Background
The cysteine-loop family of ligand gated ion channels
(LGIC) is comprised of receptors with pentameric quaternary
structure and at least two ligand binding sites present at
the subunit interfaces [ 1 2 ] . This receptor family is
characterized by the presence of a critical disulfide loop
structure within the binding site and an integral ion
selective channel. LGIC receptors are found in both the
peripheral and central nervous systems. Members of this
family include the acetylcholine receptors [ 3 4 ] , the
γ-amino butyric acid type A receptor (GABA
A R) [ 5 ] , and the glycine receptor
(GlyR) [ 6 ] .
The first subunit of the 5-HT
3 R was cloned in 1991 [ 7 ] . The
sequence of this subunit was shown to be highly homologous
to LGIC receptors and thus identified the 5-HT
3 R receptor as another member of this
superfamily [ 7 8 9 ] . Similar to other LGIC receptors,
more than one subtype has been identified. Two splice
variants of an A subunit (long and short forms), and a
single B subunit have been cloned [ 10 11 12 13 ] . Both
the long and short forms of the A subunit are capable of
forming functional homomeric receptors [5-HT
3AL R and 5-HT
3AS R] although some differences between
an agonist and partial agonist activity have been observed
[ 14 ] . A third subtype is formed by a combination of the
A and B subunits to produce a heteromeric receptor of
unknown stoichiometry [ 10 ] . Heteromeric receptors are
pharmacologically and functionally distinct from the
homomeric 5-HT
3AL and 5-HT
3AS receptors [ 11 ] .
5-HT
3 Rs are distributed throughout the
central and peripheral nervous system, playing a
significant role in phenomenon such as anxiety, emesis and
alcoholism. Antagonists to 5-HT
3 Rs are clinically efficacious in the
treatment of chemotherapy-induced emesis [ 15 ] and recent
studies on human subjects have suggested their potential
application in the treatment of early onset alcoholism [ 16
17 ] .
Hibert
et al proposed an early model for the
antagonist pharmacophore of the 5-HT
3 R [ 18 ] . According to this model,
all 5-HT
3 R antagonists contain an aromatic
ring, a carbonyl oxygen or bioisosteric equivalent, and a
basic nitrogen. According to Hibert's model, the basic
nitrogen is located 5.2A° from the centre of the aromatic
ring and approximately 1.7A° above plane of the ring. The
carbonyl oxygen and the aromatic ring are coplanar and
separated by a distance of 3.3A°. Recent studies have
expanded on this model to include another lipophilic region
and a second hydrogen bonding interaction two atoms away
from the first [ 19 20 21 ] . A compound that contains all
five pharmacophoric regions was synthesized by Orjales
et al [ 22 ] . This compound
(1-(phenylmethyl)-2-piperizinyl benzimidazole or
lerisetron) is shown in figure 1and is a potent 5-HT
3 R antagonist. Functional groups on
this compound capable of forming interactions with the
receptor are the distal amino group, a benzimidazole and a
benzyl group in the N1 position of the benzimidazole. While
Lerisetron contains no carbonyl group, the second nitrogen
contained in the benzimidazole heterocycle could act as
bioisostere of this functional group [ 22 ] . Orjales
demonstrated the importance of the N-benzyl group by
synthesizing several N1 substituted analogs of Lerisetron.
Removal of the N-benzyl group produced a 80-fold decrease
in affinity, indicating a role for this group in
interacting with the 5-HT
3 R. Other studies have supported this
observation and suggest a more specific electrostatic
interaction [ 23 ] .
While structure-activity relationship studies and
molecular modeling have led to the development of a
detailed pharmacophore model, determining specific point
interactions between 5-HT
3 antagonists and binding site amino
acids has proven difficult. Mutagenesis studies have
identified the interaction of amino acids W89 and R91 in
the binding of 5-HT
3 R ligands [ 24 23 ] . Studies
conducted in our laboratory have identified three
additional putative binding site residues (Y140, Y142, and
Y152) [ 25 ] . W89 and R91 are present in a conserved
region of LGIC receptors often referred to as loop D [ 24
26 ] . Similarly, Y140, Y142 and Y152 are located in the
region homologus to E loop region of nicotinic AchR.
In this study, we have endeavoured to identify the amino
acids interacting with the different functional groups
present on the lerisetron molecule in order to develop a
model for interaction of this compound with the 5-HT
3 R. Using site directed mutagenesis in
combination with analogs of lerisetron, we have identified
amino acids that appear to interact selectively with the
terminal amino group, the N-benzyl group and the aromatic
benzimidazole.
Results
Functional activity of lerisetron
Whole cell patch-clamp experiments were performed to
test the functional activity of lerisetron. No response
was observed when lerisetron was applied alone (data not
shown). When co-applied with 5-HT, lerisetron inhibited
the absolute magnitude of the response with no apparent
alteration of the response profile (Figure 2). The
combination of several identical inhibition experiments
produced a K
i value of 0.2 ± 0.03 nM for
lerisetron inhibition of the 5-HT induced response. These
data correspond well with previously reported data for
this compound [ 22 ] and verify the competitive
antagonist action of lerisetron. Analogs of lerisetron
have been shown to inhibit 5-HT
3 Rs in a similar manner [ 23 ] .
Importance of the N-benzyl and distal piperazine
nitrogen to binding of lerisetron
The K
i value for lerisetron inhibition of [
3H]-granisetron binding to wildtype receptors was
determined to be 0.8 ± 0.19 nM (Figure 3and Table 1).
This value agrees with previously published data for this
compound. The K
i values for analogs 1 and 2 under
identical conditions are 25 ± 3.2 nM and 320 ± 82 nM
respectively (Figure 4A, Table 1). The observed change in
K
i represents the decreases in binding
energy resulting from removal of the N-benzyl group
(analog 1) and the distal piperazine nitrogen (analog
2).
Identification of amino acids interacting with
Lerisetron
In order to determine the nature of the amino acids
interacting with the distal amino and N-benzyl groups of
lerisetron, we constructed 5-HT
3AS Rs containing mutations at W89,
R91, Y140, Y142 and Y152. Figure 3shows inhibition of [
3H]-granisetron binding by lerisetron on wildtype and
mutant receptors. For most amino acids, an alanine
substitution was constructed in order to effectively
remove any amino acid interaction with the ligand. For
W89, an alanine substitution has been shown to prevent
binding of [ 3H]granisetron; therefore a less severe
mutation was constructed.
The W89F mutation produces a 18-fold change in K
d for [ 3H]-granisetron binding (18 ±
2 nM) and the W89Y mutation produces a 5.8-fold change in
K
d (5.7 ± 0.7 nM). Mutation of amino
acid R91 to alanine produced a 5-fold change in K
d for [ 3H]granisetron binding (4.9 ±
0.7 nM) (Table 1). These data agree well with previously
reported values [ 24 ] . Alanine mutations at the
tyrosine positions Y140, Y142 and Y152 also produced
minor increases in K
d for [ 3H]granisetron binding (2.7 ±
0.19 nM, 4.5 ± 0.5 nM and 7.8 ± 1.1 nM respectively) [ 25
] (Table 1).
Only small changes in K
i for lerisetron were observed for the
Y140A mutation while the Y142A and Y152A mutations
produced large increases in the K
i (Table 1and Figure 3). For W89F and
W89Y, the changes in K
i observed for lerisetron were much
smaller than for the alanine mutations at Y142A and
Y152A, as would be expected for the less severe nature of
these mutations. The changes were, however significant (p
< 0.001 in both cases) and are similar to the changes
in K
d reported for [ 3H]granisetron. The
increase in K
i on the W89F mutant receptor was 4.8
± 0.56 fold and the increase in K
i on the W89Y receptor was 3.6 ± 0.4
fold. The R91A mutant produced an increase in K
i of 7.6 ± 1.5 fold as compared to the
wildtype receptor. These data indicated potential
interactions of lerisetron with W89, R91, Y142 and
Y152.
Mutation of W89
As mentioned above, the lack of [ 3H]-granisetron
binding to W89A mutant receptors necessitated the use of
W89F and W89Y mutations to analyze functional group
interactions. The effects of these mutations on the K
i for analogs 1 and 2 are shown in
figure 4B, 4Cand Table 1. Analog 1 inhibited [
3H]-granisetron binding to W89F receptors with a K
i of 170 ± 54 nM (7 ± 3.2 fold
increase, p < 0.001) and W89Y receptors with a K
i of 81 ± 14 nM (3.2 ± 0.6 fold
increase, p < 0.001). This reflects a significant
increase in K
i and reflects a potential interaction
of analog 1 with W89. The strength of this interaction is
apparently similar to the strength of the interaction
with [ 3H]-granisetron and lerisetron since the magnitude
of the change is similar in both cases. Analog 2 also
showed a significant increase in K
i as a result of the W89F and W89Y
mutations. The magnitude of the change for W89F (5.1 ±
1.3 fold, p < 0.05) was similar to that observed for
lerisetron and analog 1. The W89Y mutation produced a 6.8
± 1.6 fold change in K
i (p < 0.05). Thus, all three
compounds appear to form similar interactions with
W89.
Mutation of R91
Mutation of R91 to alanine (R91A) resulted in a
significant, but small increase in K
i for lerisetron of 7.6 ± 1.5 fold (p
< 0.01). Figure 4Dshows the inhibition of [
3H]-granisetron binding by analogs 1 and 2 at R91A mutant
receptors. No significant change in K
i was observed on these receptors for
either analog 1 (0.9 ± 0.28 fold) or analog 2 (0.56 ±
0.14 fold) (Table 1) as compared to the wildtype
receptor.
Mutation of Y142
Mutation of Y142 to alanine produced one of the
largest observed changes in K
i for lerisetron (Figure 3and Table
1). The K
i obtained for lerisetron was 130 ± 28
nM, reflecting a change of 160 ± 37 fold compared to
wildtype receptors. The K
i value for analog 1, in contrast,
increased only 6.8 ± 2.3 fold (p < 0.01) as a result
of this mutation (Figure 4Eand Table 1). The K
i for analog 2 showed a similar change
of 17 ± 0.77 fold (p < 0.01). While these K
i values are significantly different
from wildtype values for each analog, the lack of larger
effects suggests that neither analog 1 nor analog 2 bind
as strongly as lerisetron to Y142.
Mutation of Y152
The Y152A mutation showed the most variability in its
effects on K
i values for lerisetron, analog 1 and
analog 2 (Figure 3, Figure 4Fand Table 1). Lerisetron
inhibited [ 3H]-granisetron binding with a K
i value of 150 ± 36; an increase of
190 ± 43 fold compared to wildtype values. The K
i value for analog 1 increased from 25
± 3.2 nM (wildtype) to 2.5 ± 0.40 μM. This change of 100
± 16 fold is slightly smaller, but not significantly
different from the relative change observed for
lerisetron. The K
i for analog 2 increased from 0.32 ±
0.08 μM on wildtype to 13 ± 4.2 μM on Y152A mutant
receptors (40 ± 12 fold increase). The increase observed
for analog 2 was significantly less than that observed
for both lerisetron and analog 1. The smaller change in K
i for analog 2 suggests that analog 2
binds weakly to Y152 while lerisetron and analog 1 bind
more tightly.
Discussion
Functional group interactions of W89
The W89F mutation produced a significant increase in K
i for all three compounds. The
magnitude of the change was similar in all cases. In
addition, the increase in K
i was identical to the increase in K
d that has been observed for [
3H]granisetron binding on this mutant [ 24 ] .
Alterations in K
i resulting from the W89Y mutation
were slightly less, however the change was again the same
for lerisetron, analog 1 and analog 2. These data suggest
that all three compounds form binding site interactions
with W89. The interaction between lerisetron and W89 is
unlikely to be via the N-benzyl functional group since
the K
i for analog 1 was also altered by
this mutation. The same argument can be made for the
distal piperazine nitrogen since the K
i for analog 2 also increased. The
portion of the molecule common to all three compounds,
the aromatic benzimidazole, is thus the most likely point
of interaction for W89.
Functional group interactions of R91
The R91A mutation increased the K
i value for lerisetron inhibition of [
3H]granisetron binding by 7.6 fold. This is a moderately
small change for an alanine mutation, particularly
considering that the smallest change in K
i for removal of a functional group on
lerisetron (the N-benzyl group) was 31 fold. It is
therefore likely that this interaction is either
extremely weak or the change in K
i is the result of a structural change
in the binding site. Previous studies concluded that R91
was an important interaction for the 5-HT
3 R agonist 5-hydroxytryptamine
(5-HT), since the K
i for 5-HT inhibition increased over
3000 fold as a result of the R91A mutation [ 24 ] . A
change in K
d for [ 3H]granisetron binding to R91A
was also observed. In order to determine whether the
N-benzyl or distal piperazine nitrogen of lerisetron was
involved in an interaction with R91, we tested both
analog 1 and 2 on R91A mutant receptors. No change in K
i was observed for either compound.
This result makes it much more difficult to assign the
correct functional group to this amino acid since it
suggests that one or both of the compounds is no longer
binding the receptor in precisely the same manner as
lerisetron. Considering the small change observed for
lerisetron binding as a result of this mutation, even a
slight reorientation of the molecule in the binding site
could result in the loss of this interaction.
Functional group interactions of Y142
The K
i values for inhibition of [
3H]granisetron binding by analogs 1 and 2 were altered
only slightly by the Y142A mutation. The magnitude of the
increase in K
i for lerisetron, however, was
considerably larger (160 fold) and is indicative of an
important interaction of the compound with Y142. The lack
of a large change in K
i for both analogs makes it difficult
to interpret this data since one or both of the compounds
appears to be interacting differently with the binding
site than lerisetron.
Analogs 1 and 2 differ from each other both in the
functional groups contained in the molecule and their
structural similarity to lerisetron. Analog 2 is most
similar in overall structure. The substitution of oxygen
for the distal amino nitrogen alters the potential
interactions formed at this position, but is likely to
have a small effect on the overall size and shape of the
molecule. Analog 1 is far less similar to lerisetron and
more similar to the 5-HT
3 R antagonist granisetron. Previous
studies have shown that the binding of granisetron is not
affected by the Y142A mutation [ 25 ] . Analog 1 may bind
more similar to granisetron than lerisetron and thus
would be unaffected by mutations at Y142. This is less
likely to be the case with analog 2.
The strength of the putative interaction at Y142 can
be identified by examining the change in binding of
lerisetron as a result of the Y142A mutation. The Y142A
mutation produced a 160 fold change in K
i . This change reflects the binding
energy lost as a result of the alanine substitution. The
observed change in Ki on wt receptors is much larger than
that observed for removal of the N-benzyl group (31
fold), but is similar to that observed for substitution
of the distal amino nitrogen in analog 2 (400 fold).
Taken together with the close structural similarity of
analog 2 to lerisetron, it can be concluded that
comparison of analog 2 and lerisetron should provide the
best means of identifying the interaction at Y142. No
change in K
i was observed for analog 2 as a
result of the Y142A mutation indicating a lack of any
significant interaction of this compound with Y142. These
data support our hypothesis that Y142 interacts with the
distal piperazine nitrogen of lerisetron. A second amino
acid may also be involved since the change in K
i for lerisetron binding as a result
of the Y142A mutation was smaller than the change
produced by substitution of the piperazine nitrogen. As
described below, one candidate for this second amino acid
is Y152.
Functional group interactions of Y152
The Y152A mutation produced increases in K
i for all three compounds although the
magnitude of the change differed. The increases in the K
i values were 190 fold for lerisetron,
98 fold for analog 1 and only 40 fold for analog 2. Thus,
analog 1 retains much of its ability to interact with
Y152 despite the absence of the N-benzyl group, while
analog 2 interacts more weakly with this amino acid.
Since the K
i for analog 1 is increased by the
Y152A mutation, it is unlikely that the N-benzyl group
interacts with Y152. The small change in K
i for analog 2 supports a partial
interaction of Y152 with the distal piperazine nitrogen
although some interaction with another group is also
apparent. This other group would be expected to be in
close proximity to the distal nitrogen. The most likely
candidate is the other nitrogen of the piperazine ring.
Thus Y152A may form a partial interaction with both
piperazine nitrogens.
Conclusions
Figure 6shows a hypothetical model of the
lerisetron-binding site supported by our observations. The
model illustrates the secondary structure of the region of
the receptor from Y140 - Y152 in a loop configuration. This
structure is supported by site-directed mutagenesis data [
25 ] as well as structural predictions obtained from other
LGIC receptors [ 31 ] . The recent determination of the
structure of a nicotinic acetylcholine binding protein [ 27
] that shares significant homology with the LGIC family
also supports a loop structure in this part of the protein.
The region from W89 through Y93 is shown as a β-sheet as
has been hypothesized based on site-directed mutagenesis
studies of this strand of the 5-HT
3 R [ 24 ] . Our data indicate the
functional groups of lerisetron that may interact with W89,
R91, Y142 and Y152.
W89 is shown interacting with the aromatic benzimidazole
group of lerisetron although the precise position of W89
relative to this group is not known. The W89 interaction
with this group is supported by the observed increase in K
i for lerisetron, analog 1 and analog 2.
Since the benzimidazole group is common to all three
compounds it is the most likely point of interaction. W89
also represents a common interaction in the binding site
for both lerisetron and [3H]granisetron.
Y142 is shown interacting with the distal piperazine
nitrogen possibly through a cation-π interaction. This
orientation of an amino group interacting with an aromatic
amino acid in a cation-π interaction has been shown for the
nicotinic acetylcholine receptor and has been hypothesized
for many LGIC receptors [ 28 29 30 26 ] . This conclusion
is based on both the magnitude of the change observed on
the wild type receptor for removal of the amino group (400
fold) compared to the effect of the Y142A mutation on
lerisetron binding (160 fold) and the lack of any major
change in K
i for analog 2 as a result of this
mutation. Our data does not support an interaction of this
amino acid with either the N-benzyl or benzimidazole
portions of lerisetron.
Y152 is shown positioned between the two piperazine
nitrogens. This conclusion is supported by the smaller
increase in K
i for analog 2 (40 fold) compared to
that observed for lerisetron (190 fold). These results
suggest a partial interaction of Y152 with the distal
piperazine nitrogen. Since some change was observed, a
second interaction is also likely. The functional group in
closest proximity to the distal piperazine nitrogen is the
other nitrogen on the piperazine ring. Another possibility
would be the N-benzyl interaction, however, since the Y152A
mutation also produced a large increase in K
i for analog 2, this conclusion is not
supported by our data.
R91 is shown as interacting with the N-benzyl group.
This is a difficult conclusion to make considering the
small effect of the R91A mutation on lerisetron binding.
The interaction is included in the model based on
structural information obtained from the crystal structure
of AChBP [ 27 ] . The region of this protein homologous to
loop E and loop D of the 5-HT
3A receptor suggests a loop structure
from Y140 to Y152 and a 3-residue turn containing a glycine
at position 147 and the β-strand from W89 through Y93
oriented as shown in Figure 6. The orientation of
lerisetron between W89 and Y142A as shown would enable the
N-benzyl group to be positioned in close proximity to R91.
If this is the case, then a small alteration in position of
analog 1 or 2 in the binding site could result in the loss
of this presumably weak interaction. The apparent
alterations in the binding site location of analog 1 would
be consistent with this hypothesis. An alternate hypothesis
would place the N-benzyl group in a different position,
interacting with another amino acid; either solely or in
concert with R91.
Our data support a binding site for lerisetron on the
5-HT
3 R that spans the D and E loop regions.
Table 2shows the sequence alignment for the 5-HT
3 R, the α7 receptor and the AChBP for
these loops. Sequence alignment of mouse 5-HT
3 AR, α7 nAchR and AChBP result in
alignment of the proposed D and E loop of the 5-HT
3 AR with corresponding regions of the
α7 nAchR and AChBP. The amino acids W89, R91 Y140, Y142 and
Y152 of the 5HT
3 A R can be aligned with W53, Q55,
L102, R104, and M114 of the AChBP (Figure 6). These amino
acids form a cluster in the proposed acetylcholine binding
domain of AChBP similar to that proposed in our model. Both
loops have been identified on the complementary face of the
binding site of the nAChR. It is unknown if lerisetron
utilizes amino acids on the principal face although none
have been identified. The model for lerisetron binding will
be further refined as its interactions with other binding
site amino acids are investigated [ 26 32 33 ] . Of
particular interest would be potential interactions of the
N-benzyl group that would account for the decrease in
binding affinity of analog 2. Additional information gained
from comparison of our model with the recent crystal
structure of the AChBP demonstrates that lerisetron can be
roughly 'fit' into the binding site such that all the
residues line up as shown in our model. While this is not
direct evidence that the model is correct, subsequent
molecular modeling of the data presented in this paper may
provide further support for our hypothesis. Our current
model provides an initial working hypothesis that can form
the basis of further investigation. Also, while it is
unclear whether the information obtained in this study can
be extended to other 5-HT
3 R ligands, a similar approach would be
useful in identifying functional group interactions for
mCPBG, 5-HT, dtC and granisetron.
Materials and Methods
Mutagenesis
Wild type 5-HT
3AS mouse receptor cDNA was derived
from N1E-115 neuroblastoma cells as previously described
[ 24 ] . Mutant receptors were constructed using
polymerase chain reaction (Quick change mutagenesis kit,
Promega). All mutations were confirmed by DNA
sequencing.
Cell culture methods and transfections
tsA201 cells (a derivative of the HEK293 cell line)
were grown in Dulbecco's modified Eagles medium (D-MEM)
containing 10% FBS and 100-units/ml
penicillin/streptomycin. Cultures were maintained in
humidified atmosphere of 5% CO
2 at 37°C. For binding studies, tsA201
cells were plated at a density of 5 × 10 6cells/75 cm
2and grown for 9 hours prior to transfection. Cells were
transfected with 10 μg murine 5-HT
3AS R cDNA using calcium phosphate
co-precipitation (New Life Technologies, NY), then
incubated 36 hours prior to harvesting. For whole cell
patch clamp experiments, tsA201 cells were plated at a
density of 0.25 × 10 6cells/27 cm 2dish and grown 12
hours prior to transfection. Cells were washed with fresh
culture medium then transfected with 10 μg 5-HT
3AS R cDNA using Qiagen Superfect
transfection reagent (Qiagen, CA). Transfected cells were
incubated with this mixture for 2.5 hours, then divided
into 35 mm culture dishes at a density of approximately 5
× 10 4cells/dish and incubated for 24 hours at 37°C
before recording.
Radioligand Binding Assay
Transfected cells were scraped from the dishes, washed
twice with Dulbecco's PBS (New Life Technologies, NY),
then resuspended in 1.0 ml PBS/100 mm dish. Cells were
either used fresh or frozen at this step until needed.
Immediately prior to use, cells were homogenized in PBS
using a glass tissue homogenizer then centrifuged at 35
000 × g for 30 minutes in a Beckman JA20 rotor. Membranes
were washed once more with PBS then resuspended in 1 ml
PBS/100 mm dish. Protein content was determined using a
Lowry assay (Sigma. Diagnostics, St. Louis, MO). Binding
assays were performed in PBS. For K
d determinations, 100 μl of homogenate
was incubated at 37°C for 1 hour with varying
concentrations of [ 3H] granisetron (NEN, MA). Specific
binding of [ 3H] granisetron was determined as the bound
[ 3H] granisetron not displaced by a saturating
concentration of a competing ligand (100 μM mCPBG or 10
μM MDL-72222). K
d values were determined by fitting
the binding data to the following equation using GraphPad
PRISM (San Diego CA): B = Bmax [L] n / ([L] n + Kn),
where θ is bound ligand, Bmax is the maximum binding at
equilibrium L is the free ligand concentration and n is
the Hill coefficient. For K
i determinations, 100 μl of homogenate
was incubated at 37°C for 2 hours with varying
concentrations of inhibitor and [ 3H] granisetron (NEN,
MA). Binding was terminated by rapid filtration onto a
GF/B filters. The IC
50 values were calculated by fitting
the data to the following equation using GraphPad PRISM
(San Diego CA): θ = 1/ (1+(L/IC
50 )), where θ is the fractional
amount of [ 3H] granisetron bound in the presence of
inhibitor at concentration L as compared to the amount of
[ 3H] granisetron bound in the absence of inhibitor. IC
50 is the concentration at which θ =
0.5. The K
i is calculated from the IC
50 value using the Cheng-Prusoff
equation.
Electrophysiological Methods
Transfected tsA201 cells were transferred to a
recording chamber and submerged in extracellular
recording buffer containing 25 mM HEPES pH 7.4, 140 mM
NaCl, 1.7 mM MgCl
2 , 5 mM KCl, 1.8 mM CaCl
2 . Patch electrodes (2-2.5 MΩ) were
filled with intracellular recording buffer containing 25
mM HEPES pH 7.4, 145 mM KCL, 2 mM MgCl
2 and 1 mM EGTA. Cells were clamped in
whole cell configuration at a holding potential of -60
mV. Currents elicited by agonist application were
measured using an Axopatch 200B amplifier (Foster City,
CA) under computer control (DataPac 2000, RUN
Technologies). Agonists and antagonists were dissolved in
extracellular solution and delivered to cells using a
rapid perfusion system (Warner Instruments, Hamden, CT).
For EC
50 determinations, responses were
normalized to the maximum response obtained from the full
agonist 5-HT and fitted to the equation Ψ= 1/1+(EC50/ [C]
n), where Ψ is the normalized current at 5-HT
concentration [C], EC50 is the concentration of 5-HT
needed to obtain half maximal activation and n is the
apparent Hill coefficient. For inhibition experiments,
cells were exposed to inhibitor alone for 30 s prior to
co-exposure with 5-HT. Inhibited responses were
calculated as a fraction of the response to 5-HT alone.
Data were plotted as the fractional response versus the
concentration of inhibitor and analysed using GraphPad
software. The IC
50 value was calculated as the
concentration of antagonist inhibiting the 5-HT evoked
response by 50%. A K
i value was calculated from the IC
50 using the Cheng-Prusoff
equation.
Synthesis of Lerisetron and its analogs
All target molecules were prepared according to a
general 2-step synthesis reported previously by Orjales
et al . with only slight
modification [ 22 23 ] . Commercially available
2-chlorobenzimidazole, in dry DMF was treated with a
slight excess of NaH, (1.1eq). After stirring for 1 hour
at room temperature, one equivalent of the appropriate
alkyl bromide was added slowly and the reaction mixture
heated under reflux for > 5 hours (the reaction was
monitored by TLC). Reaction product was partitioned
between water and methylene chloride; organic layer was
dried (Na
2 SO
4 ) and concentrated in vacuum. The
solid residue was purified by Flash chromatography, which
afforded the corresponding N-substituted
2-Chlorobenzimidazole intermediates in good yield. The
final step involved a nucleophilic substitution of the
2-chloro group by piperazine at high temperatures. The
reaction was performed neat using 4-10 fold excess
piperazine and typically heated for a short period only,
(30-45 min). Similar work-up afforded a residue that was
purified by either crystallization or chromatography. The
yields ranged from 40-95%. All compounds were
characterized by NMR, MS, HRMS, and /or elemental
analysis or were identical to literature reports.
Materials
D-MEM, Penicillin-Streptomycin, fetal bovine serum,
and Trypsin were obtained from New Life Technologies.
5-HT and MDL-72222 were obtained from RBI. [
3H]-granisetron (84 Ci/mmol) was purchased from New
England Nuclear.
