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ThesisItem Open Access Computational analysis of potential phytochemicals interaction with COVID-19 main protease(CCSHAU Hisar, 2022-07) Nisha; Ahalawat, NavjeetThe global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2020 has affected almost every country with around 543 million confirmed cases worldwide. Currently to fight against the infection, multiple vaccines and several types of drugs alone or with combination have been used in many countries for emergency use. Some severe side effects of repositioned drugs are a cause for concern and their efficacy is still under evaluation. Antiviral therapeutics based on phytochemicals that have been reported to have more tolerable side effects can serve as a reliable alternative to synthetic antiviral drugs for the inhibition of viral replication and penetration. The main protease of SARS-CoV-2 is one of the highly potent and vital targets to design and develop antiviral drugs for the inhibition of COVID-19 contamination. Computational approach helps to identified many drugs that can target viral proteins Mpro. We generated a phytochemical library containing 2453 phytochemicals which have been reported as having antiviral activity through deep literature study.The virtual molecular docking results reveals that five bioactive compounds; theaflavate C, theaflavin-3,3-O-digallate, amentoflavone, hinokiflavone, theaflavin3-gallate have a higher binding affinity toward COVID-19 main protease (MPro) with the binding energy of -10.1, -9.9, -9.7, -9.7 and -9.7 Kcal/mol. But these phytochemicals didn’t follow Lipinski rule of five, so we selected more four phytochemicals having ADMET properties. The main protease docked complexes with phytochemicals; theaflavate C, theaflavin-3,3-O-digallate, amentoflavone, hinokiflavone, theaflavin3-gallate, 10-methoxycamptothecin, caribine, sesamin and licoisoflavanone were used for MD simulation of 20 ns time period using Gromacs package. The MD results were analysed with respect to RMSD, RMSF, Rg, SASA, hydrogen bond and principal component analysis. The resulting trajectories of converged period of MD were further exploited in MM-P/G/BSA calculations to derive accurate estimates of binding free energies. The study provides a basic foundation and suggests that the seven phytochemicals, viz. theaflavate C, theaflavin-3,3-O-digallate, amentoflavone, hinokiflavone, theaflavin3-gallate, caribine, and licoisoflavanone serve as potential inhibitors in regulating the Mpro protein’s function and controlling viral replication and may assist the development of effective anti-COVID-19 drugs.ThesisItem Open Access In-silico identification of miRNA in Rice (Oryza sativa L.)(CCSHAU,HiSAR, 2020) Bhavya; Sudhir KumarRice (Oryza sativa L.) is the world’s single most important and essential crop and a primary food source for half of the world’s populace. An aggregate of 49% calories consumed by the human population come from rice. Micro RNAs (miRNA) are single-stranded non coding RNA molecules of about 21-23 nucleotides in length which regulate gene expression. The main function of miRNAs is to down-regulate the gene expression. The twelve chromosomes of Oryza sativa was downloaded from the International Rice Genome Sequencing Project (IRGSP), after that they transcribed into RNA and then they were cut into fragments by using split command. The output of split command was subjected as input for palindrome finding in palindrome.pl script. Only four mismatches were allowed. The secondary structures and MFE of output of palindrome.pl script were predicted using RNAfold program. The sequences whose energy were less than -40kcal were sorted and identified as putative miRNA. These putative miRNA were compared against miRNA database in miRBase. Total 61 miRNAs and their 255 putative targets were identified. These target genes have regulatory functions in some certain biological processes.ThesisItem Open Access In-silico Prediction of microRNAs and their targets in Wheat (Triticum aestivum L.)(CCSHAU, 2019) Bishnoi, Stela; Sudhir KumarWheat (Triticum aestivum L.), a staple crop which constitutes major part of human diet, belongs to family Poaceae or Gramineae family. As a major constituent of human diet it is therefore one of the most widely cultivated food grain in not only India but the world as well. In India it is the grain with second largest consumption after rice. The miRNAs play a major biological role by modifying the expression levels of a diverse repertoire of genes in a sequence in a dependent manner which is attained at either transcriptional or post-transcriptional level. The Nucleic acids sequences of the 21- chromosomes of Triticum aestivum were downloaded from the International Wheat Genome Sequences Consortium. After downloading wheat genome sequence from IWGSC, the DNA sequence is transcribed into RNA sequence and later split through split.pl which gave an output of 138 fragments. Then after finding out palindromes of fragments using palindrome finding perl script (palindrome.pl). The fragments with at most four mismatches were stored in a file. Secondary structure was predicted using RNAfold server. The sequences are sorted out which have MFE of secondary structure less than -40kcal, and considered as putative miRNAs. From BLAST there identified 699 new miRNAs having 111175 targets. These target genes have regulatory function in certain biological processes.ThesisItem Open Access Structural and Molecular Dynamics Studies of UDP Glucose Pyrophosphorylase Dimerization in Rice (Oryza sativa L.)(CCSHAU, 2019) Mamatha Y S; Sudhir KumarUDP-Glucose pyrophosphorylase (UGPase) (EC 2.7.7.9) belongs to family Glycosyltransferace clan (PF01702) which is present in plants as well as animals. UGPase is involved in sucrose synthesis as a catalyzing agent in the reaction, Mg+2-UTP + Glu-1-P PPi + UDP-Glu. It catalyzes both forward and reverse reaction depending on the metabolic status of the tissue. Crystal structure of UGPase shows that it have three domains with N-terminal domain at one end, catalytic domain which have nucleotide binding loop present at the center and the C-terminal domain include insertion loop at another end. C-terminal group is involved in dimer formation and stabilization of protein. Monomer is the active form in most of the parasitic and a plant UGPase. Modelling and dynamic study can uncover the interaction forces involved in UGPase activity. A 469 amino acids long rice UGPase was retrieved from NCBI and further aligned using BLAST program to identify templates for comparative structure prediction. Modelling of UGPase peptide by Modeller9.20, Swiss-model server and I-TASSER server predicted Model1, Model2 and Model3 respectively. All models were subjected to energy minimization using GROMOS96 force field and structure assessment by QMEAN. All models were further verified, validated and evaluated using WHATIF and SAVES server. The RMSD of models on superimposition with template was found to be less than 1.0 Å. Models were further refined using GROMACS-2019, a molecular dynamic (MD) code designed for highperformance simulation of large biomolecular systems. Dimers were generated by using protein-protein docking tools with predicted models. These generated dimers were further evaluated and analyzed. Dimer analysis provides the information about the hydrogen bonding in between the peptides of protein. Solvent accessible area calculated for the residues, which are present in ligand binding site. Solvent accessible area in dimer found to be less compared to monomer. The study showed that protein losses its activity due to dimerization.ThesisItem Open Access Virtual high throughput screening of sodium channel blockers as potential insecticide leads(CCSHAU, 2018) Naina Kumari; Sudhir KumarSodium channels are integral transmembrane proteins responsible for the initiation and propagation of action potential. Sodium channel consists of α and β subunits. α subunit is responsible for voltage dependent ion conductance whereas β subunit is responsible for membrane localization. By the use of virtual high throughput screening (molecular docking), potential inhibitors of voltage gated sodium channel were identified. vHTS is a computational method for screening in silico collection of compound libraries. Using vHTS, the binding affinity of the compounds from in silico library and target receptor was predicted. The crystal structure of insect sodium channel (5X0M) consisting of 1553 amino acids was retrieved from RCSB PDB and the active site and binding pocket were identified by CastP tool.. On the basis of substructure similarity of well-known inhibitors of sodium channel, 2,06,404 small molecules/ligands were obtained from ZINC15 database. Out of 2, 06,404 ligands, 1,65,437 were selected on the basis of Lipinski’s rule of 5. Molecular docking of the ligands with the sodium channel protein was performed using USCF DOCK6. 4,854 Docked ligands were obtained on the basis of acceptable binding energy score. The ligands were further filtered by their ADMET properties prediction through vNN-ADMET and TEST. Out of 2,06,404 ligands, 47 ligands were identified as non-toxicant and potential insect sodium channel blockers. The interaction study of the 47 accepted leads with the sodium channel protein revealed that ARG 1138, ARG 1120, GLU 1435, LEU 1224, ILE 1152, ASP 1412, GLU 1123, LEU 1224 and TYR 1430 are the key residues involved in interaction through hydrogen bonding, electrostatic interactions or vander waals forces.ThesisItem Open Access Computational modelling and molecular dynamic simulation of glutamate decarboxylase of wheat (Triticum aestivum L.),(CCSHAU, 2018) Jakhar, Ritu; Sudhir kumarGlutamate decarboxylase (GAD) catalyses the decarboxylation of glutamate amino acid to γ – amino butyrate (GABA) in presence of Pyridoxal phosphate cofactor. GABA build up happens after GAD activation in plants in response to various biotic and abiotic stress such as hypoxia, temperature shock, water stress, salinity stress, acidosis, virus infection and mechanical manipulation. GAD assembled into dimer and subsequently in hexamer for activation. In plants, GAD dimerisation and activation is associated with binding of CaM and C-terminal of GAD. The N-terminal residues are required for the assembly and stabilization of hexameric state of GAD. Modelling and dynamics study can uncover the interaction forces involved in GAD activity. A 500 AA long wheat GAD sequence was retrieved from UniProtKB and further aligned using BLAST program to identify templates for comparative structure prediction. Modelling of GAD peptide (monomer subunit) by Modeller9.19 and Phyre2 server provided Model1 and Model2, respectively. Model1 was generated in two fragments for N- and C- terminal with Modeller9.19 and joined using Chimera visualization tool. Both models were subjected to energy minimisation using GROMOS force field and structure assessment by GROMOS, QMEAN and ANOLEA. Both models were further verified, validated and evaluated using WHATIF and SAVES server. The RMSD of models on superimposition with the template was found to be less than 2.0 Å. Models were further refined using NAMD, a molecular dynamics (MD) code designed for high-performance simulation of large biomolecular systems. Out of two models, Model1 was predicted better model than Model2 on the basis of RMSD between initial and simulated model.ThesisItem Open Access Computational modeling and molecular dynamic simulation of pyrophosphatase of rice (Oryza sativa L.)(CCSHAU, 2018) Manisha; Sudhir KumarInorganic pyrophosphatase plays a significant role in various processes in plants. It causes chalkiness and hydrolytic breakdown of ADP-glucose in plastidal compartment. It has significance in lipid metabolism, calcium absorption, DNA synthesis and biochemical transformations. The sequence of inorganic pyrophosphatse was retrieved from NCBI and template was identified using BLASTP. With 84% query coverage and 71% identity 4LUG was selected as template. Modeller 9.19 and RaptorX were used for computational modeling. Predicted models were refined by energy minimization with GROMOS force field from Swiss-pdb Viewer. Minimum energy calculated for Modeller 9.19 and RaptorX predicted models were -2394.489KJ/mol and -7365.312KJ/mol respectively. The structures were assessed by GROMOS, ANOLEA and QMEAN graphs. More favourable region was shown by GROMOS and ANOLEA as compare to QMEAN. WHATIF server programs were used for structures optimization and validation. Bond length Z-score, bond angle Z-score, coarse packing quality and Ramachandran Z-score, were approximately 0.4, 1.2, -0.9 and 0.1 respectively. SAVES server programs score for PROVE, VERIFY3D and ERRAT were approximately 4.2%, 81% and 91% respectively. Ramachandran plot calculated by PROCHECK showed approximately 94% amino acid in core and 6% in allowed region. The models visualization showed coils were dominantly present in both the structures. RMSD for the structures was less than 0.5. Explicit solvent molecular dynamic simulation was done by VMD and NAMD software. The total energy and RMSD graphs calculated after simulation were stable for the structures. Structure superimposition with template showed significant conserved region between template and predicted structures. RMSD calculated after simulation was less than 0.5 Å against for both models template. The model predicted by RaptorX was found better as compared to Modeller 9.19 predicted model.ThesisItem Open Access In silico characterization of WRKY transcription factor in rice (Oryza sativa L.)(CCSHAU, 2017) Simerpreet; Sudhir KumarRice, being a model plant for genomic research of grass species can pave a way for characterization of WRKY transcription factors involved in DNA binding. WRKY protein is 60-70 long amino acid sequence present at N-terminal end and/or C-terminal end along with zinc finger like motif. WRKY TFs are involved in many plant processes e.g. plant growth and senescence, physiological, biological processes, regulation of transcription etc. Thus, present study aimed the in silico characterization of WRKY TF of rice from GRASSIUS database for homology search, analysis of conserved motifs and phylogenetic relatedness with WRKY family of maize, sorghum and sugarcane. Total of 369 WRKY protein sequences of rice, maize, sorghum and sugarcane were retrieved from GRASSIUS database of transcription factor. They were analyzed for the presence of WRKY domain using SMART and HMMER database. True WRKY proteins sequences after removal of partial and unwanted domains were subjected to homology search using BLAST which revealed that maize WRKY proteins have highest identity to OsWRKY proteins instead of sorghum and sugarcane WRKY proteins. Conserved residues were identified in multiple sequence alignment along with conserved WRKY motif and zinc finger motif which helps in specific binding activity. All species belong to poaceae family, their phylogenetic analysis revealed many orthologs and paralogs indicating the evolutionary significance and indicating presence of WRKY domains before divergence of species. Maximum orthologs were shown by rice and maize while in case of SbWRKY and ScWRKY, ortholgs obtained were on the basis of alignment of proteins on the basis of conserved region only. These results clearly indicates that rice is more closely related to maize than sorghum and sugarcane due to the ancient events like gene loss events which causes more divergence and gene duplication, tandem duplication which relates the genes between other species. Conserved motifs analysis using MEME tool identified conserved motifs in the sequences and provided motif distribution map. Genes having same motif distribution have a possibility of functioning in same conditions. The present study therefore, opens up possibility for further investigation of functional characterization WRKY regulatory proteins by wet lab experimentation with potential for rice improvement.ThesisItem Open Access Phylogeny and homology modeling of starch synthases of rice (Oryza sativa L.)(CCSHAU, 2017) Palak Singh; Sudhir KumarThe research work entitled “Phylogeny and homology modelling of starch synthase of rice (Oryza sativa L.)” was conducted keeping in mind the importance of starch in production and improvement in rice quality as well as in various food and non-food sectors, it is necessary to understand the basic proteins involved in the synthesis of starch in rice granule. The study explains the basic structural features and relationships of the Starch Synthase (SS) enzyme in rice with the other plants for the same enzyme. SS in rice consists of 11 genes grouped into five isoforms namely GBSS, SSI, SSII, SSIII and SSIV. Total of 28 GBSS and 50 SS (SSI to SSV) isoform sequences in rice as well as in other plants were retrieved from NCBI and were aligned using CLUSTALW program. The phylogenetic analysis of SSs in rice with other plants was done using MEGA7 and PHYLIP softwares and best fit tree was obtained by bootstrapped parsimony method. Phylogenetic consensus trees clearly depicted separate clusters for each isoforms and within each clusters of starch synthase isoforms (GBSS, SSI, SSII, SSIII, and SSIV) monocots and dicots were found to have two distinct clusters. Homology modelling of five isoforms was done by three softwares Modeller9.18, Swiss Model server and RaptorX server. Models obtained were then subjected to energy minimisation using Gromos force field and assessed by GROMOS and ANOLEA graph in which Swiss Model Server models were found best and hence were used for further studies. All the five selected models (GBSSII, SSI, SSII, SSIII and SSIV) of Swiss Model server were verified, validated using WHATIF server and were evaluated using SAVES server. Superimposition of target-template of each isoform showed conserved structural similarity, especially for ligand binding regions. The RMSD for all models on superimposition with the template was found to be less than 2.0 Å.
