2.1. Retrieval of sequence and ligands
The sequence of a human cannabinoid receptor (CNR1 Accession Number: P21554) was retrieved from the universal protein database 10. Totally 472 residues are availed in CNR1. Target residue is starting from MKSIL and end with SAEAL. Ligand molecules of cyanobacterial compounds taken from chemspider database 11.
2.2. Homology modeling
Homology modelling finds the 3D structure of a protein based on its sequence similarity to one or more proteins of known structure. This method support the observation of the structural conformation of a target which is more highly conserved than its amino acid sequence. Homology modelling is finds the following steps: template identification, sequence alignment, BLAST homology search, model building and validation 12. This model with the lowest energy was selected for further refinement, which comprises the loop refinement and energy minimization using OPLS 2005 force field in Schrodinger Prime 13. The minimized model was then validated using Ramachandran plot, generated from PROCHECK at the SAVES version 4 server (Structure Analysis and Verification Server; (http:// services.mbi.ucla.edu/SAVES/)14. This model was optimized prior to docking using the Protein Preparation Wizard in Schrodinger Maestro Suite 2014 15.
2.3. Preparation of Target molecule
The CNR1 is prepared by using protein preparation wizard in Maestro v10.2. It was mainly involved in the process like to add missing hydrogen atoms, arranging bond order assignments, charge states an orientation of various groups 15.
2.4. Validation of binding site
Binding site process is an essential role of target. Because all the ligand molecules are binding with the target binding cavity. Here effective and suitable ligand molecules get favorable on drug preparation. These sites were validated with the help of site score. It means that the active residues are available and get a larger volume of the binding pocket 16.
2.5.Preparation of Ligands
Totally 20 cyanobacterial bioactive selected from this molecular docking studies such as Symplocamide A (Chemspider Id: 23314421), Pompanopeptin B (Chemspider Id: 27023335), Lyngbyastatin 5 (Chemspider Id: 23076610), Lyngbyabellin D(Chemspider Id: 9106209), Hoiamide A(Chemspider Id: 28185012), Nostocylopeptide A(Chemspider Id: 215110), Lyngbyabellin H(Chemspider Id: 23314421) and Cryptophycin 327(Chemspider Id: 23314421). These ligands are prepared with LigPrep tool. Ligand preparation includes a series of steps that perform conversions from 2D to 3D and apply corrections to the structure, produce ionization states at biological pH, generate possible tautomers, optimize the geometrics and finally minimize the ring conformations were represented in Fig.1 17.
2.6. Molecular docking
Docking studies were carried out in modeled CNR 1 protein and some cyanobacterial ligands by using commercial software Schrodinger suite version 10.2 Maestro. This study shown efficient ligand molecule against target. For instance Shubani et al, using the software of Schrodinger version Masetro 9.0 13. But we are using the advanced level of tool.
2.7.Prime/MM-GBSA binding-free energy computation
The selected eight cyanobacterial compounds were further analyzed by Prime/MM-GBSA method to calculate the free energy of ligand binding in the receptor-ligand complex. This method was follwed by Kakarala et al, 18. The total free energy of binding is calculated as follows:
?G bind = G complex – (G protein + G ligand)
Where G = MME (molecular mechanics energies) + GSGB (SGB solvation model for polar solvation) + GNP (nonpolar solvation. MM-GBSA values were evolved as the tool of Prime application 15.
2.8.Molecular dynamic simulations
A protein- ligand complex molecular dynamic simulations were implemented Desmond program v 3.8 (https://www.schrodinger.com/desmond)19. A CNR1 is a membrane protein with ligand so I set up a membrane in a predefined model by POPC 300K (Palmitoyl oleoyl phosphatidyl choline) (Vytautas Gapsys et al., 2013)20 and the CNR1 protein with ligand complex are solvated by TIP4P (Tripathi et al., 2013)21 with OPLS-2005 force field parameters by using all calculations. The orthorhombic periodic bounding box of the size 10Å3, after the system was neutralized by adding appropriate counter-ions followed by addition 0.15M of salt resemble of the physiological condition. Before the MD simulation running process the system was relaxed by performing a series short, restrained and non-restrained solute minimization methods followed by 1.2ps simulation steps using NPT ensembles. After the MD simulation were implemented 10 ns using the NPT ensembles. Trajectories were established inside maestro atmosphere and the results were evaluated through RMSD interaction.22.