Dengue is a viral infection caused by the dengue virus, which is transmitted to humans primarily through the bites of infected female Aedes aegypti and Aedes albopictus mosquitoes (Aguiar et al. 2022; Ferreira-de-Lima and Lima-Camara 2018). This disease is a significant public health concern, especially in tropical and subtropical regions of Southeast Asia, the Pacific Islands, the Caribbean, and Latin America. According to the World Health Organization, dengue affects more than 390 million individuals yearly, with approximately 67% of combined benign and malignant cases are concentrated in Asian nations (Ma and Cheng 2022). In the Philippines alone, the country’s health department reported 220,705 dengue-related cases in 2022, with a case fatality rate of 0.3% and an increase of 182% cases reported compared to the same period in 2021 (Villanueva 2023). Currently, there are no direct and specific treatments that could suppress the progression of the dengue virus inside the human body (Wong et al. 2022). Supportive care remains the primary approach, focusing on symptom management and fluid replacement. However, this supportive care is not sufficient; the disease may later progress into a severe one, which could become fatal to a dengue patient (Ghetia et al. 2022; Harapan et al. 2020). This critical gap in antiviral therapy necessitates the development of innovative approaches to combat the virus.

From a molecular perspective, dengue virus is a single-stranded RNA virus classified within the Flavivirus genus of the Flaviviridae family (Endale et al. 2021). DENVs can be categorized into four serotypes, namely DENV-1, DENV-2, DENV-3, and DENV-4 (Ullah et al. 2023); though all serotypes are genetically similar, DENV-3 has been observed to have a more widespread distribution compared to the rest of the serotypes (Bravo et al. 2014; Dieng et al. 2021; Rimal et al. 2023; Sy et al. 2023; Tchetgna et al. 2021). A dengue virus serotype contains an 11kb RNA genome that also houses a significant protein called NS5. It is a multifunctional protein vital for viral replication and transcription that also serves a dual role as an RNA-dependent RNA polymerase (RdRp) and an RNA methyltransferase (MTase) enzyme (Bhatnagar et al. 2021). The RdRp domain enables the replication of the viral genome, a vital step in the virus’s life cycle. It acts as a molecular photocopier, ensuring the virus can reproduce within host cells (Osawa et al. 2023). Furthermore, NS5’s RNA MTase enzyme activity adds a protective cap structure to the viral RNA, preventing its degradation and enhancing translational efficiency (Feracci et al. 2023). Inhibiting the functions of NS5 could disrupt viral replication, providing a potential avenue for treatment.

Since the prehistoric period, plants have been used by the common folks to alleviate different diseases and ailments; with the help of today’s modern technology, medicinal plants are shown and used to be the major composition of drugs today. These biological organisms contain biologically active compounds called phytochemicals. These molecules have a role of being an antioxidant that serves as protective layer for cells to protect them from the free radicals that can cause illness (Park 2023). In the present, at least eight out of ten common drugs containing potent phytochemicals that are derived from plants are being used to treat infections and inflammations, cardiovascular diseases, and cancer (Thomas 2021). Given its natural components, capability to combat free radicals, and ability to inactivate pathogenic proteins, many species of conventional and novel medicinal plants are being given spotlight for further research and therapeutic development (Zhang et al. 2023).

The Philippines is home to some of the medicinal plants renowned for their bioactive compounds and therapeutic potential targeting the DENV NS5 protein (Cordero and Alejandro 2021). Anacardic acid, known for its inhibitory effects against viruses, is a crucial compound potentially hindering the functions of DENV NS5 (Dayrit et al. 2021). Chloroquine, historically used to treat malaria, has shown potential antiviral effects against dengue (Tomasiak et al. 2023). Methyl gallate, a polyphenol with antioxidant and antiviral properties, may also play a crucial role in inhibiting replicating the multiplication of the dengue virus in human cells (Rodrigo et al. 2020). Carica papaya (papaya) is a common edible fruit that has two specific phytochemical compounds found antiviral properties. Kaempferol, a flavonoid, exhibits a range of biological activities, including antiviral effects, making it a candidate for interfering with DENV NS5 functions. Myricetin is known for its antiviral potential, which can inhibit various viruses (Liang et al. 2023). Psidium guajava (guava) is also recognized for its antiviral potential, and two specific compounds derived from this plant are of interest in dengue treatment. Suramin, a known antiviral agent, has been studied for its efficacy against various viruses (Arabyan et al. 2021; Badshah et al. 2021). Cianidanol, a compound similar to Catechin, is another antiviral component found in P. guajava leaves (Lim et al. 2020). Musa paradisiaca (banana), a staple in Philippine cuisine, can be another source of potential dengue virus inhibitors; Quercetin, a flavonoid derived from the fruit, is known for its antiviral effects, making it a candidate for inhibiting DENV NS5 and preventing viral replication (Udaya Rajesh and Sangeetha 2023). Lastly, Andropogon citratus (lemon grass) presents Citral, a compound with recognized antiviral properties (Gao et al. 2020).

A recent study from Johnson & Johnson (J&J) have begun producing the antiviral compound JNJ-1802, or Mosnodenvir as registered in National Center for Biotechnology Information (NCBI) PubChem, in 2019. The JNJ-1802 inhibits and targets NS3 and NS4B to stop the virus from replicating and creating new viral RNA. Its testing phase is administered orally comes in vials containing 250 mg and 4000 mg of white to off-white powder. The said testing was conducted in the countries with high cases of dengue: Philippines, Thailand, Peru, Brazil and Colombia (Goethals et al. 2023). The outcome of the testing has provided limitations due to the side effects like grade 2 rash in some patients making it less effective when taken in higher doses (Global Data 2023). J&J’s project to develop anti-viral drug to combat dengue is still under evaluation. As of date, there is still no approved antiviral drug against dengue.

To speed up the early stages of drug development, in silico or computational approaches have become invaluable. Candidate selection and molecular docking of different potential plant phytochemical inhibitors allow for the identification of potential drug candidates and the prediction of their binding affinities with the target dengue protein. Molecular docking is a current established standard method in assessing phytochemical inhibition of a potential drug component using virtual computations (Kaur et al. 2019; Manojkumar et al. 2024). Thus, this study aims to identify potential plant molecule inhibitors of the dengue virus NS5 protein and analyze the level of phytochemical inhibition and interactions to a DENV serotype that could potentially help contribute to the future development of antiviral drugs for dengue. This study also aims to identify novel phytochemical compounds with superior therapeutic potential compared to Mosnodenvir for the treatment of dengue.

NS5 Protein Sequence Retrieval

The NS5 protein sequence of the Dengue virus were obtained through Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank database at https://www.rcsb.org, specifically the 3D crystalline structures of DENV-2 NS5 (PDB ID: 5ZQK) and DENV-3 NS5 (PDB ID: 4V0Q). NCBI Basic Local Alignment Search Tool Protein (blastp) analysis, derived from https://blast.ncbi.nlm.nih.gov/Blast.cgi?%20PROGRAM=blastp, was conducted to compare the DENV-2 and DENV-3 structures and to evaluate their possible proteomic dissimilarity.

Generation of Compound Library

A thorough review of the relevant scientific literature was conducted to compile a comprehensive library of compounds known to possess inhibitory properties against the NS5 protein. Different medicinal plants native in the Philippines were also selected for the study due to their documented antiviral properties in established articles.

Retrieval of Ligand Molecule

The identified molecules from the compound library were canvassed through NCBI PubChem via https://pubchem.ncbi.nlm.nih.gov/. The corresponding 3D structures were then retrieved and converted into Protein Data Bank with partial charge Q and atom type T (.pdbqt) file format.

Molecular Docking of Protein and Ligands

The molecular docking of protein and ligand molecules was performed using the software Python Prescription (PyRx), openly sourced at https://pyrx.sourceforge.io/. The program was chosen as the software of choice as it utilizes AutoDock Vina and Python as its core languages, commonly used by different researchers globally for in silico due to its efficiency in docking (Vieira and Sousa 2019). The protein structure was loaded into the program and converted into a macromolecule to make it suitable for docking analysis. Subsequently, the ligands were then added to the program and their respective energies were minimized as part of the pre-processing procedure. The energy minimization parameters were as follows: force field was set as “uff”, optimization algorithm was set as “conjugate gradients”, total number of steps was configured to “2000”, number of steps for update was configured to “1”, and it shall stop if the energy difference is less than 0.1. Afterwards, the ligands with minimized energies were then converted into .pdbqt file format. The protein and ligands were selected before proceeding to resize the grid box around the active site residues and running it through Vina Wizard to begin docking. During these simulations, the binding sites for ligand inhibition were predicted systematically. The product of the molecular docking simulation was the formation of a protein-ligand complex.

Structural Modeling of Ligand-Protein Complex

PyMOL, openly sourced at https://pymol.org/2/, and Discovery Studio Visualizer, sourced at https://discover.3ds.com/discovery-studio-visualizer-download, are software that were used to visualize the docked protein-ligand complexes which resulted in obtaining the pharmacophore model that represents 3D molecular features that were used for effective binding to the NS5 protein. In addition, this was used as one of the reference points during the identification of potential NS5 inhibitors from the compound library.

Binding Affinity Analysis of Ligand-Protein Complex

The docked protein-ligand complex was analyzed regarding their binding affinity through PyRx and PyMOL. In identifying the optimal binding site, root mean square deviation (RMSD) was used to quantify the average distance of atoms of a docked ligand the NS5 protein which is significant for the effectiveness of potential inhibitors (Castro-Alvarez et al. 2017). Binding affinity was also assessed the stability of complex formed and to determine the boundedness of two wherein the higher binding affinity indicates a stronger and more stable interaction, often associated with better biological activity and therapeutic potential (Pantsar and Poso 2018).

Filtering and Ranking of Ligand Molecules

The inhibitor compounds were filtered to ensure that non- effective compounds were not ranked with the most effective ones using the calculated RMSD. The compounds that are effective were ranked based on their respective binding energy values. Compounds with the most negative value were selected as the most favorable and have potentially effective interactions to ensure that those with the highest binding affinity for NS5 proteins would be selected for recommendation for further analysis and experimental validation, thus providing a foundation for potential antiviral drug development.

Target Identification and Protein Sequence Retrieval

In search of the best DENV serotype to use for molecular docking, blastp analysis and crystalline structures from Protein Data Bank of NS5 DENV-2 and NS5 DENV-3 are reported (Table 1). The blastp results of query protein sequences of 4V0Q and 5ZQK, shown in Figs. 1 and 2 respectively, have met the parameter that includes the overall similarity, strength of the match, extensive alignment, and an E-value of 0.0 that signifies low probability of the similarity occurring by chance (Al-Khayyat and Al-Dabbagh 2016). Among all serotypes, DENV-3 showed high alignment scores and low E-values indicating substantial conservation of amino acid sequences, making DENV-3 the candidate for molecular docking.

Figure 1. Amino Acid Sequence of 4V0K. The figure illustrates the amino acid sequence of the 4V0Q protein as derived using the Discovery Studio Analyzer. The colors indicate residual hydrophobicity, with hydrophobic residues represented in yellow, hydrophilic residues in violet, and neutral residues in green. Beta strands are denoted by thick blue arrows, while alpha helices are represented by solid red cylinders

Figure 2. Amino Acid Sequence of 5ZQK. The figure illustrates the amino acid sequence of the 5ZQK protein as derived using the Discovery Studio Analyzer. The colors indicate residual hydrophobicity, with hydrophobic residues represented in yellow, hydrophilic residues in violet, and neutral residues in green. Beta strands are denoted by thick blue arrows, while alpha helices are represented by solid red cylinders

Table 1 . Blastp hit results of the DENV-2 and DENV-3 proteins.

Max score	Total score	Query cover	E-value	Percent identity
1520	1520	99%	0.0	79.66%

Preparation of Phytochemical Molecules

Plants produce phytochemical molecules, which are biologically active substances with antioxidant capabilities (Kumar et al. 2023). Supported by various scientific literature and generated ligands in NCBI Pubchem, there are a total of 12 phytochemical molecules with Mosnodenvir as the ligand of comparison (Table 2).

Table 2 . Molecular docking of plant phytochemical ligands against selected DENV proteins.

Plant phytochemical	Molecular formula	Plant	Reference
Anacardic acid	C22H36O3	Vitex negundo	Tomasiak et al. 2023
Chloroquinone	C6H3ClO2	Vitex negundo	Rodrigo et al. 2020
Methyl gallate	C8H8O5	Vitex negundo	Liang et al. 2023
Kaempferol	C15H10O6	Carica papaya	Arabyan et al. 2021; Badshah et al. 2021
Myricetin	C15H10O8	Carica papaya	Arabyan et al. 2021; Badshah et al. 2021
Cianidanol	C15H14O6	Psidium guajava	Lim et al. 2020
Quercetin	C15H10O7	Musa paradisica	Udaya Rajesh and Sangeetha 2023
Citral	C10H16O	Andropogon citratus	Gao et al. 2020
10-Gingerol	C21H34O4	Zingiber officinale	Levita et al. 2018
6-Shogaol	C17H24O3	Zingiber officinale	Levita et al. 2018
Carvacrol	C10H14O	Origanum amboinicus	Giatropoulos et al. 2022
Allicin	C6H10OS2	Allium sativum	Rouf et al. 2020

Molecular Docking of Protein and Ligand Molecules

Molecular docking, a computational method for studying protein-ligand interactions, utilizes thermodynamic principles, notably Gibbs free energy (ΔG), to predict binding affinities. Gibbs free energy (ΔG) evaluates a system’s potential to perform work under constant temperature and pressure, indicating the driving forces within thermodynamics. In this method, protein-ligand binding results in a negative ΔG at equilibrium under constant pressure and temperature, signifying the association. The magnitude of this negative change determines the stability of the complex and the ligand’s binding affinity (Du et al. 2016). The formula for calculating binding energy is presented as Eq. 1, where it involves the free energies of the complex (G_complex), protein (G_protein), and ligand (G_ligand).

Anacardic acid, present in Anacardium occidentale (cashew) nuts, exhibits diverse beneficial effects such as antioxidative, antiparasitic, antibacterial, and anti-inflammatory properties (Tomasiak et al. 2023). It also demonstrates inhibition of inflammation and oxidative stress (Chen et al. 2023), primarily targeting the RdRp domain by interacting with specific amino acids: Asn405, Lys401, Val402, Ala421, and Phe485. Additionally, it forms alkyl bonds and a π-alkyl interaction with Val603, Trp477, and Arg481 as shown in Table 3. Derived from Vitex negundo, this phytochemical molecule has a comparatively lower binding affinity, suggesting limited potential as an NS5 protein inhibitor as presented in Table 2.

Table 3 . Molecular docking interactions of plant phytochemical ligands with the DENV-3 NS5 protein.

Plant phytochemical	Inhibited site	Amino acid involved	Interactions
Anacardic acid	RdRp domain	Asn405, Lys401	Conventional hydrogen bond
		Val402	π-Sigma interaction
		Ala421, Phe485	Alkyl bond
		Val603, Trp477, Arg481	Alkyl bond, π-Alkyl interaction
Chloroquinone	RdRp domain	Thr360	π-Sigma interaction
		Arg540, Ala259	Conventional hydrogen bond
		Gly258	Carbon-hydrogen bond
Methyl gallate	MTase domain	Asn69	Conventional hydrogen bond
		Pro298	π-Alkyl interaction
		Arg352	Alkyl bond
Kaempferol	MTase domain	Glu296, Asn69	Conventional hydrogen bond
		Glu67	Conventional hydrogen bond, π-sigma
		Val66, Pro298, Leu94	π-Alkyl interaction
		Ile72	π-Sigma interaction
		Lys96	Unfavorable donor-donor interaction
Myricetin	RdRp domain	Glu356	Conventional hydrogen bond
		Arg540	Conventional hydrogen bond, π-cation interaction
		Gly258	Carbon-hydrogen bond
		His52	π-π Interaction
		Asp256	π-Anion interaction
		Ala535	π-Sigma interaction
		Lys357	π-Alkyl interaction
		Lys689	Unfavorable donor-donor interaction
Cianidanol	MTase domain	Thr214, Asp146	Unfavorable donor-donor interaction, unfavorable Acceptor-acceptor interaction
		Lys180	π-Cation interaction
		Gly148, SAH1051, ACT1884	Conventional hydrogen bond
		Leu182, Glu216, Trp87, Ser150, Glu149, Gly58, Ser59, Ser56, Gly86, Arg57	Van der Waals forces
Quercetin	MTase domain	ACT1887	Conventional hydrogen bond
		Ile691	Conventional hydrogen bond, amide-π stacked interaction
		Glu49	π-Cation interaction
		Arg47	π-Anion interaction
		Pro692	π-Alkyl interaction, π-Sigma interaction
		Thr50	Carbon-hydrogen bond
Citral	MTase domain	Ser31	Conventional hydrogen bond
		Leu27, Phe242, Tyr28, Leu20	Alkyl bond
10-Gingerol	RdRp domain	Asn492	Carbon-hydrogen bond
		Gln602, Lys401	Conventional hydrogen bond
		Val603	π-Sigma interaction, alkyl bond
		Arg481	π-Alkyl interaction
		Val402, Ala421	Alkyl bond
6-Shogaol	MTase domain	Lys95, Gln351	Conventional hydrogen bond
		Lys355	π-Sigma interaction, alkyl bond
		Pro298	π-Alkyl interaction
		Ile72, Arg581	Alkyl bond
Carvacrol	MTase domain	Ile94, Ile72	Alkyl bond
		Pro298	π-Alkyl interaction
		Val66	π-Sigma interaction
		Glu67	Conventional hydrogen bond
Allicin	MTase domain	Lys355, Arg352, Phe348, Pro582	Alkyl bond
		Glu67	Carbon-hydrogen bond
Mosnodenvira	RdRp domain	Glu356	Halogen bond
		Tyr119	Conventional hydrogen bond, π-π interaction
		Asn682	Carbon-hydrogen bond
		Arg540, Thr261	Conventional hydrogen bond
		Lys357, Val687, Pro363	Alkyl bond
		Arg362	π-Alkyl interaction
		His263	Carbon-hydrogen bond, π-alkyl interaction

^aMosnodenvir is the primary ligand for comparison for all plant phytochemicals (Goethals et al. 2023)..

Chloroquinone, a derivative of quinine, is extensively used for malaria treatment and various infections, including autoimmune diseases (Colson et al. 2020). Table 3 presents that it targets the RdRp domain, interacting with amino acids Thr360, Arg540, Ala259, and Gly258. As per Table 2, it is derived from the plant Vitex negundo, however, its binding affinity is also relatively weak, making it less effective and reliable as an NS5 protein inhibitor.

Methyl gallate, as per Table 2, is mainly derived from Vitex negundo and is abundant in natural plants. It also boasts numerous biological functions including anti-tumor, anti-inflammatory, antioxidant, hepatoprotective, and anti- microbial activities (Liang et al. 2023). It inhibits the MTase domain through interaction with amino acids including: Asn69 with a conventional hydrogen bond, Pro298 with a π-alkyl interaction, and Arg352 with an alkyl bond as shown in Table 3.

Kaempferol, isolated from Camellia sinensis and Carica papaya(Table 2), demonstrates anti-inflammatory and anticarcinogenic effects (Badarau et al. 2022). It exhibits effectiveness against bacteria such as E. coli, B. subtilis, and K. pneumoniae. As presented in Table 3, inhibition occurs at the MTase domain, involving interactions with amino acids Glu296, Asn69, Glu67, Val66, Pro298, Leu94, Ile72, and Lys96. Kaempferol is noted for its high binding affinity and considered as the highest among all plant phytochemical molecules, particularly in inhibiting the NS5 protein.

Myricetin, an antioxidant found in fruits and vegetables, boosts antioxidant enzyme levels, suppresses inflammation by inhibiting cytokines, and may reduce mortality rates. It shows promise in combating viral infections by disrupting DNA replication pathways, particularly inhibiting the RdRp domain (Agraharam et al. 2022). Based on Table 3, some of the key amino acids involved in this inhibition include: Glu356, Arg540, Gly258, and His52, engaging in various interactions like hydrogen bonds, π-cation, and π-π interactions. As presented in Table 2, it is derived from Carica papaya and exhibits high binding affinity, making it an effective inhibitor of the NS5 protein, together with Kaempferol and 6-Shogaol.

Cianidanol, a flavonoid antioxidant derived from Psidium guajava as seen in Table 2, demonstrates promising pharmaceutical potential against SARS-CoV-2 infection. Its excellent binding and ADME properties make it a candidate for therapeutic use pending further validation through experimental and clinical trials (Srivastava et al. 2020). Inhibition occurs at the MTase domain, involving interactions with various amino acids, including Thr214, Asp146, Lys180, Gly148, and other amino acids, through hydrogen bonds, π-cation, and van der Waals forces as presented in Table 3.

Quercetin is a bioactive flavonoid with antioxidant properties found in plants. It exhibits diverse biological activities including antiviral, antidiabetic, anti-inflammatory, and vasodilating effects. It can also serve as a supplement to combat free radicals in the body (Anand et al. 2016). Inhibition occurs at the MTase domain, involving interactions at various amino acids: ACT1887 with a conventional hydrogen bond, Ile691 with a conventional hydrogen bond and an amide-π stacked interaction, Glu49 with a π-cation interaction, Arg47 with a π-anion interaction, Pro692 with a π-alkyl and π-sigma interaction, and Thr50 with a carbon hydrogen bond (Table 3).

Citral is a natural plant compound known for its aroma, flavor, and insect-repelling properties, and has also been recognized for its antimicrobial and antiviral effects. Research revealed its ability to inhibit viral activities of murine norovirus, herpes simplex virus 1, and influenza under certain conditions (Gilling et al. 2014). Inhibition occurs at the MTase domain, with Ser31 forming a conventional hydrogen bond, and Leu27, Phe242, Tyr28, and Leu20 forming an alkyl bond (Table 3).

10-Gingerol, found in Zingiber officinale, exhibits potent anti-inflammatory, anti-cancer, and antioxidant properties, with even greater effects reported compared to other ginger compounds (Levita et al. 2018). Inhibition occurs at the RdRp domain, involving Asn492 forming a carbon hydrogen bond, Gln602 and Lys401 forming a conventional hydrogen bond, Val603 forming a π-sigma interaction and an alkyl bond, Arg481 forming a π-alkyl interaction, and Val402 and Ala421 forming an alkyl bond (Table 3).

6-Shogaol, also found in Zingiber officinale, exhibits potent anti-inflammatory properties (Bischoff-Kont and Fürst 2021). Inhibition occurs at the MTase domain, with Lys95 and Gln351 forming a conventional hydrogen bond, Lys355 forming a π-sigma interaction and an alkyl bond, Pro298 forming a π-alkyl interaction, and Ile72 and Arg581 forming an alkyl bond (Table 3). Based on its binding affinity, it is considered the phytochemical molecule with the highest binding affinity after Kaempferol, indicating that it interacts well with and inhibits the NS5 protein.

Carvacrol is a monoterpenoid found in aromatic plants like oregano and thyme, exhibits various biological activities including antioxidant, antimicrobial, and anticancer properties (Sharifi-Rad 2018). Inhibition occurs at the MTase domain. Specifically, Ile94 and Ile72 form an alkyl bond, Pro298 forms a π-alkyl interaction, Val66 forms a π-sigma interaction, and Glu67 forms a conventional hydrogen bond (Table 3).

Allicin, derived from Allium sativum, possesses antimicrobial, antioxidant, and antiviral properties. It can disrupt viruses like reticulo-endotheliosis virus by penetrating cell membranes and inhibiting enzymes (Wang 2017). Inhibition at the RdRp domain involves amino acids Lys355, Arg352, Phe348, and Pro582 forming an alkyl bond, while Glu67 forms a carbon hydrogen bond (Table 3). Based on its binding affinity, it is the lowest among all phytochemical molecules, indicating that it has the weakest interaction and most limited potential as an inhibitor of NS5 protein.

Mosnodenvir, as the ligand of comparison, is notable that based on its properties; it inhibits the site located at RdRp domain. As shown in Table 4, the amino acid and its interactions observed includes: Glu356 with a halogen bonding, Tyr119 with conventional hydrogen bond, π-π interaction, Asn682 with carbon hydrogen bond, Arg540, Thr261 with conventional hydrogen bond, and further amino acids and interactions can be found in Table 3. Based on its binding affinity compared to the different phytochemicals, Mosnodenvir only falls next right after Kampferol.

Table 4 . Binding affinity results for plant phytochemical ligands of the DENV-3 NS5 protein.

Phytochemical	Lowest upper bound RMSD (Å)	Lowest lower bound RMSD (Å)	Binding affinity(kcal/mol)
Anacardic acid	0	0	-5.1
Chloroquinone	0	0	-4.5
Methyl gallate	0	0	-6.4
Kaemferol	0	0	-10.0
Myricetin	0	0	-7.5
Cianidanol	0	0	-6.9
Quercetin	0	0	-7.5
Citral	0	0	-5.2
10-Gingerol	0	0	-5.2
6-Shogaol	0	0	-7.7
Carvacrol	0	0	-6.5
Allicin	0	0	-4.4
Mosnodenvira	0	0	-8.7

^aMosnodenvir is the primary ligand for comparison for all plant phytochemicals (Goethals et al. 2023)..

Structural Modelling and Its Binding Affinities

The 3D pharmacophore structures of the protein-ligand complex of plant phytochemicals in addition with Mosnodenvir are illustrated in Fig. 3, facilitating subsequent in-depth analysis of both RMSD values and binding affinity. The figures present the NS5 protein highlighted in yellow alongside the docked ligand molecules in red. These depictions provide insight into the spatial arrangement of chemical features that contribute to the ligand’s binding affinity (Rimac et al 2021).

Figure 3. Pharmacophore Structures of Plant Phytochemical-NS5 Complexes. The illustration depicts a compilation of the formed protein-ligand complexes after molecular docking. (A–L) The NS5 protein is depicted in yellow, and the phytochemical ligands of interest are depicted in red. (M) To highlight the ligand for comparison, the protein-ligand complex of Mosnodenvir is depicted in a darker shade of yellow

Phytochemicals and Mosnodenvir molecular docking based on significant efficacy were ranked in Tables 5 alongside the ranked binding affinities with the NS5 protein of the dengue virus. RMSD values and binding affinities in kcal/mol provide insights to each compound inhibitory potential. Two RMSD types were presented in Table 4: lowest upper bound and lowest lower bound. Both the lower and upper bound RMSD yield a result of 0 in the unit of Å (Ångström), indicating similar structures. This depicts that the lower the binding energy, the stronger the binding affinity.

Table 5 . Ranking of plant phytochemical ligands based on significant computed affinity efficacy.

Rank	Phytochemicals	Lowest upper bound RMSD (Å)	Lowest lower bound RMSD (Å)	Binding affinity (kcal/mol)
1st	Kaempferol	0	0	-10.0
-	Mosnodenvira	0	0	-8.7
2nd	6-Shogaol	0	0	-7.7
3rd	Myricetin	0	0	-7.5
10th	Anacardic acid	0	0	-5.1
11th	Chloroquinone	0	0	-4.5
12th	Allicin	0	0	-4.4

^aMosnodenvir is the primary ligand of comparison for all plant phytochemicals (Goethals et al. 2023)..

Among the phytochemical molecules, Kaempferol showed the highest binding affinity, followed by 6-Shogaol and Myricetin. In contrast, the phytochemical molecules with the lower binding affinity includes: Anacardic acid, Chloroquinone, and Allicin as the lowest among them. Overall, among all the tested phytochemical ligand molecules, Kaempferol exhibited the highest binding affinities compared to Mosnodenvir, suggesting strong interaction with the dengue NS5 protein and indicating its potential as a potent phytochemical inhibitor against the dengue virus.

The authors express gratitude to all contributors for their significant role in completing this study. The authors acknowledge the Far Eastern University - Department of Medical Technology for their support throughout the study process. This study was also supported by the Department of Science and Technology - Science Education Institute (DOST-SEI) of the Philippines.

The authors have no financial or personal conflicts of interest to declare.