In-silico drug designing of novel Morpholino based Physcion drug candidate and investigation of inhibition effects on Covid-19 RNA Dependent -RNA polymerase non structural protein 12 (nsp 12) with ADMET study
Recent explosion of highly fatal pandemic corona virusCovid-19 in human population. The Covid-19 is a positive sense single stranded enveloped virus which belongs to the Coronaviridae family required non-structural proteins 12 (nsp12), a RNA dependent-RNA polymerase as an important machinery for the viral genome replication and transcription processes. There are various RNA polymerase inhibitors are currently using in clinical activities to treat Covid-19 infections but their treating efficacy is not up to much impressive particularly in aged people. In this study, we docked Morpholino based physcion drug candidate against RNA polymerase target (PDB ID : 6NUR). We designed drug candidate using Chemsketch software and further it was proceeded to molecular docking using AutoDock Vina 4.0 software. UCSF Chimera software was used for visualization of 3-Dimensional structure of ligand -protein docked pose. Moreover the docked drug candidate was checked for ADMET properties. Hence, this study supports the emergence of developing an efficient new drugs to combat Covid-19 infections. From this computational study we identified the designed drug candidate have high potential of inhibition of virus RNA Dependent -RNA polymerase minimum binding energy of -8.76. To identify the inhibition potential of designed ligand, we used Remdesivir resulted minimum binding energy of -7.25 as a positive control. These findings supports emergency discovery of anti-viral drug candidate to combat Covid-19 infections all over the world.
Introduction<!>Protein Target Preparation<!>Ligand Designing<!>Protein optimization & Energy Minimization<!>Hydrogen Bond Analysis<!>Ligand Designing<!>Figure No.1 :<!>Energy Minimization<!>Molecular Docking algorithm
<p>Based on the statistics from World Health Organization (WHO), Covid-19 novel viral strain has been emerged first in Wuhan, China and represents a pandemic infections to public health. Initially, the infection was unable to identify causing agent and then the Chinese Center for Disease Control and Prevention (CDC) and local CDCs found illness is because of novel virus from coronavirus (CoV) family. They are large family of positive sense single stranded and many animals are acting as reservoirs for those viruses. From current studies, the therapeutic methods to cure this infection are supportive only and there is no available vaccine to prevent this infection in the human community [1]. The transmission of COVID-19 occurred by human to human with an aggressive 1 to 4 th degree infections in human community. Currently there approximately 22,000 deaths has been reported in all over the world.</p><p>From the data received from December 2019, an emergence of COVID-19 infections in human health, the anti-viral drug Remdesivir was reported by World Health Organization (WHO). Previous case of Ebola virus, Remdesivir was used to inhibit RNA-Dependent RNA polymerase which replaces Adenosine Tri Phosphate (ATP) by polymerization process before chain termination and it was considered as the nucleotide analog [2]. In-vitro assay of Remdesivir exhibited 1.76 µM (Effective Concentration) against corono virus [3]. Currently, the Remdesivir was administrated in COVID-19 infected patients to control the disease states. But there efficacy is not up to the mark in aged peoples. Hence there is a requirement of new drugs to combat and control the COVID-19 aggressive nature in community of human population in whole world. The aspartate amino acid residue present on beta turn of protein structure acting as the highly conserved domain and enables them to access nucleotide channel [5,6].</p><p>Morpholino backbone block binding of other complementary molecules in RNA molecule using complementary nucleotide analog. Additionally the structure of morpholino resembles the structure of naturally occurred nucleotide and exhibited resistant to nuclease activity.</p><p>Physcion is a naturally available bioactive molecules by plants, endophytic fungi which is a derivative of anthraquinone. It exhibits various biological activities such as laxative, anti-hepatic, anti-inflammatory, anti-microbial and anti-cancer activity (nasopharyngeal carcinoma) in in-vivo assays [7]. In order to find new antiviral compound, physcion was evaluated to inhibit viral replication, anti-absorption and virucidal activities. Further it was identified as inhibitor against CVB4 and RSV infections [8].</p><p>Hence, from our study, the molecular docking results are suggest to use as anti-polymerase therapeutic drug candidate against the novel strain of Coronavirus 2019 infections.</p><!><p>The crystalline structure of protein target (RNA Dependent -RNA Polymerase) was not available in the Protein Data Bank Databases. To modelled targeted protein, Sequences in FASTA format was retrieved from NCBI public database. The retrieved sequence was used to build a protein model by using SWISS-MODEL serve.</p><p>Template search with BLAST and HHBlits has been performed against the SWISS-MODEL template library. The target sequence was searched with BLAST against the primary amino acid sequence contained in the SMTL. An initial HHblits profile has been built using the procedure outlined in [9], followed by 1 iteration of HHblits. For each identified template, the template's quality has been predicted from features of the target-template alignment. The templates with the highest quality have then been selected for model building [10][11][12]. The existed protein model IN PDB (Id: 6NUR) was selected as the template for further docking study based on sequence similarity.</p><!><p>The Morpholino based -physcion drug candidate was designed by using Chemsketch free software and further selected for molecular docking study [Figure No.1].</p><p>Moreover, the designed ligand structures closely resembles the structure of Remdesivir with slight modification in replacement of nucleoside analog with morpholino analog. The modelled targeted protein was proceeded to virtual screening of potential drug candidate against COV2 2019 virus infection inhibition.</p><!><p>The targeted protein was opened with word pad to remove the coordinates of other ligands which is not a part of protein and hence consider as a hetero atom [HETATM].</p><p>For Protein optimization, all the hetero atoms were removed till before the END of our protein. Now the optimized protein is save and run for the further studies. The energy minimization was done by using Swiss PDB Viewer software.</p><p>After ligand drawing with the help of chemsketch software and the structures were saved as MDL Molfiles [V2000]. Open Babel software is used for converting the .mol file pdb format of designed ligand for proceeding the docking study. AutoDock Vina 4.0 is used in this project for docking study. Target protein were open as read molecule and converted to PDBQT format using MGL Tools (version -1.5.6rc3). The polar hydrogen atoms, kollman charge was added. A systematic analysis of ligand binding poses generated by AutoDock Vina shows that the highest accuracy is achieved when the dimensions of the search space are 2.9 times larger than the radius of gyration of a docking compound [13]. The grid size was generated automatically with the help of installed MGL tools to increase the accuracy of docking studies. Docking with AutoDock Vina 4.0 employ the Lamarckian genetic algorithm to generate a better conformations of a lead compound. Totally Ten docking runs were performed. The scoring of the generated docking poses and ranking of the ligands is based on the AutoDock Vina RMSD (Root Mean Square Deviation) empirical scoring function approximating the binding affinity in kcal/mol. The docked ligands were arranged in an order of minimum binding energy and the pdb files were visualized by using UCSF Chimera (3D visualizing software).</p><!><p>The hydrogen bond formation contributes the strong stability of protein-ligand complex and its important in enzymatic catalysis reaction to stabilize a ligand in a binding pocket of target protein. The analysis of hydrogen bond to determine the hydrogen bond donor -acceptor pair. More the aminoacids residues in a protein, the more number of hydrogen bond formation. The hydrogen bond analysis was done by using Protein-Ligand Interaction Profiler (PLIP) web tool [14].</p><!><p>Computer aided drug designing have became an efficient method to identify new potential drug candidates using various Bioinformatics tools. This methodology create an impact in drug discovery process with a significant low cost development in less time duration. Then the designed compound was synthetically made using synthetic chemistry processes and further investigating their in-vivo bioactivity screening, pharmacokinetic properties.</p><!><p>The above ligand was designed by using Chemsketch free software.</p><!><p>The energy minimization was run with steepest descent method instead of using MD simulation in order to change the protein coordinates in such a way as to reduce the potential energy. The energy minimized model was opened with word pad in order to remove the spdbv coordinates it put during energy minimization run of steepest method with 10.000Å cutoff. The spdbv coordinates removed energy minimized is the final refined model for further studies.</p><!><p>By using the cygwin command prompt software we create GLG file and DLG file to generate the RMSD value for the identification lead compound which showing minimum binding energy with the docked ligands [Table No</p>