Binding of mazindol and analogs to the human serotonin and dopamine transporterss

Article abstract of DOI:10.1124/mol.113.088922

ABSTRACT Mazindol has been explored as a possible agent in cocaine addiction pharmacotherapy. The tetracyclic compound inhibits both the dopamine transporter and the serotonin transporter, and simple chemical modifications considerably alter target selectivity. Mazindol, therefore, is an attractive scaffold for both understanding the molecular determinants of serotonin/ dopamine transporter selectivity and for the development of novel drug abuse treatments. Usingmolecularmodeling and pharmacologic profiling of rationally chosen serotonin and dopamine transporter mutants with respect to a series of mazindol analogs has allowed us to determine the orientation of mazindol within the central binding site. We find that mazindol binds in the central substrate binding site, and that the transporter selectivity can be modulated through mutations of a few residues in the binding pocket. Mazindol is most likely to bind as the R-enantiomer. Tyrosines 95 and 175 in the human serotonin transporter and the corresponding phenylalanines 75 and 155 in the human dopamine transporter are the primary determinants of mazindol selectivity. Manipulating the interaction of substituents on the 7-position with the human serotonin transporter Tyr175 versus dopamine transporter Phe155 is found to be a strong tool in tuning the selectivity of mazindol analogs and may be used in future drug design of cocaine abuse pharmacotherapies. Copyright

Full text of DOI:10.1124/mol.113.088922

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http://molpharm.aspetjournals.org/content/suppl/2013/11/08/mol.113.088922.DC1.html
1521-0111/85/2/208–217$25.00
MOLECULAR PHARMACOLOGY
http://dx.doi.org/10.1124/mol.113.088922
Mol Pharmacol 85:208–217, February 2014
Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics
Binding of Mazindol and Analogs to the Human Serotonin and
Dopamine Transporterss
Kasper Severinsen, Heidi Koldsø, Katrine Almind Vinberg Thorup, Christina Schjøth-Eskesen,
Pernille Thornild Møller, Ove Wiborg, Henrik Helligsø Jensen, Steffen Sinning,
and Birgit Schiøtt
Laboratory of Molecular Neurobiology, Translational Neuropsychiatry Unit, Department of Clinical Medicine, Aarhus University
Hospital, Risskov, Denmark (K.S., K.A.V.T., P.T.M., O.W., S.S.); and iNANO and inSPIN Centers (H.K., B.S.) and Department of
Chemistry (C.S.-E., H.H.J.), Aarhus University, Aarhus, Denmark
Received August 2, 2013; accepted November 8, 2013
ABSTRACT
Mazindol has been explored as a possible agent in cocaine substrate binding site, and that the transporter selectivity can be
addiction pharmacotherapy. The tetracyclic compound inhibits
both the dopamine transporter and the serotonin transporter,
and simple chemical modifications considerably alter target
selectivity. Mazindol, therefore, is an attractive scaffold for
both understanding the molecular determinants of serotonin/
dopamine transporter selectivity and for the development of
novel drug abuse treatments. Using molecular modeling and phar-
macologic profiling of rationally chosen serotonin and dopamine
transporter mutants with respect to a series of mazindol analogs
has allowed us to determine the orientation of mazindol within the
modulated through mutations of a few residues in the binding
pocket. Mazindol is most likely to bind as the R-enantiomer.
Tyrosines 95 and 175 in the human serotonin transporter and
the corresponding phenylalanines 75 and 155 in the human
dopamine transporter are the primary determinants of mazindol
selectivity. Manipulating the interaction of substituents on the
7-position with the human serotonin transporter Tyr175 versus
dopamine transporter Phe155 is found to be a strong tool in
tuning the selectivity of mazindol analogs and may be used in
central binding site. We find that mazindol binds in the central future drug design of cocaine abuse pharmacotherapies.
hypothesis has been verified by knock-in of a cocaine-
Introduction
insensitive DAT (Chen et al., 2006; Thomsen et al., 2009).
Abuse of psychostimulants is a huge burden to society,
Several approaches to treat cocaine addiction have been tried;
resulting in financial, criminal, and health problems (Dutta
however, none of these has led to compounds used clinically
et al., 2003). Cocaine is one of the most powerful reinforcers
(Kharkar et al., 2008), and an urgent need for the develop-
known to date, often leading to dependence and abuse. Co-
ment of drugs against cocaine abuse remains.
caine is known to inhibit all three human monoamine trans-
The tetracyclic compound mazindol was first synthesized in
porters: the serotonin (hSERT), norepinephrine, and dopamine
1968 (Fig. 1). Like cocaine, mazindol binds to all human
transporters (hDATs). The most important site of action for
cocaine is the hDAT, which has led to the “dopamine hy-
pothesis” (Kuhar et al., 1991) stating that cocaine binds to the
transporter and blocks the reuptake of dopamine, leading to
monoamine transporters (Javitch et al., 1984; Angel et al.,
1988; Eshleman et al., 1999) and has been shown to be useful
as an appetite suppressant and has therefore been used
against obesity (Acri, 2008).
an increased concentration of this neurotransmitter in the
Mazindol functions with a favorable rapid rate of onset and
synaptic cleft. Consequently, enhancement and prolonged
binds with a higher affinity to hDAT than cocaine without
stimulation of the dopaminergic system occur (Kuhar et al.,
having significant side effects (Berger et al., 1989; Eshleman
1991), causing the strongly reinforcing effect of cocaine. This
et al., 1999; Chen and Reith, 2002), leading to the speculation
that mazindol could be used in the treatment of cocaine
This work was supported by the Lundbeck Foundation; the Carlsberg
addiction. Promisingly, mazindol reduces craving and eupho-
ria in cocaine-abusing patients in short-term studies (Berger
et al., 1989), although results from longer-term studies are not
significantly better than those observed in placebo-treated
patients (Stine et al., 1995). Mazindol was shown to cause
psychostimulation in monkeys, which might be assigned to a
different route of administration, pointing to pharmacokinetics
Foundation; the Novo Nordisk Foundation; the Danish Natural Science
Research Council; and the Danish National Research Foundation [Grant
DNRF59]. Computations were possible through allocations of time at the
Centre for Scientific Computing (Aarhus, Denmark).
K.S., H.K., K.A.V.T., C.S.-E., P.T.M., and S.S. contributed equally to this
work.
dx.doi.org/10.1124/mol.113.088922.
s
This article has supplemental material available at molpharm.
aspetjournals.org.
ABBREVIATIONS: hDAT, human dopamine transporter; hSERT, human serotonin transporter; IFD, induced fit docking; LeuT, Aquifex aolicus
leucine transporter; PaMLAC, paired mutation ligand analog complementation; QM, quantum mechanics; QPLD, quantum mechanics polarized
docking.
208

Computational and Biochemical Analysis of Mazindol Orientation
209
Fig. 1. (A) Chemical structures and atom numbering of mazindol and analogs used in this study, and (B) chemical structures of protonated serotonin,
dopamine, and cocaine.
as an important aspect of abuse potential in addition to DAT
inhibition (Chait et al., 1987; Dutta et al., 2003).
The two enantiomers of mazindol and its analogs exist in
a dynamic equilibrium between three isomers (the keto and
the R and S–ol forms, respectively) with the R or S–ol being
the only relevant forms at physiologic pH (Houlihan et al.,
2002). These two enantiomers may have markedly different
protein binding affinities, which cannot easily be measured
experimentally due their presumed easy interconversion. It
was necessary to computationally treat the two enantiomeric
–ol forms separately, which predicts that the two enantiomers
of mazindol are orientated in similar ways in both proteins.
The difference in affinity between the transporters can
thereby be assigned to the difference in amino acid composi-
tion in the binding pocket at primarily two positions and not
to differences in ligand binding. This allows us to present the
first biochemically validated binding mode of mazindol and
analogs inside the central binding site of hSERT and hDAT
pointing to R-mazindol as the biologically relevant enantio-
mer, which may facilitate progress toward the development of
antiabuse drugs.
Mazindol is selective toward hDAT over hSERT and has
been used as a starting molecule in the synthesis of different
functionalized analogs by Houlihan et al. (2002). Novel
analogs were synthesized and tested in this study (Fig. 1).
Here we explore the binding of mazindol to hDAT and hSERT
homology models to determine the molecular determinants of
affinity and selectivity. Inhibitory potencies of mazindol and
eight analogs (Fig. 1) for hSERT and hDAT mutants were
explored. Of the original mazindol analogs from Houlihan
et al. (2002), three have a significant preference for wild-type
hDAT over wild-type hSERT; compounds 1, 2, and 3 show
a more than 100-fold preference for hDAT, whereas analog 4
only shows 36-fold selectivity. The novel analogs reported
here were chosen to aid experimental validation of the mazindol
binding mode and they generally show lower potency; however,
two analogs show increased selectivity for hSERT. By muta-
genesis of a few residues lining the central binding pocket of
hSERT, we show that the selectivity of analog 2, and to some
extent analog 3, can be reversed from being predominantly
hDAT selective to equipotent against hSERT and hDAT. This
tendency can be rationalized from computational studies by
means of induced fit docking (IFD) (Sherman et al., 2006) and
quantum mechanics polarized ligand docking (QPLD) (Cho
et al., 2005) calculations of mazindol into homology models of
hDAT and hSERT.
Materials and Methods
The synthesis of analogs 1–4 is described by Houlihan et al. (2002).
Organic Synthesis
Mazindol analogs 5, 6, and 8 were prepared by procedures
analogous to those described by Houlihan and Parrino (1982). See
the Supplemental Experimental Procedures for details.

210
Severinsen et al.
The hSERT homology model used in the present study is a further
refined model based on our previously validated model of hSERT
with serotonin bound (Celik et al., 2008b), including the loop opti-
mization described by Koldsø et al. (2010, 2013b). The hDAT homol-
ogy model was constructed by the use of LeuT and the optimized
extracellular loop 2 of hSERT as templates and built in MODEL-
LER9v5 (Fiser et al., 2002; Eswar et al., 2007) as previously
described (Koldsø et al., 2013b). During the modeling procedure of
hDAT, 20 models of the protein were built. The main validation
criteria for these models were as follows: 1) the model quality as
measured by the probability density function, molpdf (Eswar et al.,
2007); 2) the stereochemical quality illustrated by a Ramachandran
plot using PROCHECK (Laskowski et al., 1993); 3) the size of the
central cavity binding site of the protein measured the solvent
accessible area method with a 1.2 Å probe in Molegro Virtual Docker
(version MVD2008.2.4; http://www.molegro.com) (Thomsen and
Christensen, 2006); and 4) the x1 angle of Asp79 in hDAT, which
should be 6gauche as was previously suggested (Jørgensen et al.,
2007; Celik et al., 2008b; Koldsø et al., 2010, 2013b).
Crystallography of Analog 5
Please see Supplemental Fig. 1 for details.
Mutagenesis
Mutations in hSERT or hDAT pcDNA inserted into the pcDNA3.1
vector were introduced by mismatched primer pairs in a polymerase
reaction using Phusion (Finnzymes, Vantaa, Finland). DNA was
purified from overnight XL10 Gold Escherichia coli cultures grown in
LB media supplemented with 200 ng/ml ampicillin using the Pure-
Yield Midiprep Kit (Promega, Madison, WI). Mutant DNA was
sequenced across the entire transporter open reading frame using
BigDye v3.1 chemistry (Applied Biosystems, Foster City, CA) ana-
lyzed on an ABI 3100 Sequencer (Applied Biosystems) to verify that
the transporter gene contained the desired mutations and that no
unwanted mutations had been introduced.
Cell Culture
The sodium ions found in the LeuT crystal structure were imported
with the same coordinates into the final hDAT and hSERT models,
and the chloride ion was manually introduced in both models at the
proposed binding site (Forrest et al., 2007; Zomot et al., 2007) followed
by a brief minimization.
Human embryonic kidney 293MSR cells (Invitrogen, Carlsbad, CA)
were cultured in Dulbecco’s modified Eagle’s medium (BioWhitaker,
Mississauga, ON, Canada) supplemented with 10% fetal calf serum
(Invitrogen), 100 U/ml penicillin, 100 mg/ml streptomycin (BioWhitaker),
and 6 mg/ml Geneticin (Invitrogen) at 95% humidity and 5% p(CO2)
at 37°C. Two days prior to the uptake assay, cells were detached by
Versene and trypsin/EDTA treatment and mixed with a preformed
complex of transporter DNA and LipofectAMINE 2000 (Invitrogen).
This transfection mix was dispensed into TC-treated white 96-well
plates (Nalgene Nunc International, Penfield, NY) at a cell density of
50–70% confluence and 0.167–0.333 mg DNA per cm2 and incubated
for 50–60 hours.
Ligand Preparation. Both enantiomers of mazindol were built
in Maestro (version 8.5; Schrödinger, LLC, New York, NY). See
Supplemental Fig. 2 for MacroModel atom types for mazindol. The
pKa values of ionizable groups were predicted by Epik (version 1.6;
Schrödinger, LLC) within the Schrödinger software. The nitrogen at
position 1, N1 (see Fig. 1 for atom labeling), had a predicted pKa value
of 9.12 and should be charged at physiologic pH. Furthermore,
because of the possibility of conjugation and charge delocalization
between N1 and N4 in the imidazoline ring, N1 and N4 were chosen to
be sp2-hybridized and thus planar. To investigate the structure of the
two enantiomers of mazindol more precisely, a quantum mechanics
(QM) optimization was made using the density functional theory
method B3LYP (Lee et al., 1988; Becke, 1993; Stephens et al., 1994)
with the 6-311G(d) basis set (Hehre et al., 1972). The QM optimized
structure deviates slightly from planarity around the charged conju-
gated nitrogen, N1, with a few degrees, which is most likely caused
by the intramolecular strain of the tricyclic ring system. The energy
minimized structure from the QM optimization was then used as the
input for a Monte Carlo conformational search, using the MCMM
methodology in MacroModel (version 9.6; Schrödinger, LLC) with the
MMFFs force field in an implicit water model. This specific force field
was selected because it has been optimized to keep sp2-hybridized
nitrogens planar (Halgren, 1996, 1999) with atom types N4 and N2
to describe the two nitrogen atoms of the imidazoline ring (see
Supplemental Fig. 2 for details). The conformational search was per-
formed to identify all low energy conformations of mazindol and the
global minimum of each enantiomer was used as the input structure
for the following IFD simulation.
Uptake Inhibition Assay
Adherent transfected cells were washed with phosphate-buffered
saline with calcium and magnesium (137 mM NaCl, 2.7 mM KCl,
4.3 mM Na2HPO4, 1.4 mM KH2PO4, 0.1 mM CaCl2, and 1 mM MgCl2,
pH 7.4) and preincubated for 25 minutes with a dilution series of the
inhibitor. Uptake was initiated by adding a mixture of 50–100 nM
[3H]serotonin or [3H]dopamine (PerkinElmer, Waltham, MA) and the
inhibitor at the same concentration as in the preincubation. Radio-
active neurotransmitter uptake was allowed to proceed for 10 minutes
at 22°C and was terminated by washing with phosphate-buffered
saline with calcium and magnesium. Aspirated cells were lysed with
Microscint 20 (Packard BioScience Co., Meriden, CT) and the accu-
mulated radioactive neurotransmitter was quantified on a Packard
Topcounter.
Data Analysis
Radioactive counts from accumulated neurotransmitters were
fitted to a sigmoidal dose-response curve in GraphPad Prism 3
software (GraphPad Software, Inc., La Jolla, CA). The resulting IC50
values were transformed to Ki values using the Cheng–Prusoff
equation. Statistical comparison of Ki values was conducted using
the t test.
IFDs with the positive charge localized on N4 were also performed
(see the summary in the Supplemental Experimental Procedures).
However, these results do not differ from the results from IFD with
N1 modeled as positively charged and thus only the results from the
latter will be analyzed in detail here.
Protein Preparation. The protein complexes were prepared for
docking calculations by the Schrödinger 2008 Suite Protein Prepara-
tion Wizard. Herein, the protonation states and bond orders of the
amino acids were initially predicted and afterward optimized. The
automatically assigned protonation states were used in most cases;
Molecular Modeling
Homology Modeling. One homology model of hDAT and one
homology model of hSERT were used in this study. Both models were
constructed using the crystal structure of the Aquifex aolicus leucine
transporter (LeuT) as a template (Yamashita et al., 2005). The however, two residues in both protein complexes were changed
alignment used between LeuT and hSERT and LeuT and hDAT,
manually. These residues were Asp524 and Glu508 in hSERT and
respectively, was the thoroughly refined one of the neurotransmitter Glu396 and Glu491 in hDAT, which, according to PROPKA 2.0
sodium symporters published by Beuming et al. (2006) and was
previously used by us with success (Celik et al., 2008b; Koldsø et al.,
2010, 2011, 2013a,b; Sinning et al., 2010; Severinsen et al., 2012).
predictions (Li et al., 2005; Bas et al., 2008), were both suggested to be
neutral at physiologic pH. The protonation state of Glu508 in hSERT
and Glu491 in hDAT as neutral is further supported by the finding

Computational and Biochemical Analysis of Mazindol Orientation
that similar Glu–Glu pairs are conserved in the NNS family as
211
Results
revealed in the LeuT crystal structure (Forrest et al., 2007). Most
histidines in hSERT were retained as the d-tautomer, except His240,
which was modeled as charged. In the model of hDAT, His165, His179,
His225, His375, and His444 were all modeled as the «-tautomer.
Subsequently, each protein was subjected to a constrained minimi-
zation within the OPLS-AA force field (Jorgensen and Tirado-Rives,
1988) and converged to a root mean squared deviation of 0.3 Å. The
refined protein structures were then used as the input structures for
the following IFD studies.
IFD Simulations. Since mazindol is significantly larger than
leucine, which was bound inside the binding pocket of the template
LeuT, it was thus necessary to include protein flexibility in the
docking simulations. The Schrödinger Suite 2008 Induced Fit Docking
protocol (Sherman et al., 2006) was used throughout this study. In the
Molecular Modeling. Since mazindol has the positive
charge delocalized over two nitrogen atoms, two binding
modes can be expected where either N1 or N4 interacts with
Asp98 in hSERT and Asp79 in hDAT assuming this ionic
interaction to be present also for mazindol, similarly to what
was previously found for 5-hydroxytryptamine/serotonin, imip-
ramine, and citalopram (Celik et al., 2008b; Andersen et al.,
2009; Koldsø et al., 2010; Sinning et al., 2010) in hSERT and
for dopamine and cocaine in hDAT (Koldsø et al., 2013b). It
was previously proposed that cocaine binds in a similar
fashion to both hDAT and hSERT (Beuming et al., 2008), and
it can thus be hypothesized that mazindol also does this. Our
IFD workflow, some side chain flexibility is introduced for residues experimental data (see below) support these findings, and
with at least one atom within a distance of 5 Å from the ligand. The
homology models of hDAT and hSERT do not contain a bound ligand
so the binding site was defined by residues previously shown to be
important for inhibitor binding, which were Asp98 and Ile172 in
hSERT (Henry et al., 2006) and Asp79 and Val152 in hDAT (Lee et al.,
2000; Beuming et al., 2008), respectively. The number of poses to save
in both the initial and the redocking stages of the IFD protocol was
changed from the default value of 20 to 100. Furthermore, the energy
window of the Prime energy in the sorting and filtering step was
changed from the default value of 30 kcal/mol to 50 kcal/mol to allow
for more diversity among the saved docking poses. The scoring
function applied in the initial soft docking was standard precision
(Friesner et al., 2004), whereas the extra precision scoring function
(Friesner et al., 2006) was applied in the redocking stage of the IFD
protocol.
Binding Site Optimization. The binding site of the best docking
poses from the IFD calculations, judged from GlideScore and Emodel,
were subsequently energy minimized in MacroModel (version 9.6;
Schrödinger, LLC) with the conjugated gradient method until conver-
gence using the OPLS-AA (Jorgensen and Tirado-Rives, 1988) force
field with no solvent. The ligand was used to define the center and
complete residues in a radius of 8 Å are allowed to move freely during
minimization. Surrounding the freely moving area, a shell with a
radius of 10 Å was constrained with a force constant of 200 kJ/mol×Å2
hereby allowing only moderate flexibility of this part of the protein
structure, leaving the rest of the protein frozen during this calcu-
lation. The refined protein/ligand complexes were used in the fol-
lowing QPLD docking simulations.
QPLD. The binding modes identified from the IFD calculations of
each enantiomer bound to both of the protein models were then
further evaluated by the Schrödinger QM-Polarized Ligand Docking
protocol. QPLD is a quantum mechanics/molecular mechanics–based
docking method (Cho et al., 2005) combining Glide (Friesner et al.,
2004) and QSite (Murphy et al., 2000). During the QPLD procedure,
the ligands are initially docked into a rigid protein using Glide. The
resulting binding modes of the ligands are then used for calculation of
partial charges of the ligand by a single-point calculation in QSite. By
this method, the effect of the polarization from the protein experi-
enced by the bound ligand is taken into account in the final docking
stage in which the partial charges obtained from the QM calculations
of the ligand are used. No protein flexibility is possible during the
QPLD calculation; however, the ligands are treated as flexible in each
of the two docking stages. Three different setups were tested for these
calculations exploring the two scoring functions in the initial docking
and final redocking stages. In all setups, the center for the grid
calculations for the quantum mechanics/molecular mechanics step
was defined by the position of the ligand in the minimized best
structure from the IFD calculations. The number of poses to be
returned in each setup is set to 20. The QM level in the QPLD protocol
is set to Accurate, which implies that the density functional theory
method B3LYP (Lee et al., 1988; Becke, 1993; Stephens et al., 1994) is
applied with the 6-31G(d) basis set.
only binding modes identified in both proteins will thus be
analyzed in detail here. One dominating binding mode of
R-mazindol in hSERT, as well as hDAT, was identified from
the docking calculations in which N1 forms an ionic in-
teraction with Asp98 in hSERT, or the equivalent Asp79, in
hDAT. This binding mode is termed R-C1 in the following
sections of this article (Fig. 2, B and E). For R-mazindol, no
poses with an ionic interaction below 4.5 Å between N4 and
Asp79 in hDAT were found. However, two poses in hSERT
were different from R-C1 and these two, termed R-C2, showed
an ionic interaction with a distance of less than 4 Å between
N4 and Asp98 in hSERT. Since the R-C2 binding mode is only
present in hSERT, it will not be analyzed further at this time.
The R-C1 binding mode of mazindol furthermore shows
a hydrogen bond between the positive N1 atom of mazindol
and the backbone carbonyl group of Phe335 in hSERT and
Phe320 in hDAT. Phe335 is one of the two aromatic residues
that form an aromatic lid on top of the binding site as
observed in the crystal structures of LeuT and other studies
(Celik et al., 2008a,b) and it is conserved among almost all
neurotransmitter sodium symporters (Yamashita et al.,
2005; Beuming et al., 2006). Furthermore, the hydroxyl
group at the spiro-center in R-C1 can form a hydrogen bond
with the backbone of Ser438 in hSERT and Ser422 in hDAT.
The chloro substituent of mazindol in R-C1 resides in a pocket
lined by residues Ile165, Ile168, Ala169, Ile172, Phe341,
Val343, and Gly442 in hSERT and Phe76, Ile148, Ser149,
Val152, Phe326, Gly327, Val328, Gly425, Gly426, and Ser429
in hDAT, all within 5 Å of the chloro group.
Comparable with observations for R-mazindol, one similar
binding mode was observed for S-mazindol in both hSERT
and hDAT with an ionic interaction between N1 and Asp98
and Asp79, respectively. This binding mode is termed S-C1.
From the IFD calculations in hDAT, another binding mode
(S-C2) was observed with distances between N4 and Asp79
falling below 4.2 Å. This cluster will not be analyzed further
because of the lack of its presence in both transporters. From
the IFD with the positive charged localized on N4, another
binding mode (S-C3), only found in hSERT, was observed.
These poses will not be analyzed further because of the
experimental data below and the absence of this binding mode
in hDAT. The S-C1 binding mode was almost identical be-
tween the two different proteins, with only a very small var-
iation in the binding pattern (compare Fig. 2, C and F). In
S-C1, the hydroxyl group is orientated toward Phe341 in
hSERT and Phe326 in hDAT. The positively charged nitrogen
in S-C1 forms charge-reinforced hydrogen bonds with Asp98

212
Severinsen et al.
Fig. 2. The binding mode of mazindol in hSERT and hDAT. (A) The homology model of hSERT. The transmembrane (TM) parts forming the binding site
are highlighted with TM1 in pink, TM3 in purple, TM6 in green, and TM8 in yellow. (B) R-Mazindol bound in hSERT is shown with selected residues
lining the binding pocket. R-C1 is shown in orange and the protein in light gray. (C) S-Mazindol bound in hSERT is shown with selected residues around
the binding pocket. S-C1 is shown in green and the protein in light gray. (D) The homology model of hDAT. The TM parts forming the binding site are
highlighted with TM1 in pink, TM3 in purple, TM6 in green, and TM8 in yellow. (E) R-Mazindol in hDAT is shown with selected residues of the binding
pocket. R-C1 is shown in orange and the protein in dark gray. (F) S-Mazindol in hDAT is shown with selected residues of the binding pocket. S-C1 is
shown in green and the protein in dark gray.
in hSERT and Asp79 in hDAT. Furthermore, the chloro mutants. The biochemical experiments were performed with
substituent in S-C1 is located in the pocket surrounded by racemic mixtures; however, this is appropriate since the two
Ala169, Ala173, and Gly442 in hSERT and Gly153, Asn157, enantiomeric forms are presumed to be readily interchange-
Ala423, and Met427 in hDAT. The statistics from the different able and to bind with similar affinities.
docking setups are listed in Supplemental Table 1 and Table 1.
The paired mutation ligand analog complementation
It is evident that the docking scores are similar for both (PaMLAC) paradigm was used to validate the binding modes
enantiomers in the dominant clusters S-C1 and R-C1 in the two of mazindol in hSERT and hDAT predicted from the docking
proteins hSERT and hDAT (see column 8 in Table 1). In studies (S-C1, R-C1; Fig. 2). PaMLAC provides a complemen-
addition, the ionic interaction distance between the N11 of the tary method in which a battery of mazindol analogs and
ligand and Asp79 and Asp98 in hDAT and hSERT, respectively, complementary protein mutants allows us to extensively
is similar among each enantiomer (see column six in Table 1). investigate the position of specific ligand-protein interaction
In summary, the “core” of mazindol, the fused tricyclic ring points. In this study, we used racemic analogs previously
system, is predicted to be located in the same orientation described by Houlihan et al. (2002) with four novel mazindol
inside both proteins for both enantiomers. The major differ- analogs (49 substituent analogs 5–8). Compound 5 was further-
ence between the predicted bindings of the two enantiomers is more crystallized and the structure was solved by crystallog-
the orientation of the hydroxyl group and the chlorophenyl raphy (see the Supplemental Experimental Procedures). The
group; thus, unequivocal answers may not be expected related analogs used to study the binding are substituted either on
to substitutions at the 49-position if both enantiomers indeed the “core” of the ligand at positions 6, 7, and 9 (see Fig. 1) or
bind to the protein. From the docking scores, it seems most at the para-position (49-position) of the phenyl group. The
likely that the two enantiomers of mazindol bind with similar mutants used were selected so that the mutated amino acid
affinities.
residue lines the putative binding pocket of the protein. The
Experimental Validation of Mazindol Orientation in measured Ki values are listed in Supplemental Tables 2–4
hSERT and hDAT. To validate the predicted binding and Tables 2, 3, and 4.
modes, structure-activity measurements were conducted for
Orientation of the Tertiary Ammonium Group of
a battery of mazindol analogs against hSERT and hDAT Mazindol. The possible ionic interaction between the charged

Computational and Biochemical Analysis of Mazindol Orientation
213
TABLE 1
Statistics for the clusters identified from the different setups applied
The mean values [S.D.] are listed for distance, GlideScore, and Emodel. Data for all poses are included in Supplemental Table 1.
Scoring Function Initial
Docking/Redocking
Distance
Distance
Asp98(Od)-N4
Cluster
Protein
Method
Poses/Total Poses
GlideScore
Emodel
Asp98(Od)-N1+
n
Å
kcal/mol
R-C1
hSERT
IFD
SP/XP
SP/SP
SP/XP
XP/XP
11/17
20/20
10/10
1/1
3.13 [0.19]
4.05 [0.71]
3.43 [0.04]
3.45 [–]
4.20 [0.25]
4.67 [0.12]
4.59 [0.03]
4.60 [–]
210.4 [1.2]
29.6 [1.1]
210.5 [0.1]
210.5 [–]
253.7 [17.9]
277.77 [15.0]
275.3 [1.7]
276.3 [–]
QPLD
QPLD
QPLD
S-C1
R-C1
S-C1
hSERT
hDAT
hDAT
IFD
SP/XP
SP/SP
SP/XP
XP/XP
4/12
11/20
6/6
2.98 [0.40]
2.70 [0.07]
2.73 [0.10]
2.72 [0.05]
4.67 [0.31]
4.60 [0.08]
4.63 [0.08]
4.65 [0.03]
28.6 [1.7]
29.3 [0.5]
29.9 [0.1]
210.0 [0.0]
233.1 [26.1]
290.1 [6.9]
268.7 [1.7]
269.4 [1.3]
QPLD
QPLD
QPLD
2/2
IFD
SP/XP
SP/SP
SP/XP
XP/XP
2/12
17/20
9/9
3.34 [0.09]
3.62 [0.35]
3.49 [0.02]
3.52 [0.00]
3.11 [0.36]
3.52 [0.06]
3.51 [0.03]
3.52 [0.01]
29.8 [0.4]
29.2 [1.9]
210.7 [0.1]
210.8 [0.1]
249.5 [9.0]
274.5 [20.0]
271.3 [5.7]
275.5 [0.7]
QPLD
QPLD
QPLD
2/2
IFD
SP/XP
SP/SP
SP/XP
XP/XP
4/24
10/20
10/10
2/2
2.94 [0.20]
2.80 [0.08]
2.78 [0.06]
2.82 [0.10]
4.77 [0.28]
4.76 [0.06]
4.74 [0.04]
4.77 [0.06]
29.3 [0.7]
210.3 [0.2]
29.9 [0.4]
29.9 [0.6]
235.9 [21.0]
284.0 [3.3]
262.1 [3.9]
265.2 [1.2]
QPLD
QPLD
QPLD
SP, standard precision; XP, extra precision.
N1 position of mazindol and Asp98 in hSERT and Asp78 in (Ki,hDAT Phe76Tyr 5 1700 nM versus Ki,hSERT Tyr95Phe 5 182 nM;
hDAT is the dominant docking poses of both enantiomers in P 5 0.0002). This shows that Tyr95 in hSERT and the
both proteins. This type of charge-reinforced interaction was corresponding Phe76 in hDAT is the primary determinant of
previously seen in other compounds that interact with hSERT mazindol selectivity, as previously shown by Barker et al.
(Celik et al., 2008b; Koldsø et al., 2010, 2011, 2013a; Sinning (1998). No other single mutant in the binding site of residues
et al., 2010), hDAT (Beuming et al., 2008), or both (Severinsen diverging between wild-type hDAT and wild-type hSERT
et al., 2012; Koldsø et al., 2013b) and the presence of a proton- (see Tables 2 and 3) shows a similar response; however,
ated ammonium group is a hallmark of high-affinity inhibitors secondary and tertiary mutations in the hSERT Tyr95Phe or
of SERT and DAT.
hDAT Phe76Tyr background, in particular hDAT Val152Ile,
hDAT Met427Leu, hSERT Ile172Val, and hSERT Leu443Met,
further enhance the response (Tables 2 and 3).
hSERT Tyr95/hDAT Phe76 Is the Primary Determi-
nant of Mazindol Selectivity for hDAT over hSERT.
Mazindol is 3.4-fold more selective for wild-type hDAT than
Location of the 6-Position by Use of Analog 1. The
for wild-type hSERT (Ki,hDAT wt 5 140 nM versus Ki,hSERT wt 5 location of the 6-position of mazindol in hSERT has been
480 nM; P , 0.0001). This selectivity can be fully reversed investigated by measuring inhibitory potencies of analog 1
to a selectivity of 0.11 when introducing the corresponding for wild-type transporters and single mutant constructs in
hDAT residue on hSERT position 95 and the correspond- uptake inhibition experiments. The affinity of analog 1 in
ing hSERT residue at the equivalent hDAT position 76 hDAT is 160-fold better than in hSERT, with Ki values of
TABLE 2
TABLE 3
Ki values from 3H-serotonin uptake inhibition experiments in wild-type
hSERT and hSERT mutations of residues in the central binding site
diverging between hSERT and hDAT
Ki values from 3H-dopamine uptake inhibition experiments in wild-type
hDAT and hDAT mutations of residues in the central binding site
diverging between hSERT and hDAT
Confidence levels are provided in Supplemental Table 2.
Confidence levels are provided in Supplemental Table 3.
Analog
Analog
hSERT Variant
Mazindol
hDAT Variant
Mazindol
1
2
3
4
1
2
3
4
nM
nM
Wild type
480
182
230
270
850
310
590
380
189
78
14,800
2000
1940
230
760
830
1560
75
840
1340
165
220
170
290
157
1140
98
2500
159
1500
610
2080
310
4100
590
Wild type
140
1700
196
92
260
210
37
17.1
52
11.5
71
68
177
71
Y95F
F76Y
A169S
15,500
10,000
6900
740
430
490
1240
830
1770
119
36
S149A
21
13.4
6.5
I172V
V152I
80
5.4
12.7
96
5.1
21
7.7
230
76
10.6
163
33
A173G
G153A
340
61
69
290
82
5700
4100
159
76
7.7
94
14.7
14.0
88
8.1
Y175F
2200
F155Y
59
16.8
101
13.4
5.6
T439A
8900
A423T
5.4
L443M
7200
M427L
13.4
5.7
T497A
4400
1240
310
A480T
Y95F_I172V
Y95F_L443M
I172V_L443M
Y95F_I172V_L443M
4300
F76Y_V152I
F76Y_M427L
V152I_M427L
F76Y_V152I_M427L
1400
550
68
230
127
7.5
204
1200
210
360
77
3200
72
270
27
250
450
44
17,900
4000
830
360
17,400
1660
2300

214
Severinsen et al.
TABLE 4
would be very strong support for a direct interaction between this
residue and the 7-position of mazindol. The hSERT Tyr175Phe
and corresponding hDAT Phe155Tyr mutations fully satisfy
this PaMLAC criterion. For hSERT, the mazindol/analog 2
selectivity shifts from 0.25 for wild-type hSERT to 4.1 for
Tyr175Phe (Ki,mazindol 5 310 nM versus Ki,2 5 75 nM; P 5
0.0045). Similarly, for hDAT, the mazindol/analog 2 selectiv-
ity shifts from 8.2 for wild-type hDAT to 0.64 for Tyr175Phe
(Ki,mazindol 5 61 nM versus Ki,2 5 96 nM; P 5 0.0029). These
mirroring reversals of selectivity are highly significant
[selectivity of 0.25 versus 4.1 (P , 0.0001) and selectivity of
8.1 versus 0.63 (P , 0.0001)] and experimentally support the
computational models, in which the 7-position of S-C1 and
R-C1 mazindol point toward hDAT Phe155 or hSERT Tyr175
and interact directly with substituents on the 7-position of
mazindol.
Location of the 9-Position by Use of Analog 3. We also
used analog 3 to elucidate the effect of the Tyr175Phe muta-
tion in hSERT compared with the reverse mutation Phe155Tyr
in hDAT to discern between model R-C1 and S-C1. In S-C1,
the 9-position of mazindol is located less than 3.5 Å from
the hydroxyl group on Tyr175 and any substituents on the
9-position would be parallel to this hydroxyl and likely to
interact. In R-C1, the ring system of mazindol is tilted away
from Tyr175 and 9-substituents are not likely to interact with
the Tyr175 hydroxyl group.
Ki values from serotonin and dopamine uptake inhibition experiments in
wild-type hDAT, wild-type hSERT, and mutants
Confidence levels are provided in Supplemental Table 4. Residues Val343, Thr439,
Leu443, and possibly Asn177 are suggested by S-C1 to interact with the 49-
substituent on mazindol, whereas A173, and possibly Ile165 are suggested by R-C1 to
interact with the 49-substituent on mazindol. Ala169 is located between the 49-
substituent on mazindol in R-C1 and S-C1.
Analog
Transporter Variant
Mazindol
5
6
7
8
nM
Wild-type hSERT
hSERT N177A
hSERT N177S
hSERT N177T
hSERT N177Q
hSERT T439A
hSERT T439S
hSERT T439V
hSERT L443S
hSERT A169S
hSERT I165A
hSERT I165S
hSERT I165T
hSERT I165N
hSERT A173S
hSERT A173L
hSERT A173M
hSERT V343S
Wild-type hDAT
0.48
5.4
1.92
0.39
0.26
0.22
1.43
0.94
0.31
0.87
1.11
3.4
12.3
2.7
6.7
5.0
5.9
3.4
21
ND
3.0
0.073
0.035
0.028
ND
0.150
0.27
0.20
ND
2.5
4.6
13.6
5.1
1.24
0.92
1.34
2.1
4.5
4.8
1.72
2.0
1.72
2.8
2.44
4.3
0.24
3.5
5.3
3.6
0.58
3.1
2.2
2.2
5.4
1.05
8.3
0.48
27
0.144
0.145
0.182
0.183
0.32
0.72
1.08
0.77
0.70
1.41
0.035
ND
6.6
9.0
4.4
17.2
15.9
1.54
0.062
ND
8.4
12.1
0.0132
ND
0.49
0.140
0.47
ND
0.55
0.94
28
14.5
3.1
3.8
11.5
35.5
1.27
ND, not determined.
The inhibitory potency of analog 3 in the wild-type hSERT
compared with mazindol is decreased 2.4-fold, with Ki values
14,800 nM in hSERT (Table 2) and 92 nM in hDAT (Table 3). of 1140 nM (analog 3) and 480 nM (mazindol). On the contrary,
When comparing the inhibitory potency of analog 1 with that the inhibitory potency of analog 3 against wild-type hDAT is
for mazindol, a 31-fold decrease is observed in hSERT, with Ki 12-fold higher for analog 3 compared with mazindol, with Ki
values of 14,800 nM (analog 1) and 480 nM (mazindol). The values of 11.5 nM (analog 3) and 140 nM (mazindol). This
opposite selectivity is observed in hDAT with a very small 1.5- analog thus clearly has a distinct selectivity for hDAT. When
fold increase in potency upon binding of analog 1 compared introducing the Tyr175Phe mutation in hSERT, the potency
with mazindol, with Ki values of 92 nM (analog 1) and 140 nM of analog 3 remains unchanged compared with wild-type
(mazindol). However, some of the lost affinity can be regained hSERT, with Ki values of 1240 nM (Tyr175Phe) and 1140 nM
in hSERT when introducing the Tyr175Phe mutation. In this (wild-type). Similarly, when introducing the corresponding
situation, the affinity of analog 1 is increased 7-fold compared mutation in hDAT, an approximately 2-fold increase in
with wild-type hSERT (Ki,Tyr175Phe 5 2200 nM versus Ki,wt 5 potency of mazindol and analog 3 is observed compared with
14,800 nM; P , 0.0001), whereas the effect of introducing the wild-type hDAT, with Ki values of 61 nM and 5.6 nM
Tyr175Phe mutation is only a limited 1.6-fold increase for (Phe155Tyr) compared with 140 nm and 11.5 nM (wild-
mazindol (Ki,Tyr175Phe 5 310 nM versus Ki,wt 5 480 nM; P 5 type), but no changes in drug selectivity are observed upon
0.0225). This indicates that the 6-position must be located in introduction of the mutation (wild-type hDAT selectivity of 12
a pocket close to Tyr175, which is also observed in the S-C1 versus hDAT Phe155Tyr selectivity of 10.8). Similarly, hSERT
and R-C1 binding models.
selectivity for analog 3 compared with mazindol remains
7-Position by Use of Analog 2. The 7-substituted analog constant upon introduction of the hSERT Tyr175Phe mutation,
2 inhibits hDAT 113-fold more potently than hSERT, with Ki with wild-type hSERT selectivity of 0.42 (480 nM/1140 nM)
values of 1940 nM in hSERT and 17.1 nM in hDAT. In versus hSERT Tyr175Phe selectivity of 0.25 (310 nM/1240 nM).
addition, the 7-position of S-C1 and R-C1 from IFD and QPLD This indicates that there is no strong interaction between the
are located in close proximity to Tyr175 in hSERT and Phe155 9-position of mazindol and Tyr175 in hSERT and that this
in hDAT (see Fig. 2). In wild-type hSERT, the introduction of residue is not conferring the different selectivity pattern
the 7-methoxy as in analog 2 yields decreased inhibitory between hSERT and hDAT with respect to 9-substituted
potency compared with mazindol corresponding to a 0.25-fold analogs, which again supports the 9-position as pointing away
selectivity (Ki,mazindol 5 480 nM versus Ki,2 5 1940 nM; P , from the pocket lined by Tyr175 in hSERT and Phe155 in
0.0001), whereas the complete opposite selectivity is observed hDAT. These findings do not support the S-C1 model and
for wild-type hDAT with a 8.2 selectivity (Ki,mazindol 5 140 nM point to the R-C1 model because they are suggested from the
versus Ki,2 5 17.1 nM; P , 0.0001). These opposite selec- computational studies, in which the 7-position, and not the
tivities can be used to orient mazindol within the binding site. 9-position, is oriented toward Tyr175 in hSERT and the cor-
Ideally, a single mutation in hSERT that could introduce the responding position Phe155 in hDAT (Fig. 2).
hDAT-like selectivity in hSERT and conversely the corre-
Substituents at the Phenyl Group. The R-C1 dockings
sponding single mutation in hDAT that could introduce a of R-mazindol in hSERT place the electrophilic 49-chloro
hSERT-like selectivity for analog 2 compared with mazindol substituent in a subpocket lined by Ile168, Ala169, Val343,

Computational and Biochemical Analysis of Mazindol Orientation
215
and partly Phe341, whereas the S-C1 docking of S-mazindol improving it 37-fold compared with wild-type hSERT. How-
in hSERT instead suggests a subpocket lined by Ala173, ever, the selectivity for the two hydrophobic analogs (5 and 6)
Gly442, Leu443, and partly Ala169. We decided to identify or the two hydrophilic analogs (7 and 8) of Ala173Leu remains
which subpocket harbors the chloro substituent to determine unchanged compared with wild-type hSERT. Similarly, the
the most likely stereochemistry of mazindol when bound to selectivity of Ala173Ser for analogs 5–8 is unchanged, sug-
hSERT.
gesting that the Ala173 side chain does not interact with
To determine the correct binding mode, we decided to focus 49-substituents, disfavoring S-C1.
on mutations of Ala169, Asn177, and Val343. Mutations of
Gly442 were deemed unsuitable to determine the correct
binding mode because mutations of a glycine within a a-helix
can have profound effects on the dynamics of the helix, and
Discussion
The IFD and the QPLD calculations of S- and R-mazindol
also because Gly442 is located approximately halfway be- in hSERT and hDAT resulted in one dominating common
tween the two subpockets. Ala169 is also located between the binding model for each enantiomer bound to the primary
two subpockets and as such cannot be used to unambiguously binding site in each of the refined homology models with an
validate S-C1 over R-C1 or vice versa, although the models ionic interaction between N1 of mazindol and Asp98 (hSERT),
would suggest that the A169 side chain would interact more or the equivalent Asp79 (hDAT). The predicted binding modes
with the 49-substituent in R-C1 than S-C1. We find that the of R-mazindol in hSERT and hDAT were similarly oriented.
introduction of a hydrophilic residue, in the form of the Furthermore, the S-mazindol binding mode was also the same
Ala169Ser mutant, does not significantly affect the potency in the two protein homology models except for a small spatial
of mazindol or analogs 5, 6, or 8 compared with wild-type displacement. In addition, the tricyclic scaffold of mazindol
hSERT, indicating that the –Cl, 2CH3, 2OCH3, or 2CH2OH molecule was located similarly in both enantiomers and in
is either unable or too large to form a hydrogen bond with the both protein structures, with all forming an ionic interaction
hydroxyl on the serine side chain. However, the introduction with Asp98 in hSERT and Asp79 in hDAT, respectively.
of the smaller hydrophilic 49-hydroxy in analog 7 allows for
From extensive structure–activity relationship studies, it
a new hydrogen bond that increases the potency of analog 7 by was possible to validate the predicted structure for mazindol
a factor of 3.4 (Ki.hSERT wt 5 12.3 nM versus Ki,A169S 5 3.6 nM; in hSERT as well as hDAT using the PaMLAC paradigm. The
P 5 0.0016) and supports the plausibility of both S-C1 and method does not only rely on isolated changes in inhibitory
R-C1 and lends support to the notion that R-C1 may be potency of analogs but as a secondary measure also links them
preferred over S-C1.
to the ability of complementary mutations to reverse these
Asn177 is located deep in the subpocket harboring the 49- changes. Thus, this approach, when successful, adds an extra
chloro group in S-C1 at a distance of more than 6 Å. It is layer of evidence that provides strong experimental data
therefore unlikely that any direct interaction between the side supporting a direct interaction between a substructure of the
chain and the 49-substituent is possible but mutation of ligand and a particular residue side chain. Conversely, if
Asn177 might change the overall hydrophilicity or volume of structural changes to the analog or mutations should induce a
this subpocket. All mutants with smaller residues (Ala, Ser, different binding mode, this is unlikely to result in PaMLAC-
Thr) at position 177 exhibit significantly increased affinity for derived conclusions at both levels of the evaluation but
the studied analogs (Table 4) but no pattern of hydrophilic instead yield incoherent patterns. For example, we used the
side chains pairing favorably with hydrophilic 49-substituents PaMLAC methodology to map three key ligand–protein
or hydrophobic side chains pairing favorably with hydropho- interactions with high certainty to arrive at an experimen-
bic 49-substituents are found. For the larger but conservative tally validated orientation of tricyclic antidepressants in
Asn177Gln mutation, the same trend of generally improved hSERT (Sinning et al., 2010). The accuracy of this orientation
affinity is observed but much less pronounced than for the was verified in a recent crystal structure (Penmatsa et al.,
Ala, Ser, or Thr mutants, indicating that increased volume of 2013) demonstrating the resolution and power of the PaMLAC
the subpocket harboring the 49-substituent in S-C1 is favor- methodology. By utilizing several analogs, we were able to
able but that no direct interaction between the side chain of identify the difference between Tyr175 in hSERT and Phe155
residue 177 and the 49-substituent can be established as in hDAT as being a key reason for the difference in affinity
predicted by both models.
between these two proteins. Mazindol and the analogs all bind
Val343 is located at the bottom of the subpocket harboring to hDAT with greater affinity than to hSERT.
the 49-Cl in the R-C1 pose at a distance of less than 3.5 Å from
We show that the 6-position is vicinal to hSERT Tyr175Phe
the chlorine, whereas the same distance is in excess of 5.5 Å just as predicted by poses R-C1 and S-C1. This means that
for S-C1. Mutation of the hydrophobic valine to the hydro- this phenyl group penetrates the aromatic lid composed of
philic serine does not in itself affect mazindol potency signif- hSERT Tyr176 and Phe335 just as has been observed for
icantly. Despite the presence of a potential hydrogen bonding other inhibitors (Koldsø et al., 2010; Sinning, et al., 2010) and
partner in Val343Ser, the selectivity for the four different suggests the same inhibitory mechanism (i.e., inability to
49-substituent analogs 5–8 compared with mazindol remains close the aromatic lid prevents translocation of the ligand
unchanged, suggesting that the side chain of Val343Ser is bound to the central substrate site). This proposal is seconded
unable to interact with the 49-substituent. This observation by Gabrielsen et al. (2012), who included R-mazindol in the
disfavors R-C1 as the correct binding mode.
utilization of a flexible docking protocol for different inhibitors
The models predict that the side chain of Ala173 should of SERT and found that one of its aromatic groups forced
have considerable impact on the selectivity for the 49-substituted Tyr176 and Phe335 apart. Furthermore, we observe that
analogs for the S-C1 pose. We observe that the Ala173Leu a substituent on the 9-position is not sensitive to hSERT
mutation has a considerable impact on mazindol potency, Tyr175 mutation as predicted from poses R-C1 and S-C1.

216
Severinsen et al.
However, the single most striking result is that it was been suggested as an anti-cocaine abuse drug (Berger et al.,
possible to reverse the selectivity of analog 2 in hSERT by 1989). Cocaine binds to hSERT with affinity that is very
introduction of the Tyr175Phe mutation. This mutation similar to mazindol; however, mazindol binds 11-fold better
makes this position in hSERT similar to the corresponding than cocaine to hDAT (Eshleman et al., 1999; Chen and Reith,
position in hDAT, and a large gain in affinity of analog 2 is 2002; Houlihan et al., 2002). Since hDAT is the main target for
observed (26-fold). Similarly, potency decreases when in- cocaine and mazindol apparently binds to the same site as
troducing the Phe155Tyr mutation in hDAT, thereby con- cocaine (Beuming et al., 2008), it might be able to compete
structing a hSERT-like architecture of the binding site at this with cocaine and displace it from the binding site. Combined
position. Consequently, we suggest that the 7-position of with this favorable profile of mazindol, our findings about
mazindol is oriented toward Tyr175 in hSERT and Phe155 in mazindol orientation may be used to develop novel ligands
hDAT as observed in S-C1 and R-C1. Mazindol must, ac- based on a mazindol core structure that have a desirable
cordingly, bind in very similar ways in both hSERT and hDAT pharmacological profile with respect to potential cocaine
and the difference in amino acid composition must be the key addiction pharmacotherapies.
difference in determining the selectivity of these types of
compounds inside the binding pocket.
Acknowledgments
The authors thank the late Professor William Houlihan for readily
sharing analogs 1–4 with them. Jacob Overgaard is also gratefully
acknowledged for help with solving crystal structures.
With the phenyl part of the tricyclic system extending out
through the aromatic lid of the binding site, the chlorophenyl
moiety is likely to be buried inside the central substrate site.
Both R-C1 and S-C1 predict this to be the case, however, with
Authorship Contributions
different location of the chloro group. The inhibition data for
Participated in research design: Severinsen, Koldsø, Wiborg,
Ala169Ser are most consistent with R-C1. However, data for
Jensen, Sinning, Schiøtt.
Val343Ser could not corroborate this observation, whereas
Conducted experiments: Severinsen, Koldsø, Thorup, Schjøth-
inhibition data for Ala173 mutants is mostly consistent with
R-C1. Therefore, the preponderance of the inhibition data
points to R-C1 as the most likely binding mode and could
therefore indicate a preference for the R-enantiomer of mazindol.
The docking results by Gabrielsen et al. (2012) suggest
an alternative orientation of R-mazindol in which the 49-
chlorophenyl protrudes out of the binding site through the
aromatic lid in an orientation that is dissimilar to both S-C1
or R-C1 in our model. These differing results could be the
consequence of the difficulties of different docking protocols to
differentiate between two similar aromatic systems, and
minor differences in the protocols might lead to the aromatic
system interchanging in the computations. These discrep-
ancies point to the importance of validation by biochemical
experiments. In biochemical experiments, we find that a
hydroxy group on the 49-position can interact favorably, likely
through a hydrogen bond, with residue 169 when mutated to
a serine. This observation points to the 49-position being
buried in the central binding site, consistent with both S-C1
and R-C1.
Eskesen, Møller, Sinning.
Performed data analysis: Severinsen, Koldsø, Thorup, Schjøth-
Eskesen, Møller, Jensen, Sinning, Schiøtt.
Wrote or contributed to the writing of the manuscript: Koldsø,
Jensen, Sinning, Schiøtt.
References
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Also worth mentioning is another disparity in the binding
site compositions between hDAT and hSERT, in which Tyr95
in hSERT corresponds to Phe76 in hDAT. The binding data
suggest that it is preferable for mazindol analogs to have a
phenylalanine at this position since mazindol and all analogs
gain affinity when making the Tyr95Phe mutation in hSERT,
whereas a decrease in affinity is observed for mazindol and all
analogs when introducing the inverted Phe76Tyr mutation
in hDAT. This indicates that the property of this aromatic
residue is a very important determinant in dictating the selec-
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Address correspondence to: Birgit Schiøtt, Department of Chemistry,
Aarhus University, Langelandsgade 140, 8000 Aarhus C, Denmark. E-mail:
birgit@chem.au.dk