International Journal of

Molecular Sciences

Article
Differential Regulation of Bilastine Affinity for Human
Histamine H1 Receptors by Lys 179 and Lys 191 via Its Binding
Enthalpy and Entropy
Hayato Akimoto 1 , Minoru Sugihara 2 and Shigeru Hishinuma 1, *

1 Department of Pharmacodynamics, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose,

Tokyo 204-8588, Japan; d206901@std.my-pharm.ac.jp
2 Pharmaceutical Education and Research Center, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose,
Tokyo 204-8588, Japan; sugihara@my-pharm.ac.jp
* Correspondence: hishi@my-pharm.ac.jp

Abstract: Bilastine, a zwitterionic second-generation antihistamine containing a carboxyl group, has

higher selectivity for H1 receptors than first-generation antihistamines. Ligand-receptor docking
simulations have suggested that the electrostatic interaction between the carboxyl group of second-
generation antihistamines and the amino group of Lys179ECL2 and Lys1915.39 of human H1 receptors
might contribute to increased affinity of these antihistamines to H1 receptors. In this study, we
evaluated the roles of Lys179ECL2 and Lys1915.39 in regulating the electrostatic and hydrophobic
binding of bilastine to H1 receptors by thermodynamic analyses. The binding enthalpy and entropy
of bilastine were estimated from the van ’t Hoff equation using the dissociation constants. These



constants were obtained from the displacement curves against the binding of [3 H] mepyramine to


membrane preparations of Chinese hamster ovary cells expressing wild-type human H1 receptors and
Citation: Akimoto, H.; Sugihara, M.;
their Lys179ECL2 or Lys1915.39 mutants to alanine at various temperatures. We found that the binding
Hishinuma, S. Differential Regulation
of bilastine to wild-type H1 receptors occurred by enthalpy-dependent binding forces and, more
of Bilastine Affinity for Human
dominantly, entropy-dependent binding forces. The mutation of Lys179ECL2 and Lys1915.39 to alanine
Histamine H1 Receptors by Lys 179
and Lys 191 via Its Binding Enthalpy
reduced the affinity of bilastine to H1 receptors by reducing enthalpy- and entropy-dependent binding
and Entropy. Int. J. Mol. Sci. 2021, 22, forces, respectively. These results suggest that Lys179ECL2 and Lys1915.39 differentially contribute to
1655. https://doi.org/10.3390/ijms the increased binding affinity to bilastine via electrostatic and hydrophobic binding forces.
22041655
Keywords: affinity; antihistamine; bilastine; enthalpy; entropy; histamine H1 receptor
Academic Editors: Paul Chazot and
Ilona Obara
Received: 22 December 2020
Accepted: 5 February 2021 1. Introduction
Published: 6 February 2021
It is known that Gq/11 -protein-coupled H1 receptors are involved in mediating allergic
and inflammatory responses in peripheral tissues and the state of arousal in the central
Publisher’s Note: MDPI stays neutral
nervous system [1–8]. Various first- and second-generation antihistamines have been
with regard to jurisdictional claims in
developed for the treatment of type I hypersensitivity, such as allergic rhinitis [9–13].
published maps and institutional affil-
Some second-generation antihistamines have zwitterionic properties owing to the presence
iations.
of carboxyl groups, which help reduce their side effects such as sedation and impaired
performance resulting from the blockade of H1 receptors in the central nervous system
via their penetration into the brain through the blood–brain barrier. Bilastine (Figure 1), a
zwitterionic second-generation antihistamine, has non-sedative properties as well as an
Copyright: © 2021 by the authors.
increased selectivity for H1 receptors than first-generation antihistamines [13–19].
Licensee MDPI, Basel, Switzerland.
The human H1 receptor possesses Asp1073.32 (superscripts indicate Ballesteros-Weinstein
This article is an open access article
numbering [20]), a highly conserved amino acid in aminergic G protein-coupled receptors,
distributed under the terms and
deep in the ligand-binding pocket, whereas Lys179ECL2 and Lys1915.39 , located at the
conditions of the Creative Commons
entrance of the ligand-binding pocket, are anion-binding sites unique to the H1 receptor
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
(Figure 2a) [21]. Ligand-receptor docking simulations based on the crystal structure of
4.0/).
human H1 receptor indicated that the carboxyl group of second-generation antihistamines,

Int. J. Mol. Sci. 2021, 22, 1655. https://doi.org/10.3390/ijms22041655 https://www.mdpi.com/journal/ijms

 COO–

Int. J. Mol. Sci. 2021, 22, 1655 2 of 8

N
+
N
such as olopatadine, levocetirizine, fexofenadine, and acrivastine, appeared to form a
N
salt bridge with Lys179ECL2 and/or Lys1915.39 . This bridge might have contributed to the
increased selectivity of carboxylated second-generation antihistamines for H1 receptors [21].
Int. J. Mol. Sci. 2021, 22, x FOR PEER Accordingly,
REVIEW ECL2 and/or Lys191
2 5.39
of 8
O the electrostatic interaction of bilastine with Lys179
may be important in the determination of its binding affinity for H1 receptors.
Figure 1. Chemical structure of bilastine. Bilastine is a zwitterionic second-generation antihista-
mine containing a carboxyl group that contributes to its non-sedative properties as well as an in-
COO–
creased selectivity for H1 receptors.

The human H1 receptor possesses Asp1073.32 (superscripts indicate Ballesteros-Wein-

N
stein numbering [20]), a highly+ conserved amino acid in aminergic G protein-coupled re-
N
ceptors, deep in the ligand-binding pocket, whereas Lys179ECL2 and Lys1915.39, located at
N
the entrance of the ligand-binding pocket, are anion-binding sites unique to the H1 recep-
tor (Figure 2a) [21]. Ligand-receptor docking simulations based on the crystal structure of
human H1 receptor indicated O that the carboxyl group of second-generation antihista-
mines, such as olopatadine, levocetirizine, fexofenadine, and acrivastine, appeared to
form a salt bridge with Lys179ECL2 and/or Lys1915.39. This bridge might have contributed
Figure1.
Figure 1. Chemical
Chemicalstructure
structureof ofbilastine.
bilastine.Bilastine
Bilastineisisaazwitterionic
zwitterionicsecond-generation
second-generationantihistamine
antihista-
to themine
increased selectivity
containing a carboxylof carboxylated second-generation antihistamines asfor H1asre-
containing a carboxyl groupgroup that contributes
that contributes to its non-sedative
to its non-sedative properties
properties as well well
as an an in-
increased
ceptors [21]. selectivity
creased Accordingly,for Hthe electrostatic interaction of bilastine with Lys179ECL2 and/or
1 receptors.
selectivity for H1 receptors.
Lys1915.39 may be important in the determination of its binding affinity for H1 receptors.
The human H1 receptor possesses Asp1073.32 (superscripts indicate Ballesteros-Wein-
stein numbering (a)[20]), a highly conserved amino acid in aminergic (b) G protein-coupled re-
ceptors, deep in the ligand-binding pocket, whereas Lys179 ECL2 and Lys1915.39, located at
Lys179ECL2
the entrance ofBilastine
the ligand-binding pocket, are anion-binding sites unique to the H1 recep-
5.39
tor (Figure 2a) [21]. Ligand-receptor docking simulations based on theLys191 crystal structure of
∆G˚(<0) = ∆H˚-T∆S˚
human H1 receptor indicated that the carboxyl group of second-generation antihista-
mines, = RTlnKd levocetirizine, fexofenadine, and4.3Å
NH2 such as olopatadine, acrivastine, appeared to
ECL2
form a salt bridge withLys179 Lys179 ECL2 and/or Lys1915.39. This bridge might have contributed
3.6Å
to the increased selectivity Lys191 of5.39
carboxylated second-generation antihistamines for H1 re-
ceptors [21]. Accordingly, the
Asp107 electrostatic
3.32 interaction of bilastine with Lys179ECL2 and/or
Lys1915.39 may be important in the determination of its3.4Å binding affinity for H1 receptors.
Bilastine
(a) Asp1073.32 (b)
COOH
Histamine H1 Receptor
Bilastine Lys179ECL2
Lys1915.39
FigureFigure
2. A schematic
2. A schematic ∆G˚(<0)
structurestructure =of∆H˚-T∆S˚
of human H1 receptor
human (a) and
H1 receptor (a)docking simulation
and docking on theon
simulation binding
the binding of
of bilastine to human
bilastine to NH human
2
H 1 receptor =
(b). RTlnK
(a) A schematic structure of human H
H1 receptor (b). (a) Ad schematic structure of human H1 receptor1 receptor is
4.3Åisshown
shown and indi-
and indicates
cates thatthat Asp107
Asp107 is aishighly
3.323.32 a highly conserved
conserved amino
ECL2amino acid in the aminergic G-protein-coupled
Lys179 acid in the aminergic G-protein-coupled
3.6Å receptors,
receptors, and Lys179ECL2 and Lys1915.39 are
and Lys179ECL2 and Lys1915.39 are
anion-binding
5.39
anion-binding
sites unique to the H1 receptor. Ligand-
sites unique to the H1 receptor. Ligand-receptor
Lys191
receptor interaction is also shown to demonstrate that the binding affinity for ligands (Kd) is deter-
interaction is also shown to demonstrate3.32 that the binding affinity for ligands (Kd ) is determined by
mined by their thermodynamic binding Asp107
forces (∆G° = ∆H° − T∆S° = RTlnKd). (b) Docking simula-
their thermodynamic binding forces (∆G◦ = ∆H ◦ − T∆S◦ = RTlnKd). 3.4Å
tion was performed to reveal the final position of bilastine binding to human(b) H1Docking
receptorsimulation
as de- was
performed to reveal the final position of bilastine binding to human
scribed in the materials and methods section. The amino group of bilastine appeared to interactH 1 receptor as described in the
3.32 to interact
Bilastine 3.32
materials
with Asp107 3.32,and
andmethods
the carboxyl section.
groupTheofamino group
bilastine of bilastine
appeared Asp107
appeared
to interact with Lys179ECL2 with
and Asp107 ,
COOH
Lys191 and
5.39. the carboxyl
Atoms group of
of nitrogen, bilastine
oxygen, appeared
carbon, to interactare
and hydrogen with Lys179
shown in ECL2
blue,and
red,Lys191 5.39 . Atoms of
gray, and
white,nitrogen, Histamine
respectively. The carbon,
oxygen, H
distancesReceptor
1 and between
hydrogenkeyareatoms of bilastine
shown in blue, and
red,amino
gray, and acidwhite,
residues are also The
respectively.
shown. distances between key atoms of bilastine and amino acid residues are also shown.
Figure 2. A schematic structure of human H1 receptor (a) and docking simulation on the binding
The binding
of bilastine to humanaffinity (Kd) of(b).
H1 receptor ligands
(a) Ais determined
schematic by the
structure ofthermodynamic
human H1 receptor binding
is shownforces
of ligands, and these are the binding enthalpy (∆H ◦ ) and entropy (∆S◦ ) (Figure 2a) [22–27].
and indicates that Asp107 is a highly conserved amino acid in the aminergic G-protein-coupled
3.32

∆H ◦ is usually associated
receptors, and Lys179ECL2 andwith Lys191binding forces via thesites
5.39 are anion-binding formation
unique toofthe
new bonds between
H1 receptor. Ligand-
receptors and ligands, such as electrostatic interaction via salt bridges, hydrogen
receptor interaction is also shown to demonstrate that the binding affinity for ligands (K bonds and
d) is deter-

van
minedderbyWaals
their thermodynamic binding∆S
interactions, whereas ◦ is usually
forces (∆G° = ∆H°characterized by binding
− T∆S° = RTlnKd). forcessimula-
(b) Docking via the
tion was performed
displacement to reveal
of ordered the final
water position
molecules of bilastine
coupled withbinding to humanofHnew
the formation 1 receptor as de-
hydrophobic
scribed in the materials and methods section. The amino group of bilastine appeared to interact
with Asp1073.32, and the carboxyl group of bilastine appeared to interact with Lys179ECL2 and
Lys1915.39. Atoms of nitrogen, oxygen, carbon, and hydrogen are shown in blue, red, gray, and
white, respectively. The distances between key atoms of bilastine and amino acid residues are also
shown.
Int. J. Mol. Sci. 2021, 22, 1655 3 of 8

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 9

interactions [22–30]. Thus, thermodynamic analyses provide important information for

evaluating the electrostatic and hydrophobic binding of bilastine with H1 receptors. In this
The
study, webinding
examinedaffinity
how(KLys179 ECL2 and
d) of ligands isLys191 5.39 might
determined by the thermodynamic
increase binding
the electrostatic and
forces of ligands,
hydrophobic and these
binding are the with
of bilastine bindingH1 enthalpy
receptors(∆H°) andthe
through entropy (∆S°)of(Figure
mutation ECL2
Lys1792a)
[22–27]. ∆H° is 5.39
and/or Lys191 usually associated with binding forces via the formation of new bonds
to alanine.
between receptors and ligands, such as electrostatic interaction via salt bridges, hydrogen
2. Results
bonds and and
van Discussion
der Waals interactions, whereas ∆S° is usually characterized by binding
2.1. Docking Simulation
forces via the displacement on theofBinding
orderedof water
Bilastine to H1 Receptor
molecules coupled with the formation of
new hydrophobic interactions
Docking simulation [22–30]. Thus,
was performed thermodynamic
to simply estimate theanalyses provideofimportant
configuration bilastine at
information for evaluating
the ligand-binding pocket the electrostatic
of human and hydrophobic
H1 receptors (Figure 2b).binding
We foundof bilastine
that thewith H1
carboxyl
receptors. In this study, we examined
group of bilastine was located between Lys179 how Lys179 ECL2 and Lys1915.39
ECL2 and Lys191 5.39 might increase the
and that the amino
electrostatic and hydrophobic
group of bilastine was close binding of bilastine
to Asp107 3.32 . Thewith H1 receptors
oxygen atom of through the mutation
the carboxyl group of
of Lys179 ECL2 and/or Lys1915.39 to alanine.
bilastine appeared to be closer to the nitrogen atom of Lys179 (3.6 Å) than that of Lys191
(4.3 Å). Since the docking simulation could not exactly predict roles of Lys179ECL2 and
2. Results
Lys191 5.39and Discussionthe binding affinity of bilastine, we then performed receptor
in regulating
2.1. Docking
binding Simulationusing
experiments 3 H] mepyramine,
on the[Binding of Bilastine atoradioligand
H1 Receptor for H1 receptors, to evaluate
actual roles of Lys179 ECL2 and Lys191 5.39 in regulating the binding affinity of bilastine via
Docking simulation was performed to simply estimate the configuration of bilastine
itsthe
at thermodynamic
ligand-binding binding
pocketforces.
of human H1 receptors (Figure 2b). We found that the car-
boxyl group of bilastine was located between Lys179ECL2 and Lys1915.39 and that the amino
2.2. Roles of Lys179wasECL2 and Lys1915.39 in the Binding Affinity for Bilastine
group of bilastine close to Asp1073.32. The oxygen atom of the carboxyl group of bilas-
To evaluate
tine appeared changes
to be closer into the
the binding
nitrogenaffinity
atom ofofLys179
bilastine(3.6for
Å)Hthan
1 receptors
that of by mutations
Lys191 (4.3
Å). Since ECL2
of Lys179 the and/or
dockingLys191 5.39 , the IC for bilastine was first obtained from the
simulation could 50 not exactly predict roles of Lys179 displace-
ECL2 and

ment curves
Lys191 for the binding
5.39 in regulating of 3 nM
the binding [3 H] mepyramine
affinity to membrane
of bilastine, we then performed preparations
receptor of CHO
bind-
cellsexperiments
expressing usingwild-type human H1 receptors (WT), Lys179 ECL2 or Lys1915.39 mutants of
ing [ H] mepyramine,
3 a radioligand for H1 receptors, to evaluate actual
WT to ECL2 and Lys1915.39 mutants of WT to
roles ofalanine
Lys179ECL2(K179A and K191A),
and Lys191 and both Lys179
5.39 in regulating the binding affinity of bilastine via its ther-
◦ ◦
alanine (K179A
modynamic forces.at 4 C–37 C (Figure 3). The Ki values for bilastine were then
+ K191A)
binding
calculated from the IC50 , as described in the materials and methods section.
2.2. Roles
Figureof Lys179 ECL2 and
4 shows the Lys191
lnKi values5.39 in the ofBinding
bilastineAffinity corresponding ∆G◦ values
for Bilastine
and their
(∆G To◦ RTlnKi ) for
= evaluate WT, in K179A, K191A, and of K179A + K191A at a standard tempera-
changes the binding affinity bilastine for H1 receptors by mutations
◦ C (298.15 K). The ± 0.08from
ture
of Lys179of 25ECL2 and/or Lys1915.39, Kthe i values
IC50 forofbilastine
bilastinewaswere 1.92
first obtained nM forthe WT (n = 4),
displace-
± nM for K179A (n = 7), 2.573 ±
ment curves for the binding of 3 nM [ H] mepyramine to membrane preparations of CHOfor
5.20 1.18 0.16 nM for K191A (n = 4), and 7.96 ± 0.76 nM
K179A
cells + K191Awild-type
expressing (n = 4). Thus,
human theHaffinity
1 receptorsof bilastine for HECL2
(WT), Lys179 1 receptors
or Lys191 was significantly
5.39 mutants of
reduced by approximately 1.3 to 4.1 times following the mutation of Lys179 ECL2 and/or
WT to alanine (K179A and K191A), and both Lys179 ECL2 and Lys191 mutants of WT
5.39 to
Lys1915.39
alanine . These
(K179A results suggest
+ K191A) at 4 °C–37 that °C Lys179 ECL2 and Lys1915.39 contribute to the increased
(Figure 3). The Ki values for bilastine were then
affinity forfrom
calculated bilastine.
the IC50, as described in the materials and methods section.

(a) WT (b) K179A

Figure 3. Cont.
Int.
Int. J.J. Mol.
Mol. Sci.
Sci. 2021, 22, x1655
2021, 22, FOR PEER REVIEW 4 of4 of
9 8

(c) K191A (d) K179A + K191A

Figure 3. Displacement curves for bilastine against the binding of [3H] mepyramine to WT (a),
K179A (b), K191A (c), and K179A + K191A (d). The binding of 3 nM [3H]mepyramine to mem-
branes containing WT (a), K179A (b), K191A (c), and K179A + K191A (d) in the presence or ab-
sence of various concentrations of bilastine was measured at 4 °C (open circles), 14 °C (closed cir-
cles), 25 °C (open triangles), and 37 °C (closed triangles), as described in materials and methods
section. The data points are the percentages of bound [3H] mepyramine, with 100% as 3 nM [3H]
mepyramine binding in the absence of bilastine. Data represent mean ± standard errors of means
of 4–7 independent experiments determined in quadruplicates. The lines are the best-fit curves to
a one-site model.
Displacement
Figure 3.3.Displacement
Figure curves
curves forfor bilastine
bilastine against
against the binding
the binding of [3H] [3 H] mepyramine
of mepyramine to WT to (a),WT (a),
K179A
Figure
(b),
4
K191A
shows
(c),
the
and
lnK
K179A
i values of bilastine and their corresponding
+ K191A (d). The binding of 3 nM [ 3 H]mepyramine
∆G°tovalues
membranes
(∆G°
K179A (b), K191A (c), and K179A + K191A (d). The binding of 3 nM [ H]mepyramine to mem- 3
= RTlnK
branes
containing i) for WT, K179A, K191A, and K179A + K191A at a standard temperature of 25 °C
containing
WT (a),WT (a), K179A
K179A (b), K191A
(b), K191A (c), and
(c), and K179AK179A + K191A
+ K191A (d)(d)in in
thethe presenceororabsence
presence ab- of
(298.15
sence of K). The
various K i values of bilastine
concentrations of bilastine were
was 1.92 ±
measured 0.08
◦ atnM
4 °C for WT
(open (n =
◦
circles),
various concentrations of bilastine was measured at 4 C (open circles), 14 C (closed circles), 25 C 4),
145.20
°C ± 1.18
(closed nM
cir- for
◦
K179A
cles),
(open25
(n (open
°C = 7), and
triangles),
2.57 ± 0.16and
triangles), nM37for
37 ◦ C (closed °CK191A
(closedas
triangles),
(ndescribed
= 4), and
triangles), 7.96 ± 0.76and
asindescribed
materialsin
nM for K179A
materials
methods and + K191A
methods
section. The data
(n
= 4).
section.Thus, the
The data affinity
points areofthebilastine for
percentages
3 H receptors
of1 bound [3H] was significantly
mepyramine, with
points are the percentages of bound [ H] mepyramine, with 100% as 3 nM [ H] mepyramine binding 3 reduced
100% as 3 nMby approxi-
[3H]
mepyramine
mately 1.3 to binding
4.1 timesin the absence of
following bilastine.
the mutation Dataofrepresent
Lys179ECL2 mean ± standard
and/or Lys191 errors of means
5.39. These results
in the absence of bilastine. Data represent mean ± standard errors of means of 4–7 independent
of 4–7 independent
suggest that Lys179experiments determined
ECL2 and Lys191 in quadruplicates.
5.39 contribute to the The lines are
increased the best-fit
affinity for curves to
bilastine.
aexperiments
one-site model.determined in quadruplicates. The lines are the best-fit curves to a one-site model.

Figure 4 shows the lnKi values of bilastine and their corresponding ∆G° values (∆G°
= RTlnKi) for WT, K179A, K191A, and K179A + K191A at a standard temperature of 25 °C
(298.15 K). The Ki values of bilastine were 1.92 ± 0.08 nM for WT (n = 4), 5.20 ± 1.18 nM for
K179A (n = 7), 2.57 ± 0.16 nM for K191A (n = 4), and 7.96 ± 0.76 nM for K179A + K191A (n
= 4). Thus, the affinity of bilastine for H1 receptors was significantly reduced by approxi-
mately 1.3 to 4.1 times following the mutation of Lys179ECL2 and/or Lys1915.39. These results
suggest that Lys179ECL2 and Lys1915.39 contribute to the increased affinity for bilastine.

Figure Changes in
Figure 4. Changes inthe
thevalues ∆G◦and
valuesofof∆G° and lnK
lnK i of
i of bilastine
bilastine byby mutations
mutations in Lys179
in Lys179 ECL2ECL2
and/or and/or
Lys191 5.39
Lys191 .. Scatter
5.39 Scatter plots
plots of
of values
values ofof ∆G°◦
∆G versus
versus lnK
lnKiiofofbilastine
bilastinefor
forWT,
WT, K179A,
K179A, K191A, and K179A
++ K191A
K191Aare areshown, which∆G
shown,ininwhich ◦ values
∆G° valueswere
werecalculated
calculatedaccording
according totothethe equation,
equation, ◦ = RTlnKi,
∆G∆G° = RTlnKi, at
aatstandard
a standard temperature
temperature of 25
of 25 ◦ °C (298.15
C (298.15 K). K). Increases
Increases in values
in values of ∆G°
of ∆G ◦ andandlnKlnK
i
i represent
represent reduc-
reductions
tions
in thein the affinities
affinities for bilastine
for bilastine by mutations
by mutations of Lys179
of Lys179 ECL2 ECL2 and/or
and/or Lys191
Lys191 . *. *p p<<0.05,
5.395.39 0.05,**
** pp <
< 0.01;
0.01;
compared to the values for WT.
compared to the values for WT.

2.3. Roles of Lys179ECL2 and Lys1915.39 in Thermodynamic Binding Forces of Bilastine

Figure 5a shows the van ’t Hoff plots used to determine the thermodynamic binding
Figure 4. Changes in the values of ∆G° and lnKi of bilastine by mutations ◦ /RT − ∆Sin Lys179 ECL2 and/or
◦ /R. Figure
forces5.39
Lys191 of. Scatter
bilastine according
plots to∆G°
of values of the versus
equation, lnK
lnKi of i = ∆Hfor
bilastine WT, K179A, K191A, and5b shows
K179A
scatter plots of values of − T∆S ◦ versus ∆H ◦ for bilastine obtained from the van ’t Hoff plots.
+ K191A are shown, in which ∆G° values were calculated according to the equation, ∆G° = RTlnKi,
Inathe
at binding
standard of bilastine
temperature of 25to°C
WT (Figure
(298.15 5b; WT), in
K). Increases negative
values ofvalues
∆G° andof lnK∆Gi ◦represent
(= ∆H ◦ reduc-
− T∆S◦ )
for bilastine wereforobtained ◦
tions in the affinities bilastine by the binding
by mutations enthalpyand/or
of Lys179 ECL2 (∆H Lys191
) and more
5.39 . * p < dominantly
0.05, ** p < 0.01;the
binding entropy
compared (−T∆S
to the values ◦ ). These results are consistent with our previous findings that
for WT.
entropy-dependent binding forces of second-generation antihistamines were significantly
higher than those of first-generation antihistamines [25].
 forces of bilastine according to the equation, lnKi = ∆H°/RT − ∆S°/R. Figure 5b shows scat-
ter plots of values of -T∆S° versus ∆H° for bilastine obtained from the van ’t Hoff plots. In
the binding of bilastine to WT (Figure 5b; WT), negative values of ∆G° (= ∆H° − T∆S°) for
bilastine were obtained by the binding enthalpy (∆H°) and more dominantly the binding
Int. J. Mol. Sci. 2021, 22, 1655
entropy (-T∆S°). These results are consistent with our previous findings that entropy-de-5 of 8
pendent binding forces of second-generation antihistamines were significantly higher
than those of first-generation antihistamines [25].

(a) (b)

Figure
Figure 5. Changes in
5. Changes inthe
thethermodynamic
thermodynamicbinding binding forces
forces of of bilastine
bilastine by mutations
by mutations ECL2 ECL2
of Lys179
of Lys179
and/or
and/or Lys191 5.39
Lys1915.39. (a)
. (a) van
van ‘t Hoff
‘t Hoff plots
plots for for bilastine:
bilastine: according
according to thetovan
the‘tvan
Hoff‘tequation,
Hoff equation,
lnKi = lnKi
∆H ◦ /RT
=∆H°/RT − ∆Sthe
− ∆S°/R, ◦ /R,slope and intercept
the slope of the vertical
and intercept axis represent
of the vertical ∆H°/R and
axis represent ◦ /R and
∆H−∆S°/R, −∆S◦ /R,
respec-
tively. (b) Scatter
respectively. plots of
(b) Scatter values
plots of −T∆S°
of values of −versus
T∆S◦∆H°: compounds
versus with a negative
∆H ◦ : compounds value of ∆H°
with a negative value of
and◦ positive value of −T∆S° are classified
◦ as enthalpy driven (H driven);
∆H and positive value of −T∆S are classified as enthalpy driven (H driven); conversely, conversely, compounds
compounds
with a positive value of ∆H° and negative value of −T∆S° are classified as entropy driven (S
with a positive value of ∆H ◦ and negative value of −T∆S◦ are classified as entropy driven (S driven).
driven). Compounds with negative values of ∆H° and −T∆S° are classified as enthalpy and en-
◦ and −T∆S◦ are classified as enthalpy and entropy driven
Compounds
tropy drivenwith(H&Snegative
driven).values of ∆Hin
Reductions values of ∆H° and −T∆S° reveal increases in the binding
(H&S
forcesdriven).
of ligands Reductions
mediated by in enthalpy
values ofand ∆H ◦entropy,
and −T∆S ◦ reveal increases in the binding forces of
respectively. The arrow indicates changes
ligands
inducedmediated by enthalpy
by the mutation and entropy,
of Lys191 respectively. The arrow indicates changes induced by the
to alanine.
mutation of Lys191 to alanine.
The mutation of Lys179ECL2 to alanine (Figure 5b; K179A) led to a reduction in the
The mutation ofbinding ECL2 to alanine (Figure 5b; K179A) led to a reduction in
Lys179forces
enthalpy-dependent (∆H°) of bilastine by 1.9 kJ/mol although not signifi-
the enthalpy-dependent binding forces (∆H ◦ ) of bilastine by 1.9 kJ/mol although not
cantly. This might explain the reduction in the affinity of bilastine by the mutation of
significantly.
Lys179ECL2 to This might
alanine. explain
Thus, Lys179 the reduction
ECL2 may have inplayed
the affinity
a roleof
inbilastine by the
maintaining themutation
electro-
of Lys179 ECL2 to alanine. Thus, Lys179ECL2 may have played a role in maintaining the
static binding forces of bilastine.
electrostatic bindingofforces
The mutation Lys191 of5.39
bilastine.
to alanine (Figure 5b; K191A) led to a marked change in
The mutation of Lys191 5.39 to alanine (Figure 5b; K191A) led to a marked change in
the binding enthalpy and entropy of bilastine compared with the expected. The entropy-
the binding binding
dependent enthalpyforces
and entropy
(-T∆S°) of of bilastine
bilastine compared with thereduced
were significantly expected.byThe
8.2 entropy-
kJ/mol,
dependent binding forces ( − T∆S ◦ ) of bilastine were significantly reduced by 8.2 kJ/mol,
which may explain the reduced affinity of bilastine following mutation. Thus, Lys1915.39
which maya explain the reduced affinitytheof bilastine following mutation. 5.39
may play crucial role in maintaining hydrophobic binding forces ofThus, Lys191
bilastine. Con-
may playenthalpy-dependent
versely, a crucial role in maintaining
binding forces the hydrophobic
(∆H°) of bilastine binding
wereforces of bilastine.
significantly Con-
increased
versely, enthalpy-dependent bindingofforces (∆H ◦ of bilastine were
5.39. ) significantly increased
by 8.1 kJ/mol due to the mutation Lys191 Thus, Lys1915.39 may play an inhibitory
by 8.1inkJ/mol due to the mutation 5.39 . Thus, Lys1915.39 may play an inhibitory
of Lys191These
role the electrostatic binding of bilastine. results are in agreement with our find-
role
ings that Lys191 might not necessarily contribute toresults
in the electrostatic
5.39 binding of bilastine. These are inbinding
electrostatic agreement
forceswith our
of car-
findings 5.39 might not necessarily contribute to electrostatic binding forces
boxylated antihistamines such as levocetirizine [26]. It should be noted that Lys179 of
that Lys191 ECL2

carboxylated antihistamines ECL2

and Lys1915.39 differentiallysuch as levocetirizine
regulated the enthalpy- [26]. It
andshould be noted that Lys179
entropy-dependent binding
and Lys191 5.39 differentially regulated the enthalpy- and entropy-dependent binding forces
forces of bilastine.
of bilastine.
The mutation of both Lys179ECL2 and Lys1915.39 to alanine led to a reduction in the
entropy-dependent binding forces (−T∆S◦ ) of bilastine by 2.2 kJ/mol although not signifi-
cantly, which might explain the reduction in the affinity of bilastine. It is most likely that
Lys179ECL2 and Lys1915.39 interacted with bilastine at the entrance of the ligand-binding
pocket of H1 receptors to increase the binding affinity of bilastine via electrostatic and
hydrophobic interactions, as it is assumed that ligands may interact with both positively
and negatively charged regions as well as hydrophobic transmembrane domains of the
receptor before reaching the final position in the ligand-binding pocket [27].
In conclusion, the study revealed that the binding of bilastine to H1 receptors occurred
by the binding enthalpy (∆H ◦ ) and the binding entropy (−T∆S◦ ) and that Lys179ECL2
Int. J. Mol. Sci. 2021, 22, 1655 6 of 8

and Lys1915.39 play a differential role in regulating the thermodynamic binding forces
of bilastine. These findings provide further insight into the mechanisms by which the
affinities of ligands for their receptors are individually regulated by electrostatic and
hydrophobic interactions.

3. Materials and Methods

3.1. Materials
[Pyridinyl-5-3 H]-mepyramine was purchased from PerkinElmer (Waltham, MA, USA).
Bilastine was purchased from Tokyo Chemical Industry (Tokyo, Japan). Chinese hamster
ovary cells (CHO-K1: RCB0285, RRID: CVCL_0214) were purchased from the RIKEN Biore-
source Center (Tsukuba, Ibaraki, Japan). The expression vectors (3×HA hH1R/pcDNA3.1(+))
for human H1 receptors tagged with three molecules of hemagglutinin (YPYDVPDYA) at
the N terminus were purchased from the Missouri S&T cDNA Resource Center (Rolla, MO,
USA). Other materials were purchased from Sigma-Aldrich (Tokyo, Japan).

3.2. Docking Simulation on the Binding of Bilastine to Human H1 Receptor

A docking engine, Sievgene, implemented in MyPresto5.0 (N2 PC, Tokyo, Japan) [31]
was used in for the present study under the following settings: Flexible ligand, rigid protein,
and no water molecule. The docking score was a modified version of the multiple active
site correction score [32]. The ligand binding site was indicated by a set of reference points,
which were the atom coordinates of the ligand in the target protein-ligand complex. The
ligand atoms were superposed to the binding site using the geometric hashing method [33]
and the optimal complex structure was obtained by using the steepest decent algorithm
with the AMBER-type molecular force field. The interactions that accounted for this method
were van der Waals, Coulomb, hydrogen bond, and hydrophobic interactions. In this study,
the target protein model was generated from the crystal structure of the human H1 receptor
(PDB:3RZE) [21]. Docking calculations were performed by placing bilastine in a random
position within 6.5Å from the binding site and optimizing the steepest descent algorithm.

3.3. Measurement of [3 H]Mepyramine Binding to Membrane Preparations

CHO cells stably expressing WT, K179A, K191A, and K179A + K191A were cultured,
and membrane preparations were obtained as described previously [25–27]. The receptor
binding assay with [3 H] mepyramine, a radioligand for H1 receptors, was performed in
accordance with the methods described previously [25–27]. Briefly, aliquots (0.1 mL) of
membrane preparations (approximately 50 µg of membrane proteins) were incubated with
3 nM [3 H] mepyramine in the presence or absence of various concentrations of bilastine for
3 h at 37 ◦ C, 24 h at 25 ◦ C and 14 ◦ C, and 7 days at 4 ◦ C in normal HEPES buffer (NaCl,
120 mM; KCl, 5.4 mM; MgCl2 , 1.6 mM; CaCl2 , 1.8 mM, D-glucose, 11 mM; and HEPES,
25 mM; pH 7.4 at 37 ◦ C; final volume 1 mL). The reaction mixture was filtered through glass
fiber filters, and the radioactivity trapped on the filters was determined by scintillation
counting. All determinations were made in quadruplicate. The protein content in the
membrane preparations was determined using a BCA protein assay kit (Pierce, Rockford,
IL, USA).

3.4. Data Analyses

All data were presented as means ± standard errors of means of at least three measure-
ments performed in quadruplicate. Statistical significance was evaluated using Student’s
t-test or analysis of variance with Bonferroni correction. Results with a value of p < 0.05
were considered significant.
The IC50 for bilastine was determined by fitting the displacement curves to the one-site
model (KaleidaGraph; Synergy Software, Reading, PA, USA):

B = 100 − P × C/(C + IC50 )

Int. J. Mol. Sci. 2021, 22, 1655 7 of 8

where B is the amount of [3 H] mepyramine bound, C is the free concentration of bilastine,

and P is the percentage of the binding sites of bilastine.
The Ki values for bilastine were estimated from the Cheng and Prusoff equation, as
follows [25–27,34]:
Ki = IC50 /(C/Kd + 1)
where Ki is the dissociation constant for bilastine, C is the free concentration of [3 H]
mepyramine, and Kd is the dissociation constant for [3 H] mepyramine.

Author Contributions: Conceptualization, S.H.; investigation, H.A. and M.S.; writing—original

draft preparation, H.A. and M.S.; writing—review and editing, S.H.; supervision, S.H. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was partially funded by KAKENHI (No. 23590119) from the Japan Society
for the Promotion of Science (JSPS).
Institutional Review Board Statement: The experimental protocols of this research were approved
by the Institutional Safety Committee for Recombinant DNA Experiments, Meiji Pharmaceutical
University (No. 1209).
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We greatly appreciate the technical assistance provided by Chizuru Akatsu,
Chihiro Kobayashi, Airi Tanaka, and Tomomi Yasuda.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

CHO Chinese hamster ovary

K179A Lys179 mutant of H1 receptor to alanine
K191A Lys191 mutant of H1 receptor to alanine
K179A + K191A Both Lys179 and Lys191 mutant of H1 receptor to alanine
WT Wild-type H1 receptor

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