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Recent advances in the understanding of the interaction of antidepressant drugs with serotonin and norepinephrine transporters

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The biogenic monoamine transporters are integral membrane proteins that perform active transport of extracellular dopamine, serotonin and norepinephrine into cells. These transporters are targets for therapeutic agents such as antidepressants, as well as addictive substances such as cocaine and amphetamine. Seminal advances in the understanding of the structure and function of this transporter family have recently been accomplished by structural studies of a bacterial transporter, as well as medicinal chemistry and pharmacological studies of mammalian transporters. This feature article focuses on antidepressant drugs that act on the serotonin and/or the norepinephrine transporters. Specifically, we focus on structure-activity relationships of these drugs with emphasis on relationships between their molecular properties and the current knowledge of transporter structure.
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Recent advances in the understanding of the interaction of antidepressant
drugs with serotonin and norepinephrine transportersw
Jacob Andersen,
a
Anders S. Kristensen,
a
Benny Bang-Andersen
b
and Kristian Strømgaard*
a
Received (in Cambridge, UK) 12th February 2009, Accepted 25th March 2009
First published as an Advance Article on the web 7th May 2009
DOI: 10.1039/b903035m
The biogenic monoamine transporters are integral membrane proteins that perform active
transport of extracellular dopamine, serotonin and norepinephrine into cells. These transporters
are targets for therapeutic agents such as antidepressants, as well as addictive substances such as
cocaine and amphetamine. Seminal advances in the understanding of the structure and function
of this transporter family have recently been accomplished by structural studies of a bacterial
transporter, as well as medicinal chemistry and pharmacological studies of mammalian
transporters. This feature article focuses on antidepressant drugs that act on the serotonin and/or
the norepinephrine transporters. Specifically, we focus on structure–activity relationships of these
drugs with emphasis on relationships between their molecular properties and the current
knowledge of transporter structure.
Introduction
The three unique, but closely related transporters for the
biogenic monoamine neurotransmitters serotonin (5-hydroxy-
tryptamine, 5-HT, 1), norepinephrine (NE, 2) and dopamine
(DA, 3) (Fig. 1) constitute the biogenic monoamine class of
neurotransmitter transporters in the human brain. The neuro-
physiological function of monoamine transporters is to
regulate the extracellular concentrations of 5-HT, NE, and
DA in the brain by performing active transport (or re-uptake)
of released monoamine transmitters from the extracellular
space into neurons or glial cells (Fig. 1). Monoamine trans-
porters are integral membrane proteins located in the plasma
membrane and utilize co-transport of Na
+
to drive mono-
amine transport.
1
The three monoamine transporters, the
serotonin transporter (SERT), the norepinephrine transporter
(NET) and, to a lesser extent, the dopamine transporter
(DAT), are important targets for a wide range of drugs used
in the treatment of mood, anxiety and behavioural disorders
Jacob Andersen received his MSc degree from the Danish University of Pharmaceutical Sciences in 2005. In 2009, he obtained his
PhD from the Faculty of Pharmaceutical Sciences, University of Copenhagen, where he studied interactions of antidepressants with
the serotonin and norepinephrine transporters under the supervision of Professor Kristian Strømgaard. He is currently continuing
these studies as a postdoctoral research fellow in the Chemical Biology group at the University’s Department of Medicinal
Chemistry.
Anders S. Kristensen graduated from Aarhus University in 1999 (MSc in Chemistry–Molecular Biology) and received his PhD in
2004 from the Danish University of Pharmaceutical Sciences, Copenhagen, after work on glutamate receptors in the group of
Professor Arne Schousboe. From 2004 to 2006, Anders S. Kristensen was a postdoctoral fellow in the laboratory of Professor Stephen
F. Traynelis at Emory University School of Medicine, Atlanta, where after he joined the Chemical Biology group at the Department of
Medicinal Chemistry, University of Copenhagen, where he is currently an Associate Professor.
Benny Bang-Andersen received his MSc in Chemical Research from University College London in 1992 under the supervision of
Professor Robin Ganellin and his MSc in Pharmacy in 1993 from the Danish University of Pharmaceutical Sciences. He subsequently
completed his PhD in medicinal chemistry in 1996 under the supervision of Professor Povl Krogsgaard-Larsen in collaboration with
Dr Klaus P. Bøgesø at Lundbeck. Since then he has worked at H. Lundbeck as a medicinal chemist, chemistry and/or drug discovery
project leader, and line manager. He is currently a Senior Principal Scientist and is working as a group leader in medicinal
chemistry.
Kristian Strømgaard received his PhD at the Danish University of Pharmaceutical Sciences in 1999 and subsequently worked as a
postdoctoral fellow in the group of Koji Nakanishi at Columbia University. He is currently a Professor of Chemical Biology at the
Department of Medicinal Chemistry, University of Copenhagen, where he works with a group of ca. 20 people with expertise in
applying chemistry and biology in studies of membrane-bound proteins in the central nervous system. Specifically the group currently
focuses on ionotropic glutamate receptors and neurotransmitter transporters.
a
Department of Medicinal Chemistry, University of Copenhagen,
Universitetsparken 2, DK-2100 Copenhagen, Denmark.
E-mail: krst@farma.ku.dk; Fax: +45 3533 6040;
Tel: +45 3533 6114
b
Lundbeck Research Denmark, H. Lundbeck A/S, Otilliavej 9,
DK-2500 Valby, Denmark
wElectronic supplementary information (ESI) available: Links to
PDB visualizations are included in the online supplementary data.
See DOI: 10.1039/b903035m
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such as major depressive disorder (MDD), generalized anxiety
disorder (GAD), and attention-deficit hyperactivity disorder
(ADHD). Furthermore, monoamine transporter drugs are
also being used in the treatment for smoking cessation and
obesity,
2,3
and SERT, NET and DAT are targets for drugs of
abuse including cocaine, amphetamine, methamphetamine
and 3,4-methylenedioxy-N-methylamphetamine (MDMA,
ecstasy).
4
Despite the clinical importance of monoamine
transporter inhibitors, little is known regarding the structural
basis of inhibitor function at monoamine transporters. This
is especially important considering the need for improved
antidepressants targeting monoamine transporters.
The monoamine transporters belong to the solute carrier 6
(SLC6) gene family,
5
also known as the neurotransmitter/sodium
symporter (NSS) family.
6
The SLC6 family is one of the largest
transporter families in the human genome and comprises
20 members with SERT, NET, and DAT forming a phylo-
genetically distinct subfamily.
7
The determination of a high-
resolution X-ray crystal structure of a bacterial homologue to
SLC6 transporters, the leucine transporter (LeuT), in 2005
8
provided a breakthrough for research directed at understanding
the structure–function relationship of SLC6 transporters, includ-
ing the human monoamine transporters. The LeuT structure has
proved an important structural template to guide experiments at
mammalian SLC6 transporters on key structural and functional
features such as location and structure of binding sites for
substrates and inhibitors. Furthermore, the structure of LeuT
provides an opportunity to understand existing structure–activity
data for monoamine transporters in a structural context. Such
structural insight can guide future medicinal chemistry efforts to
develop new drugs directed at this class of transporters.
In this feature article, we provide an overview of the current
knowledge of the structure–activity relationship (SAR) for
antidepressants acting on SERT and/or NET and relate this
to recent structural advances. We focus on selected examples
of compounds from different classes of marketed anti-
depressants and summarize how these compounds together
with mutational studies have provided an insight into drug–
transporter interaction. For other aspects of the SLC6 trans-
porters, including more detailed discussion on the different
drug classes targeting the transporters and general reviews of
the impact of the crystal structure of LeuT, we refer to a
number of excellent reviews published in recent years.
4,9–16
Inhibitors of monoamine transporters
as antidepressants
There is substantial evidence for a link between affective
disorders, including depression, and imbalances in monoaminergic
neurotransmission in the brain.
17–20
Most antidepressant
Fig. 1 A. Schematic representation of a simplified monoaminergic
synapse. Monoamine transmitters (5-HT, DA, or NE) are stored in
vesicles in the presynaptic terminal, mediated by vesicular monoamine
transporters (VMATs; red). For all three transmitter systems, the released
transmitters exert their action mainly through activation of G protein-
coupled receptors (GPCRs; light blue) with the only exception being the
5-HT
3
receptor (green), which is a ligand-gated ion channel. The actions
of 5-HT, NE and DA are terminated mainly by re-uptake into the
presynaptic neuron via monoamine transporters (blue), and re-sequestered
into vesicles by VMATs. B. Structures of the biogenic, monoamine
neurotransmitters, serotonin (1), norepinephrine (2) and dopamine (3).
Fig. 2 Structures of prototypical TCAs. The structures of the TCAs are generally rather conserved, with a tricyclic scaffold of either a six- or
seven-membered ring flanked by two phenyl moieties. The central ring is then substituted with an aminopropyl chain, with the amino group having
one or two methyl groups. The latter is an important determinant for SERT/NET selectivity.
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drugs are modulators of monoaminergic neurotransmission,
and they can generally be divided into three classes:
(i) monoamine oxidase inhibitors (MAOIs), (ii) monoamine
receptor antagonists, and (iii) monoamine transporter inhibi-
tors. Most MAOIs are associated with a number of severe
side-effects and are considered a last-line-of-defence used in
patients not responding to other antidepressants. Hence, the
mainstay of presently prescribed antidepressants are mono-
amine transporter inhibitors and, to a much lesser extent,
monoamine receptor antagonists such as the a 5-HT
2
receptor
antagonist trazodone
21
and mirtazapine,
22
an antagonist of
adrenergic a
2
, 5-HT
2
and 5-HT
3
receptors. In this review we
focus entirely on the monoamine transporter inhibitors, which
is by far the most prescribed class of drugs for treating
depression.
The first generation of monoamine transporter inhibitors
developed as antidepressants was the tricyclic antidepressants
(TCAs). Following the seminal discovery of the antidepressant
effects of imipramine (4, Fig. 2),
23,24
a series of closely related
derivatives were developed (Table 1 and Fig. 2). Although the
Table 1 Overview of monoamine transporter inhibitors approved as antidepressants and their pharmacological profile at human SERT, NET
and DAT
Drug name and classification Company
a
Patent
year
First
reference
Approval
year
b
K
i
/nM
c
hSERT hNET hDAT
TCAs
Imipramine Geigy 1951 1954 1951 1 37 8500
Trimipramine Rhone Poulenc 1959 1961 1966 149 2450 1690
Amitriptyline Hoffmann-La Roche 1961 1960 1961 4 35 3780
Opipramol Geigy 1961 1952 1961 NA
i
NA
i
NA
i
Desipramine Geigy 1962 1959 1964 18 1 3190
Nortriptyline Geigy 1962 1962 1963 18 4 1140
Protriptyline Merck 1962 1964 1966 20 1 2100
Dosulepin SPOFA 1962 1962 1969 9 46 5310
Butriptyline Ayerst 1962 1962 1974 1360 5100 3940
Clomipramine Geigy 1963 1961 1970 0.3 38 2190
Doxepin Pfizer 1963 1962 1969 68 30 12100
Maprotiline Ciba 1966 1969 1974 5800 11 1000
Amoxapine Lederle Laboratories 1968 1967 1980 58 16 4310
Lofepramine LEO AB 1970 1969 1980 70 5 18000
Amineptine Laboratories Servier 1978 1977 1983 NA
i
NA
i
NA
i
SSRI
Zimelidine Astra 1971 1976 1982 152 9400 11700
Fluoxetine Eli Lilly 1974 1974 1987 1 240 3600
Paroxetine Ferrosan 1977 1977 1992 0.1 40 490
Fluvoxamine Solvay 1977 1977 1994 2 1300 9200
Citalopram H. Lundbeck 1977 1977 1989 1 4070 28100
Sertraline Pfizer 1983 1983 1991 0.3 420 25
Escitalopram H. Lundbeck 1989 1992 2001 1
j
410000
k
410000
k
SNRI
Sibutramine Abbott 1982 1988 1997 NA
i
NA
i
NA
i
Venlafaxine Wyeth 1984 1990 1993 9 1060 9300
Milnacipran Forest Laboratories 1985 1985 2009 420
d
77
d
6100
d
Duloxetine Eli Lilly 1988 1990 2004 1
g
8
g
240
g
Desvenlafaxine Wyeth 1984 1990 2008 40
e
558
e
4100000
e
NRI
Viloxazine ICI 1974 1974 1977 17300 155 4100000
Mirtazapine
h
Organon 1975 1982 1994 4100000 4600 4100000
Atomoxetine Eli Lilly 1984 1984 2002 152
f
5
f
658
f
Reboxetine Pharmacia 1991 1991 1999 242
e
3
e
410000
DRI
Bupropion Burroughs Wellcome 1969 1979 1985 9100 52000 520
NRI/DRI
Nomifensine Hoechst AG 1969 1971 1977 1010 16 56
SRI+
Trazodone Angelini 1965 1970 1972 160 8500 7400
a
The company submitting the original patent application or biologic license application to the United States Federal Drug Agency or European
equivalent for approval of a drug for the treatment of depression.
b
Year of approval as new molecular entity by the United States Federal
Drug Agency or European equivalent.
c
Values taken from Tatsumi et al. (1997) unless otherwise indicated.
53 d
Chen et al. (2008) (data
for milnacipran).
181 e
Deecher et al. (2006).
73 f
Greenhill et al. (2002).
182 g
Bymaster et al. (2001).
183 h
Antagonistic activity at the a
2
adrenergic
receptor is thought to be an important component of the antidepressant mechanism; thus mirtazapine can be considered an NRI+.
184 i
NA, no
data available for human cloned receptors.
j
Sanchez et al. (2004).
58 k
J. Andersen, unpublished data.
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TCAs represented a major therapeutic breakthrough in anti-
depressive therapy, their broad activity across a variety of
receptors including the 5-HT
2A
, histaminergic H
1
, adrenergic
a
1
and muscarinic M
1
receptors,
25,26
associates the use of
TCAs with a number of severe side-effects. As it became clear
that the clinical effect of TCAs was attributed to their activity
at monoamine transporters, medicinal chemistry efforts
throughout the 1960s and 1970s were focused on developing
a new generation of inhibitors with improved selectivity for
these transporters.
27
This resulted in the selective serotonin
re-uptake inhibitors (SSRIs), such as citalopram (12) and
fluoxetine (14) (Fig. 3), which have an increased selectivity
towards SERT over NET and DAT and little or no affinity for
other proteins (Table 1). This led to fewer side-effects without
compromising the clinical efficacy, and today SSRIs are the
most prescribed class of antidepressants.
28
Later the combined
serotonin and norepinephrine re-uptake inhibitors (SNRIs)
emerged, which are potent inhibitors of both SERT and NET,
and are thought to have an improved antidepressant efficacy
and a faster onset of action compared to SSRIs (Table 1).
29
Duloxetine (18) and venlafaxine (19) (Fig. 3) are prominent
members of the SNRI family and have been developed for the
treatment of MDD and anxiety disorders such as GAD,
respectively.
30,31
Inhibition of SERT is a common feature of
TCAs, SSRIs, and SNRIs, and is thought to be a fundamental
requirement for their antidepressant activity. However, the
selective norepinephrine re-uptake inhibitors (NRIs, Table 1)
atomoxetine (23) and reboxetine (24) (Fig. 3), have also been
developed against depression despite having little or no SERT
activity, although these drugs are primarily prescribed for
treatment of ADHD.
32,33
The immediate pharmacological effect of monoamine trans-
porter inhibition is an acute increase of concentrations
of extracellular monoamines in the brain.
34
However, the
therapeutic effects require longer periods (43 weeks) of treat-
ment suggesting that induction of long-term adaptive changes
in monoaminergic and other neurotransmitter systems are
required to accomplish a clinical effect.
35
Additionally, a
significant proportion of patients do not receive full symptom
remission when treated with antidepressants.
36,37
To improve
these shortcomings, several augmentation principles to SERT
inhibition have been pursued, which has led to the identifica-
tion of compounds with a fine-tuned pharmacological profile
by including agonistic or antagonistic effects on monoamine
receptors or other receptor systems in addition to SERT
inhibition (often denoted SRI+ compounds, Table 1).
11
Another emerging class is the ‘‘triple action’’ inhibitors that
target SERT, NET and DAT at the same time;
38
but the first
drug from these new generations of monoamine transporter
inhibitors has yet to be approved.
Structure–activity relationship studies of
antidepressant drugs
In the following discussion of SAR studies, we have focused
on how subtle modifications of selected SSRIs, SNRIs and
NRIs can lead to dramatic changes in the selectivity profile
towards SERT and NET. Such structurally closely related
compounds with distinct selectivity profiles are particularly
useful for an ample understanding of the relationship between
structural modification and selectivity towards SERT and
NET and can be applied as valuable pharmacological tools
Fig. 3 Structures of prototypical SSRIs, SNRIs and NRIs. The upper row list SSRIs that are marketed drugs, and are currently the most
prescribed drugs for the treatment of depression. The middle and lower rows show both structurally and pharmacologically related drugs which,
however, have equal activity at NET and SERT, (SNRIs), or have shown selectivity for NET, (NRIs).
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in studies of these transporters. Employing inhibitors with
different structural scaffolds can further reinforce the general
understanding of how antidepressants interact with the trans-
porters at the molecular level, and such studies might reveal
differences in binding modes of different antidepressants.
We focus on SAR studies of selected TCAs, SSRIs, SNRIs
and NRIs, rather than cover all the numerous ligands targeting
SERT and NET. Other compounds of principal interest that
are not covered here include 2-(2-dimethylaminomethyl-
phenylsulfanyl)-5-methyl-phenylamine (MADAM, 25) and
3-amino-4-(2-dimethylaminomethyl-phenylsulfanyl)benzonitrile
(DASB, 26) (Fig. 4), which have high potency and selectivity
towards SERT and their [
11
C]-labeled versions are used as
positron emission tomography (PET) ligands.
39–41
They have
also served as templates for SAR studies demonstrating that
subtle modifications change their pharmacological profile
from SSRI towards SNRI and further to NRI.
42–44
In addi-
tion, cocaine has served as a template for numerous SAR
studies to elucidate structural requirements for selectivity
between SERT, NET and DAT. While cocaine is a potent
inhibitor of all three monoamine transporters,
45
the rewarding
properties and abuse potential of the psychostimulant are
believed to be mediated through DAT.
46,47
Structural
modification of the cocaine scaffold has led to the identifica-
tion of novel SSRIs and NRIs, in addition to inhibitors with
high selectivity for SERT and DAT over NET.
48,49
TCAs
The first TCA drug on the market was imipramine (4,Fig.2),
developed by Geigy from modification of the 6-6-6 tricyclic ring
structure known from the antipsychotic drug chlorpromazine.
50
Clomipramine (6) and amitriptyline (8) are, together
with imipramine (4), prototypical TCAs with the same basic
dimethylamino group, but with slightly different tricyclic
systems. Imipramine (4) and clomipramine (6) have a dibenzo-
azepine ring system, whereas amitriptyline (8) contains a
dibenzocycloheptene scaffold and an ethylidene moiety at
the 5-position. These minor structural differences are not
of crucial importance for their biological activity, as
clomipramine, imipramine and amitriptyline are equipotent
inhibitors of SERT and NET.
26
All three drugs, 4,6and 8, are metabolized in humans to
their corresponding N-desmethyl analogues desipramine (5),
norclomipramine (7) and nortriptyline (9) (Fig. 2), which are
believed to be highly important for the therapeutic action of
these drugs.
51
Desipramine (5) and nortriptyline (9) both
display preference for NET relative to SERT, but they also
display affinity for monoaminergic receptors, similar to the
other TCAs. Nortriptyline (9) is further metabolized to
desmethylnortriptyline, which is a 10-fold weaker inhibitor
of NET compared to nortriptyline (9).
52
This demonstrates the
importance of the monomethylamino group for potent NET
inhibition, which is further substantiated by the TCAs
protriptyline (10) and maprotiline (11) having preference
for NET relative to SERT.
53
Maprotiline (11), which is a
9,10-ethyl bridged derivative of 9,10-dihydroanthracene,
displays particularly high selectivity for NET,
53
demonstrating
the significance of the tricyclic ring structure for the
NET/SERT selectivity.
Citalopram
Citalopram (12) and talopram (27) (Fig. 5) are structurally
closely related, but have distinct pharmacological profiles,
with citalopram being a prototypical SSRI, while talopram
is a potent and selective NRI. The two compounds have the
same phenyl substituted phthalane skeleton, as well as an
aminopropyl chain and differ only by four substitutions on
this scaffold.
54
We have recently carried out a systematic SAR
study of citalopram (12), talopram (27) and 14 analogues in
which each of the four positions distinguishing citalopram
from talopram were varied independently.
55
It was assumed
that a SAR study of these analogues would provide a detailed
understanding of the remarkable selectivity of citalopram (12)
and talopram (27) at SERT and NET, respectively. Indeed, we
found that two of the four positions are main determinants for
the biological activity, namely the cyano group on citalopram
(12) and the dimethyl substituents on the phthalane ring on
talopram (27) (Fig. 5). This dimethyl group was found to be
the primary factor in reducing the activity towards SERT,
whereas the cyano substituent is the major determinant for
enhancing SERT activity.
55,56
The fluorine and N-methyl
substituents are less important factors for SERT inhibition.
Similarly, NET activity is enhanced with the presence of the
dimethyl group and/or the absence of the cyano substituent.
However, the number of methyl substituents on the amino
group is important for NET activity, as analogues with
secondary amines in all cases are significantly more potent
than the corresponding tertiary amino analogues. This is in
accordance with observations from TCAs where inhibitors
having a secondary amine are more potent NET inhibitors
compared to their corresponding tertiary amine derivatives.
26
Interestingly, one of the analogues in this series, 28 (Fig. 5), is
Fig. 4 Structures of MADAM and DASB. These compounds are
extensively used as radioligands for positron emission tomography
(PET) imaging of SERT and NET in the brain.
Fig. 5 SAR studies of citalopram (12) and talopram (27), potent and
selective inhibitors of SERT and NET, respectively, and the inter-
mediate 28 which is an inhibitor of both SERT and NET.
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an SNRI, illustrating the transition from an SSRI (citalopram, 12)
to an SNRI (28) and further to an NRI (talopram, 27) by only
delicate modifications of the same scaffold.
The stereochemistry of citalopram has great importance for
the biological activity, and the SERT inhibition resides in the
(S)-enantiomer (escitalopram, 13), which is 27–170 fold
more potent than the (R)-enantiomer.
57,58
In addition the
(R)-enantiomer has been found to modulate the effect of
the (S)-enantiomer, which has prompted the development
of the (S)-enantiomer, escitalopram, as an improved anti-
depressant. Escitalopram (13) has been designated as an
allosteric serotonin re-uptake inhibitor (ASRI) and has shown
an improved efficacy and response ratio
59
and a faster onset of
action.
60,61
Fluoxetine and atomoxetine
Fluoxetine (14), marketed under the trade name Prozac, was
one of the first SSRIs discovered,
62
using 3-phenoxy-3-phenyl-
propylamine as a starting point for the development of
antidepressants. Researchers at Lilly prepared a number of
analogues resulting in nisoxetine (29, Fig. 6), a highly selective
and potent NRI. Later, the analogues were tested for selective
inhibition of serotonin re-uptake, and fluoxetine (14) was
found to be the most potent and selective SERT inhibitor.
63
Unlike citalopram, the SSRI activity of fluoxetine (14)is
independent of the stereochemical configuration, and the
(R)- and (S)-enantiomers are equipotent to the racemic
mixture.
64
The two structures of fluoxetine (14) and nisoxetine
(29) clearly illustrate that very subtle modifications of the same
chemical skeleton can change the inhibitor selectivity from
SERT to NET; here substitution of the para-trifluoromethyl
group in fluoxetine (14) for an ortho-methoxy in nisoxetine
(29, Fig. 6). Interestingly, combining the para-trifluoromethyl
and the ortho-methoxy group, led to a compound (30) with
equal potency towards SERT and NET, thus being an SNRI
(Andersen et al.
65
). Furthermore, the inhibitory activity of
nisoxetine (29) towards NET can be improved by substituting
the ortho-methoxy group with an ortho-methyl group, with the
(R)-enantiomer (atomoxetine, 23, Fig. 2) having the highest
inhibitory activity.
33
Recently, derivatives of atomoxetine (23) where the amino-
propyl chain is constrained in an aliphatic 5- or 6-membered
ring system (exemplified by compound 31, Fig. 7), retain NET
potency, but they have an improved NRI selectivity profile
compared to atomoxetine (23).
66–68
Similarly, SERT potency
was retained when the amino ethyl side chain of fluoxetine (14)
was embedded in a piperidine ring.
69
Combining structural
modifications of the phenoxy and alkyl amino moieties of
fluoxetine (14), as in compound 32 which is an SNRI, indicate
that the major determinant of SERT/NET selectivity resides in
the phenoxy moiety. However, modification of the phenyl
group of fluoxetine (14) also affects the selectivity profile, as
substitution of this moiety for a thien-2-yl group (33) leads to
an SNRI, even though the para-trifluoromethyl group is still
present.
70
Thus, the thien-2-yl is not acting as a bioisostere of
the phenyl group, but rather increases favourable interactions
with NET.
Duloxetine
Duloxetine (18) was also discovered by researchers at Lilly and
the racemic form was published in 1988.
31
The (S)-enantiomer
was chosen for further studies and subsequently named
duloxetine. Structurally, duloxetine is the 1-naphthyl deriva-
tive of 33 (Fig. 7) and its pharmacological profile makes it an
SNRI.
31
In contrast to citalopram (12)
71
and fluoxetine (14)
72
modification of the amino substituents of duloxetine (18) have
profound impact on the selectivity profile, as replacement of
the N-methylamino group with an N,N-dimethylamine
changes the selectivity profile from SNRI to SSRI.
70
Thus,
similarly to that observed for talopram (27), desipramine (5)
and nortriptyline (9) and their tertiary amine derivatives,
26,55
inhibitory potency at NET is significantly increased with a
secondary amine compared to a tertiary amine.
Venlafaxine and desvenlafaxine
Venlafaxine (19) and the active O-desmethyl metabolite,
desvenlafaxine (20), are both SNRIs and have similar potency
towards SERT and NET.
26,30,73
Interestingly, the (S)-enantiomer
of venlafaxine is a selective inhibitor of SERT, whereas the
(R)-enantiomer has SNRI properties similar to the racemic
mixture.
74
This could indicate a general tendency, namely that
SERT and NET have opposite stereochemical preferences,
75
however further studies are required to examine whether this is
a general trend for other types of racemic inhibitors.
Fig. 7 Key compounds in SAR studies of atomoxetine (23), duloxetine
(18), venlafaxine (19) and milnacipran (21), where their pharmaco-
logical activity as either SNRIs or NRIs is indicated.
Fig. 6 SAR studies of fluoxetine (14) and nisoxetine (29), potent and
selective inhibitors of SERT and NET, respectively, and the inter-
mediate 30 which is an inhibitor of both SERT and NET.
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Embedding the tertiary amine of venlafaxine (19) in a piper-
azine ring eliminated inhibitory activity at SERT and NET.
76
However, replacing the ortho-methoxy group of this
compound with a meta-trifluoromethoxy group gave a potent
and selective NET inhibitor (34, Fig. 7).
76
Milnacipran
Milnacipran (21) is structurally different from the other NET
and SERT inhibitors, with a unique cyclopropyl skeleton, and
has recently been used as a template in several SAR studies. In
these studies, primarily the phenyl ring and the tertiary amino
moiety have been modified, and these structural modifications
have led to increased potency towards both SERT and
NET, and the SNRI profile has generally been retained.
77–80
However, in some cases a modified selectivity profile
was achieved. Substitution of one of the N-ethyl groups in
milnacipran (21)foranN-phenyl group (35) selectively decreased
the activity towards SERT leading to 100-fold selectivity for
NET over SERT (Fig. 7).
81
Interestingly, other modifications
of the tertiary amine, including substitution of the N,N-diethyl
groups for aliphatic chains and heterocyclic rings did not
change the selectivity for SERT and NET.
81
Thus, the replace-
ment of an N-ethyl with an N-phenyl is so far the only
modification that can distinguish between SERT and NET,
suggesting the aromatic ring has unfavourable interactions
with SERT while the interaction with NET resembles that of
the N-ethyl substituent. Recently, the (1S,2R)-enantiomer of
milnacipran, which has improved affinity for both SERT and
NET, has been approved as an antidepressant.
78
Summary
Here we have focused on SAR studies of selected anti-
depressants, which relate to their selectivity for SERT and
NET. It is clear from these studies that the considerable
differences in selectivity often can be ascribed to subtle
changes in the same chemical scaffold. This is particularly
pronounced in the case of fluoxetine (14) and nisoxetine (29),
but also noteworthy for citalopram (12) and talopram (27).
Apparently, one would consider such similar molecules to
bind in a similar mode in the binding pocket of SERT and
NET and that the subtle differences in their structure reflects
corresponding differences in the binding pockets of SERT and
NET.
56
However, there are indications that the stereo-
chemistry of these similar structures plays a decisive role in
their selectivity, and further studies should elucidate this in
more detail. Additionally, very little is known regarding the
molecular determinants for this selectivity in SERT and NET,
but with the advent of seminal structural information
from LeuT, such studies are now possible, where rational
mutational analysis combined with modelling studies might
provide this information.
The ‘alternating access’ model for substrate
translocation
Understanding how antidepressant drugs inhibit transporter
function requires detailed information on the structural mecha-
nism of transport. Monoamine transporters are assumed to
operate as dynamic proteins that undergo a series of confor-
mational changes while exercising transport. The most widely
accepted model for this mechanism is the ‘alternating access’
model first proposed by Jardetzky
82
in 1966, and implies that
the transporters shuttle between a series of at least two major
protein conformations: (i) An ‘outward facing’ conformation
where substrate and ion binding sites are exposed to the
extracellular medium and (ii) an ‘inward facing’ conformation
that exposes the same substrate binding site to the intracellular
medium to where substrate and ions can dissociate (Fig. 8).
The activity of SERT, NET and DAT is dependent on
extracellular Na
+
ions that need to bind to the transporters in
order to initiate the alternating access transport mechanism.
Specifically, it is believed that one (for SERT and NET) or two
(for DAT) Na
+
-ions co-translocate with one substrate mole-
cule per transport cycle and that Na
+
thus is a co-substrate for
SERT, NET and DAT.
83
This coupled transport of Na
+
is the
energy source for transport of monoamines that allows SERT,
NET and DAT to transport monoamines against their
chemical gradient.
1
Moreover, transport is also dependent
on Cl
,
84
and a recent study on DAT showed that both
intracellular and extracellular Cl
facilitates transport turn-
over, suggesting that Cl
acts as a co-factor rather than a
co-substrate (Fig. 8).
85
This role of Cl
most likely also applies
to SERT and NET. For all monoamine transporters, the
maximal transport rate is approximately one substrate
molecule per second being transported with K
m
values around
0.5 to 2 mM.
Initial structure and function relationships in
monoamine transporters
Prior to the arrival of the structure of LeuT, structural studies
for SERT, NET and DAT were limited to sequence analysis
of transporter amino acid sequences and indirect evidence
derived from biochemical experiments. Monoamine trans-
porters were predicted to consist of 12 transmembrane
domains (TMDs) connected through intra- and extracellular
loops with intracellular amino- and carboxy-terminal
Fig. 8 The alternating access model for monoamine transporter
function. Transporters shuttle through different conformational states
that leads to alternating exposure of the binding sites for substrate (red
oval) and co-transported ions (Na
+
, purple diamond; Cl
, yellow
triangle; K
+
, green hexagon) to the extra- or intracellular medium. In
contrast to NET and DAT, SERT is thought to require a cytoplasmic
K
+
-ion to bind before the re-orientation step from can occur.
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domains.
86,87
Identification of putative residues that form the
binding pockets for substrates and antidepressants have
mainly been derived from extensive pharmacological and
mutational studies, and key conclusions from such studies
are discussed in the following paragraphs and summarized in
Fig. 9.
Radioligand competition binding experiments have demon-
strated that TCAs and SSRIs are displaced from SERT in a
concentration-dependent manner by 5-HT.
88–91
This supports
a model in which SSRIs and TCAs bind in a competitive
manner within or in close proximity to the substrate binding
site. However, this behaviour can also be explained by the
existence of a distinct antidepressant site on the transporter
whose occupation is mutually exclusive with substrate site
occupation.
90
Chemical labelling experiments support this
interpretation, proposing the existence of two distinct binding
sites on SERT, one for TCAs and one for citalopram (12) and
fluoxetine (14),
92
and also suggest a differentiation between
sites for TCA and 5-HT binding.
93
Radioligand binding
studies have substantiated these findings,
94
and two distinct
binding sites have furthermore been proposed for the proto-
typical TCA imipramine (4).
95–97
Thus, radioligand binding
has not led to a consensus regarding the location or the
number of antidepressant binding sites on SERT and NET.
Identification of specific residues located within or in close
proximity to the putative binding pocket for substrates and
inhibitors has mostly relied on mutagenesis studies. A widely
used approach has been the substituted-cysteine accessibility
method (SCAM), where cysteines are introduced in different
positions and selectively derivatized with thiol reactive com-
pounds, to provide information on protein structure.
98–100
Several SCAM studies on SERT
101–109
and NET
110
have
verified the overall 12 TMD topology as well as identified a
number of amino acid positions as potential candidates for
binding site residues.
SERT has been cloned from a number of different species,
111–119
and the inter-species differences in ligand affinity have been
exploited to identify key residues for antidepressant selectivity.
Transporter cross-species chimeras have been created and
characterized and specific domains critical for differences in
inhibitor pharmacology have been elucidated. Typically, these
domains contain a limited number of species-diverging
residues; thus greatly reducing specific candidate residues.
For example, Barker et al.
120
found that imipramine (4) was
a more potent inhibitor of human SERT (hSERT) compared
to rat SERT (rSERT), and used hSERT/rSERT chimeras to
identify inter-species differences in TMDs 11 and 12 to be
responsible for the discrimination of imipramine potency.
Subsequently the effect was tracked down to a single residue
(Phe586 in hSERT; Val586 in rSERT) by site-directed
mutagenesis,
121
suggesting that this residue is important for
recognition of imipramine. Another notable example of the
use of species scanning mutagenesis includes the use of the
fruitfly Drosophila melanogaster SERT (dSERT) and hSERT
chimeras to pinpoint a specific residue in TMD 1 (Tyr95 in
hSERT; Phe90 in dSERT) to be a key determinant for the
inter-species difference in citalopram (12) potency.
122
The
same aromatic residue has been shown to discriminate
between derivatives of 5-HT, thereby implying that this
residue is important for substrate recognition.
123
Comparison
of hSERT and dSERT also led to the identification of a
residue in TMD 3 (Ile172 in human SERT) as an important
determinant for recognition of inhibitors. A large decrease in
antidepressant potency for a number of antidepressants was
observed when substituting Ile172 in hSERT for a methionine
(the identity in dSERT), but remarkably there was no effect on
substrate binding or transport.
124
Analysis of chicken SERT
(gSERT) and hSERT chimeras and subsequent site-directed
mutagenesis verified that Ile172 and Phe586 in addition to
Ala169 have an important role in recognition of antidepressants,
116
while comparison of hSERT and bovine SERT (bSERT)
revealed Met180 and Phe513 to be responsible for the higher
potency of several antidepressants at hSERT compared with
bSERT.
125
Recently, by comparing SERT from zebrafish
(drSERT) and hSERT, three residues in TMD 10 (Ala505,
Leu506 and Ile507) were found to be important for the binding
of TCAs.
119
These three residues have previously been
identified as key determinants for differences in the allosteric
effect of (R)-citalopram comparing hSERT and gSERT,
suggesting that these residues influence inhibitor binding
through an allosteric mechanism.
126
In contrast to the inter-species differences in SERT, the
affinity for many inhibitors does not differ between NET from
various species.
127,128
However, TCAs have a much higher
affinity for NET compared to the closely related DAT. Taking
advantage of the different pharmacological profiles, chimeras
of human NET and DAT were used to locate the major
determinants for NET/DAT differences in TCA affinity in
TMDs 6 and 8 (Fig. 9).
129
Using the same approach, the
primary determinants of NET selectivity for the TCAs
desipramine (5) and nortriptyline (9) (Fig. 2) and the NRI
nisoxetine (29) were identified within TMDs 4–7, and sub-
sequently pinpointed to three residues (Phe316, Val356 and
Gly400).
130,131
Important residues have also been identified in site-directed
mutagenesis studies without preceding identification of
important domains from chimeric transporter constructs.
A mutational scanning of SERT revealed that mutation of
Thr178 in TMD 3 reduced the potency of citalopram (12) and
imipramine (4).
132
Furthermore, substitution of Thr276 with
an aspartate reduced the potency of the SSRIs citalopram (12)
and sertraline (16).
133
Similarly, mutation of Leu114 and
Fig. 9 Schematic overview of monoamine transporter topology.
Residues in SERT (blue) and NET (red) controlling antidepressant
binding are indicated. The principal transmembrane domains (TMDs)
thought to form the substrate and inhibitor binding sites are high-
lighted in gray.
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Gly117 in TMD 2 of NET, in addition to Thr268 and Tyr271
in TMD 5 significantly decreased the potency of desipramine
(5) and nisoxetine (29).
134–136
Furthermore, combining the
L114S mutation with a mutation in TMD 10 (L469F), gave
an even further decrease in affinity for desipramine (5).
137
In summary, a large number of mutagenesis studies of
SERT and NET have identified several amino acid positions
at which mutations can influence inhibitor potency. As shown
in Fig. 9, these positions are scattered across almost all TMDs.
However, those residues that appear to be key determinants
for inhibitor potency in the sense that mutations here produce
more than 10-fold loss-of-potency for a given inhibitor all
localize to TMD 1, 3 and 8, suggesting that it is these domains
that form one or more binding sites for inhibitors.
Crystal structure of a bacterial homologue of
monoamine transporters
The high resolution X-ray crystal structure of a prokaryotic
homolog to the mammalian SLC6 transporters, LeuT,
8
repre-
sents a major advance in structure and function studies of
monoamine transporters. LeuT originates from the thermo-
philic bacterium Aquifex aeolicus, the natural habitat of which
is hot springs with growth-temperature maxima near 95 1C;
hence the proteome of this bacterium is extremely stable.
138
The transporter was initially crystallized in complex with the
substrate leucine,
8
and hereby gave the first structural insight
into the substrate binding site of a SLC6 transporters
(Fig. 10). Furthermore, the LeuT structure confirmed the
predicted 12 TMD topology for SLC6 transporters, and
showed that residues in TMDs 1, 3, 6 and 8 form a substrate
binding site in the core of the transporter (Fig. 10). Interest-
ingly, TMDs 1 and 6 both contain non-helical regions near the
centre of the transporter which enables several consecutive
residues to participate in substrate coordination. In addition
to leucine, two Na
+
ions (denoted Na1 and Na2) were
found in the binding cavity. Na1 is directly coordinated
to the substrate, thus providing a structural basis for the
tight coupling between transport of substrate and ions. An
unexpected internal structural repeat relating TMDs 1–5 to
TMDs 6–10 by an inverted mirror symmetry perpendicular to
the plane of the membrane was found in LeuT. The structure
Fig. 10 Structure of LeuT in complex with leucine. A: The first
crystal structure of LeuT (PDB code 2A65). TMDs 1, 3, 6 and 8 form
the substrate binding site and are shown in cyan. The substrate leucine
is shown in yellow and residues forming the putative extra- and
intracellular gates are shown as sticks. The regions containing the
putative extra- and intracellular gates are indicated with punctuated
boxes above and below the substrate binding site. B: Close-up view of
the extracellular gate formed by Arg30 (TMD 1) and Asp404 (TMD 10)
interacting via a pair of water molecules. The extracellular gate is
further supported by a cation–pinteraction between the positively
charged guanidinium group of Arg30 and the aromatic ring of Phe253
(TMD 6), which is layered on top of the substrate binding site.
C: Close-up view of the intracellular gate formed by a salt-bridge
between Arg5 (N terminus) and Asp369 (TMD 8) near the cytoplasmic
side of the protein. Arg5 also forms a cation–pinteraction with Tyr268
(TMD 6) and a hydrogen bond with Ser267 (TMD 6).
Fig. 11 Structural differences between LeuT in the outward-facing and occluded conformation. A–B: Overlay of crystal structures of LeuT in
complex with leucine, representing the occluded conformation, (PDB code 2A65; shown in grey) and in complex with tryptophan, representing the
‘outward facing’ conformation (PDB code 3F3A; shown in brown). Leucine is shown in yellow to illustrate the location of the substrate binding
pocket. TMD 11 is removed for clarity in A. TMDs 1, 2, 3, 8 and 10 shown as cylinders and the side chain of Phe253 is shown as sticks from a side
view (Bleft) and from a top view (Bright). TMDs 1, 2 and 6 are rotated outwards in the ‘outward facing’ conformation relative to the occluded
conformation. Movement of the aromatic side chain of Phe253 controls access to the binding site in the ‘outward facing’ conformation.
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revealed a conformation in which the substrate is occluded
from solution on both the extracellular and cytoplasmic sides
of the transporter. Recently, Gouaux and colleagues solved
the high resolution structures of LeuT in complex with a range
of different amino acids including tryptophan.
139
Tryptophan
is a competitive inhibitor of LeuT and interestingly, the
transporter adopts a different conformation when tryptophan
is bound compared to when a substrate is bound. The main
difference is an outward rotation of the extracellular end of
TMDs 1, 2 and 6 leaving the substrate binding site more
exposed to the extracellular medium (Fig. 11). A second
tryptophan molecule was found located B4A
˚above the
primary binding site in the ‘outward facing’ conformation.
The amino and carboxylic acid moieties of the second trypto-
phan molecule coordinated Arg30 and Asp404 and thereby
disrupted the salt-bridge forming the extracellular gate in the
‘occluded’ substrate bound conformation. LeuT is evolution-
arily distant from the mammalian SLC6 transporters and
shares only 20–25% over all sequence identity with human
SERT, DAT and NET.
140
However, several functionally
important regions, including the substrate binding site,
contain substantially higher sequence identity with the
mammalian transporters.
140
Therefore, LeuT initially appeared
to be a good structural template for key regions in SERT, NET
and DAT, including the binding sites for substrate and inhibi-
tors. This has so far been verified by a series of LeuT-guided
studies on mammalian SLC6 transporters, including human
SERT, NET, and DAT, that successfully have identified and
characterized binding sites for substrates,
141–145
ions
146,147
and
inhibitors
143,148,149
as well as structural models for the transport
mechanism.
150
The structural mechanism underlying the
‘alternating access’ model
During the translocation cycle, the transporters adopt different
conformations to facilitate the movement of substrates from
the extracellular space into the cytoplasm. The crystal struc-
tures of LeuT in complex with the substrate, leucine, and the
competitive antagonist, tryptophan, show two different con-
formations of the transporter, namely an ‘occluded’ conforma-
tion and an ‘outward facing’ conformation, respectively.
8,139
This most likely represents two conformational states in the
alternating access transport cycle (Fig. 8 and Fig. 12). Currently
there are no structures available that represent the ‘inward
facing’ or the ‘occluded unbound’ conformation within the
SLC6 family.
Utilizing the internal symmetry in the topology of LeuT,
Forrest et al.
150
have recently constructed an experimentally
validated structural model of the ‘inward facing’ conformation
of SERT, which essentially is an inverted form of the ‘outward
facing’ conformation. Comparison of the ‘outward facing’
crystal structure of LeuT with the constructed ‘inward facing’
model reveals a simple mechanism for the conformational
change between these conformations, which is in agreement
with the alternating access model first proposed more than
40 years ago.
82
Recent crystal structures of a sodium/galactose
symporter (vSGLT),
151
a benzyl-hydantoin transporter
(Mhp1)
152
and a betaine transporter (BetP)
153
revealed that
these transporters, although belonging to distantly related
transporter families, have a pseudosymmetrical core architec-
ture similar to that found in LeuT, including a strikingly
similar motif of two discontinued TMD helices around the
substrate binding site. Thus, four structurally homologous
transporters in four different conformations are available,
the ‘outward facing’ (LeuT
139
and Mhp1
152
), the ‘outward
facing occluded’ (LeuT
8
and Mhp1
152
), the ‘occluded’
(BetP
153
) and the ‘inward facing occluded’ (vSGLT
151
)
(Fig. 12). Due to their common topology, these structures
are potentially useful templates for constructing models which
can provide structural insight into the translocation mecha-
nism in addition to substrate and inhibitor binding.
154,155
Different conformational states are poised to influence
access to the inhibitor binding sites and the structure of these.
Ibogaine and cocaine, two hallucinogenic inhibitors of the
monoamine transporters, have been proposed to stabilize
monoamine transporters in an ‘inward facing’ and an
‘outward facing’ conformation, respectively.
109,148,156,157
Fig. 12 Crystal structures of LeuT and structurally homologous transporters. A–F:Top: Schematic representation of the conformations of the
crystallized transporters. Co-crystallized substrate/inhibitor and Na
+
-ions are shown in red and magenta, respectively. Bottom: Substrates/
inhibitors are shown in yellow and Na
+
-ions as magenta spheres in the crystal structures. A: LeuT co-crystallized with tryptophan and two
Na
+
-ions (PDB code 3F3A). B: Mhp1 co-crystallized with Na
+
(PDB code 2JLN). C: Mhp1 co-crystallized with benzyl-hydantoin and one
Na
+
-ion (PDB code 2JLO). D: LeuT co-crystallized with leucine (PDB code 2A65). E: BetP co-crystallized with betaine (PDB code 2W8A).
F: vSGLT co-crystallized with galactose (PDB code 3DH4).
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Interestingly, closely related analogues of cocaine have been
shown to stabilize the DAT in a different conformation, which
has been linked to the reduced stimulant in vivo effect of the
analogues compared to cocaine.
157–159
The molecular basis for
these observations is unknown, but they suggest that monoamine
inhibitors are not compounds that upon binding simply just
block the access of a substrate to the binding site, but also can
promote significant conformational changes in the transporter.
However, for antidepressants it is still unknown if these drugs
interact with the transporters in a conformation associated with
the alternating access transport mechanism or whether binding
arrests the transporter in an inhibitor-specific conformation.
Such information is of great relevance for ongoing and future
efforts aiming to build structural models for the basic mechanism
underlying the molecular action of antidepressants.
The substrate binding site in SERT and NET
The X-ray crystal structure of LeuT provided the first
structural insight into the substrate binding site of SLC6
transporters. Previous studies have suggested that the binding
site for the substrate in SERT is formed, at least in part, by
residues in TMD 1 and 3,
102,123,160–162
which is in agreement
with the LeuT structure. In contrast, prior to the arrival of
LeuT there were no indications that TMDs 6 and 8 con-
tributed to the binding site.
Using the LeuT structure as a template, several 3D struc-
tural models of the substrate binding site in monoamine
transporters have appeared. Two models of the 5-HT binding
site in SERT
143,163
have been generated based primarily on
previous mutagenesis data, whereas a recent model was
experimentally validated by mutating positions around the
predicted binding site (Fig. 13).
141
Proposed ligand–SERT
interactions were systematically examined by rational substitu-
tion of predicted interacting side chains with residues contain-
ing a side chain incapable of forming the proposed contact to
the functional group on the substrate. The interaction was
then re-established by pairing a substrate analog containing a
reciprocal modification at the functional with the mutated
residue. This approach circumvents the risk of identifying
indirect effects, which is always a concern in conventional
mutagenesis studies. The experiments supported a model in
which Asp98 anchored the primary amine of 5-HT, whereas
the hydroxyl group of 5-HT was in close proximity to Thr439.
An elegant example of a similar experimental paradigm was
provided by Barker et al.
160
who focused on the role of Asp98
in substrate recognition in SERT. They applied 5-HT deriva-
tives gramine and dimethyltryptamine, which differ only by a
methylene group in their amine-containing side chain, and the
shorter-chain gramine was 10-fold weaker than dimethyl-
tryptamine as a SERT inhibitor (Fig. 14). Assuming that the
amino group of dimethyltryptamine interacted with the
carboxylate group of Asp98, the authors hypothesized that
extending the side chain of Asp98 by one methylene group
(D98E) would establish an ionic interaction with the amino
group of the shorter gramine. Indeed, it was found that
gramine became a more potent inhibitor at the D98E mutant,
strongly suggesting that the amino group of tryptamines,
including 5-HT, interacted directly with the carboxylate group
of Asp98 (Fig. 14). The equivalent residue in DAT, Asp79, has
been proposed to have similar interactions with dopamine.
164
The aspartate in SERT and DAT corresponds to a glycine
(Gly24) in LeuT located in the unwound region of TMD 1 in
the leucine binding site.
8
This implies identical location of the
substrate binding sites in LeuT and the human monoamine
transporters, and verifies LeuT as a valid structural model for
mammalian transporters.
The antidepressant binding site in SERT and NET
Highlighting residues in LeuT that are homologues to residues
in SERT and NET identified as being important for anti-
depressant recognition show that these are scattered across the
LeuT structure (Fig. 9 and Fig. 15). Some are located within or
near the substrate binding site, while others are buried within
the structure or located on surface areas facing the membrane.
Thus, immediate interpretation of existing mutational data on
the basis of the LeuT structure does not provide a clear picture
of the location of the antidepressant binding site in SERT and
NET (Fig. 9 and Fig. 15). The main reason for this complexity
is that differentiation between direct and indirect effects in
mutagenesis studies is inherently difficult. Specifically, it is
likely that a significant proportion of the identified SERT and
NET residues influence antidepressant binding in an indirect
Fig. 13 Homology models of SERT with bound ligands. SERT in complex with 5-HT (A) and (S)-citalopram (B). Key residues on TMDs 1, 3 and
8 (shown as grey helices) are highlighted. Ligands are shown in yellow and sodium ions as magenta spheres. Models were kindly provided by
Dr Birgit Schiøtt, Department of Chemistry, Aarhus University (5-HT) and Anne-Marie Jørgensen, Lundbeck Research Denmark, H. Lundbeck A/S
[(S)-citalopram].
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manner. For example, mutation at a position outside of an
inhibitor binding site might perturb the structure of the inhibitor
binding site by a long-range allosteric effect that decreases
affinity or induce a shift in the equilibrium between the
conformational states of the transporter, hereby decreasing
temporal accessibility to the inhibitor binding site. Therefore,
mutational data cannot be interpreted unambiguously without
performing additional experiments, for example by pairing site-
directed mutagenesis with ligand derivatives as described above.
Recently, we identified the conservative mutation of Ser438
to a threonine (S438T), thus only introducing a methyl group
into SERT, to have a large impact on the inhibitory potency of
several inhibitors.
165
The equivalent residue to Ser438 in
LeuT, Ser355, is located in the substrate binding site,
8
and a
recent study has confirmed that Ser438 has a key role in the
recognition of 5-HT in the substrate binding site of SERT.
141
The S438T mutation resulted in a large decrease (up to 2000-fold)
in inhibitory potency for several antidepressants including the
SSRIs citalopram (12) and paroxetine (15) in addition to the
TCAs imipramine (4), clomipramine (6) and amitriptyline (8).
A dimethyl substituted propylamine chain was identified as a
common structural feature of the compounds greatly affected
by the S438T mutation. We speculated that a steric clash
between one of the methyl groups on the propylamine
chain and the introduced methyl group in the S438T
mutant could account for the dramatic effect (Fig. 14). Char-
acterization of derivatives of citalopram (12), imipramine (4),
clomipramine (6) and amitriptyline (8) having a monomethyl
propylamine chain, showed that these compounds were
significantly less affected by the S438T mutation compared
to their dimethyl congeners. This striking pattern, that intro-
duction of a methyl group into the protein with the S438T
mutant could be fully reversed by reciprocal modification of
the inhibitor, established Ser438 as an important contact point
for antidepressants having a propylamine chain (Fig. 14).
Given the location of Ser438 in the substrate binding site, this
supports a model in which the antidepressant’s binding site is
overlapping with the substrate binding site.
Supportive of these findings, previous mutational studies
have identified three residues located in the substrate binding
site of SERT (Tyr95, Asp98 and Ile172) as major determinants
for the inhibitory potency of antidepressants. Located in the
unwound region of TMD 1, Asp98 has been suggested to
coordinate the primary amine of 5-HT.
141,160,163
Similarly, the
carboxylate group of Asp98 is believed to anchor the amine
found in all SSRIs and TCAs.
160
Recently, Henry et al.
124
reported that substitution of Ile172 for a methionine (the
identity in dSERT) in hSERT decreased the potency of several
antidepressants (as much as 1000-fold), and further combining
the I172M and Y95F mutations resulted in a 10 000-fold loss
of citalopram (12) potency. The reverse mutation in dSERT
(M167I) increased the potency of inhibitors, substantiating
that the large effect induced by the mutation is caused by a
direct perturbation of the SERT–inhibitor interactions.
Interestingly, the I172M and S438T mutations affect anti-
depressants differently. The inhibitory potency of fluoxetine
Fig. 15 Residues in LeuT homologous to SERT and NET residues
controlling antidepressant binding. The alignment by Beuming et al.
140
was used to map the amino acid positions in LeuT corresponding to
the positions in SERT (red) and NET (blue).
Fig. 14 Schematic showing the proposed interaction between 5-HT derivatives and Asp98 (A) and TCAs and SSRIs with S438 in SERT (B).
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(14) is decreased by the I172M mutation but unaffected by the
S438T mutation. In contrast, S438T induces a significant
increase in the K
i
-values for paroxetine (15) and amitriptyline
(8), but I172M has no effect on these inhibitors. The potency
of citalopram (12), sertraline (16) and clomipramine (6)is
severely decreased by both mutations. Ile172 and Ser438 are
located in TMDs 3 and 8, respectively, on opposite sides of the
substrate binding site. The differential effects induced by
I172M and S438T indicate that antidepressants have distinct
binding modes in SERT, and might reflect that the inhibitors
stabilize different conformations of the transporter.
Until now few structure-based models of antidepressant
binding to monoamine transporters have yet been reported.
Using the crystal structure of LeuT as a template for SERT, a
model of the putative escitalopram (13) binding site has been
constructed guided by existing mutational data and SAR
studies and further refined by molecular dynamics simulations
(Fig. 13).
143,149
In this model, the binding site for escitalopram
(13) is overlapping with the substrate binding site and explains
aspects of existing SAR data reasonably well. The model was
not validated by independent experiments, but a comprehen-
sive mutational analysis of escitalopram (13) is currently being
carried out in our laboratories specifically for this purpose.
Recently, two groups independently addressed the location of
the binding site for antidepressants in LeuT. Co-crystal structures
of LeuT and three different TCAs, imipramine (4), desipramine
(5) and clomipramine (6) revealed a surprising binding site in the
outer vestibule of LeuT distinct from the substrate binding
site.
166,167
Based on mutations of ortholog residues in the human
monoamine transporters, Zhou et al.
167
proposed that the
primary effects of TCAs and possibly SSRIs are mediated by
binding in this region in SERT. To investigate this further, we
performed a comprehensive mutational analysis of the equivalent
vestibule in SERT, and found that the inhibitory potency of
SSRIs and TCAs were affected only to a negligible extent.
165
Together with evidence from other studies, these data suggest
that the TCA binding site found in LeuT is not equivalent to the
antidepressant binding site in mammalian transporters.
168–170
In addition to the high-affinity binding site, several studies
have showed that TCAs and SSRIs bind to a low affinity
allosteric site in SERT.
94,95,171–175
Allosteric modulation is a
general mechanism for the control of protein function and
allosteric modulators bind to regulatory sites distinct from the
primary binding site on the protein and produce conforma-
tional changes that modify the protein function. The low
affinity allosteric binding site on SERT has particularly been
examined with citalopram (12). Specifically, it has been
suggested that (R)-citalopram antagonizes the effect of
(S)-citalopram in vivo,
58,176,177
possibly by attenuating the
association rate, and thereby the action of (S)-citalopram
(13).
178
Nine residues located in four distinct segments in
TMDs 10–12 have been proposed to be involved in the
allosteric effect mediated by (S)-citalopram (13).
126
These
residues are clustered on the membrane-facing surfaces of
TMDs 10–12 but do not form a distinct binding site.
126,143
TMDs 11 and 12 have been proposed to be part of an
oligomerization interface,
179
and the identified residues there-
fore potentially linking conformational changes induced by
activation of the primary binding site in one monomer to the
primary binding site in an adjacent monomer by long-range
allosteric effects. However, recent studies showed that the
allosteric effect was conserved for (R)- and (S)-citalopram in
the Y95F–I172M mutant, which essentially eliminates binding
of antidepressants to the primary binding site in SERT.
180
Thus, it seems unlikely that interactions between the primary
binding site in two adjacent monomers can account for the
allosteric mechanism. The recently identified TCA site in LeuT
could serve as an allosteric binding site in the mammalian
transporter, but the nine residues identified as being involved
in the allosteric mechanism are located more than 10 A
˚from
this site. Hence, further studies are required to elucidate a
putative role of this vestibule in the allosteric regulation of the
monoamine transporters.
Conclusion
Compounds that selectively block SERT and/or NET have
found unparalleled use in the treatment of depression and
related diseases. Although these drugs and the genes for SERT
and NET have been known for more than two decades, progress
in elucidating the molecular mechanism underlying their func-
tion has been sparse. A major obstacle has so far been lack of
structural information for SERT and NET. The recent arrival of
structural information on a bacterial transporter related to
SERT and NET has opened new opportunities for breaking
down this barrier. Combining the knowledge obtained from
SAR and mutagenesis studies with structural models for SERT
and NET based on crystallographic data from bacterial trans-
porters seems to provide a solid basis for construction of three
dimensional models of inhibitor binding sites in the monoamine
transporters. Such models will provide an imperative foundation
for rational design of novel inhibitors with improved properties,
which might lead to new and improved drugs. Specifically, these
models hold great promise for rapidly advancing our under-
standing of important issues such as the molecular basis for
selective inhibition of SERT and NET. It has been apparent that
very subtle modifications in the structures of inhibitors targeting
these transporters have profound effect on the selectivity profile,
as illustrated by fluoxetine/nisoxetine and citalopram/talopram.
However, the residues in SERT and NET responsible for the
observed selectivity are essentially unknown. SERT and NET
are highly homologous and the novel structural information has
provided an opportunity for guiding future studies to identify
such residues in SERT and NET. An analysis of the proposed
antidepressant binding sites in SERT and NET shows that a
dozen residues are not conserved between the two transporters.
Studies are currently ongoing that explore whether the marked
differences in pharmacological selectivity of the closely related
inhibitors citalopram/talopram and fluoxetine/nisoxetine can be
assigned to these residues.
Acknowledgements
We thank Drs Klaus P. Bøgesø, H. Lundbeck A/S and Mark
Lansdell, Pfizer for critical reading and excellent suggestions
to the manuscript and the Drug Research Academy, Faculty
of Pharmaceutical Sciences, University of Copenhagen,
Denmark (PhD scholarship to J.A.).
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... Desipramine (DMI) is a Food and Drug Administration (FDA)approved antidepressant first developed in 1962 [5] which has been reported to block the onset of MPT in the liver [6]. Numerous antidepressant drugs have been shown to affect autophagy in a variety of tissues [7]. ...
... Although DMI was initially developed for the treatment of depression, it is clear that this tricyclic agent is multi-functional. Besides its blockage of the reuptake of norepinephrine and serotonin in the brain [5], DMI has been known to inhibit acid sphingomyelinase (aSMase) [19] and acid ceramidase [20]. In mouse livers, it has been proposed that imipramine, a metabolic precursor of DMI [21], blocks aSMase and attenuates I/R injury [22]. ...
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We investigated the cytoprotective effect of desipramine (DMI) during in vitro simulated ischemia/reperfusion (I/R) of rat hepatocytes. Primary hepatocytes isolated from male Sprague-Dawley rats were subjected to 4 h of anoxia at pH 6.2 followed by normoxia at pH 7.4 for 2 h to simulate ischemia and reperfusion, respectively. During simulated reperfusion, some hepatocytes were reoxygenated using media containing 5 μM DMI. Necrotic cell death and the onset of mitochondrial permeability transition (MPT) were assessed using fluorometry and confocal microscopy. Changes in autophagic flux and autophagy-related proteins (ATGs) were analyzed by immunoblotting. DMI was shown to substantially delay MPT onset and suppress I/R related cell damage. Mechanistically, DMI treatment during reperfusion increased the expression level of the microtubule-associated protein 1A/1B-light chain 3 (LC3) processing enzymes, ATG4B and ATG7. Genetic knockdown of ATG4B abolished the cytoprotective effect of DMI. Together, these results indicate that DMI is a unique agent which enhances LC3 processing in an ATG4B-dependent way.
... Our experiments suggest that amino acid transporters provide amino acids that are critical for the de novo synthesis of monoamine neurotransmitters. Since inhibitors of monoamine transporters have been widely used as antidepressants, amino acid transporters specific for monoamine neurotransmitter synthesis (such as TADR) may provide new treatment options for neurological diseases associated with the dysregulation of monoamine neurotransmitters (Andersen et al., 2009). ...
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Neurotransmitters are generated by de novo synthesis and are essential for sustained, high-frequency synaptic transmission. Histamine, a monoamine neurotransmitter, is synthesized through decarboxylation of histidine by Histidine decarboxylase (Hdc). However, little is known about how histidine is presented to Hdc as a precursor. Here, we identified a specific histidine transporter, TADR (Torn And Diminished Rhabdomeres), which is required for visual transmission in Drosophila . Both TADR and Hdc localized to neuronal terminals, and mutations in tadr reduced levels of histamine, thus disrupting visual synaptic transmission and phototaxis behavior. These results demonstrate that a specific amino acid transporter provides precursors for monoamine neurotransmitters, providing the first genetic evidence that a histidine amino acid transporter plays a critical role in synaptic transmission. These results suggest that TADR-dependent local de novo synthesis of histamine is required for synaptic transmission.
... Desipramine is a tricyclic antidepressant sold under the brand name Norpramin and was first patented in 1962. 31 The typical adult dose of desipramine is 100 mg to 200 mg/day. In more severely ill patients, the dosage may be gradually increased to 300 mg/day if necessary. ...
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Background: Multiple pharmacogenomic studies have identified the synonymous genomic variant rs7853758 (G>A, L461L) and the intronic variant rs885004 in SLC28A3 as statistically associated with a lower incidence of anthracycline-induced cardiotoxicity (AIC). However, the true causal variant(s), the cardioprotective mechanism of this locus, the role of SLC28A3 and other solute carrier (SLC) transporters in AIC, and the suitability of SLC transporters as targets for cardioprotective drugs has not been investigated. Methods: Six well-phenotyped, doxorubicin-treated pediatric patients from the original association study cohort were re-recruited and human induced pluripotent stem cell-derived cardiomyocytes were generated. Patient-specific doxorubicin-induced cardiotoxicity (DIC) was then characterized using assays of cell viability, activated caspase 3/7, and doxorubicin uptake. The role of SLC28A3 in DIC was then queried using overexpression and knockout of SLC28A3 in isogenic hiPSCs using a CRISPR/Cas9. Fine−mapping of the SLC28A3 locus was then completed after SLC28A3 resequencing and an extended in silico haplotype and functional analysis. Genome editing of potential causal variant was done using cytosine base editor. SLC28A3−AS1 overexpression was done using a lentiviral plasmid-based transduction and was validated using stranded RNA-Seq after ribosomal RNA depletion. Drug screening was done using the Prestwick drug library ( n = 1200) followed by in vivo validation in mice. The effect of desipramine on DOX cytotoxicity was also investigated in eight cancer cell lines. Results: Here, using the most commonly used anthracycline, doxorubicin, we demonstrate that patient-derived cardiomyocytes recapitulate the cardioprotective effect of the SLC28A3 locus and that SLC28A3 expression influences the severity of DIC. Using Nanopore¬-based fine-mapping and base editing we identify a novel cardioprotective SNP rs11140490 in the SLC28A3 locus which exerts its effect by regulating an antisense long noncoding-RNA ( SLC28A3-AS1 ) that overlaps with SLC28A3 . Using high-throughput drug screening in patient-derived cardiomyocytes and whole organism validation in mice, we identify the SLC competitive inhibitor desipramine as protective against DIC. Conclusions: This work demonstrates the power of the human induced pluripotent stem cell model to take a SNP from a statistical association through to drug discovery, providing human cell-tested data for clinical trials to attenuate DIC.
... Although there are a diverse arrays of receptors that mediate the functional effects of 5-HT (2), its activity is extinguished by just one molecular species, the Na + /Cl − -coupled serotonin transporter (SERT) (3). In accord with the crucial role that 5-HT plays in mood states, selective serotonin reuptake inhibitors that act on SERT are some of the most widely used medications to treat depression, obsessive compulsive disorder, fibromyalgia, and anxiety disorders (4). SERT is also the target of a number of different abused and illicit psychostimulants, including cocaine and amphetamines (5). ...
Article
The serotonin transporter (SERT) terminates serotonin signaling by using sodium and chloride gradients to drive reuptake of serotonin into presynaptic neurons and is the target of widely used medications to treat neuropsychiatric disorders. Despite decades of study, the molecular mechanism of serotonin transport, the coupling to ion gradients, and the role of the allosteric site have remained elusive. Here, we present cryo–electron microscopy structures of SERT in serotonin-bound and serotonin-free states, in the presence of sodium or potassium, resolving all fundamental states of the transport cycle. From the SERT-serotonin complex, we localize the substrate-bound allosteric site, formed by an aromatic pocket positioned in the scaffold domain in the extracellular vestibule, connected to the central site via a short tunnel. Together with elucidation of multiple apo state conformations, we provide previously unseen structural understanding of allosteric modulation, demonstrating how SERT binds serotonin from synaptic volumes and promotes unbinding into the presynaptic neurons.
... However, IMI was also found to exhibit a large number of side effects including neurological, pulmonary, and gastrointestinal complications, as well as toxicity 27 . Selective serotonin reuptake inhibitors (SSRIs) were developed to bind SERT with high affinity and specificity, resulting in better tolerance and fewer side effects 28,29 . However, many individuals who are prescribed SSRIs also still report a myriad of side effects including sexual dysfunction, weight loss or gain, anxiety, nausea, headaches, dizziness, insomnia, and dry mouth, many of which pose barriers to adherence 30 . ...
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Depression is a common mental disorder. The standard medical treatment is the selective serotonin reuptake inhibitors (SSRIs). All characterized SSRIs are competitive inhibitors of the serotonin transporter (SERT). A non-competitive inhibitor may produce a more favorable therapeutic profile. Vilazodone is an antidepressant with limited information on its molecular interactions with SERT. Here we use molecular pharmacology and cryo-EM structural elucidation to characterize vilazodone binding to SERT. We find that it exhibits non-competitive inhibition of serotonin uptake and impedes dissociation of [ ³ H]imipramine at low nanomolar concentrations. Our SERT structure with bound imipramine and vilazodone reveals a unique binding pocket for vilazodone, expanding the boundaries of the extracellular vestibule. Characterization of the binding site is substantiated with molecular dynamics simulations and systematic mutagenesis of interacting residues resulting in decreased vilazodone binding to the allosteric site. Our findings underline the versatility of SERT allosteric ligands and describe the unique binding characteristics of vilazodone.
Chapter
Neurotransmitter signaling is tightly controlled by transporters, which serve to terminate neurotransmission by high-affinity uptake of their cognate neurotransmitter from extracellular fluid into nerve terminals, and in some instances, into glia. Here we focus on five members of the solute carrier 6 (SLC6) family, the serotonin (5-HT), dopamine (DA), norepinephrine (NE), gamma-aminobutyric acid (GABA) and glycine transporters, SERT, DAT, NET, GAT and GlyT, respectively. Their crucial role in maintaining homeostasis of 5-HT, DA, NE, GABA and glycine signaling make them prime targets for therapeutics used to treat a broad spectrum of disorders ranging from major depression, anxiety, attention deficit hyperactivity disorder to obesity, epilepsy and pain. The monoamine transporters, SERT, DAT and NET, are also key sites of action for numerous drugs of abuse, including amphetamine, cocaine and their congeners. In addition, we discuss a subset of the SLC22 family of organic cation transporters (OCTs), OCT1, OCT2 and OCT3, as well as the SLC29 member, the plasma membrane monoamine transporter (PMAT). OCTs and PMAT are emerging as important players in monoaminergic signaling and are promising targets for development of novel therapeutics. We provide a general overview of these transporters, and then focus on their pharmacology and therapeutic applications.
Preprint
Full-text available
Neurotransmitters are generated by de novo synthesis and are essential for sustained, high-frequency synaptic transmission. Histamine, a monoamine neurotransmitter, is synthesized through decarboxylation of histidine by Histidine decarboxylase (Hdc). However, little is known about how histidine is presented to Hdc as a precursor. Here, we identified a specific histidine transporter, TADR (Torn And Diminished Rhabdomeres), that is required for visual transmission in Drosophila . TADR and Hdc co-localized to neuronal terminals, and mutations in tadr reduced levels of histamine, thus disrupting visual synaptic transmission and phototaxis behavior. These results demonstrate that a specific amino acid transporter provides precursors for monoamine neurotransmitters, providing the first genetic evidence that a histidine amino acid transporter plays a critical role in synaptic transmission. These results suggest that TADR-dependent local de novo synthesis of histamine is required for synaptic transmission.
Article
Cancer cells require a massive supply of nutrients, including sugars and amino acids-the upregulation of transporters for each nutrient contributes to meet the demand. Distinct from glucose transporters, amino acid transporters include ones whose expression is specific to cancer cells. For example, LAT1 (SLC7A5) displays protein expression mostly limited to the plasma membrane of cancer cells. The exceptions are the placental barrier and the blood-brain barrier, where immunohistochemical and mass spectrometric studies have shown LAT1 expression, although their levels are supposed to be lower than those in cancers. The expression of LAT1 has been reported in cancers from various tissue origins, where high LAT1 expression is related to the poor prognosis of patients. LAT1 is essential for cancer cell growth because the pharmacologic inhibition and knockdown/knockout of LAT1 suppress the proliferation of cancer cells and the growth of xenograft tumors. The inhibition of LAT1 suppresses protein synthesis by downregulating the mTORC1 signaling pathway and mobilizing the general amino acid control (GAAC) pathway in cancer cells. LAT1 is, thus, a candidate molecular target for the diagnosis and therapeutics of cancers. 18F-labeled 3-fluoro-l-α-methyl-tyrosine (FAMT) is used as a LAT1-specific PET probe for cancer detection due to the LAT1 specificity of α-methyl aromatic amino acids. FAMT accumulation is cancer-specific and avoids non-cancer lesions, including inflammation, confirming the cancer-specific expression of LAT1 in humans. Due to the cancer-specific nature, LAT1 can also be used for cancer-specific delivery of anti-tumor agents such as l-para-boronophenylalanine used for boron neutron capture therapy and α-emitting nuclide-labeled LAT1 substrates developed for nuclear medicine treatment. Based on the importance of LAT1 in cancer progression, high-affinity LAT1-specific inhibitors have been developed for anti-tumor drugs. JPH203 (KYT0353) is such a compound designed based on the structure-activity relationship of LAT1 ligands. It is one of the highest-affinity inhibitors with less affecting other transporters. It suppresses tumor growth in vivo without significant toxicity in preclinical studies at doses enough to suppress tumor growth. In the phase-I clinical trial, JPH203 appeared to provide promising activity. Because the mechanisms of action of LAT1 inhibitors are novel, with or without combination with other anti-tumor drugs, they could contribute to the treatment of cancers that do not respond to current therapy. The LAT1-specific PET probe could also be used as companion diagnostics of the LAT1-targeting therapies to select patients to whom therapeutic benefits could be expected. Recently, the cryo-EM structure of LAT1 has been solved, which would facilitate the understanding of the mechanisms of the dynamic interaction of ligands and the binding site, and further designing new compounds with higher activity.
Chapter
Major depression and bipolar disorder can rob a person of the potential to enjoy life and to participate productively in society. Although disease-causing genes have yet to be identified, depression is heritable but likely requires environmental triggers to manifest. Depression presents with structural and functional brain changes that include atrophy of the hippocampus, enlargement of the amygdala, and reduced signaling via the monoamine transmitters, serotonin and norepinephrine. Changes in neurotransmission are the result of increased glutamate release from the hippocampus onto neurons in the limbic system. This is accompanied by long-lasting structural changes in dendrites and synapses, including reduced hippocampal neurogenesis. Current treatments inhibit monoamine degradation or reuptake and are no more effective than placebo or behavioral therapies in many cases. The recent finding that the anesthetic ketamine is highly effective in treating the acute phase of treatment-resistant depression provides hope that new, more effective drugs targeting glutamate as opposed to monoamines may be on the horizon.
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The utilization of fluorescent ligands to study the monoamine transporters (MATs) has increased our knowledge of their function and distribution in live cell systems. In this study, we extend SAR for nisoxetine and talopram as parent compounds, to identify high affinity rhodamine-labeled fluorescent probes for the norepinephrine transporter (NET). Nisoxetine-based fluorescent probe 6 demonstrated high binding affinity (K i = 43 nM) for NET and an overall selectivity compared to the other transporters for dopamine (DAT; K i = 1540 nM) and serotonin (SERT; K i = 785 nM) in competitive radioligand binding assays. Using confocal microscopy, compound 6 was shown to stain both NET and SERT, but not DAT, at low nanomolar concentrations, in transporter-expressing cells.
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New data on the pharmacology of tricyclic antidepressants (TCAs), their affinities for human cloned CNS receptors and their cytochrome P450 enzyme inhibition profiles, allow improved deductions concerning their effects and interactions and indicate which of the TCAs are the most useful. The relative toxicity of TCAs continues to be more precisely defined, as do TCA interactions with selective serotonin reuptake inhibitors (SSRIs). TCA interactions with monoamine oxidase inhibitors (MAOIs) have been, historically, an uncertain and difficult question, but are now well understood, although this is not reflected in the literature. The data indicate that nortriptyline and desipramine have the most pharmacologically desirable characteristics as noradrenaline reuptake inhibitors (NRIs), and as drugs with few interactions that are also safe when coadministered with either MAOIs or SSRIs. Clomipramine is the only available antidepressant drug that has good evidence of clinically relevant serotonin and noradrenaline reuptake inhibition (SNRI). These data assist drug selection for monotherapy and combination therapy and predict reliably how and why pharmacodynamic and pharmacokinetic interactions occur. In comparison, two newer drugs proposed to have SNRI properties, duloxetine and venlafaxine, may have insufficient NRI potency to be effective SNRIs. Combinations such as sertraline and nortriptyline may therefore offer advantages over drugs like venlafaxine that have fixed ratios of SRI/NRI effects that are not ideal. However, no TCA/SSRI combination is sufficiently safe to be universally applicable without expert knowledge. Standard texts (e.g. the British National Formulary) and treatment guidelines would benefit by taking account of these new data and understandings. British Journal of Pharmacology (2007) 151, 737–748; doi:10.1038/sj.bjp.0707253
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RESULTS: Compared with citalopram, escitalopram-treated patients showed significantly higher response rates and increased mean change from baseline in MADRS at weeks 1 and 8. The superiority of escitalopram over citalopram was more pronounced in very severely depressed patients. This superiority was further shown to increase with degree of severity of the depression. The robustness of meta-analysis results was supported by sensitivity analyses. The clinical superiority of escitalopram versus citalopram is consistent with the results of preclinical pharmacological studies. CONCLUSION: Escitalopram was shown to be an effective therapeutic treatment for MDD, presenting significant advantages over citalopram. (Int J Psych Clin Pract 2003; 7: 259/268)
Article
Various α-aryloxy-benzyl derivatives of ethanolamine and morpholine were synthesized and tested as potential antidepressant agents. A morpholine derivative, the 2-[α-(2-ethoxy-phenoxy) benzyl]morpholine, showed outstanding activity in the antireserpine test if compared with imipramine, desipramine and viloxazine. This compound is a selective reuptake inhibitor for noradrenaline (NA) as well as an antagonist of the presynaptic α2-NA receptors.
Article
Synopsis Mirtazapine is a tetracyclic antidepressant with a novel mechanism of action; it increases noradrenergic and serotonergic neurotransmission via blockade of central α2-adrenergic auto- and heteroreceptors. The increased release of serotonin (5-hydroxytryptamine; 5-HT) stimulates serotonin 5-HT1 receptors because mirtazapine directly blocks 5-HT2 and 5-HT3 receptors. The enhancement of both noradrenergic- and 5-HT1 receptor-mediated neurotransmission is thought to be responsible for the antidepressant activity of mirtazapine. In short term (5 to 6 weeks) clinical trials in patients with depression. mirtazapine produces clinical improvements significantly superior to those of placebo, similar to those of tricyclic antidepressants (TCAs) [amitriptyline, clomipramine and doxepin] and possibly superior to those of trazodone. Short term clinical tolerability data suggest that mirtazapine produces fewer anticholinergic-, adrenergic- and serotonergic-related adverse events than TCAs. In rare cases, mirtazapine, in common with many antidepressants, was associated with potentially serious changes in haematological parameters (e.g. agranulocytosis and neutropenia). The drug appears to be safe in overdose and possesses a very low propensity for inducing seizures. Comparisons with other classes of antidepressants are needed to determine the relative position of mirtazapine in clinical practice. However, preliminary data indicate that mirtazapine, with its novel mechanism of action, is a promising addition to currently available options for the treatment of depression. Pharmacodynamic Properties In vitro neurochemical studies have demonstrated that mirtazapine blocks central α2-adrenergic auto- and heteroreceptors, but has no effect on noradrenaline (norepinephrine) reuptake. The affinity of the drug was 10-fold higher for central presynaptic α2-adrenoceptors than for central postsynaptic and peripheral α2-adrenoceptors, and 30-fold higher for α2-adrenoceptors than for α1-adrenoceptors. Microdialysis and neurophysiological experiments as well as behavioural studies performed in rats support the α2-adrenoceptor antagonist properties of mirtazapine. Receptor binding studies have shown that mirtazapine has a high affinity for serotonin 5-HT2 and 5-HT3 receptors, central and peripheral histamine H1 receptors and a low affinity for 5-HT1, dopaminergic and muscarinic cholinergic receptors. Its activity at serotonin receptor subtypes has been confirmed in animal behaviour models. Mirtazapine activates 5-HT1 receptor—mediated serotonergic neurotransmission by enhancing the stimulatory effect of the noradrenergic system on serotonergic cell firing (an α1-adrenoceptor-mediated effect) as well as antagonising the inhibitory effect of the noradrenergic system on serotonin release (an α2-adrenoceptor-mediated effect). Electrophysiological experiments have demonstrated that mirtazapine enhances serotonergic transmission through blockade of presynaptic α2-adrenoceptors. The drug does not inhibit serotonin reuptake. Pharmacokinetic Properties The bioavailability of mirtazapine is approximately 50%. Peak plasma concentrations are reached within 2.2 to 3.1 hours after single oral doses of 15 to 75mg and are dose-dependent. Mirtazapine is extensively metabolised in the liver; up to 85% of the drug is eliminated in the urine (up to 4% as unchanged drug) and the remaining 15% is eliminated in the faeces. The mean elimination half-life of mirtazapine is approximately 22 hours, making it suitable for once-daily administration. Therapeutic Potential In randomised double-blind comparative trials including patients with major depression, short term (5 to 6 weeks) therapy with mirtazapine was significantly more effective than placebo, as effective as amitriptyline, clomipramine and doxepin, and at least as effective as trazodone. Results from a meta-analysis of 5 comparative trials in which 60% of patients were hospitalised with severe depression [mean baseline 17-item Hamilton Depression Rating Scale (HAMD) score ≥25] revealed no significant differences between mirtazapine and amitriptyline. The responder rates (≥50% decrease in HAMD score from baseline) at 6 weeks and study end-point were 70 and 61 %, respectively, for mirtazapine and 73 and 64%, respectively, for amitriptyline. In a comparative trial in older outpatients (mean age 61 to 63 years), reductions in rating scale scores of depression and the percentage of responders tended to be higher in mirtazapine than in trazodone recipients. Tolerability The tolerability profile of mirtazapine is based on results from short term (5 to 6 weeks) comparisons with placebo and other antidepressants; no longer term data are available. Drowsiness (23 vs 14%), excessive sedation (19 vs 5%), dry mouth (25 vs 16%), increased appetite (11 vs 2%) and bodyweight gain (10 vs 1%) occurred significantly more frequently with mirtazapine in placebo-controlled trials. Analysis of blood pressure, heart rate and symptoms of sexual dysfunction indicated no significant differences between mirtazapine and placebo recipients. In a meta-analysis, mirtazapine appeared to be better tolerated than amitriptyline, with significantly fewer patients experiencing anticholinergic (dry mouth, constipation, and abnormal accommodation and vision), cardiac (palpitations and tachycardia) and neurological (tremor and vertigo) adverse events. Mirtazapine was at least as well tolerated as clomipramine, doxepin and trazodone in comparative trials and appeared to be associated with slightly lower incidences of anticholinergic and neurological adverse events than these drugs. Clinical trial and postmarketing surveillance data suggest that mirtazapine has a very low potential for inducing seizures. Excessive but transient somnolence was the only symptom noted in 10 patients taking an overdose (up to 315mg) of mirtazapine. Mirtazapine is infrequently associated with clinically relevant changes in laboratory parameters. Granulocytopenia and elevated alanine aminotransferase levels have been reported; most were mild in severity and returned to normal values with continued administration of mirtazapine. Elevated cholesterol levels (mean 3 to 4%) have also been reported. Dosage and Administration The recommended starting dosage of mirtazapine is 15 mg/day for 4 days, then 30 mg/day for 10 days. If effective, the drug should be continued unchanged at this dosage or, in patients assessed as insufficiently improved, the daily dosage may be further increased to 45 mg/day. In patients with hepatic or renal insufficiency, careful dosage titration as well as regular and close monitoring for adverse events is recommended. Concomitant use of mirtazapine and diazepam or alcohol (ethanol) may also impair cognitive and/or motor performance.
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Mirtazapine is a tetracyclic antidepressant with a novel mechanism of action; it increases noradrenergic and serotonergic neurotransmission via blockade of central α 2-adrenergic auto- and heteroreceptors. The increased release of serotonin (5-hydroxytryptamine; 5-HT) stimulates serotonin 5-HT 1 receptors because mirtazapine directly blocks 5-HT 2 and 5-HT 3 receptors. The enhancement of both noradrenergic- and 5-HT 1 receptor-mediated neurotransmission is thought to be responsible for the antidepressant activity of mirtazapine. In short term (5 to 6 weeks) clinical trials in patients with depression, mirtazapine produces clinical improvements significantly superior to those of placebo, similar to those of tricyclic antidepressants (TCAs) [amitriptyline, clomipramine and doxepin] and possibly superior to those of trazodone. Short term clinical tolerability data suggest that mirtazapine produces fewer anticholinergic-, adrenergic- and serotonergic-related adverse events than TCAs. In rare cases, mirtazapine, in common with many antidepressants, was associated with potentially serious changes in haematological parameters (e.g. agranulocytosis and neutropenia). The drug appears to be safe in overdose and possesses a very low propensity for inducing seizures. Comparisons with other classes of antidepressants are needed to determine the relative position of mirtazapine in clinical practice. However, preliminary data indicate that mirtazapine, with its novel mechanism of action, is a promising addition to currently available options for the treatment of depression.
Current antidepressants modulate monoaminergic neurotransmission via interaction with receptors, transporters and/or metabolism. Drug discovery programs focus mostly on orthosteric drugs that compete with the endogenous ligand for its primary binding site. There is growing interest in allosteric modulators that act via nonorthosteric binding sites. Allosteric modulation of the serotonin transporter is likely to contribute to the therapeutic effect of the selective 5-HT reuptake inhibitor, escitalopram. Therapeutic perspectives of allosteric modulation of monoamine transporters are discussed.
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