Pro-Apoptosis Activity of Pogostemon cablin Benth. Against Nasopharyngeal Carcinoma through the BCL-2 Inhibition Signaling Pathway: A Computational Investigation

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Makara Journal of Science Makara Journal of Science

Volume 27

Issue 3 September Article 6

9-25-2023

Pro-Apoptosis Activity of Pogostemon cablin Benth. Against Pro-Apoptosis Activity of Pogostemon cablin Benth. Against Nasopharyngeal Carcinoma through the BCL-2 Inhibition Nasopharyngeal Carcinoma through the BCL-2 Inhibition Signaling Pathway: A Computational Investigation

Signaling Pathway: A Computational Investigation

Aigia Syahraini

Department of Biology, Faculty of Mathematics and Sciences, Universitas Syiah Kuala, Banda Aceh 23111, Indonesia

Essy Harnelly

Department of Biology, Faculty of Mathematics and Sciences, Universitas Syiah Kuala, Banda Aceh 23111, Indonesia

Feri Eko Hermanto

Bioinformatics Research Centre, Indonesian Institute of Bioinformatics, Malang 65162, Indonesia, [email protected]

Follow this and additional works at: https://scholarhub.ui.ac.id/science Part of the Bioinformatics Commons, and the Cancer Biology Commons Recommended Citation

Recommended Citation

Syahraini, Aigia; Harnelly, Essy; and Hermanto, Feri Eko (2023) "Pro-Apoptosis Activity of Pogostemon cablin Benth. Against Nasopharyngeal Carcinoma through the BCL-2 Inhibition Signaling Pathway: A Computational Investigation," Makara Journal of Science: Vol. 27: Iss. 3, Article 6.

DOI: 10.7454/mss.v27i3.1484

Available at: https://scholarhub.ui.ac.id/science/vol27/iss3/6

This Article is brought to you for free and open access by the Universitas Indonesia at UI Scholars Hub. It has been accepted for inclusion in Makara Journal of Science by an authorized editor of UI Scholars Hub.

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**Pro-Apoptosis Activity of Pogostemon cablin Benth.**

Against Nasopharyngeal Carcinoma through the BCL-2 Inhibition Signaling Pathway: A Computational Investigation

Aigia Syahraini

^1,2

, Essy Harnelly

^1,2

, and Feri Eko Hermanto

^3,4*

1. Department of Biology, Faculty of Mathematics and Sciences, Universitas Syiah Kuala, Banda Aceh 23111, Indonesia

2. ARC-PUIPT Nilam Aceh, Universitas Syiah Kuala, Banda Aceh 23111, Indonesia 3. Bioinformatics Research Centre, Indonesian Institute of Bioinformatics, Malang 65162, Indonesia

4. Faculty of Animal Sciences, Universitas Brawijaya, Malang 65145, Indonesia

*E-mail: [email protected]

Received December 29, 2022 | Accepted August 7, 2023

Abstract

Resistance to chemotherapy and radiotherapy frequently emerges in the later stage of nasopharyngeal carcinoma (NPC) tumorigenesis. The decreased response of NPC to radiotherapy and chemotherapy occurs owing to the inhibition of cancer cell apoptosis by the B-cell lymphoma-2 (BCL-2) protein. Thus, inhibiting BCL-2 protein may become a powerful approach to eliminate NPC through apoptosis regulation. Meanwhile, Pogostemon cablin is reported to exhibit anticancer properties, but there are limited studies on its use for NPC treatment. The objective of this study is to investigate the potential bioactive compounds in P. cablin as anti-apoptosis BCL-2 protein inhibitors using in-silico approach. Natural compounds from P. cablin were retrieved from the KNApSAcK database and screened for inhibitory effects on BCL-2 protein via molecular docking coupled with molecular dynamics. It was found that apigenin, rhamnetin, and apigenin 7- (6″-p-coumarylglucoside) showed potential inhibitory properties against BCL-2 protein based on binding affinity and interaction chemistry. The highest binding affinity was recorded for apigenin 7-(6″-p-coumarylglucoside) at −9.9 kcal/mol, followed by rhamnetin and apigenin at −7.2 kcal/mol. These compounds are also bound to the inhibitory sites of BCL-2 and venetoclax, mainly by hydrophobic bonds and Van der Waals interactions. Nevertheless, molecular dynamics simulations revealed that apigenin 7-(6″-p-coumarylglucoside) had unstable conformation and binding to BCL- 2. In summary, this study demonstrated that P. cablin has excellent potency as an alternative or complementary therapy against radiotherapy and chemotherapy resistance of NPC, mainly through rhamnetin and apigenin.

Keywords: apigenin, B-cell lymphoma-2 inhibitor, nasopharyngeal carcinoma, pro-apoptosis, rhamnetin

Introduction

Nasopharyngeal carcinoma (NPC) is a typical head and neck squamous cell carcinoma emerging from the Rosenmüller fossa in the nasopharynx, which is a transitional area from cuboid epithelium to squamous epithelium [1]. NPC is common in southern China, Southeast Asia, and northern Africa but rare in western countries [2]. Chemotherapy and radiotherapy are two common treatments for NPC. Unfortunately, resistance to chemotherapy and radiotherapy has become a notable problem in NPC treatment as a result of the decreased response of tumors to radiotherapy or chemotherapy treatment, primarily due to the inhibition of cancer cell apoptosis by B-cell lymphoma-2 (BCL-2) protein [3].

BCL-2 is an anti-apoptotic protein that regulates apoptosis or programmed cell death [4]. In the case of

NPC, BCL-2 overexpression is highly associated with tumor cell differentiation [5] and patient mortality [6].

This event occurs due to the inhibition of apoptosis and results in faster tumor growth with decreased tumor response to radiotherapy [7]. Therefore, controlling the activity of BCL-2 by limiting its activity can be a promising way to ameliorate the pathogenesis of NPC [8].

Natural products as an alternative or complementary therapy were found to improve the pathological conditions of patients undergoing NPC treatment [9, 10]. In addition, it was shown that almost half of all human pharmaceuticals are derived from natural plant compounds [7], which exhibit the positive potential of natural products to be developed as alternative or complementary therapy for patients with NPC. Among the least-studied herbs, Pogostemon cablin has numerous biological activities to

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Pro-Apoptosis Activity of Pogostemon cablin Benth. 209

Makara J. Sci. September 2023  Vol. 27  No. 3

improve physiological homeostasis, including its curative role in prostate [11], colorectal [12], endometrial [13], and blood [14] cancer. Nevertheless, to the best of our knowledge, there is no study evaluating the activity of P. cablin against NPC. Several compounds found in P.

cablin are associated with the antiproliferative effect through the induction of cell cycle arrest and apoptosis via extrinsic and intrinsic pathways [11–14]. Owing to the absence of information related to the apoptosis modulator of P. cablin in NPC through suppression of BCL-2 protein activity, this study aimed to explore that modulatory activity computationally.

Methods

Compound data mining. The three-dimensional structure of BCL-2 protein with Protein Data Bank identifier (PDB ID) 6O0K was obtained from the Research Collaboratory for Structural Bioinformatics (RCSB PDB) (https://www.rcsb.org/) in .pdb format.

The active compounds of P. cablin were obtained from the KNApSAcK database [15], and the 3D compound structures were downloaded through PubChem in .sdf format. Venetoclax was used as a control molecule according to the previous literature [16].

Molecular docking. Molecular docking analysis was performed using AutoDock Vina [17] in PyRx 0.9.5 software [18]. BCL-2 protein was prepared using Discovery Studio version 16 to remove the previously attached ligand and water molecules. Meanwhile, ligands were prepared using Open Babel [19] integrated with PyRx to minimize their energy and then converted as PyRx ligands. The BCL-2 protein was set as a rigid macromolecule, while the compounds were treated as flexible molecules [20]. Docking was targeted into the active sites of BCL-2 protein [16] with grid settings as follows: grid center (x, y, z) of −12.454, 2.703, and

−9.653 with dimensions (x, y, z) of 19.696, 22.680, and 31.748. The complex with binding affinity lesser than

−7.0 kcal/mol was selected for ligand–residue interaction analysis using Discovery Studio 2019 [21]. Compounds exhibiting a remarkable number of interactions with the active site of BCL-2 protein were directed into molecular dynamics analysis [21, 22].

Molecular dynamics. Molecular dynamics simulations were performed for 50 ns with the AMBER14 forcefield [23] using the following parameters: pH 7.4, 0.9% NaCl concentration, 0.997 water density, 1 bar pressure, and 310 K temperature with cubic grid shape. All simulations were performed in YASARA software version 21 [24] in 2000 timesteps with simulation box size as mentioned in the supplementary file (Table S1). Binding energy fluctuations were also calculated using the Poisson–Boltz- mann surface (PBS) method [25] in YASARA macros in accordance with the previously described equation and method [26].

Estimation of absorption, distribution, metabolism, excretion, and toxicity. Compounds evaluated using the molecular docking and molecular dynamics were assessed for potential absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties according to the prediction by pkCSM [27].

Results and Discussion

Molecular docking. Molecular docking was conducted on the 26 bioactive compounds against the BCL-2 protein. Nine compounds have the lowest binding affinity (≤ −7.0 kcal/mol), which describes the most potent compound to interact with BCL-2. The lowest ligand binding affinity between nine compounds was apigenin 7-(6″-p-coumarylglucoside) at −9.9 kcal/mol. However, all the bioactive compounds were less firmly bound than venetoclax as the common inhibitor of BCL-2.

Fortunately, some compounds exhibited numerous hydrogen bonds accompanied by hydrophobic bonds and some Van der Waals interactions. Apigenin 7-(6″-p- coumarylglucoside) had the most interactions, primarily through Van der Waals interaction. Although ombuin had the most hydrogen bonds, it had the lowest number of hydrophobic bonds. Interestingly, rhamnetin formed adequate hydrogen, hydrophobic bonds, and Van der Waals interactions with all of the interacted residues found in venetoclax (Table 1).

In addition, the hydrophobic surface of the binding site contributes to the stability of protein–ligand interaction.

Therefore, the hydrophobicity map of the binding site was also evaluated. Generally, the compounds from P.

cablin have a similar hydrophobicity map compared to that of venetoclax. The interaction of these molecules with BCL-2 was characterized by high hydrophobicity, particularly for apigenin 7-(6″-p-coumarylglucoside), rhamnetin, and apigenin (Figure 1). The surface hydrophobicity of proteins, which also commonly serve as ligand binding pocket, contributes to its structure’s protein–ligand stability and flexibility [28]. Along with polar interaction, the proteins’ hydrophobic surface accommodates its thermodynamic stability [29].

Therefore, the binding of the compounds to the hydrophobic regions of the proteins allows interaction stabilization to perform its bioactivity.

Some hydrogen bonds in the protein–ligand complex also affects the stability and energy requirements of the protein–ligand interaction [30]. The hydrogen bond performs a robust and attractive force between the protein and ligand through bond formation through their functional groups, such as carboxyl, amino, or hydroxyl groups [31]. Remarkably, the hydrogen bond comprises the primary interaction type of inhibitor of proteins in the PDB database [32], suggesting the role of this interaction in the discovery of a protein antagonist. Thus, due to their similar binding characteristics – including affinity, residue

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binding sites, interaction type, and hydrophobicity mapping – in comparison to Venetoclax, apigenin,

rhamnetin, and apigenin 7-(6″-p-coumarylglucoside) were selected for molecular dynamics simulations.

Table 1. Binding Affinities of the Compounds from P. cablin that have Lower Binding Energy than −7 kcal/mol

Compound

Binding Affinity (kcal/mol)

Hydrogen Bond Hydrophobic

Bond Van der Waals Others

Venetoclax −12.1

ASN 143; ASP 111; ASP 103;

GLY 145; ALA 100; TYR 202.

ARG 146; TYR 108; LEU 137;

VAL 133; MET 115; PHE 112;

VAL 156; PHE 104; ALA 149;

VAL 148.

PHE 198; TRP 144;

GLU 136; PHE 153;

GLU 152; SER 105;

ARG 107.

N/A

Cycloseychellene −7.1 N/A

TYR 202; ALA 100; PHE 198;

VAL 148; PHE 104.

TRP 144; GLY 145;

ASP 103; TYR 108;

ARG 107.

N/A

δ-Patchoulene −7.3 N/A

PHE 104; VAL 156; PHE 112;

ALA 149; MET 115; LEU 137.

ASP 111; TYR 108;

SER 105; GLU 152;

PHE 153; VAL 133.

N/A

Apigenin 7-(6″-p-

coumarylglucoside) −9.9 ASP 103; PHE 104.

MET 115; ALA 149; VAL 148.

VAL 133; ASP 111;

PHE 112; VAL 156;

SER 105; GLU 152;

PHE 153; TYR 108;

ASN 143; ARG 107;

ALA 100; GLY 145;

ASP 140; LEU 137;

GLU 136.

ARG 146.

Ombuin −7.1 LEU 137; ARG

146; ASP 111.

ALA 149; MET 115.

GLU 136; ASN 143;

PHE 104; PHE 153;

VAL 156; GLU 152;

PHE 112; GLU 114.

N/A

α-Guaiene −7.1 N/A

MET 115; PHE 104; VAL 156;

PHE 153; LEU 137; ALA 149;

VAL 133.

GLU 114; ASP 111;

PHE 112; SER 105;

GLU 152.

N/A

Rhamnetin −7.2 PHE 104; ASP

103.

ARG 146; ALA 149; VAL 148.

ASN 143; LEU 137;

ALA 100; ARG 107;

TYR 108; GLY 145.

N/A

Pachypodol −7.2 ALA 100.

ARG 146; GLY 145; VAL 148;

ALA 149; PHE 104.

ASN 143; LEU 137;

TYR 108; ARG 107;

PHE 198.

ASP 103.

β-Caryophyllene −7.5 N/A

PHE 104; VAL 156; MET 115;

PHE 153; ALA 149; LEU 137;

VAL 133.

GLU 152; PHE 112;

ASP 111. N/A

Apigenin −7.2 ALA 100.

GLY 145; ARG 146; ALA 149;

PHE 104; VAL 148.

ARG 107; TYR 108;

ASN 143; LEU 137. ASP 103.

Notes: The amino acids in bold describe a similar binding site with venetoclax. Hydrophobic bond refers to the interactions involving π-hydrophobic (π–π stacked, π–π T-shaped, and amide π-stacked), alkyl hydrophobic (alkyl), and mixed π/alkyl hydrophobic (π-sigma and π-alkyl). Others means unfavorable bonds. N/A means not available.

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Figure 1. Hydrophobicity Map of the Binding Site of the BCL-2 Receptor Bound with the Ligands: Venetoclax (a), Cyclo- seychellene (b), δ-patchoulene (c), Apigenin 7-(6″-p-coumarylglucoside) (d), Ombuin (e), α-guaiene (f), Rhamnetin (g), Pachypodol (h), β-caryophyllene (i), and Apigenin (j)

Earlier works using molecular docking approaches reported the inhibitory properties of apigenin against BCL-2. Sekar et al. revealed that apigenin bound to BCL- 2 with a binding energy of −7.8 kcal/mol, but the specific binding site was not identified [33]. Similarly, Ghani et al. observed binding between apigenin and BCL-2 with a binding energy of −7.2 kcal/mol, but the binding site and hydrogen bonding differed from the present study [34].

This could be due to variations in grid box position and the absence of a native ligand or control inhibitor for reference. Gyebi et al. investigated the apigenin analog, apigenin 7,4′-dimethyl ether, that showed different interaction sites and no hydrogen bonding compared to the current study [35]. They used the navitoclax binding site as the reference inhibitory position. However, no studies have reported the inhibitory activities of rhamnetin and apigenin 7-(6″-p-coumarylglucoside) against BCL-2. This study aims to be the first to explore the potential of these compounds as BCL-2 inhibitors.

Although in-silico analysis did not show lower binding affinities of P. cablin’s active compounds to the BCL-2 protein compared to venetoclax, the combination of multiple compounds in P. cablin may results in a synergistic effect, leading to potent anticancer activity.

Natural substances were observed to work together to target specific proteins or pathways, enhancing their effectiveness [36]. For example, natural substances were found to have an anticancer effect on annexin A2, a multifunctional tumor-associated protein [37]. Similarly, for P. cablin, its extract disrupted the cell cycle and

induced apoptosis in colorectal cancer. Its combination with the chemotherapeutic agent 5‐fluorouracil produced an even more pronounced effect [12]. This suggests that natural compounds from P. cablin hold great promise for cancer treatment as their potential synergistic activity can effectively induce apoptosis, making them highly effective in killing cancer cells.

Molecular dynamics simulation. To investigate the stability of the contact between proteins and P. cablin compounds, molecular dynamics simulations were performed on apigenin, apigenin 7-(6″-p-coumarylglucoside), and rhamnetin, which displayed promising binding from the molecular docking analysis.

Equilibrium state was realized in all simulated complexes along the simulation periods (supplementary file (Figure S1)). The findings showed that the binding of P. cablin’s compounds did not significantly impact the stability of the backbone atoms of the protein, as indicated by the root-mean-square deviation (RMSD) of the backbone atoms (Figure 2a). However, the RMSD of the ligand structures indicated that apigenin and rhamnetin had similar stability to the venetoclax (Figure 2b).

Interestingly, only rhamnetin remained stably bound to BCL-2, with minimal movement compared to the other compounds (Figure 2c). Moreover, the instability of the other compounds had less effect on the binding energy fluctuations (Figure 2d), the number of hydrogen bonds in the complexes (Figure 2e), and the root-mean-square fluctuation (RMSF) of each protein residue (Figure 2f).

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The data from the molecular dynamics simulations described the potential inhibitory activities of apigenin and rhamnetin, but not with apigenin 7-(6″-p- coumarylglucoside), through their interactions and structural stabilities. Polar and nonpolar interactions along with ligand stereochemistry may have influenced the stability of compound structure and binding with proteins [32,38]. The stability of venetoclax supported this phenomenon compared to P. cablin’s compounds according to the RMSD of the ligand structure and ligand movement (Figures 2b and c). Compared to the other complexes, many polar and nonpolar interactions were formed in the venetoclax–BCL-2 complex (Table 1), which contributes to the binding stability of venetoclax to BCL-2. However, slight differences were found in the complex of BCL-2 with P. cablin’s compound in terms of their interaction chemistry. As a consequence, this phenomenon may describe the impact of stereochemistry in the stability of the P. cablin–BCL-2 interaction.

Nevertheless, the binding energy fluctuations and total hydrogen bonds in each complex showed no significant differences, suggesting that these three compounds could be excellent candidates as BLC-2 antagonists. Hydrogen bonds formed between the ligand and the protein serves to maintain a compact structure, where the flexibility of the protein residues also comes into play as they are the ones that will form bonds with the ligand molecules. The binding of these compounds not only influence the amino acid fluctuations but also supports the stability of the interaction to inhibit BLC-2 activity. A higher RMSF suggests increased flexibility, thus increasing the potential to interact with ligand molecules. The RMSF values of the three simulated ligands were close to that of

venetoclax; although there are considerable fluctuations in residues ASP 35, ARG 110, and ARG 183, they do not have a major effect because they are not in the active site of the protein. Thus, apigenin and rhamnetin may be effective compounds in the anticancer mechanism of P.

cablin, mainly by suppressing the anti-apoptosis mechanism.

ADMET properties of the screened compounds.

Apigenin and rhamnetin (Figure 3) were found to be effective BCL-2 inhibitors through molecular docking and dynamics simulations. ADMET analysis was next performed to evaluate their drug-likeness properties using pharmacokinetic parameters (Table 2). From the pkCSM prediction, both compounds exhibited good solubility in water and permeability through the gastrointestinal tract, although rhamnetin had lower permeability across intestinal cells. Nonetheless, considering that drug absorption is influenced by factors such as efflux transport and metabolism beyond Caco-2 permeability [39], rhamnetin still retains a favorable likelihood for its absorption properties.

Distribution analysis revealed excellent distribution volume (VDss) and low fraction unbound scores for both compounds, suggesting their efficient distribution through the bloodstream [40, 41]. However, they exhibited low permeability across the central nervous system (CNS). Fortunately, this characteristic is less significant as the target is not located in the CNS, where the blood–brain barrier (BBB) and the blood–

cerebrospinal fluid barrier highly limit the distribution of certain compounds in the CNS.

Figure 2. Molecular Dynamics Simulations on the Interaction of BCL-2 with the Selected Compounds: RMSD Backbone Atoms (a), RMSD Ligand Conformations (b), RMSD Ligand Movements (c), Binding Energy Fluctuations (d), Number of Hydrogen Bonds (e), and RMSF (f)

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Figure 3. Two-dimensional Structures of Apigenin and Rhamnetin as the Best Predicted Compounds in P. cablin to Bind with BLC-2

Table 2. ADMET Properties of Apigenin and Rhamnetin Based on the pkCSM Prediction

Model Name Unit Apigenin Rhamnetin

Absorption

Water solubility Numeric (log mol/L) −3.329 −3.212

Caco-2 permeability Numeric (log Papp in 10⁻⁶cm/s) 1.007 −0.361

Intestinal absorption (human) Numeric (% Absorbed) 93.25 80.214

Distribution

VDss (human) Numeric (log L/kg) 0.822 0.419

Fraction unbound (human) Numeric (Fu) 0.147 0.073

BBB permeability Numeric (log BB) −0.734 −1.345

CNS permeability Numeric (log PS) −2.061 −3.235

Metabolism

CYP2D6 substrate Categorical (Yes/No) No No

CYP3A4 substrate Categorical (Yes/No) No No

CYP1A2 inhibitor Categorical (Yes/No) Yes Yes

CYP2C19 inhibitor Categorical (Yes/No) Yes No

CYP2C9 inhibitor Categorical (Yes/No) No No

CYP2D6 inhibitor Categorical (Yes/No) No No

CYP3A4 inhibitor Categorical (Yes/No) No No

Excretion

Total clearance Numeric (log ml/min/kg) 0.566 0.473

Renal OCT2 substrate Categorical (Yes/No) No No

Toxicity

AMES toxicity Categorical (Yes/No) No No

Oral rat acute toxicity (LD50) Numeric (mol/kg) 2.45 2.453

Oral rat chronic toxicity (LOAEL) Numeric (log mg/kg bw/day) 2.298 2.679

Hepatotoxicity Categorical (Yes/No) No No

Minnow toxicity Numeric (log mM) 2.432 1.885

Apigenin and rhamnetin showed favorable metabolic activity against different cytochrome enzyme isoforms.

They were not identified as substrates for CYP2D6 and CYP3A4, suggesting improved pharmacokinetic properties, including lower clearance and longer half-life, which can enhance efficacy and reduce toxicity [42].

Additionally, both compounds were predicted to inhibit CYP1A2, which is associated with stable plasma concentrations and potential benefits in increasing toxicity against cancer cells and reducing adverse symptoms when co-administered with chemotherapeutic agents [43].

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Apigenin and rhamnetin were evaluated for their excretion properties based on total clearance and renal OCT2 substrate parameters. The results indicated that both compounds are likely to be excreted without excessive accumulation and have fewer potential contraindications [27]. Moreover, toxicity estimations suggested low potential toxicity for both compounds.

They showed no toxic effects in AMES toxicity, hepatotoxicity, and Minnow toxicity assays, supported by low predicted toxic doses in oral rat acute and chronic toxicity studies. These predictions were supported by existing literature stating apigenin and rhamnetin as low toxic natural-derived compounds with broad bioactivities [44, 45]. In summary, both apigenin and rhamnetin demonstrate excellent potential as BCL-2 inhibitors with favorable ADMET properties. However, further research is required to validate these findings and develop precise formulations to enhance their bioavailability.

Conclusion

Among several active compounds from P. cablin, apigenin, apigenin 7-(6″-p-coumarylglucoside), and rhamnetin have the best binding energies and interaction chemistry. However, apigenin 7-(6″-p-coumarylglucoside) exhibited unstable conformation and binding during molecular dynamics analysis. Therefore, apigenin and rhamnetin were the most potent compounds as BCL-2 inhibitors to ameliorate the NPC conditions. Both compounds exhibit strong interactions with BCL-2 according to the binding energy, binding site, structural integrity, and interaction stability.

Acknowledgment

The authors thank to Prof. Nashi Widodo from Depart- ment of Biology, Brawijaya University, for providing the resources and licenses to perform molecular dynamics analysis.

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