Evaluation of binding affinity of S eenthil kudineer with enzyme RNA- dependent RNA polymerase of dengue virus through molecular docking In- silico approach

ETHEL SHINY.S¹, BHARATH CHRISTIAN.C.B.S², GOMATHI.P ³

¹ Associate Professor & HOD( I/c), Department of Gunapadam – Marunthiyal,

² Assistant Professor, Department of Maruthuvam,

³ Assistant Professor, Department of Varmam, Pura Maruthuvam and Sirappu Maruthuvam,

^1,2,3 Santhigiri Siddha Medical College, Thiruvananthapuram, Kerala.

Seenthil Kudineer (SK) a classical siddha formulation is used to treat various fevers which cause moderate to severe acute hemorrhagic symptoms as is indicated in the text. The research article has shown the following bioactive compounds such as Tinosporide, Santalic acids, Gingerenone-A, Vasicinone, Beta-santalol, Rutin, Geniposide, Thymol, Ascorbic acid and Gamma-Himachalene. Molecular docking is a great approach in current trends to identify the possibility of pharmacological effects of medicinal compounds which could be exerted over their corresponding protein targets which are relevant for the disease. Docking simulations were performed using the Lamarckian genetic algorithm (LGA) and the Solis & Wets local search method ( Solis and Wets, 1981) against the target protein Dengue virus NS5 RNA dependent RNA polymerase (2J7U) which is responsible for viral replication and transcription. The binding energy observed were of the order: -10.28 Kcal/mol (Rutin), - 7.90Kcal/mol (Geniposide),

-7.14 Kcal/mol(Gingerenone), - 6.09 Kcal/mol(Santalic acids),-5.91 Kcal/mol (Gamma Himachalene), -5.80 Kcal/mol(Beta - Santalol), -5.43Kcal/mol(Tinosporide), - 5.32 Kcal/mol(Ascorbic acid), -5.12 Kcal/mol(Vasicinone) and -4.22 Kcal/mol(Thymol), respectively towards for the target 2J7U. These findings confirm that the Siddha formulation Seenthil Kudineer has some potent activity against the symptoms produced by Dengue virus.

Key words : Seenthil Kudineer, Dengue , Molecular docking, Siddha medicine.

1.Introduction :

Throughout the world, dengue ranks as most important mosquito borne viral disease in the world. WHO estimates that 50-100 million dengue infections occur in each year and almost half of the world’s population lives in the countries, where dengue is endemic. Close to 75% of global population exposed to dengue are in Asia-Pacific region.There are no antiviral medicines available for dengue. Current conventional management focuses on fluid replacement and management of other metabolic consequences of viral infection.A major problem in fight against virus is rapid adaptation and development of drug resistance as well as emergence of new hybrid viruses. In past few years natural remedies came more and more in centre of interest. Recent studies showing antiviral potential of plant extracts against viral strains resistant to conventional antiviral agents have challenged modern drug discovery practices and deem a very careful look towards exploring natural antiviral components of medicinal plants and exploring traditional medical formulations used in treatment of viral diseases.

Molecular docking is the preclinical and a in-silico approach can be done before starting the pharmacological and the clinical study which can be helpful one to assessing the future outcome whether positive or negative in the management of the particular disease.Eventhough Molecular docking studies are considered as a preliminary study, it is significant with other Pre-clinical studies done by the Research scholars from various fields including in-vivo immuno-modulatory activity against viral infection in backyard chicken and in-vivo safety studies in wistar rats.

Dengue fever is an acute febrile illness caused by virus belonging to Flaviviridae family. Four well defined dengue viruses identified are DENV-1, DENV-2, DENV-3 and DENV-4. Each of them has a distinct genetic structure. Dengue virus is transmitted to humans through bites of infective Ades mosquitos- Ades aegyptii, Ades albopictus. Dengue fever is characterized by biphasic fever, myalgia, arthralgia and rashes. Dengue haemorrhagic fever (DHF) is characterized by abnormality in haemostasis and by marked leakage of plasma from the capillaries. The later may lead to Dengue shock syndrome (DSS).

In siddha medicine, the Pithasuram mentioned in the classical siddha text book, Yugi Vaidhiya Chinthamani can be correlated with Dengue fever.Most of the symptoms in the pitha suram can be matched with the signs and symptoms of dengue clinical presentation as follow.

Table.1. Pitha suram vs Dengue Fever ( A Correlation view)

2.Materials and Methods :

2.1. Source of drug

The Sastric drug Seenthil Kudineer (SK) was prepared as per the Siddha classical text book Gunapadam mooligai vaguppu.

The drug has the following 8 herbals such as Tinospora cordifolia (Seenthil),

Santalum album(Santhanam), Zingiber officinale (Chukku),

Sida cordifolia (Sittramutti), Cyprus rotundus (Korai kizhangu),

Hedyotis corymbosa (Parpadagam), Plectranthus vettiveriodes ( Vilamichuver) and Chrysopogon zizanioides( Veetiver).

Table.2. List of herbs present in the SK formulation with their phytochemicals

2.2. Molecular Docking

Molecular Docking analysis performed with a commonly well-known established software Auto dock Gridfree a very convenient and excellent screening tool for identifying binding energy between the 3D structures of each ligand and target proteins.

The target protein PDB ID: 2J7U was selected, a Gridfree docking performed, and the binding energies of each ligand found.

Figure.1.Structure of Dengue virus NS5 RNA dependent RNA polymerase protein

2.3. Receptor Structure :

Crystalline structure of the target protein Dengue virus NS5 RNA dependent RNA polymerase (2J7U) was retrieved from protein data bank and protein clean-up process was done and essential missing hydrogen atom were being added.

Different orientation of the lead molecules with respect to the target protein was evaluated by Autodock program and the best dock pose was selected based on the interaction study analysis.

2.4. Objective of Molecular Docking :

Binding of phytocomponents with the core amino acids (Arg-737, Arg-729 and Ser-710) of the targets by forming hydrogen bond will hinder the function of the enzyme RNA-dependent RNA polymerase since these are the prime mediators for dengue viral replication. Thereby phytocomponents which inhibit this enzyme may act as a potential therapeutic agent for management of dengue fever can be assessed by this method.

2.5. Methodology :

Docking calculations were carried out for retrieved phytocomponents against target enzyme Dengue virus NS5 RNA dependent RNA polymerase (2J7U). Essential hydrogen atoms, Kollman united atom type charges, and solvation parameters were added with the aid of AutoDock tools ( Morris, Goodsell et al., 1998).

Affinity (grid) maps of ×× Å grid points and 0.375 Å spacing were generated using the Autogrid program (Morris, Goodsell et al., 1998). AutoDock parameter set- and distance-dependent dielectric functions were used in the calculation of the van der Waals and the electrostatic terms, respectively.

Docking simulations were performed using the Lamarckian genetic algorithm (LGA) and the Solis & Wets local search method ( Solis and Wets, 1981). Initial position, orientation, and torsions of the ligand molecules were set randomly.

All rotatable torsions were released during docking. Each docking experiment was derived from 2 different runs that were set to terminate after a maximum of 250000 energy evaluations. The population size was set to 150. During the search, a translational step of 0.2 Å, and quaternion and torsion steps of 5 were applied.

3. Observation and Inference :

Table.3. 2D and 3D Structure of Selected Ligands with active components in

Each herbs used in the Seenthil Kudineer preparation

Table.4. Ligand Properties of the Compounds selected for Molecular docking

Table.5. Summary of the molecular docking studies of compounds against

Dengue virus NS5 RNA dependent RNA polymerase - PDB 2J7U

Figure.2. Docking Poses , 2D interaction plot analysis and Hydrogen bond plotting with core amino acid a nalysis of all the compounds of the molecular docking studies of Seenthil kudineer against Dengue virus NS5 RNA dependent RNA polymerase - PDB 2J7U

D OCKING POSE

Tinosporide with Dengue virus NS5 RNA dependent RNA polymerase - PDB 2J7U

Tinosporide with target -

2D Interaction Plot Analysis

Tinosporide with target-

Hydrogen bond plotting with core

amino acid Analysis

D OCKING POSE

Santalic acid with Dengue virus NS5 RNA dependent RNA polymerase -

PDB 2J7U

Santalic acid with target -

2D Interaction Plot Analysis

Santalic acid with target -

Hydrogen bond plotting with core amino acid Analysis

DOCKING POSE

Gingerenone-A with Dengue virus NS5 RNA dependent RNA polymerase - PDB 2J7U

Gingerenone-A with target -

2D Interaction Plot Analysis

Gingerenone-A with target -

Hydrogen bond plotting with core amino acid Analysis

DOCKING POSE

Vasicinone with Dengue virus NS5 RNA dependent RNA polymerase - PDB 2J7U

Vasicinone with target -

2D Interaction Plot Analysis

Vasicinone with target -

Hydrogen bond plotting with core amino acid Analysis

DOCKING POSE

Beta-santalol with Dengue virus

NS5 RNA dependent RNA polymerase -

PDB 2J7U

Beta-santalol with target -

2D Interaction Plot Analysis

Beta-santalol with target -

Hydrogen bond plotting with core amino acid Analysis

DOCKING POSE

Rutin with Dengue virus NS5 RNA dependent RNA polymerase -

PDB 2J7U

Rutin with target -

2D Interaction Plot Analysis

Rutin with target -

Hydrogen bond plotting with core amino acid Analysis

DOCKING POSE

Geniposide with Dengue virus

NS5 RNA dependent RNA polymerase - PDB 2J7U

Geniposide with target -

2D Interaction Plot Analysis

Geniposide with target -

Hydrogen bond plotting with core amino acid Analysis

DOCKING POSE

Thymol with Dengue virus NS5 RNA dependent RNA polymerase

- PDB 2J7U

Thymol with target -

2D Interaction Plot Analysis

Thymol with target -

Hydrogen bond plotting with core amino acid Analysis

DOCKING POSE

Ascorbic acid with Dengue virus NS5 RNA dependent RNA polymerase

- PDB 2J7U

Ascorbic acid with target -

2D Interaction Plot Analysis

Ascorbic acid with target -

Hydrogen bond plotting with core amino acid Analysis

DOCKING POSE

Gamma- Himachalene with Dengue virus NS5 RNA dependent RNA polymerase -

PDB 2J7U

Gamma- Himachalene with target -

2D Interaction Plot Analysis

Gamma- Himachalene with target -

Hydrogen bond plotting with core amino acid Analysis

Table.6. Amino acid Residue Interaction of Lead and Standard against

Dengue virus NS5 RNA dependent RNA polymerase PDB 2J7U

3.1.Observations of Amino acid Residue Interaction of Lead and Standard against

Dengue virus NS5 RNA dependent RNA polymerase PDB 2J7U :

Total of 10 bioactive lead compounds were retrieved from the herbs present in the siddha formulation - Seenthil Kudineer. From reported data the phytochemicals such as Tinosporide, Santalic acids, Gingerenone-A, Beta-santalol, Rutin, Geniposide, Ascorbic acid and Gamma-Himachalene reveals maximum of 2- 3 interactions that accounts of 100 % of the occupancy with the core active amino acid residues present on the target dengue NS5 RNA dependent RNA polymerase enzyme.

4. Conclusion :

Based on the results of the computational analysis it was concluded that the bio-active compound’s like Tinosporide, Santalic acids, Gingerenone-A, Vasicinone, Beta-santalol, Rutin, Geniposide, Thymol, Ascorbic acid and Gamma-Himachalene in Seenthil kudineer, possess significant binding against the amino acid residues Arg-737, Arg-729 and Ser-710 present on the active site thereby it was concluded that these compounds may exerts promising anti-viral by inhibiting the enzyme Dengue NS5 RNA dependent RNA polymerase.

Thereby phytocomponents which inhibit the target may act as a potential therapeutic agent for management of dengue viral infection by inhibiting the viral entry into target cellular pathway and replication.

Based on the findings of docking score values we can strongly suggest this Siddha poly herbal formulation Seenthil Kudineer for the better management of Dengue. Further preclinical and clinical trials have to be conducted in order to know the exact mechanism and efficacy of Seenthil Kudineer in Dengue infection management.

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