Bivalent ligands for the serotonin 5-HT3 receptor

Article abstract of DOI:10.1021/ml2000388

The serotonin 5-HT₃ receptor is a ligand-gated ion channel, which by virtue of its pentameric architecture, can be considered to be an intriguing example of intrinsically multivalent biological receptors. This paper describes a general design a

Full text of DOI:10.1021/ml2000388

LETTER
pubs.acs.org/acsmedchemlett
Bivalent Ligands for the Serotonin 5-HT3 Receptor
Andrea Cappelli,*,† Monica Manini,† Marco Paolino,† Andrea Gallelli,† Maurizio Anzini,† Laura Mennuni,‡
Marta Del Cadia,§ Francesca De Rienzo,§,^ M. Cristina Menziani,§ and Salvatore Vomero†
†Dipartimento Farmaco Chimico Tecnologico and European Research Centre for Drug Discovery and Development,
Universitꢀa di Siena, Via A. Moro, 53100 Siena, Italy
‡Rottapharm Madaus, Via Valosa di Sopra 9, 20052 Monza, Italy
§Dipartimento di Chimica, Universitꢀa di Modena e Reggio Emilia, Via Campi 183, 41100 Modena, Italy
^Centro S3, CNR-Istituto di Nanoscienze, Via Campi 213A, 41125 Modena, Italy
S Supporting Information
b
ABSTRACT: The serotonin 5-HT3 receptor is a ligand-gated
ion channel, which by virtue of its pentameric architecture, can
be considered to be an intriguing example of intrinsically
multivalent biological receptors. This paper describes a general
design approach to the study of multivalency in this multimeric
ion channel. Bivalent ligands for 5-HT3 receptor have been
designed by linking an arylpiperazine moiety to probes showing
different functional features. Both homobivalent and hetero-
bivalent ligands have shown 5-HT3 receptor affinity in the
nanomolar range, providing evidence for the viability of our
design approach. Moreover, the high affinity shown by homo-
bivalent ligands suggests that bivalency is a promising approach
in 5-HT3 receptor modulation and provides the rational basis for applying the concepts of multivalency to the study of 5-HT3
receptor function.
KEYWORDS: Serotonin receptors, 5-HT3, bivalent ligands, multivalency, molecular modeling
erotonin 5-HT3 receptor (5-HT3R) belongs to the family of
the ligand-gated ion channels (LGICs) and shows a penta-
In a previous paper, we described both the results of the
exploration of the receptor pocket interacting with the c-edge
(positions 3 and 4) of the quipazine quinoline nucleus by means
of a systematic approach based on lipophilic probes (primarily
alkyl chains of different length in compounds 34DSQ) and the
development of tacrine-related heterobivalent ligand 1. This
compound showed nanomolar potency both for 5-HT3R and
for human AChE and represented the first example of a rationally
designed high affinity 5-HT3R ligand showing nanomolar AChE
inhibitory activity.7 More recently, we reported a noncovalent
approach to dynamic multivalent complexes composed of a
hydrophobic urea adamantyl-functionalized poly(propylene imi-
ne) dendrimer, a solubilizing oligo(ethylene glycol)-based guest
molecule, and an arylpiperazine heterobivalent 5-HT3R ligand
(2a,b).8 This work provided new perspectives for obtaining
dynamic and adjustable noncovalent complexes with pharmaco-
logical potential. However, the stability of the supramolecular
complexes in the biological environment should be further
increased to allow for the in vivo evaluation of the construct.
Thus, the exploration of multivalency in 5-HT3R function by
means supramolecular constructs involves the preparation of
S
meric structure similar to that of nicotinic acetylcholine (nACh),
glycine, and type A γ-aminobutyric acid (GABAA) receptors.1,2
By virtue of its multimeric architecture, 5-HT3R represents an
intriguing example of intrinsically multivalent biological recep-
tors. Initially, a single 5-HT3R subunit (5-HT3A) was cloned, and
subsequently, other 5-HT3R subunits (5-HT3B-5-HT3E) have
been identified.1,2 The 5-HT3A cDNA forms functional homo-
pentameric cell surface receptors, whereas 5-HT3B was reported
to form functional 5-HT3Rs when expressed together with
5-HT3A but not when expressed alone.1,2 The three-dimensional
structure of 5-HT3R has not yet been determined at an atomic
level, but a large amount of site-directed mutagenesis experi-
ments have been performed to gather information about struc-
ture and function,1ꢀ3 and the data obtained have been used in the
building of 3D receptor homology models.4
The research activity performed by the pharmaceutical com-
panies in the development of 5-HT3R antagonists has produced a
large number of drug molecules showing remarkable efficacy in
preventing acute chemotherapy-induced nausea and vomiting. Our
interest in the development of 5-HT3R ligands started from the
study of arylpiperazine derivatives related to quipazine (Scheme 1)
and has since produced a considerable amount of information on
the interaction of this class of ligands with their receptor.5,6
Received: February 8, 2011
Accepted: May 9, 2011
Published: May 09, 2011
r
2011 American Chemical Society
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Scheme 1. Development of Arylpiperazine-Based Bivalent
5-HT3R Ligands 1 and 2a,b
Scheme 2. Synthesis of Homobivalent Ligands 3aꢀfa
a Reagents: (i) H2N-CH2-Y-(X-CH2CH2)n-NH2, (C2H5)3N, CH2Cl2.
(ii) H2N-CH2-Y-(X-CH2CH2)n-NH-COOC(CH3)3, CH2Cl2, (C2H5)3N.
(iii) CF3COOH. (iv) CH2Cl2, (C2H5)3N. (v) N-methylpiperazine. Spacers:
3a and 9a: Y = (CH2)2, X = CH2, and n = 1; 3b and 9b: Y = (CH2)3,
X = CH2, and n = 1; 3c and 9c: Y = CH2, X = CH2, and n = 2; 3d and 9d:
Y = CH2, X = O, and n = 2; 3e, 8e, and 9e: Y = CH2, X = O, and n = 7; 3f, 8f,
and 9f: Y = CH2, X = O, and n = 9.
Homobivalent ligands 3aꢀf were synthesized as depicted in
Scheme 2, while the synthesis of the remaining target compounds
included in Table 1 is described in the Supporting Information.
Key intermediates 9aꢀf were obtained from acyl chloride 77
following two different pathways. When short spacers are
involved, compound 7 was reacted with the suitable diamine to
give chlorinated homodimers 9aꢀd, while longer homodimers
were obtained starting from amides 8e,f by deprotection and
coupling with 7. The nucleophilic substitution with N-methylpi-
perazine completed the reaction sequence to target homodimers
3aꢀf.
Figure 1. Design of arylpiperazine bivalent ligands 3ꢀ5. The elements
in red are the pharmacophoric groups, which allow the arylpiperazine
moiety (anchor) to interact with the 5-HT3R binding site; magenta
indicates the probe responsible for the interaction with a biological
target (biofunctional probe, MPQC and Tacrine) or for a particular
chemical property (chemofunctional probe, UAA); blue indicates the
spacer.
new guest molecules showing an increased interaction strength
with the host.
The affinity of compounds 3ꢀ6 for the 5-HT3R was measured
by means of displacement studies performed with radiolabeled
granisetron specifically bound to the 5-HT3R in rat cortical
membranes9 (see the Supporting Information), and the results
are summarized in Table 1.
The rational design of tailored bivalent ligands requires a deep
understanding of their interaction with the receptor. Therefore, in
the present paper, we report on the results of a study focused on the
characterization of the interaction of the 5-HT3R with bivalent
ligands assembled by (a) an optimized arylpiperazine 5-HT3R
ligand (playing the role of an anchor in interacting with the main
binding site of the 5-HT3R), (b) a spacer showing different length,
and (c) a probe showing different stereoelectronic and functional
features. On the basis of their functional features, the probes can be
classified into chemofunctional [e.g., the ureido acetic acid (UAA)
moiety of heterobivalent guests 2a,b] or biofunctional [a group
capable of interacting with receptors or enzymes, e.g., tacrine in
heterobivalent ligand 1, or the same arylpiperazine anchor
(MPQC) in the corresponding homobivalent ligands] (Figure 1).
The results suggest that the 5-HT3R is capable of accommo-
dating bivalent ligands showing different spacers and probes,
which modulate the affinity in a significant manner. Homobiva-
lent ligands 3 are the most potent bivalent ligands in each
subgroup characterized by the same spacer, and most notably,
they are more potent than the corresponding reference mono-
valent ligands 6aꢀf bearing a t-butoxycarbonylamino probe.
Moreover, the spacer's features appear to affect the interaction
of the different probes. Thus, the heptamethylene tether appears
to behave as the optimal spacer (see RP values in Table 1), and
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Table 1. 5-HT3R Binding Affinities of Bivalent Ligands 3ꢀ5 and Reference Monovalent Ligands 6
compd
probea
spacer (met. equiv.)b
Ki (nM)c
RPd
3a
3b
3c
3d
3e
3f
MPQC
MPQC
MPQC
MPQC
MPQC
MPQC
tacrine
tacrine
tacrine
UAA
ꢀ(CH2)6ꢀ (6)
ꢀ(CH2)7ꢀ (7)
ꢀ(CH2)8ꢀ (8)
28 ( 7.1
2.7 ( 0.7
67 ( 15
7.7 ( 1.5
27 ( 8.2
15 ( 2.4
71 ( 22
5.6f
10
1
25
3
ꢀCH2ꢀCH2ꢀOꢀCH2ꢀCH2ꢀOꢀCH2ꢀCH2ꢀ (8)
ꢀCH2ꢀCH2ꢀOꢀ(CH2ꢀCH2ꢀO)6ꢀCH2ꢀCH2ꢀ (23)
ꢀCH2ꢀCH2ꢀOꢀ(CH2ꢀCH2ꢀO)8ꢀCH2ꢀCH2ꢀ (29)
ꢀ(CH2)6ꢀ (6)
10
5
4a
4be
4c
5be
5de
5e
5f
13
1
ꢀ(CH2)7ꢀ (7)
ꢀ(CH2)8ꢀ (8)
69 ( 17
26g
141g
12
1
ꢀ(CH2)7ꢀ (7)
UAA
ꢀCH2ꢀCH2ꢀOꢀCH2ꢀCH2ꢀOꢀCH2ꢀCH2ꢀ (8)
ꢀCH2ꢀCH2ꢀOꢀ(CH2ꢀCH2ꢀO)6ꢀCH2ꢀCH2ꢀ (23)
ꢀCH2ꢀCH2ꢀOꢀ(CH2ꢀCH2ꢀO)8ꢀCH2ꢀCH2ꢀ (29)
ꢀ(CH2)6ꢀ (6)
5
UAA
106 ( 18
154 ( 21
178 ( 32
18 ( 3.1
127 ( 23
132 ( 25
89 ( 18
71 ( 17
4
UAA
6
6a
6b
6c
6d
6e
6f
NH-Boc
NH-Boc
NH-Boc
NH-Boc
NH-Boc
NH-Boc
10
1
ꢀ(CH2)7ꢀ (7)
ꢀ(CH2)8ꢀ (8)
ꢀCH2ꢀCH2ꢀOꢀCH2ꢀCH2ꢀOꢀCH2ꢀCH2ꢀ (8)
ꢀCH2ꢀCH2ꢀOꢀ(CH2ꢀCH2ꢀO)6ꢀCH2ꢀCH2ꢀ (23)
ꢀCH2ꢀCH2ꢀOꢀ(CH2ꢀCH2ꢀO)8ꢀCH2ꢀCH2ꢀ (29)
7
7
5
4
a See Figure 1. b Methylene equivalents. c Each value is the mean ( SEM of three determinations and represents the concentration producing half the
maximum inhibition of [3H]granisetron (final concentration, 1 nM) specific binding to rat cortical membranes. d Relative potency with respect to the
compound bearing the heptamethylene spacer (expressed as the ratio between the Ki value of the compound and that of the analogue showing the same
probe and the heptamethylene spacer). e Compounds 4b and 5b,d are cited in the introduction as 1 and 2a,b, respectively. f See ref 7. g See ref 8.
either its shortening to the hexamethylene or the elongation to
the octamethylene counterparts decreases the 5-HT3R affinity of
about 1 order of magnitude in compounds 3 and 4. The
replacement of two methylene units in the octamethylene
spacers of homobivalent ligand 3c with two oxygen atoms as in
3d produces an increase in potency of about 1 order of
magnitude, whereas the spacer elongation leading to 3e,f gen-
erates a subtle affinity modulation. When long spacers are
considered, a possible fluctuation in the extrareceptorial aqueous
environment can be assumed, but the structureꢀaffinity relation-
ships in the compounds 3e,f bearing the longest spacers (23 or 29
methylene equivalents, respectively) appear to suggest that the
probe could interact with different environments.
The homology model of the homomeric 5-HT3AR described
in a previous paper4 was used as an instrument to check the
receptor's ability to accommodate bivalent ligands. The proto-
nated structures of homobivalent ligands with different length
spacers (3aꢀd, see Table 1) were docked into the interface of the
5-HT3A-A dimer. In all cases, the main poses of the ligand's
anchor moiety lie within the serotonin binding site and share
similar features: The charged head of the piperazine moiety
establishes a charged-reinforced H-bond interaction with the
Glu129 side chain, the nearby amide NH is hydrogen-bonded to
the hydroxyl side chain of Tyr153, and the ligand aromatic
moiety establishes π-interactions with Trp183 and Trp90.10ꢀ12
On the contrary, the docking poses of the arylpiperazine probe
can be clustered into two main groups. The first putative bind-
ing site for the probe is located below the known active site, close
to the membrane (Figure 2d), and is characterized by both
aromatic and polar residues: Tyr73, Tyr88, and Phe130, which
last has been proved to be a key residue for receptor function,13
and Ser177, which establishes H-bond interaction with the
piperazine ring.
The second putative binding site, instead, was found in the
upper extracellular region of the dimer interface (in the opposite
direction with respect to the membrane, Figure 2c) and is
characterized by Asp189, which forms a charge-reinforced
H-bond with the charged head of the piperazine moiety, and
polar and aromatic residues, such as Tyr143 and Gln151, whose
role in antagonist binding, particularly granisetron, was previously
proven by single-point mutagenesis experiments.8b,11,12
Although all of the studied homobivalent ligands can dock into
these two putative binding pockets, they show binding peculia-
rities worthy of note. The heptamethylene spacer ligand (3b), in
its most frequent low energy poses, shows a stretched out spacer,
and its probe is found to be lying into one of the two putative
probe binding pockets (Figure 2c,d). These poses may indicate
the presence of a secondary or allosteric binding site, as pre-
viously pointed out by Barbosa et al.,4 which could explain the
greater affinity of this homobivalent ligand for the 5-HT3R. On
the contrary, the homobivalent ligand with the shortest hexam-
ethylene spacer (3a) prefers the docking pose where both of the
cationic amines of the piperazine moieties interact with Glu129
in a pincerlike manner (Figure 2a), while the ligand with the
longer octamethylene spacer (3c) prefers a pose where the probe
extends outside the receptor surface (Figure 2b). The molecular
dynamic simulation performed on this latter docking pose of 3c
shows that the probe progressively moves from the receptor
interface toward a polar surface area near loop C provoking a
partial unfolding of the loop (mainly due to the formation of
persistent H-bonds between the probe and the side chains of
Ser227 and Glu225 on the receptor surface). This might be
responsible for the misplacement of the anchor moiety, which
moves away from Glu129 (a key residue for the agonist/
antagonist binding), preventing the formation of the important
charged-reinforced H-bond interaction, thus explaining the
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Figure 2. Docking of compounds 3aꢀc into the 5-HT3A-A receptor dimer: binding mode of the probe in a pincerlike manner (a), on the receptor
surface (b), in the upper extracellular region (c), and close to the membrane (d).
Figure 3. Docking of the arylpiperazine probe on the 5-HT3A receptor surface: charged surface of the putative sites in the extracellular receptor portion
(a), above the loop C (b), and near the membrane (c).
lower affinity of compound 3c, which is 25 times less active than
3b. Finally, it is worth noting that the docking poses of the
homobivalent ligand 3d are different from those of 3c, which is
characterized by the same spacer length in methylene equiva-
lents. In the most populated low energy clusters, the orientation
of compound 3d is very similar to that of the heptamethylene spacer
ligand (3b); therefore, the probe does not extend outside the
receptor surface but lies into one of the two putative probe
binding pockets (Figures 2c,d). In fact, the replacement of two
methylene units in the octamethylene spacers of homodimer 3c
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with two oxygen atoms in the homodimer 3d stabilizes the
complex by favoring the interactions between the oxygen atoms
in the spacer and the interface residues. These results could
justify the significant increase in potency (about 1 order of
magnitude) of the homobivalent ligand 3d with respect to
compound 3c.
The results of the molecular dynamics simulations suggest the
possibility that the probe of homobivalent ligands with longer
spacers (e.g., 23 or 29 methylene equivalents) reaches other
binding sites in the same receptor. To check this hypothesis, the
compound with the longest spacer (3f, see Table 1) was manually
docked into the 5-HT3R dimer: Although the spacer was forced
to adopt an unrealistic all-extended conformation, the probe
could not reach the adjacent binding interface. However, the
probe of these long bivalent ligands may either intercept active
sites of other receptors or dock into other probable binding
pockets on the same receptor surface.
studies implies that multivalency in 5-HT3R could involve
receptor domains different from the main binding site. The high
affinity shown by homobivalent ligands 3aꢀf suggests that
bivalency is a promising approach in 5-HT3R modulation.
Because bivalency can be considered the first step of multi-
valency, the results obtained provide the rational basis for the
application of the concepts of multivalency to the study of
5-HT3R function.
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental details for the
b
synthesis and the characterization of compounds 3ꢀ5 and their
intermediates and the computational methods and the experi-
mental procedures used in binding assays. This material is
available free of charge via the Internet at http://pubs.acs.org.
In the attempt to discover new possible binding sites, rigid
docking of the probe was performed on the whole receptor
surface. The results showed the presence of three main surface
areas capable of accommodating the arylpiperazine probe
(Figure 3).
’ AUTHOR INFORMATION
Corresponding Author
*Tel: þ39 0577 234320. Fax: þ39 0577 234333. E-mail:
cappelli@unisi.it.
The main pose of the probe is found in the extracellular
portion of the receptor, near the channel entrance, opposite to
the membrane (Figure 3a). Here, the probe occupies a pocket
that shows features very similar to those of the serotonin site. In
fact, the main residues involved in ligand binding are aromatic or
negatively charged: Asn49 (whose carboxylic group is hydrogen-
bonded to the cationic head of the piperazine ring), Glu98
(whose side chain forms a H-bond interaction with the ligand
amino group), Tyr50, Phe99, and Trp102 (which stabilize the
complex interacting with the aromatic group).
Funding Sources
Thanks are due to Italian MIUR for financial support.
’ ACKNOWLEDGMENT
Prof. Stefania D'Agata D'Ottavi's careful reading of the manu-
script is acknowledged.
The second putative surface site shows different features. It is a
polar pocket (Pro63, Ile187, Asn191, and Gln188) surrounded
by a few positively charged residues: Arg 61, Arg196 (whose
guanine group is hydrogen-bonded to the cationic head of the
piperazine ring), and Lys62 (which forms a H-bond with the
ligand carbonyl group, Figure 3b). This secondary site is localized
in a surface region just above loop C, very near the active site;
therefore, it may be exploited by bivalent ligands with a relatively
short spacer, as in the case of compound 3c discussed above. In
addition, as this site is positively charged, it may easily accom-
modate negative chemofunctional probes, such as the deproto-
nated ureido acetic moiety of compounds 5e,f. Finally, the least
important pose shows the ligand probe lying in a surface pocket
near the membrane formed by Ile242, Asn175, Asp172, Val173,
Tyr167, and Gln174 (Figure 3c). Therefore, depending on the
spacer length, a bivalent ligand probe may interact with one of the
putative sites on the surface or reach other receptors and act as a
biofunctional or chemofunctional ligand on the basis of its
stereoelectronic and functional features.
In conclusion, the study of the interactions of the 5-HT3R with
arylpiperazine ligands led to conceive a design approach to
bivalent ligands in which an arylpiperazine moiety is linked by
means of a spacer to a probe showing different functional
(chemofunctional or biofunctional) features. The high 5-HT3R
affinity shown by homobivalent and heterobivalent ligands
provides evidence of the viability of our approach, which can
be considered of broad applicability in the design of bivalent
ligands tailored for specific applications. Moreover, the existence
on the receptor surface of three potential accessory binding sites
for the arylpiperazine moiety shown by the molecular modeling
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