In Silico analysis of compounds characterized from Pseudarthria viscida root with TNF-α

Tumour necrosis factor is a polypeptide cytokine involved in inflammation and the acute phase response. TNF-alpha is present in larger quantities in persons with rheumatoid arthritis. Direct inhibition of TNF-α by the commercial biological agents has produced significant advances in rheumatoid arthritis treatment and validated the extra-cellular inhibition of this proinflammatory cytokine as an effective therapy. However, viable leads molecule that inhibits TNF-α have not been reported . Bioinformatics is seen as an emerging field with the potential to significantly improve how drugs are found brought to the clinical trials and eventually released to the marketplace. Computer-Aided Drug Design (CADD) is a specialized discipline that uses computational methods to stimulate drug-protein interaction. Discovery studio 2.1 provides a set of protocols for predicting and analyzing the interaction between protein and ligands. Molecular Docking experiments were carried out for the compounds identified from Pseudarthria viscida root extract with TNF-alpha using Accelry’s DISCOVERY STUDIO 2.1. Docking is a method which predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex. Out of 13 compounds characterized from Pseudarthria viscida, only one of them docked with Tumour necrosis factor α.


INTRODUCTION
Tumour necrosis factor alpha (TNF α) is a pleotropic inflammatory cytokine produced by the immune system that suppresses tumour cell proliferation.Subsequent studies established that TNF α is a key mediator of inflammation (Warren et al., 1988;Vasanthi et al., 2007).It is a proinflammatory cytokine capable of inducing multiple signaling cascades that can serve in host defense and paradoxically contribute to inflammatory tissue injury (Mu Culloch et al., 2006).The elevated level of TNF α has been reported in arthritic patients and in experimentally induced arthritis (Philippe et al., 1997).
Molecular Docking continues to hold great promise in the field of Computer Aided Drug Design (CADD) which screens small molecule by orienting and scoring them in the binding site of a protein.It is a study of how two (or) more molecular structure, fit together.In other words, the problem in like solving a 3 dimensional puzzles.The action of a harmful protein in human body may be prohibited by finding an inhibitor, which binds to that particular protein.Novel ligands for enzymes of known structure were designed and their interaction energies were calculated using the scoring function (Irwin et al., 2002).
Secondary metabolites are the substances, which are produced by plants as defense chemicals.It includes alkaloids, flavonoids, essential oils, phenols, terpenes etc.These metabolites are sought after because they are known to exhibit number of biological activities that promotes health effects (Saxena, 2001).Plants have played a remarkable role in healthcare since the ancient times.Traditional plant based medicines still exert a great deal of importance to people living in developing countries and also leads to the discovery of new drug candidates (Orhan et al.,2007).The plants were initially used in unmodified form, later as extracts, and in the 19 th century, advances in chemistry made it possible to isolate the active compounds of some medicinal plants.A large number of the pharmaceutical agents used today contain natural compounds, including those with various modification of the original molecule (Kinghorn, 2001).In addition, bioactive plant compounds have served as templates for several synthetic drugs, and as precursors used in the production of semi-synthetic drugs (Newman et al, 2003;Verdine, 1996;Wessjohann, 2000).Historically, Willow bark (Salix) was used for its antiinflammatory properties, which later resulted in isolation and synthesis of pure compound salicylic acid, and its acetylated derivative acetyl salicylic acid, commonly known as Aspirin (Vane and Botting, 1996).

Ligand preparation
The three dimensional structures of compounds taken for binding analysis were downloaded in .sdfformat from PubChem database.Hydrogen bonds were added and the energy was minimized using CHARMm force field.Lipinski properties such as Molecular weight, XLog P, number of hydrogen bond donors and acceptors for the compounds were obtained from PubChem (shown in Table : 1)

Protein preparation
The PDB is a key resource in areas of structural biology, is a key repository for 3D structure data of large molecules.The molecule which taken is TNF α for our consideration.The PDB ID 2TNF and a resolution factor is 1.40 A˚ and the method of incorporation is X-ray diffraction method.The ligand and crystallographic water molecules were removed from the protein; and the chemistry of the protein was corrected for missing hydrogen.Crystallographic disorders and unfilled valence atoms were connected using alternate conformations and valence monitor options.Following the above steps of preparation, the protein was subjected to energy minimization using the CHARMm Force field.

Docking studies
The docking method used in this study is LigandFit.To perform docking process the modeled protein, a protocol called "Dockligands" (LigandFit) is selected among those listed under receptor-ligand interaction protocol cluster.Each ligand compound is given as input in the parameter meant for "input ligands" and the protocol was run for each of the inhibitors selected for the study.The various conformations for ligand in this docking procedure were generated by Monte Carlo trials.The final energy refinement of the ligand pose (or) pose optimization in ligandfit occurs by Broyden-Flecher Gold Farbshanno (BFGS) method.The Dock score of the best poses docked in to the enzyme for all the 13 compounds is calculated.

RESULTS AND DISCUSSION
The crystal structure of Tumour Necrosis Factor  with PDB ID 2TNF having structural weight 52000.04 is retrieved from PDB.The resolution factor is 1.40 A and the method of incorporation is X-ray diffraction method.The structure of 2TNF has in total 3 chains viz.A, B and C. All the chains are sequence unique.The structure of TNF has one helices and twelve stands.1% of the structure comprising 3 aminoacid residues belongs to helical part and 48% of structure comprising 75 amino acid residues belongs to strands.Out of different compounds (ligands) taken for docking analysis only one compound docked with the protein turmour necrosis factors alpha.Docked pose of the compound with protein (Tumour necrosis factor α ) is presented in Figure 1.The dockscore values includes Ligscore 1&2 (Mayo et al., 1990); Piecewise Linear Potential (PLP)-PLP1 (Krammer et al ,2005;Gehlhaar et al., 1995) and PLP 2 (Gehlhaar et al.,1995); Jain (Jain 1996) ; Potential of Mean Force-PMF (Mugge and Martin YC, 1999); PMF04 (Mugge, 2006), Ligand internal energy and dockscore obtained using LigandFit protocol of Discovery studio 2.1.
The ligand rutin docked with protein turmour necrosis factor with 22.035 dock score value and formed two hydrogen bonds.The atoms involved in forming hydrogen bond between rutin and protein (TNFα) are presented in Table : 2   Table. 2 To ensure that the ligand orientation obtained from the docking studies was likely to represent valid and reasonable binding modes of the inhibitors, the ligand Fit program docking parameters had to be first validated for the crystal structure's active site.Protein utilities and health protocol of Discovery's studio was used to find out the active site contains amino acids such as Ser 9, Lys 11, Ala 156, Leu 157 etc. Results of docking showed that the LigandFit determined the optimal of the docking inhibitor, exactly to these active sites.
Here top ranked ligand is taken for binding affinity studies.The validation process consisted of two parts; 1. Hydrogen bond details of the top -ranked docked pose.3 2. Prediction of binding energy between the docked ligand and the enzyme using various score calculated using Discovery studio

CONCLUSION
In appreciating the inflammatory process, it is important to understand the role of chemical mediators.These are the substances that tend to direct the inflammatory response.These inflammatory mediators come from plasma proteins or cells including mast cells, platelets, neutrophils and monocytes / macrophages.They are triggered by bacterial products or host proteins.Chemical mediators bind to specific receptors on target cells and can increase vascular permeability and neutrophil chemotaxis, stimulate smooth muscle contraction, have direct enzymatic activity, induce pain or mediate oxidative damage.Most mediators are short-lived but cause harmful effects.One such a mediator is TNF α.In silico molecular docking in one of the most powerful techniques to discover novel ligand for proteins of known structure and thus play key role in structure based drug.Investigators often use docking computer programs to find the binding affinity for molecules that fit a binding site on the protein.
Hence in this present work we have carried out in silico molecules docking to analyze the binding properties of the mediator called TNF α with 13 different compounds reported from root of Pseudarthria viscida.The wet analysis carried out by us showed very good result with regard to anti-inflammatory property of this plant extract.So the present study may act as supportive evidence that substanciate property of this plant extract may because of the inhibiting ability of Rutin identified from this plant with TNF α.