Molecular docking and panicolytic effect of 8-prenylnaringenin in the elevated T-maze

Article abstract of DOI:10.1248/cpb.c14-00569

The purpose of this study was to investigate the effects of the chronic administration of a racemic mixture of 8-prenylnaringenin (8-PN) on rats submitted to the elevated T-maze (ETM) model of generalized anxiety and panic disorders. The selective serotonin (SERT) reuptake inhibitor fluoxetine was used as a positive control. Rat locomotion was assessed in a circular arena following each drug treatment. The administration of racemic 8-PN for 21 d in rats increased one-way escape latencies from the ETM open arm, indicating a panicolytic effect. To evaluate the interactions of 8-PN with monoamine transporters, a docking study was performed for both the R and S configurations of 8-PN towards SERT, norepinephrine (NET) and dopamine transporters (DAT). The application of the docking protocol showed that (R)-8-PN provides greater affinity to all transporters than does the S enantiomer. This result suggests that enantiomer (R)-8-PN is the active form in the in vivo test of the racemic mixture.

Full text of DOI:10.1248/cpb.c14-00569

December 2014
Regular Article
Chem. Pharm. Bull. 62(12) 1231–1237 (2014)
1231
Molecular Docking and Panicolytic Effect of 8-Prenylnaringenin
in the Elevated T-Maze
Mariane Cristovão Bagatin,a Camila Santos Suniga Tozatti,a,† Layara Akemi Abiko,a,‡
Diego Alberto dos Santos Yamazaki,a Priscila Rebeca Alves Silva,b Leonardo Martins Perego,b
Elisabeth Aparecida Audi,b Flavio Augusto Vicente Seixas,c Ernani Abicht Basso,a and
,a
Gisele de Freitas Gauze*
b
a Departamento de Química, Universidade Estadual de Maringá; Departamento de Farmacologia e Terapêutica,
c
Universidade Estadual de Maringá; Maringá, PR 87020–900, Brazil: and Departamento de Bioquímica,
Universidade Estadual de Maringá; Umuarama, PR 87506–370, Brazil.
Received August 6, 2014; accepted September 5, 2014
The purpose of this study was to investigate the effects of the chronic administration of a racemic mixture
of 8-prenylnaringenin (8-PN) on rats submitted to the elevated T-maze (ETM) model of generalized anxiety
and panic disorders. The selective serotonin (SERT) reuptake inhibitor fluoxetine was used as a positive
control. Rat locomotion was assessed in a circular arena following each drug treatment. The administration
of racemic 8-PN for 21d in rats increased one-way escape latencies from the ETM open arm, indicating a
panicolytic effect. To evaluate the interactions of 8-PN with monoamine transporters, a docking study was
performed for both the R and S configurations of 8-PN towards SERT, norepinephrine (NET) and dopamine
transporters (DAT). The application of the docking protocol showed that (R)-8-PN provides greater affinity
to all transporters than does the S enantiomer. This result suggests that enantiomer (R)-8-PN is the active
form in the in vivo test of the racemic mixture.
Key words animal model; anxiety; panic; molecular docking; monoamine transporter’s inhibitor
Anxiety disorders, such as generalized anxiety disorder, than coumestrol, genistein and daidzein)6,7) being able to bind
panic disorder, obsessive compulsive disorder and phobias, to both α- and β-oestrogen receptors.8) Additionally, 8-PN is
affect one eighth of the world population and have become an also recognized for its action as an inflammation inhibitor,9) in
important area of interest in psychopharmacology research.1,2) angiogenesis10) and against cancer cell proliferation.11,12)
Genetic and neurobiological similarities between anxiety and
depressive disorders have been investigated.
Some studies have examined the effects of phytoestrogens
on brain and behavior.13–17) Lephart et al. investigated the
Antidepressant compounds are considered the first choice influence dietary phytoestrogens on anxiety-related behaviors.
treatment of anxiety and depression disorders. Blockage of Animal fed with a phytoestrogen-rich diet (genistein–daid-
serotonin (SERT), norepinephrine (NET) and dopamine trans- zein) showed a significant reduction of anxiety-like indicators
porters (DAT), which cover the majority of antidepressant in the elevated plus-maze when compared to control animal.
drug targets, increases the level of these neurotransmitters
Besides that the flavanoid 8-prenilnaringenin present a
in the synaptic cleft. Thus, the biogenic amine concentration strutuctural similarity with 1,3,7-trihydroxy-2-(3-methylbut-
in the central nervous system (CNS) is maintained by these 2-enyl)-xanthone (Fig. 1), a xanthone extract of Kielmeyera
transporters.3)
coriacea steams, popularly known as “Pau Santo.” This xan-
Selective serotonin reuptake inhibitors (SSRIs) and 5-HT/ thone proved to be a prototype drug useful in mood disorders
NE reuptake inhibitor (SNRI)4) are well tolerated and have a such as anxiety, panic and depression, or may indeed to be a
better side effect profile than the traditional tricyclic antide- beneficial adjunctive treatment, improving the efficacy of anti-
pressant (TCA) class,3) but all of them have limitations, such depressant drugs and/or accelerating the effects of these drugs
as delayed therapeutic improvement. For this reason, continual in patients with mood disorders.18)
efforts have been made to develop new efficacious drugs with
In light of the above, the aim of the present work was
earlier therapeutic effect onset and better side effect profiles.5) to synthesize and investigate the anxiolytic and panico-
8-Prenylnaringenin (Fig. 1), together with xanthohumol lytic effects of 8-PN on rats subjected to the elevated T-maze
and isoxanthohumol, is a member of a large group of prenyl-
ated chalcones and flavanones isolated from common hop
(Humulus lupulus). Their major source in human diet is beer,
as hop female flowers are used as a flavouring agent and as
a beverage preservative. 8-Prenylnaringenin (8-PN) has been
identified as the most potent plant phytoestrogen (more active
The authors declare no conflict of interest.
† Present address: Instituto Federal de Mato Grosso do Sul-Campus Coxim;
Coxim, MS 79400–000, Brazil.
‡ Present address: Instituto de Química, Universidade de São Paulo; São
Fig. 1. Structure of 8-Prenylnaringen (1) and 1,3,7-Trihydroxy-2-(3-
Paulo, SP 05508–000, Brazil.
methylbut-2-enyl)-xanthone (2)
© 2014 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: gfgbandoch@uem.br

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(ETM) test. Moreover, to evaluate the interactions of 8-PN Fig. 2A show that the ETM inhibitory avoidance latency was
with monoamine transporters, we performed a docking study not affected by any of the drug treatments. RM-ANOVA
for both R and S configurations of 8-PN towards SERT, NET showed a significant effect of trial [F(2.40)=47.82, p=0.001],
and DAT.
but no significant effect of treatment [F(2.20)=1.048, p=0.36]
or a significant treatment X trial interaction [F(2.40)=0.80,
p=0.52]. Figure 2B shows that 8-PN (10mg/kg) and fluoxetine
Results and Discussion
Chemistry Flavanoid 8-PN (1) was synthesized in race- (15mg/kg) significantly increased the ETM one-way escape
mic form using a convenient four-step sequence from com- latency. RM-ANOVA showed a significant main effect of
mercially available ( )-narigenin (3) following the procedure treatment [F(2.20)=4.91, p=0.018] but no significant effect of
described by Humpel et al.19) (Chart 1). This method is based trial F(2.40)=0.19, p=0.82] and no treatment X trial interac-
in method described by Gester et al.20) but includes several tion [F(4.40)=1.58, p=0.198]. Post-hoc comparisons showed
improves, as no chromatographic steps are needed and the that 8-PN (10mg/kg) significantly increased escape 2 latency
amount of catalyst is drastically reduced. First, the chemose- (p=0.03) and fluoxetine increased escape 3 latency (p=0.02)
lective acetylation of C(7) and C(4′) phenolic hydroxyl groups when compared to the control group, which was interpreted as
of 3 was carried out following installation of the requisite pre- a panicolytic-like effect.
nyl ether moiety at C(5). Mitsunobu reaction of 4 with prenyl
In Fig. 3, the ANOVA shows that none of the drug treat-
alcohol smoothly delivered the desired substrate 5 for the key ments used significantly affected the distance travelled by the
sigmatropic event. Next, europium(III)-catalysed Claisen– rats in the circular arena [F(2.20)=1.43, p=0.26].
Cope rearrangement of the prenyl group from C(5) to C(8)
The ETM test is the only test that involves two tasks that
position occurred. The obtained product 6 is directly used are performed by the same rat: inhibitory avoidance and one-
for step 4. Finally, the diacetate was subjected to potassium way escape. For the first one, the rat is placed at the distal end
carbonate-catalysed hydrolysis for deprotection of the C(7) of the enclosed arm and the time to withdraw from this arm
and C(4′) phenolic hydroxyl groups.
with the four paws is measured in three successive trials. For
Pharmacological Evaluation The results illustrated in the escape task, the rat is placed at the end of one of the open
Chart 1. Synthetic Route of 8-PN
Fig. 2. Effects (Mean S.E.M.) of Repeated Administration with Vehicle (Control Group), Fluoxetine or ( ) 8-PN on Inhibitory Avoidance in Rats
(Panel A) and One-Way Escape Latencies (Panel B) in ETM Test (n=7–9)
*p<0.05 compared to the control group.

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Fig. 4. Structures of the Ligands Evaluated in This Study
binding pocket of leucine in the LeuT crystal structure. The
principal ligand-receptor interactions were analysed and the
best orientation for each model and both 8-PN enantiomers
were compared (Fig. 5).
Figures 5A and B show the docking orientation of (R)-8-PN,
(S)-8-PN and (R)-fluoxetine (model) in SERT. The trifluo-
rophenyl moiety of fluoxetine is stabilized through van der
Waals contact with residues TYR175, TYR176 and PHE335.
Fig. 3. Effects (Mean S.E.M.) of Repeated Administration of ( )
8-PN, Fluoxetine or Vehicle (Control) for 21d on the Distance Travelled
by Rats for 300s in the Circular Arena (n=7–8), p>0.05, Compared to
the Control Group
arms and the withdrawal latency is measured three times. The aromatic ring that contains this group has stacking inter-
Pharmacological validation has shown that the drug profile actions with TYR175 and ILE179. The hydrogen of the proton-
of inhibitory avoidance is similar to that of generalized or ated amino group forms hydrogen bonds with GLU493.
anticipatory anxiety, while the drug response of escape is akin
For enantiomer (R)-8-PN (Fig. 5A), one can see that the pre-
to that of panic disorder.21) As a consequence, the ETM is con- nyl group is stabilized by van der Waals contact with TYR176,
sidered a sound animal model of both anxiety and panic.22,23) TYR175 and PHE335, the same residues that stabilize the
Regarding panic, performance of the one-way escape in the trifluoromethyl group in the standard. Additionally, three hy-
ETM has been shown to be impaired by several antidepressant drogen bonds can be observed, one between the carbonyl and
drugs following chronic, but not acute, administration, paral- LYS399, another between hydroxyl 7 of ring A and TYR175,
leling the therapeutic response.21)
and the other between hydroxyl 4′ of ring B and ASP400. For
The administration of racemic 8-PN for 21d to rats submit- enantiomer (S)-8-PN (Fig. 5B), the prenyl group is in a region
ted to the ETM consistently impaired the escape indication of defined by ILE179, LYS399 and ASP400, in contrast to what
panicolytic effect. The acquisition of inhibitory avoidance was occurs for enantiomer (R)-8-PN and the trifluoromethyl group
not affected by the drugs, showing no anxiolytic effect. These in fluoxetine. Additionally, hydrogen bonds were observed
effects are probably not due to unspecific motor effects, since between hydroxyl 7 and 4′ of rings A and B with LYS399 and
the same drug treatments failed to significantly affect locomo- TYR176, respectively.
tion, measured in the circular arena. SSRI fluoxetine, used as
Figures 5C and D show the active site of transporter nor-
positive control in our study, confirmed the panicolytic effect epinephrine (NET) complexed with standard (S,S)-reboxetine
in the ETM observed in another study. Fluoxetine has been and enantiomers (R)-8-PN and (S)-8-PN, respectively. The
proven to be effective in treating panic disorder and shown aromatic rings of the standard are stabilized by van der Waals
to increase extracellular 5-HT in dorsal periaqueductal grey contact with TRP80, ALA77, ARG81 and ASP473. The amine
(dPAG).24)
group makes a hydrogen bond with THR381. The prenyl
group of enantiomer (R)-8-PN (Fig. 5C) is stabilized by van
de Waals contact with ALA77, ARG81 and ASP 473, which
are the same residues that stabilize the aromatic rings of the
standard, while the prenyl group of (S)-8-PN (Fig. 5D) is sta-
bilized by van der Waals contact with THR470, THR381 and
GLU382.
Figure 5E shows the active site of the dopamine transporter
(DAT) complexed with standard (S)-bupropion and enantiomer
(R)-8-PN. The aromatic ring of the standard is stabilized by
van de Waals contact with ALA81, ILE159 and PHE155, and
the tert-butyl group with LYS384 and TYR88. In (R)-8-PN,
the prenyl group is stabilized by van der Waals contact with
PHE472, THR473 and LYS384; furthermore, there is a hy-
drogen bond between ARG85 and hydroxyl 4′ of ring B. The
molecular docking of enantiomer (S)-8-PN showed that it does
not overlap in the active site of this transporter effectively.
Table 1 compares the calculated (Autodock) and experimen-
Acute treatment with the same doses of 8-PN (10mg/kg)
produced no effects in rats submitted to ETM (results not
shown). Our results confirm existence of a latency period until
the onset of the therapeutic action. This effect is also observed
for other compounds with antidepressant profile, a period of
about 21d is necessary for adaptive changes in serotonin re-
ceptors located in the raphe nucleus, as well as raising differ-
ent post-synaptic receptors.25)
Molecular Docking In order to rationalize the tendency
of activity of the compounds under study, molecular docking
of (R)-fluoxetine, (S)-bupropion, (S,S)-reboxetine (Fig. 4) and
of both 8-PN enantiomers (Fig. 1) was performed with the ho-
mology models of SERT, NET and DAT constructed by Ravna
et al.26) It is important to point out that pharmacological tests
were conducted with the racemic mixture of the enantiomers.
The molecular docking assays were performed for each enan-
tiomer to verify the influence of the 8-PN configuration in the
binding mode with the transporters.
) of
tal (Binding Database) values of inhibition constants (Ki
The compounds were docked in the central binding pocket each of the ligands with their respective targets.
of SERT, NET and DAT, which corresponds to the substrate The Ki values reported in the Binding Database (literature)

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Fig. 5. Docking Orientation, Ligands Are Shown in Thick Tube and Residues in Thin Tube: (A) (R)-8-PN and (R)-Fluoxetine and (B) (S)-8-PN and
(R)-Fluoxetine in SERT; (C) (R)-8-PN and (S,S)-Reboxetine and (D) (S)-8-PN and (S,S)-Reboxetine in NET; (E) (R)-8-PN and (S)-Bupropion into DAT
show that (R)-fluoxetine has greater affinity to SERT, followed is the most probable target of (R)-fluoxetine. The e-values of
by DAT, which is confirmed by the lower theoretical Ki values this analysis indicate the probability of a false positive result,
given by Autodock. However, this ligand can also bind to therefore, the lower this value, the greater the reliability of the
NET with smaller affinity (greater Ki).
The prediction of the drug targets based on the ligand docking data agree with the experimental results.
structures by SEA predictions indicated that the SERT protein There are no data in the literature that indicate that (S,S)-
results. Summing up, these results show that the theoretical

December 2014
1235
Table 1. Comparison of the Calculated (Autodock) and Experimental (Binding Database) Inhibition Constants (Ki) Values of Each of the Ligands with
the Irrespective Targets and with the Score Function (pKD) Calculated with the Polscore Program
SERT
NET
DAT
Target drug
SEA predictions TARGET (e-value)
Autodock Ki Binding DB Ki Autodock Ki Binding DB Ki Autodock Ki Binding DB Ki
(nM)
(nM)
(nM)
(nM)
(nM)
(nM)
(R)-Fluoxetine
6470
0.7–2.0
16780
850–2186
7000
11–6670
SERT (3.1×10−31
)
NET (6.8×10−23
NET (4.7×10−36
)
(S,S)-Reboxetine
4490
—
1090
3060
0.3–15.8
12540
829
—
)
SERT (1.3×10−20
)
DAT (5.5×10−6
DAT (8.9×10−17
NET (2.0×10−3
)
(S)-Bupropion
12710
>10000
940–10000
441–871
)
)
R-8-Prenylnaringenin
S-8-Prenylnaringenin
1310
7290
—
—
2460
6110
—
—
1890
*
—
*
Oestrogen receptor β (8.1×10−3);
affinity: 57nM
Oestrogen receptor α (2.6×10−2);
affinity: 68nM
*No significant results.
reboxetine can bind to SERT or DAT; however, NET is known This effect is also observed for other compounds with antide-
to be its main target. Again, the theoretical Ki values from pressant profile.
Autodock confirmed this observation. The small theoretical
The docking simulations performed in this study were
Ki calculated for the (S)-bupropion showed that this ligand has guided by the crystallographic pose of ligand (R)-fluoxetine,
greater affinity to DAT, again in agreement with the literature. a compound with experimentally demonstrated binding to the
This validation shows that the reproducibility of the in three transporters evaluated in this study. Furthermore, the
silico experimental data based on classical ligands of receptors excellent correlation between the experimental and theoreti-
SERT, NET and DAT provides reliable theoretical results and cal data obtained for the docking of ligands (S,S)-reboxetine
thus allows the use of the docking protocol presented here in and (S)-bupropion leads to the conclusion that (R)-8-PN has
the evaluation of the interaction of other ligands with these greater affinity to the evaluated transporters than enantiomer
protein targets.
(S)-8-PN and, therefore, the former must be the active form in
The data in Table 1 show that (R)-8-PN presents greater the racemic mixture tested in vivo.
affinity (lower inhibition constant) for all transporters (SERT,
NET and DAT) than enantiomer (S)-8-PN, indicating that
(R)-8-PN interacts more effectively with the residues of the
active sites of the transporters.
Experimental
Chemistry Flavanoid 8-prenylnaringenin (8-PN) was
synthesized in racemic form using a convenient four-step
These results suggest that the panicolytic effect obtained sequence from commercially available ( )-narigenin (Sigma-
with the racemic form of 8-PN in the ETM test can be at- Aldrich, U.S.A.) following the procedure described by Humpel
tributed mainly to the R enantiomer employed. It is known et al.19) by distillation. The product is dried in vacuo at 40°C
that the activity of a drug is often related to only one of its overnight.
enantiomeric forms, such as is the case of the antidepressant
Pharmacology: Animals Male Wistar rats (State Univer-
escitalopram. Clinical and preclinical studies have shown that sity of Maringá) weighing 230–300g were housed in groups
escitalopram interacts more actively with SERT and that it is of five in Plexiglas-walled cages at a room temperature of
more efficacious than racemic citalopram.27,28) (R)-Citalopram 22 1°C, with alternating 12h:12h light/dark cycles (lights
counteracts the actions of escitalopram, and it has been hy- on from 07:00 to 19:00h) and free access to food and water,
pothesized that this antagonistic effect is mediated via interac- except during testing. The experimental procedures adopted
tion with the allosteric binding site on SERT.29)
were approved by the Committee of Ethical Conduct in the
Reboxetine, a potent and selective NET inhibitor ligand, is Use of Animals in Experiments of the State University of
marketed as a racemic mixture of (R,R)- and (S,S)-reboxetine. Maringá (072/2010-CEAE) and the recommendations for Bio-
It is a non tricyclic antidepressant drug, the (S,S) enantiomer medical Research Involving Animals (CIMS) (Geneva, 1985)
being two times more potent in receptor binding and in in were observed.
vivo models of norepinephrine re-uptake inhibition than the
Drugs The following drugs were used: 8-PN, fluoxetine
(Sigma, U.S.A.) or vehicle. All drugs were dissolved in sterile
(R,R) enantiomer.30,31)
Therefore, further pharmacological studies could be per- saline with 2% Tween 80. The SSRI, fluoxetine, was used as a
formed only with enantiomer (R)-8-PN to increase the panico- positive control.
lytic effect observed for the racemic mixture of 8-PN.
Apparatus The ETM was made of wood and has three
arms of equal dimensions (50×12cm). One arm, enclosed by
40-cm high walls, was perpendicular to two opposed open
Conclusion
This study showed that ( )8-PN promoted a specific pani- arms. To avoid falls, the open arms were rimmed with 1-cm
colytic effect on ETM test. Our results too confirm existence high Plexiglas. The whole apparatus was elevated 50cm above
of a latency period until the onset of the therapeutic action. the floor. Locomotion was measured inside a wooden circular

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arena (70cm diameter) with 30-cm high walls. Brightness at B3LYP/6–31++g(d,p) level.
the level of the maze arms and open-field centre was 60lx.
Preparation of the Receptors The structures of the
Procedure The ETM is an anxiety model that evokes two transporters of serotonin (SERT), dopamine (DAT) and
defensive responses in the same rat, namely, inhibitory avoid- norepinephrine (NET) modelled in Apo form were kindly
ance latency from the closed arm (baseline, avoidance 2 and provided by Ravna et al.26) The active sites were identified
3) and one-way escape latency from the open arm (escape 1, 2 through the overlapping of the three-dimensional structure
and 3), which have been related to generalized anxiety disor- of the LeuT submitted to crystallography with (R)-fluoxetine
der and panic disorder, respectively. Locomotion was assessed (PDB: 3GWV)42) in the three modelled transporters. Ligand
in the open field following each drug treatment as a control (R)-fluoxetine was added to the transporter structures by geo-
for nonspecific motor effects.
metric docking. LeuT from Aquifex aeolicus (pdb: 3GWV)
After 21d of treatment with 8-PN (10mg/kg), fluoxetine was chosen as a geometric docking mold for fluoxetine be-
(15mg/kg) or the vehicle were administered by gavage and be- cause this protein belongs to the Sodium Neurotransmitter
havioural tests were performed. The doses of fluoxetine were Symporter Family (SNF), the same as the transporters SERT,
chosen based on their effects in previous studies performed in DAT and NET (protein family id: PF00209), according to
the ETM.32) The doses of 8-PN were chosen based on in vivo evaluation performed by server Pfam.43) Furthermore, pdb
study using the adult rat model33) and in a previous study per- 3GWV is the only structural mold linked to (R)-fluoxetine that
formed in the ETM with a similar compound.34) On the 20th was available.
day of treatment, the animals were gently handled for 5min
(R)-Fluoxetine-receptor complexes were submitted to 60000
and pre-exposed to one of the open arms of the ETM for energy minimization steps by conjugated gradient for the re-
30min. The open arm exit was closed with a wooden barrier moval of any steric hindrance with the NAMD2 program.44)
mounted between the central area of the maze and the proxi- The objective of docking (R)-fluoxetine to the three receptors
mal end of the arm. It has been shown that such pre-exposure was to serve as a docking guide for other ligands which had
makes the escape task more sensitive to antipanic drugs, as it unknown bonding mechanisms.
shortens the withdrawal latencies from the open arm during
Ligand Docking The docking protocol was validated by
redocking assays of (R)-fluoxetine in the three minimized
the test.35) The ETM test was performed 24h later.
The ETM test started with the inhibitory avoidance task. complexes. The protocol was considered valid when the rmsd
Each animal was placed at the distal end of the enclosed calculated from the overlapping of the best pose onto the
ETM arm facing the intersection. The time that the rats took ligand was smaller than an average of 0.5Å in four assays of
to leave this arm with four paws was recorded (baseline la- each complex, in order to avoid false positive results.
tency). This measurement was repeated in two subsequent
The docking simulations for the standards (R)-fluoxetine,
trials (avoidance 1 and 2) at 30-s intervals. Thirty second after (S,S)-reboxetine and (S)-bupropion and for enantiomers
the last avoidance trial, the rats were placed at the end of the (R)-8-PN and (S)-8-PN were performed with the AutoDock
open arm to which they had been previously exposed and the 4.2.3 program implemented at the interface PyRx 0.9,45) ap-
latency to leave this arm with four paws was recorded in three plying the redocking-validated protocol (hybrid Lamarckian
consecutive trials (escape 1, 2 and 3) at 30-s intervals. A cut- Genetic Algorithm).
off time of 300s was established for avoidance and the escape
The energy evaluation grid was chosen according to the
latency. Thirty second after the ETM test, the animals were crystallographic structure of (R)-fluoxetine for each receptor
placed inside the circular arena for 5min to evaluate locomo- and was centred on coordinates (X=27.980, Y=20.251 and
tion. The total distance travelled was recorded with a video Z=24.870) in SERT, (X=28.301, Y=21.269 and Z=20.420) in
tracking system (Ethovision; Noldus, Holland) for analysis.
DAT and (X=25.639, Y=20.555 and Z=20.010) in NET, with
The results observed in the ETM and circular arena were grid points in the x, y and z axes set to 50×50×50 and sepa-
submitted to one-way ANOVA. When appropriate, the Dun- rated by 0.375Å. The initial population size and maximum
can post hoc test was used. The significance level was set at number of energy evaluations were set to 10. The docked
p<0.05. The Statistica Six Sigma software was used for statis- results within an rmsd of 2.0Å were clustered and the final
tical analysis.
results of each ligand were selected considering both the
Molecular Docking: Ligand Preparation Standards embedded empirical binding free energy evaluation and the
(R)-fluoxetine, (S,S)-reboxetine, (S)-bupropion (Fig. 4) were clustering analysis.
selected according to literature data36–39) and confirmed as
Energy Minimization The lowest energy poses obtained
the actually desired transporters using the SEA search tool.40) by docking the ligands (S,S)-reboxetine and (S)-bupropion
The three-dimensional structures of these compounds were and enantiomers (R)-8-PN and (S)-8-PN were exported, in-
obtained from the ZINC database (codes ZINC01530638, corporated into the receptor structures and submitted again
ZINC00002284 and ZINC00020228, respectively). It is note- to 60000 energy minimization steps with program NAMD2.
worthy that at pH 7.0, all standards have positively charged In the energy minimization procedures, the field force
nitrogen; the protonated version of the structures were used in CHARMM C35b2–C36a2 was adopted for the proteins, while
the molecular docking calculations.
for the ligands, it was generated in the same format by the
The enantiomers structures (R)-8-PN and (S)-8-PN were SwissParam server.46)
generated with the Gaussian 09 program.41) The stable ge-
Energy minimization was simulated with the complexes
ometries of the compounds were obtained by calculating immersed in a box with water at least 10Å from the protein
the potential energy surface (PES) through the HF/3-21G outermost surface. Either Na+ or Cl− counter ions were added
level of theory. The most stable geometries were optimized in appropriate amounts to neutralize the system charges. The
by density functional theory (DFT) calculations with the temperature and the pressure were adjusted to 300K and 1

December 2014
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25) Pineyro G., Blier P., Pharmacology Rev., 51, 534–579 (1999).
26) Ravna A. W., Sylte I., Dahl S. G., J. Mol. Model., 15, 1155–1164
(2009).
27) Sánchez C., Bergqvist P. B., Brennum L. T., Gupta S., Hogg S.,
Larsen A., Wiborg O., Psychopharmacology (Berl.), 167, 353–362
(2003).
atm.
After energy minimization, the protein–ligand complexes
were redocked using the same parameters used in docking,
giving rmsd values between 0.4 and 1.5Å, which made the
final results reliable.
28) Sánchez C., Bøgesø K. P., Ebert B., Reines E. H., Braestrup C.,
Psychopharmacology (Berl.), 174, 163–176 (2004).
Acknowledgments The authors thank the CNPQ for a
scholarship (M.C.B), a fellowship (E.A.B) and the financial
support. We also thank Ravna et al. by the structures of the
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