Submitted : 20-12-2019 Revised : 20-02-2021 Accepted : 24-03-2021 Trad.
Med.
January-April 2021 Vol.
, p 1-12 ISSN-p : 1410-5918 ISSN-e : 2406-9086 Screening and Prediction of Potential Compounds from Virgin Olive Oil Acting on Proteins Associated with Cancer Disease Achmad Rodiansyah1,2* Department of Biology.
Universitas Negeri Malang.
Indonesia 2 Department of Biotechnology.
School of Life Sciences and Technology.
Institut Teknologi Bandung.
Indonesia
ABSTRACT
Virgin olive oil contains phenolic compounds that were potential for anti-inflammatory and cancer Computational biology is a beneficial method to understand how this compound can affect the biological process in humans.
This research is conducted by the potential screening of VOO compounds, constructing the pharmacological network and enrichment, and docking simulation.
The enrichment result showed that the EGFR.
BRAF.
MAPK1.
CCND1, and MDM2 protein have multiple cancer contributions and related pathways.
The docking simulation result showed that the interaction of EGFR-luteolin.
BRAFluteolin.
MAPK1-luteolin.
CCND1-apigenin, and MDM2-1-hydroxypinoresinol has the highest binding Further research with the in-vitro methods are required to check the reliable mechanisms of each compound to their protein target.
Keywords: Bioinformatics.
Cancer.
Molecular docking.
Network pharmacology.
Olive oils.
Phenolics INTRODUCTION The virgin olive oil (VOO) is the main product of olive trees (Olea europaea subs.
Eureupae.
This tree is a symbolic species in the Mediterranean region.
Genomic research with this tree is important for facilitating study in metabolisms, developmental and physiological process, which is the research could contribute to improving the economic values of this tree (Cruz et , 2.
Extra VOO contains dominant phenolic compounds (Visioli and Bernardini, 2.
, which those compounds have different classes, such as phenyl ethyl alcohols, steroids, phenolic acids, hydroxy-isochromans, lignans and, flavonoids.
The phenolics .
lso known as polyphenol.
from a plant have some benefits in the human body to prevent various diseases.
They work to bind several proteins, which may lead to a specific disease, and they have also antioxidants activity against free-radical (Preedy and Watson, 2.
the in-vitro studies, they can modulate intracellular signaling pathways.
VOO compounds, like hydroxytyrosol, tyrosol, and other minor compounds, have been the focus of research to see its effect in the biological process (Serreli and Deiana, 2.
Recent studies in-vitro and in-vivo with VOO compounds have been done.
The recent studies, hydroxytyrosol from VOO has been reported could induce cell cycle arrest and apoptosis on various *Corresponding author: Achmad Rodiansyah Email : ach.
rodiansyah@gmail.
cells, such as colon cancer cells, bladder cancer and cholangiocarcinoma (Coccia et al.
, 2016.
Li et al.
Lypez de las Hazas et al.
, 2.
Pinoresinol may also probably have antitumor activity in breast cancer cells (Lypez-Biedma et al.
, 2.
, and p-HPE-EDA can inhibit colon cancer cells with inhibition of AMP-activation protein kinase (AMPK) and cyclooxygenase-2 (COX-.
(Khanal et al.
, 2.
In silico and in vitro study showed that oleuropein confirmed could inhibit mTOR, which is responsible for tumor cellsAo properties on breast cancer (Corominas-Faja et al.
, 2.
World
(WHO)
mentioned that cancer which causes the death of 6 million death in 2018, is a group of diseases with abnormal cell growth that can invade other organs.
The mechanisms in this disease are very complicated.
Researchers mention that this disease is mainly caused by a gene mutation affecting cell functions, carcinogenic chemical compounds, and unhealthy lifestyle (Hassanpour and Dehghani, 2.
Genetic materials and proteins play an essential role in this groupAos diseases.
with bioinformatics, it will more easily explore potential clinical applications and improve diagnosis, therapies, and cancer diseases prognosis (Wu et al.
, 2.
Computational network biology is a new research field that involves theory and applications to describe a moleculeAos interaction on living cells.
this field also contributes to accelerating molecular biology, pharmacology, and genetics studies (Ni et al.
This method is acceptable for integrating Traditional Medicine Journal, 26.
, 2021 | DOI : 10.
22146/mot.
Achmad Rodiansyah Table I.
Bioactive compounds in virgin olive oil PubChem ID Compound Name alpha-Linolenic acid Linoleic acid Oleic acid Palmitic acid Stearic acid Tyrosol .
-HPEA) Hydroxytyrosol .
,4-DHPEA) 1-.
-Methoxy-4-hydrox.
-phenyl-6,7-dihydroxy-isochroman 1-Acetoxypinoresinol 1-Hydroxypinoresinol 1-Phenyl-6,7-dihydroxy-isochroman 3,4-DHPEA-EA Apigenin Luteolin p-Hpea-eda Pinoresinol and analyzing big data.
with this network model, the extensive information of the biological system could be more easily to be understanding (Ideker and Nussinov, 2017.
MaAoayan, 2.
METHODOLOGY
Preparation of ligand and protein target VOO bioactive compounds were collected from the PubChem database .
ttps://pubchem.
gov/) based on relevant literature (Alavi and Golmakani, 2017.
Basiricy et al.
, 2019.
Rupani, 2014.
Waterman, 2007.
Zalejska-Fiolka et al.
, 2.
Finally, 16 bioactive compounds used in this study were listed in Table I.
The 3D structure and canonical SMILES of all compounds were downloaded from the PubChem database.
Canonical SMILES compounds were used for identifying the potential target proteins on the Phrammapper web server .
ttp://lilab-ecust.
pharmmapper/) and Swiss Target Prediction web server .
ttp://w.
ch/).
Phrammapper web server is a platform for identifying the potential protein target from bioactive compounds with statistical method calculation include more than 7000 target pharmacophores (X.
Liu et al.
, 2010.
Wang et al.
The specification for searching target proteins was set as Druggable Pharmacophore Models .
2017,16.
(Liang et al.
, 2.
Like a Phrammapper webserver.
SwissTargetPrediction is an accruable webserver to predict the target proteins of bioactive compounds (Gfeller et al.
this web server has been updated for efficient prediction of protein targets (Daina et al.
The result from Phammapper and SwissTargetPrediction webserver was saved as csv format and used to construct network compound-protein interactions.
Network construction and analysis The network compound-protein interaction was constructed by Cytoscape software and its plugins .
ttps://cytoscape.
org/).
Excel files from Pharmmapper and SwissTargetPrediction that contains compound-protein interaction data were imported to Cytoscape software with the menu Auimport network form file systemAy.
The compound table was selected as a Ausource nodeAy, and the protein target was selected as a Autarget nodeAy.
The following analysis was used ClueGO CytoscapeAos plugins to show the interpretation of list genes on the metabolical process (Bindea et al.
The ClueGO pluginAos setting performs as pathways with p-values O 0.
001, and the kappa score was set as 0.
The Gene Ontology databases were used from the Kyoto Encyclopedia Genes Genomes (KEGG) Wikipathways.
The selected target proteins from the ClueGO enrichment were used for docking simulation for determining ligand-protein Protein Preparation and Molecular docking The ClueGO results relating to cancer diseases were selected as a target protein for molecular docking.
The target protein used for molecular docking were retrieved from Protein Data Bank .
ttps://w.
org/).
The 3D structure of proteins used in this study was listed as follows: EGFR-PDB.
id 6LUD (Kashima et al.
BRAF-PDB.
id 6NSQ (Assadieskandar et al.
Traditional Medicine Journal, 26.
, 2021 Screening and Prediction of Potential Compounds from Virgin Olive Oil Figure 1.
The network interaction from Cytoscape of 16 compounds of VOO with their target proteins 2.
MDM2-PDB.
id 3LNJ (M.
Liu et al.
, 2.
MAPK/ERK-PDB.
id 5NHH (Ward et al.
, 2.
, and CCND1 6P6G (Guiley et al.
, 2.
Those target proteins were sterilized from waters and ligands PyMOL .
vailable https://pymol.
org/2/).
The ligands used for molecular docking simulation were converted to pdbqt format using open babel on PyRx software .
vailable in https://pyrx.
io/), and the reverse docking simulation was performed using Vina Wizard in PyRx (Dallakyan and Olson, 2015.
Trott and Olson, 2.
The docking results were visualized and evaluated using PyMOL software.
The interaction between amino acid residues and ligands in 2D view were visualized using BIOVIA DiscoveryStudio 2019 software .
vailable in https://w.
com/).
RESULT AND DISCUSSION
Preparation of ligand and network data Sixteen potential ligands from VOO were collected from PubChem, and the list of protein targets from Phrammapper and SwissTargetPrediction Each compound interacted with various proteins that may contribute to many functions on biological pathways to build the compoundproteins network.
Network construction with Cytoscape software showed 1639 nodes and 4016 A node represents the target proteins and VOO compounds, while an edge represents the interaction of compoundsAetarget proteins on the biological pathways (Figure .
The VOO compounds labeled with red nodes in Figure 1 interact with blue nodes labeled as target proteins.
Luteolin, 1-Hydroxypinorisenol, apigenin, linoleic Traditional Medicine Journal, 26.
, 2021 acid, pinoresinol, oleic acid, stearic acid, 1Acetoxypinorisenol, palmitic acid, p-Hpea-eda, and alpha-linolenic acid are located on central of nodes target protein, and other compoundAos nodes are located on outside of central interaction.
Table panel protein target interactions from the Cytoscape network were saved as an Excel file.
All protein was filtering and grouping to determine their biological pathways using ClueGO CytoscapeAos plugin.
Network construction and analysis ClueGO analysis with KEGG and gene ontologies database showed 26 nodes and 38 edges with three pathways (Figure 2.
, and WikiPathways database 35 nodes and 59 edges with four pathways (Figure 2.
ClueGOAos plugin pathways were listed in Table 2.
this result showed that VOO compounds are associated with various pathways, especially in tumor and cancer.
The VOO compounds interact with various proteins that work on several biological pathways and are interconnected with its pathway or other From the KEGG database and WikiPathways database enrichment, it has known that VOO compoundsAo target protein was probably related to cancer disease and tumor with a p-value O of 0.
A significant test of the p-value is vital for decision-making.
A very small p-value indicated that the hypothesis is probably correct (Panagiotakos, 2.
These enrichment pathways indicated that they have a high confidence value.
All pathways from ClueGO are related to each other indicated that the several nodes of the gene connect with all pathways in Figure 2 .
arked with a red circl.
Glioma is the one type of tumours disease that occurs in the Central Achmad Rodiansyah .
Figure 2.
ClueGO grouping shows the biological pathway from the list of the genes from the network.
KEGG database.
Wikipathways database.
Table II.
ClueGO enrichment pathways with KEGG pathways database and WikiPathways database Source Database KEGG ontologies KEGG ontologies KEGG ontologies Wikipathways Wikipathways Wikipathways Wikipathways Pathway Prostate cancer Bladder cancer Glioma Glioblastoma Bladder cancer Oncostatin M signalling pathway Matrix metalloproteinases Nervous System (CNS) and spinal cord.
this disease is a common type of primary malignant brain tumor derived from glial cells (Ernest and Sontheimer, 2.
Glioblastoma or glioblastoma multiforme (GBM) is a subclass of glioma disease (JOVUEVSKA et al.
, 2.
Oncostatin M (OSM) is an important pathway associated with the biological process and cellular responses.
This pathway is vital for clinical and biomedical therapeutic on the human disease Colour Avocado green Turquoise Dull green Blue Turquoise Green Dull green P-Value 77 x 10-9 13 x 10-6 60 x 10-6 66 x 10-8 28 x 10-6 49 x 10-6 37 x 10-6 (Dey et al.
, 2.
OSM pathway has been reported associating with cancer cellAos plasticity (Junk et al.
also contribute to breast tumor specifically mediated by OSMR (Underhill-Day and Heath, 2.
and prostate cancer (Godoy-Tundidor et al.
Prostate cancer and bladder cancer are double cancer primary cancer with high frequency this data suggest that the patients diagnosed with bladder or prostate cancer should be followed second malignant urologic diagnosis Traditional Medicine Journal, 26.
, 2021 Screening and Prediction of Potential Compounds from Virgin Olive Oil (Kinoshita et al.
, 2.
Disturbing the OSM pathway may is potential for cancer cell treatment (Caffarel and Coleman, 2014.
Stroeder et al.
The Matrix metalloproteinases (MMP.
, is a group of enzymes responsible for the degradation of extracellular matrix protein during organogenesis and normal tissue replacement.
This group of enzymes is also associated with oral cancer (Sorsa et al.
, 2.
Based on ClueGO enrichment, it is shown that this pathway has a connection with bladder cancer and OSM.
The interruption of this protein activity could lead to various diseases (Laronha and Caldeira.
From KEGG and Wikipathways database enrichment, five genes contribute to OCM, glioma, bladder, and prostate cancer pathways.
Those genes are CCND1 encodes G1/S-specific cyclin-D1 protein.
MAPK1 encodes dual specificity mitogenactivated protein kinase 1.
MDM2 encodes E3 ubiquitin-protein ligase Mdm2.
EGFR encodes epidermal growth factor receptor protein.
BRAF encodes serine/threonine-protein kinase B-RAF.
These proteins were chosen for molecular docking simulation docked with the compound from VOO as the ligand to know their interaction.
They could be evaluated to be used as therapeutic compounds for treating cancer disease.
The CCND1 (Cyclin D.
gene has a function in the regulation of CDK kinase in the cell cycle.
when this gene had the mutation and overexpressed, it can promote a various type of cancers disease in humans, such as breast cancer, endometrial cancer, colon cancer, and prostate cancer (Fu et al.
, 2004.
Ikeda et al.
, 2013.
MorenoBueno et al.
, 2003.
Xu and Lin, 2.
This gene is also used as a biomarker in breast cancer (Lundberg et al.
, 2.
Cyclin D1 protein functions as cyclin-dependent-kinase (CDK) in a subunit of CDK4 or CDK6 to regulate the cell cycle from G1 to S phase transition.
They act as apoptosis regulators interacting with tumor suppressor protein retinoblastoma (R.
to interrupting the cell cycle.
CCND1 also acts on chromatin recruitment, mitochondrial biogenesis, and DNA Damage Response (DDR) (Fu et al.
, 2004.
Massaguy, 2004.
Pestell, 2.
When this gene act as an oncogene caused by point mutation, local DNA rearrangements, or chromosomal translocation, then they could overexpress in a cell, and that cell will have rich of complex CDK-cyclin.
so, it could stimulate the progression in cell cycles, stimulate tumorigenesis, and metastases even in the absence of growth factor (Fu et al.
, 2004.
Hardin et al.
, 2012.
Kim and Diehl, 2.
Traditional Medicine Journal, 26.
, 2021 The murine double minute 2 (Mdm.
gene encodes a protein acting as a negative regulator of the p53 tumor suppressor.
These genesAo overexpression occurs in many tumors (Iwakuma and Lozano, 2003.
Senturk and Manfredi, 2.
Mdm2 most commonly associated with liposarcoma compared with breast cancer and bladder cancer in MD Anderson phase 1 clinic (Dembla et al.
, 2.
, and Mdm2 amplification not in most tumor types, that is just in the small subset in types tumor (Kato et al.
, 2.
The Mdm2 interacts with p53 protein to make p53 inactive, and this mdm2-p53 interaction can cause apoptosis failure (Hardin et al.
, 2.
Activation of p53 protein is critical to protect the propagation cells from damaged DNA with oncogenic mutations and control the cell cycle (Moll and Petrenko.
Blocking the Mdm2-p53 and Mdm-non p53 interaction is a promising cancer therapeutic strategy (Shangary and Wang, 2008.
Wang et al.
BRAF encodes Raf protein and MAPK1/2 gene-encoded mitogen-activated protein kinase 1/2 or Extracellular Signal-Regulated Kinases (ERK.
protein which play on signal transduction in the MAPK signaling pathway.
This protein is known as signal transduction which works on the Ras-Raf-MEK-ERK signaling for cell proliferation, cell differentiation, cell metastasis, cell survival, and apoptosis (Mansfield et al.
, 2018.
Mebratu and Tesfaigzi, 2009.
Thatcher, 2.
Activating this pathway starts with growth factor family protein like Epidermal Growth Factor (EGF) binds their receptor to phosphorylate Raf protein as downstream of Ras on the MAPK signaling pathway (Ursem et al.
, 2.
Altered of this signaling pathway or mutation in genes encoding the protein in this pathways have been reported correlated and detected in tumors and cancer disease, including in pituitary tumourigenesis, cervical cancer tissue, and significantly correlated on breast cancer with axillary lymph node metastasis (Suojun et al.
, 2012.
Manousaridis et al.
2013 Jan 1.
Li et al.
, 2015: 1.
AACR Project GENIE Shao et al.
, 2.
The inhibitors with targetting on these kinases protein could treat malignant tumors.
this inhibitor would be promising and challenging in future research (Liu et al.
, 2018.
Suojun et al.
, 2.
National Comprehensive Cancer Network (NCCN) also recommending BRAF testing gene for diagnosis of colorectal cancer (CRC) (Ursem et al.
, 2.
Molecular Docking Simulation Computational methods recently used in the biotechnology and pharmaceutical industries were Achmad Rodiansyah Figure 3.
Docking result of the compounds with selected protein from KEGG and WikiPathways database Table i.
Docking score from control compounds Ligand
Osimertinib
Vandetanib
Encorafenib
Vemurafenib
AMG-232
Idasanutlin Vemurafenib Encorafenib CDK4 inhibitor PubChem Id beneficial for drug discovery with high success and accuracy and reduced costs (Parenti and Rastelli.
Suortti, 1.
Reverse docking is a promising drug prediction technique that acts on protein-related disease as inhibitors (Kharkar et al.
, 2.
this method has excellent drug design and drug discovery success.
The docking simulation result showed that VOO compounds have various docking scores to target proteins (Figure .
The bar chart in Figure 3 shows the docking simulation score from VOO compounds with their target proteins.
The deep-blue chart is an EGFR an orange chart is a BRAF protein.
a yellow
Protein
EGFR
EGFR
BRAF
BRAF
MDM2
MDM2
MAPK
MAPK
CCND1
Score .
cal/mo.
Label Red Magenta Red Magenta Red Magenta Red Magenta Red chart is a MAPK protein.
a grey chart is an MDM2 protein, and a blue chart is a CCND1 protein.
Luteolin from VOO has the highest docking score, 9 kcal/mol on EGFR protein and -9.
5 kcal/mol on BRAF protein.
1-Hydroxypinoresinol has the highest docking score with -7.
0 kcal/mol on MDM2 Apigenin has the highest docking score 7 kcal/mol on CCND1 protein.
on MAPK protein, luteolin has the highest docking score of -9.
0 kcal/mol, respectively (Figure .
Compared to the control compound, only the docking score from Luteolin-BRAF and luteolinMAPK were more stable.
The docking score from control compounds was listed in Table i.
Traditional Medicine Journal, 26.
, 2021 Screening and Prediction of Potential Compounds from Virgin Olive Oil Figure 4.
Molecular interaction VOO ligands with the target proteins.
EGFR.
BRAF.
MDM2.
MAPK.
CCND1.
Binding affinity is a critical aspect of drug design to produce potential ligands with high binding affinity to the target protein and low target binding affinity to non-targeted protein (Kairys et al.
, 2.
The Gibbs energy of binding .
or binding affinity is value to define a strong interaction between two molecules, so this is a crucial quantity for study molecule interaction (Vangone et al.
, 2.
The type of bond on reverse docking is critical on binding affinity as a docking score, especially the hydrogen bond in ligandprotein interaction.
The 2D visual interaction of the VOO ligands colored with green within their target protein can be seen in Figure 4, and the list of amino acid residues can be seen in Table IV.
The results from molecular docking showed that the most of the ligands occupied the vital region of the protein.
This interaction could potentially disrupt the work of target protein, like preventing phosphorylation and inhibiting protein A ligandsAo effectiveness to interfere with proteinsAo action can be predicted by the docking score from the inhibitor ligand to the protein.
Many factors that contribute to binding affinity score as follow: the role of water, the existence of H-bonds.
Traditional Medicine Journal, 26.
, 2021 the different types of the bind of ligand-protein interaction include ionic interactions.
Van der Waals hydrophobic interaction.
Pi-alkyl bond, and Pi-sulfur bond.
those factors must be considered to evaluate docking results (Arthur and Uzairu, 2019.
Pantsar and Poso, 2.
However, not all parameters can be calculated on this docking simulation, so an in-vitro and in-vivo study must be carried to determine the reliable responses (Pintilie and Stefaniu, 2.
The illustration of VOO compounds to inhibit the target protein can be seen in Figure 5.
From this illustration, the VOO compounds probably can work on several fields on the cells, like on the extracellular-membrane layer, cytoplasm, and nucleus cells field.
EGFR protein is located on membrane cell.
MAPK1 and BRAF protein work on the cytoplasm.
MDM2 and CCND1 protein work on the nucleus.
CONCLUSION
The VOO compounds have multiple protein targets on various pathways, especially in cancers and tumors.
Five proteins that act on multiple Achmad Rodiansyah Figure 5.
The illustration of VOO compounds work on their target proteins in the cell (The illustration was created with BioRender.
(Available at: https://biorender.
Table IV.
Amino acid residues from molecular docking simulation with the highest score and the information domain from UniProt
Protein
EGFR
VOO ligand
BRAF
MDM2
1-Hydroxypinoresinol
MAPK1
Luteolin
CCND1
Apigenin Amino acid residues LEU718.
GLY719.
VAL726.
ALA743.
LYS 745 GLU762.
LEU792.
MET793.
PRO794.
GLY796.
MET798.
LEU844.
THR854.
ASP855
ILE463.
VAL471.
ALA481.
VAL482,
LYS483.
GLU501.
VAL504.
LEU505,
THR508.
ILE513.
LEU514.
ILE527,
THR529.
GLN530.
TRP531.
CYS532,
LEU567.
PHE583.
GLY593.
ASP594,
PHE595
LEU54.
LEU57.
GLY58.
ILE61.
MET62,
TYR67.
GLN72.
HIS73.
VAL93.
VAL75,
HIS96.
ILE99.
TYR100
ILE31.
GLY32.
GLU33.
GLY34.
VAL39,
ALA52.
LYS54.
ILE84.
GLN105,
ASP106.
LEU107.
MET108.
GLU109,
THR110.
LYS114.
LEU156.
CYS166,
ASP167
ASN174.
ILE178.
ILE177.
HIS181,
VAL212.
GLY214.
LEU217.
ARG218,
PRO220.
ASN222
Domain UniProt Uniprot Id: P00533 Region: 712-972 (Protein kinase Uniprot Id: P15056 Region: 457-717 (Protein kinase Uniprot Id: Q00987 Region: 1-110 (USP interaction domai.
Uniprot Id: P28482 Region: 25-313 (Protein kinase Region: 105-108 (Inhibitor binding Uniprot Id: P24385 Region: 2-208 (Interaction regio.
Region: 2-19 (Region for RPLP0 & TCF.
Region: 150-360 (Region for TCF.
Traditional Medicine Journal, 26.
, 2021 Screening and Prediction of Potential Compounds from Virgin Olive Oil central pathways from KEGG and WikiPathways database were MAPK1.
BRAF.
EGFR.
MDM2, and CCND1.
The docking simulation showed that the luteolin compound was stable with EGFR protein and BRAF protein, with the docking score reaching 9 kcal/mol and -9.
5 kcal/mol, 1-hydroxypinoresinol was stable to interact with MDM2 protein, reaching a score of 0 kcal/mol.
apigenin and stearic acid were stable to interact with CCND1 protein with docking score reaching -6.
7 kcal/mol.
Also.
MAPK protein is stably interacting with luteolin, which has a docking score of -9.
0 kcal/mol.
Those compounds, especially luteolin, probably have the potential for therapeutic on various cancers and tumors.
These docking simulation results also report that luteolin has a stronger binding affinity than the control compound for interfering with the BRAF and MAPK The in-vitro and in-vivo study must be carried out to validate the specific response from ligands on proteins involved in cancer pathways.
ACKNOWLEDGEMENT
The author is grateful to his previous affiliation, "Universitas Negeri Malang.
Indonesia", to facilitate internet connection and give a permit to access articles and software used in this study.
REFERENCES