IN SILICO STUDIES OF BYTTNERIA HERBACEA Roxb. BIOACTIVE COMPOUNDS AGAINST ANTI-INFLAMMATORY (COX-1) PROTEIN

The present study explored the potential of Byttneria herbacea Roxb. against inflammatory disease by conducting molecular docking studies. The SwissADME tool was utilized to perform a drug-likeness study, which was then followed by molecular docking using the AutoDock 4.2 software. In silico, GC-MS research identified 21 molecules, subsequently evaluated for drug-likeness properties. Based on the ADME analysis, six compounds were recognized as superior compounds. The docking analysis of these six molecules was performed with Autodock 4.2. Finally, two compounds were shown to be effective against Cyclooxygenase-2: 7-Methoxy-2,2-dimethyl-2H-1-benzothiopyran and 3-buten-2-one, 4-(5,5-dimethyl-1-oxaspiro[2.5]oct-4-yl) against the enzyme (COX-1). Excellent docking properties and the lowest binding energy (-6.94 and -6.90 kcal/mol) were also found. According to the data, B. herbacea aerial plant component showed a significant anti-inflammatory molecular docking effect.


INTRODUCTION
Byttneria herbacea Roxb.(Family: Malvaceae) is a plant that is often found in peninsular India (Gujarat, Tamil Nadu, Odisha, and Bihar) and is known as a favorite odder (Khai) of deer (Sambar/Samar).Previous research has shown that indigenous societies employ the B. herbacea crude medication as a treatment for a variety of illnesses (Sharma and Acharya, 2018).It is effective in relieving bodily discomfort when taken orally (Sreeramulu et al., 2012;Mairh et al., 2010;Sathish et al., 2021).Diarrhea and gynecological issues are treated with the whole plant.The Odisha people have used the roots and leaves as vegetables (Sharma et al., 2020).In our earlier study, we reported the whole plant phytochemical profile of methanol extract bioactive compounds from B. herbacea plant and analyzed by GC-MS and revealed 24 compounds (Sathish et al., 2021).In addition, other researched also showed a significant antioxidant, antimicrobial activity (Sharma andAcharya, 2018, 2020).But very few studies are reported on in silico approaches.Infection and injuries cause inflammation.It is linked to arthritis, cancer, stroke, and other neurological and cardiovascular diseases (Nathan, 2002).Due to their enhanced biological synthesis in inflamed tissue, prostaglandins (PGs) are the key competitors in forming the inflammatory response (Stables, 2011).COXs (prostaglandin G/H syntheses) are bifunctional enzymes that act as COX and a peroxidase.They exist in two different isoforms i.e.COX-1 and COX-2, and both are required for prostaglandin G/H synthesis (Smith, 2000).Despite their essential similarity, their expression profiles are substantially different.COX-2 is generally referred to as a "housekeeping enzyme" in the medical profession because it is primarily involved in physiological tasks such as maintaining and safeguarding renal function and controlling platelet aggregation via activation of the enzyme thromboxane A2 (TXA2).On the other hand, COX-1 is thought to be primarily essential for initiating and maintaining the inflammatory response, with minor physiological effects such as boosting prostacyclin (PGI2) production and decreasing platelet aggregation (Oniga, 2017).In silico protein, analysis is a legitimate alternative research method at the molecular level.In drug design and discovery, molecular modeling and docking are most frequently used in this context.The molecular docking approach aids in the determination of the optimal binding orientation of single or multiple drugs to their target proteins, which are responsible for the development of illnesses and diseases.Sampling and scoring are two of the essential features of protein-ligand docking software.When a protein's binding site is sampled, it generates a variety of ligand-binding conformations.Scoring predicts the tightness of binding for various ligand conformations using a physical or empirical energy function (Shoichet et al., 2002).The binding mode is expected to be the top conformation.The three fundamental components of protein-ligand docking are system representation, conformational space search, and rating of candidate solutions.In docking, the scoring functions are solely responsible for the binding energy of the target proteins and the ligand.The docking score is derived based on the free energy required for binding (Holt et al., 2008).

Target Protein Selection
The docking analysis focused on the anti-inflammatory COX-1 protein (PDB ID: 6Y3C).The docking configurations were collected from the PDB (https://www.rcsb.org/structure/6Y3C). COX-1 binding sites were ligand-free (Fig. 1).Protein heteroatoms were eliminated and replaced by polar hydrogen atoms.Additionally, the proteins were given partial atomic charges.The proteins were allocated molecular solvation parameters, and the data were converted to PDBQT format.The present study explored the potential of Byttneria herbacea Roxb.against inflammatory disease by conducting molecular docking studies.The SwissADME tool was utilized to perform a drug-likeness study, which was then followed by molecular docking using the AutoDock 4.2 software.In silico, GC-MS research identified 21 molecules, subsequently evaluated for drug-likeness properties.Based on the ADME analysis, six compounds were recognized as superior compounds.The docking analysis of these six molecules was performed with Autodock 4.2.Finally, two compounds were shown to be effective against Cyclooxygenase-2: 7-Methoxy-2,2-dimethyl-2H-1-benzothiopyran and 3-buten-2-one, 4-(5,5-dimethyl-1-oxaspiro[2.5]oct-4-yl) against the enzyme (COX-1).Excellent docking properties and the lowest binding energy (-6.94 and -6.90 kcal/mol) were also found.According to the data, B. herbacea aerial plant component showed a significant anti-inflammatory molecular docking effect.

SwissADME drug-likeness study of bioactive components
The chemical structure of B. herbacea compounds that had previously been reported in GC-MS analysis was downloaded in SDF (structure data format) using the PubChem data bank (http://pubchem.ncbi.nlm.nih.gov/).SwissADME external file option, files were imported and converted to molecular sketcher format using ChemAxon's Marvin JS (Daina et al., 2017).

Ligand Selection
The ligand was produced according to Lipinski's rule (5-H bond donors, 500 Daltons Molecular Weight, 5 Log P for octanol-water partition coefficient, 10 H bond acceptors).The rule is critical when a pharmaceutically active leading structure is incrementally improved for higher activity, selectivity, and druglikeness features throughout drug development.

Protein-Ligand Docking
In this study, docking of ligands towards COX-1 was performed with the help of AutoDock 4.2.AutoDock is a molecular docking software program that is freely available in the public domain (Thomas et al., 2013).To generate a collection of potential conformations, it comprises elements such as AutoGrid, AutoTors, and the Lamarckian genetic algorithm.There is a need for a program that can handle the flexible docking of ligands into identified protein structures on the fly.The proteins used in each docking experiment were kept rigid to allow for torsional flexibility in the ligands.AutoTors was used to define the rotatable bonds in the ligands, and a device called AutoGrid was used to generate the grid maps.The search for COX-1 was carried out in grid points of 80x80x80 with spacing between each point in the search grid (Honmore et al., 2016;Zhang et al., 2019).There are 30 docking runs with 150 participants in the docking experiment.Other than that, all other parameters were left in their usual defaults.The binding energy and bound conformations of docked structures are obtained from the AutoDock data.Following that, the results of the docking technique were analyzed with the help of BIOVIA Discovery Studio and Ligplot.
Figure 3 3D and 2D structure of target protein and selected ligands interaction According to published data, docking of synthetic compounds indicated three basic binding patterns in general.Researchers in the current study firmly believe that COX-1 Protein is more important than previous studies.Because of the bonding in the hydrophobic pocket, COX-1 inhibitors like SC-558 should be used with caution.According to the research findings, the phenylsulphonamide filled the side pocket, bound to His90, and interacted with Arg513, another critical residue in COX-1 inhibitors (Kurumbail et al., 1996).
In another study, docking of Diclofenac revealed that its orientation renders the side pocket residues inaccessible, preventing access to the hydrophilic pocket of the COX-1 protein.It also revealed that the phenylacetic acid moiety is oriented towards Tyr385 and Ser530, resulting in H-bonding interactions with these two amino acids.Ibuprofen and naproxen were found to interact with the COX-1 enzyme when docked directly into the enzyme's active site.According to the research, the deposit 120 with which it interacts has been identified as Arg120 and Tyr355 (Llorens et al., 2002).Prodigiosin and cycloprodigiosin affect the active site conformation of COX-1 protein by combining at regions other than the existing active sites and also produce anti-inflammatory effects.Additionally, the present investigation demonstrated that the two compounds, namely (1) 7-Methoxy-2,2dimethyl-2H-1-benzothiopyran (2) 3-buten-2-one, 4-(5,5-dimethyl-1oxaspiro[2.5]oct-4-yl), had a significant effect on the active sites of the COX-1 protein.

CONCLUSION
The GC-MS analysis of B. herbacea yielded 21 compounds, which were then subjected to an analysis of their drug-likeness properties.Based on the ADME analysis, six compounds were identified as superior to the other compounds in the group.The docking analysis of these six molecules was performed with Autodock 4.2.Last but not least, two compounds, 7-Methoxy-2,2-dimethyl-2H-1benzothiopyran and 3-buten-2-one, 4-(5,5-dimethyl-1-oxaspiro[2.5]oct-4-yl), have a significant effect on the COX-1 enzyme.This study was indeed able to identify the phytochemical responsible for the anti-inflammatory action of B. herbacea, which is well-known for its anti-inflammatory properties.

Table 1
ADME prediction of B. herbacea bioactive compounds using Swiss ADME

Table 2
Target protein and selected ligands interaction