Differential Regulation of Bilastine Affinity for Human Histamine H1

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; [email protected] 2 Pharmaceutical Education and Research Center, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan; [email protected] * Correspondence: [email protected]

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- ECL2 5.39 generation antihistamines and the amino group of Lys179 and Lys191 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 3 constants were obtained from the displacement curves against the binding of [ 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 reduced the affinity of bilastine to H receptors by reducing enthalpy- and entropy-dependent binding and Lys 191 via Its Binding Enthalpy 1 ECL2 5.39 and Entropy. Int. J. Mol. Sci. 2021, 22, forces, respectively. These results suggest that Lys179 and Lys191 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 (Figure1), 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. 3.32 The human H1 receptor possesses Asp107 (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:// (Figure2a) [ 21]. Ligand-receptor docking simulations based on the crystal structure of creativecommons.org/licenses/by/ 4.0/). human H1 receptor indicated that the carboxyl group of second-generation antihistamines,

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

N + such as olopatadine,N levocetirizine, fexofenadine, and acrivastine, appeared to form a salt bridgeN 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 REVIEW ECL2 2 5.39of 8 Accordingly,O the electrostatic interaction of bilastine with Lys179 and/or Lys191 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 itsCOO non-sedative– properties as well as an increased 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 receptors, deep in the ligand-bindingN pocket, whereas Lys179ECL2 and Lys1915.39, located at N the entrance of the ligand-binding pocket, are anion-binding sites unique to the H1 receptor (Figure 2a) [21]. Ligand-receptor docking simulations based on the crystal structure of

human H1 receptor indicatedO that the carboxyl group of second-generation antihistamines, such as olopatadine, levocetirizine, fexofenadine, and acrivastine, appeared to form a salt bridge with Lys179ECL2 and/or Lys1915.39. This bridge might have contributed FigureFigure 1.1. ChemicalChemical structurestructure ofof bilastine.bilastine. BilastineBilastine is is a a zwitterionic zwitterionic second-generation second-generation antihistamine antihista- to the increased selectivity of carboxylated second-generation antihistamines for H1 re- containingmine containing a carboxyl a carboxyl group group that contributes that contributes to its non-sedativeto its non-sedative properties properties as well as aswell an as increased an in- ECL2 ceptorscreased [21]. selectivityAccordingly, for H the1 receptors. electrostatic interaction of bilastine with Lys179 and/or 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 receptors, deep in the ligand-binding pocket, whereas Lys179ECL2 and Lys1915.39, located at Bilastine Lys179ECL2 the entrance of 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- = RTlnK mines,NH2 such as olopatadine,d levocetirizine, fexofenadine, and 4.3Åacrivastine, appeared to ECL2 form a salt bridge withLys179 Lys179ECL2 and/or Lys1915.39. This 3.6Åbridge might have contributed 5.39 to the increased selectivityLys191 of carboxylated second-generation antihistamines for H1 receptors [21]. Accordingly,Asp107 the 3.32electrostatic interaction of bilastine with Lys179ECL2 and/or 3.4Å Lys1915.39 may be important in the determination of its binding affinity for H1 receptors. Bilastine (a) Asp1073.32 (b) COOH Histamine H1 ReceptorBilastine Lys179ECL2

Lys191 5.39

FigureFigure 2. A schematic 2. A schematic structure∆ structureG˚(<0) of human = of∆H human˚- HT1∆ receptorS˚ H1 receptor (a) and (a )docking and docking simulation simulation on the on binding the binding of of bilastine to human H1 receptor (b).= (RTa) lnA Kschematic structure of human H1 receptor is shown bilastine toNH human2 H1 receptor (b). (a) Ad schematic structure of human H1 receptor4.3Å is shown and indi- and indicates that Asp1073.323.32 is a highly conservedECL2 amino acid in the aminergic G-protein-coupled cates that Asp107 is a highly conservedLys179 amino acid in the aminergic G-protein-coupled3.6Å receptors, receptors, and Lys179ECL2 ECL2 and Lys1915.39 5.39 are anion-binding sites unique to the H1 receptor. Ligand- and Lys179 and Lys191 areLys191 anion-binding5.39 sites unique to the H1 receptor. Ligand-receptor receptor interaction is also shown to demonstrate that the binding affinity for ligands (Kd) is deter- interaction is also shown to demonstrate that the binding afﬁnity for ligands (K ) is determined by mined by their thermodynamic bindingAsp107 forces3.32 (∆G° = ∆H° − T∆S° = RTlnKd). (b) Dockingd simula- their thermodynamic binding forces (∆G◦ = ∆H◦ − T∆S◦ = RTlnK3.4Åd). (b) Docking simulation was tion was performed to reveal the final position of bilastine binding to human H1 receptor as de- scribedperformed in the materials to reveal and the methods ﬁnal position section. of The bilastine amino binding group toof humanbilastine H appeared1 receptor to as interact describedBilastine in the 3.32 3.32 with Asp107materials3.32, andand methodsthe carboxyl section. group The of aminobilastin groupe appeared of bilastine to interactAsp107 appeared with toLys179 interactECL2 withand Asp107 , COOH Lys191and5.39. theAtoms carboxyl of nitrogen, group ofoxygen, bilastine carbon, appeared and hydrogen to interact are with shown Lys179 in ECL2blue,and red, Lys191 gray, and5.39. Atoms of white,nitrogen, respectively.Histamine oxygen, The carbon,distances H1 Receptor and between hydrogen key areatom showns of bilastine in blue, and red, amino gray, and acid white, residues respectively. are also The

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 affinity (Kd) of ligands is determined by the thermodynamic binding forces of bilastine to human H1 receptor (b). (a) A schematic structure◦ of human H◦1 receptor is shown ofand ligands, indicates and that these Asp107 are3.32 the is bindinga highly conserved enthalpy (amino∆H ) andacid in entropy the aminergic (∆S ) (Figure G-protein-coupled2a) [ 22–27] . ◦ ∆receptors,H is usually and Lys179 associatedECL2 and with Lys191 binding5.39 are anion-binding forces via the sites formation unique to of the new H1 receptor. bonds between Ligand- receptorsreceptor interaction and ligands, is also such shown as electrostatic to demonstrate interaction that the binding via salt bridges,affinity for hydrogen ligands (K bondsd) is deter- and vanmined der by Waals their thermodynamic interactions, whereas binding∆ forcesS◦ is usually(∆G° = ∆H characterized° − T∆S° = RTln byKd). binding (b) Docking forces simula- via the displacementtion was performed of ordered to reveal water the final molecules position coupled of bilastine with binding the formation to human of H new1 receptor hydrophobic as described 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.

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interactions [22–30]. Thus, thermodynamic analyses provide important information for evaluating the electrostatic and hydrophobic binding of bilastine with H1 receptors. In this study,The we binding examined affinity how ( Lys179Kd) of ligandsECL2 and is Lys191determined5.39 might by the increase thermodynamic the electrostatic binding and ECL2 forceshydrophobic of ligands, binding and these of bilastine are the withbinding H1 enthalpyreceptors ( through∆H°) and the entropy mutation (∆S°) of (Figure Lys179 2a) [22–27].and/or Lys191∆H° is 5.39usuallyto alanine. associated with binding forces via the formation of new bonds between receptors and ligands, such as electrostatic interaction via salt bridges, hydrogen bonds2. Results and andvan Discussionder Waals interactions, whereas ∆S° is usually characterized by binding forces2.1. Docking via the Simulation displacement on the of Binding ordered of water Bilastine molecules to H1 Receptor coupled with the formation of new hydrophobicDocking simulation interactions was performed[22–30]. Thus, to simply thermodynamic estimate the analyses configuration provide of important bilastine at informationthe ligand-binding for evaluating pocket the of human electrostatic H1 receptors and hydrophobic (Figure2b). binding We found of bilastine that the with carboxyl H1 receptors.group of bilastineIn this study, was locatedwe examined between how Lys179 Lys179ECL2ECL2 andand Lys191Lys1915.395.39 mightand that increase the amino the electrostaticgroup of bilastine and hydrophobic was close binding to Asp107 of bilastine3.32. The with oxygen H1 receptors atom of through the carboxyl the mutation group of ofbilastine Lys179ECL2 appeared and/or toLys191 be closer5.39 to to alanine. 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.Lys191 Results5.39 andin regulatingDiscussion the binding affinity of bilastine, we then performed receptor 3 2.1.binding Docking experiments Simulation usingon the [BindingH] mepyramine, of Bilastine ato radioligandH1 Receptor for H1 receptors, to evaluate ECL2 5.39 actualDocking roles of simulation Lys179 wasand performed Lys191 toin simply regulating estimate the bindingthe configuration affinity of of bilastine bilastine via atits the thermodynamic ligand-binding binding pocket forces.of human H1 receptors (Figure 2b). We found that the carboxyl group of bilastine was located between Lys179ECL2 and Lys1915.39 and that the amino ECL2 5.39 group2.2. Roles of bilastine of Lys179 was closeand Lys191to Asp1073.32in. theThe Binding oxygen Affinity atom of for the Bilastine carboxyl group of bilastine appearedTo evaluate to be changes closer into thethe bindingnitrogen affinity atom of of Lys179 bilastine (3.6 for Å) H than1 receptors that of byLys191 mutations (4.3 ECL2 5.39 Å).of Lys179Since the and/ordocking Lys191 simulation, the coulIC50d fornot bilastine exactly waspredict first obtainedroles of fromLys179 theECL2 displace- and Lys191ment curves5.39 in regulating for the binding the binding of 3 nM affinity [3H]mepyramine of bilastine, we to membranethen performed preparations receptor ofbind- CHO ECL2 5.39 ingcells experiments expressing using wild-type [3H] mepyramine, human H1 receptors a radioligand (WT), Lys179for H1 receptors,or Lys191 to evaluatemutants actual of rolesWTto of alanineLys179ECL2 (K179A and Lys191 and K191A),5.39 in regulating and both Lys179the bindingECL2 andaffinity Lys191 of bilastine5.39 mutants via its of ther- WT to ◦ ◦ modynamicalanine (K179A binding + K191A) forces. at 4 C–37 C (Figure3). The Ki values for bilastine were then calculated from the IC50, as described in the materials and methods section. ECL2 5.39 ◦ 2.2. RolesFigure of Lys1794 shows theand lnLys191Ki values in the of Binding bilastine Affinity and theirfor Bilastine corresponding ∆G values ◦ (∆G To= evaluateRTlnKi) forchanges WT, in K179A, the binding K191A, affinity and of K179A bilastine + K191Afor H1 receptors at a standard by mutations tempera- ◦ ofture Lys179 of 25ECL2C and/or(298.15 Lys191 K). The5.39, Kthei values IC50 for of bilastine bilastine was were first 1.92 obtained± 0.08 from nM forthe WTdisplace-(n = 4), ment5.20 ± curves1.18 nMfor thefor K179Abinding ( nof= 3 7), nM 2.57 [3H]± mepyramine0.16 nM for K191Ato membra (n =ne 4), preparations and 7.96 ± 0.76 of CHO nM for cellsK179A expressing + K191A wild-type (n = 4). Thus, human the H affinity1 receptors of bilastine (WT), Lys179 for HECL21 receptors or Lys191 was5.39 mutants significantly of ECL2 WTreduced to alanine by approximately (K179A and K191A), 1.3 to 4.1 and times both following Lys179ECL2 the and mutation Lys1915.39 of mutants Lys179 of WTand/or to 5.39 ECL2 5.39 alanineLys191 (K179A. These + K191A) results suggest at 4 °C–37 that °C Lys179 (Figure 3).and The Lys191 Ki valuescontribute for bilastine to the were increased then calculatedaffinity for from bilastine. the IC50, as described in the materials and methods section.

(a) WT (b) K179A

Figure 3. Cont.

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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 membranes containing WT (a), K179A (b), K191A (c), and K179A + K191A (d) in the presence or absence of various concentrations of bilastine was measured at 4 °C (open circles), 14 °C (closed circles), 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. 3 FigureFigure 3. 3. DisplacementDisplacement curves curves forfor bilastine bilastine against against the binding the binding of [3H] of mepyramine [ H] mepyramine to WT to(a), WT (a), Figure 4 shows the lnKi values of bilastine and their corresponding ∆G° values (∆G° K179AK179A ( (bb),), K191A K191A ( (cc),), and and K179A K179A + + K191A K191A ( (dd).). The The binding binding of of 3 3 nM nM [3 [H]mepyramine3H]mepyramine to to mem- membranes branescontaining= RTln containingKi) for WT WT, (a WT), K179AK179A, (a), K179A (b K191A,), K191A (b), K191A and (c), K179A and(c), and K179A + K179A K191A + K191A + K191Aat a (standardd )(d in) in the the presencetemperature presence or or absence ab- of 25 °C of sencevarious(298.15 of concentrationsvariousK). The concentrations Ki values of bilastine of bilastineof bilastine was measured were was measured1.92 at ± 4 0.08◦C at (open nM 4 °C for circles),(open WT circles), 14(n ◦=C 4),(closed 14 5.20 °C (closed± circles), 1.18 nMcir- 25 for◦C cles),(openK179A 25 triangles), °C(n (open= 7), and2.57triangles), 37 ± ◦0.16C (closed and nM 37 for triangles),°C K191A (closedas triangles),(n described = 4), and as in described7.96 materials ± 0.76 in and nMmaterials methods for K179A and section. methods + K191A The data (n section. The data points are the percentages of1 bound [3H] mepyramine, with 100% as 3 nM [3H] points= 4). Thus, are the the percentages affinity of of bilastine bound [3H] for mepyramine, H receptors with was 100% significan as 3 nMtly [3H] reduced mepyramine by approxi- binding mepyramine binding in the absence of bilastine. Data representECL2 mean ± standard errors5.39 of means inmately the absence 1.3 to 4.1 of bilastine.times following Data represent the mutation mean ± ofstandard Lys179 errors and/or of means Lys191 of 4–7. These independent results ofsuggest 4–7 independent that Lys179 experimentsECL2 and Lys191determined5.39 contribute in quadruplicates. to the increased The lines are affinity the best-fit for bilastine. curves to 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 approximately 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.

◦ ECL2 Figure 4. ChangesChanges in in the the values values of of ∆∆GG° andand ln lnKiK ofi ofbilastine bilastine by bymutations mutations in Lys179 in Lys179ECL2 and/orand/or 5.395.39 ◦ Lys191 .. Scatter Scatter plots plots of of values values of of ∆GG° versusversus ln lnKKi iofof bilastine bilastine for for WT, WT, K179A, K179A, K191A, and K179A ++ K191AK191A areare shown,shown, in in which which∆ ∆GG◦°values values were were calculated calculated according according to to the the equation, equation,∆G ∆◦G=° RT= RTlnKlni,K ati, at a standard temperature of ◦25 °C (298.15 K). Increases in values of ◦∆G° and lnKi represent reduc- a standard temperature of 25 C (298.15 K). Increases in values of ∆G and lnKi represent reductions ECL2 5.39 intions the in afﬁnities the affinities for bilastine for bilastine by mutations by mutations of Lys179 of Lys179ECL2 and/or and/or Lys191 Lys1915.39.*. *p p< < 0.05, 0.05, ****p < 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

Figure5a shows the van ’t Hoff plots used to determine the thermodynamicECL2 binding Figure 4. Changes in the values of ∆G° and lnKi of bilastine by mutations◦ in Lys179◦ and/or forces5.39 of bilastine according to the equation, lnKi = ∆H /RT − ∆S /R. Figure5b shows Lys191 . Scatter plots of values of◦ ∆G° versus ln◦ Ki of bilastine for WT, K179A, K191A, and K179A +scatter K191A plots are shown, of values in which of − T∆∆GS° valuesversus were∆H calculatedfor bilastine according obtained to the from equation, the van ∆G° ’t = Hoff RTln plots.Ki, ◦ ◦ ◦ atIn a thestandard binding temperature of bilastine of 25 to °C WT (298.15 (Figure K). 5Increasesb; WT), negativein values of values ∆G° and of ln∆KGi represent(= ∆H reduc-− T∆S ) ◦ tionsfor bilastine in the affinities were for obtained bilastine byby mutations the binding of Lys179 enthalpyECL2 and/or (∆H Lys191) and5.39 more. * p < dominantly0.05, ** p < 0.01; the comparedbinding entropyto the values (−T for∆S WT.◦). These results are consistent with our previous findings that entropy-dependent binding forces of second-generation antihistamines were significantly higher than those of first-generation antihistamines [25].

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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 forces of bilastine according to the equation, lnKi = ∆H°/RT − ∆S°/R. Figure 5b shows scatter 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)

ECL2 FigureFigure 5.5. Changes in in the the thermodynamic thermodynamic binding binding forces forces of ofbilastine bilastine by mutations by mutations of Lys179 of Lys179ECL2 5.395.39 and/orand/or Lys191 Lys191 . (.(a) avan) van ‘t Hoff ‘t Hoff plots plots for forbilastine: bilastine: according according to the to van the ‘t van Hoff ‘t equation, Hoff equation, lnKi = lnKi =∆H∆°/HRT◦/RT − ∆−S°/R,∆S the◦/R, slope the slopeand intercept and intercept of the vertical of the verticalaxis represent axis represent ∆H°/R and∆H −∆◦/SR°/Rand, respec-−∆S◦/R, respectively.tively. (b) Scatter (b) Scatter plots of plots values of values of −T∆ ofS° −versusT∆S◦ ∆versusH°: compounds∆H◦: compounds with a negative with a negativevalue of ∆ valueH° of ∆andH◦ andpositive positive value value of −T of∆−S°T are∆S ◦classifiedare classified as enthalpy as enthalpy driven driven (H 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- ◦ − ◦ Compoundstropy driven with(H&S negative driven).values Reductions of ∆H in andvaluesT of∆ S∆Hare° and classified −T∆S° reveal as enthalpy increases and in entropy the binding driven ◦ ◦ (forcesH&S driven). of ligands Reductions mediated by in enthalpy values of and∆H enandtropy,−T respectively.∆S reveal increasesThe arrow in indicates the binding changes forces of ligandsinduced mediated by the mutation by enthalpy of Lys191 and entropy, to alanine. respectively. The arrow indicates changes induced by the mutation of Lys191 to alanine. The mutation of Lys179ECL2 to alanine (Figure 5b; K179A) led to a reduction in the ECL2 enthalpy-dependentThe mutation of binding Lys179 forcesto (∆ alanineH°) of bilastine (Figure 5byb; 1.9 K179A) kJ/mol led although to a reduction not signifi- in ◦ thecantly. enthalpy-dependent This might explain binding the reduction forces (∆inH the) ofaffinity bilastine of bilastine by 1.9 kJ/mol by the althoughmutation notof significantly.Lys179ECL2 to Thisalanine. might Thus, explain Lys179 theECL2 reduction may have in played the affinity a role of inbilastine maintaining by the the mutation electro- ECL2 ECL2 ofstatic Lys179 binding forcesto alanine. of bilastine. Thus, Lys179 may have played a role in maintaining the electrostaticThe mutation binding of forces Lys191 of5.39 bilastine. to alanine (Figure 5b; K191A) led to a marked change in 5.39 the bindingThe mutation enthalpy of Lys191and entropyto of alanine bilastin (Figuree compared5b; K191A) with the led expected. to a marked The change entropy- in thedependent binding binding enthalpy forces and entropy(-T∆S°) of bilastinebilastine comparedwere significantly with the reduced expected. by The 8.2 entropy-kJ/mol, ◦ dependentwhich may bindingexplain forcesthe reduced (−T∆ Saffinity) of bilastine of bilastine were following significantly mutation. reduced Thus, by 8.2Lys191 kJ/mol,5.39 5.39 whichmay play may a explaincrucial role the reducedin maintaining affinity the of bilastinehydrophobic following binding mutation. forces of Thus, bilastine. Lys191 Con- mayversely, play enthalpy-dependent a crucial role in maintaining binding forces the hydrophobic(∆H°) of bilastine binding were forces significantly of bilastine. increased Con- ◦ versely,by 8.1 kJ/mol enthalpy-dependent due to the mutation binding of forces Lys191 (∆5.39H. )Thus, of bilastine Lys191 were5.39 may significantly play an inhibitory increased 5.39 5.39 byrole 8.1 in kJ/mol the electrostatic due to the binding mutation of bilastine. of Lys191 These. Thus, results Lys191 are in agreementmay play with an inhibitoryour find- roleings inthat the Lys191 electrostatic5.39 might binding not necessarily of bilastine. contribute These to resultselectrostatic are in binding agreement forces with of car- our 5.39 findingsboxylated that antihistamines Lys191 might such not as necessarilylevocetirizine contribute [26]. It should to electrostatic be noted binding that Lys179 forcesECL2 of ECL2 carboxylatedand Lys1915.39 antihistamines differentially such regulated as levocetirizine the enthalpy- [26]. Itand should entropy-dependent be noted that Lys179 binding 5.39 andforces Lys191 of bilastine.differentially regulated the enthalpy- and entropy-dependent binding forces 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 significantly, 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-5-3H]-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 (N2PC, 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 [3H]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 3 binding assay with [ 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 [3H] 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 measurements 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 [3H] 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) 3 where Ki is the dissociation constant for bilastine, C is the free concentration of [ H] 3 mepyramine, and Kd is the dissociation constant for [ 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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