DENV protein is made up both structural and non-structural proteins.³⁶ Envelop (E), capsid (C), and membrane proteins are included to the structural ones. Envelop is located outside the virus and contains three ectodomains and transmembrane segment.³⁷ Thus, this protein is in charge to recognize immune cell. DENV via its E protein will attach to several host receptors such as heparin sulphate, b-integrin, and Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN) or Cluster of Differentiation 2019 (CD209).³⁸ Meanwhile, the capsid protein (C) is involved in crucial functions of multiple processes such as structural maintenance, virus assembly, and viral genome release.⁷ Recent study showed that C protein shuttles from and to the nucleus of infected cell.⁸ Moreover, both of the structural proteins can be used as key targets for drug development.^6,8,39

Other protein group that discovered in DENV is non-structural protein. This group is classified to NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 that are transcribed by 11,000 bases genome.^40,41 NS2B, NS3, and NS5 are common proteins for classical targets dengue drug development.⁴² NS2B/NS3 is one of the primary targets of dengue antiviral development in recent years.⁴³ It is trypsin-like serine protease which splits dengue polyprotein into the the separated proteins necessary for viral replication. NS3 plays essential roles for viral growth and maturation post-translation. It is boosted by NS3 that contributed as cofactor for cleavage process.⁹ Besides, NS5 is the most enormous non-structural protein (±100 kDa) and highly conserved in DENV genome. It has key play in innate immune response impairment during evasion as well as RNA synthesis, capping, and methylation from its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent RNA polymerase (RdRp).^10,11,44 This finding raises the possibility of developing antiviral targeting NS2B/NES3 and NS5 as DENV drugs.

G. atroviridis showed numerous health properties as spices and herbal plant. There are 38 phytochemical compounds found from various parts of this plant based on the prior studies.¹¹ However, limited databases retrieved from PubChem webserver made this study only examine 24 compounds to asses for drug-likeness analysis. Based on the drug-likeness analysis, only 6 phytochemical compounds fulfilled the criteria. In order to analyze the toxicity, the filtered compounds passed with one or no violation of 5 criteria from pkCSM toxicity prediction. After that, selected compounds docked to investigate the interaction stability. The result showed that atroviridin was the most promising pyranoxanthone compounds as anti-DENV inhibits E, NSB/NS3, and NS5 proteins. The binding affinity scores were lower than the selective synthetic drug. Meanwhile, quercetin as a part of flavonoid compounds performed more positive result than ST-148 as the control for C protein. The lowest or the most negative binding affinity is needed to support the stability interaction during the cellular process and has capability as probable inhibitors.⁴⁵

Atroviridin revealed as the most effective compound inhibitor of E protein compared to drug control. In comparison, atroviridin formed π-donor hydrogen bond with Asn390 from subunit A. There are other chemical interactions including hydrophobic (π-π stacked and π-alkyl) and van der Waals (vdw). Meanwhile, NITD448 demonstrated other chemical interactions including carbon hydrogen and conventional hydrogen bonds that known as the strongest chemical interaction.⁴⁶ However, the halogen interaction of fluorine and chlorine made this complex satisfied. Halogen can sustain inter- and intramolecular ligand-protein interaction and affect molecular folding but it is weaker than hydrogen bonds. As a result, 20% of drugs that approved by Food and Drug Administration (FDA) were halogenated compounds.⁴⁷ On the other hand, some of fluoride in biological interactions show that fluoride is highly oxidative that generate of reactive oxygen species, cell necrosis, and apoptosis.⁴⁸ Because of that, the binding affinity of NITD448 had a higher binding than the atroviridin. Both of the compounds exhibited unfavorable donor-donor interactions and not form any interactions towards active sites (Figure 1). But they share same interaction with Trp391 of subunit A with multiple hydrophobic interactions to help inhibit virion recognition to host cell with aromatic and electron clouds.⁴⁹

According to the docking analysis, ST-448 displayed the most negative one among other compounds. ST-148 had hydrogen (conventional), hydrophobic (π-σ, π-π T-shaped, amide-π stacked, alkyl, and π-alkyl), electrostatic (π-cation), and unfavorable interactions. There is miscellaneous interaction towards Trp69 from subunit B namely π-sulphur. This interaction provides aromatic compounds that interact with single sulphur atom.⁵⁰ Sulphur gains specific function in biological activities consists folding stability and intermolecular interaction that larger than expected from vdw contacts.⁵¹ Another research reported that S-arene interactions were preferred over O-arene ones due to the non-covalent bonds.⁵² Meanwhile, quercetin formed only 3 hydrogen (conventional), 3 hydrophobic (amide-π stacked and π-alkyl), an electrostatic (π-anion), and a vdw interactions. Though quercetin did not possess unfavorable bonds, fewer groups of interaction provided more positive or less satisfied binding affinity via molecular docking.²⁰ Overall, neither chemical interaction with active sites from atroviridin nor quercetin interacted more intensively with various amino acid and C protein subunits.

NSB2/NS3 shares co-dependency to activate protease activities in dengue evasion.⁴² ARDP0006 as control synthetic drug showed higher binding affinity than the atroviridin from G. atroviridis. It made this compound is preferred as anti-DENV drug candidate. Atroviridin has 3 conventional hydrogen (Lys74, Leu85, and Ala164), hydrophobic (both π-sigma and π-alkyl interact to Leu76), and one unfavorable donor-donor interaction (Trp83). Unfavourable interaction may indicate the presence of repulsive forces between ligand and target protein. It will affect to the more positive results after molecular screening.^53-55 Next, ARDP0006 demonstrated only 2 kinds of interactions: conventional hydrogen bonds (2 interactions against Lys74 and one interaction against Trp83) and π-sigma interaction (Leu76). Atroviridin as well as ARDP0006 indicated interactions with amino acid residues from subunit B and share same receptor toward Lys74, Leu76, and Trp83 with various bonds. Despite the lack of contacts with catalytic triad and unfavourable bond, atroviridin still revealed best inhibitory activity across the different interactions.⁵⁶

Based on the molecular docking, atrovridin also developed the most efficient inhibitor for another non-structural protein, NS5. Conventional hydrogen bonds and π-alkyl assist stabilization of ligand-protein complex and triggering inhibitory responds against genome replication and disabling the innate immunity.³⁴ The results of control ligand showed the interaction against active site Asp663 via conventional hydrogen bonds. Besides, it possessed more hydrogen bonds and one hydrophobic interaction. Therefore, unfavourable donor-donor towards Ser710 made the binding affinity and desirable in computational study lower.⁵⁴

Protein flexibility can be indicated by measuring the amplitude of atomic movements when it was simulated.⁵⁷ In this analysis, the protein flexibility was represented by RMSF value. Molecular dynamics showed that the most effective inhibitors of selected phytochemical compounds from G. atroviridis are significantly stable based on the predominantly RMSF value that fall between 1-3 Å.⁵⁸ However, several residue indexes from 4 selected ligand-target protein complexes demonstrated RMSF value >3 Å.

NITD448 is DENV fusion inhibitor that might attach to the ג-OG pocket in DENV E protein. But the antiviral effects only were observed during the initial viral entrance.⁵⁹ Besides, ST-148 proposed to impede viral assembly and release range in DENV-1, -3, and -4. The antivirus potentially beneficial work occurred after post infection and post entry stage.^38,60 Following that, ARDP0006 was identified suppress DENV-2 replication via virtual screening and cell culture.⁶¹ In addition, SAH in combination with sinefungin, compound 10, and guanosine monophosphate (GMP) failed to block NS5 with good progress due to the cell non-permeability.⁶² Some of the lack potential of synthetic antivirals above is required to address some of inhibitory functioned compounds towards specific target of DENV both targeting structural and non-structural proteins.^38,44 G. atroviridis has the potentials to be the next natural anti-DENV drugs. In silico analysis through molecular docking revealed that atroviridin has the most effective potential against E, NS2B/NS3, and NS5 proteins. Furthermore, in vitro and in vivo analyses still required further to confirm anti-DENV efficiency.