Exploring Therapeutic Potential of Nutraceutical Compounds from Propolis on MAPK1 Protein Using Bioinformatics Approaches as Anti-Coronavirus Disease 2019 (COVID-19)

. This study explores the potential of propolis, a natural substance, as a gene therapy for treating COVID-19. Despite the advent of COVID-19 vaccines, their side effects pose new health challenges. Utilizing network pharmacology, this research identifies propolis compounds through various databases and assesses their ability to target proteins associated with COVID-19. MAPK1 emerges as a potential therapeutic target, and molecular docking reveals Broussoflavonol F, Glyasperin A, and Sulabiroins as promising compounds with strong binding affinities, i.e., - 9.0, -9.0, and -8.8 kcal/mol, respectively, exceeding the native ligand (-7.2 kcal/mol). Molecular Dynamics displays stable complex behavior, with backbone RMSD values consistently below 4 Angstroms and RMSF simulations showing minimal fluctuations within ±2 Angstroms error. Moreover, MM-PBSA analysis further supports the strong binding of Broussoflavonol F, Glyasperin A, and Sulabiroins A, with relative binding energies of -122.82±89.65, 131.48±95.39, and -155.97±111,37 kJ/mol, respectively. These results indicate that propolis has potential as an anti-COVID-19 agent, primarily through inhibiting the MAPK1 pathway. However, further research is needed to validate these results and develop practical applications for COVID-19 therapy. This study underscores the significance of network pharmacology and computational models in understanding propolis mechanisms, offering potential directions for future research and treatment strategies against COVID-19.


Introduction
COVID-19, an acute respiratory infectious disease, is attributed to the viral pathogen known as SARS-CoV-2 [1].Common symptoms of COVID-19 include cough, sore throat, fever, arthralgia, myalgia, lethargy, and headache [2].Patients with comorbidities may experience acute respiratory distress syndrome, shock, metabolic acidosis, and multiple organ failure [3]- [6].As of November 28, 2022, the global tally of confirmed COVID-19 cases was at 636,440,663, with a corresponding count of 6,606,624 deaths.Despite the development of numerous COVID-19 vaccines and the subsequent initiation of large-scale vaccination campaigns, several vaccines have demonstrated efficacy against SARS-CoV-2 [7].Although COVID-19 vaccines have been developed and large-scale vaccination campaigns have begun, several vaccines have shown efficacy against SARS-CoV-2.However, the side effects of these vaccines will be a new health problem for humans [8].These issues encompass thrombocytopenia and vaccine-induced immune thrombosis (VITT), Bell's palsy, and hypersensitivity myocarditis.Hence, investigating genes proves to be a more efficient approach, exhibiting less incidence of adverse effects [9].Various health interventions have been implemented to mitigate the incidence of COVID-19 infections.These interventions include using masks, adhering to physical distancing measures, practicing proper hand hygiene, and administering vaccines [10].Herbal medicine constitutes a category of naturally occurring substances employed as adjunctive treatment for COVID-19 [11].Numerous natural constituents have been documented to possess inhibitory properties against the coronavirus [12].The antiviral action of Propolis, a resinous substance bees produce, has been documented in scientific literature.This activity is attributed to phenolic chemicals, flavonoids, and aromatic acid esters within Propolis [13].Propolis has demonstrated inhibitory effects against the varicella-zoster virus, herpes virus, and human immunodeficiency virus, particularly concerning its antiviral action [14].Preclinical investigations have documented the interactions between Propolis and various target proteins associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 [15].Moreover, several clinical investigations have provided evidence regarding the possible beneficial impacts of propolis and honey products on the elimination of the SARS-CoV-2 virus and the alleviation of patient symptoms [10].Nevertheless, the current body of knowledge regarding prospective biomarkers for COVID-19 remains significantly limited, with a shortage of comprehensive understanding regarding the underlying mechanisms involved [16][17][18].The objective of this study was to stimulate an intellectually stimulating discussion on the potential use of Propolis for the management of COVID-19, employing modern advancements in computer-assisted drug design, namely network pharmacology [19].Network pharmacology is a discipline that falls under systems biology theory, incorporating many approaches like high-throughput omics data analysis, computer virtual computing, and network database retrieval [20].Systematic biological network analysis is a prevalent strategy in modern-day research [21].This emerging field of study can forecast potential pharmacological targets from a holistic standpoint, thereby enhancing the efficacy of the drug development procedure.The discipline of network pharmacology has successfully surmounted past challenges associated with single-target, single-drug investigations.It has garnered considerable momentum in the identification of active constituents and comprehension of efficacious approaches [22].The organization of pharmacological and structural health networks is aligned with the complete qualities of Propolis, which contributes to the advancement of our comprehension of the intricate mechanisms encompassing many ingredients, targets, and pathways in herbal therapy [23][24].Furthermore, this study examined genetic material derived from publicly available bioinformatics sources.Moreover, molecular docking, a technique based on the fundamental principles of ligand-receptor interactions, is a versatile technology employed to elucidate the mechanisms by which chemical compounds interact with their molecular targets [25][26].Consequently, it is widely utilized in pharmaceutical research and development.Molecular dynamics (MD) simulations elucidate the dynamic characteristics of biomolecules at the atomic level, offering a dependable representation of their structural soundness [27].The present study holds promise for establishing a theoretical framework that can facilitate the development of novel pharmaceutical interventions and elucidate the underlying mechanisms implicated in the pathogenesis of COVID-19.This study employed network pharmacology, molecular docking, and molecular dynamics (MD) modeling methodologies to investigate the active ingredients, molecular targets, and significant biological pathways associated with the anti-COVID-19 capabilities of Propolis.

2.
Materials and Methods

Enrichment Analysis
The Database for Annotation, Visualization, and Integrated Discovery (DAVID database, https://david.ncifcrf.gov/)was used for enrichment analysis and visualization of Gene Ontology (GO), Biological Process (BP), and KEGG pathways of overlapping Propolis gene targets.The findings of combinations of proteins associated with Propolis and COVID-19 were then filtered using the parameter P-value <0.05 to quantify statistical differences and target potential [42]- [44].The Gene Ontology of Biological Process and KEGG target data were shown using the Bioinformatics website platform (http://www.bioinformatics.com.cn)[45]- [48] and GraphPad Prism 9.0.0.

Identification of Core Targets of Propolis against for COVID-19
Targets identified using Gene Ontology of Biological Process and KEGG were collected and analyzed using STRING Database Ver.11.5 (https://string-db.org/)network map and .tsvextension data to conduct an in-depth study of protein-protein interaction (PPI) between Propolis and COVID-19 [49], [50].Topological characteristics were examined using the Network-Analyzer settings of the Cytoscape program [51].The anticipated target proteins and associated target proteins were excised and examined using Cytoscape software (https://cytoscape.org/,Version 3.7.2) to investigate the pharmacological mechanism of Propolis in COVID-19.The Cytoscape CytoHubba plugin was used to investigate protein interactions.Maximal Clique Centrality (MCC), Density of Maximum Neighborhood Component (DMNC), Maximum Neighborhood Component (MNC), Degree Centrality, Edge Percolated Component (EPC), BottleNeck, EcCentricity, Closeness, Radiality, Betweenness, Stress, and ClusteringConff are among the 12 techniques presented [52]- [54].After comparing the data, the MAPK1 gene were discovered in 11 of the 12 techniques tested for Molecular Docking Analysis.

Molecular Docking
The molecular docking procedure was performed with the Autodock Vina algorithm implemented in the PyRx program, version 0.9.9 [55].The crystal structures of the MAPK1 protein target, specifically the PDB ID: 3SA0 [56], were obtained from the RCSB Protein Data Bank (http://www.rcsb.org/)[57].The Macromolecules and Propolis compounds were sourced from the PubChem database, accessible at https://pubchem.ncbi.nlm.nih.gov/[58].The protein structures were generated with Autodock tools-1.5.7.This process encompassed the elimination of water molecules and heteroatoms, the introduction of hydrogen polar groups, the consolidation of non-polar groups, and the integration of gasteiger charges.The protein structures generated were then stored in a file format referred to as pdqt format.Before initiating molecular docking, the protein structure was confirmed by redocking the co-crystallized ligand within the protein's binding area.The utilization of Autodock Vina, a software utility accessible in PyRx version 0.9.9, facilitated the accomplishment of this task.The selection of binding sites was conducted by utilizing a grid box centered on the ligand for each protein in Autodock.The grid box was built to enclose the protein's active region, MAPK1, utilizing the XYZ coordinates (-14.992,14.3, and 41.122).The grid was configured with dimensions of 20x20x20.The LigRMSD (https://ligrmsd.appsbio.utalca.cl/)value of 0.74 Å was employed to calculate the root mean square deviation between crystalline ligands before and following re-docking [59].This deviation must remain confined to the range of 2Å.The process by which a ligand can form a binding interaction with a receptor is widely recognized in scientific literature as affinity.The BIOVIA Discovery Studio Visualizer presents data in a three-dimensional format [60].The LigPlot software was employed to examine the interactions between proteins and ligands in PDB files containing encrypted docking data [61], [62].The present study involved a comprehensive analysis of three compounds exhibiting superior binding affinity alongside a control consisting of the Native Ligand.These compounds were subjected to rigorous investigation using Molecular Dynamics.

Molecular Dynamics
The simulations utilized YASARA version 21 software, employing the AMBER14 forcefield [63], [64].The parameters of the simulation are as follows: The experimental conditions included a pH of 5.0, a NaCl concentration of 0.9%, a water density of 0.997, a pressure of 1 atm, a running time of 20 ns, a temperature of 310°K, and a cubic grid form.The determination of bond energy stability or molecular mechanics energies, in conjunction with the Poisson-Boltzmann (MMPBSA) approach, was performed using the Poisson-Boltzmann (PBS) method [65].This analysis was conducted on the macros md_analyzebindingenergy.mcr obtained from YASARA [66].

Identifying Propolis targets and intersection COVID-19
Comprehensive set of 1438 proteins associated with COVID-19 was acquired from the GeneCards webserver.Additionally, 313 proteins related to Propolis were obtained through STITCH, TargetNet, and Swiss Target Prediction.Subsequently, an intersection analysis was performed, resulting in the identification of 74 proteins that were common to both propolis compounds and COVID-19-related proteins.These findings were visually represented using a Venn diagram (Figure 1).

Biological Function and Pathway Enrichment Analysis
The biological functions of the 74 probable protein targets were determined using Gene Ontology and KEGG pathway enrichment analysis.In the GO study, 399 GO keywords were discovered, including 62 Molecular Functions (MF), 282 Biological Processes (BP), and 55 Cellular Components (CC), where these data were collected using a p-value threshold of (<0.05).The top ten BP words are ordered by p-value and the quantity of protein listed (Figure 2).The main GO terms BP are related to protein phosphorylation, response to xenobiotic stimulus, response to drug, peptidyl-serine phosphorylation, positive regulation of gene expression, negative regulation of apoptotic process, positive regulation of neuron apoptotic process, cellular response to reactive oxygen species, peptidyl-threonine phosphorylation, positive regulation of transcription, DNA-templated.Furthermore, the Coronavirus disease -COVID-19 pathway from Propolis is shown by the KEGG pathway enrichment analysis, which is the focus of this research topic, to be significantly associated with 74 potential pathways (Table 1).

Components-Targets Network Construction of COVID-19
To determine a protein's crucial involvement in COVID-19, proteins from the Top 10 GO BP and 20 KEGG were gathered, and up to 26 proteins were evaluated using STRING analysis and protein network topology.The interactive protein-related protein from Propolis to COVID-19 was assessed with the Tool Cytoscape program and the CytoHubba plug-in, with at least two CytoHubba techniques used as a cutoff in this procedure.Table 2 shows that the twelve CytoHubba techniques contain a total of 19 proteins.Among the 19 proteins, MAPK1 is at the top with its appearance in eleven different methods: Betweenness Method, Stress Method, Degree Method, EPC, Closeness Method, Radiality Method, MCC Method, BottleNeck Method, MNC Method, EcCentrality Method, and DMNC Method.In addition, AKT1, JUN, and RELA were present in ten methods, HSP90AA1 and LCK were present in nine methods, IL2 in eight methods, EGFR and PIK3R1 in seven methods, MAPK8 in six methods, APP in four methods, CASP3, CDK1, and FOS in three methods, and last AR, NOS2, PRKCE, PTPN1, and TNF proteins were present in only two methods.To see this in Figure 3. is a visual representational proof for the occurrence of screened genes in the various CytoHubba methods.We used VinaWizard algorithm in the PyRx software version 0.9.9 to perform molecular docking to validate further the binding affinity of the propolis compound and MAPK1 protein, where Norathyriol is the Native Ligand in MAPK1.Based on the results of docking, where Norathyriol, which acts as a Native Ligand, has a docking score (-7.2 kcal/mol).Meanwhile, out of a total of 21 propolis compounds, three compounds had the highest binding affinity, namely Broussoflavonol F (-9 kcal/mol), Glyasperin A (-9 kcal/mol), and Sulabiroins A (-8.8 kcal/mol).To see the bonds inside, Native Ligand (Norathyriol) forms 4 (Glu33, Met108, Asp106, and Gln105) Hydrogen Bonds and 8 (Gly32, Val39, Gly34, Lys54, Leu156, Ala52, Leu107, and Ile84) Hydrophobic Interaction.Then in the three compounds with the highest Binding Affinity, Broussoflavonol F forms 3(Asp167, Ala35, and Lys114) Hydrogen Bonds, 15(Ile165, Leu156, Gln105, Met108, Val39, Asp106, Ile31, Ala52, Asp111, Ser153, Glu33, Gly34, Glu71, Lys54, and Asn154) Hydrophobic Interaction, Glyasperin A triple (Ala35, Glu71, and Met108) Hydrogen Bond and 14 (Tyr36, Arg67, Lys54, Leu156, Ile84, Asp106, Ala52, Leu107, Gln105, Ile31, Lys114, Gly34, Val39, and Asp167) Hydrophobic Interaction.Finally, the Sulabiroins A compound has 10 (Glu71, Asp167, Ser153, Asn154, Leu156, Val39, Ala52, Ile31, Lys114, and Lys54) Hydrophobic Interaction.Based on the literature from the PDB source, it is stated that the residues Gln105, Asp106, and Met108 are the active sites for the bond with the Native Ligand (norathyriol) [56].This is directly proportional to the results obtained for the compounds Broussoflavonol F and Glyasperin A. Twentyone docked propolis compounds were selected for the top three propolis compounds based on their binding affinity score will conducted Molecular Dynamics to evaluate RMSD, RMSF, and binding energy calculation along simulation 20ns.Based on this research, the pathway mechanisms involved in propolis and COVID-19 have been studied from the collected literature, where the TNF signaling pathway can cause disruption in the localization of the zonula occludens-1 (ZO-1) junction and downregulation of ZO expression.-1, as well as other members of the claudin family in the lung, which functionally regulate the unblocking of TJs [67].Through this pathway, Propolis can protect the alveolar endothelial/epithelial barrier by maintaining the integrity of the connections between cells, which ultimately reduces COVID-19 by improving lung ventilation function [68].

Conclusion
This research systematically analyzes the therapeutic targets, signaling pathways, and propolis mechanisms that can potentially be applied in the treatment of COVID-19 based on the Network Pharmacology approach and Molecular Docking and Molecular Dynamics verification.The therapeutic efficacy of propolis against COVID-19 is most likely mediated through the MAPK1 protein, where the compounds Broussoflavonol F, Glyasperin A, and Sulabiroins A from propolis were found to interact with MAPK1 through Molecular Docking and Molecular Dynamics.In addition, KEGG enrichment analysis illustrates that propolis can simultaneously play a role in various pathways in COVID-19, one of which is the Coronavirus disease pathway -COVID-19.Our research suggests that Broussoflavonol F, Glyasperin A, and Sulabiroins A may be helpful in COVID-19 therapy.These findings may provide a reference basis for clinical application and further exploration of the mechanism by which compounds in propolis affect COVID-19.Apart from that, MAPK1 as a potential therapeutic target in COVID-19 therapy has yet to be widely discussed so that this gene could be a suitable candidate.This needs to be done to provide a promising reference for future research.

Table 2 .
List of the genes present from twelve different methods of the CytoHubba plugin Cytoscape