MATERIALS AND METHODS
3.1.1DATABASE ( S )
The databases are used to seek and obtain informations that related to the research such as mark proteins, ligands and drug. These informations are all come from different databases based on their type and construction. Since all these databases are dependable, well-managed and ever up-to-date hence the cogency of the constructions obtained are non in uncertainty.
184.108.40.206RESEARCH COLLABORATORY FOR STRUCTURAL BIOINFORMATICS ( RCSB ) PROTEIN DATA BANK ( PDB )
Figure 3.2.1 Home page of RCSB Protein Data Bank
The RCSB Protein Data Bank is a publically available biological macromolecular resource Centre. It contains all known and supported protein
molecules with merely confirmed construction are allowed in this database. Hence, provides a dependable beginning of protein information. The constructions of the molecules in PDB are normally presented in a 3D construction format which makes it easy for molecular moorage procedures. Furthermore, RCSB Protein Data Bank besides allows us to download the informations in PDB format which is a cosmopolitan format for 3D protein construction. Besides we can see the information in FASTA format. FASTA format is convenience when it comes to sequence analysis as it enables us to straight transcript and paste the sequence for BLAST or even phylogeny trials.
Figure 3.2.2 Home page of ZINC Database
ZINC Database is a little molecule database. This is where the 3D construction of ligand is obtained. Currently ZINC Database provides more than 35 million compounds although some of the contents are purchase-only but this database is free-to-use for public. It allows us to download files in multi-format such as MOL, SMILES and PDB.
Figure 3.2.3 Home page of DrugBank
The DrugBank database is a publically alone bioinformatics and cheminformatics drug resource that combines elaborate drug informations together with comprehensive drug information. Currently DrugBank contains 7759 drugs with more than 1600 FDA-approved drugs. This database allows user to download drug informations in assorted format including MOL, SMILES and PDB.
Online tools are used to prove the relevant informations before they were undergoes the docking simulation. Those trials are including Lipinski’s Rules trial, active-sites anticipations trial and sequence alignment trial. These trials are of import in order to find the cogency of the construction and were carried out to both mark proteins and ligands. Though there is one online tool that is used to transport out the docking simulation as opposed to the standalone package.
Figure 3.3.1 Home page of Molinspiration
Molinspiration is an on-line tool that offering free services for computation of of import molecular belongingss such as logP, polar surface country, figure of H bond givers and acceptors and others. Or in other word Molinspiration is used to transport out the Lipinski’s Rule trial and bioactivity score ligand every bit good as the drug. This trial will find whether the ligand or the drug is suited for unwritten ingestion.
Figure 220.127.116.11 Home page of SwissDock
SwissDock is an on-line tool that offers docking service to foretell the molecular interactions that may happen between a mark protein and a little molecule or ligand. SwissDock is based on the moorage package EADock DSS, whose algorithm consists of many adhering manners that are generated either in a box ( local moorage ) or in the locality of all mark pits ( blind docking ) .
Figure 18.104.22.168 Home page of DogSiteScorer
DogSiteScorer is an machine-controlled pocket sensing and analysis tool which can be used for protein druggability appraisal. Predictions with DoGSiteScorer are based on deliberate size, form and chemical characteristics of automatically predicted pockets, incorporated into a support vector machine for druggability appraisal.
Standalone package is used to transport out the docking simulation between mark protein and ligand or drug. Furthermore, some standalone package besides used to see the 3D construction of the mark protein, ligand, drug and the merchandise of docking which is protein-ligand composite.
Figure 22.214.171.124 Graphic User-Interface of AutoDock Tools
AutoDock Tools is free educational-purpose package that can imitate a moorage procedure and foretell how ligands are interacting with the supermolecules. It has a constitutional AutoDock Vina circuit board that will be used to execute the docking simulation. Autodock Vina performs flexible ligand moorage, so the optimum geometry of the ligand will be determined during the moorage. Consequence of the docking procedure will be analysed by sing the tonss of RMSD and adhering affinity.
126.96.36.199PYMOL MOLECULAR VIEWER
Figure 188.8.131.52 Graphic User-Interface of PyMOL
PyMOL is stand-alone downloadable molecular spectator package. In this undertaking, it was used as the most of import tool for the important portion of research which is the structural alignment measure. However, for some research, due to certain processs limitation the research workers need to develop a new tool to be used as a PyMOL plugin. When this package is opened, it will give user a spectator window and its Molecular Graphics System window on the top where all the scenes can be configured. In the spectator window, the action button were all listed on the bottom right in alphabet codifications such as the missive S to demo the list of aminic acid residues on the exact location ; the cutoff keys of traveling the protein construction, and such.
Figure 184.108.40.206 Graphic User-Interface of UCSF Chimera
UCSF Chimera is a plan for visual image and probe of the compound constructions and their informations. The consequences can be in images or lifes were available to be downloading freely for users. This package was used to change over MOL2 file informations to.pdb format of compound. Some of the informations that can be obtained from UCSF Chimera are ViewDock which were used to testing of the orientations of docked ligand and Movie tools for rematch the flights of compound.
3.2.1GENERAL OVERVIEW OF METHODS
3.2.2 DATA Mining
During the informations excavation stage, all the informations relevant to the research were collected. First, 3D construction of Adenomatous polyposis coli protein was searched in RCSB Protein Data Bank and found. Then the construction was downloaded into the computing machine in.pdb format with a codename of 3NMW. Following, we searched for ligand construction which is in our survey was the Lupeol construction in little molecules database, ZINC Database. There were a few Lupeol structures available but we chose the 1 that normally found in Arabidopsis thaliana. The file is downloaded in.mol2 format. Last we searched for the currents drug which is the Celecoxib in DrugBank. After found the drug we downloaded the file in.mol format.
3.2.3Target PROTEIN ( APC ) Preparation
Before traveling through the moorage simulation, the APC protein was undergoes a few trials and was prepared for a smooth, accurate moorage consequence. First of all, after downloaded the protein construction from RCSB Protein Data Bank into the computing machine in.pdb format, the construction was so submitted to DogSiteScorer where its possible active sites on the protein surface are calculated based on the 3D co-ordinates of the protein. The consequences were generated within five to ten proceedingss as it was depends on the server’s burden. The consequences are besides sent to the user’s electronic mail.
Then we opened AutoDock Tools package and load the APC protein into it. By utilizing the characteristics available in the AutoDock Tools, the H2O molecules were removed from the construction. Later we added H on the supermolecules every bit good as merged all the non-polar in all those supermolecules. After that we added Kollman charges to the molecule and the entire Kollman charges added was shown at the same clip. Afterward, we detected the root of the ligand in the construction and changed the tortuosity roots into rotatable if the bonds were non-rotatable. Besides, we chose the tortuosity for the molecule. Last, we saved the construction file as in.pdbqt format as compatible for AutoDock Vina.
The downloaded Lupeol construction was submitted to an on-line tool called Molinspiration to cipher the Lipinski’s Rules of Five which consists of the computation of logP, figure of H bond giver, Numberss of H bond acceptors, molecular weight and rotational bonds. Then the construction was loaded into UCSF Chimera to be converted into.pdbqt format as compatible for AutoDock Vina.
Similar to Lupeol construction, the downloaded Celecoxib construction was submitted to an on-line tool called Molinspiration to cipher the Lipinski’s Rules of Five which consists of the computation of logP, figure of H bond giver, Numberss of H bond acceptors, molecular weight and rotational bonds. Then the construction was loaded into AutoDock Tools to be converted into.pdbqt format as compatible for AutoDock Vina and.mol2 format as compatible for SwissDock.
Last measure and the most of import portion of the methods is the docking simulation. For this peculiar survey we were utilizing 2 different docking package but with same flexible moorage mechanism. The package is AutoDock Vina and SwissDock.
First, both.pdbqt format files of APC protein construction and Lupeol were placed in a booklet. Then two chief feasible files of AutoDock Vina which are “vina.exe” and “vina_split.exe” were placed into the same booklet along with constellation file named “conf.txt” . The constellation file is consists of all the puting demand to run the moorage simulation including the parametric quantities of the grid size, receptor, ligand and end product file. The constellations were as follows:
Figure 3.3.6 Configuration file
The moorage procedures were run manually and it was done from the bid prompt of the computing machine ‘s operating system. The same stairss were repeated with APC protein construction and Celecoxib utilizing the same scene as default to acquire accurate and just consequences.
In SwissDock, foremost APC protein construction and Lupeol were uploaded to the SwissDock waiter. The APC protein construction was in.pdb format while the Lupeol was in.mol2 format. After the relevant information in the entry signifier has been filled up, the occupation was so submitted as all the parametric quantities and puting were all automatically configured by the SwissDock itself. The docking procedure was taking a few hours depending on the server’s burden and occupation question. Last the consequences were sent through the user’s electronic mail.