prepared in
saline at a concentration of 10 mM. The cDNA plasmids for human α1-
glycine receptor expression were kindly provided by Prof. Joe Lynch
(The University of Queensland, Brisbane, Australia). Capped cRNA
transcripts were synthesized in vitro using a mMESSAGE mMACHINE kit
from Ambion (Austin, TX, USA) and analyzed on a 1.2% formaldehyde
agarose gel to check the size and quality of the transcripts.
2.2. Data analysis
Average values were calculated as the mean of 6–8
experiments ± SEM. Throughout this study, n defines the number of
oocytes or number of samples tested in each experiment. Statistical
significance was analyzed using paired t-test. The criterion for statis-
tical significance was set at a P value of less than 0.05.
2.3. Cell line studies
Human embryonic kidney HEK-293 cell line (ATCC CRL1573) was
kindly donated by Prof. Dr. Christa Müller (Pharmaceutical Institute,
Pharmaceutical Chemistry I, University of Bonn, Germany).
Neuroblastoma IMR-32 cell line was provided by Department of
Oncogenomics, Academisch Medisch Centrum, Amsterdam, Holland
(Cheng et al., 1995).
2.3.1. Antiproliferative assay against HEK-293
The HEK-293 cells were seeded in 96-well flat-bottomed microtiter
plate at a concentration of 1 × 104 cells/well and cultured in 200 μl/
well of Dulbecco’s Modified Eagle’s Medium − DMEM (Gibco,
Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS),
100 mg mL−1 streptomycin and 100 U mL−1 penicillin in a humidified
atmosphere (5% CO2, 95% air) at 37 °C for 24 h to reach 60%
confluence. The stock solutions of 2 and 5 in DMSO (25 mM) were
diluted into the fresh growth medium and added to the wells in the
concentration range 0.1–100 μM. Final DMSO concentration did not
exceed 1%. After 48 h of incubation, the EZ4U (Biomedica, Vienna,
Austria) labeling mixture (20 μl) was added to each well and the cells
were incubated under the same conditions for 5 h. The absorbance of
the samples was measured using a microplate reader (EnSpire,
PerkinElmer, Waltham, MA USA) at 492 nm. The reference compound
doxorubicin (DX) was used during this study as a cytotoxic standard
against HEK-293 cells.
2.3.2. Antiproliferative assay against IMR-32
The IMR-32 cell line was seeded in 96-well flat-bottomed microtiter
plate at a concentration of 1 × 104 cells/well and incubated under the
same conditions as described above. The stock solutions of 2, 5 and 6-
OHDA in DMSO (25 mM) were diluted into the fresh growth medium
and added to the cells in concentration range 1–100 μM and incubated
for 48 h. Final DMSO concentration did not exceed 1%. Next, the EZ4U
assay was performed in the same way as described above.
2.3.3. Neuroprotection test
The Mitochondrial ToxGlo™ Assay and protocols were provided by
Promega (Madison, WI, USA). The neuroblastoma IMR-32 cell line was
seeded in 96-well white microtiter Plate 24 h before experiments at a
concentration 100 000 cells/ml. The cells were incubated in 200 μl of
DMEM (Gibco, Invitrogen Ltd, Paisley, UK) supplemented with 10%
FBS in a humidified atmosphere (5% CO2) at 37 °C. Next, the old media
were removed and the 2, 5 at the concentrations 50 μM, and 6-OHDA at
the concentration 100 μM dissolved in DMEM supplemented with 10%
FBS and 4.5 g/L glucose were added to the plate. The cells were
incubated at 37 °C in a humidified and CO2-supplemented incubator for
90 min. After addition of 20 μl fluorescence-based Cytotoxicity Reagent
(Promega) the plate was mixed (500 rpm) and incubated at 37 °C (5%
B. Sadek et al.
Fig. 1. Chemical structures of phenylalanine derivatives tested in the current study.
CO2) for next 30 min. The fluorescence was measured at 485 nmEx/
530 nmEm by microplate reader (EnSpire, PerkinElmer, Waltham, MA
USA) The 65 μM of digitonin (DGT) was used as a positive necrosis
control. Then, the assay plate was adjusted to room temperature
(5–10 min) and 100 μl of Adenosine triphosphate (ATP) Detection
Reagent (Promega) was added to each well. After orbital shaking
(500–700 rpm) for 1–5 min, the bioluminescence was measured by
microplate reader (EnSpire, PerkinElmer, Waltham, MA USA).
2.4. Data analysis
Statistical significance was analyzed by GraphPad Prism™ software
(version 5.01, San Diego, CA, USA) using One-way ANOVA and
Bonferroni's Multiple Comparison Post Test. The experiments were
performed in triplicate wells. Statistical significance was set at a P value
of less than 0.05.
2.5. In-vivo STR-induced seizure
2.5.1. Animals
Inbred male Wistar rats (Central Animal Facility of the UAE
University) of body weight 180–220 g were used for this study. The
animals were retained in an air-conditioned animal facility room with
controlled temperature (24 °C ± 2 °C) and humidity (55% ± 15%)
under a 12-h light/dark cycle.
The animals were allowed free access to food and water. The
experiments of this study were carried out between 9 and 12 AM, and
all procedures were performed according to the guidelines of the
European Communities Council Directive of November 24, 1986 (86/
609/EEC) and were previously approved for epilepsy study by the
College of Medicine and Health Sciences, United Arab Emirates
University (Institutional Animal Ethics Committee, approval number;
A9-14). In the present study and according to previously used experi-
mental procedures, STR was utilized to induce convulsions (Sadek
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Epilepsy Research 138 (2017) 124–131
et al., 2014b, 2016a,b,c, 2015b, 2014c, 2013; Sadek and Stark, 2015).
First, different doses of the proconvulsant compound STR have been
examined (in six to eight rats per dose) to define the minimum dose that
induces convulsions in all treated animals (Branco Cdos et al., 2012;
Coppola et al., 2010; Loscher, 2011; Sancheti et al., 2013; Sowemimo
et al., 2011). This minimal proconvulsant dose was then applied to
screen potential activities of the test compounds 2 and 5. Consequently,
STR (3.5 mg/kg, i.p.) was administered to all experimental test groups
(six to eight rats per group), ie, saline and treated rats. Saline, VPA
(300 mg/kg, i.p.) (Loscher, 2011) or tested ligands 2 and 5 (5, 10, and
15 mg/kg, i.p.) were injected 30 min prior to STR (3.5 mg/kg, i.p.)
administration, and rats were immediately observed for 30 min (ex-
periment period) for any convulsive signs. The convulsion signs were
observed, and graded scores have been used to assess the severity of
convulsions according to the following scale: score 0 = no seizures,
score 1 = eye or facial twitches, score 2 = convulsive waves across the
body, score 3 = myo-clonic jerks or rearing, score 4 = turn over on to
one side position, and score 5 = turn over on to back position,
generalized tonic-clonic seizures, or die during the experiment period.
The animals were divided into groups of seven rats. Animals in the
positive control group were injected with VPA at a dose of 300 mg/kg,
this being the minimal dose of VPA that protected animals against STR-
induced seizures without mortality in rats. Animals in the experimental
groups were administered test compounds at doses of 5, 10, or 15 mg/
kg, i.p., 30 min before the STR treatment. The dose of STR used in this
study formed convulsions (score 4–5) in 100% of animals with
mortality, and this dose was used according to previously described
experimental protocols (Sadek et al., 2016a,c, 2015b; Serdiuk et al.,
2014). In further two control experiments, two groups of seven animals
were injected with test ligands 2 and 5 (5, 10, and 20 mg/kg, i.p.)
30 min prior to saline administration, and rats were immediately
observed for 30 min (experiment period) for any convulsive signs.
B. Sadek et al. Epilepsy Research 138 (2017) 124–131

Fig. 2. Effect of phenyl alanine derivatives (1–8) on human α1-glycine receptor-mediated

ion currents. GLY, glycine; STR, strychnine. Comparison of the extent of inhibition caused
by 30 μM glycine (GLY), 10 μM of strychnine (STR), and 10 μM of each test compound
after 10 min application time. Each bar represents the normalized mean ± SEM from 6 to
8 cells. **P < 0.005.

2.5.2. Data analysis

For statistical comparisons, the software package SPSS 20.0 (IBM
Middle East, Dubai, UAE) was used. All data were expressed as the
mean ± standard error of the mean. The effects of test compounds on
epilepsy were analyzed using One-way analysis of variance (ANOVA)
with Treatment (vehicle or test compound) and Dose (test compound)
as the between-subjects factor. In case of a significant main effect, post
hoc comparisons were performed with Bonferroni’s test. The criterion
for statistical significance was set at a P-value of < 0.05.

Fig. 3. α1-GLYR-mediated ion currents is blocked by ligand 2 and potentiated by ligand

3. Results 5. (A) Records of currents activated by glycine (GLY, 30 μM) in control conditions (left),
during co application of 10 μM STR and GLY (30 μM) after10 min pretreatment
3.1. Effects of ligands 1-8 on α1-GLYR-mediated ion currents with10 μM STR (middle), and15 min following STR washout (right). (B) Records of
currents activated by glycine (GLY, 30 μM) in control conditions (left), during co
In un-injected oocytes (n = 5), glycine (GLY) at the highest application of 10 μM (2) and GLY after10 min pretreatment with 10 μM (2) (middle),
and15 min following (2) washout (right). (C) Records of currents activated by glycine
concentration used in this study (30 μM) did not cause detectable
(GLY, 30 μM) in control conditions (left), during co application of 10 μM (5) and GLY
currents (data not shown). On the other hand, application of 30 μM GLY after10 min pretreatment with 10 μM (5) (middle), and 15 min following (5) washout
for 3–4 s induced rapidly activated inward-currents in oocytes injected (right).
with cRNA encoding the human α1-GLYR. These α1-GLYR-mediated
inward-currents were completely inhibited by 10 min bath application
of 10 μM STR (n = 7) (Figs. 2 and 3 A ), indicating that currents were
mediated by the activation of α1-GLYR. Effects of phenylalanine
derivatives 1–8 at a concentration of 10 μM on the function of α1-
GLYR are shown in Fig. 2. The results show that ligands 1–8 did not
induce any detectable current, when they were applied alone for 10 min
at the concentration of 10 μM (n = 7–9). Ligands 1, 3, 4, 6–8 in the
concentration of 10 μM did not cause a significant change in the
maximum amplitudes of α1-GLYR-mediated currents (Fig. 2). However,
10 min bath application of ligand 2 significantly inhibited these
currents (Figs. 2 and 3 B). Interestingly, ligand 5 when tested in the
same concentration (10 μM) for 10 min significantly potentiated the
function of GLYR (Figs. 2 and 3 C).

Fig. 4. Antiproliferative test against normal human HEK-293 cell line. Values represent
3.2. Antiproliferative assay against HEK-293 the mean of n = 4 experiments. ***P < 0.001.

To evaluate in vitro toxicity of ligands 2 and 5 the normal human
embryonic kidney HEK-293 cell line and formazan based EZ4U anti-
proliferative assay were used. The cells were incubated with the
presence of ligand 2 and ligand 5 in the concentration range
0.1–100 μM for 48 h. The cytostatic compound DX was used as a
reference. As a result, no antiproliferative activity of compound 5 was
observed. For compound 2, very weak antiproliferative effect in
compare to DX was observed only at the highest concentration
(100 μM) (Fig. 4).
3.3. Antiproliferative assay against IMR-32
To determine the neurotoxicity and most suitable concentrations for
further neuroprotection studies, the antiproliferative effect of neuro-
toxin 6-OHDA and selected ligands 2, 5 against neuroblastoma IMR-32
cells was also studied using EZ4U assay. The 6-OHDA, and phenylala-
nine derivatives were examined in three concentrations: 1, 10 and
100 μM. As was shown in Fig. 5, compounds 2 and 5 did not show any
antiproliferative activity after 48 h of incubation with IMR-32, even in

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B. Sadek et al. Epilepsy Research 138 (2017) 124–131

Fig. 5. Antiproliferative test against neuroblastoma cell line IMR-32. Values represent the
mean of n = 4 experiments. ***P < 0.001.

the highest used doses 100 μM, whereas 6-OHDA significantly inhibited
IMR-32 proliferation at 100 μM. Thus, for the neuroprotection studies
the 50 μM concentrations of ligands 2 and 5 and 100 μM of 6-OHDA
were selected.
Fig. 7. ATP level assessment after 130 min of IMR-32 cells incubation with 6-OHDA
(100 μM), 2 (50 μM), 5 (50 μM) and after simultaneously addition of 50 μM of
phenylalanine derivatives with 100 μM of 6-OHDA. Values represent the mean of n = 3
experiments. ***P < 0.0001, *P < 0.05.

ated next by measuring of ATP level in cells using bioluminescent

method. The generated bioluminescent signal was proportional to the
3.4. Neuroprotective effects of compounds 2 and 5
amount of ATP present in cells. The incubation of 6-OHDA and 6-OHDA
together with ligand 5 decreased significantly (P < 0.05) the ATP level
For the neuroprotection study we used Mitochondrial ToxGlo™
in the IMR-32 cell line, in compare to the control cells. These results
Assay. This test employs a sequential-addition, multiplexed chemistry
showed that compound 5 was not able to inhibit 6-OHDA toxic effect on
for predicting necrosis and potential mitochondrial dysfunction in cells
mitochondria. However, slight but not statistically significant decrease
as a result of the short-term xenobiotic exposure. The IMR-32 cell line
of ATP level in cells treated by 2 with 6-OHDA was observed indicating
was incubated with ligands 2 or 5 (50 μM) together with neurotoxin 6-
that ligand 2 appeared to inhibit moderately the 6-OHDA activity on
OHDA (100 μM) for 90 min to determine their potential neuroprotec-
mitochondria (Fig. 7). Moreover, both tested ligands 2 and 5 did not
tive effects. The tested ligands were also incubated separately without
decrease significantly the ATP level in IMR-32 cells, confirming
6-OHDA for assessing their potential influence on cell membrane
previously obtained data indicating no toxicity of ligands 2 and 5
integrity and mitochondria activity. The necrosis effect was assessed
(Figs. 4, 5 and 7).
first by fluorometric quantification of dead cell protease activity
associated with necrosis. DGT was used as a positive necrosis control.
As depicted in Fig. 6, in comparison with the control cells containing
3.5. Effects of ligands 2 and 5 pretreatment on STR-induced convulsions
media only and positive control with DGT, none of tested phenylalanine
derivatives at 50 μM and 6-OHDA at 100 μM concentration caused
In STR-induced seizure model, ligand 5 (5 mg/kg, i.p.) provided
necrosis after short-term 90 min incubation. Similarly, no necrosis
moderate protective activity only after 30 min of observation
effect was observed, if tested compounds were co-applied with
[F(1,12) = 6.12; P < 0.05], whereas acute systemic administration of
100 μM of 6-OHDA.Thus, these results indicated different from necrosis
ligand 5 in a dose of 10 and 15 mg/kg, i.p. exhibited moderate
cytotoxic mechanism of 6-OHDA on IMR-32 cells and confirmed no
protection against STR-induced convulsion when compared to the
toxic effect of 2 and 5.
saline-treated group after 10, 20, and 30 min of observation [all P-
The effect of tested ligands on the mitochondrial activity and their
values > 0.5] (Fig. 8). Notably, the reference drug VPA (300 mg/kg,
potential protection from 6-OHDA mitochondrial toxicity were evalu-
i.p.) provided full protection when compared with saline-treated group
after 10, 20, and 30 min with [F(1,12) = 148.41; P < 0.05],
[F(1,12) = 1156; P < 0.05], and [F(1,12) = 653.42; P < 0.05], respec-
tively. Moreover, the results show that ligand 2 (5, 10, and 15 mg/kg,
i.p.) neither provided proconvulsant nor anticonvulsant activity when
compared to saline-treated group (all P-values > 0.5) (Fig. 8). Notably,
neither Saline + Saline vs. 2 (5 and 10 mg/kg, i.p.) + Saline, nor
Saline + Saline vs. 5 (5, 10, and 15 mg/kg, i.p.) + Saline differences
were significant (all P-values > 0.05) (Fig. 9). However, acute systemic
administration of ligand 2 (15 mg/kg, i.p.) exhibited slight proconvul-
sant effect when compared with saline- treated group after 10, 20, and
30 min with [F(2,11)(2.11) = 6.12; P < 0.05], [F(2,11) = 10.57;
P < 0.05], and [F(2,11) = 4.24; P < 0.05], respectively (Fig. 9). How-
ever, the convulsions induced after acute systemic administration of
ligand 2 (15 mg/kg, i.p.) is significantly lower than that provided by
STR (3.5 mg/kg, i.p.) after 10, 20, and 30 min with [F(2,11) = 26.27;
P < 0.001], [F(2,11) = 87.63; P < 0.001], and [F(2,11) = 38.46;
Fig. 6. Necrosis assessment after 90 min of IMR-32 cells incubation with 6-OHDA
P < 0.001], respectively (Fig. 9).
(100 μM), 2 (50 μM), 5 (50 μM) and after simultaneously addition of 50 μM of
phenylalanine derivatives with 100 μM of 6-OHDA. Values represent the mean of n = 3
experiments. ***P < 0.001.

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B. Sadek et al.
Fig. 8. Effect of acute systemic pretreatment of 2 and 5 on STR-induced convulsions in
male adult rats. VPA (300 mg/kg, i.p.) and ligands 2 and 5 (A–C in a dose of 5, 10, and
15 mg/kg, i.p., respectively) were injected 30 min before STR (3.5 mg/kg, i.p.) treat-
ments. Values are expressed as the mean ± SEM (n = 7). *P < 0.05 and **P < 0.001
vs (STR-saline)-treated group. #Full protection. Abbreviations: STR, strychnine; VPA,
valproic acid; SEM, standard error of the mean.
Fig. 9. Effect of acute systemic pretreatment of 2 and 5 on saline-treated male adult rats.
STR (3.5 mg/kg, i.p.) and ligands 2 and 5 (A, B, and C in a dose of 5, 10, and 15 mg/kg,
i.p., respectively) were injected 30 min before observation time. Values are expressed as
the mean ± SEM (n = 7). *P < 0.05 vs saline-saline-treated groups. #P < 0.001 vs
STR-saline)-treated groups. Abbreviations;: STRstrychnine; SEMstandard error of the
mean.
4. Discussion
GLY receptors are known to play important roles in epilepsy, pain
transmission, and motor coordination in central as well as peripheral
nervous systems (Bedet et al., 2006; Eichler et al., 2008; Kirchner et al.,
2003; Meier and Grantyn, 2004; Mori et al., 2002; Song et al., 2006;
Zhang et al., 2008). Based on several previous clinical findings that
patients would benefit from a facilitated glycinergic inhibition, the
central role of GLYRs in modifying temporal lobe epilepsy has been
proposed,
In the current study, a series of unnatural racemic phenylalanine
derivatives (1–8) bearing a carboxyl or nitro group −has been explored
on the functional properties of the human α1-GLYR expressed in
Xenopus oocytes. When tested in a concentration of 10 μM, compounds
1, 3, 4, 6–8 −failed to cause a significant change in the amplitude of
α1-GLYR-mediated currents (Fig. 2). However, ligands 2 and 5
functionally interacted with human α1-GLYRs. Interestingly, ligand 2
(10 μM) significantly inhibited the GLY-induced currents (Figs. 2 and 3
B), whereas ligand 5 when tested in the same concentration signifi-
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Epilepsy Research 138 (2017) 124–131
cantly potentiated the α1-GLYR-mediated effects (Figs. 2 and 3 B).
Interestingly, none of compounds 2 and 5, previously designed as
ligands of ionotropic glutamate receptors, showed significant affinity
for native AMPA, KA, NMDA or homomeric GluK1 kainate receptors
(Szymanska et al., 2009).
From a structural point of view, two particular differences in the
structure of both compounds appear to be of special importance for the
control of ligand activity on human α1-GLYRs. Accordingly, the amino
acid 2 containing biphenyl moiety provided the most promising
antagonistic effect on α1-GLYRs, while separation two phenyl rings
by methylene linker in the analogue 5 resulted in the highest potentia-
tion effect (Figs. 2 and 3 C). The second difference lies in moving away
of the carboxyl function in the ligand 2 compared to 5. Both modifica-
tions significantly influence the spatial shape of the whole molecule and
place the carboxyl group in various positions regarding the amino acid
moiety. The location of the carboxyl substituent seems to be crucial for
high affinity for α1-GLYRs of the ligand 2 and is not reproduced in any
other compound within the investigated series; the similar situation can
be observed in case of the compound 5. As it is shown in our studies,
none of the positional isomers of 2 and 5 showed any significant affinity
for α1-GLYRs (Figs. 1 and 2, ligands 1 and 8). Furthermore, replace-
ment of the acidic substituent with a nitro group (6, 7), or removal of
an additional aromatic ring (3, 4) resulted in a failure to provide
affinity at α1-GLYRs. Accordingly, a role could also be played by other
additional substituents in the respective test compound, e.g. −Cl, for
compound 3; −Cl and −NO2, for compound 4; or two −Cl substitu-
ents, for compounds 6 and 7. These results are in agreement with a
previous study in which the pharmacological properties of the respec-
tive α-amino acid at the α1-GLYRs are critically determined by the size
and polarity of a certain substitution position (Schmieden and Betz,
1995).
In further experiments, compared to the reference compound
doxorubicin (DX), ligand 5 did not show any antiproliferative activity
after 48 h of incubation with HEK-293 cells whereas for 2 very weak
effect, but only at the 100 μM was observed (Fig. 4). Moreover, ligands
2 and 5 did not act on neuroblastoma IMR-32 cell line viability in the
concentration range 1–100 μM, whereas 6-OHDA significantly inhibited
its proliferation −when applied at 100 μM (Fig. 5). Consequently,
50 μM concentration of the respective ligand and 100 μM toxic
concentration of 6-OHDA were selected for neuroprotection studies.
As depicted in Fig. 6 none of tested phenylalanine derivatives and 6-
OHDA produced necrosis. Next, the effect of ligands 2 and 5 on the
mitochondrial activity and 6-OHDA induced mitochondrial toxicity
were assessed by measuring of ATP level in cells. Results confirmed that
selected phenylalanine derivatives are safe at the 50 μM and do not
have significant effect on mitochondrial functions (Fig. 7). Regarding
the neuroprotection activity, the comparison of the ATP level in cells
treated with 6-OHDA and 6-OHDA together with 2 or 5 showed that
compound 5 did not possess neuroprotective properties. However,
ligand 2 appeared to inhibit moderately toxic 6-OHDA activity on
mitochondria according to the measured ATP levels (Fig. 7).
In a further in vivo experiment and among promising ligands 2 and
5, the results showed that acute systemic administration of ligand 5 (10
and 15 mg/kg, i.p.) provided some appreciable protection against STR-
induced convulsions in male adult rats, whereas the standard AED VPA
(300 mg/kg, i.p.) exhibited full protection following 10–30 min time
observation (Fig. 8). Moreover, ligand 2 (5, 10 and 15 mg/kg, i.p.) and
the lower dose of ligand 5 (5 mg/kg, i.p.) failed to provide protection
against STR-induced convulsions throughout observation time (Fig. 8).
Interestingly, the results observed for ligand 5 (10 and 15 mg/kg, i.p.)
are in agreement with previous studies in which anticonvulsant effects
were obtained at the same dose range for several α-amino acid
derivatives (De Caro et al., 2014; Paruszewski et al., 2001; Torregrosa
et al., 2015). Moreover, a previous study showed that glycine potenti-
ates the anticonvulsant effect of diazepam and concluded that the
potentiating effect involves a direct pharmacodynamic interaction
B. Sadek et al.
between these two compounds (Peterson and Frye, 1987). Furthermore,
the latter in vivo findings for ligand 5 further confirmed our in vitro
results for its observed significant potentiating effects on the α1-GLYR-
induced currents (Figs. 1–3), since the mechanism underlying STR-
induced convulsions is thought to be credited mainly to its blocking
effect on glycine receptors in the brain and in the spinal cord (Sadek
et al., 2016a,b; Sowemimo et al., 2011). Importantly, the results for
ligands 2 and 5 were further confirmed as a moderate proconvulsant
effect was observed for ligand 2 (15 mg/kg, i.p.) in saline-treated male
adult animals whereas no appreciable effect was obtained for ligand 5
(5, 10, and 15 mg/kg, i.p.) (Fig. 9).
5. Conclusion
The results indicate that phenyl alanine derivatives carrying
selected hydrogen bond acceptors can constitute a new group of
molecules with micromolar potencies for human α1-GLYRs. The
structure-activity relationships revealed that one particular substituent
position seemed to be crucial for control of ligand activity. The
biphenyl derivative 2 provided the most promising antagonistic effect
on α1-GLYRs, while its structural analogue 5 displayed the highest
potentiation effect. Moreover, ligand 5 (potentiator of human α1-
GLYRs) provided moderate in vivo protection, whereas ligand 2
(inhibitor of human α1-GLYRs) failed to show anti(pro)convulsant
effect when tested in STR-induced convulsion model in male adult rats.
Furthermore, the safety studies showed ligands 2 and 5 as non-toxic. A
very weak antiproliferative effect in comparison to DX was only
observed for ligand 2 against HEK-293 cell line at the highest
concentration used. Additionally, neither necrosis, nor modulated
mitochondrial toxicity in IMR-32 were observed for 2 and 5 at
50 μM, while ligand 2 showed weak neuroprotective properties. In
light of the present findings, we conclude that the novel ligands 2 and 5
with modulatory effects on human α1-GLYRs can be used as a potential
template for further drug design. However, further investigations
addressing additional in vitro as well as in vivo pro(anti)convulsant
effects of ligands 2 and 5 are warranted to exclude potentials of off-
target effects and to confirm the possible utility of α1-GLYR modulators
as AEDs.
Acknowledgements
The authors cordially thank Prof. Joe Lynch of the University of
Queensland, Australia for providing the cDNA for human α1-GLYR.
Bassem Sadek and Murat Oz were supported by intermural funds from
the College of Medicine and Health Sciences and the Office of Graduate
Studies and Research, UAE University. The financial support of this
research by the Ministry of Science and Higher Education in Poland
(Jagiellonian University statutory project K/ZDS/005594) as well as the
National Science Centre Poland (DEC-2014/15/B/NZ7/00908) is grate-
fully acknowledged.
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