Favorable binding of Quercetin to α-Synuclein as potential target in Parkinson disease: An Insilico approach

 

Himadri Shekhaar Baul¹, Muniyan Rajiniraja²*

¹Department of Biomedical Science, School of Bio-Sciences and Technology, VIT University, Vellore- 632014, Tamil Nadu, India

²Department of Biotechnology, School of Bio-Sciences and Technology, VIT University, Vellore- 632014, Tamil Nadu, India

ABSTRACT:

The aim of the research is to identify possible flavonoid for the effective docking into α-Synuclein in Parkinson’s disease (PD) computationally.  Flavonoids are evolving as the potential cure for the PD. α-Synuclein is one of the potential drug target involving in the death of dopaminergic neurons of Substantia Nigra Pars Compacta (SNPC) of basal ganglia in PD. In our study, molecular docking analysis were carried out on flavonoids like quercetin, epigallocatechin gallate (EGCG) and acacetin to investigate their inhibitory role and binding capability with α-Synuclein. Molecular docking experiments were performed using AutodockVina program. The Protein-Ligand interaction analysis result showed that the favorable interaction with only on quercetin and not with others. This was comparatively a strong ligand to show significant interactions with α-Synuclein with lowest binding energy. The cluster analysis of the docked conformations out of 100 runs, quercetin shows comparable higher numbers (31 conformations) with α-Synuclein. Molecular interaction of quercetin and its analogs with the potential drug target of Parkinson’s disease provides a wide scope for drug designing to combat Parkinson’s disease.

 

 

INTRODUCTION:

Neuronal degeneration from SNPC on basal ganglia is one the principle cause of PD¹. Consequently, there is loss of dopamine production leading to severe motor symptoms. As PD is an age onset disorder, the rectification is not possible in early age². The deposition of misfolded a-synuclein is the central cause for neuronal degeneration. Further studies proved that other factors such as mutations³, dysfuntion of mitochondria⁴, neuroinflammation⁵, and increased production of reaction oxygen species (ROS) ⁶ are also linked with this disease.

 

Mutation of SNCA gene leads to the production of abnormal a-synuclein, which fails to fold properly and results in misfolded protein. This misfolded protein lead to lewy body formation which induces mitochondrial stress and neuroinflammation resulting in cell death. So, targeting the misfolded a-synuclein will provide a possible cure for PD.

 

The pre-existing therapy for parkinson’s disease with L-DOPA has drawbacks, leading to dyskinesia⁷. This demerit has led to the emergence of the development of new drugs for PD that can target a-synuclein. Phytochemicals like flavonoids has shown some positive effect on neuronal survivality both in vivo and in vitro by different mechanisms. Flavonoids such as quercetin⁸and EGCG⁹ has shown pronounced effects on inhibiting a-synuclein fibrillation which actually lead to neuro protection. Our study is mainly concerned with docking analysis of the flavonoids such as EGCG, acacetin and quercetin with a-synuclein in order to know the computationally predicted interacting amino acids which may lead to prevention of a-synuclein fibrillation.

 

MATERIALS AND METHODS:

2.1  Tools / software’s:

Tools used for our studies are Argus lab 4.0.1¹⁰, Pymol1.7.4.5 visualization tool (www.pymol.org), Auto dock docking tools with aMGL software packages version 1.5.6rc3¹¹ and Autodock Vina¹².

 

2.2   Ligand preparation:

The ligands were acquired from PubChem database (https://pubchem.ncbi.nlm.nih.gov) viz. epigallocatechin gallate (EGCG) (PubChem CID: 65064), acacetin (PubChem CID: 5280442) and quercetin (PubChem CID: 5280343) and were drawn using ChemSketch 11.0¹³. ArgusLab 4.0.1 was used to optimize all the ligand molecules with universal force field (UFF)(Fig. 2).

 

2.3  Protein preparation:

The protein three dimensional (3D) structure such as α-synuclein (PDB ID: 1XQ8) was retrieved from Protein Databank (www.rcsb.org/pdb/home/home.do). Removal of all water molecules and hetero atoms from the crystal structures was done using Pymol software. The Gasteiger charge calculation method was employed followed by adding of partial charges to the ligand atoms prior to docking¹⁴ using Autodock vena. Finally, the .pdbqtfile of the protein is employed for our docking experiment.]

 

2.4   Identification of binding site residues:

The binding site residues for α-synuclein¹⁵were identified from the literatures associated for the crystal 3D structure of PDB (Table 1).

 

Table 1. Binding site residues of the protein targets along with the grid box parameters were shown.

Protein	Binding site residues	Centre grid box (points)	Grid size (points)
a-synuclein	Leu38, Tyr39, Val40, Lys43 and Lys45	238.739 x 77.814 x 10.589	80 x 80 x 80

2.5  Grid box preparation and docking:

Docking of EGCG, acacetin and quercetin on α-synuclein were performed with the help of AutodockVina. The grid box (Table 1) was set with the help of Auto Dock tools. The Lamarckian Genetic Algorithm was used during docking for the investigation of best conformational space with a population size of 150 individuals. The maximum numbers of generation and evaluation were set at 27,000 and 2,500,000, respectively for 100 runs. Other default parameters were set to run docking process¹⁶.

 

 

RESULTS AND DISCUSSION:

Pymol (www.pymol.org) is used for the visualization of the interaction between a-synuclein and flavonoids and their analysis was done (Table 2).

 

Table 2. Predicted interacting amino acids of the protein with the ligand molecules were shown

Ligands Proteins	EGCG	Acacetin	Quercetin
a-Synuclein	Glu35, Gly36, Lys45, Lys43, Leu38 and Val40	Lys32, Val40, Lys43 and Lys45	Lys43, Lys45, Val40 and Lys32

 

Amino acid residues, which are involved H-bond interaction with the respective ligands, were indicated in bold while others are involved in electrostatic interactions.

 

Binding energies (BE) (Fig. 1A), Mean Binding Energy (MBE), Standard Deviation (SD) and Inhibitory Constant (IC) (Table 3) of the flavonoids with a-synuclein was compared. It has been found that BE and MBE is lower in case of EGCG with a-synuclein followed by that of quercetin and acacetin. IC values revealed that quercetin can inhibit a-synuclein more potentially than acacetin and EGCG due to its low value.

 

 

Table 3. Mean binding energy (MBE) ± standard deviation (SD) and inhibitory constant (IC) of different docked protein targets with flavonoids were shown

Flavonoids	EGCG		Acacetin		Quercetin
Parameters Protein Targets	MBE (kcal/mol) ± SD	IC (µM)	MBE (kcal/mol) ± SD	IC (µM)	MBE (kcal/mol)  ± SD	IC (µM)
a-Synuclein	-8.92 ± 0.56	118.13	-5.16 ± 0.29	59.22	-6.82 ± 0.55	4.71

 

The number of conformations in the selected cluster (maximum in numbers) has been compared on the basis of each ligand-protein interactions (Fig. 1B). However, at RMS tolerance of 4Å, the cluster was chosen on the basis of low B.E and highest number of conformations in cluster. Quercetin has the highest number of conformation compared to that of acacetin and EGCG.

 

 

Figure 1:

 

A) Comparative binding energies of best docked conformation and A) number of conformations in the cluster analysis with the tolerance of 4Å between α-Synuclein with quercetin, acacetin and EGCG are shown.

 

3.1 Docking of flavonoids with protein target:s

The interaction between the a-synuclein and the flavonoids namely quercetin, acacetin and EGCG (Figs. 2A, 2B, 2C respectively) takes place in the loop and adjacent a-helix, which suggests that, these flavonoids could possibly arrest the mobilization of a-synuclein, blocking its abnormal function and aggregation and led the misfolded a-synuclein towards degradation, preventing neurodegeneration.

 

 

 

Figure 2.

 

The ligand-protein interaction between flavonoids and a-synuclein visualized using Pymol. Colour scheme: Flavonoids are indicated in green sticks and interacting amino acids are indicated in blue sticks. Hydrogen bond interactions of a-synuclein with (A) Quercetin (B) Acacetin and (C) EGCG.

 

To find out whether the flavonoids (Quercetin, Acacetin and EGCG) has any inhibitory effect on a-synuclein, is our main aim. Our docking study shows the binding of flavonoids with α-synuclein at the major binding sites comprising the loop, which is crucial for any protein to exhibit its dynamic nature. Moreover, quercetin had shown the favorable binding with a-synuclein. a-synuclein was the main target for PD as it initiates various other processes leading to cell death. Development of drugs targeting α-synuclein aggregation was a challenge. However, the α-synuclein can be prevented on the basic level by down-regulating its synthesis from SNCA gene. It has been seen that up-regulating the parkin expression can prevent α-synuclein pathology in cell resulting in reduced cell death in animal models¹⁷. Moreover, up-regulating Hsp70¹⁸ and activating lysosomal degradation¹⁹ are also the potential targets for reducing α-synuclein aggregation. However, direct blocking of α-synuclein aggregation could be the possible therapeutic target. Another source for the oxidative stress is mitochondrial dysfunction which is also found to be initiated by a-synuclein²⁰. Moreover, a-synuclein dysfunction also leads to neuroinflammation²¹. Overall, targeting a-synuclein with flavonoids will potentially minimizes the neuronal cell death in PD.

 

CONCLUSION:

Docking studies are crucial for drug designing and drug discovery. Our findings suggest that quercetin can be a basic resource for drug designing and drug discovery in PD. Further work has to be done to check whether quercetin and its derivative compounds are targeting the a-synuclein in vitro as well as in vivo.

 

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Received on 08.08.2017         Modified on 24.08.2017

Accepted on 13.09.2017      © RJPT All right reserved