Keywords

Neurotransmitter, Dopamine, G-protein, Schizophrenia, Homology Modelling, Docking, Interaction.

Introduction

Schizophrenia is a debilitating disease in which unusual behavior and experiences are seen [1]. In this work DRD3 receptor [2] are virtually screened against the compounds of Valeriana wallichii, Asparagus racemosus and Acorus calamus.

D(3) dopamine receptor (DRD3)

D(3) dopamine receptor is coded by the DRD3 gene. It responds to the chemical messenger (neurotransmitter) dopamine to trigger signals within the nervous system, including signals involved in producing physical movement [3]. This receptor is expressed in phylogenetically older regions of the brain. This suggests that this receptor plays a role in cognitive and emotional functions. DRD3 gene is associated with essential tremor. A DRD3 gene variant seen in some families affected by this disorder results in a dopamine receptor D3 protein. This variant binds more strongly to dopamine, resulting in a stronger response to the neurotransmitter and possibly causing the involuntary shaking seen in essential tremor [4]. Dopamine receptors are implicated in many neurological processes, like motivation, pleasure, cognition, memory and learning. Abnormal dopamine receptor signaling and dopaminergic nerve function is noted in several neuropsychiatric disorders. Coupled to inhibitory Gproteins, dopamine receptors have an inhibitory effect on neurotransmission when bound by an agonist. Many neuroleptic drugs are antagonists of the dopamine receptors. This class of drug is used to treat psychotic disorders, such as schizophrenia. [3, 4].

Valeriana wallichii

Valeriana wallichii is native to India (Himalayas). It is an important Indian medicinal plant & is used for its benefits in calming down the nervous system. It relieves stress and anxiety and also fights depression. The compounds identified from the plant are acetoxy valerenic acid, acevaltrate, baldrinal, bornyl acetate, bornyl isovalerate, fenchene, β- sitosterol, calarene, homobaldrinal, isovaltrate, valeranone, valerenal, valerenic acid, valepotriate, valtrate, valtroxal and xanthorrhizol (Fig. 1) [5].

Acorus calamus

Acorus calamus or sweet flag has been long known for its medicinal value and is cultivated in India for this reason. The rhizome possesses anti-spasmodic, carminative and anthelmintic properties and is used for the treatment of epilepsy and mental ailments. The compounds identified from the plant are 1α, 2β, 3γ, 19α- tetrahydroxyurs-12en-28-oicacid-28-O{-β-Dglucopyranosyl (1→2)} β- D- galactopyranoside, 2,3- dihydro-4,5,7-trimethoxy-1- ethyl-2-methyl-3-(2,4,5- trimethoxy phenyl)indene, 2,4,5-trimethoxy benzaldehyde, 2,6-diepishyobunone, 3β, 22α, 24, 29-tetrahydroxyolean-12-en-3-O-{-β-Darabinosyl(1→3)}-β-D-arabinopyranoside, 4, 5, 8- trimethoxyxanthone-2-O-β-D-glucopyranosyl(1→2)- O-β-D-galactopyranoside, acoradin, acoragermacrone, acoramone(1,2,4 –trimethoxy-5(2-propanoyl) benzene, β-sitosterol, Calamusenone, cis-asarone(cis-1,2,4 – trimethoxy-5(2- propenyl) benzene, galangin, γ-cisasarone(cis-1,2,4 –trimethoxy-5(2- propenyl) benzene, isoeugenol methyl ether, isocalamendiol limonene, preisocalamendiol, shyobunone, thujane and Z-3- (2,4,5-trimethoxy phenyl)-2-propenal. (Fig. 2) [6].

Asparagus racemosus

Asparagus racemosus’s tubers are candied and eaten as a sweetmeat. It is one of the constituent of medicated oils for external application in nervous and rheumatic affections. The important constituents of Asparagus racemosus are sarsasapogenin, shatavarin, rhamnose, asparagamine and racemosol [7].

Methods

Selection of template modeling

The DRD3 sequence with accession number AAI28123 was taken the National Center for Biotechnology Information (NCBI) and using Basic Local Alignment and Search Tool (BLAST) search engine against Protein Data Bank (PDB) the following template was selected and its crystal structure was downloaded from PDB.

  • 2YOOA: Turkey Beta1 Adrenergic Receptor With Stabilising Mutations And Bound Partial Agonist Dobutamine [E-value-3e-24]
  • 3EMLA: The 2.6 A Crystal Structure Of A Human A2a Adenosine Receptor Bound To Zm241385 [Evalue-7e-16] Using homology modeling the 3D structure of DRD3 protein was generated by modeller [8].

The 3d structures of the components of Valeriana wallichii, Asparagus racemosus and Acorus calamus were drawn using chemsketch and saved as *.mol file [9].

Glide Ligand Docking

Glide Ligand Docking of the compounds of Valeriana wallichii, Asparagus racemosus and Acorus calamus was done with the DRD3 protein using Maestro9.1 software [10].

Protein Preparation

By using Protein Preparation Wizard of Maestro9.1 the modeler generated protein can uploaded for optimization & energy minimization.

Active Site Generation

he active site residues were determined by SiteMap [11].

Receptor Grid Generation

The receptor grid can be generated from the Receptor Grid Generation panel.

Ligand Preparation

The compounds of each plant are opened in the workspace and saved as a single database file. This file is opened in LigPrep. LigPrep is tool to prepare high quality 3D structure for large number of molecules taking input as 2D or 3D structures and giving output as a single, low energy 3D structure.

Ligand Docking

The Glide→Ligand Docking Module takes the receptor from the output of Receptor Grid Generation panel and ligands from the LigPrep output.

Results & Discussion

Homology Modelling & Protein Preparation

Amino acid sequence of DRD3 (accession number AAI28123) was retrieved from NCBI’s database. Homology modelling of DRD3 protein was carried out using the software Modeller9.9. The templates used for DRD3 mutation protein were 3PBLA, 2Y00A, 3EMLA (downloaded from PDB) and five models (3D structures) of the DRD3 mutation protein were generated [8]. Structural Analysis & Verification Server (SAVES)’s Procheck module generated Ramachandran Plot [11]. This Ramachandran Plot was used to generate the values of DRD3 protein obtained in favoured, allowed and disallowed region (Table 1, Fig. 1). As per the Ramachandran Plot, Model 4 was selected as the best protein since it is having maximum number of residues in the favoured region and least residues in the disallowed region (Fig. 1, 2).

Table 1: Values of DRD3 protein obtained in favoured, allowed and disallowed region using Ramachandran Plot (SAVES server).

Number of residues in favoured region Number of residues in additional allowed region Number of residues in generously allowed region Number of residues in dis-allowed region
Model 1 323 (91.0%) 28 (7.9%) 2 (0.6%) 2 (0.6%)
Model 2 326 (91.8%) 23 (6.5%) 2 (0.6%) 4 (1.1%)
Model 3 326 (91.8%) 23 (6.5%) 1 (0.3%) 5 (1.4%)
Model 4 326 (91.8%) 26 (7.3%) 1 (0.3%) 2 (0.6%) (selected)
Model 5 326 (91.8%) 25 (7.0%) 0(0.0%) 4 (1.1%)

Fig 1 – Ramachandran Plot of DRD3 mutation protein model #4

Fig 2 – DRD3 protein model #4(visualization in PYMOL)

The modeler generated protein is not suitable for immediate use in docking or other molecular modeling calculations, hence we go for protein preparation. Protein Preparation: Using Protein Preparation Wizard of Maestro, the best modeled protein was uploaded and optimized and minimized [12]. LigPrep is a collection of softwares used to prepare high quality, all-atom 3D structures for large number of drug-like molecules, starting with 2D/3D to 3D which can be saved either in SD or Maestro format [13]. The compounds of the plants V. wallichii, A. racemosus and A. calamus were sketched using chemsketch [9], opened in the maestro workspace and converted to a single file. This prepaired structure was used as input in LigPrep to produce a single, lowenergy, 3D structures with correct chiralities for each successfully processed input structures. Using Glide (Grid-based Ligand Docking with Energetics), we can perform ligand database screening and high-accuracy docking [10]. Glide searches for favorable interaction between the ligands & the protein. The ligand docking jobs cannot be performed until the receptor grids have been generated. The receptor grid requires a “prepaired” structure (i.e. the output of Protein Preparation Wizard). SiteMap module of Maestro was used to determine the active site region [14] and as per the output sitemap_site_4 with SiteScore 1.075 (2nd highest score) and size 99 was used to determine the active site of the modelled protein.

MET134, HIS137, TYR138, THR130, GLY141, THR142, GLN144, SER146, ASP127, ILE126, VAL203, ARG149, THR63, VAL150, MET153, ALA123, CYS122, LEU199, PRO200, ALA156, LEU119, VAL157, LEU160, THR115, ALA161, VAL164

Using the Site tab the active site residues to the protein are assigned. The receptor grid was set up and active site residues for the proteins were assigned by using the Receptor Grid Generation panel [10]. Glide searches for favorable interactions between one or more ligand molecules and a receptor molecule, usually a protein. Glide can be run in rigid or flexible docking modes; the latter automatically generates conformations for each input ligand. In flexible docking Glide generates conformations internally during the docking process and hence used in this experiment [10]. Hence, in Glide→Ligand Docking we screen the compounds of each plant against DRD3 receptor. The protein prepared using Protein Preparation Wizard. The Ligand Docking was carried out using the above prepared receptor and ligand. Using Glide’s XP Visualizer the final result of Ligand Docking was viewed. The Glide extra precision (XP) Visualizer panel provides a way to visualize and analyze the results of a Glide XP docking run. The XP visualizer provides 3D visualization for XP terms. Information for this visualizations is read from the from the descriptor file, which is generated by Glide XP, descriptor information. To generate this, it is needed to select XP in Ligand Docking’s settings [10]. The output of Ligand Docking for the compounds of Valeriana wallichii against DRD3 receptor [Table 2, Fig. 3]; The output of Ligand Docking for the compounds of Asparagus racemosus against DRD3 receptor [Table 3, Fig. 4]; The output of Ligand Docking for the compounds of Acorus calamus against DRD3 receptor [Table 4, Fig. 5].

Table 2: Docking results of the compounds of Valeriana wallichii

Ligand Docking score/ glide g score (kcal/mol) Doner Distance in Å Interaction
β-sitosterol -6.72 MET153 2.04 MET153(OH)…O(UNK)
isovaltrate -6.14 ARG149 1.993 ARG149(OH)…O(UNK)
acevaltrate -5.68 ARG149 2.012 ARG149(OH)…O(UNK)
valtroxal -5.67 ARG149 1.952 ARG149(OH)…O(UNK)
valtrate -5.65 ARG149 2 ARG149(OH)…O(UNK)

Table 3: Docking results of the compounds of Asparagus racemosus.

Ligand Docking score/ glide g score (kcal/mol) Doner Distance in Å Interaction
shatavarin -6.62 ARG148 2.631 ARG148(OH)…O(UNK)
ARG149 2.271 ARG149(OH)…O(UNK)
2.287
2.451
THR130 1.886 THR130(OH)…O(UNK)
racemosol -6.04 ARG149 2.641 ARG149(OH)…O(UNK)
rhamnose -5.3 ARG149 2.627 ARG149(OH)…O(UNK)
THR130 2.508 THR130(OH)…O(UNK)

Fig. 3: Interaction of DRD3 receptors with compounds of Valeriana wallichii

Fig. 4: Interaction of DRD3 receptors with compounds of Asparagus racemosus

Table 4: Docking results of the compounds of Acorus calamus

Ligand Docking score/ glide g score (kcal/mol) Doner Distance in Å Interaction
4, 5, 8-trimethoxyxanthone-2-O-β-D-glucopyranosyl(1-2)-O-β-D-galactopyranoside -6.72 ARG149 2.155 ARG149(OH)…O(UNK)
2.644
THR130 2.333 THR130(OH)…O(UNK)
1.897
β-sitosterol -6.21 MET153 2.472 MET153(OH)…O(UNK)
3α, 22β, 24, 29-tetrahydroxyolean-12-en-3-O-{-β-D-arabinosyl(1-3)}-β-D-arabinopyranoside -6.06 VAL195 2.059 VAL195(OH)…O(UNK)
1α, 2β, 3γ, 19α-tetrahydroxyurs-12en-28-oicacid-28-O{-β-D-glucopyranosyl (1-2)} β- D- galactopyranoside -5.49 THR130 1.876 THR130(OH)…O(UNK)
galangin -4.74 ALA123 2.217 ALA123(OH)…O(UNK)
acoradin -4.59 ARG149 2.106 ARG149(OH)…O(UNK)
Z-3-(2,4,5-trimethoxy phenyl)-2-propenal -4.33 ARG149 2.007 ARG149(OH)…O(UNK)

Fig. 5: Interaction of DRD3 receptors with compounds of Acorus calamus.

Conclusions

DRD3 protein showed good interaction with all the above ligands. Hence, all the above ligands could be tested in-vitro and in-vivo for their efficacy in treating the disorder.