Biomedical Engineering Reference
In-Depth Information
The existence of an allosteric-binding site on SERT and its possible relevance for the effect
of antidepressants has been known since the early 1990s, and among the SSRIs only citalopram
and to a lesser extent paroxetine exert an effect via this low-afi nity binding site. The dual action
on both the allosteric and the primary-binding site results in an increased dissociation half-life of
escitalopram from its primary-binding site. The (
R
)-enantiomer has a three times weaker allosteric
effect, and it signii cantly reduces the association rate for [
3
H] escitalopram binding to SERT in low
(40-80 nM) concentrations. These effects of the (
R
)-enantiomer may be important in relation to its
inhibition of the (
S
)-enantiomer in citalopram.
18.3.1.4 The SSRI Pharmacophore and SERT Homology Model
Despite very different molecular structures, the SSRIs all bind to SERT. As information about the
3D structure of the transporter is lacking, development of a pharmacophore model was of major
interest in the early 1990s. Thus, a pharmacophore model of the SSRIs was developed at Lundbeck
based on extensive conformational studies and superimpositions of SSRIs and other reuptake inhib-
itors (Figure 18.7a). The model operates with three i tting points, namely the centroids of the two
aromatic rings and a site point positioned 2.8 Å from the nitrogen atom in the direction of the lone
pair. The nitrogen site point mimics a hypothetical hydrogen-binding atom on SERT, most likely the
carboxy group of Asp 98 (see also discussion in the following text). The use of a nitrogen site point
as i tting point in this model gave a very good superimposition of all key SSRIs (i.e., escitalopram,
(
S
)- and (
R
)-l uoxetine, (1
S
,4
R
)-sertraline, and (3
S
,4
R
)-paroxetine), which was not possible when
the basic nitrogen atoms were superimposed. Additionally, many SSRIs have aromatic substituents
(cyano, tr il uoromethyl, chloro, methylendioxo, etc.) that all, in this model, occupy the same volume
marked in yellow. Hence, this volume of the transporter is allowed for SSRIs but not for NRIs. On
the contrary, the volume marked in white dei nes a forbidden volume for SSRI ligands. Protrusion
of ligands into this volume allows for design of NRIs (Figure 18.7a).
The pharmacophore model has been validated with a number of 5-HT and NE reuptake inhibi-
tors in addition to the compounds in Figure 18.6. Importantly, the model explains the more than
100-fold stereoselectivity of the citalopram enantiomers. Thus, it is possible to i nd a conformation
of the (
R
)-enantiomer that is superimposable with the proposed bioactive conformation of esci-
talopram, but the conformational energy penalty is 2.8 kcal/mol, which corresponds closely to a
100-fold afi nity difference. The enantiomers of l uoxetine display no stereoselectivity at SERT, and
they can be i tted to the model with no differences in conformational energy in accordance with
their equipotency.
An x-ray crystal structure of a leucine transporter (LeuT
Aa
), which is a bacterial homolog of SERT,
from
Aquifex aeolicus
has offered an opportunity to build a more reliable homology model of SERT
as compared to earlier transporter models based on more distantly related proteins. In Figure 18.7b,
the secondary structure of the LeuT
Aa
is illustrated with the primary-binding site for leucine marked
with a triangle, and in Figure 18.7c the corresponding homology model of SERT built at Lundbeck
is shown. The homology model of SERT is colored and oriented in a similar manner as LeuT
Aa
in
Figure 18.7a and with escitalopram docked into the proposed primary-binding site. In Figure 18.7d
a “close up” of the primary-binding site with bound escitalopram is shown. The “close up” is taken
from the intracellular side looking up into the transporter. From this illustration it is possible to see
the putative interaction points between escitalopram and SERT, notably the hydrogen bond between
the basic amine of escitalopram and Asp 98. It is also possible to see the putative hydrophobic
interaction between the two aromatic rings of escitalopram and the hydrophobic amino acid Ile 172.
Importantly, it has been shown in a number of mutation studies that Asp 98 and Ile 172 are essential
for the binding of escitalopram to SERT giving validity to this model.
The nine amino acids (shown in white in Figure 18.7c) that have been shown to be involved in the
allosteric binding of escitalopram on SERT are located in TM 10, 11, and 12 near the C-terminal.
Very little is known about the interaction of escitalopram with this putative-binding site. However,
it is highly likely that the allosteric effect is important for the superior effect of escitalopram as an
antidepressant, especially in patients with severe depression.
