LIU Jian-Zhi HOU Yan-Jun LIU Jing-BoZHENG Liu-Ping CAI Kai-Cong

a (Department of Otorhinolaryngology, Fujian Medical University Union Hospital, Fuzhou 350001, China)

b (College of Chemistry and Materials Science, Fujian Normal University, Fuzhou 350007, China)

c (Fujian Provincial Key Laboratory of Theoretical and Computational Chemistry, Xiamen 361005, China)

d (Fujian Province University Key Laboratory of Green Energy and Environment Catalysis, Ningde Normal University, Ningde 352100, China)

1 INTRODUCTION

Vestibular system is an important part of balance system in our bodies, which can provide many advanced perceptive functions such as feeling control,space orientation and memory.Some symptoms like dizziness, nausea, vomiting and nystagmus will appear if vestibular system is partly or fully diseased[1-5].Clinically, meniere disease, benign positional paroxysmal vertigo (BPPV), etc.[6-9]are commonly observed peripheral vertigo diseases.When this symptom appears, a self-compensation process(vestibular compensation) will be started up[10,11].This compensation is the important rehabilitation basis for balance disorder patients[12].Histamine(HIS) has been known as an important neurotransmitter in the chemical mechanism of vestibular compensation[13,14].So far, the well known HIS receptors can be divided into four types, i.e.H1, H2,H3and H4, which all belong to G-protein coupled receptors.Postsynaptic receptors (H1and H2) and presynaptic receptor (H3) can all be expressed in neurons of central nervous system.According to the evaluation results from the biological model of HIS and its receptor in brain interactions[15], HIS is a weak agonist of H1receptor and a strong antagonist of H3receptor[16], however, no interaction can be found between HIS and H2receptors.The combination of HIS with H1receptor should be activated by a self feedback process[17,18], while its combination with H3receptor needs a negative feedback process[19,20].Then HIS can be generated and released.Self vestibular compensation is a time-consuming process for organs, thus the rehabilitation of patients would need a long time.

The design of new drugs with similar pharmacological properties as HIS for the acceleration of self vestibular compensation process is really important.Betahistine is a kind of drug with similar pharmacological properties as histamine, its antagonism will be generated when interacting with H3receptor, and the synthesis and release of HIS will be accelerated,thus leading to the raising of blood flow in cochlea and the excitation of I-type cell and rebalancing the vestibular system more quickly.In addition, the interactions between betahistine and H1receptor can improve egersis, which has been proved by the animal experiment.The experimental results show that the sensorimotor function of cat can be greatly improved after the use of betahistine, which suggests that betahistine is an important factor for the body recovery[21].

Betahistine has been clinically widely used for peripheral vertigo and vestibular disease[22].The commonly used betahistine drugs include betahistine methanesulfonate (BMT) and betahistine hydrochloride (BHD), which have different salinization groups.These drugs are imidazole-free, which overcomes the disadvantages of higher water-solubility and worse permeability to blood-brain barrier in imidazolecontaining medicines.Specially, the clinic cross reaction and extrapyramidal symptoms led by imidazole-containing medicines can also be dispelled.The main metabolites of these two betahistine drugs consist of 2-pyridylacetic acid, 2-pyridylethyamin and 2-pyridylalcohol[23].When betahistine works on vestibular organs, blood flow will increase to improve the dysaemia of inner ear[19,24,25], endolymphatic hydrops and pressure in the inner ear will be lightened, and static potential in vestibular organs will be lowered[26].However, how these drugs take effect on improving vestibular compensation process and the pharmacodynamic properties of these two kinds of betahistine drugs still remain mystery.In this work, geometry optimizations and normal mode analysis were performed for BHD and BMT to gain insight into their structural features, and the drug metabolism processes could be monitored by means of vibrational frequency and line shape.The molecular docking method was introduced for the docking interactions between structure-optimized betahistine drugs and H1, H3receptors, and ZDock Score values and hydrogen bonds between ligand and receptor were utilized as the criteria for the interaction strength between HIS, BMT, BHD and H1, H3receptors, respectively.This work will provide theoretical guides for the discovery of drugreceptor interaction mechanisms and the therapeutic evaluations, which would be beneficial for the drug choice during the curing of vestibular disease.

2 CALCULATION METHODS

2.1 Quantum chemical calculations

The structures of HIS, BHD and BMT were downloaded from Chem Spider database[27], and undergone geometry optimization and normal mode analysis at the B3LYP/6-311++G(d,p) level of theory by using Gaussian 09 software[28].

2.2 Molecular docking

The molecular interactions between ligands (HIS,BHD, and BMT) and receptors (H1, PDB ID: 3RZE;H3, PDB ID: 5CXV)[29]were conducted by using ZDock method[30,31]in Discovery Studio 2.5 package[32,33].ZDock has been widely used in rigid docking of biomolecules, in which fast Fourier transform technique was utilized to search the translational and rotational spaces of ligand and receptor.The conformations of ligand and receptor would not be changed, only the relative locations and postures would be adjusted[34,35].Considering the docking efficiency and accuracy, angular step size was set to 15°, RMSD cutoff for clustering was set to 6 ?, and interface cutoff was set to 9 ?.

During the molecule docking, 2000 different poses of ligand/receptor complex could be generated, and then be divided into different clusters according to the RMSD cutoff value.The first cluster contains the most poses.The larger the cluster number is, the less poses they contain.Among these 2000 poses, the most possible pose was selected as the docking target, which was determined by the ZDock Score.The ZDock Score takes shape complementation, static potential, energies into account to estimate the ligand/receptor interaction.Higher ZDock Score value indicates better docking efficiency.The reliable docking results could be obtained by judging from the ZDock Score.

3 RESULTS AND DISCUSSION

3.1 IR spectra simulation and structural characterization

The optimized geometries of HIS, BHD and BMT are shown in Fig.1.BHD and BMT both tend to present two layers feature.Salinization groups locate at one layer, and betahistine occupies another layer.Among these two layers, atoms with more electronegativity and electropositivity locate at opposite sites, thus the configurations were stabilized by intermolecular interactions.The optimized geometries of HIS suggest that its structure is quite similar with betahistine.

The calculated IR spectra of HIS, BHD and BMT are also shown in Fig.1.For HIS, strong peak at 831.5 cm-1is the vibration motion of H–C–N–H torsion at the branched chain, and peak at 2967.2 cm-1corresponds to the C–H (linking to N atom in the side chain) stretching vibration.Those peaks at 3023.3 and 3067.4 cm-1can be attributed to the C–H stretching motion connecting the iminazole rings.For BHD, strong Cl–H stretching vibration appears at 1749.6 cm-1.The stretching vibration of N(from branched chain) –H(from hydrochloride) can be observed at 2526.7 cm-1.For BMT, vibration peaks at 3140.9 and 3323.8 cm-1can be assigned to the stretching vibration of S–O bonds.The simulated IR spectra of these three compounds differ greatly due to the presence of salinization groups, and these three compounds can be identified quickly by means of characteristic absorption peaks.

Fig.1. Optimized structures and calculated infrared spectra of HIS (A/B), BHD (C/D), and BMT (E/F)

3.2 Molecular docking

During the docking between optimized ligand and H1receptor, 53 clusters are obtained for BMT/H1complexes, 42 clusters are found for BHD/H1complexes, and 24 clusters are generated for HIS/H1complexes, respectively.As for the docking between optimized ligand and H3receptor,18 clusters are obtained for HIS/H3complexes, and 35 clusters are produced for BHD/H3complexes and BMT/H3complexes, respectively.The results show that, HIS, BHD and BMT all combine themselves into receptors at proper sites in the cavities, and the cluster numbers can indicate the possibility of drugs'entry into cavities.BHD and BMT tend to form more clusters with H1or H3receptors than HIS, and BMT can form 11 more clusters with H1receptor than BHD, while the formed cluster numbers are the same for H3receptor.

The docking pose with the highest ZDock Score is considered as the most stable one.In the case of docking with H1receptor (Fig.2), the ZDock Score calculated for HIS, BHD and BMT are 4.4, 5.6, and 7.6, respectively.In the case of docking with H3receptor (Fig.2), the ZDock Scores are 4.6, 5.7,and 7.7.The results show that during the docking with H1and H3receptors, BMT presents the highest score, BHD exhibits similar but slightly lower score,and HIS is the lowest.

Fig.2. Docking poses for the ligands and receptors

Hydrogen bonds formed between ligands and receptors can greatly stabilize the complexes.The hydrogen bond cutoff distance was set as 3 ? in this work.As for the stable docking poses of ligands with H1receptor, no hydrogen bond can be found between HIS and H1receptor, indicating that the interaction between them is really weak.There is one hydrogen bond observed for BHD, where the chlorine atom in BHD forms a short hydrogen bond(2.65 ?) with oxygen in threonine (sequence No.194) of the H1receptor.In contrast, eight strong hydrogen bonds can be found, three of which are formed in BMT itself and five between the BMT and H1receptor, and the shortest hydrogen bond(2.30 ?) among all of them is formed by hydroxyl group of tyrosine (sequence No.108) in the H1receptor and the oxygen in BMT.Comparing the stability of docking poses with H3receptor, there are three hydrogen bonds between HIS and H3receptor, among which the strongest one (2.79 ?)appears between the nitrogen of arginine (sequence No.171) in H3receptor and hydrogen in HIS.No intermolecular hydrogen bond is found between BHD and H3receptor, while chlorine and hydrogen atoms in BHD form two hydrogen bonds with the shortest distance of 1.91 ?.As for BMT and H3receptor, two hydrogen bonds are present inside BMT itself with the shortest distance of 1.70 ? and one occurs between BMT and H3receptor.The results show that BMT can form the most stable complex with H1and H3receptors considering the number and length of hydrogen bonds.In order to illustrate the classification of 2000 docking poses, a 3D diagram was made for all the docking poses,according to the correlations between cluster,density and Zdock score (Fig.3).

Fig.3. 3D point plot of interactions between ligands and receptors

The results show that BMT is the most suitable for the combination with H1and H3receptors, and the formed complex poses could be stabilized by intermolecular hydrogen bond.Although BHD's performance comes second, BMT and BHD are both superior to HIS in the curing of vestibular impairment.Therefore, these two drugs can promote the recovery of central nervous system and sensorimotor ability.

4 CONCLUSION

Vestibular compensatory drugs' development is important for the vestibular impairment disease and the structural characterization of drug, and how they interact with organ needs to be revealed for further development of new and more efficient drugs.Nowadays, the widely used BHD and BMT drugs have similar pharmaceutical behavior as HIS, partly because their structural features are quite alike.The simulated IR results show that, the vibrational peaks of three compounds differ greatly, so they can be distinguished quickly and be in situ monitored during drug metabolism.

The molecular docking results show that BHD and BMT both can improve the vestibular compensation process than HIS, however, their interaction strengths with receptors differ greatly due to their different salinization groups, and BMT exhibits better performance in the docking patterns with H1and H3receptors.The obtained results open up a new theoretical way for the drug's characterization and gain insights into the drug/receptor interaction strength, mechanism and curing efficiency estimation,which would be really helpful for further drug developments.

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