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. 2024 Apr 25;12(1):35. doi: 10.1007/s40203-024-00207-2

Identifying citrus limonoids as a potential fusion inhibitor of DENV-2 virus through its in silico study and FTIR analysis

Satyajit Das 1, Geetartha Sarma 2,✉, Nithin J Panicker 2, Partha P Sahu 2
PMCID: PMC11045700  PMID: 38680655

Abstract

Dengue virus type 2 (DENV-2) is an arthropod-borne deadly RNA human pathogen transmitted through the mosquito Aedes. The DENV-2 roots viral infection by facilitating entry with its envelope glycoprotein to the receptor protein Dendritic-cell-specific ICAM3-grabbing non-integrin (DC-SIGN) through membrane fusion. Here, an organizational path is reported for inhibiting the transition due to fusion activation and by blocking the residues of the DC-SIGN–E-Glyco protein complex through citrus limonoids with its antiviral effect. Based on lower binding affinity obtained with E-glycoprotein, and based on ADMET and drug-likeness study, limonin was selected as having effective interaction with DC-SIGN–E-glycoprotein complex in comparison to other citrus limonoids. The FTIR spectra performed with the limonin–E-glycoprotein sample provide evidence of hydrogen bond formation that indicates the formation of a strong limonin–E-glycoprotein conjugate. Further, the strong physical interaction between DC-SIGN and small limonin molecules in comparison to that of E-glyco with DC-SIGN assures the development of immunity against DENV-2.

Supplementary Information

The online version contains supplementary material available at 10.1007/s40203-024-00207-2.

Keywords: DENV-2, Molecular docking, Citrus limonoids, ADMET, Drugs-likeness

Introduction

The Dengue virus (DENV) is a deadly single positive-stranded RNA virus of the genus Flavivirus causing severe dengue haemorrhagic fever (Rodenhuis-Zybert et al. 2010; Organization et al. 2009). DENV infection is more prevailed in children than in adults, especially in the Indian subcontinent and in Southeast Asia (Carlos et al. 2005; Guzmán et al. 2002; Kittigul et al. 2007; Nakao et al. 1989; Ismail and Jusoh 2017). Dengue infections are triggered by four virus serotypes that are closely related namely DENV-1, DENV-2, DENV-3, and DENV-4. The structure of the dengue virus as shown in Fig. 1a has mainly a nucleocapsid core consisting of viral genome and C protein where it is surrounded by a viral envelope embedded with E and M protein layers (Kuhn et al. 2002). Significant genetic diversity exists within each serotype, causing complications in the development of vaccines (Normile 2013; Dighe et al. 2019). The DENV genome is encoded with three structural proteins (C-capsid protein, M-membrane protein, E-envelope protein) and seven non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5) with small non-coding regions on both the 5′ and 3′ ends (Fig. 1b) (Rodenhuis-Zybert et al. 2010; Guzmán et al. 2002; Hanley and Weaver 2010; Guzman et al. 2010; Chandrasekaran et al. 2019; Perera and Kuhn 2008). For virus fusion toward binding to the host receptor, Envelope glycoprotein (E-glyco) plays an important role (Kuhn et al. 2002; Mir et al. 2016). The envelope protein structure has three domains: domain I (DI) having N terminal central domain, domain I (DII) having the fusion (or dimerization) with the hydrophobic fusion peptide and domain III (DIII) having the putative receptor-binding domain. The “kl” hairpin forms the part of the “hinge” connecting DI and DII (Modis et al. 2003; Bhardwaj et al. 2001; Bressanelli et al. 2004). The DIII is involved in receptor binding (Crill and Roehrig 2001). The envelope protein occurs at the tip of DII as the dimeric structure with highly preserved fusion peptide hidden in a hydrophobic pocket gives protection from the interaction with cellular membranes (Mir et al. 2016; Rey et al. 1995; Zhang et al. 2004).

Fig. 1.

Fig. 1

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a Composition of DENV. b Structure showing Dengue virus genome encodes with the structural and non-structural protein (Švajger et al. 2010). c Flowchart presenting the steps to screen citrus phytochemicals. d DC-SIGN stricture (Švajger et al. 2010) e penetration of DENV into transmembrane of human cell through DC-SIGN receptor

The lower pH triggers the rearrangement in the E-protein owing to the protonation of highly conserved histidine residues (Kampmann et al. 2006; Fritz et al. 2008; Stiasny et al. 2007). The Envelope fusion protein is exposed and summits the E-protein for initiating the fusion process in interaction through the host endosomal membrane. The hinge region helps in bringing the fusion peptide in close with the host membrane (Mir et al. 2016; Zhang et al. 2004; Modis et al. 2004). In acidic medium, kl β-hairpin participates in fusion through conformational changes. At the edge between DI and DII, kl loop forms a channel with its dimer close to the holes in the hydrophobic channel in the presence of a small molecule. The Hydrophobic interface unlocks in the absence of a molecule that changes the kl loop. This shifting permits DII to hinge away from its dimer partner. As a result, it plans the fusion peptide at the tip toward the membrane of the target cell. Hence the changes in conformational at acidic environment predict the binding of molecules where the ligand-binding pocket in opened kl hairpin conformation may inhibit the infection preventing the early fusion process (Mir et al. 2016; Kwon et al. 2010; Yasuhara-Bell et al. 2010).

The related receptor nature of DENV on the surface of the cell is still not clear (Knipe et al. 2007). Among various receptors—heat shock protein 70, HSP90, GRP78, mannose receptor, DC-SIGN and CD14-associated protein, the DC-SIGN is the human Dendritic-Cell-Specific ICAM3-grabbing non-integrin used as a receptor molecule mediating infection with all serotypes of DENV (Shah et al. 2013).

Citrus limonoids (CLs) are highly oxygenated terpenoid phytochemicals of the secondary metabolites category that are found in citrus fruits showing numerous biological activities such as anti-viral, anti-cancer, anti-microbial, anti-oxidant, anti-diabetic properties, etc. (Gualdani et al. 2016). More than 300 limonoids have been isolated so far and categorized among which limonin is the first highly oxygenated triterpenoid dilactone. It is also collectively considered as the standard conveyor of CLs that have great potential for traditional medicinal uses and current nutraceutical products (Arias and Ramón-Laca 2005; Patil et al. 2009; Codoñer-Franch and Valls-Bellés 2010; Zhao et al. 2012). CLs also possess anti-carcinogenic activity. On carcinogenic delta retrovirus human T-cell leukemia/lymphoma virus type 1 (HTLV1), the potency of limonin and nomilin has been found as inhibitors (Balestrieri et al. 2011). Limonexic acid shows a potential inhibitor against the hepatitis B virus (Zhao et al. 2012). The limonin exhibits promising antiviral properties in constraining the anti-Newcastle disease virus (NDV) (Abd et al. 2019). Considering the anti-viral activities of limonoids and studying the inhibition process of Envelope protein in the binding pocket, the proposed work is to interrupt the process of fusion activation with a receptor at the very early stage of interaction by searching the potential small molecules such as CLs and lead compounds. The small molecules limonoids were interacted in the ligand-binding pocket of the Envelope glycoprotein of DENV-2 virus through molecular docking using suitable docking tool. The limonoids inserted at this position can inhibit the fusion process by interrupting further conformational changes. The protein complex of DC-SIGN–E glyco protein has been made through molecular docking interaction and studied the interaction of selected phytochemicals with the protein complex that successfully blocks conserved residues present at DC-SIGN–E-glyco complex interface.

The potential interaction of small molecules with the active protein residues is estimated by using computational simulation. The outcome of interaction assessing the ligand-binding energy indicates its selection towards finding potential inhibitant (Abdolmaleki et al. 2017; Zheng et al. 2013). Here computational simulation using Autodock Tools allows recognizing the active sites of the protein structure in finding out the small molecules to be interacted (Trott and Olson 2010). This study focuses on screening and identifying CL molecules that interact with the ligand-binding pocket of the Envelope glycoprotein and DC-SIGN E glycoprotein to prevent DENV and DC interaction.

The CLs having antiviral properties have been screened by using molecular docking with E-glycol and with DC-SIGN–E glyco complex and by studying their medicinal chemistry and Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) properties. These molecules obstruct the fusion process of the virus more effectively by preventing conformational rearrangement in E-glycoprotein while blocking the hinge region movement and the residues of DC-SIGN. In comparison to other citrus limonoids, limonin shows more effective interaction with E-glycoprotein as per strong binding with E-glyco protein, ADMET and drug-likeness study. The strong interaction between E-glycoprotein and limonin was validated with Fourier Transform Infrared Spectroscopy.

Materials and methods

Ligand (Citrus phytochemicals) and protein preparation

In the present study, the biologically active 11 chemicals from citrus limonoids (Limonin, nomilin, Obacunone, obacunone, Deacetylnomilin, limonexic acid, Isolimonexic acid, Citrusin, Ichangin, Isoobacunoic acid, Deoxylimonin, Limonin 17-β-d-glucoside) were selected considering their antiviral medicinal properties (Supplementary Table S1). The smiles strings files of the selected citrus phytochemicals were obtained from (https://pubchem.ncbi.nlm.nih.gov/). We have optimized the chemical structures of small molecules and converted and saved them in the PDB format.

The Crystallography structure of DENV-2 Envelope Glycoprotein protease of serotypes 2 in composite with n-octyl-β-d-glucoside (βOG) was acquired from Protein Data Bank (PDB) (www.rcsb.org) in.pdb format (PDB id: 1OkE). In the process of pocket selection, a hydrophobic pocket near the hinge region was targeted for docking and inhibition (Modis et al. 2004). The hydrophobic pocket was occupied by a small βOG molecule of E protein.

Molecular docking between CLs with the Envelope (E) glycoprotein

Molecular docking simulations were performed using AutoDock Vina 1.5.6 program to find out the binding affinity (BA) of selected CLs with the E-glycoprotein of DENV serotype 2. The scheme in Fig. 1c shows the screening and docking steps of Citrus phytochemicals. By using Auto Dock tools, the protein structure and CLs in their PDB form were converted into PDBQT format. Polar hydrogen atoms and Gasteiger charges were inserted into the ligands. The dimensions of the grid box of protein for the docking-specific site were taken at size_x = 40, y = 40, z = 40. The exhaustiveness was fixed at 8 for docking purposes. Lamarckian genetic algorithm (LGA) was employed for simulation in docking. The docked results obtained as the output of Autodock Vina tool were further analyzed using BIOVIA Discovery studio visualizer.

Molecular docking to make DC-SIGN–E-glyco complex

DC-SIGN is made of four domains: a carbohydrate recognition domain (CRD), seven to eight extracellular neck repeats, a trans-membrane domain and a cytoplasmic domain (Shah et al. 2013) (Fig. 1d). Molecular docking between DC-SIGN receptor and E-glyco is performed using ClusPro27 online server (https://cluspro.bu.edu/home.php) to form DC-SIGN–E-glyco protein–protein complex. For the estimation of cluster scores and binding affinity the computation equation for force is written as

E=0.40ER+-0.40EA+600EE+1.00ED

where ER = repulsive force, EA = attractive force, EE = electrostatic forces and ED = interaction forces extracted from the decoys as the reference state (DARS) which are determined using molecular docking study (Kozakov et al. 2017; Kozakov et al. 2013).

Then molecular docking between DC-SIGN–E-glyco complex and screened limonoids ligands was carried out by using AutoDock Vina 1.5.6 program to estimate the performance of ligands on the complex in terms of binding energy.

Drug likeliness and ADMET properties estimation

Drug likeliness of the compounds was investigated for the screening of the phytochemicals in identifying the best possible inhibitors in the fusion process of E-glyco to the receptors. The drug-likeliness properties were studied for screening of small molecules based on Lipinski’s rule of five (rule of five (RO5)). In silico ADMET study of selected Citrus limonoids has been done for Pharmacokinetics properties using the online tool ‘http://biosig.unimelb.edu.au/pkcsm/prediction’. The pkCSM AMES test attained a correctness of 83.8% compared to other available methods like ToxTree49 having an accuracy of 75.8%. The Pearson correlation coefficients range from 0.6 to 0.9 as presented by regression models of pkCSM. For most data sets, pkCSM has presented a statistically major enhancement in prediction in comparison with available methods (Pires et al. 2015). Drug likeliness, medicinal chemistry and friendliness of small molecules were studied using SwissADME, a free web tool (Daina et al. 2017).

Molecular docking between DC-SIGN and screened citrus phyto-ligands

Then molecular docking between DC-SIGN protein and screened phyto-ligands was carried out by using AutoDock Vina 1.5.6 program to estimate the binding energy performance of ligands on DC-SIGN receptor.

FTIR spectroscopy

For the analysis of different functional groups of limonin, Fourier Transform Infrared (FTIR) Spectroscopy (IMPACT 410, NICOLET, USA) was performed. Further chemical interaction of limonin with envelope surface glycoprotein protease of dengue was estimated by analyzing the change of absorption peaks of functional groups of conjugate samples of the above chemicals using FTIR spectroscopy.

Results and discussion

Screening of antiviral citrus phytochemicals

By using the Auto dock Vina computational approach, the selected phytochemicals have interacted with the binding pocket of the Envelope protein. The Mol Dock results in terms of binding affinities (BA) are shown in Table 1. The dock score of all citrus phytochemicals against Envelope protein shows BA lower than − 8.3 kcal/mol. Here five best phytochemicals—limonin (BA = − 8.5 kcal/mol), nomilin (BA = − 8.8 kcal/mol), limonexic acid (BA = − 8.8 kcal/mol) Isolimonexic acid (BA = − 9.4 kcal/mol), citrusin (BA = − 8.9 kcal/mol) were selected based on higher binding Affinity estimated through molecular computation for further study.

Table 1.

The Binding Affinity of Citrus phytochemicals binding at the active sites of the E-glycoprotein observed through Auto dock Vina computation

Sl no. Ligand Molecular formula Binding affinity (kcal/mol)
1 Limonin C26H30O8  − 8.5
2 Nomilin C28H34O9  − 8.8
3 Obacunone C26 H30O7  − 8.1
4 Deacetylnomilin C26H32O8  − 7.9
5 limonexic acid C26H30O10  − 8.8
6 Isolimonexic acid C26H30O10  − 9.4
7 Citrusin C28H34O11  − 8.9
8 Ichangin C26H32O9  − 7.9
9 Isoobacunoic acid C26H32O8  − 7.8
10 Deoxylimonin C26H30O7  − 8.2
11 Limonin 17-β-d-glucoside C32H42O14  − 7.4
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The selected phytochemicals were studied and again screened based on TPSA (topological polar surface area) and drug likeliness as shown in (Table 2). Drug likeliness properties of selected ligands have been studied based on adherence to Lipinski’s rule of five or rule of five (RO5) following five parameters—Mol. Weight (MW in g/mol), Hydrophobicity parameter (log p) which is the ratio of organic to aqueous phase concentration, HB (D): Hydrogen bond donor, HB (A): Hydrogen Bond Acceptor, Drug likeness score. As per Lipinski’s rule of five, the permitted values are MW < 500, log p < 5, HB (D) < 5, HB (A) < 10, and 0 < drug-likeness score < 0.8 (Lipinski 2004).

Table 2.

Drug-likeness properties of selected ligands based on adherence to Lipinski’s rule of five

Sl. no. Name of the ligands Mol. weight (g/mol) Log p HB (D) HB (A) Drug likeness score TPSA, Å2
1 Limonin 470.518 2.55 0 8 0.55 104.57
2 nomilin 514.571 2.97 0 9 0.55 121.64
3 Obacunone 454.51 3.17 0 7 0.55 95.34
4 Deacetylnomilin 472.53 2.53 1 8 0.55 115.57
5 limonexic acid 502.51 1.35 1 10 0.55 137.96
6 Isolimonexic acid 502.51 1.32 1 10 0.55 137.96
7 Citrusin 546.56 1.65 1 11 0.17 155.03
8 Ichangin 488.53 1.72 2 9 0.55 135.80
9 Isoobacunoic acid 472.53 2.70 1 8 0.56 115.57
10 Deoxylimonin 454.51 2.91 0 7 0.55 92.04
11 Limonin 17-β-d-glucoside 650.67 0.29 5 14 0.11 214.95
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Hydrophobicity parameter Log P: Octanol–water partition coefficient value, HB (D): Hydrogen bond donor, HB (A): Hydrogen Bond Acceptor. TPSA: total polar surface area

Although the molecular weight of a drug plays a dynamic role in the drug’s oral bioavailability, for the compound to classify as poor or good oral bioavailability, the 500 cut-offs may not be significant. As per Lipinski’s rule of molecular weight, only limonin is qualified as the other four compounds—nomilin, limonexic acid, Isolimonexic acid and citrusin have MW > 500. Regarding the physicochemical aspect, parameters such as partition coefficient and solubility play an important role in oral bioavailability. All of the phytochemicals have calculated Log P values from 0.29 to 3.7 (Table 2). Further, among the top 5 selected compounds (based on BA), Citrusin was screened out due to having a TPSA score of 155.03 Å2 more than 140 Å2 (a standard value to qualify the oral bioavailability) (Daina et al. 2017). The Compound Citrusin also had more numbers of HBA violating the rule of five (RO5). Other four phytochemicals namely Isolimonexic acid, limonexic acid, Nomilin and Limonin have TPSA below 140 Å2 obeying the parameters of drug-likeness and also satisfying the limitations of lipophilicity, hydrophobicity and polarity but number of HBA of both Isolimonexic and limonexic acid is of 10 which is just permitted value. Also, molecular weight of both is more than the permitted value of 500 (Lipinski 2004). Similarly molecular weight of nomilin is more than the permitted value violating Lipinski’s rule of five. The ADMET-associated properties were analyzed for the phytochemicals isolimonexic acid, limonexic acid, nomilin and limonin by using Pkcsm server prediction. Out of the above phytochemicals, Limonexic acid shows AMES toxicity as per prediction causing oxidative DNA damage. Oxidative DNA damage indicates it is mutagenic and carcinogenic causing cancer (Guardia and Lleonart 2014). Witnessing this property, limonexic acid is screened out for further study as a fusion inhibitor for DENV-2 virus. Now, limonin is selected for interaction with DC SIGN–E-glycoprotein complex and DC-SIGN receptor for their performance at the active sites of the protein targets of DENV-2. As per the biological activity study (Supplementary Table S1), limonin is more effective than other limnoids for the development of immunity in the human body.

E-glyco-ligand interaction

The compounds isolimonexic acid, nomilin and limonin have higher bindings to the active binding sites with the hydrogen bond and bond interactions (Table 3). The process of evaluation of results in identifying potential inhibitors depends upon the binding affinity. It also depends on the number of bonding interactions like conventional hydrogen bonds between limonoid ligands and the target protein.

Table 3.

Interaction of Screened Limonoids with E-glyco showing the binding affinity, Bond Length and amino acid residues as calculated through Autodock vina computation and visualized through Bio Discovery Studio Visualizer

Limonoids BA (k cal/mol) Bonding Type Amino acid residues of E-glyco with bonding sites of Ligand BD (Å) Binding between ligands and amino acid sites of E-glyco portein
Isolimonexic acid  − 9.4 HB LYS B:246: HZ2: IS01:09 2.531 graphic file with name 40203_2024_207_Figa_HTML.gif
HB ARG A:2: HH12:-IS01:09 2.213
HB ARG A:2: HH22-:-IS01:09 1.85
HB LYS B:247: HZ2-: IS01:05 1.988
HB ISO1: H21-ASN B:103: OD1 2.568
HB GLY A:28: HN-: IS01:03 2.090
Nomilin  − 8.8 HB LEU A:277: HN-: nom1:03 2.768 graphic file with name 40203_2024_207_Figb_HTML.gif
HB ASN A:276: HD21-: nom1:01 3.078
HB ASN A:276: HD22-: nom1:01 2.76
HB LYS B:247: HZ1-: nom1:01 2.445
HB LYS B:247: HZ1-: nom1:02 2.38
HB ARG A:2: HH12-: nom1:07 2.173
Pi-Anion ASP A:154: OD2-: nom1 3.551
Limonin  − 8.5 HB LYS B:247: HZ1-: lim1:01 2.905 graphic file with name 40203_2024_207_Figc_HTML.gif
HB LYS B:247: HZ2-: lim1:01 2.51
HB Lim1:03-GLU A:44: OE2 3.219
HB LYS B:246: HZ3-: Lim1:02 1.978
HB Lim1:03-LEU A: 45: O 3.103
HB GLY A:28: HN-: Lim1:03 2.051
Pi-Anion GLU A:161: OE2-: Lim1 4.490
Pi-Alkyl ILE A:46-: Lim1 4.577
C-H GLY A:156: CA-: Lim1:08 3.416
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BA: Binding Affinity, HB: Hydrogen Bond, BD: Bonding Distance, C-H: Carbon–Hydrogen

Isolimonexic acid ligand

Binding interaction with Isolimonexic acid ligand indicates hydrogen bonding with LYS B:246, LYS B:247, ARG A:2, GLY A:28, ASN B:103 and GLY A: 28 amino acid residues in E-glycoprotein active sites (Table 3). The ribbon representation of interaction in Fig. 2a shows conformal changes occurred in fragment structures after binding of ligand with amino acid residues of E-glycoprotein structure (Fig. 1b). Figure 2a shows a stick representation of interaction with E-glycoproetin amino acid residues indicating strong hydrogen bonds with the binding affinity of − 9.4 kcal/mol.

Fig. 2.

Fig. 2

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Small molecules binding pose for A Isolimonexic acid, B Nomilin, C Limonin with the amino acid residues of Envelop Glycoprtein of DENV-2

Nomlin ligand

The interaction with nomilin ligand reveals hydrogen bonding with LEU A: 277, ASN A: 276, LYS B: 247, ARG A: 2 and Pi-Anion bonding with ASP A: 154 amino acid residues in E-glycoprotein active sites (Table 3). Figure 2b shows conformal changes in fragment structures after the binding of nomilin with amino acid residues of E-glycoprotein structure (Fig. 2b). Figure 2a represents a stick diagram of interaction with E-glycoproetin amino acid residues having less hydrogen bonding. As a result, binding affinity is less than that with Isolimonexic acid ligand.

Limonin ligand

The binding interaction with limonin ligand shows hydrogen bonding with LYS B: 247, GLU A: 44, LYS B: 246, LEU A: 45, GLY A: 28 amino acid residues of E-glycoprotein. It also possessed Pi-Anion bonding to GLU A: 161, Pi-Alkyl to ILE A: 46 and C–H bond to GLY A: 156 (Table 2). The representation of interaction in Fig. 2c indicates conformal changes after the binding of limonin with amino acid residues of E-glycoprotein structure. The binding affinity of the interaction with limonin is less than that with Isolimonexic acid due to having less hydrogen bonding with amino acid residues of E-glycoprotein as seen in fragment structure of interaction in Fig. 2c.

The inhibition process of fusion to the receptor at the initial period of viral entry is advantageous. The envelope protein facilitates the virus attachment to a receptor in the fusion process. In comparison to nomilin and limonin, isolimonexic acid shows more hydrogen bonds and less bond lengths with amino residue sites of E-glycoprotein showing more effective interaction with isolimonexic acid (Table 3).

Effectiveness of isolimonexic acid, nomilin and limonin on DC-SIGN and E-glyco protein complex

The DENV-2 first penetrates through receptor protein DC-SIGN of the cellular membrane and attachment features in finding its way to the cytoplasm (Fig. 1e). The acidic environment activates conformational changes on the E protein dimers to become fusogenic trimers. Then the genomes get released into the cytoplasm in Fig. 1e through the pores (Guardia and Lleonart 2014). Here, molecular docking between E-glyco protein of DENV-2 and DC-SIGN receptor protein is performed to form protein complex by using ClusPro web tool (Kozakov et al. 2017; Kozakov et al. 2013) (Fig. 3a) showing three hydrogen bonding with amino acid residues (two with B: SER 274 and one with A: GLN 233) of E-glycoprotein, one P-Anion bonding of amino acid residue (B: GLU 257) of E-glycoprotein and two P-Alkyl bonding with amino acid residues (B: LYS 123 and B: LYS 202) of E-glycoprotein where DC-SIGN protein acts as acceptor. The most predominant interactions in DC-SIGN–E-glyco complex are hydrogen bonds with amino acid residue SER 274 of E-glyco. On the basis, blocking SER 274 of E-Protein and its surrounding preserved interfacing residues in protein complex can inhibit dengue virus attachment to its candidate receptor. The hydrogen bond lengths between amino acid residues of E-glyco and human DC-SIGN are below 3 Å exhibiting their strong interaction.

Fig. 3.

Fig. 3

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Ribbon assembly representation a formation of DC-SIGN–E-glycoprotein complex through molecular docking b interaction of limonin with protein complex through molecular docking c interaction of nomilin with protein complex through molecular docking d interaction of isolimonexic acid protein complex through molecular docking e mechanism for DENV penetration through E-glycoprotein binging with DC sign and limonin interaction with DC-SIGN

Molecular docking scores for limonin, nomilin and isolimonexic acid ligand with E-glyco–DC-SIGN protein complex were computed through Autodock vina software and shown in Table 4. The limonin in Fig. 3b shows two Carbon Hydrogen bonding with SER B 274 with bond lengths of 2.30 Å and 2.7674 Å (Table 4) whereas nomilin in Fig. 3c exhibits hydrogen bond with residue VALA with bond distance 1.9445 Å and isolimonexic acid in Fig. 3d shows one hydrogen bond with residue HIB B 26 and bond length of 3.56 Å (Table 4). The other bonds of limonin are Pi alkyl with GLU B: 257 residues and Pi anion bonds LYS B 123 and LYS B 202 (Table 4). The limonin attributes more reactive Hydrogen bonding than that of the other screened limonoids nomilin and isolimonexic acid and also bonds with the same amino acid residues of E-glyco protein. Hence the result shows that limonin having a better binding affinity of − 8.8 kcal/mole than the other two molecules is more effective on protein complexes indicating more probability of binding with Protein complexes than that of other molecules. Limonin attributes more number of reactive functional groups than E-glycoprotein due to smaller molecular weight providing more probability of interaction with DC-SIGN (Fig. 3e). Moreover, Limonin shows more absorption and diffusion capability contributing more tendency of physical binding with receptor protein DC-SIGN in comparison to that of E-glycoprotein (Supplementary Table S2). Hence it indicates that limonin may successfully block conserved residues present at DC-SIGN protein interacting with E-glycoprotein for inhibiting the DENV-2 in human cell membrane (Fig. 3e).

Table 4.

Interaction of Limonoids Isolimonexic acid, Nomilin, and Limonin with (DCSIGN–E-glyco) complex obtained through Auto dock Vina computation showing the binding affinity, Bond Length and amino acid residues

Ligands BA (kcal/mol) Bonding type Amino acid Residues of E-glyco with bonding sites of Ligand BD (Å) Binding between ligands and amino acid sites of DCSIGN–E-glyco protein complex
Isolimonexic acid  − 8.6 Carbon Hydrogen Bond HIS B 261: CE1: ISO1:01 3.56 graphic file with name 40203_2024_207_Figd_HTML.gif
Nomilin  − 8.0 Carbon Hydrogen Bond VAL A 252: HN-nom1:01 1.945 graphic file with name 40203_2024_207_Fige_HTML.gif
Pi-Alkyl VAL A 250: nom1 5.075
Limonin  − 8.8 Carbon Hydrogen Bond SER B 274: HN-UNK0: O 2.30 graphic file with name 40203_2024_207_Figf_HTML.gif
Carbon Hydrogen Bond SER B 274: HG: UNK0: O 2.767
Pi-Anion GLU B:257: OE2: UNK0 3.792
Pi-Alkyl LYS B 202: UNK0 4.995
Pi-Alkyl LYS B 123: UNK0 5.283
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BA, binding affinity, BD, bonding distance

Molecular docking of limonin, nomilin and isolimonexic acid with DC-SIGN

Molecular docking of DC-SIGN was carried out with limonin, nomilin and isolimonexic acid by using AutoDock Vina 1.5.6 computation (Supplementary Table S3). The Limonin shows two hydrogen bonding (HB) with DC-SIGN amino acid residues CYS369: HN1, one HB with ASN370: HN1, one HB with HIS 278: CD and one HB GLY 352: O whereas nomilin ligand exhibits one HB with amino acid residues ASP355: HN, one HB with ASN370: OD1 and P-Alkyl bond with LEU371. The isolimonexic acid has one HB with HIS278: HD1, one HB with ASP355: HN, one HB with LYS368: HZ3, one HB with ASN370: HD21, one HB with HIS278: CE1 and one HB with GLU353: O (Supplementary Table S3). Here isolimonexic acid shows more hydrogen bonding with DC-SIGN receptor in comparison to Limonin and nomilin but the probability of limonin interaction with DC-SIGN is more than the other two ligands isolimonexic acid and nomilin. The ribbon assembly representation in Fig. 4a shows strong hydrogen bonding of amino acid residues with limonin. When DENV enters the cell membrane, the physical binding between limonin and DC-SIGN exhibits a rejection to accept the E-glycoprotein molecule of DENV by DC-SIGN due to having absorption of small molecular weight limonin (Fig. 4b). Hence it develops strong immunity to resist the penetration of DENV through multifunctional DC-SIGN receptor of cell membrane.

Fig. 4.

Fig. 4

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Ribbon assembly representation of limonin and DC-SIGN obtained by molecular docking b mechanism showing limonin received by DC-SIGN and bounced back of DENV2 due to after accepting limonin by DC-SIGN due to smaller molecular wright

ADMET and drug likeness study

The major challenge in the identifying process of phytochemicals as drugs is the assessment of ADMET properties of compounds. The interpretation of ADMET outcomes is based on computed values compared with marginal values (Supplementary Table S2). Caco-2 permeability is high if > 0.90, intestinal absorption is poor if the value is less than 30%, the human volume of distribution (VDss) is low if it is below 0.71 L/kg and if above 2.81 L/kg, the human volume of distribution is considered high. For BBB permeability, the Blood Brain Barrier will be crossed if logBB > 0.3 and poorly distributed in blood blood–brain for logBB < 1. For the central nervous system, compounds with logPS > − 2 penetrate through the Central nervous system whereas for logPS < -3 it seems the compound is unable to penetrate through the CNS. For a compound to measure T. pyriformis toxicity, a predicted value > 0.5 μg/L dignified toxic and for minnow toxicity it is considered as high acute toxicity if the value of logLC50 < 0.3. Low Skin permeability if log Kp > − 2.5 (Pires et al. 2015). The predicted ADMET-associated properties of the phytochemicals isolimonexic acid, nomilin and limonin for different models were summarized in (Supplementary Table S2). The ADMET results show constructive results for most of the parameters as shown for the screened phytochemicals. The ADMET-associated properties of screened phytochemicals were compared with those of other phytochemicals (Supplementary Tables S4s and S5). Lipophilicity is an important drug property that impacts drug distribution and metabolism and excretion process (Supplementary Table S4). Increased lipophilicity promotes off-target binding leading to an increase in binding to unwanted cellular targets (Hughes et al. 2008). The lipophilicity values of the study include Log Po/w (iLOGP), Log Po/w (XLOGP3), Log Po/w (WLOGP), Log Po/w (MLOGP), Log Po/w (SILICOS-IT). The values of lipophilicity (log Po/w) for the screened molecules show a preference for molecules to be associated with the lipid phase and will likely permeate biological membranes spontaneously.

The water solubility of citrus limonoids was further studied with the SwissADME tool using topological methods such as ESOL model (Delaney 2004), model adopted from Ali model (Ali et al. 2012) and the solubility predictor developed by SILICOS-IT (Pires et al. 2015) as shown in (supplementary Table S5). The water solubility of Limonin and Nomilin shows moderately soluble in water whereas for other molecules isolimonexic acid it showed soluble in water. The predictions of drug-likeness, the rules of Ghose filter (Ghose et al. 1999) and Veber’s Rule (Veber et al. 2002) were carried out for all showing no violation of limonin in drug-likeness study (supplementary Table S6).

Synthetic accessibility (SA) signifies the ease of synthesis of drug. The SA for molecules is assessed in various areas of the drug discovery process using an SA score between 1 (easy to make) and 10 (very difficult to make) (Veber et al. 2002; Ertl and Schuffenhauer 2009). The SA score for all selected molecules was in the range from 6 to 7 (Supplementary Table S7) exhibiting the ease of synthesis on a scale of 1–10. The phytochemical Isolimonexic acid, nomilin and limonin show zero alerts in finding characteristics of the PAINS molecules yielding false positive biological output irrespective of the protein target (Baell and Holloway 2010) obtained by SwissADME evaluation (Supplementary Table S7). Hence from the above studies, limonin is the preferred antiviral phytochemical in DENV disease in humans exhibiting no violation in terms of ADMET and medicinal chemistry-associated properties (Baell and Holloway 2010).

FTIR spectra

For the preparation of the interaction sample for FTIR spectroscopy, 0.001 mg of S-glycoprotein was mixed in 2 ml of 4 milli-molar limonin solution for 10 min in an ultra-sonication chamber. For experimental verification of the interaction of limonin and E-glycoprotein, FTIR spectroscopy was performed after interacting S-glycoprotein sample with limonin indicating broad peaks of OH−1 in comparison to OH−1 peak at 3450 cm−1 obtained with limonin sample showing evidence of hydrogen bond (Fig. 5). The peak at 3450 cm−1 is broadened after interaction of limonin with E-glycoprotein revealing hydrogen bonding between limonin and E-glyco protein indicating the formation of the protein complex. A small peak of limonin–E-glycoprotein conjugate sample at 1600 cm−1 confirms the presence of NH groups, whereas NH vibration peaks do not prominently appear in FTIR of only limonin samples. The broad C–N stretching peaks have been seen at 1150 cm−1 in comparison to that of only the limonin sample indicating interaction between limonin and E-glycoprotein (Omar et al. 2020). The absorption peaks at 2920 cm−1 reveal the presence of β-furan ring in the sample (Qin et al. 2018) and the peak at 1395 cm−1 indicates the occurrence methyl groups in both samples (Derdar et al. 2019) showing chemical functional groups of limonin.

Fig. 5.

Fig. 5

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FTIR results of limonin and E-glycoprotein–limonin conjugate sample

Conclusion

Here we have performed Molecular docking analysis of citrus limonoids into E-glycoprotein of DENV-2 (a deadly RNA virus spread especially over the Indian subcontinent). The Isolimonexic acid, limonexic acid, Citrusin, nomilin and limonin molecules have been identified as potential inhibitors based on Binding Affinity (BA > − 8.4 kcal/mole). Considering Lipinski’s rule of five as per drug likeliness and also ADMET study, limonin was screened. We have also performed the effectiveness study of the above molecules on (DC-SIGN–E-glycoprotein complex (made through molecular docking). Here, limonin shows strong hydrogen bonding and physical interaction with complex having a binding affinity of − 8.5 kcal/mole. Further due to the smaller molecular weight of limonin, its strong physical interaction between DC-SIGN and in comparison to that with E-glyco protein assures the prevention of bonding of E-glyco protein with conserved residues with DC-SIGN protein that inhibits the spread of DENV-2. Experimentally FTIR spectroscopy confirms a strong hydrogen bonding interaction of E-glyco protein with limonin. The strong physical interaction between DC-SIGN and small limonin molecules in comparison to that of E-glycoprotein with DC-SIGN promises the growth of immunity against DENV2. Further, the results are beneficial in articulating tactics using conventional medicine and in the optimization of drug design against DENV-2. The experimental biological estimations for validation of the in-silico results through RAT model are beneficial in articulating tactics using conventional medicine and in optimization of drug design against DENV-2.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file 1 (DOCX 150 kb) (150.6KB, docx)

Acknowledgements

The authors like to thank the Department of Electronics and Communication Engineering, Tezpur University for providing facilities to do the research work.

Author contributions

Satyajit Das: Conceptualization, methodology, investigation, writing—original draft preparation. Geetartha Sarma: investigation, writing—reviewing and editing Nithin Joseph Panicker.: Investigation, writing—reviewing and editing, Partha P. Sahu: supervision, writing—reviewing and editing.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

All data generated or analyzed during this study are included in this published article (and its supplementary information files).

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Supplementary Materials

Supplementary file 1 (DOCX 150 kb) (150.6KB, docx)

Data Availability Statement

All data generated or analyzed during this study are included in this published article (and its supplementary information files).

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