Identification of the binding sites and selectivity of sarpogrelate, a novel 5HT 2 antagonist, to...

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Life Sciences 73 (2003) 193–207

Identification of the binding sites and selectivity of sarpogrelate,

a novel 5-HT2 antagonist, to human 5-HT2A, 5-HT2B and

5-HT receptor subtypes by molecular modeling
2C
Mamunur Rashida, Philippe Manivetb,c, Hiroaki Nishiod, Jaturong Pratuangdejkulb,c,Mazen Rajabb,c, Masaji Ishiguroe, Jean-Marie Launayb,c, Takafumi Nagatomoa,*

aDepartment of Pharmacology, Niigata University of Pharmacy and Applied Life Sciences, 5-13-2 Kamishinei-cho,

Niigata 950-2081, JapanbCR C. Bernard ‘‘Pathologie Experimentale et Communications Cellulaires’’, IFR Jules Marrey,

Service de Biochimie et de Biologie Moleculaire, Hopital Lariboisiere, 75475 Paris Cedex 10, FrancecE.A. ‘‘Biologie des maladies a prions et regulations cellulaires’’, Laboratoire de Biologie Cellulaire,

UFR des Sciences Pharmaceutiques et Biologiques, 4 Avenue de l’Observatoire, 75006 Paris, FrancedDepartment of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University,

Hiroshima 729-0292, JapaneSuntory Institute for Bioorganic Research, 1-1-1 Wakayamadai, Shimahon-cho, Mishima-gun, Osaka 618-8503, Japan

Received 14 November 2002; accepted 17 December 2002

Abstract

The aim of the present study was to investigate the binding sites interactions and the selectivity of sarpogrelate

to human 5-HT2 receptor family (5-HT2A, 5-HT2B and 5-HT2C receptor subtypes) using molecular modeling.

Rhodopsin (RH) crystal structures were used as template to build structural models of the human serotonin-2A and

-2C receptors (5-HT2AR, 5-HT2CR), whereas for 5-HT2BR, we used our previously published three-dimensional

(3D) models based on bacteriorhodopsin (BR). Sarpogrelate, a novel 5-HT2R antagonist, was docked to the

receptors. Molecular dynamics (MD) simulations produced the strongest interaction for 5-HT2AR/sarpogrelate

complex. Upon binding, sarpogrelate constraints aromatic residues network (Trp3.28, Phe5.47, Trp6.48, Phe6.51,

Phe6.52 in 5-HT2AR; Phe3.35, Phe6.51, Trp7.40 in 5-HT2BR; Trp

3.28, Phe3.35, Phe5.47, Trp6.48, Phe6.51, Phe6.52 in 5-

HT2CR) in a stacked configuration, preventing activation of the receptor. The models suggest that the structural

origin of the selectivity of sarpogrelate to 5-HT2AR vs both 5-HT2BR and 5-HT2CR comes from the following

results: (1) The tight interaction between the antagonist and the transmembrane domain (TMD) 3. Asp3.32

0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved.

doi:10.1016/S0024-3205(03)00227-3

* Corresponding author. Tel.: +81-25-268-3170; fax: +81-25-268-1280.

E-mail address: [email protected] (T. Nagatomo).

M. Rashid et al. / Life Sciences 73 (2003) 193–207194

neutralizes the cationic head and interacts simultaneously with carboxylic group hydrogen of the antagonist

molecule. (2) Due to steric hindrance, Ser5.46 (vs Ala5.46 in 5HT2B and 5HT2C) prevents sarpogrelate to enter

deeply inside the hydrophobic core of the helix bundle and to interact with Pro5.50. (3) The side chain of Ile4.56 (vs

Ile4.56 in 5HT2BR and Val4.56 in 5HT2CR) constraints sarpogrelate to adjust its position by translating toward the

strongly attractive Asp3.32. These results are in good agreement with binding affinities (pKi) of sarpogrelate for

5-HT2 receptor family expressed in transfected cell.

D 2003 Elsevier Science Inc. All rights reserved.

Keywords: Sarpogrelate; Molecular modeling; 5-HT2 receptor family; Antagonist; Selectivity

Introduction

There are many physiological, behavioral and cognitive functions involved through the interaction

between the biogenic amine serotonin (5-Hydroxytryptamine, 5-HT) and a large family of serotonin

receptor on the membrane of both neurones and non-neuronal cells that belong to the super-family of

G-protein coupled receptor [16]. These receptors are divided in seven classes (5-HT1 to 5-HT7, except

5-HT3 that is a ionic channel) and more than 14 molecularly identified 5-HT receptor subtypes, some

with splice variants and others with isoforms created by mRNA editing [8].

The 5-HT2 serotonin receptors (5-HT2R) are of significant clinical interest because of their potential

involvement in cardiovascular function and in certain mental disorders. 5-HT2R family contains 5-HT2A,

5-HT2B and 5-HT2C subtypes sharing similar intron-exon distribution [3], high primary sequence

homology (68–79% in the transmembrane segments), pharmacology, and all stimulate phospholipase C

activity, leading to increased phosphoinositide hydrolysis in the cell. It is of now-a-days interest to give

more emphasis for identification of more selective ligands to fully elucidate the functional role of

different subtypes. Molecular modeling studies are useful in as much as they may allow us to understand

the activity and selectivity of currently existing agents, and furthermore, may aid in the design of

completely novel therapeutic agents [37].

Sarpogrelate, a 5-HT2 antagonist, was introduced as a therapeutic agent for the treatment of ischemic

diseases associated with thrombosis [17]. Sarpogrelate inhibits thrombus formation [13,39], lowers

platelet aggregation, [14,23], inhibits both serotonin-induced coronary artery spasm [21] and contraction

[12] in porcine model as well as vascular smooth muscle cell proliferation [30]. Presently these

pathophysiological effects are claimed to be mediated by 5-HT2A receptors. Based on radio-ligand

binding and functional studies, it was shown that sarpogrelate exibit specificity toward 5-HT2 receptors,

since it has lack of significant 5-HT1, 5-HT3, 5-HT4, a1-, a2- and h-adrenoreceptor, histamine H1, H2

and muscarinic M3 antagonistic activity [20,24,26]. However, the structural origin of the selectivity of

sarpogrelate to 5-HT2AR vs 5-HT2B and 5-HT2C receptor subtypes has not yet been reported. In the

present study, we thus intend to precise the selectivity and binding sites of sarpogrelate to 5-HT2A,

5-HT2B and 5-HT2C receptor subtypes by molecular modeling. Using bovine rhodopsin X-ray structure

(1HZX) [31] as template, 3D structural models of the human serotonin 5-HT2AR and 5-HT2CR were

constructed. For 5-HT2BR we used our previously published 3D model [19]. The receptors were then

complexed with sarpogrelate by molecular modeling techniques and submitted to MD simulations. The

antagonist-receptor complexes were then used to examine sarpogrelate binding sites and to determine key

residues that may be important for specifying sarpogrelate 5-HT2AR selectivity.

M. Rashid et al. / Life Sciences 73 (2003) 193–207 195

Materials and methods

Radioligand binding experiments

5-HT2A receptor binding assay in CHO-K1 cells expressing a cloned human 5-HT2A receptor was

performed using [3H]ketanserin as a radioligand. CHO-K1 cells stably transfected with a plasmid

encoding the human serotonin 5-HT2A receptor are used to prepare membranes in modified Tris–HCl

buffer (50 mM Tris–HCl, pH 7.7) using standard techniques [4]. In competition binding experiments, a

30 mg aliquot of membrane protein is incubated with 0.5 nM [3H]ketanserin for 60 minutes at 25 jC.For saturation binding experiments, concentrations of radioligand ranged from 0.01 nM to 2.0 nM. Non-

specific binding is estimated in the presence of 1.0 AM mianserin. Membranes are filtered and washed 3

times and the filters are counted to determine [3H]ketanserin specifically bound. Ki was calculated using

the Cheng–Prusoff equation [9]. Ki values are expressed as pKi (� log Ki).

5-HT2B receptor binding assay in CHO-K1 cells expressing a cloned human 5-HT2B receptor was

performed using [3H]LSD as a radioligand. CHO-K1 cells stably transfected with a plasmid encoding

the human serotonin 5-HT2B receptor are used to prepare membranes in modified Tris–HCl buffer (50

mM Tris–HCl, pH 7.4, 4 mM CaCl2, 0.1% ascorbic acid) using standard techniques [4]. In com-

petition binding experiments, a 30 mg aliquot of membrane protein is incubated with 1.2 nM [3H]LSD

for 60 minutes at 37 jC. For saturation binding experiments, concentrations of radioligand ranged

from 0.05 to 8.0 nM. Non-specific binding is estimated in the presence of 10 mM serotonin. Mem-

branes are filtered and washed 3 times and the filters are counted to determine [3H]LSD specifically

bound. Ki was calculated using the Cheng–Prusoff equation [9]. Ki values are expressed as pKi

(� log Ki).

5-HT2C receptor binding assay in CHO-K1 cells expressing a cloned human 5-HT2C receptor was

performed using [3H]mesulergine as a radioligand. CHO-K1 cells stably transfected with a plasmid

encoding the human serotonin 5-HT2C receptor are used to prepare membranes in modified Tris–HCl

buffer (50 mM Tris–HCl, pH 7.7, 0.1% ascorbic acid, 10 AM pargyline) using standard techniques [4].

In competition binding experiments, a 3.2 mg aliquot of membrane protein is incubated with 1.0 nM

[3H]mesulergine for 60 minutes at 25 jC. For saturation binding experiments concentrations of

radioligand ranged from 0.05 to 8.0 nM. Non-specific binding is estimated in the presence of 1 AMmianserin. Membranes are filtered and washed 3 times and the filters are counted to determine

[3H]mesulergine specifically bound. Ki was calculated using the Cheng–Prusoff equation [9]. Ki values

are expressed as pKi (� log Ki).

Protein assay

Protein contents of the membrane concentration were measured by the method of Bradford [5] using

bovine serum albumin as the standard.

Data analysis

Kd (dissociation constants) and Bmax (the density of 5-HT2 receptors binding sites) were determined

using nonlinear regression analysis. Nonlinear regression analysis of saturation and competition binding

assay were performed using Prism (GraphPad Software, San Diego, CA, USA). Data were presented as


the mean F standard error of mean (SEM). Differences between groups were analyzed using a one-way

ANOVA, to obtain a p value.

Drugs

The drugs used and their sources were as follows: sarpogrelate from Mitsubishi Chemical Corporation

(Tokyo, Japan); ketanserin, miancerin and serotonin from Research Biochemicals International (Mary-

land, USA); [3H]ketanserin (88 Ci mmol� 1) and [3H]LSD (76.2 Ci mmol� 1) from NEN Life Science

Products (USA); [3H]mesulergine (74 Ci mmol� 1) from Amersham Pharmacia Biotech (England).

Molecular modeling

3D models of the TMD bundle of the three 5-HT2R family member were carried out by using

homology modeling technique using criteria and procedures which took into account sequence

conservation [1,25], degree of residue polarity [40], and incorporated a large number of experimental

constraints as reviewed elsewhere [2,34]. All bioamine-GPCR sequences presently available were

Fig. 1. Transmembrane domains multiple sequence alignment of human 5-HT2AR, 5-HT2BR, 5-HT2CR and bovine rhodopsin.

Most conserved residue of each TMD among GPCR super-family is shaded in gray. Residues are numbered according to a

consensus numbering scheme [2], and each helix N-terminal starting residue is numbered according to SWISSPROT genome

databank numbering scheme (5-HT2AR P28223, 5-HT2BR P41595, 5-HT2CR P28335, bovine rhodopsin P02269). Residues

involved in sarpogrelate binding are depicted in bold. Residues that are making electrostatic interactions with sarpogrelate are

indicated by circle. For clarity, only the TMD sequences of the template and of the three studied 5-HT2Rs are presented in

Fig. 1.


aligned with RH and BR using the CLUSTALX software [32]. For clarity, only the TMD sequences of

the two templates, BR and RH, and the three studied 5-HT2R family are represented in Fig. 1. The

residues are numbered according to both the SwissProt database and to Ballesteros and Weinstein [2], in

order to facilitate comparison between the three subtypes of 5-HT2R.

Model refinements, energy minimization, and molecular dynamics (MD) simulations were performed

with the CHARMM program [7]. A 20-A cut-off was applied to non-bond interactions. All graphical

manipulations were done with the Insight II modeling package (Accelrys Inc, San Diego, CA). Both

graphical interface and calculations were performed on Silicon Graphics O2 stations.

To minimize errors inherent to the generation of receptor 3D-models, for each receptor, five different

protocols of simulation were performed by assigning different initial velocities, leading in fine to fifteen

receptor models. For 5-HT2BR, we used our previously published 3D model [19]. The 5-HT2AR and

5-HT2CR models were built on the basis of the backbone coordinates of 1HZX [31] file from the

Protein Data Bank. Side-chains of the receptors were positioned using the Biopolymer module of Insight II

software. Interhelix contacts were adjusted by taking into account data from site-directed mutagenesis

experiments. After 700 ps of constrained MD simulations, average structures resulting from 200 ps

of free MD simulations were energy-minimized. Conformational stability of the generated 5-HT2AR,

5-HT2BR and 5-HT2CR models were systematically assessed by following the root mean square

deviations (RMSDs) of the cartesian coordinates of the backbone atoms at all steps of the simulations.

The obtained relaxed and equilibrated receptors were further used for the docking of sarpogrelate.

Sarpogrelate parameters for CHARMM force field were optimized by performing quantum chemistry

conformational analysis of sarpogrelate. Calculations were done at the DFT/6-31 + G** level. The 3D

models of 5-HT2 receptors were finally complexed to sarpogrelate by using the AutoDock 3.0 program

[22]. The most stable receptor/antagonist drug complexes were then submitted to 300 ps MD at 300 K

and finally analyzed.

Results

Radioligand binding experiments for 5-HT2 receptor family

In saturation binding experiments, specific binding of [3H]ketanserin, [3H]LSD and [3H]mesulergine

were determined using the cloned membranes of the 5-HT2A, 5-HT2B and 5-HT2C receptor subtypes,

respectively. The affinity constant (Kd) and the receptor density (Bmax) for all the membranes are

shown in the Table 1. Both sarpogrelate and ketanserin monophasically inhibited this bindings. Table 2

shows the binding affinity (pKi) for these two 5-HT2 antagonists at the three subtypes. Both the

Table 1

Saturation binding parameters for human 5-HT2 receptor family

Receptors Radioligand Kd (nM) Bmax (fmol mg� 1 protein� 1)

5-HT2A (3) [3H]ketanserin 0.2 F 0.005 510.0 F 62.0

5-HT2B (3) [3H]LSD 2.1 F 0.2 1100.0 F 62.0

5-HT2C (3) [3H]mesulergine 1.1 F 0.1 4900.0 F 843.0

The data shown indicate mean F SEM and numbers in parentheses indicate number of experiments.

Table 2

Binding affinities (pKi) of sarpogrelate and ketanserin for human 5-HT2 receptor family in CHO-K1 cells

Receptors Sarpogrelate (pKi) Ketanserin (pKi)

5-HT2A 8.52 F 0.12 (3) 9.67 F 0.12 (4)

5-HT2B 6.57 F 0.12 (3) 6.55 F 0.09 (4)

5-HT2C 7.43 F 0.03 (3) 7.39 F 0.11 (4)

The data represented the mean F SEM. Number in the parentheses indicated the number of experiments. pKi = � log (Ki),

where Ki is in nM.


antagonists showed high binding affinity to 5-HT2A subtype vs 5-HT2B and 5-HT2C subtypes. However,

ketanserin showed 10 fold higher binding affinity to 5-HT2A subtype in comparison with sarpogrelare.

Sarpogrelate displayed higher binding affinity to 5-HT2A subtype than 5-HT2B and 5-HT2C subtypes.

The affinities of sarpogrelate and ketanserin to both 5-HT2B and 5-HT2C receptor subtypes were almost

similar.

Molecular modeling

The observation of the modeled binding sites of sarpogrelate in the three receptors subtypes shows a

strong interaction between antagonist and receptors (Figs. 3 and 4). Those interactions involve a large

number of residues (listed in Table 3) belonging to the TMDs 3, 4, 5, 6, and 7. In the three complexes

Table 3

List of the interacting residues with 5-HT2AR, 5-HT2BR and 5-HT2CR

TMDs 5HT2AR 5HT2BR 5HT2CR

VdW Elc VdW Elc VdW Elc

3 Trp3.28 Trp3.28 Val3.33 Asp3.32 Trp3.28 Asp3.32

Ile3.29 Asp3.32 Phe3.35 Leu3.31

Asp3.32 Asp3.32

Val3.33 Phe3.35

Ser3.36

Ser3.39

Met3.41

4 Ile4.56 Leu4.51 Ile4.52

Ser4.53

Val4.56

5 Ser5.46 Gly5.42 Ala5.46

Phe5.47 Ala5.46 Phe5.47

Thr5.49 Pro5.50

6 Trp6.48 Trp6.48 Phe6.51 Trp6.48

Phe6.51 Thr6.54 Phe6.51

Phe6.52 Leu6.58 Phe6.52

7 Val7.39 Glu7.36 Gly7.42 Ser7.46

Tyr7.43 Ile7.37

Trp7.40

Residues are numbered according to a consensus numbering scheme [2]. Receptor transmembrane domains (TMDs). Van der

Waals contacts (VdW). Electrostatic interactions (Elc).


sarpogrelate takes a similar orientation inside the helix bundle, parallel to the membrane plane. The

hydrophobic part of the antagonist molecule that is defined by two 6-membered rings (rings A and B,

Fig. 2), is tightly bound to residue side chains of the hydrophobic pocket delimited by TMDs 4, 5 and 6

through a large number of Van der Waals interactions. The charged part of sarpogrelate interacts mostly

with polar and charged residues belonging to TMD 3 and 6 for 5-HT2AR, TMD 3 for 5-HT2BR and TMD

3 and 7 for 5-HT2CR, through electrostatic interactions.

Electrostatic interactions

In the three receptor subtypes/antagonist complexes, a strong electrostatic interaction is formed in the

course of all simulations between the cationic head of sarpogrelate and the carboxylate group of Asp3.32

(Fig. 4) that is the major counter-ion for 5-HT binding to 5-HT2Rs [11,18,29,33,38].

In the 5-HT2AR, the number of electrostatic interactions established with sarpogrelate is higher than in

the two other subtypes. Indeed, in addition to the interaction with the cationic head (hydrogen atom of

the trimethyl ammonium) of sarpogrelate, Asp3.32 (acidic function oxygen) interacts with hydrogen H1

atom of carboxylate group of the antagonist. The peptide bond oxygen of Trp3.28 interacts with the

hydrogen atom of the trimethyl ammonium group of sarpogrelate and finally, indole NH group hydrogen

of Trp6.48 interacts with the carboxylate group oxygen O2 of sarpogrelate (Figs. 3a and 4a).

In the 5-HT2BR and the 5-HT2CR, sarpogrelate interacts with acidic function of Asp3.32 (Fig. 4b and

4c). Moreover, in the 5-HT2CR, carboxylate group oxygen O1 of sarpogrelate is H-bonded by Ser7.46 OH

group. In 5-HT2AR and 5-HT2BR, interactions with Ser7.45 and Ser7.46 are not possible due to the axial

position of sarpogrelate.

Fig. 2. Sarpogrelate chemical structure. Ox (x = 1–6), oxygen atom numbering scheme.

Fig. 3. Stereo view of the binding site of sarpogrelate in 5-HT2AR (a), 5-HT2BR (b), 5-HT2CR (c) receptor, view orthogonal to

the membrane plane from the extracellular side of the cell.


Hydrophobic interactions

Whatever the receptor subtype considered, most of the Van der Waals contacts with hydrophobic

residues are done by atoms of the two 6-membered rings A and B of sarpogrelate. A major difference of

Fig. 4. Two dimensional plots of sarpogrelate/5-HT2AR (a), -/5-HT2BR (b), -/5-HT2CR (c) complexes. Hydrogen atoms were not

represented for clarity of the picture. Spiky semicircle around interacting atoms represent Van der Waals interactions. Small

lines between interacting heavy atoms symbolize hydrogen bonds. Residues are numbered according to a consensus numbering

scheme [2]. Schematic diagrams were obtained with the help of LIGPLOT software [35].


sarpogrelate binding among the three receptor subtypes appears in the ligand bundle axial position

(orthogonal to the membrane plane). In the 5-HT2AR, sarpogrelate is localized near the extracellular

edge of the receptor, whereas it is deeply embedded inside the hydrophobic binding pocket delimited by

TMD 4, 5 and 6, in the 5HT2CR and in a lesser extend in the 5HT2BR. This difference seems to be due to

mostly for the nature of key residues present in TMD 4 and 5. In 5HT2AR, the side chain of Ser5.46 (vs

Ala5.46 in 5HT2BR and 5HT2CR,) prevents the methoxybenzyl rings (ring B, Fig. 2) of sarpogrelate to

enter deeply inside the hydrophobic binding pocket. Due to steric hindrance, the methyl position of the

methoxy group of ring B prevents the formation of a hydrogen bond between OH group of Ser5.46 and

the methoxy oxygen O6 of sarpogrelate. During the course of molecular dynamics trajectory, driving

forces guided the ligand toward TMD 4. In the 5-HT2AR, to avoid steric clash and in order to adjust Van

der Waals contacts with Ile4.56 side chain (vs Ile4.56 in 5HT2BR and Val4.56 in 5HT2CR), sarpogrelate is

constrained to move away from Ile4.56 and Ser5.46 toward the extra-cellular side of the receptor and to

translate toward the strong attractive and negatively charged Asp3.32. The presence of a shorter length

side chain Val residue at locus 4.56 in the 5-HT2CR, makes possible the interaction of sarpogrelate with

Ile4.52 and Ser4.53.

In 5HT2CR, sarpogrelate is deeply embedded in the helix bundle and the methoxy group of aromatic

ring B can interact with Pro5.50. Additional contacts are done with Ala5.46 and Phe5.47. In 5HT2BR,


sarpogrelate is less embedded than in 5-HT2CR. The interaction with Pro5.50 is not possible since the

translation of sarpogrelate is blocked by the presence of the Thr5.49 side-chain (vs Ile5.49 in 5HT2AR and

5HT2CR).

Interactions with aromatic residues

Sarpogrelate promotes the rearrangement by direct contacts of residues belonging to the aromatic

network upon binding, including in 5-HT2A/2CR, Phe5.47, Trp6.48, Phe6.51 and Phe6.52 and in 5-HT2BR,

only Phe6.51. The conformational changes undergone by those residue side-chains are not compatible

with activation of the receptor. Indeed, we previously described the structural modification promoted by

5-HT docking in 5-HT2BR [19]. Upon 5-HT binding, rotation of the Phe6.52 aromatic ring affected the

aromatic network within the hydrophobic pocket delimited by TMDs 4, 5, 6, and 7. The two contacted

aromatic residues, Phe6.52 and Trp6.48, adopted a ‘‘T-shape’’ oriented structure. In the case of

sarpogrelate, aromatic residues are constrained in a stacked conformation. The two biphenyl rings A

and B of sarpogrelate constrain the side-chains of those amino acids by hydrophobic interactions and

prevent the propagation of sequential side-chain rotation by maintaining stacked the ring plane of

aromatic residues.

We note also in the three complexes that some aromatic residues (Trp3.28, Trp6.48, Phe6.51 in 5-HT2AR;

Phe3.35, Phe6.51, Trp7.40 in 5-HT2BR; Phe3.35, Trp3.28, Trp6.48, Phe6.51 in 5-HT2CR) define an ‘‘ aromatic

box ’’ that surrounds and stabilizes the cationic head of sarpogrelate and maintains the antagonist

molecule in a favorable orientation to interact with Asp3.32.

Discussion

In this report, computer modeling of the 5-HT2R family allowed us to define putative binding sites of

sarpogrelate in the three subtypes and to speculate on the possible 5-HT2AR binding selectivity. The

relevance of this theoretical approach was estimated by comparing the structural predictions made on the

basis of the sole computations models to radioligand binding assays data measuring affinity of

compounds with pKi.

Sarpogrelate affinity for the three subtypes

The binding of sarpogrelate in our three 5-HT2R subtypes 3D models shows the participation

of a higher number of interacting residues, mainly through hydrophobic interactions, in 5-HT2A/

2CR than in 5-HT2BR (Table 3). In 5-HT2AR, sarpogrelate makes electrostatic interactions with

three residues (Asp3.32, Trp3.28 and Trp6.48). At first approximation, this suggests a greater affinity

of sarpogrelate for 5-HT2AR followed by 5-HT2CR and finally 5-HT2BR. Those data are in good

agreement with experimentally determined binding affinities of sarpogrelate for 5-HT2R family

expressed in transfected cell. pKi values of sarpogrelate for 5-HT2AR, 5-HT2BR and 5-HT2CR,

are respectively 8.52, 6.57, 7.43 (Table 2). Sarpogrelate is a highly active on 5-HT2AR (pKi =

8.52).

In the three receptor models, Asp3.32 is involved in the binding site of sarpogrelate. Previous

published molecular modeling studies of 5-HT2R family member/antagonist complexes have shown


the existence of such a pattern of interactions between TMD 3, through highly conserved Asp3.32

and ligand molecule [11,18,29,33,38]. Asp3.32 neutralizes the cationic head of sarpogrelate and

allows the antagonist molecule to prevent the fixation and the action of agonist molecules.

Sarpogrelate and aromatic residues network

Hibert et al. [15] reported that the two highly conserved aromatic residues Trp6.48 and Phe6.52

are directly involved in the binding of several neurotransmitters (5-HT, dopamine and adrenaline)

to their corresponding receptors. They also speculated that the side chain conformations of these

hydrophobic residues probably changed during the binding process. Such changes could directly

affect the conformation of the adjacent helices, in particular in the vicinity of proline residues, and

of other helices by propagation along the backbone of interacting conserved aromatic residues.

Site-directed mutagenesis experiments also supported the crucial role of the two above aromatic

residues [10,28,36]. Six conserved aromatic residues (Trp3.28, Phe3.35, Trp4.50, Phe6.44, Trp6.48, and

Trp7.40) are in hydrophobic interaction and belong to this aromatic network. We have previously

demonstrated that in 5-HT2BR/5-HT complex, upon 5-HT binding, a shift of Phe6.52 induces a

displacement of the Trp6.48 side chain. Consequently, the other aromatic amino-acids in the

hydrophobic core of 5-HT2BRs are submitted to conformational changes. In our present 3D

models, whatever the subtype considered, the two phenyl rings of sarpogrelate are embedded in the

hydrophobic core of the receptors delimited by TMD 4, 5, 6 and interact tightly with aromatic

residues. Phe6.52 and Trp6.48 side chains are constraints and can undergo a conformational change

upon sarpogrelate binding. This structural configuration explains the low RMSD of the backbone

atoms found between the starting and final MD conformations of 5-HT2AR and in a lesser extent

for 5-HT2CR.

5-HT2AR selectivity

Radioligand binding assay showed that sarpogrelate had higher binding affinity to human 5-

HT2AR than 5-HT2BR and 5-HT2CR. The sarpogrelate pKi values at 5-HT2AR, 5-HT2BR and 5-

HT2CR show a moderate selectivity for 5-HT2AR vs 5-HT2CR (10 fold) and a high selectivity for

5-HT2AR vs 5-HT2BR (100 fold) (Table 2). Our modeling results are in good agreement with

experimental data. Indeed, bearing in mind that standard MD simulation algorithms do not allow

simulations of the protein folding processes and calculations of theoretical pKi, it can be used to

appreciate the role of key residues in the compound selectivity and interpret experimental data. At

first approximation, receptor affinity can be appreciated by the number of interacting residues and

the strength of the interactions. The number and strength of interactions are easily given by listing

effective Van der Waals and electrostatic interaction between the ligand and the receptor. The

selectivity can be evaluated by comparing and analyzing the binding modes of the different

complexes.

We have shown above that axial position of sarpogrelate in the helix bundle is different for the

three 5-HT2R subtypes. This orientation could explain the difference of affinity between the three

receptors subtypes and also the selectivity of sarpogrelate for 5-HT2AR. The position of sarpogrelate

in the 5HT2A/2CR is mainly influenced by residues at the loci 4.56 and 5.46. In 5-HT2AR, the side

chain of Ser5.46 prevents sarpogrelate to deeply enter inside the helix bundle and to make a contact


with Pro5.50 due to steric hindrance between OH group of Ser5.46 and the methoxy oxygen O6 of

sarpogrelate. The side chain of Ile4.56 constraints sarpogrelate to adjust its position by translating

toward the strongly attractive and negatively charged Asp3.32. In 5-HT2CR, Ala5.46 and Val4.56, two

shorter side-chain residues allow 5-HT2CR to interact down the helix bundle axis to Pro5.50. Since

5-HT2BR 3D model was built from a different 3D crystallized template, a small but significant

difference appears in TMD 5. The methyl group of Thr5.49 side-chain interacts with phenyl ring B

of sarpogrelate and not with Pro5.50.

Ser5.46 is not conserved among all the serotonin receptor families. Indeed, among the 90 aligned

receptors, a Ser residue is present at this locus only in five different serotonin receptors (human, pig,

Rhesus macaque 5-HT2AR ; drosophila 5-HT7R and silk moth 5-HTR) and a Thr residue carrying a

hydroxyl group is present in seven different serotonin receptors (drosophila 5-HT1AR and 5-HT1BR ;

noctuid moth 5-HTR ; human, rat and mouse 5-HT6R ; coenorhabditis 5-HTCE2LR). In the other receptor

sequence, an Ala residue is present. A long side chain hydrophobic residue may change the binding

profile of sarpogrelate. Those observations are reinforcing the hypothesis that sarpogrelate 5-HT2AR

selectivity may depend mostly on the presence of a polar residue or a long hydrophobic side-chain

residue at locus 5.46. It is interesting to note that the OH group hydrogen of Ser5.46 can not make a

hydrogen bond with the methoxy oxygen of sarpogrelate as we could expect. Indeed, the long side chain

of Ile4.56 prevents the methoxy group of sarpogrelate and the OH group of Ser5.46 to adjust their position

to optimally interact.

Comparison of sarpogrelate with other antagonist/5-HT2R family complexes

Locus 7.45 has been previously described as determinant for selective binding of ketanserin to the

5HT2AR vs 5-HT2CR [18]. The presence of a polar residue at locus 7.46 seems also to influence

interaction with locus 7.45. In our 3D models, locus 7.46 participates to antagonist binding and increases

5-HT2CR selectivity vs 5-HT2AR. Neither locus 7.45 nor locus 7.46 is participating to sarpogrelate

binding in the 5-HT2A/2BR 3D model due to the receptor axial position of the molecule due to their

positions in the helix bundle axis.

Comparison of experimental binding affinities of sarpogrelate and ketanserin shows that both ligand

are strongly active on 5-HT2AR. pKi values for sarpogrelate and ketanserin were 8.52 and 9.67 for

5-HT2AR, 6.57 and 6.55 for 5-HT2BR, 7.43 and 7.39 for 5-HT2CR, respectively (Table 2). We can

note more pronounced 5-HT2AR affinity (10 fold) and selectivity for ketanserin.

Sarpogrelate binding mode in our 5-HT2R family 3D models are different from recently published

ketanserin related compound like aminoalkylbenzo and heterocycloal [6]. In this study, antagonist

compounds were predicted to bind with TMD 2, 3 and 7 and have no contact with TMD 4, 5 and 6.

Affinity of ketanserin for Ser5.46 to Ala 5-HT2A mutant receptor has been also tested in order to

control that helix 5 is not participating to ketanserin binding [27]. However, only the lowest energy

conformation of the complexes was retained for study, selected from ligand docking software protocol.

No MD simulation was applied to those ligand/receptor configurations to test the availability of the

antagonist binding mode. Sarpogrelate is not related to the same family of compounds than ketanserin

and present a particular chemical structure, i.e., a very flexible aromatic part (ring A and B) that

makes feasible the docking of the molecule perpendicular to the bundle axis. This can explain the

difference obtained with ketanserin binding, parallel to the bundle axis since the latter molecule is

somewhat rigid.


Conclusion

Our molecular modeling investigations are useful to predict that the sarpogrelate binding site of 5-

HT2R family involves residues belonging to TMDs 3, 4, 5, 6 and 7. We also provide first structural

hypothesis on the 5-HT2AR selectivity of sarpogrelate that may be further examined by site-directed

mutagenesis experiments and radioligand binding assays. Important determinants for selective binding

appear to be assumed mostly by Trp3.28, Asp3.32, Ile4.56, Ser5.46. Those results are useful to axe research

in the way to improve 5-HT2AR vs 5-HT2BR/5-HT2CR selectivity by modifying the chemical structure of

sarpogrelate, and the present findings of the selectivity of sarpogrelate towards 5-HT2AR vs 5-HT2BR

and 5-HT2CR emphasises its clinical implications.

Acknowledgements

This research was in part supported by a grant from the Promotion and Mutual Aid Corporation for

Private Schools of Japan.

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Identification of the binding sites and selectivity of sarpogrelate, a novel 5HT 2 antagonist, to...

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