In silico docking analysis of selective bioactive compounds of Phyllanthus acidusaqueousfruit extractagainst MAPK1

Introduction and Aim: Phyllanthus acidus L.Skeels (Family: Phyllanthaceae) or Star Gooseberry which bears small, edible, juicy, sour, yellow berries fruit is known as a “liver tonic” in ayurvedic medicine. However, the behavior of the plant fruit or its constituents in cell apoptosis/cell survival is unknown. Hence, the purpose of thepresent study was to perform an in silico docking of selective bioactive compounds of aqueous extract of fruit of P.acidus (PAFAE) against MAPK1. Mitogen activated protein kinase is a family of serine threonine specific protein kinases- MAPK1/ERK1/2, JNK1-3, p38MAPK and ERK5.Activation ofMAPK1 promotes cell survival in certain tissues by inhibiting proapoptotic proteins and by stimulating anti apoptotic factors. 
  
Methodology: In silico docking studies was carried out using bioinformatics tools.The active compounds (Trihomovitamin D3; 2Z,6Z,8Z,12E Hexadecatetraenoic acid, Methyl prednisolone, Hydroxysalmeterol and Tridesacetoxykhivorin) ofP.acidus aqueous fruit extract were docked against MAPK1 resulting in receptor-ligand complex. 
  
Results: The binding energy is correlated with the probability of affinity and stable bound between ligand and its receptor. 
  
Conclusion: The molecular docking study of selective bioactive compounds of PAFAE with MAPK1 protein revealed that Tridesacetoxykhivorinand Methyl Prednisolone, is having good interaction in favorable pose with MAPK1 as shownfrom theireffective binding energy(-7.79kcal/mol and -7.19 kcal/mol), strong bond length and interactions with active site of MAPK1.


INTRODUCTION
tar gooseberry (Phyllanthus acidus. L) is a crisp, sour, juicy edible fruit. The plant is found throughout Asia, Caribbean, Central and South America. The plant has wide range of its use from culinary to medicinal. Conventionally, it is used in the treatment of liver disease, bronchitis, urinary tract disease, rheumatism, constipation, diabetes, etc., (1). Further, the plant is reported to possess antimicrobial, anti-inflammatory, anti-nematodal, hypolipidemic, hypoglycemic, antihypertensive, antioxidant and hepatoprotective properties (2)(3)(4)(5)(6)(7)(8)(9). However, the antiapoptotic activity of the plant remains unexplored as per literature, which is indeed significant in the context of "oxidative stress induced cell death", an implication of several chronic diseases. Fig. 1: Phyllanthus acidus. L Cellular signaling pathways that involve protein kinases play critical roles in determining the balance between cell death and survival (10). One such pathway is mitogen-activated protein kinase (MAPK) signaling pathway. MAPK are serine/threonine specific protein kinases that respond to varied extracellular stimuli like mitogens, osmotic stress, heat shock, proinflammatory cytokines, growth factors, etc. and regulate various cellular activities including development, differentiation, proliferation, inflammatory responses and apoptosis. The three well-known MAPK pathways in mammalian system are extracellular signal-regulated kinases (ERKs1/2), c-Jun N-terminal kinases (JNKs1/2/3), and p38-MAPKs. MAPK1 or ERK1/2, plays an "antiapoptotic role" by the downregulation of proapoptotic proteins and upregulation of anti-apoptotic proteins through both transcriptional and posttranslational mechanisms (11).
Hence, the aim of this work was to carry out an in silico docking of selective compounds of aqueous extract of fruit of P. acidus (PAFAE) against MAPK1, a prime protein target involved in cell survival and apoptosis; so as to find the best compound/phytochemical among the selected that could target MAPK1 and therefore will be useful for controlling oxidative stress induced cell death. S Nowadays, the use of bioinformatic tools, before doing wet lab activities to determine the binding of datasets of small molecules to known receptors is a major component of drug discovery as it is cost effective and time saving (12)(13)(14).
They were further docked with MAPK1 using automated docking software (16). Before docking, the 3D structure of both ligands and the target protein in pdb format were retrieved using the following bioinformatics tools:

UniProt
UniProt is a comprehensive, high-quality, freely accessible resource of protein sequence and functional information. (http://www.uniprot.org/). The sequence of the target protein (MAPK1) was retrieved from UNIPROT database.

PDB
The Protein Data Bank (PDB) is a repository for the 3-D structural data of large biological molecules, such as proteins and nucleic acids (http://www.rcsb.org/pdb/home/home.do).
The crystal structure of ERK2 (MAPK1) was retrieved from PDB database.

PubChem
PubChem is the world's largest collection of freely accessible chemical information. (http://pubchem.ncbi.nlm.nih.gov/). The threedimensional structure of the bioactive compounds selected for the present study were retrieved from PubChem database in SDF and MOL formats.

ACD/ChemSketch
ACD/ChemSketch is an easy-to-use, molecular structure drawing application used worldwide. (https://www.acdlabs.com). The structure of some of the compounds/ligands under study which were not found in PubChem, were drawn using ACD Chemsketch.

Open Babel Converter
Open Babel converter is used for converting various chemical file formats. (http://openbabel.org/docs /current/Introduction/goals.html). In the present study, the compound structure saved in.mol format are converted to .pdb format using Open Babel molecular converter tool.

Autodock
AutoDock is molecular modeling simulation software used especially for protein-ligand docking studies (http://autodock.scripps.edu/resources/tools). In the present study, the binding efficiency between the ligands and the target protein was identified using Autodock.

Discovery Studio Visualizer
Molecular visualization is a key aspect of the analysis and communication of modeling studies. The hydrogen bond interaction between compound and MAPK1 were visualized using Discovery Studio Visualizer. (http://accelrys.com/products/discoverystudio/visualization.html).

Protein sequence retrieval: MAPK1
The sequence of the target protein MAPK1 was retrieved from UNIPROT database and its ID is P28482. Gene name is MAPK1 from organism Homo sapiens (Human).

Structure retrieval
The Crystal structure of ERK2 (MAPK1) was retrieved from PDB database and its PDB ID is 2OJJ. The structure of MAPK1 visualized using RASMOL tool ( Figure 4). Pink Color coil indicates -helix; Yellow color arrow indicates -sheets; White color indicates turns.

Preparation of ligands
Five bioactive compounds/ligands to be used for the present docking study were identified via LCMS from PAFAE and were selected based on their pharmacological property. The structure and molecular formula of the compounds of PAFAE is shown in Table 1.

Molecular docking analysis
Using AutoDock 4.2, molecular docking analysis was performed so as to ascertain the binding conformation of the protein-ligand complex. The binding conformation would in turn help to reveal the binding energy of the MAPK1 and the selected ligands (17).

DISCUSSION
After successful docking of the bioactive compounds one by one with the target protein, the receptor/ligand complex models generated was analyzed based on hydrogen bond interactions, binding energy and orientation of the docked compound within the active site of the target protein, MAPK1 (18,19,20). Thus, the binding energy of Tridesacetoxykhivorin and Methyl Prednisolone with MAPK1, as obtained from analysis of molecular docking supports a possible antiapoptotic role of PAFAE compounds. It is suggested so, based on the finding that MAPK1 inhibits apoptosis upon activation although the mechanism varies with cell types. MAPK1 promotes cell survival by inhibiting pro apoptotic proteins (BAD) and by activating anti apoptotic factors like BCL2 and MCL1 (21).

CONCLUSION
In conclusion, the results of the present study clearly demonstrated the in silico molecular docking studies of Methyl Prednisolone, Tridesacetoxykhivorin, Hydroxy salmeterol, 2Z,6Z,8Z,12E Hexadecatetraenoic acid and Trihomovitamin D3 from PAFAE with MAPK1 and their binding interactions. From the present in silico analysis, it could be inferred that the secondary metabolites, Tridesacetoxykhivorin and Methyl Prednisolone of Phyllanthus acidus aqueous fruit extract (PAFAE) are capable of interacting with active binding site of the c target protein MAPK1 with maximum energy and may serve as a natural therapeutic agent in treating oxidative stress induced cell death in chronic diseases. However, these results are only preliminary screening just to facilitate subsequent in vitro and in vivo studies and thus warrants further investigation.