Molecular Network Simulation of Bawang Ada’ (Eleutherine americana Merr.) from Dayak Lundayeh in North Kalimantan Tackle various Viral Infection Targeting Key Protein

Viol Dhea Kharisma^1,2, Priscilla Listiyani², Ahmad Affan Ali Murtadlo²,

Rizal Adistya Putra Pradana², ANM Ansori³, Alexander Patera Nugraha⁴, Rollando Rollando⁵,

Raden Joko Kuncoroningrat Susilo⁶, Suhailah Hayaza⁶, Sofya Olegovna Budagova⁷,

Gregory Vadimovich Nadvodnyk⁷, Ivan Gennadievich Lebedev⁸,

Zaira Nadirovna Khalibekova⁹, Igor Vladimirovich Rzhepakovsky¹⁰, Maksim Rebezov^11,12, Vikash Jakhmola¹³, Hery Purnobasuki¹, Dwi Kusuma Wahyuni^1*

¹Department of Biology, Faculty of Science and Technology, Universitas Airlangga, Surabaya, Indonesia.

²Computational Virology Research Unit, Division of Molecular Biology and Genetics,

Generasi Biologi Indonesia Foundation, Gresik, Indonesia.

³Professor Nidom Foundation, Surabaya, Indonesia.

⁴Department of Orthodontics, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia.

⁵Pharmacy Department, Faculty of Science and Technology, Ma Chung University, Malang, Indonesia.

⁶Department of Nanotechnology Engineering, Faculty of Advance Technology and Multidiscipline,

Universitas Airlangga, Surabaya, Indonesia.

⁷Faculty of Dentistry, Stavropol State Medical University, Stavropol, Russian Federation.

⁸Medical and Preventive Faculty, Rostov State Medical University, Rostov-on-Don, Russian Federation.

⁹Faculty of Medicine, Stavropol State Medical University, Stavropol, Russian Federation.

¹⁰Medical and Biological Faculty, North Caucasus Federal University, Stavropol, Russian Federation.

¹¹Department of Scientific Research, V. M. Gorbatov Federal Research Center for Food Systems, Moscow,Russian Federation.

¹²Faculty of Biotechnology and Food Engineering, Ural State Agrarian University,

Yekaterinburg, Russian Federation.

¹³Uttaranchal Institute of Pharmaceutical Sciences, Uttaranchal University, Dehradun, Uttarakhand, India.

*Corresponding Author E-mail: dwi-k-w@fst.unair.ac.id

ABSTRACT:

Viral replication inhibition strategies are needed to prevent pandemics through the latest therapeutic agent designs. A viral infection occurring over a wide area is called a pandemic. The strategy of inhibiting virus replication is used to tackle the pandemic Viruses can trigger negative regulation of apoptosis in host cells for viral survival. Apoptosis can reduce viral load and inhibit viral replication. Several types of viruses can evade the immune response through upregulation of various anti-apoptotic proteins, which allows this research to explore specific types of anti-apoptotic proteins in host cells for the design of candidate therapeutic agents.Medicinal plants from the Dayak Lundayeh tribe in North Kalimantan have potential for health, the antiviral potential of these plants has not been identified. This study aims to reveal the potential of the bioactive compounds from Bawang Ada' as antivirals with a molecular mechanism through apoptosis with an in silico approach.The in silico method used in this study consisted of ligand preparation, druglikeness analysis, pathway prediction, docking, and molecular interaction.Bawang Ada' acts as the best antiviral candidate through the activity of Erythrolaccin and Isoeleutherin compounds in inhibiting antiapoptotic proteins consisting of GSK3B and AKT1. We recommend the binding sites Val70, Leu132, Ile62, Leu188, Asp200, and Cys199 (GSK3B) and Leu210, Leu264, Tyr272, Asp292, Trp80, Lys 268, Val270, and Ser205 (AKT1) for further research as antiviral target development.

KEYWORDS: Apoptosis Agonist, Antiviral, Bawang Ada’, Bioinformatics,Eleutherine americana.

INTRODUCTION: 

Viral infection is the process of virus multiplication in cells through a replication cycle. Host cells are cells that are infected and produce viruses¹. Viruses can hijack several functional cell proteins to continue the replication process². Viruses infect cells by interacting with specific receptors and fusion through membranes to reach functional organelles such as ribosomes or the nucleus³. Protein synthesis is used by viruses to form functional proteins, the process is in the assembly stage. Generally, virus replication consists of attachment, penetration, replication, assembly, maturation, and release⁴. Expression of early genes in viruses can trigger pathogenesis mechanisms such as evasion of the immune response, inflammation, and specific symptoms such as fever, cough, rash, etc. Cases of viral infections such as COVID-19, MERS, Zika, Ebola, and Infulenza occur in large areas or are pandemics⁵. Influenza triggered the first pandemic in 1918 called the Spanish Flu and the 2019 pandemic⁶. The virus was identified as a zoonotic origin consisting of birds and pigs⁷. Inhibition of viral functional proteins by a therapeutic agent can fight infection and prevent the process of replication of a type of virus.

Viral infection can trigger extrinsic signaling of the apoptotic pathway through cell-surface death receptor mediation from the tumor necrosis factor family. Apoptosis is one of the three pathways of programmed cell death due to viral infection⁸. Viruses can trigger negative regulation of apoptosis in host cells for viral survival. Apoptosis can reduce viral load and inhibit viral replication. The mechanism of the intrinsic pathway for apoptosis signaling is mediated by BAX and BAK in the outer mitochondrial membrane to trigger MOMP⁹. Apoptosis signaling via extrinsic mediation is mediated by TNF-related apoptosis-inducing ligand (TRAIL) receptors, TNF receptor 1 (TNF-R1), Fas (also called CD95 or Apo-1 or TNFRSF6), DR6 and DR3¹⁰. Several types of viruses can evade the immune response through upregulation of various anti-apoptotic proteins, which allows this research to explore specific types of anti-apoptotic proteins in host cells for the design of candidate therapeutic agents.

Biodiversity is very much owned by Indonesia such as traditional medicine from natural ingredients consisting of a mixture of plants, animals and specific minerals which have been used for generations for alternative medicine^11,16.

The culture of the surrounding community, which consists of various ethnic groups, triggers the preservation of the use of traditional medicines from certain types of plants^12,17,18. Dayak Lundayeh tribe in North Kalimantan also uses medicinal plants such as Bawang Ada' (Eleutherine americana Merr.) as an alternative treatment as an anti-inflammatory and antimicrobial^13,14. The results of chromatographic identification from previous studies showed Eleutherine americana Merr. has bioactive compounds consisting of Hongconin, Eleutherol, Isoeleutherin, Elecanacin, and Erythrolaccin¹⁵. The potential of the bioactive compounds from Bawang Ada' as antivirals and the molecular mechanisms of these compounds have not been revealed much through research. This study aims to reveal the potential of the bioactive compounds from Bawang Ada' as antivirals with a molecular mechanism through apoptosis with an in silico approach.

METHOD:

Ligan Preparation:

Information retrieval such as 3D structure with Canonical SDF, CID, and SMILE files of the bioactive compounds from Bawang Ada' (Eleutherine americana Merr.) consisting of Hongconin, Eleutherol, Isoeleutherin, Elecanacin, and Erythrolaccin were obtained from PubChem database (https://pubchem.ncbi.nlm.nih.gov/). The ligand minimization process is performed through OpenBabel v2.4.1 to increase the flexibility of ligand atoms and convert SDF files to PDB^19,20,21.

Druglikeness Analysis:

Prediction of drug-likeness aims to determine the drug-like molecule properties of query compound with several parameters through the SWISS ADME (http://www.swissadme.ch/)^22,23. Druglikeness parameters used in this study, such as Lipinski, Ghose, Veber, Egan, and Muegge for compound bioavailability and ability analysis of compounds to pass through the cell membrane^24,25. Prediction of the probability of the compound as an apoptotic agent was peformed through PASSOnline (http://www.pharmaexpert.ru/passonline/) with medium confidence or Pa>0.3^26,27,28.

Pathway Prediction and Key Protein Retrieval:

The query and target interactions are predicted through SWISS Target Prediction (http://www.swisstargetprediction.ch/), the target data is then predicted via STRING-DB v11 (https://string-db.org/cgi/input.pl) for identification protein interactions in Homo sapiens. Key protein refers to the type of protein as a negative regulator of apoptosis in various viral infections. The 3D protein target structure with PDB format obtained through RCSB, then sterilized in PyMol v2.5 software to remove water molecule and native ligands^29,30.

Blind Docking Simulation:

After knowing some of the target proteins that contribute to the process of apoptosis when a specific virus infection occurs, then molecular docking of the Bawang Ada' compound was performed on the target using the PyRx v0.8.8 software. The blind docking was used in this study to screen the level of activity of Bawang Ada' bioactive compounds when bound to target. Binding affinity is the level of energy binding ability of a ligand to a target protein in triggering the formation of a biological response such as inhibition^31,32,33.

Chemical Interaction:

3D ligand-protein complex is displayed using the PyMol v2.5 software. by structural selection and coloring. Weak bond interactions are formed when a ligand binds to a specific domain of the target protein and triggers a biological response, several types of weak bond interactions consist of hydrogen, hydrophobic, Van der Waals, electrostatic, and pi alkyl. Weak bond interactions in this study were analyzed with LIGPLOT v.4.5.3, the software can identify hydrogen and hydrophobic bond interactions^34,35.

RESULT AND DISCUSSION:

Dayak Lundayeh tribe in North Kalimantan also uses medicinal plants such as Bawang Ada' (Eleutherine americana Merr.) as an alternative treatment as an anti-inflammatory, antipyretic and antimicrobial¹³.The results of chromatographic identification from previous studies showed Eleutherine americana Merr. has bioactive compounds consisting of Hongconin, Eleutherol, Isoeleutherin, Elecanacin, and Erythrolaccin¹⁵.The compounds from Bawang Ada' consist of Hongconin (CID: 10108147, SMILE: CC1C2= C(C3=C(C=CC=C3OC)C(=C2C(=O)C(O1)C)O)O), Eleutherol (CID: 120697, SMILE: CC1C2=C(C= C3C=CC=C(C3=C2O)OC)C(=O)O1), Isoeleutherin (CID: 4483892, SMILE: CC1CC2=C(C(O1)C)C (=O)C3=C(C2=O)C=CC=C3OC), Elecanacin (CID: 5491405 SMILE: CC1CC23C(CC2O1)C(=O) C4=C(C3=O)C=CC=C4OC), and Erythrolaccin (CID: 9817337 SMILE: CC1=CC(=C(C2=C1C(=O) C3=C(C2=O)C=C(C=C3O)O)O)O) obtained from database. Query compounds as drug-like molecules are predicted to have the same properties as drugs. Bioavailability shows the ability of a drug molecule referring to the percentage value that can circulate in the body. Drug-like molecule compounds can bind directly to targets at positions within the cell or in the cytoplasmic region. All compounds from Bawang Ada' are drug-like molecules because they follow the druglikeness parameter which consists of Lipinski, Ghose, Veber, Egan, and Muegge with a bioavailability score of 0.55 (Table 1).

Probability prediction as apoptotic agonist with medium confidence (Pa>0.3) on Bawang Ada' showed Hongconin (Pa: 0.412 and Pi: 0.068), Eleutherol (Pa: 0.484 and Pi: 0.043), Isoeleutherin (Pa: 0.531 and Pi: 0.035 ), Elecanacin (Pa: 0.526 and Pi: 0.036), and Erythrolaccin (Pa: 0.862 and Pi: 0.005), all compounds have Pa>0.3 values and are potential apoptotic agonists. Identification of the target intractor (Int) in Bawang Ada' compound showed a high probability with a percentage of ≥80%. The selection results for the identification of key proteins for antivirals showed that Elecancin had a total of 79 interactors, Eleutherol 36 interactors, Erythrolaccin 74 interactors, Hongconic 25 interactors, and Isoeleutherin 67 interactors. Compounds from Bawang Ada' have a total of >100 protein interactors with a false discovery rate (FD)<0.1 for apoptosis agonist (Figure 1). Identification of the key proteins similarity showing AKT1 and GSK3B play a role in the initiation of viral diseases and negative regulation for apoptotic agonist pathway, both proteins are probable as major targets for antivirals to trigger agonist apoptotic signaling pathways. PIK3CB and PIK3CA were not used for further analysis because they were only interactors for Isoeleutherin. (Table 2).

Table 1: The result of druglikeness from Bawang Ada’ bioactive compound

Compound	Druglikeness Parameters						Probable
	Lipinski	Ghose	Veber	Egan	Muegge	Bioavailability Score
Hongconin	Yes	Yes	Yes	Yes	Yes	0.55	Drug-like Molecule
Eleutherol	Yes	Yes	Yes	Yes	Yes	0.55	Drug-like Molecule
Isoeleutherin	Yes	Yes	Yes	Yes	Yes	0.55	Drug-like Molecule
Elecanacin	Yes	Yes	Yes	Yes	Yes	0.55	Drug-like Molecule
Erythrolaccin	Yes	Yes	Yes	Yes	Yes	0.55	Drug-like Molecule

Table 2: Network prediction of Bawang Ada’ bioactive compound and key protein in viral infection

Compound	Biological Process	False Discovery Rate	Interactors	Viral Diseases	Key Protein
Elecancin	Positive regulation of cell death	3,25e-09	17	HPV, Influenza A, Herpesvirus, EBV, HCV, HBV, HTLV-1	AKT1, GSK3B
	Regulation of apoptotic process	1,12e-08	23
	Regulation of cell death	1,78e-09	25
	Positive regulation of apoptotic process	3,58e-07	14
Eleutherol	Regulation of apoptotic process	8,02e-05	14	EBV, HBV, HPV	GSK3B, HDAC1
	Regulation of programmed cell death	2,62e-05	15
	Positive regulation of apoptotic process	0,0031	7
Erythrolaccin	Regulation of apoptotic process	5,77e-13	29	EBV, HBV, Measles, HPV, HCV, HTLV-1	GSK3B
	Regulation of programmed cell death	1,20e-13	30
	Positive regulation of apoptotic process	7,59e-08	15
Hongconic	Regulation of cell death	4,73e-06	16	HBV, Measles, HPV, Influenza A, EBV, HCV, Herpesvirus	AKT1
	Positive regulation of cell death	0,00021	9
Isoeleutherin	Regulation of cell death	7,24e-10	29	EBV, Influenza A, HCV, Measles, HBV, HPV, HTLV-1	AKT1, PIK3CB, GSK3B, PIK3CA
	Regulation of apoptotic process	2,75e-09	27
	Positive regulation of apoptotic process	0,00044	11

Figure 1: Graphical summary of pathway assessment on key protein interactors of Bawang Ada' compounds.

Glycogen synthase kinase-3 beta (GSK-3β or GSK3B) is the name of the ability to increase signaling of glucose and glycogen metabolism, GSK3B also has multifunctional functions such as being a key regulator in cell signaling, growth metabolism, and transcription factors for death or survival in organism³⁶.The role of GSK3B in cases of viral infection is inversely related, such as as a negative regulator for T cell proliferation and function^37,40. GSK3B as part form GSK-3 plays a role in the initiation of phosphorylation of viral N structural proteins (viral assembly), viral transcription, and replication^38,39,41.AKT1 or protein kinase B is a type of serine/threonine kinase as a mediator in cell survival and death signals, these protein was identified to be able to inhibit pro-apoptotic protein activity through a phosphorylation mechanism. AKT1 can trigger viral processes such as viral entry and viral protein synthesis, several studies have reported anti-apoptotic protein activity has been increased by AKT1 activating when a virus infection occurs in the host cell.Inhibition of GSK3B and AKT1 can trigger increased apoptosis through the TRAIL signaling pathway^42,43.This study aims to reveal the potential of the bioactive compounds from Bawang Ada' as antivirals with a molecular mechanism through apoptosis with an in silico approach.

Molecular docking aims to identify ligand activity on targets through binding affinity values^44,45. Binding affinity is the bond energy formed in the ligand-protein complex referring to Gibbs' law, the more negative the potential to trigger biological activity such as inhibitors^46,47,48. This study used docking simulations to identify the activity of compounds from Bawang Ada' as key protein inhibitors consisting of GSK3B and AKT1 and types of chemical interactions.The docking grid positions in this study are Center (Å) X:13.812 Y:-13.266 Z:-13.393 Dimensions (Å) X:50.435 Y:66.320 Z:66.183 for AKT1 and Center (Å) X:27.796 Y:-6.5708 Z :-38.051 Dimensions (Å) X:76.368 Y:60.256 Z:63.973 for GSK3B. Erythrolaccin had a more negative binding affinity (-9.0 kcal/mol) with the same position of the GSK3B binding site at Val70, Leu132, Ile62, Leu188, Asp200, and Cys199. Isoeleutherin has a more negative binding affinity (-9.9 kcal/mol) with the same position of AKT1 binding site Leu210, Leu264, Tyr272, Asp292, Trp80, Lys 268, Val270, and Ser205.3D structure of the protein-ligand is performed through transparent surfaces, cartoons, and sticks, single color selection on the protein and the colors of the C, H, O atoms on the ligand (Figure 2)Erythrolaccin and Isoeleutherin can be better inhibitor candidates than other compounds because they have a more negative binding affinity and hydrophobic and hydrogen bond interactions (Figure 3). Inhibition of GSK3B and AKT1 activity can trigger apoptosis in virus-infected cells.

Table 3: The results of compound binding affinity score and molecular interaction simulation

Compound	Binding Affinity (kcal/mol)		Chemical Interaction
	GSK3B	AKT1	GSK3B	AKT1
Erythrolaccin	-9,0	No Interaction	Hydrophobic:Val70, Gly63, Asn64, Ala83, Leu132, Ile62, Leu188, Asp200, Cys199, Tyr134 Hydrogen:Lys85, Val135, Asp133	No Interaction
Eleutherol	-8,3	No Interaction	Hydrophobic: Leu132, Val70, Ile62, Cys199, Leu188, Ala83 Hydrogen:Lys85, Asp200	No Interaction
Isoeleutherin	-8,2	-9,9	Hydrophobic:Tyr134, Ala83, Val135, Leu132, Cys199, Pro136, Ile62, Thr138, Leu188, Asp200, Val70, Lys85	Hydrophobic:Ile290, Thr211, Leu210, Leu264, Tyr272, Asp292, Trp80, Lys 268, Val270 Hydrogen:Ser205
Elecanacin	-7,5	-9,0	Hydrophobic:Asn64, Asp200, Val70, Leu132, Thr138, Cys199, Ala83, Leu188, Ile62, Val135	Hydrophobic:Asp292, Tyr272, Gln79, Trp80, Leu264, Ser205, Val271, Val270, Lys268, Leu210
Hongconin	No Interaction	-9,5	No Interaction	Hydrophobic:Leu264, Leu210, Ile290, Tyr272, Trp80, Asp292, Lys268, Val270 Hydrogen:Ser205

Figure 2: Molecular complex from AKT1 (red) and GSK3B (blue) docking simulation with Bawang Ada' compound. Ligand binding positions are shown via black circles. (A) AKT1_Elecanacin, (B) AKT1_Hongconin, (C) AKT1_Isoeleutherin, (D) GSK3B_Elecanacin, (E) GSK3B_Eleuthreol, (F) GSK3B_Erythrolaccin, (G) GSK3B_Isoeleutherin.

Figure 3: Molecular interaction of Bawang Ada' compounds with AKT1 and GSK3B. Hydrophobic (green line) and hydrophobic bonds (red line)  have been identified.

CONCLUSION:

Erythrolaccin and Isoeleutherin of Bawang Ada' (Eleutherine americana Merr.) is a good antiviral candidate. through a molecular mechanism of inhibiting the activity of anti-apoptotic proteins key such as GSK3B and AKT1. Erythrolaccin and Isoeleutherin have a more negative binding affinity and form hydrogen-hydrophobic interactions in the target domain. We recommend the binding sites Val70, Leu132, Ile62, Leu188, Asp200, and Cys199 (GSK3B) and Leu210, Leu264, Tyr272, Asp292, Trp80, Lys 268, Val270, and Ser205 (AKT1) for further research as antiviral target development.

ACKNOWLEDGMENT:

This work was supported by the Taman Husada Graha Famili Surabaya (Medicinal Plant Garden) and PT. Intiland Development Tbk.

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Received on 08.01.2023            Modified on 17.04.2023

Accepted on 27.05.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(5):1961-1967.

DOI: 10.52711/0974-360X.2024.00311