Molecular Docking Studies of Selected Flavonoids on Inducible Nitric Oxide Synthase (INOS) in Parkinson’s Disease

 

H. S. Baul¹, M. 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:

Objectives: The urgency to discover and develop drugs combating Parkinson’s Disease is immense. The loss of dopaminergic neurons in Substantia Nigra Pars Compacta (SNPC) has been the main cause of PD, which occurs due to oxidative stress and neuroinflammation. Inducible Nitric Oxide Synthase (iNOS) has shown to be associated with the activation of microglia promoting neuro inflammation and oxidative stress. In our study, molecular docking analysis was carried out on flavonoids like epigallocatechin gallate (EGCG), acacetin and quercetin obtained from different plant sources to investigate their inhibitory potential and binding capability with iNOS. Molecular docking experiments were performed using Autodock Vina program. The Protein-Ligand interaction analysis showed the favorable interactions flavonoids (EGCG, acacetin and quercetin) with iNOS efficiently, among which quercetin is comparatively a strong ligand to show remarkable interactions. A potential molecular interaction of flavonoids with iNOS provides a wide scope for drug designing which aids therapeutic strategy to combat Parkinson’s disease.

 

 

 

INTRODUCTION:

The death of dopaminergic neurons from SNPC of basal ganglia is the central cause of PD. It results in mild to severe motor dysfunctions [1]. Other than motor symptoms that are exhibited prominently, there are non-motor symptoms, which are undertreated and under-recognized, such as slow motor transit constipation [2]. In early stage of life, PD cannot be detected prominently as it is an age onset neurodegenerative disorder [3]. The most widely accepted cause of neuro-degeneration is the inclusions of misfolded a-synuclein resulting in formation of lewybodies [4].

 

 

 

However, recent researchers have found that various other factors like mitochondrial stress [4], elevated reactive oxygen species [5] and inflammatory process [6] have also a prominent role in PD pathogenesis.

 

Till date, the most understandable cytotoxic mechanisms initiated by proinflammatory cytokines in PD areiNOS activation that facilitates the nitric oxide (NO) formation which produces neurotoxity by signaling mechanisms [7]. Studies on parkinsonian patients reveal the increased density of iNOS expressing glial cells in SNPC compared to the control patients [8]. Besides, iNOS has been linked with microglial activation inducing inflammatory response resulting in neural cell death [9]. Thus iNOS, can be a potential target for PD.

 

The dopaminergic therapy which is used to treat PD has many drawbacks because after some years of therapy, the unresponsiveness of L-dopa prevails which can lead to dyskinesia [10]. The drawbacks have encouraged developing new possible therapies that can combat PD reducing neural cell death particularly targeting neuro-inflammation by inhibiting iNOS. However, flavonoid like EGCG [11], acacetin [12] and quercetin [13] has also shown to modulate the iNOSexpression in different experimental studies. The main aim of our study is to dock the flavonoids like EGCG, acacetin and quercetinon iNOS and analyze the inhibitory potential of three flavonoids to iNOS computationally which can further lead the way of drug discovery for PD.

 

MATERIALS AND METHODS:

Tools and software’s:

Materials which are used for carrying out the experiments arecomputer system (Intel i3 processor); Databases (PubChem, Protein Data Bank), Argus lab 4.0.1 [14], Pymol molecular graphic system, version 1.7.4.5 (www.pymol.org), Auto Dock tools [15], a free graphic user interface of MGL software packages version 1.5.6rc3 and Autodock Vina [16].

 

Ligand preparation:

The chosen ligands namely EGCG (PubChem CID: 65064), acacetin (PubChem CID: 5280442) and quercetin (PubChem CID: 5280343) is retrieved from PubChem database (https://pubchem.ncbi.nlm.nih.gov) and ChemSketch 11.0 [17] is used for drawing the ligands. Ligand molecule optimization has been done with the help of Argus lab 4.0.1 [14] followed by energy minimization by universal force field (UFF). The file has been converted to .pdbqt format and taken to perform molecular docking.

 

Protein preparation:

The crystal structures of iNOS (PDB ID: 2NSI) was retrieved from RCSB Protein Data Bank (www.rcsb.org/pdb/home/home.do). The protein wasoptimized using Autodock tools [15]. Water molecules and hetero atoms were removed from the crystal structure using PyMOL molecular graphic system, version 1.7.4.5 (www.pymol.org).The file has been converted to .pdbqt format and taken to perform molecular docking.

 

Grid box preparation and docking:

Molecular docking has been carried out with EGCG, acacetin and quercetin on iNOS with the help of AutodockVina [18]. Grid boxparameters (Table 1) were madefor docking and Lamarckian Genetic Algorithm was usedto investigate the best conformational space for the ligand with a population size of 150 individuals during docking. All the parameters of docking were set as default.

 

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)
iNOS	His477, Phe 476, Asp382, Trp461, Glu479, Glu377, Ile462, Trp463, Cys200, Trp372, Phe369, Trp194, Trp346, Tyr373, Arg388, Arg266 and Gln263	238.739 x 77.814 x 10.589	80 x 80 x 80

 

RESULTS AND DISCUSSION:

The molecular interactions between the flavonoids and Inos (Table 2) were obtainedand Pymol software (www.pymol.org) was used for visualization.

 

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

Ligands Protein	EGCG	Acacetin	Quercetin
iNOS	Trp461, Met480, Asn482 and Ile462	Ser118, Ile119, Ser124 and Met480	Ser118, Trp461, Ser124, Thr109, Ile119 and Asn482

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

 

Comparison of binding energy (BE) (Fig. 1a), mean binding energy (MBE), standard deviation (SD), inhibitory constant (IC) (Table3) and number of conformations (Fig. 1b)of each interaction between flavonoids and iNOS had been done.It was seen that acacetin have the highest number of conformations with lowest BE compared to that of EGCG and quercetin. Quercetin showed the possible inhibition of iNOS as it has the lowest inhibitory constant values compared to that of EGCG and acacetin.

 

 

 

 

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

Flavonoids	EGCG		Acacetin		Quercetin
Parameters Protein	MBE (kcal/mol)±SD	IC (µM)	MBE (kcal/mol)±SD	IC (µM)	MBE (kcal/mol)±SD	IC (µM)
iNOS	-7.56±0.67	976.61	-7.21 ± 0.41	2.24	-7.47 ± 0.47	1.52

 

 

 

 

 

Fig. 1: a) Comparative binding energies of best docked conformation and b) number of conformations in the cluster analysis with the tolerance of 4Å between iNOS withEGCG,acacetin and quercetin are shown.

 

Docking of flavonoids with iNOS:

Quercetin has shown strong interactions with iNOS (Fig. 2c) as it is interacting with two different loops and one ß-pleated sheet. This binding could possibly block the function of iNOS and the production of nitric oxide will be hampered. Moreover, the production of Reactive Oxygen Species (ROS) and lipid peroxidation inside the dopaminergic neurons will be prevented resulting in neuroprotection. EGCG and acacetin also shown to bind with loop portion of iNOS (Fig. 2a and 2b), indicating a possible inhibition of iNOS leading to neuroprotection.

 

Flavonoids can be found in variety of medicinal plants [19]. Quercetin has the significant role for curing the various diseases like, antianaphylactic, antiarthritic, analgesic, allergenic and antivenomous and others [20]. iNOS, which is linked with the synthesis of NO has been possibly inhibited by quercetin with lowest IC value. iNOS has a major role in the pathogenesis of PD by inducing oxidative stress and neuroinflammation by activating the proinflammatory cytokines in dopaminergic neurons. Mechanisms that can potentially activate NO-induced dopaminergic toxicity are still to be understood clearly. Some of the mechanisms have been proposed based on the experimental findings. Firstly, nitrosylation induced by NO on proteins, can alter the protein functions [21]. This mechanism has been evidenced in mechanism in the observation where there is increased 3-nitrotyrosine when immunostaining in Lewy bodies of PD patients [22]. Secondly, evidence of the disruption of iron homeostasis by NO is been observed respectively by up and downregulating the expression of ferritin and transferrin receptor [23] leading to high iron concentration in SNPC resulting in generation of toxic free radicals by Fenton reaction. However, silencing of iNOS has also shown to be neuroprotective in 6-OHDA models of PD [24]. It has been seen that in basal ganglia of PD patients, altered NO signaling occurs [25]. Hence, targeting iNOS could be the potential therapy for PD. The inhibition of the iNOS was our main concern. In our study, all the flavonoids have shown possible interactions with iNOS, which still remain to be established experimentally.

 

 

Fig.2: Docked complex was visualized using Pymol software. Colour scheme: Flavonoids are indicated in green sticks and interacting amino acids are indicated in blue sticks. Hydrogen bond interaction of iNOS with (a) EGCG, (b) Acacetin and (c) Quercetin

 

CONCLUSIONS:

Flavonoids and its analogues are efficient in treating various diseases. Quercetin is one of the flavonoid was targeting majority of proteins in various diseases. In our docking study, the possible binding of quercetin with iNOS can lead to act as a potential flavonoid candidate for the Parkinson’s disease.

 

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Accepted on 12.05.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(8): 3685-3688.