INTRODUCTION
Dengue virus (DENV) is the causative agent of den- gue fever which is currently an increasing health prob- lem in many countries. It is a member of the Flavivirus genus of the Flaviviridae family. DENV is transmitted to humans by the bite of Aedes aegypti or Ae. albopictus female mosquitoes and causes the full spectrum of infec- tion by any one of five serologically related but geneti- cally distinct virus serotypes (DEN 1-5)1–7. In several cases, the disease progresses to serious medical condi- tions such as dengue hemorrhagic fever8–10 which causes bleeding, low blood platelets count and blood plasma leak- age, or dengue shock syndrome defined by a massive in- crease in the systemic capillary permeability with conse- quent hypovolemia and dangerously low blood pressure11–14. DENV has a single-stranded RNA genome that is pack- aged by the virus capsid protein in the host-derived lipid bilayer and surrounded by 180 copies of two glycopro-
In silico evaluation of inhibitory potential of triterpenoids from Azadirachta indica against therapeutic target of dengue virus, NS2B-NS3 protease
Vivek Dhar Dwivedi
1–2, Indra Prasad Tripathi
1& Sarad Kumar Mishra
21Faculty of Science and Environment, Mahatma Gandhi Chitrakoot Gramodaya Vishwavidyalaya, Chitrakoot, Satna, Madhya Pradesh;
2Department of Biotechnology, Deen Dayal Upadhyaya Gorakhpur University, Gorakhpur, Uttar Pradesh, India
ABSTRACT
Background & objectives: NS2B-NS3 protease (NS2B-NS3pro) of dengue virus (DENV) is the prime therapeutic target for the development of anti-dengue drug to combat the DENV infection, which is currently an increasing health problem in many countries. In the area of antiviral drug discovery, numerous reports on the antiviral activity of various medicinal plants against dengue viruses have been published. Neem plant (Azadirachta indica) is one among those medicinal plants which is reported to show potential antiviral activity against DENV. But active principle of neem plant extract which has inhibitory potential against DENV NS2B-NS3pro is not yet reported.
The aim of the present study was to explore the inhibitory potential of five triterpenoids from neem plant, viz.
nimbin, desacetylnimbin, desacetylsalannin, azadirachtin and salannin, against DENV NS2B-NS3pro.
Methods: The molecular 3D structural data of DENV NS2B-NS3pro and selected triterpenoids of neem plant were collected from protein databank (PDB ID: 2VBC) and PubChem database respectively. The molecular docking approach was employed to find out the in silico inhibitory potential of the five triterpenoids against DENV NS2B- NS3pro.
Results: The molecular docking results showed that nimbin, desacetylnimbin and desacetylsalannin have good binding affinity with DENV NS2B-NS3pro, while azadirachtin and salannin did not show any interaction with the target protein. It was observed that the DENV NS2B-NS3pro binding energy for nimbin, desacetylnimbin and desacetylsalannin were –5.56, –5.24 and –3.43 kcal/mol, respectively.
Interpretation & conclusion: The findings attained through this study on the molecular interaction mode of three neem triterpenoids and DENV NS2B-NS3pro can be considered for further in vitro and in vivo validation for designing new potential drugs for DENV infection.
Key words Azadirachtin; DENV; desacetylnimbin; desacetylsalannin; drug designing; neem; nimbin; NS2B-NS3pro; salannin
teins. The RNA genome of DENV is about 11000 bases that encode only 10 proteins. Three of these are struc- tural proteins, viz. capsid protein (C), membrane precur- sor protein (prM) and envelope protein (E), which form the coat of the virus and deliver the RNA to target cells;
while seven of them are nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 that coordi- nate the assembly of new viruses once the virus gets in- side the host cell15–17. The non-structural protein NS3 con- tains a protease (NS3pro) and a helicase (NS3hel) domain.
The NS3pro domain mediates the processing of polyprotein in specific site. The NS3pro domain, the member of S7 family of serine proteases depends upon a cofactor, NS2B to turn into their fully active form. NS2B-NS3pro of DENV has already been recognized as a potential therapeutic target for the development of new antiviral compounds against DENV18–23.
Therefore, there is a need for screening the new po- tent inhibitors from various sources using different strat-
egies for the development of effective drugs against DENV. In the area of drug discovery, medicinal plants are one of the most popular sources of effective natural lead compounds. They contain a variety of phytochemicals that can be used as drug against different diseases and infections. There have been numerous reports on the antiviral activity of various plants against dengue viruses16, 25. Neem plant (Azadirachta indica) is one among those medicinal plants, which possesses potential antiviral activity against DENV26. More than 135 phytochemicals of diverse structure including triterpenoids have been isolated from various parts of neem plant, but only few of them have been studied for their specific medicinal properties27–28. Triterpenoids are terpenoid derivatives of triterpene molecules which ex- hibit a wide range of biological properties, including an- ticancer, antibacterial, antifungal, antimalarial, and anti- viral activities. The triterpenoids present in neem like salannin, nimbin, desacetylnimbin, desacetylsalannin, 3- tigloylazadirachtol, azadirachtin, salanninolide, 3-acetyl- 1-tigloyl azadirachtin, azadirachtin I, azadirachtin D, etc.
are known for their specific medicinal activities29. The antiviral activity of triterpenoids from neem plant against DENV NS2B-NS3pro has not yet been reported.
In the present study, five triterpenoids from neem plant, namely salannin, nimbin, desacetylnimbin, desacetylsalannin and azadirachtin have been evaluated for their potential inhibitory activity against DENV NS2B- NS3pro using in silico molecular docking approach. The present study was undertaken to identify an effective and potential drug candidate against dengue infection in view of the fact that till now there is not a single drug available for dengue infection.
MATERIAL & METHODS Receptor and ligands structure
The crystal structure of complete NS2B-NS3 mol- ecule fused to 18 residues of the NS2B cofactor was re- trieved from Protein Data Bank (PDB–Available from http://www.rcsb.org) using the PDB code: 2VBC. This crystal structure was identified and reported by Luo et al30 at a resolution of 3.15 Å. This structure contains two domains: serine protease N-terminal domain and the ATPase/helicase domain located at the C terminus of NS2B-NS3. The serine protease N-terminal domain of NS2B-NS3 was selected for docking studies with small molecules of interest. The 3D structure of triterpenoids of neem plant (azadirachtin, desacetylnimbin, desacetylsalannin, nimbin, and salannin) were searched and downloaded from PubChem database (Available from
http://pubchem.ncbi.nlm.nih.gov) in SDF format. All the visualizations of molecular structure were performed with Pymol31 and Ligplot32–33 as they are very powerful softwares, for all purpose of molecular visualizations.
Molecular docking
MTiAutoDock web server34 was employed to per- form molecular docking between NS2B-NS3pro of DENV and selected triterpenoids from neem plant. AutoDock allows to dock compounds into a binding site defined by the user or blind docking using AutoDock 4.235. The se- lected ligand molecules and the protein NS2B-NS3 serine protease of DENV were converted in SDF and PDB for- mat respectively, and uploaded into MTiAutoDock server for autonomous docking. The uploaded protein structure in PDB format in MTiAutoDock is automatically cleaned and preprocessed: all hetero atoms (HETATMs) are re- moved and hydrogen atoms are added to the structure using molecular graphics laboratory (MGL) tools. Fur- ther, the calculated active site in the crystal structure of protein was demarcated by using the list of residues (HIS 51, ASP 75 and SER 135) which form a catalytic triad in the binding site of the DENV NS2B-NS3pro.
RESULTS Molecular docking
Molecular docking of small molecules into the bind- ing site of therapeutic target protein is a technique com- monly used in the area of drug discovery and molecular interaction studies36–38. The inhibitory activity of small molecules in cell line experiments does not give compre- hensive information concerning the binding orientation of ligands with the target protein. But in silico molecular docking studies give a straightforward description about the molecular interaction of the compounds with the ac- tive site of the protein39. Hence, docking analysis was performed to predict the inhibitory potential of selected neem triterpenoids against DENV NS2B-NS3pro and also to determine the amino-acid residues of DENV NS2B- NS3pro involved in interaction with triterpenoids. Five chemically diverse neem triterpenoids including azadirachtin, desacetylnimbin, desacetylsalannin, nimbin and salannin (Figs. 1 a–e) were selected to study their effect on dengue NS2B-NS3pro. These small molecules were docked into the receptor structure to form complexes.
Docking generated several structures and those having high docking scores were selected for further studies. The molecular docking results showed that nimbin, desacetylnimbin and desacetylsalannin have good binding affinities with DENV NS2B-NS3pro, while
azadirachtin and salannin did not show any interaction with the target protein. Our finding substantiates the re- port of Parida et al26, in which they have reported that azadirachtin is not capable of inhibiting the replication of dengue type-2 through their in vitro and in vivo studies. It was observed that the DENV NS2B-NS3pro binding en- ergies for nimbin, desacetylnimbin and desacetylsalannin were –5.56, –5.24 and –3.43 kcal/mol, respectively. This provides the evidence that the ligand molecules have more affinity to the active site of protein and can be used as efficient and potential inhibitor or drug molecule.
Interaction analysis of protein-ligand complexes The characterization of interactions in protein-ligand complexes is essential for research in the area of struc- tural bioinformatics and drug discovery40. It is crucial for complete understanding of the molecular mechanisms of biological systems41. The interaction analysis of NS2B- NS3pro and nimbin complex (Fig. 2) revealed that four residues of DENV NS2B-NS3pro, viz. His51, Asp75, Ser135 and Asn152 are involved in the formation of hy- drogen bonds between nimbin and NS2B-NS3pro, while six residues, viz. Val36, Arg73, Pro132, Gly133, Gly153 and Val154 residues are involved in the hydrophobic in- teractions. Hydrogen bonding and hydrophobic interac- tions are key players in stabilizing energetically-favoured ligands, in an open conformational environment of pro- tein structures42. However, it is still poorly understood
how the binding parameters associated with these inter- actions facilitate a drug-lead to recognize a specific tar- get and improve drug’s efficacy43–44. The interaction plot of NS2B-NS3pro and desacetylnimbin complex as shown in Fig. 3, illustrated that in total four hydrogen bonds are involved between desacetylnimbin and NS2B-NS3pro
Figs. 1 (a–e):2D structures of neem triterpenoids from PubChem database: (a) azadirachtin; (b) desacetylnimbin; (c) desacetylsalannin; (d) nimbin; and (e) salannin.
Fig. 2:2D interaction plot of DENV NS2B-NS3proand nimbin complex generated through Ligplot programme.
Fig. 3: 2D interaction plot of desacetylnimbin and DENV NS2B- NS3pro complex.
pocket residues, Arg54, Gly133 and Asn152 respectively, in which all residues formed only one hydrogen bond except Arg54 which formed two hydrogen bonds. In to- tal 8 amino acid residues; Val36, Trp50, His51, Val72, Arg73, Asp75, Pro132, and Ser135 of NS2B-NS3pro pocket were found to be involved in hydrophobic inter- action with desacetylnimbin. From Fig. 4 of DENV NS2B-NS3proand desacetylsalannin complex, it can be deduced that only hydrophobic interactions were found between NS2B-NS3proand desacetylsalannin. Seven resi- dues (Trp50, His51, His54, Val72, Arg73, Asp75 and Asn152) of the NS2B-NS3pro pocket were found to be involved in the formation of these weak interactions.
DISCUSSION
Dengue is a complicated arthropod-borne viral infec- tion with wide spectrum of clinical signs and symptoms in humans. Each year, there are ~50 million dengue in- fections and ~500,000 individuals are hospitalized with dengue haemorrhagic fever, mainly in Southeast Asia, the Pacific and the Americas45. Increase in DENV cases is an important medical issue for which neither a special- ized vaccine nor an efficient drug available in the market.
NS2B-NS3 protease of DENV plays a vital role in ge- nome replication and viral RNA synthesis, through the cleavage of the viral polyprotein precursor to discharge individual non-structural macromolecules and a C-termi-
nal NTPase-dependent RNA helicase18, 30, 46. Hence, NS2B-NS3 protease is considered as a therapeutic target for the discovery and designing of potential lead com- pounds to combat the DENV infection. The hydrogen bonding between the ligand and one of the catalytic triad (His-51, Asp-75, and Ser-135) residues of NS3 protease could interrupt electron transfer between the carboxyl group of Asp-75 and nitrogen atom on imidazole group of His-51, possibly disrupting the ability of His-51 to activate nucleophilic attack of hydroxyl group (ß-OH) of Ser-135, which is essential for the initiation of proteoly- sis39, 47. Five putative substrate binding residues (Asp- 129, Phe-130, Tyr-150, Asn-152 and Gly-153), conserved among the Flavivirus, form the small part in the ß- sheet which is common in all serine proteases39, 48.
In this study, triterpenoids nimbin and desacetyl- nimbin showed hydrogen bond interactions with the cata- lytic triad and some of the substrate binding residues while the desacetylsalannin showed only hydrophobic interac- tions with the catalytic triad and some of the substrate binding residues. In an earlier study, it has been reported that the triterpenoids and other active compounds present in the neem plant have potential antiviral activity against coxsackie B group of viruses49. In an in silico study, nimbin has been discovered as effective against the envelope protein of four serotypes of DENV (dengue1–4)50. In another in vitro study, the antiviral ac- tivity of triterpenoid saponin, isolated from a Brazilian plant (s21) has been described against herpes simplex virus type I51. Findings of the present study suggest that nimbin, desacetylnimbin and desacetylsalannin triterpenoids commonly found in neem plant, possess very good binding affinity with DENV NS2B-NS3pro and can be considered as potential inhibitors of DENV NS2B- NS3pro for the development of highly effective and po- tential drugs against dengue virus infection.
CONCLUSION
The present study was undertaken to find out the in silico inhibitory potential of five triterpenoids (nimbin, desacetylnimbin, desacetylsalannin, salannin, and azadirachtin) found in neem plant against DENV NS2B- NS3pro. This study revealed nimbin, desacetylnimbin and desacetylsalannin as three potential broad-spectrum in- hibitors against DENV NS2B-NS3pro. The findings at- tained through this study on the molecular interaction mode of three neem triterpenoids and DENV NS2B- NS3pro can be considered for further in vitro and in vivo validation for designing new potential drugs against DENV infection. The above study is highly significant,
Fig. 4:2D interaction plot of DENV NS2B-NS3proand desacetyl- salannin complex.
as till now there is no efficient medicine available to com- bat dengue infection.
Conflict of interest
All the authors declare that they have no conflict of interest.
ACKNOWLEDGEMENTS
Authors acknowledge the support provided by De- partment of Biotechnology, D.D.U. Gorakhpur Univer- sity, Gorakhpur, Uttar Pradesh, India.
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Correspondence to: Dr Sarad Kumar Mishra, Department of Biotechnology, Deen Dayal Upadhyaya Gorakhpur University, Gorakhpur–273 001, Uttar Pradesh, India.
E-mail: [email protected]
Received: 2 December 2015 Accepted in revised form: 29 March 2016
