In silico Molecular docking of Luteolin from Momordica charantia for dementia in Alzheimer’s disease
Tamilanban T*, Naveen Kumar V, Narayanan J, Prathusa S, Dhivya N, Manasa K
Department of Pharmacology, SRM College of Pharmacy, SRM Institute of Science and Technology, Kattankulathur-603203, Chennai, Tamailnadu, India.
*Corresponding Author E-mail: veera.srm@gmail.com
ABSTRACT:
Objective: In the present study, luteolin was isolated from Momordica charantia and characterized by IR and NMR spectroscopy, undergone in silico docking to study its anti-Alzheimer’s role. In silico molecular docking is a computational system employed to predict the interaction vitality between two particles and the best introduction of ligand that shapes a complex with general least vitality. Methods: In this Protein Ligand Docking, we have used HEX 8.0.0 as a Docking Software. The receptors (PDB ID: 4PQE, 1P0I, 5O3L, 1J1C, 3NYJ, 2FJZ) were retrieved from the RCSB Protein Data Bank (PDB) Database while the ligand structure was retrieved as mol format from ChEBI database and it was converted as .pdb format using Chimera 1.12 and docking was done according to the specified parameters. Results: The molecular docking process for the luteolin was performed with the receptors and the E-value was determined. Further, the result of the binding site of ligand with the target proteins was interpreted by using Chimera 1.12 software. Conclusion: Luteolin was proved to have good affinity for target protein and it is having multitarget activity that is responsible for AD.
KEYWORDS: Momordica charantia, Luteolin, In silico molecular Docking, PDB database, ChEBI database, Hex 8.0.0 Software.
INTRODUCTION:
Alzheimer’s disease (AD) is a condition of neurodegeneration with amnesia like cognitive disability as primary symptoms and progressed by memory blackout, plaque of beta amyloid and formation of neurofibrillary tangles1,2. A senile plaques major component, the amyloid-beta will activate caspases by activating the death receptors of cell surface. In this manner, it may bring about to pathology by assisting tau hyper-phosphorylation, distorting mitochondria function, and provoking calcium dysfunction. Four genes namely APP, PS1, PS2 and ApoE are mutated and are linked to the enhanced production of Abeta42 in familial AD3,4. Also, susceptibility to endoplasmic reticulum stress formed due to the flavin adenine dinucleotide (FAD)-linked PS1 mutation5.
Figure 1- M. charantia fruit
Figure 2- Structure of Luteolin
Luteolin is chemically 3′,4′,5,7-tetrahydroxyflavone, a flavonoid from Momordica charantia with anti-inflammatory, anti-tumor, antioxidant and neuroprotection activity6. It is present in Petroselinum crispum, Thymus vulgaris, Mentha balsamea, Ocimum basilicum, Apium graveolens and Cynara scolymusin large amounts7. Luteolin has anti-inflammatory properties as it inhibits lipopolysaccharide-induced pro-inflammatory cytokines production in the epithelial cells of intestine, bone marrow-derived dendritic cells in mouse, inhibits the lipopolysaccharide (LPS)-induced production of TNF-α and nitric oxide (NO) in an activated macrophage-like cell line8,9. Also, luteolin was studied for its anti-amnesic effects against beta amyloid protein induced toxicity10,11. Luteolin obtained from Lonicera japonica, a Chinese herb has shown antipyretic and detoxifying actions12. This study deals with employing and assessing the bioactivity of the luteolin compound isolated from M. charantia in neuroprotection by performing in vitro assays and anti-oxidant studies. Previous studies of Momordica species had revealed that luteolin is abundant in the plant parts like fruits, seeds and the morphological stages like callus and multiple shoots. In this study, by using the whole ripe fruits of MC, the luteolin was isolated.
MATERIALS AND METHODS:
Collection of plant material:
Momordica charantia ripen fruits were collected from a local market, authenticated (Voucher specimen number-PARC/2017/2894). The MC fruits were air-dried and cut into small pieces, further sifted to a coarse powder and stored in air-tight containers until use.
Extraction method and Isolation process13,14
The Momordica charantia coarse powder was initially refluxed for 24 hours with methanol (80%,v/v) and further subjected to sequential extraction with the ethyl acetate and 7% Suphuric acid (2 hours) separately. The obtained filtrate was further extracted for three times using ethyl acetate, obtained three layers of ethyl acetate were re-dissolved with ether (2-5 ml) in order to obtain the flavanoid, luteolin11. Analysis of luteolin was performed by TLC with benzene:acetic acid:water at 125:72:3, v/v) by spraying FeCl3 at 1100C. The compounds was detected by UV Lamp on TLC at 426 and 440 nm.
Characterization of Luteolin:
Solubility15
Luteolin was soluble in alkaline solutions and methanol. When water is added to the luteolin, it was slightly soluble.
Determination of Melting point16
Melting point was determined by using a Mel-Temp Capillary apparatus accoutered with the digitated thermometer. Luteolin sample was heated by rising the temperature gradually and this helps to observe the accurate melting point.
Chemical Test17
Flavonoids have phenolic or methoxyl derivatives, were dissolved in alkalis and give yellow solution and turned colourless when acid was added. The intensity of their yellow colour increases with the rise in pH and number of hydroxyl groups.
FT-IR of Luteolin:
The IR spectra of isolated chemical constituent were recorded using a FT-IR (Vertex 70, Bruker, Rheinstetten, Germany). The dried luteolin was completely mixed with potassium bromide powder and pressed the pellet into a size of 1 mm, determined by Fourier Transform-Infra Red spectrometer. The recorded spectrum was in the 500–4000cm-1 wavenumber range.
1H-NMR of Luteolin:
1H-NMR analysis of isolated chemical constituent was obtained from NMR Shift DB 300MHz NMR SPECTROMETER using Chloroform-D1 (CDCl3).
Molecular Docking Studies:
The molecular interactions between the target proteins of biological interest and the lead molecule by molecular docking studies had become of much significance in the drug discovery field18,19. The binding interaction of luteolin with the selected targets has not been revealed till now. Therefore, the interaction studies of luteolin with the selected targets are proposed in this work.
Bioinformatics tools employed in this study:
RCSB PDB (Protein Data Bank) database - The pdb file format is a text like file format provides the protein and nucleic acid structures description and annotation, describes the molecular three-dimensional structures20,21.
ChEBI database:
Chemical Entities of Biological Interest is a 'small molecular entities' dictionary which is freely available22.
Hex 8.0.0 software - Hex is a program of molecular graphics interaction for displaying and calculating viable protein and DNA molecule pairs docking modes. It can also calculate the protein-ligand docking by implementing the 3D shapes of the molecules, if the ligand could superimpose the molecule pairs23.
Chimera 1.12 software (Molecular Modeling System) - UCSF Chimera is a program, highly extensible for interactive analysis and visualization of structure of molecules including docking results24.
Preparation of target proteins:
The protein targets were selected and downloaded as. pdb file format from RCSB PDB (Protein Data Bank database) for the docking process. The PDB ID for selected protein targets are 4PQE (Human Acetylcholinesterase crystal structure), 1P0I (Human butyrylcholinesterase crystal structure), 5O3L (Alzheimer's disease brain comprising paired helical filament), 1J1C (tau protein kinase I with ADP binary complex structure of human), 3NYJ (APP E2 domain structure analysis) and 2FJZ (Alzheimer's APP Cu binding domain (residues 133 to 189) in the form of 'small unit cell', structure like metal-free). The obtained protein structure of 4PQE (length: 543 amino acids), 1P0I (length: 529 amino acids), 5O3L (length: 730 amino acids), 1J1C (length: 840 amino acids), 3NYJ (length: 207 amino acids) and 2FJZ (length: 59 amino acids) had been determined using X-ray diffraction at a resolution of 2.9 Å, 2 Å, 3.4 Å, 2.1 Å, 3.2 Å and 1.61 Å respectively25-30.
Preparation of ligand molecule:
The Luteolin (isolated chemical constituent) structure was downloaded as .mol file from ChEBI database (ChEBI ID-CHEBI: 15864). Further that .mol file was converted the format as .pdb file by the usage of Chimera 1.12 software which is used for docking analysis.
Docking analysis:
The ligand structure was optimized and it was docked with the targeted proteins such as PDB ID 4PQE, 1P0I, 5O3L, 1J1C, 3NYJ and 2FJZ using the software called Hex 8.0.0. The default parameters were employed for the docking process. The docked complexes were visualized in Chimera 1.12 software to discover the interaction of luteolin with the protein targets 31.
RESULTS AND DISCUSSION:
The flavone, luteolin was extracted from the ripened fruits of Momordica charantia; obtained as light yellow crystalline substance with a melting point of 3300 C (capillary method). Luteolin turns colourless upon addition of acids, whereas changes to pale yellow colour by adding alkaline solutions.
The FT-IR of the isolated flavonoid (figure 3) reveals that the presence of different functional groups like in the peak of absorption at 1164 cm-1 (C–O–C stretching), 1655 cm-1 (C=O stretching and C=C stretching).
Figure 3- Fourier Transform-Infra Red Spectrum of Luteolin
1H-NMR spectral analysis reveals Chemical shift value at δ 6.26 (1H,d,J=2.0 Hz), 6.40 (1H,s), 6.43 (1H,d,J= 2.0Hz), 6.76 (1H,dd,J=8.4,0.5 Hz), 7.50 (1H,dd,J=1.7,0.5 Hz), 7.75(1H,dd,J=8.4,1.7 Hz). (Figure 4)
Figure 4- 1H NMR spectral representation of luteolin
In silico Molecular Docking Results:
A higher negative E-value represents the active binding between the ligand molecule and the target protein32,33,34. The calculated E-value of luteolin with different targets is given in Table 1.
Table 1-Interaction energies between luteolin and targeted proteins using software Hex 8.0.0
|
PDB ID for Target Proteins |
E-value(kcal mol-1) |
|
4PQE |
-193.7 |
|
1P0I |
-199.1 |
|
5O3L |
-209.4 |
|
1J1C |
-187.2 |
|
3NYJ |
-176.4 |
|
2FJZ |
-169.8 |
Docking of Human Acetylcholinesterase (PDB ID: 4PQE) with luteolin:
Acetylcholinesterase is a family of hydrolase enzyme 25. The docked complexes of luteolin with 4PQE having interaction of energy (E-value) was found to be -193.7 kcal mol-1. The figure 5 shows that the two binding interactions of residue Asparagine 233 in 4PQE with the positions of O5 and C14 in the luteolin molecule having 1.685 Å and 2.347 Å bond length respectively and also there are three binding interactions of residue Tryptophan 532 in 4PQE with the isolated molecule position of O4, C2 and C8 and having 2.310 Å, 2.653 Å and 2.764 Å and bond length respectively. The yellow coloured line shown in the following images which represents the bond formation between the ligand molecule and the target protein.
Docking of Human butyryl cholinesterase (PDB ID: 1P0I) with luteolin:
Butyrylcholinesterase is also comes under a family of hydrolase enzyme26. The docked complexes of luteolin with 1P0I having E-value was found to be -199.1 kcal mol-1 which was little more active bounded when compared with 4PQE. The result shows the total 3 contact points of Leucine 403, Cysteine 400 and Threonine 523 residues in 1P0I with C11, O5 and O6 flavone molecule position respectively having 2.869 Å, 2.239 Å and 1.733 Å bond length respectively.
Docking of Alzheimer's disease brain having paired form of helical filament structure (PDB ID:5O3L) with luteolin:
5O3L is a structural tau protein with 306-378 residues with cross -ᵝ/ᵝ-helix structure and defines the tau aggregation seed27. The docked complexes of luteolin with 5O3L having E-value was found to be -209.4 kcal mol-1 which was the most active bounded when compared with all the other target proteins such as 4PQE, 1P0I, 1J1C, 3NYJ and 2FJZ. The result shows that the two binding interactions of Glutamate 338 residue in the target 5O3L with the positions C4 and C1 of the luteolin molecule having 2.454 Å and 2.284 Å bond length respectively and also there are another two binding interactions of residue Valine 339 in 5O3L with the positions C14 and O5 of the ligand molecule having 2.531 Å and 1.792 Å bond length respectively.
Docking of Tau protein kinase I with ADP binary complex structure for human (PDB ID: 1J1C) with luteolin:
1J1C is classified under transferase. TPK I (Human tau protein kinase I or GSK3 beta (glycogen synthase-kinase 3 beta) is a protein kinase of serine or threonine that participated in the Alzheimer’s disease28. The docked complexes of luteolin with 1J1C having E-value was found to be -187.2 kcal mol-1. The result shows the three clash points of Arginine 720 residue in 1J1C with the position of ligand molecule C2, C8 and O4 having 2.688 Å, 2.902 Å and 2.574 Å bond length respectively.
Docking of APP in Alzheimer’s disease brain E2 domain (PDB ID: 3NYJ) with luteolin:
APP is linked to type 1 AD and its oligomeric protein structure having a major role in generation of amyloid beta peptides. Studies proved that APP forms dimers in the cell by three domain regions. 3NYJ is classified like a protein fibril29. The docked complexes of luteolin with the target protein 3NYJ having interaction energies (E-value) was found to be -176.4 kcal mol-1. The result shows that the oneclash point of residue Arginine 441 in 3NYJ with the position of ligand molecule C1 has 1.889 Å bond length and also there is a one contact point of residue Histidine 438 in 3NYJ with the position of luteolin C2 having 2.885 Å bond length.
Docking of Alzheimer's APP Cu-binding domain (residues 133 to 189) in the form of 'small unit cell', which is metal-free (PDB ID: 2FJZ) with luteolin:
2FJZ is classified like metal binding protein. APP can act as a metalloprotein. APP can modulate Cu-transport via Cu-binding domain (CuBD), there by depletes Amyloid beta levels, indicating that a copper mimetic may have therapeutic significance30. The docked complex of luteolin with 2FJZ having energy of interaction (E-value) was found to be -169.8 kcal mol-1. The result shows that the one clash point of Arginine 180 residue which was present in the target 2FJZ with the position of C12 in the luteolin molecule having 2.853 Å bond lengths.
Figure 5: Docking poses of luteolin with 4PQE
Table 2: Interpretation for the docked complexes using the software Chimera 1.12.
CONCLUSION:
Luteolin isolated from Momordica charantia (Bitter gourd) was characterized and was predicted by in silico docking analysis to bind with acetylcholinesterase enzyme, butyrylcholinesterase enzyme, tau protein, tau protein-kinase I enzyme, APP. This study reveals that the flavonoid, luteolin may enhance the acetylcholine level in neurons by binding to acetylcholinesterase and butyrylcholinesterase enzymes that are responsible for acetylcholine metabolism and reveals that the ability of luteolin to prevent the tau protein hyperphosphorylation by inhibiting tau protein kinase enzyme and formation of amyloid beta senile plaque aggregation at the cellular level. Thus, docking analysis of luteolin concludes that luteolin has multi-target action for Alzheimer’s disease by using the interaction energy values and docked molecules binding site interpretation. The therapeutic importance of luteolin in Alzheimer’s disease must be further confirmed by performing in vitro and in vivo studies (behavioral experiments).
ACKNOWLEDGEMENT:
Authors are grateful to the Dean, Vice Principal, SRM College of Pharmacy and management of SRMIST, for providing funding and necessary facilities to carry out this work.
FUNDING SOURCE:
The study was supported by the Vice-Chancellor Fund, SRMIST.
CONFLICTS OF INTERESTS:
The authors declare that they are no conflicts of interest in sending the manuscript.
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Received on 09.07.2019 Modified on 28.08.2019
Accepted on 12.10.2019 © RJPT All right reserved
Research J. Pharm. and Tech 2020; 13(5): 2381-2386.
DOI: 10.5958/0974-360X.2020.00428.X