Mechanistic study of the inhibition of monoamine oxidase-B by quercetin as the potential therapeutic strategy for Parkinson’s Disease: An in silico approach

Abstract

Parkinson’s Disease (PD) is one of the most fatal neurodegenerative disease, characterized by the loss of dopaminergic neurons from the Substantia Nigra Pars Compacta (SNPC) of basal ganglia in brain. The loss of dopaminergic neurons is due to various cellular threats, among which, the oxidative stress is one of the major cause. The occurrence of oxidative stress is due to the activity of Monoamine oxidase-B, which generates reactive oxygen species (ROS) leading to DNA damage, resulting in apoptosis. On the other hand, in vivo studies revealed the potential effect of quercetin to inhibit MAO-B in brain. But the molecular mechanism of this inhibition is unclear. Here, we have studied the inhibitory mechanism of quercetin with MAO-B through in silico approach, where we have done molecular docking analysis of quercetin with MAO-B with the help of Autodock Vina and further studied the docked complex with the help of molecular dynamic simulation (MDS) studies using GROMACS 5.1.2. We found that the active sites of MAO-B are involved while interacting with quercetin, suggesting the potential inhibition of MAO-B. Moreover, the MDS study reveals that, the docked complex is more stable than free protein as the former contains more hydrogen bonds. The mechanistic understanding of the interaction between quercetin and MAO-B will generate new strategies to improve quercetin based drugs for inhibiting MAO-B and provides new scope for drug designing and discovery which potentially aids novel therapeutic strategies to combat PD.

Keywords

Parkinson’s Disease oxidative stress monoamine oxidase-B quercetin molecular docking molecular dynamic simulation

1. Introduction

Figure 1.

Role of quercetin in attenuation of the activity of MAO-B during the pathogenesis of PD.

The degeneration of the dopaminergic neurons in SNPC of basal ganglia is one of the major pathological features of PD. Loss of dopaminergic neurons causes decreased dopamine production which further causes, motor dysfunctions characterized by tremors, bradykinesia, postural abnormality, and rigid body. The pathogenesis of PD is mainly due to the deposition of $\alpha$ -Synuclein in cytoplasm which leads to the formation of lewy bodies and induces oxidative stress (Fig. 1). Oxidative stress is one of the major cause in triggering neuronal cell death via mitochondrial dysfunction and neuro-inflammation [1]. During oxidative stress, a protein linked with the catabolic pathway of dopamine called MAO-B, acts as a key molecular player in the production of ROS, which leads to the activation of different apoptotic pathways [2]. Thus, inhibiting the MAO-B overactivation could be the possible way to manage this deadly neurodegenerative disease. Recently, in vivo studies in mouse brain mitochondria have shown the pronounced inhibition of MAO-B by quercetin by modulating the levels of 5-hydroxytryptamine (5-HT). However, the MAO-B activity of mouse intestinal mitochondria is unaffected by quercetin. This suggests that quercetin is weak but safe inhibitory molecule for MAO-B [3]. There are several drugs that are currently available for PD treatment (Table 1). One of the well-known drug is Sinemet (a combination of levodopa and carbidopa) which is mainly used to manage the symptoms like slow movement, rigid body and stiffness. However, prolonged administration of sinemet has shown to initiate dyskinesia. Moreover, PD patients who administered with levodopa for 3–5 years may develop confusion, restlessness or unusual movements within a few hours of dose. On the other hand, dopamine agonists like ropinirole, pramipexole and rotigotine are showing positive effects and thereby minimized the disease symptoms. However, continuous consumption of dopamine agonists had shown to exert short term side effects viz., nausea, vomiting, dizziness, hallucinations and confusion. Thus, to avoid the side-effects of presently available anti-parkinsonian drugs, the emergence of the development of flavonoid based drugs by in silico approach is needed. Previously, there are several pharmacology based in silico studies conducted for PD. Uenaka et al. identified 47 species of FDA approved drugs by extracting candidate drugs for PD by employing GWAS-data and in silico databases. In their study “drug A” showed potential neuroprotective effect in vitro and in vivo PD model and was concluded to be a disease modifying agent for PD [4]. In our study we have analyzed the interaction of quercetin with MAO-B with the help of molecular docking, to understand the mechanistic insights of this potential inhibition and further analyzed the best docked complex to study the complex stability with the help of MDS.

Table 1

List of drugs for PD available in the market

Drug name	Side effects
Sinemet (Levodopa and Carbidopa)	Restlessness, confusion, or unusual movements within a few hours of taking the medicine.
Dopamine agonists: ropinirole (Requip), pramipexole (Mirapex), and rotigotine (Neupro).	Nausea, vomiting, dizziness, light-headedness, confusion, and hallucinations.
Amantadine (Symmetrel)	Confusion and memory problems.
Trihexyphenidyl (Artane) and benztropine (Cogentin)	Medications can harm memory and thinking, especially in older people.
Selegiline (Eldepryl, Zelapar)	Nausea, dizziness or fainting, and stomach pain.
Rasagiline (Azilect)	Headache, joint pain, indigestion, and depression

2. Materials and methods

2.1 Tools and software’s

Materials that were used for our experiments are hp computer (Intel i3 processor), Databases (PubChem, Protein Data Bank), Argus lab 4.0.1 [5], PyMOL molecular graphic system, version 1.7.4.5 (www.pymol.org), Auto Dock tools, a free graphic user interface of MGL software packages version 1.5.6rc3 [6] and Autodock Vina [6, 7], GROMACS 5.1.2 (Groningen, Netherland) [8].

2.2 Ligand and protein preparation

Quercetin (PubChem CID: 5280343) was retrieved from PubChem database (https://pubchem.ncbi.nlm.nih.gov) and is drawn using ChemSketch 11.0 [9] followed by optimization of the ligand molecule with the help of Argus lab 4.0.1 [5] and energy minimization by universal force field (UFF) (Fig. 1). The crystal structure of MAO-B (PDB ID: 1GOS) is retrieved from RCSB Protein Data Bank (www.rcsb.org/pdb/home/home.do). The rotatable bonds in the protein were treated as non-rotatable as well as the bonds in the ligands are made flexible for the efficient docking. The Gasteiger charge calculation method is employed followed by adding of partial charges to the ligand atoms prior to docking. By using PyMOL molecular graphic system, version 1.7.4.5 (www.pymol.org), removal of all water molecules and hetero atoms from the crystal structure is done.

2.3 Prediction of binding site residues

Identification of the binding site residues MAO-B is analyzed using SPRITE and ASSAM [10] where the PDB structure of MAO-B is uploaded.

2.4 Molecular docking study

Docking experiment have been done with quercetin on MAO-B with the help of Autodock Vina. Parameters of the grid box were set with the help of Auto Dock tools, a free graphic user interface of MGL software packages (version 1.5.6rc3) [6, 7]. The process of docking used the Lamarckian Genetic Algorithm during docking for investigate the best conformational space for the ligand with a population size of 150 individuals. The maximum numbers of generation and evaluation are set at 27,000 and 2,500,000, respectively. Other parameters are set as default.

2.5 Molecular dynamic simulation

MDS has been performed using MAO-B as well as ligand-protein complex with the help of GROMACS 5.1.2 package (Groningen, Netherland) [8] along with GROMOS96 force field which is used during MDS [11]. For MDS, the conformation of the ligand-protein complex having lowest binding energy was used. Here in our study, quercetin-MAO-B has shown the perfect docking as it contains more conformation with least binding energy. GROMACS program was used to create the topology parameters for the protein (MAO-B). Dundee PRODRG server (Scotland, UK) [12] was used to build the topology parameters of the ligand (quercetin). The MD production was subjected to run at a 300 K temperature and 1 bar pressure for 5 ns.

3. Results

The molecular docking of quercetin with MAO-B was done and the interacting amino acids were visualized on Pymol software (www.pymol.org). Moreover, the mean binding energy (MBE) and inhibitory constant (IC) of quercetin-MAO-B interaction was also analyzed followed by the comparison of mean binding energy (MBE), standard deviation (SD) and inhibitory constant (IC) (Table 2). However, at RMS tolerance of 4Å, the cluster was chosen on the basis of low binding energy (BE) and highest number of conformations. This cluster has been chosen to study the binding interaction of quercetin and MAO-B.

3.1 Docking of quercetin with MAO-B

In our results, we found that, quercetin interacts with MAO-B through H-bonds, involving the amino acids Gly13, Ser15, Arg36, Arg42 and Thr43 (Fig. 2). Interestingly, these amino acids are associated with active site of MAO-B which is crucial for the catalytic activity of MAO-B, suggesting the successful inhibition of MAO-B by quercetin. Moreover, the interaction between quercetin and MAO-B exhibits lower MBE of $-$ 9.75 kcal/mol with low inhibitory constant of 54 $\mu$ M, indicating the potent inhibition of MAO-B by quercetin.

Table 2
Predicted interacting amino acids of the protein with the ligand molecules were shown along with mean binding energy (MBE) and inhibitory constant (IC)

MAO-B
Quercetin	Interacting amino acids	MBE (kcal/mol) $\pm$ SD	IC ( $\mu$ M)
	Gly13, Ser15, Arg36, Arg42 and Thr43	$-$ 9.75 $\pm$ 0.17	54.48

Figure 2.

Quercetin is colored in green and red stick; interacting amino acids are in blue stick; (A) Region of the MAO-B bound with quercetin. (B) Interaction amino acids of MAO-B with quercetin through H-bonds.

3.2 MDS of quercetin with MAO-A

MDS results were used to analyze the stability of quercetin-MAO-B complex, which provides the scope for drug designing and discovery. The RMSD plot obtained from the simulation studies (Fig. 3A) compares the stability of the free protein (MAO-B) and quercetin-MAO complex, where it was observed that both MAO-B and quercetin-MAO-B complex attain stability after a simulation time period of 1.5 ns. This prolonged stabilization of quercetin-MAO-B complex indicates the possibility of the ligand-protein complex formation, which favourable for the inhibition. Moreover, hydrogen bond plot compares the number of H-bonds existing in free protein as well as in ligand-protein complex. In our results, the hydrogen bond plot (Fig. 3B) shows that, at 2 ns to 2.5 ns the quercetin-MAO-B complex attains more stability than MAO-B by forming more hydrogen bonds. This result also reflects the nature of the ligand-protein complex as the formation of more hydrogen bond indicates the increased stability.

Figure 3.

MDS results of MAO-B and quercetin-MAO-B complex. Color scheme: black line indicates MAO-B and red line indicates quercetin-MAO-B complex. (A) RMSD plot of protein and ligand-protein complex. Note that, after 1.5 ns of simulation time, the stability of the protein and ligand-protein complex is achieved. (B) Hydrogen bond plot of protein and ligand-protein complex. Note that, the stability of the ligand-protein complex increases at 2 ns to 2.5 ns by forming more hydrogen bonds.

4. Discussion

Oxidative stress is one of the major cause in the pathogenesis of PD, where MAO-B acts as the main factor as its activity leads to the generation of ROS [1]. As per the previous studies conducted, the lewy body formation due to the aggregation of mutated and misfolded $\alpha$ -synuclein led to the over activation of MAO-B. Overactivation of MAO-B in turn elicits the uncontrolled generation of ROS, which activates the apoptotic precursors resulting in neuronal cell death. This suggest that, inhibiting the activity of MAO-B provides the possible therapeutic strategy to combat this deadly disease. In our study, quercetin has shown to inhibit MAO-B by binding at the active site with low binding energy and inhibitory constant. Our understanding is mainly focused on the mechanism of the inhibitory action of quercetin on MAO-B, which has already been proven with animal models. Moreover, quercetin has also shown to inhibit $\alpha$ -Synucleinfibrillization[13, 14] and iNOS activity [15, 16], which further indicates the potency of quercetin based drugs in the treatment of PD. Again, it is important for a ligand-protein complex to attain stability in order to exhibit its action favorably. To understand the nature of stability of our best docked complex, we have done MDS studies. In our MDS studies, we found that quercetin-MAO-B complex is much stable compared to protein alone. The stability of the complex is due to the formation of more hydrogen bonds which further indicates the strong binding of quercetin with MAO-B. Thus, our results provide a clear understanding on mechanism of the inhibition of MAO-B by quercetin.

5. Conclusion

From our studies, we have analyzed and understood the structural mechanism of the inhibition of MAO-B by quercetin with the help of in silico studies. However, Quantitative Structure-Activity Relationship (QSAR) studies still needs to be done in order to generate the pharmacophore, to design quercetin based drugs, which will potentially combat PD in future.

Footnotes

Acknowledgments

We thank Vellore Institute of Technology (VIT), Vellore for providing the necessary space for conducting our work.

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