A substituted anilino enaminone acts as a novel positive allosteric modulator of GABAA receptors in the mouse brain

Article abstract of DOI:10.1124/jpet.110.173740

A small library of anilino enaminones was analyzed for potential anticonvulsant agents. We examined the effects of three anilino enaminones on neuronal activity of output neurons, mitral cells (MC), in an olfactory bulb brain slice preparation using whole

Full text of DOI:10.1124/jpet.110.173740

0022-3565/11/3363-916–924$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
JPET 336:916–924, 2011
Vol. 336, No. 3
173740/3670166
Printed in U.S.A.
A Substituted Anilino Enaminone Acts as a Novel Positive
Allosteric Modulator of GABAA Receptors in the Mouse Brain
Ze-Jun Wang, Liqin Sun, Patrice L. Jackson, Kenneth R. Scott, and Thomas Heinbockel
Department of Anatomy, College of Medicine (Z.-J.W., L.S., T.H.) and Department of Pharmaceutical Sciences (K.R.S.),
Howard University, Washington, District of Columbia; and Department of Pharmaceutical Sciences, University of Maryland
Eastern Shore, Princess Anne, Maryland (P.L.J.)
Received August 5, 2010; accepted December 7, 2010
ABSTRACT
A small library of anilino enaminones was analyzed for potential indirect action through the elevation of tissue GABA levels.
anticonvulsant agents. We examined the effects of three anilino Neither vigabatrin (a selective GABA-T inhibitor) nor 1,2,5,6-
enaminones on neuronal activity of output neurons, mitral cells tetrahydro-1-[2-[[(diphenylmethylene)amino]oxy]ethyl]-3-
(MC), in an olfactory bulb brain slice preparation using whole- pyridinecarboxylic acid hydrochloride (NNC-711) (a selective
cell patch-clamp recording. These compounds are known to be inhibitor of GABA uptake by GABA transporter 1) eliminated the
effective in attenuating pentylenetetrazol-induced convulsions. effect of KRS-5ME-4-OCF3 on neuronal excitability, indicating
Among the three compounds tested, 5-methyl-3-(4-trifluoro- that the inhibitory effect of the enaminone resulted from direct
methoxy-phenylamino)-cyclohex-2-enone (KRS-5Me-4-OCF3) activation of GABAA receptors. The concentration-response
showed potent inhibition of MC activity with an EC50 of 24.5 curves for GABA are left-shifted by KRS-5Me-4-OCF3, demon-
␮M. It hyperpolarized the membrane potential of MCs accom- strating that KRS-5Me-4-OCF3 enhanced GABA affinity and
panied by suppression of spontaneous firing. Neither ionotropic acted as a positive allosteric modulator of GABAA receptors.
glutamate receptor blockers nor a GABAB receptor blocker The effect of KRS-5Me-4-OCF3 was blocked by applying a
prevented the KRS-5Me-4-OCF3-evoked inhibitory effects. In benzodiazepine site antagonist, suggesting that KRS-5Me-4-
the presence of GABAA receptor antagonists, KRS-5Me-4- OCF3 binds at the classic benzodiazepine site to exert its
OCF3 completely failed to evoke inhibition of MC spiking ac- pharmacological action. The results suggest clinical use of
tivity, suggesting that KRS-5Me-4-OCF3-induced inhibition enaminones as anticonvulsants in seizures and as a potential
may be mediated by direct action on GABAA receptors or anxiolytic in mental disorders.
pounds. Structurally, these compounds are uniquely differ-
Introduction
ent from currently available antiepileptic drugs (Edafiogho et
A diverse series of anilino enaminones has been synthe-
al., 1992, 2007, 2009; Foster et al., 1999; Abdel-Hamid et al.,
sized and investigated as potential anticonvulsant com-
2002; Kombian et al., 2005). This class of enaminones has
shown good to moderate protection in the traditional preclin-
ical animal models, the subcutaneous pentylenetetrazol test
and the maximal electroshock seizure test. Their anticonvul-
sant activities are comparable with those of some clinically
used agents in animal models of seizures with a minimal side
effect profile as well as a wider margin of safety than con-
ventional antiepileptic drugs such as carbamazepine, val-
proate, and phenytoin (Mulzac and Scott, 1993; Eddington et
This work was supported in part by the National Institute of Health Na-
tional Institute of General Medical Sciences [Grant S06-GM08016]; the Na-
tional Institutes of Health National Institute of Neurological Disorders and
Stroke [Grant U54-NS039407]; and the Howard University New Faculty Re-
search Program, the Howard University Health Sciences Faculty Seed Grant
Program, the Whitehall Foundation.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.173740.
ABBREVIATIONS: NMDA, N-methyl-D-aspartate; ACSF, artificial cerebrospinal fluid; MOB, main olfactory bulb; MC, mitral cell; KRS-5Me-4-
OCF3, 5-methyl-3-(4-trifluoromethoxy-phenylamino)-cyclohex-2-enone; KRS-5Me-4-F, 3-(4-fluoro-phenylamino)-5-methyl-cyclohex-2-enone;
KRS-5Me-3-Cl, 3-(3-chloro-phenylamino)-5-methyl-cyclohex-2-enone; DMSO, dimethyl sulfoxide; D-AP5, L-2-amino-5-phosphonopentanoic ac-
id; CNQX, 6-cyano-7-nitroquinoxaline-2-3-dione; gabazine (SR-95531), 2-(3-carboxypropyl)-3-amino-6-(4 methoxyphenyl)-pyridazinium bromide;
LY367385, (S)-(ϩ)-␣-amino-4-carboxy-2-methylbenzeneacetic acid; CGP55845, (2S)-3[[(1S0–1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phe-
nylmethyl) phosphinic acid; (R)-baclofen, (R)-4-amino-3-(4-chlorophenyl) butanoic acid; NNC 711, 1,2,5,6-tetrahydro-1-[2-[[(diphenylmethylene)
amino]oxy]ethyl]-3-pyridinecarboxylic acid hydrochloride; vigabatrin, (Ϯ)-␥-vinyl GABA; flumazenil, 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo
[1,5-a][1,4]benzodiazepine-3-carboxylic acid, ethyl ester; (S)-SNAP 5114, 1-[2-[tris(4-methoxyphenyl)methoxy]ethyl]-(S)-3piperidinecarboxylic acid; ANOVA,
analysis of variance; AMPA, ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; GABABR, GABAB receptor; GAT, GABA transporter; GABA-T, GABA
transaminase.
916

Anilino Enaminone Activity through GABAA Receptor
917
al., 2000). The unique pharmacophoric structure of the ani- The functional modulation of these proteins may change
lino enaminones and the variety of bioactivities provide an synaptic input and neuronal excitability. Thus, in this study,
excellent opportunity for developing new drugs.
we used acute slices of the mouse MOB and electrophysio-
We reported previously the anticonvulsant activity of ani- logical recordings from MCs to determine the effects of
lino enaminones in vivo and the possible mechanisms of enaminones on the activity of MCs and the mechanisms
action of these compounds by which they elicit their response underlying the inhibitory or excitatory actions of these com-
(Edafiogho et al., 1992; Mulzac and Scott, 1993; Ananthalak- pounds. Our results show that enaminone compounds with
shmi et al., 2007). The anilino enaminone E139 inhibited
excitatory postsynaptic currents in the rat nucleus accum-
bens and hippocampus by enhancing extracellular GABA
levels (Kombian et al., 2005; Ananthalakshmi et al., 2007)
and inhibiting tetrodotoxin-sensitive sodium currents to
modulate excessive firing in individual neurons (Anan-
thalakshmi et al., 2006). A study aimed at elucidating the
essential structural parameters necessary for anticonvulsant
activity found that some benzylamino enaminones, which
possess a similar chemical structure to anilino enaminones
with benzyl-substitution at the NH-moiety, produced anti-
convulsant effects in rats and mice neurons by suppressing
glutamate-mediated excitation and action potential firing
(Edafiogho et al., 2006). The different substitutions at the
NH-moiety change the target protein to which enaminones
bind. These studies indicate that enaminones with similar
chemical structure may possess different modes of action.
Here, we hypothesize that the substituted site in enaminones
may contribute to the mode of action of these compounds. To
study the structure-activity relationships of enaminones,
three enaminone compounds with non-ortho-substituted cy-
clohexenone were synthesized and used to determine the
mechanism of their anticonvulsant action.
Epileptic seizures result from poorly controlled neuronal
activity at a seizure focus and the subsequent spread of
electrical excitation in brain circuits (Rall and Schleifer,
1990). It is not surprising that most effective antiseizure
medications have been demonstrated to inhibit neuronal ex-
citability through modulating the function of several types of
proteins such as sodium channels, NMDA receptors, and
GABA receptors (Rho and Sankar, 1999). The excitability of
neurons in the brain is an integral of intrinsic membrane
conductances and synaptic inputs. Both excitatory and inhib-
itory inputs regulate the resting excitability (Traynelis and
Dingledine, 1988). Output neurons such as mitral cells (MCs)
non-ortho-substituted cyclohexenone suppress neuronal ex-
citability through activation of GABAA receptors and display
the characteristics of a positive allosteric modulator.
Materials and Methods
Synthesis of Anilino Enaminones. Anilino enaminones 5-methyl-
3-(4-trifluoromethoxy-phenylamino)-cyclohex-2-enone (KRS-5Me-4-
OCF3), 3-(4-fluoro-phenylamino)-5-methyl-cyclohex-2-enone (KRS-
5Me-4-F), and 3-(3-chloro-phenylamino)-5-methyl-cyclohex-2-enone
(KRS-5Me-3-Cl) were recently synthesized. The mono methyl anilino
enaminones (3) were prepared from the 5-methylcyclohexane-1,3-dione
(2) form by the decarboxylation of 4-carbo-tert-butoxy-5-methylcyclo-
hexane-1,3-dione (1) and were refluxed with appropriate substituted
aniline derivatives under standard conditions (Fig. 1) (Eddington et al.,
2003). The chemical structures of the anilino enaminones are shown in
Fig. 2. The ␤-hydroxy keto tert-butoxy ester was prepared as reported
previously (Friary et al., 1973; Edafiogho et al., 1992; Scott et al., 1993).
The enaminone structures were confirmed via NMR analyses at 400
MHz.
Slice Preparation. Wild-type mice (C57BL/6J; The Jackson Lab-
oratory, Bar Harbor, ME) were used in agreement with Institutional
Animal Care and Use Committee and National Institutes of Health
guidelines. Juvenile (16–25 days old) mice were decapitated, and the
MOBs were dissected out and immersed in artificial cerebrospinal
fluid (ACSF) at 4°C as described previously (Heinbockel et al., 2004).
Horizontal slices (400 ␮m thick) were cut parallel to the long axis
using a vibratome (Vibratome Series 1000; Ted Pella, Redding, CA).
After 30 min at 30°C, slices were incubated in a holding bath at room
temperature (22°C) until use. For recording, a brain slice was placed
in a recording chamber mounted on a microscope stage and main-
tained at 30 Ϯ 0.5°C by superfusion with oxygenated ACSF flowing
at 2.5 to 3 ml/min.
Electrophysiological Recording and Data Acquisition. Vi-
sually guided recordings were obtained from cells in the mitral cell
layer with near-infrared differential interference contrast optics and
a BX51WI microscope (Olympus Optical, Tokyo, Japan) equipped
in the mouse main olfactory bulb (MOB) display their neu- with a camera (C2400-07; Hamamatsu Photonics, Hamamatsu, Ja-
pan). Images were displayed on a Sony Trinitron Color Video moni-
tor (PVM-1353MD; Sony Corp., Tokyo, Japan). Recording pipettes
(5–8 M⍀) were pulled on a Flaming-Brown P-97 puller (Sutter In-
strument Co., Novato, CA) from 1.5-mm o.d. borosilicate glass with
filament. Seal resistance was routinely Ͼ1 G⍀, and liquid junction
potential was 9 to 10 mV; reported measurements were not corrected
for this potential. Data were obtained using a Multiclamp 700B
amplifier (Molecular Devices, Sunnyvale, CA). Signals were low-pass
Bessel-filtered at 2 kHz and digitized on computer disc (Clampex
10.1; Molecular Devices). Data were also collected through a Digi-
ronal activity as spontaneous action potential firing, which
can be modulated by intrinsic membrane receptors as well as
synaptic inputs (Shepherd et al., 2004; Ennis et al., 2007). In
the rodent MOB, MCs express high levels of different recep-
tors such as GABA receptors (GABAA, GABAB), ionotropic
and metabotropic glutamate receptors (NMDA, AMPA,
metabotropic glutamate receptor 1, kainate), and serotonin
receptors (5-HT1A, 5-HT2A/C). Most of these receptor proteins
are thought to be strongly epilepsy-related (McNamara,
1996; Snell et al., 2000; Wang et al., 2002; Ennis et al., 2007). data 1440A Interface (Molecular Devices) and digitized at 10 kHz.
Fig. 1. Synthesis of aniline enaminones.
Condition a is H2SO4, ⌬; condition b is ⌬,
substituted amine.

918
Wang et al.
Fig. 2. Chemical structure of aniline enami-
none analogs. The difference among the three
mono methyl compounds is the anilino sub-
stitution represented by meta-chloro; para-
fluoro, and para-trifluoro. E139 is a para-
bromo anilino enaminone derivative with
ortho-methyl ester substituted moiety at cyc-
lohexenone, and compound 30 is the benzyl-
amino analog.
Holding currents were generated under control of the Multiclamp olfactory receptor neurons, send excitatory projections to olfac-
700B Commander.
tory cortical areas, and receive strong feedback inhibition pri-
marily through reciprocal dendrodendritic synapses with local
interneurons (Shepherd et al., 2004; Ennis et al., 2007). MCs
generate spontaneous action potentials (1–6 Hz) in slices. Here,
we made use of the intrinsic properties of MCs such as sponta-
neous firing, membrane potential, and membrane conductance
to test the effect of enaminones on MC activity and determine
the possible binding target of enaminones.
Bath application of either KRS-5Me-3-Cl or KRS-5Me-4-F
modulated the spike rate of MCs (Fig. 3). Compared with
control conditions, 20 ␮M KRS-5Me-3-Cl slightly reduced the
MC firing rate (in control: 3.1 Ϯ 0.5 Hz; in drug: 2.6 Ϯ 0.5 Hz;
n ϭ 5; p Ͻ 0.05; paired t test). The other compound, KRS-
5Me-4-F (20 ␮M), decreased MC firing from 4.8 Ϯ 0.8 to 4.0 Ϯ
0.7 Hz (n ϭ 5; p Ͻ 0.05; paired t test) and slightly hyperpo-
The ACSF consisted of 124 mM NaCl, 3 mM KCl, 2 mM CaCl2, 1.3
mM MgSO4, 10 mM glucose, 26 mM sucrose NaHCO3, 1.25 mM
NaH2PO4 (pH 7.4, 300 mOsm), saturated with 95 O2/5% CO2 (mod-
ified from Heyward et al., 2001). The standard pipette-filling solu-
tion consisted of 125 mM K gluconate, 2 mM MgCl2, 10 mM HEPES,
2 mM Mg2ATP, 0.2 mM Na3GTP, 1 mM NaCl, and 0.2 mM EGTA.
Chemicals and Solutions. Drugs were bath-perfused at the final
concentration as indicated by dissolving aliquots of stock in ACSF.
The three enaminone compounds that we tested, KRS-5Me-4-OCF3,
KRS-5Me-4-F, and KRS-5Me-3-Cl, were recently synthesized. All
enaminones were dissolved in DMSO to make 20 mM stock solution
(final concentration of DMSO in bath Ͻ0.1%). For all experiments,
the drugs were applied by bath perfusion. Control recordings showed
that 0.1% DMSO had no detectable effects on the firing rate and
membrane potential. The following drugs were also bath applied:
L-2-amino-5-phosphonopentanoic acid (D-AP5), CNQX, gabazine, (S)-
(ϩ)-␣-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385),
(2S)-3[[(1S0–1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl]
(phenylmethyl) phosphinic acid (CGP55845), (R)-baclofen, 1,2,5,6-
tetrahydro-1-[2-[[(diphenylmethylene)amino]oxy]ethyl]-3-pyridin-
ecarboxylic acid hydrochloride (NNC 711), vigabatrin, flumazenil,
and 1-[2-[tris(4-methoxyphenyl)methoxy]ethyl]-(S)-3piperidinecar-
boxylic acid [(S)-SNAP 5114]. Chemicals and drugs were supplied by
Tocris Bioscience (Ellisville, MO) and Sigma-Aldrich (St. Louis, MO).
Numerical data are expressed as the mean Ϯ S.E.M. Tests for
statistical significance were performed using paired Student’s t tests
and one-way ANOVA followed by the Bonferroni test for multiple
comparisons.
Results
Recordings were obtained from 166 MCs with whole-cell
recordings in mouse MOB slices from 94 animals. All re-
corded cells showed measurable responses to enaminone
KRS-5Me-4-OCF3. MCs were identified visually by their
soma location and relatively large soma size and their input
resistance (297 Ϯ 19.2 M⍀, n ϭ 46). The membrane potential
of MCs in this study was Ϫ52.2 Ϯ 0.7 mV (n ϭ 46).
Enaminones KRS-5Me-3-Cl and KRS-5Me-4-F Slightly
Depressed Activity of Mitral Cells. MCs are principal neu-
rons and play a crucial role in processing sensory information in
Fig. 3. Anilino enaminones depressed the spiking activity of MCs. A,
normalized bar graph shows inhibition of spiking of MCs in response to
bath application of KRS-5Me-4-OCF3, KRS-5Me-3-Cl, and KRS-5Me-4-F.
Responses to enaminones were normalized with respect to control condi-
tions. ء, p Ͻ 0.05; ءءء, p Ͻ 0.001. B, original recording from a represen-
tative MC illustrated the inhibition in firing rate and hyperpolarization
MOB. They receive direct synaptic inputs from the axons of after application of KRS-5Me-4-OCF3.

Anilino Enaminone Activity through GABAA Receptor
919
larized the membrane potential by Ϫ0.4 Ϯ 0.1 mV (n ϭ 5; p Ͻ had no any additional effect on KRS-5Me-4-OCF3-induced
0.05; paired t test). suppression of MC activity (p Ͼ 0.05; ANOVA and Bonferroni
KRS-5Me-4-OCF3 Inhibited Spontaneous Spiking of post hoc analysis).
Mitral Cells and Hyperpolarized the Membrane Poten-
D-AP5 is a potent NMDA receptor antagonist. Blockade of
tial. For the concentration tested, KRS-5Me-4-OCF3 showed both AMPA/kainate and NMDA receptors antagonizes the
a difference in potency of inhibition of MC activity compared excitatory activities of ionotropic glutamate receptors. In the
with the above two compounds (Fig. 3A). KRS-5Me-4-OCF3 presence of both CNQX and D-AP5, KRS-5Me-4-OCF3 re-
(20 ␮M) reversibly decreased MC firing from 4.4 Ϯ 0.4 to duced the firing rate (in CNQX ϩ D-AP5: 3.4 Ϯ 0.4 Hz; in
2.9 Ϯ 0.3 Hz (n ϭ 50; p Ͻ 0.001; paired t test). The reduction CNQXϩD-AP5 plus KRS-5Me-4-OCF3: 2.2 Ϯ 0.40 Hz; n ϭ 4;
of the firing rate was accompanied by hyperpolarization of p Ͻ 0.05; paired t test) and hyperpolarized MCs by Ϫ0.9 Ϯ 0.2
the MC membrane potential by Ϫ0.9 Ϯ 0.2 mV (n ϭ 50; p Ͻ mV (n ϭ 4; p Ͻ 0.05; paired t test) (Fig. 4). These values were
0.001; paired t test). Figure 3B illustrates the inhibitory not significantly different from the values of KRS-5Me-4-
effect in an original recording from a MC.
OCF3-induced suppression recorded in ACSF control condition
Even though we did not perform a detailed comparison, the (see Fig. 3A) (p Ͼ 0.05; determined by ANOVA and Bonferroni
findings indicated that, at 20 ␮M, KRS-5Me-4-OCF3 was the post hoc analysis). These results showed that ionotropic gluta-
most potent compound compared with the other two enami- mate receptors were not involved in KRS-5Me-4-OCF3-induced
nones, KRS-5Me-3-Cl and KRS-5Me-4-F, in terms of depress- MC inhibition.
ing spiking activity of MCs. Therefore, the enaminone KRS-
The KRS-Induced Inhibition of MC Excitability Was
5Me-4-OCF3 was selected for the remainder of the study to Not Influenced by GABAB Receptors. GABAB receptors
characterize the cellular actions of an enaminone and the (GABABRs) are restricted mostly to the glomerular layer in
mechanism underlying its inhibitory effect on neuronal the MOB (Bowery et al., 1987; Chu et al., 1990). GABABRs
activity.
are metabotropic transmembrane receptors for GABA and
Ionotropic Glutamate Receptors Were Not Involved linked via G proteins to potassium channels (Chen et al.,
in the KRS-5Me-4-OCF3-Induced Inhibition of Neuro- 2005). Activation of GABABRs can stimulate the opening of
nal Activity. Ionotropic glutamate receptors play a critical Kϩ channels that will hyperpolarize the neuron, quiet down
role in the regulation of neuronal excitability in the MOB excitable cells, and hence stop neurotransmitter release. In
(Ennis et al., 2007). Blockade of ionotropic glutamate recep- the MOB, activation of GABABRs has been observed to re-
tors may result in neuronal inhibition. To determine whether duce MC excitability (Palouzier-Paulignan et al., 2002; Isaac-
the inhibitory effect of the anticonvulsant agent KRS-5Me-4- son and Vitten, 2003).
OCF3 was mediated through interaction with ionotropic glu-
In the presence of the GABABR antagonist CGP55845 (10
tamate receptors, we examined the effects of neuronal inhi- ␮M), the KRS-5Me-4-OCF3-evoked modulation of MC firing
bition evoked by KRS-5Me-4-OCF3 in the presence of AMPA/ persisted (in CGP55845: 3.7 Ϯ 0.6 Hz; in CGP55845 plus
kainate and NMDA receptor inhibitors. In the presence of KRS-5Me-4-OCF3: 2.8 Ϯ 0.5 Hz; n ϭ 5; p Ͻ 0.05; paired t
CNQX (10 ␮M), a potent AMPA/kainate receptor antagonist, test), and MCs were hyperpolarized Ϫ1.0 Ϯ 0.2 mV (n ϭ 5;
the inhibitory effects of KRS-5Me-4-OCF3 persisted as seen p Ͻ 0.05; paired t test). Figure 5A shows a representative
by a reduction of the firing rate (in CNQX: 3.2 Ϯ 0.56 Hz; in
CNQX plus KRS-5Me-4-OCF3: 2.3 Ϯ 0.50 Hz; n ϭ 4; p Ͻ 0.05;
paired t test) and hyperpolarization of MCs by Ϫ0.8 Ϯ 0.2 mV
(n ϭ 4; p Ͻ 0.05; paired t test) (Fig. 4). In comparison with the
results shown in Fig. 3A, these values indicated that CNQX
Fig. 5. GABAB receptors were not involved in KRS-5Me-4-OCF3-evoked
inhibition. A, original recording from an MC during KRS-5Me-4-OCF3
application in the ACSF condition and in the presence of GABABR an-
tagonist CGP55845. The upper trace (i) illustrates the effect of KRS-5Me-
4-OCF3 on spiking of an MC and the effect of adding CGP55845. The
upper trace is shown at an extended time scale in the middle (ii) and
lower trace (iii). B, the normalized results showed the persistence of
KRS-5Me-4-OCF3-evoked inhibitory effects on spiking of MCs in the
presence of the GABABR antagonist CGP55845 (10 ␮M) and the GABABR
agonist (R)-baclofen (50 ␮M). ء, p Ͻ 0.05; ءءء, p Ͻ 0.001. The data for the
effect of KRS-5Me-4-OCF3 on spiking of MCs were normalized with
respect to ACSF, CGP88545, or baclofen alone.
Fig. 4. Neither AMPA/kainate nor NMDA receptor antagonists blocked
the KRS-5Me-4-OCF3-induced inhibition of MCs. A, KRS-5Me-4-OCF3-
induced suppression of neuronal firing recorded in a representative MC
in the presence of CNQX. B, the normalized and averaged results showed
the persistence of KRS-5Me-4-OCF3-induced inhibitory effects on spon-
taneous spiking of MCs in the presence of either CNQX or CNQX plus
D-AP5. ء, p Ͻ 0.05.

920
Wang et al.
recording from one MC. No significant difference was ob- (Laurie et al., 1992; Persohn et al., 1992). In the presence of
served for KRS-5Me-4-OCF3-evoked inhibition in the con- GABA, KRS-5Me-4-OCF3 reduced the MC firing rate to 0.7 Ϯ
trol condition (see Fig. 3A) and in the presence of 0.2 Hz (n ϭ 4; p Ͻ 0.01; paired t test). The enhancement of
CGP55845 (p Ͼ 0.05; determined by ANOVA and Bonfer- the KRS-5Me-4-OCF3-evoked inhibition in the presence of
roni post hoc analysis).
increased extracellular GABA levels (50 ␮M) suggested that
Compared with control conditions, the GABABR agonist KRS-5Me-4-OCF3-evoked inhibition may be mediated by di-
(R)-baclofen (50 ␮M) evoked a strong decrease in the firing rect activation of GABA receptors rather than by increased
rate of MCs (in control: 2.6 Ϯ 0.4 Hz; in baclofen: 1.3 Ϯ 0.3 GABA levels. Potentially, increased endogenous GABA levels
Hz; n ϭ 4; p Ͻ 0.01; paired t test) and hyperpolarization of could result from increasing GABA release or reducing
MCs by Ϫ1.6 Ϯ 0.7 mV (n ϭ 4; p Ͻ 0.05; paired t test). In the GABA reabsorption and GABA degradation in the slice.
presence of the GABABR agonist baclofen, KRS-5Me-4-OCF3
Blockade of GABAA receptors can result in overexcitation
further reduced the MC firing rate to 0.7 Ϯ 0.1 Hz (n ϭ 4; p Ͻ of neurons. In the presence of the GABAA receptor antagonist
0.05, paired t test) and hyperpolarized MCs by Ϫ0.9 Ϯ 0.3 mV picrotoxin, the inhibition of MC activity by KRS-5Me-4-OCF3
(n ϭ 4; p Ͻ 0.05, paired t test). In comparison with the was completely blocked (in picrotoxin: 6.1 Ϯ 1.0 Hz; in picro-
inhibitory effects of KRS-5Me-4-OCF3 in control conditions toxin plus KRS-5Me-4-OCF3: 6.2 Ϯ 1.0 Hz; n ϭ 9, p Ͼ 0.05;
(percentage of control firing; see Fig. 3A), the effects of KRS- paired t test) (Fig. 6). Gabazine is another selective GABAA
5Me-4-OCF3 recorded in the presence of (R)-baclofen [per- receptor antagonist. Likewise, in the presence of gabazine,
centage of values recorded in (R)-baclofen] did not signifi- the KRS-5Me-4-OCF3-induced inhibition of MC activity was
cantly change (p
Ͼ 0.05 determined by ANOVA and completely abolished (in gabazine: 5.4 Ϯ 0.7 Hz; in gabazine
Bonferroni post hoc analysis). These results indicated that plus KRS-5Me-4-OCF3: 5.7 Ϯ 0.7 Hz; n ϭ 6, p Ͼ 0.05; paired
the inhibitory effect of KRS-5Me-4-OCF3 persisted irrespec- t test) (Fig. 6B). These results indicated that blockade of
tive of GABABR activation or blockade, suggesting that nei- GABAA receptors prevented KRS-5Me-4-OCF3-evoked inhi-
ther a GABABR antagonist nor an agonist influenced the bition of MC activity and suggested that KRS-5Me-4-OCF3
enaminone-induced MC inhibition.
acted through enhanced GABA levels or direct action on
Blockade of GABAA Receptor Reversed KRS-In- GABAA receptors.
duced Inhibition of MC Excitability. GABAARs play an
Further evidence for the involvement of GABAA receptors
important role in regulating MC excitability (Laurie et al., came from measurements of MC ionic currents induced by
1992; Panzanelli et al., 2005) by suppressing neuronal activ- KRS-5Me-4-OCF3 with or without blockade of GABAA recep-
ity. Bath application of GABA (50 ␮M) dramatically de- tors using the antagonist gabazine. In voltage-clamp mode at
creased the firing rate of MCs (in control: 5.0 Ϯ 0.8 Hz; in a holding potential of Ϫ60 mV, KRS-5Me-4-OCF3 produced
GABA: 1.7 Ϯ 0.3 Hz; n ϭ 6; p Ͻ 0.001; paired t test) and an outward current in MCs of 14.7 Ϯ 3.5 pA (n ϭ 5; the
hyperpolarized MCs by Ϫ1.1 Ϯ 0.3 mV (n ϭ 6; p Ͻ 0.05; steady-state current at Ϫ60 mV in KRS-5Me-4-OCF3 was
paired t test), indicating that GABA induced a large direct measured and subtracted from that in ACSF). In the pres-
inhibition of MC activity via GABA receptors (Fig. 6B). The ence of gabazine, KRS-5Me-4-OCF3-induced outward cur-
potent inhibition by GABA is consistent with previous re- rents were blocked (Ϫ0.2 Ϯ 1.6 pA; n ϭ 4; the current at Ϫ60
ports showing that GABA receptors are abundant in MCs mV in KRS-5Me-4-OCF3 plus gabazine was subtracted from
that in gabazine).
Enhancement of Extracellular GABA Levels Did Not
Block the KRS-5ME-4-OCF3-Induced Inhibition of
Neuronal Excitability. The above results (Fig. 6) showed
that inhibition of MC firing evoked by KRS-5Me-4-OCF3
could be blocked by GABAA receptor antagonists. This result
suggested that the enaminone either binds to GABAA recep-
tors to produce the inhibitory effects or acts to enhance ex-
tracellular GABA levels. Extracellular GABA levels are in
part controlled by GABA reuptake and degradation (Errante
et al., 2002). GABA released from synaptic terminals may be
removed from the extracellular space by GABA uptake back
into synaptic terminals and/or into glial cells by plasma
membrane transporters (GATs). Subsequently, the captured
GABA is degraded by the enzyme GABA transaminase
(GABA-T). To test the possible involvement of endogenous
GABA in the KRS-5Me-4-OCF3-evoked neuronal inhibition,
we examined the role of the enzyme GABA-T and membrane
transporters, GATs, in enaminone-mediated inhibition of MC
Fig. 6. KRS-5Me-4-OCF3-evoked inhibition of MC spiking activity was
activity.
blocked by GABAA receptor antagonists. A, original recording obtained
Bath application of vigabatrin (200 ␮M), an irreversible
from a MC showed that picrotoxin (20 ␮M) blocked 20 ␮M KRS-5Me-4-
and selective GABA-T inhibitor that results in extracellular
accumulation of GABA in the synaptic cleft, induced a reduc-
tion in MC firing rate (in control: 4.3 Ϯ 0.5 Hz; in vigabatrin:
3.4 Ϯ 0.4 Hz; n ϭ 4; p Ͻ 0.05; paired t test). In the presence
of vigabatrin, bath application of KRS-5Me-4-OCF3 (20 ␮M)
OCF3-evoked suppression of neuronal firing. B, the normalized and av-
eraged results showed KRS-5Me-4-OCF3-evoked inhibitory effects on
spiking of MCs in the presence of the GABAA receptor agonist GABA (50
␮M) and antagonist picrotoxin (20 ␮M) and gabazine (5 ␮M). ءء, p Ͻ 0.01;
ءءء, p Ͻ 0.001. The data for the effect of GABA or KRS-5Me-4-OCF3 on
spiking of MCs were normalized with respect to control condition.

Anilino Enaminone Activity through GABAA Receptor
921
resulted in further reduction of the firing rate (in vigabatrin: 4-OCF3 produced firing inhibition (in NNC711 ϩ vigabatrin:
3.5 Ϯ 0.5 Hz; in vigabatrin plus KRS: 2.0 Ϯ 0.2 Hz; n ϭ 7; p Ͻ 3.8 Ϯ 0.5 Hz; in NNC-711 ϩ vigabatrin plus KRS: 2.1 Ϯ 0.4
0.001; paired t test) accompanied by hyperpolarization of the Hz; n ϭ 9; p Ͻ 0.001; paired t test) and hyperpolarization by
membrane potential (⌬Vm ϭ Ϫ0.8 Ϯ 0.3 mV; n ϭ 7; p Ͻ 0.05; Ϫ0.7 Ϯ 0.2 mV (n ϭ 9; p Ͻ 0.05; paired t test) (Fig. 7C). These
paired t test) (Fig. 7, A and C). The persistence of the KRS- results indicated that GABA reuptake transporters, GATs, or
5Me-4-OCF3 effect in the presence of vigabatrin indicated GABA transaminase (GABA-T) did not block KRS-5Me-4-
that enaminone-evoked neuronal inhibition was not medi- OCF3-evoked neuronal inhibition, suggesting that extracel-
ated by GABA-T.
lular enhancement of GABA levels did not contribute to the
To determine whether enaminones interacted with GATs pharmacological effects of KRS-5ME-4-OCF3.
to regulate GABA reuptake and whether GATs modulate
Substituted Anilino Enaminone Exhibited Charac-
KRS-5Me-4-OCF3-evoked neuronal inhibition, we applied teristics of a Positive Allosteric Modulator of GABAA
NNC-711, a potent and selective inhibitor of GABA uptake by Receptor. We showed that KRS-5Me-4-OCF3 significantly
GAT-1, and (S)-SNAP 5114, a selective inhibitor by GAT-3 enhanced the inhibitory effect of GABA on MC activity (Fig.
and GAT-2. Bath application of NNC-711 (10 ␮M) reduced 6B), i.e., the enaminone inhibited the firing rate to 2.9 Ϯ 0.3
MC spiking (in control: 4.4 Ϯ 0.5 Hz; in NNC-711: 3.8 Ϯ 0.3 Hz in ACSF (Fig. 3) and it suppressed the firing rate to 0.7 Ϯ
Hz; n ϭ 5; p Ͻ 0.05; paired t test) but did not significantly 0.2 Hz in the presence of 50 ␮M GABA (Fig. 6B) (p Ͻ 0.05;
modulate the membrane potential (⌬Vm ϭ Ϫ0.38 Ϯ 0.09 mV; determined by ANOVA and Bonferroni post hoc analysis).
n ϭ 5; p Ͼ 0.05). In the presence of NNC-711, KRS-5Me-4- The enhancement of the inhibitory effect of bath-applied
OCF3 further reduced the firing rate (in NNC-711: 3.9 Ϯ 0.3 GABA suggested that KRS-5Me-4-OCF3 acted as a positive
Hz; in NNC-711 plus KRS: 2.3 Ϯ 0.2 Hz; n ϭ 22; p Ͻ 0.001; allosteric modulator of GABAA receptors.
paired t test) and hyperpolarized MCs by Ϫ0.8 Ϯ 0.1 mV (n ϭ
It has been established that the GABAA receptor is the
22; p Ͻ 0.01). Likewise, bath application of (S)-SNAP 5114 main target for positive allosteric modulators such as benzo-
(20 ␮M) reduced MC spiking (in control: 3.5 Ϯ 0.6 Hz; in diazepines (Mo¨hler et al., 2002; Munro et al., 2008; Fisher,
SNAP: 2.7 Ϯ 0.4 Hz; n ϭ 4; p Ͻ 0.05; paired t test) and 2009). By binding at a site distinct from the GABA binding
hyperpolarized membrane potential (⌬Vm ϭ Ϫ0.6 Ϯ 0.1 mV; site and by increasing GABA affinity for the GABAA receptor,
n ϭ 4; p Ͻ 0.05). In the presence of (S)-SNAP 5114, KRS- positive allosteric modulators facilitate an augmentation of
5Me-4-OCF3 further reduced the firing rate (in SNAP: 3.0 Ϯ GABAA receptor function. To examine the concentration de-
0.5 Hz; in SNAP 5114 plus KRS: 1.8 Ϯ 0.3 Hz; n ϭ 7; p Ͻ pendence of the inhibitory effect of KRS-5Me-4-OCF3 on neu-
0.001; paired t test) and hyperpolarized MCs by Ϫ0.9 Ϯ 0.1 ronal activity and to test whether KRS-5Me-4-OCF3 behaved
mV (n ϭ 7; p Ͻ 0.05). The results indicate that the inhibitory like a positive allosteric modulator the concentration-re-
effects of KRS-5Me-4-OCF3 persisted in the presence of sponse relationships of KRS-5Me-4-OCF3 and GABA in the
GABA reuptake inhibitors (Fig. 7, B and C).
absence and presence of KRS-5Me-4-OCF3 (0, 5, and 20 ␮M)
The effects of KRS-5Me-4-OCF3 on MC activity were also were measured (Fig. 8). The averaged inhibitory effects
tested in the presence of both the GABA reuptake inhibitor evoked by varying concentrations of the enaminone (Fig. 8A)
NNC-711 (10 ␮M) and the GABA-T inhibitor vigabatrin (100 and GABA (Fig. 8B) were well fit by the Hill equation and
␮M). Under this condition, application of 20 ␮M KRS-5Me- allowed us to estimate an EC50. Based on a fitted Hill coef-
ficient (n) value of 1.11 in Fig. 8A, it seemed that the stoi-
chiometry of drug and receptor interaction was 1:1. The in-
hibitory effect of KRS-5Me-4-OCF3 on neuronal activity was
concentration-dependent with the estimated value of 24.5
␮M EC50. Figure 8B shows that GABA evoked concentration-
dependent inhibition, and the concentration-response curves
were left-shifted by KRS-5Me-4-OCF3. The shift of the con-
centration-response relationships suggested that KRS-5Me-
4-OCF3 mostly likely bound at non-GABA binding sites on
the GABA receptor. The EC50 of GABA fitted by the Hill
equation was 28.8 ␮M for GABA only, 19.9 ␮M for GABA
plus 5 ␮M KRS-5Me-4-OCF3, and 10.5 ␮M for GABA plus 20
␮M KRS-5Me-4-OCF3. The affinity of GABA for GABA bind-
ing sites was enhanced by KRS-5Me-4-OCF3, which sug-
gested an action of KRS-5Me-4-OCF3 as a positive allosteric
modulator of the GABAA receptor. The property provides a
cellular mechanism that accounts for the anticonvulsant ef-
fects of KRS-5Me-4-OCF3 in vivo.
The Enhancement of GABA by KRS-5Me-4-OCF3 Is
through Binding at the Classic Benzodiazepine Site.
Fig. 7. Neither an inhibitor of GABA reuptake nor an inhibitor of the
Previous in vivo studies reported that enaminones show po-
GABA degradation enzyme GABA-T blocked KRS-5Me-4-OCF3-evoked
tent anticonvulsion effects in chemical-induced epilepsy an-
imal models but fewer side effects such as sedation, drowsi-
ness, and dizziness (Mulzac and Scott, 1993; Eddington et al.,
2003). Based on these in vivo results and our in vitro results,
we hypothesized that KRS-5Me-4-OCF3 might bind to ben-
MC inhibition. A, original recording from a MC showing that spiking
inhibition evoked by KRS-5Me-4-OCF3 persisted in the presence of viga-
batrin (200 ␮M). B, original recording from a MC showing that spiking
inhibition evoked by KRS-5Me-4-OCF3 persisted in the presence of NNC-
711 (10 ␮M). C, summary of results from normalized and averaged data.
ء, p Ͻ 0.05; ءءء, p Ͻ 0.001.

922
Wang et al.
itory potency depended on the chemical structure and con-
centration of the enaminone. Among the three compounds, at
the concentration tested, KRS-5Me-4-OCF3 showed the most
potent inhibition of spiking of MCs and evoked hyperpolar-
ization of the membrane potential. These results are consis-
tent with previous in vivo results that KRS-5Me-4-OCF3 is
the most potent anticonvulsant agent (Eddington et al.,
2003). Neither excitatory ionotropic glutamate receptors
(NMDA and non-NMDA receptors) nor inhibitory GABAB
receptors were involved in KRS-5Me-4-OCF3-evoked inhibi-
tion of neuronal activity. The KRS-5Me-4-OCF3-induced in-
hibition of activity was abolished by GABAA receptor antag-
onists, suggesting that the inhibition may act directly
through activation of GABAA receptors or indirectly through
an increase of extracellular GABA levels. Our results showed
that neither blockade of GABA reuptake nor blockade of
GABA-T influenced KRS-5Me-4-OCF3-evoked neuronal inhi-
bition, indicating that the inhibition by KRS-5Me-4-OCF3
was mediated through direct activation of GABAA receptors.
The left-shift of the concentration-response relationship of
enaminone KRS-5Me-4-OCF3 in the presence of GABA im-
plies that KRS-5Me-4-OCF3 binds to a site distinct from the
GABA binding site to enhance GABA activity. This property
indicates that KRS-5Me-4-OCF3 acted as a positive allosteric
modulator of the GABAA receptor. Thus, our results suggest
that KRS-5Me-4-OCF3 could be a potential medication as anxi-
olytic, anticonvulsant, anesthetic, and sedative-hypnotic.
Previously, nucleus accumbens and coronal hippocampal
slices in the rat brain have been used to study anticonvulsant
Fig. 8. The concentration-response curves of KRS-5Me-4-OCF3-evoked
inhibition and GABA in the presence of KRS-5Me-4-OCF3. A, the KRS-
5Me-4-OCF3-evoked change in spiking rate was normalized to the control
condition, and then averaged. Each point was the mean value Ϯ S.E.M. of
four to seven cells. The line is fit for the data to the Hill equation: y ϭ Y0 enaminone suppression of excitatory synaptic transmission
ϩ Axn/(Kna ϩ xn), where y is the inhibition of spiking rate, x is concentra-
and epileptiform activity (Kombian et al., 2005; Ananthalak-
shmi et al., 2007). The MOB is anatomically different from
tion of drugs, Y0 is minimal inhibition, A is maximal inhibition, Kd is the
apparent dissociation constant for agents, and n is the Hill coefficient. Kd
nucleus accumbens and hippocampus and provides three ad-
vantages to test cellular mechanisms of anilino enaminone
action. First, epilepsy-related proteins such as GABAA recep-
tors, sodium channels, ionotropic glutamate receptors, and
metabotropic glutamate are expressed in MCs. Second, MCs
show the property of spontaneous spiking, and epilepsy-re-
lated proteins participate in the regulation of neuronal spik-
ing. Third, the excitability of MCs can be regulated by syn-
aptic input. Thus, MCs in MOB slices serve as a good model
for testing the bioactivity of enaminone compounds and ex-
ploring the mechanisms underlying their activity. The strat-
egy we used was to 1) test the effects of synthesized enami-
nones on neuronal activity and 2) determine the cellular
basis of their pharmacological actions.
Mechanism Underlying the Suppression of Neuronal
Activity. The subcutaneous pentylenetetrazol seizure model
identifies compounds that inhibit the GABA antagonistic
effects of pentylenetetrazol or raise the seizure threshold
(Stables and Kupferberg, 1997). Studies have shown that a
number of enaminone compounds display inhibition against
glutamate-mediated excitatory synaptic transmission by
modulation of GABAergic transmission (Kombian et al.,
2005; Ananthalakshmi et al., 2006). Based on the above
findings and the results we present, we hypothesize the ex-
and n were estimated using a Marquadt nonlinear least-squares routine.
B, shift of the concentration-response curves of GABA in the presence of
KRS-5Me-4-OCF3 at different concentrations (0, 5, and 20 ␮M). The lines
are fits for the data to the Hill equation.
zodiazepine sites to exert its pharmaceutical actions. There-
fore, an antagonist of the benzodiazepine site was used to
ascertain whether the enhancement of GABA by KRS-5Me-
4-OCF3 was mediated at the classic benzodiazepine site.
Bath application of flumazenil (10 ␮M), a benzodiazepine
site antagonist, slightly increased MC spiking from 3.3 Ϯ 0.6
Hz (in ACSF) to 3.8 Ϯ 0.8 Hz (n ϭ 6; p Ͻ 0.05; paired t test),
but did not significantly modulate the membrane potential
(⌬Vm ϭ Ϫ0.02 Ϯ 0.08 mV; n ϭ 6; p Ͼ 0.05). In the presence
of flumazenil, KRS-5Me-4-OCF3 failed to evoke the inhibi-
tory effect on the firing rate (in flumazenil: 3.7 Ϯ 0.7 Hz; in
flumazenil plus KRS: 3.7 Ϯ 0.8 Hz; n ϭ 19; p Ͼ 0.05; paired
t test) and on the membrane potential (Vm ϭ 0.01 Ϯ 0.1 mV;
n ϭ 19; p Ͼ 0.05). The blocked inhibitory effects of KRS-5Me-
4-OCF3 by flumazenil suggested that KRS-5Me-4-OCF3
binds at the benzodiazepine site to exert its pharmacological
actions.
Discussion
Here, we provide the first report that the enaminone KRS- istence of an essential pharmacophore within the enaminone
5Me-4-OCF3 acted as a novel positive allosteric modulator to structure that possibly interacts with the GABA receptor,
decrease neuronal activity via direct regulation of GABAA which is significant for achieving anticonvulsant activity.
receptors. Our study showed that the anilino enaminone Even though the exact site and structural requirements for
KRS-5Me-4-OCF3 and its analogs displayed inhibitory ef- optimal binding are unknown, we believe, because of the
fects on neuronal activity with different potencies. The inhib- molecular similarities between the enaminone analogs, the

Anilino Enaminone Activity through GABAA Receptor
923
compounds we tested share a common binding pocket on the diazepine sites to exert its pharmaceutical actions. There-
GABA receptor, which explains the probability of eliciting fore, in addition to the reported pharmacological action of
similar biological responses.
KRS-5Me-4-OCF3 as anticonvulsant, it is reasonable to spec-
The three compounds that we tested in the present study ulate that KRS-5Me-4-OCF3 might display an anxiolytic ef-
have been reported previously for their different potency of fect in a clinical setting.
anticonvulsion in vivo (Eddington et al., 2003). The com-
pound KRS-5Me-4-OCF3, which is the most potent anticon-
vulsant in vivo (Eddington et al., 2003), possessed the most
potent inhibitory effect on neuronal activity in vitro. The
consistency of the neuronal inhibition in vitro and the anti-
convulsant activity in vivo suggests that the anticonvulsant
activity of KRS-5Me-4-OCF3 results from preventing overex-
citability in epilepsy. The consistent in vitro and in vivo
results also suggest that recording in MCs is an appropriate
means for elucidating the bioactivity of enaminones.
A Specific Substituted Site in the Chemical Struc-
ture of Enaminones May Be Required for Targeting
GABAA Receptors and Conferring Anticonvulsant Ac-
tivity. The three enaminones studied in the present article,
KRS-5Me-4-OCF3, KRS-5Me-4-F, and KRS-5Me-3-Cl (Fig.
2), differed in their ability to suppress neuronal activity at
the concentration tested. Our results indicated that a para-
substitution of the phenyl group with –OCF3 evoked the most
potent suppression of neuronal excitability, whereas a para-
or meta-substitution of the phenyl group with fluoro, chloro
decreased the potency of the inhibitory activity. This result
suggests that a substitution in the phenyl group most
strongly influences the potency of the inhibitory action of
enaminones. The importance of the substitution group was
also demonstrated in benzylamino enaminones in which un-
substituted benzylamine analog compound 30 (Fig. 2) showed
the most potent activities in anticonvulsion and excitatory
synaptic depression (Edafiogho et al., 2006).
The mechanism underlying the neuronal inhibition by the
anilino enaminone KRS-5Me-4-OCF3 is different from recent
evidence obtained for another anilino enaminone, E139 (Fig.
2). The suppression of excitatory synaptic transmission
evoked by E139 may be indirectly mediated through en-
hancement of GABA levels (Kombian et al., 2005). The most
striking difference in the chemical structure of E139 and
KRS-5Me-4-OCF3 is in the ortho-substitution of the cyclo-
hexenone moiety. E139 has an ortho-substitution of the cyc-
lohexenone moiety, whereas the three compounds we tested
in this study were not ortho-substituted in cyclohexenone.
Our results indicate that nonsubstituted enaminones in or-
tho-cyclohexenone such as KRS-5Me-4-OCF3 act as a positive
allosteric modulator of GABAA receptors. Based on the chem-
ical structure and our bioactivity analysis, we postulate that
the ortho-site of cyclohexenone plays an important role in
determining the interaction with a target protein. A study on
benzylamino enaminones, which possess a similar structure
to anilino enaminones, demonstrates a completely different
cellular mechanism of action on excitatory synaptic depres-
sion and anticonvulsion (Edafiogho et al., 2006). Benzyl-
amino enaminone compound 30 was found to depress gluta-
mate-mediated excitatory synaptic transmission. In addition,
enaminones without ortho-substitution of cyclohexenone that
target GABAA receptors were described in several patented
enaminones (Reitz et al., 1999; Yohannes et al., 2003). There-
fore, we presume that substituted enaminones with differ-
ent site substitutions may form different pharmacophores
targeting specialized proteins. The chemical and pharma-
cological analysis of the structure-response relationship
may provide a new means for guiding rational drug design
for potential anticonvulsant and anxiolytic compounds.
Both GABAA and GABAB receptors are present in the
MOB. They participate in the regulation of MC excitability in
distinct ways. GABAA receptors directly regulate the excit-
ability of MCs, whereas GABAB receptors mediate the regu-
lation of MC excitability via presynaptic inhibition in the
MOB (Shepherd et al., 2004; Ennis et al., 2007). Antiepileptic
drugs such as benzodiazepines are known to interact with
GABAA receptors (Rall and Schleifer, 1990). This supports
our results that the enaminone KRS-5Me-4-OCF3 does not
interact with GABAB receptors. Rather it interacts directly
with GABAA receptors to decrease neuronal activity of MCs.
Enaminones with different chemical structure have been
reported to display distinct mechanisms underlying their
neuronal inhibition and anticonvulsant effects. Another an-
ticonvulsant enaminone, E139 (Fig. 2), was reported to sup-
press excitatory synaptic transmission by enhancing extra-
cellular GABA levels (Kombian et al., 2005) and blocking
tetrodotoxin-sensitive sodium channels and, thereby, directly
inhibiting postsynaptic neuronal excitability (Ananthalak-
shmi et al., 2006). Meanwhile, several enaminones with
chemical moieties different from KRS-5Me-4-OCF3 were de-
scribed to interact with GABAA receptors (Reitz et al., 1999;
Yohannes et al., 2003; Hogenkamp et al., 2007). Other
enaminone derivatives that probably target GABA receptors
were provided by using the comparative molecular field anal-
ysis and comparative molecular similarity techniques, which
can generate models to define the specific structural and
electrostatic features essential for enhanced binding of
enaminones to the putative GABA receptor (Jackson et al.,
2009).
The GABAA receptor is a ligand-gated ion channel respon-
sible for mediating the effects of GABA, the major inhibitory
neurotransmitter in the brain. The GABAA receptor complex
has been reported to have distinct binding sites for GABA,
benzodiazepines, barbiturates, ethanol (Santhakumar et al.,
2007), inhaled anesthetics, and neuroactive steroids. Positive
allosteric modulators enhance the affinity of GABA for the
binding site. Allosteric modulators of GABAA receptors such
as benzodiazepines, neuroactive steroids, and barbiturates
have been identified that are useful as anxiolytics, anticon-
vulsants, anesthetics, and sedative-hypnotics. Previous in
vivo studies reported that enaminones show potent anticon-
vulsion effects in chemical-induced epilepsy animal models
but fewer side effects such as sedation, drowsiness, and diz-
ziness (Mulzac and Scott, 1993; Eddington et al., 2003).
Based on these in vivo results and our results, we hypothe-
sized and confirmed that KRS-5Me-4-OCF3 binds to benzo-
Authorship Contributions
Participated in research design: Wang and Heinbockel.
Conducted experiments: Wang and Sun.
Contributed new reagents or analytic tools: Jackson and Scott.
Performed data analysis: Wang and Heinbockel.

924
Wang et al.
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17:133–140.
Kombian SB, Edafiogho IO, and Ananthalakshmi KV (2005) Anticonvulsant enami-
nones depress excitatory synaptic transmission in the rat brain by enhancing
extracellular GABA levels. Br J Pharmacol 145:945–953.
Laurie DJ, Seeburg PH, and Wisden W (1992) The distribution of 13 GABAA
receptor subunit mRNAs in the rat brain. II. Olfactory bulb and cerebellum.
J Neurosci 12:1063–1076.
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Wrote or contributed to the writing of the manuscript: Wang, Jack-
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Other: Heinbockel acquired funding for the research.
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