Budhayash Gautam

Metabolic pathway analysis and molecular docking analysis for identification of putative drug targets in Toxoplasma gondii: novel approach.

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that can infect a wide range of warm-blooded animals including humans. In humans and other intermediate hosts, toxoplasma develops into chronic infection that cannot be eliminated by host’s immune response or by currently used drugs. In most cases, chronic infections are largely asymptomatic unless the host becomes immune compromised. Thus, toxoplasma is a global health problem and the situation has become more precarious due to the advent of HIV infections and poor toleration of drugs used to treat toxoplasma infection, having severe side effects and also resistance have been developed to the current generation of drugs. The emergence of these drug resistant varieties of T. gondii has led to a search for novel drug targets. We have performed a comparative analysis of metabolic pathways of the host Homo sapiens and the pathogen T. gondii. The enzymes in the unique pathways of T. gondii, which do not show similarity to any protein from the host, represent attractive potential drug targets. We have listed out 11 such potential drug targets which are playing some important work in more than one pathway. Out of these, one important target is Glutamate dehydrogenase enzyme; it plays crucial part in oxidation reduction, metabolic process and amino acid metabolic process. As this is also present in the targets of tropical diseases of TDR (Tropical disease related Drug) target database and no PDB and MODBASE 3D structural model is available, homology models for Glutamate dehydrogenase enzyme were generated using MODELLER9v6. The model was further explored for the molecular dynamics simulation study with GROMACS, virtual screening and docking studies with suitable inhibitors against the NCI diversity subset molecules from ZINC database, by using AutoDock-Vina. The best ten docking solutions were selected (ZINC01690699, ZINC17465979, ZINC17465983, ZINC18141294_03, ZINC05462670, ZINC01572309, ZINC18055497_01, ZINC18141294, ZINC05462674 and ZINC13152284_01). Further the Complexes were analyzed through LIGPLOT. On the basis of Complex scoring and binding ability it is deciphered that these NCI diversity set II compounds, specifically ZINC01690699 (as it has minimum energy score and one of the highest number of interactions with the active site residue), could be promising inhibitors for T. gondii using Glutamate dehydrogenase as Drug target.

Molecular modeling, dynamics studies and virtual screening of Fructose 1, 6 biphosphate aldolase-II in community acquired- methicillin resistant Staphylococcus aureus (CA-MRSA)

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has recently emerged as a nosocomial pathogen to the community which commonly causes skin and soft-tissue infections (SSTIs). This strain (MW2) has now become resistant to the most of the beta-lactam antibiotics; therefore it is the urgent need to identify the novel drug targets. Recently fructose 1,6 biphosphate aldolase-II (FBA) has been identified as potential drug target in CA-MRSA. The FBA catalyses the retro-ketolic cleavage of fructose-1,6-bisphosphate (FBP) to yield dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P) in glycolytic pathway. In the present research work the 3D structure of FBA was predicted using the homology modeling method followed by validation. The molecular dynamics simulation (MDS) of the predicted model was carried out using the 2000 ps time scale and 1000000 steps. The MDS results suggest that the modeled structure is stable. The predicted model of FBA was used for virtual screening against the NCI diversity subset-II ligand databases which contain 1364 compounds. Based on the docking energy scores, it was found that top four ligands i.e. ZINC01690699, ZINC13154304, ZINC29590257 and ZINC29590259 were having lower energy scores which reveal higher binding affinity towards the active site of FBA. These ligands might act as potent inhibitors for the FBA so that the menace of antimicrobial resistance in CA-MRSA can be conquered. However, pharmacological studies are required to confirm the inhibitory activity of these ligands against the FBA in CA-MRSA.

Insight into trichomonas vaginalis genome evolution through metabolic pathways comparison

Trichomonas vaginalis causes the trichomoniasis, in women and urethritis and prostate cancer in men. Its genome draft published by TIGR in 2007 presents many unusual genomic and biochemical features like, exceptionally large genome size, the presence of hydrogenosome, gene duplication, lateral gene transfer mechanism and the presence of miRNA. To understand some of genomic features we have performed a comparative analysis of metabolic pathways of the T. vaginalis with other 22 significant common organisms. Enzymes from the biochemical pathways of T. vaginalis and other selected organisms were retrieved from the KEGG metabolic pathway database. The metabolic pathways of T. vaginalis common in other selected organisms were identified. Total 101 enzymes present in different metabolic pathways of T. vaginalis were found to be orthologous by using BLASTP program against the selected organisms. Except two enzymes all identified orthologous enzymes were also identified as paralogous enzymes. Seventy-five of identified enzymes were also identified as essential for the survival of T. vaginalis, while 26 as non-essential. The identified essential enzymes also represent as good candidate for novel drug targets. Interestingly, some of the identified orthologous and paralogous enzymes were found playing significant role in the key metabolic activities while others were found playing active role in the process of pathogenesis. The N-acetylneuraminate lyase was analyzed as the candidate of lateral genes transfer. These findings clearly suggest the active participation of lateral gene transfer and gene duplication during evolution of T. vaginalis from the enteric to the pathogenic urogenital environment.

Potential therapeutic drug target identification in Community Acquired-Methicillin Resistant Staphylococcus aureus (CA-MRSA) using computational analysis.

The emergence of multidrug-resistant strain of community-acquired methicillin resistant Staphylococcus aureus (CA-MRSA) strain has highlighted the urgent need for the alternative and effective therapeutic approach to combat the menace of this nosocomial pathogen. In the present work novel potential therapeutic drug targets have been identified through the metabolic pathways analysis. All the gene products involved in different metabolic pathways of CA-MRSA in KEGG database were searched against the proteome of Homo sapiens using the BLASTp program and the threshold of E-value was set to as 0.001. After database searching, 152 putative targets were identified. Among all 152 putative targets, 39 genes encoding for putative targets were identified as the essential genes from the DEG database which are indispensable for the survival of CA-MRSA. After extensive literature review, 7 targets were identified as potential therapeutic drug target. These targets are Fructose-bisphosphate aldolase, Phosphoglyceromutase, Purine nucleoside phosphorylase, Uridylate kinase, Tryptophan synthase subunit beta, Acetate kinase and UDP-N-acetylglucosamine 1-carboxyvinyltransferase. Except Uridylate kinase all the identified targets were involved in more than one metabolic pathways of CA-MRSA which underlines the importance of drug targets. These potential therapeutic drug targets can be exploited for the discovery of novel inhibitors for CA-MRSA using the structure based drug design (SBDD) strategy.

Prediction of miRNA targets, affected proteins and their homologs in Glycine max.

microRNAs are small noncoding RNA gene products about 20-24nt long that are processed by Dicer from precursors with a characteristic hairpin secondary structure. As miRNAs affect the morphology of plants and animals by the posttranscriptional regulation of genes involved in critical developmental events, it has been proposed that precise regulation of miRNAs activity during various stages of growth and in specific cell types is of central importance for normal plant development. In our work we focus on the plant miRNAs and predict the miRNA targets, affected proteins by miRNA and miRNA homologs of Glycine max. Our analyses were based on sequence complementarities between miRNAs and mRNAs. As a result, we predicted 573 targets for 44 mature miRNAs sequences among 69 mature miRNAs sequences were published in database. Study of affected proteins revealed that for very less number of miRNAs, protein products are known and they mostly involved in diverse physiological process like as element of photosynthesis system. Homology analyses for miRNAs suggested that 22 miRNAs of Glycine max show 418 miRNA homologs for different plant species

Virtual screening of AmpC/β‐lactamase as target for antimicrobial resistance in Pseudomonas aeruginosa

AmpC is a group I, class C ‐lactamase present in most Enterobacteriaceae and in Pseudomonas aeruginosa and other nonfermenting gram-negative bacilli. The β‐lactam class of antibiotics is one of the most important structural classes of antibacterial compounds and act by inhibiting the bacterial D ,D - transpeptidases that are responsible for the final step of peptidoglycan cross-linking. Our main aim in the study is to screen possible inhibitors against AmpC / β ‐ lactamase (an enzyme responsible for antimicrobial activity in Pseudomonas aeruginosa), through virtual screening of 1364 NCI (National Cancer Institute) diversity set II compounds. Homology Model of AmpC / β ‐ lactamase was constructed using MODELLER and the Model was validated using PROCHECK and Verify 3D programs to obtain a stable structure, which was further used for virtual screening of NCI (National Cancer Institute) diversity set II compounds through molecular Docking studies using Autodock. The amino acid sequence of the β ‐ lactamase was also subjected to ScanProsite web server to find any pattern present in the sequence. After the prediction of 3-dimensional model of AmpC/ β‐lactamase, the possible Active sites ofβ ‐ lactamase were determined using LIGSITEcsc and CastP web servers simultaneously. The Docked complexes were validated and Enumerated based on the Autodock Scoring function to pick out the best inhibitor based on Autodock energy score. Thus from the entire 1364 NCI diversity set II compounds which were Docked, the best four docking solutions were selected (ZINC12670903, ZINC17465965, ZINC11681166 and ZINC13099024). Further the Complexes were analyzed through LIGPLOT for their interaction for the 4 best docked NCI diversity set II compounds. Thus from the Complex scoring and binding ability it is deciphered that these NCI diversity set II compounds could be promising inhibitors for Pseudomonas aeruginosa using AmpC /β ‐ lactamase as Drug target yet pharmacological studies have to confirm it.

An Approach for Identification of Novel Drug Targets in Streptococcus pyogenes SF370 Through Pathway Analysis

Streptococcus pyogenes is one of the most important pathogens as it is involved in various infections affecting upper respiratory tract and skin. Due to the emergence of multidrug resistance and cross-resistance, S. Pyogenes is becoming more pathogenic and dangerous. In the present study, an in silico comparative analysis of total 65 metabolic pathways of the host (Homo sapiens) and the pathogen was performed. Initially, 486 paralogous enzymes were identified so that they can be removed from possible drug target list. The 105 enzymes of the biochemical pathways of S. pyogenes from the KEGG metabolic pathway database were compared with the proteins from the Homo sapiens by performing a BLASTP search against the non-redundant database restricted to the Homo sapiens subset. Out of these, 83 enzymes were identified as non-human homologous while 30 enzymes of inadequate amino acid length were removed for further processing. Essential enzymes were finally mined from remaining 53 enzymes. Finally, 28 essential enzymes were identified in S. pyogenes SF370 (serotype M1). In subcellular localization study, 18 enzymes were predicted with cytoplasmic localization and ten enzymes with the membrane localization. These ten enzymes with putative membrane localization should be of particular interest. Acyl-carrier-protein S-malonyltransferase, DNA polymerase III subunit beta and dihydropteroate synthase are novel drug targets and thus can be used to design potential inhibitors against S. pyogenes infection. 3D structure of dihydropteroate synthase was modeled and validated that can be used for virtual screening and interaction study of potential inhibitors with the target enzyme.

Molecular Dynamic Simulation and Inhibitor Prediction of Cysteine Synthase Structured Model as a Potential Drug Target for Trichomoniasis

In our presented research, we made an attempt to predict the 3D model for cysteine synthase (A2GMG5_TRIVA) using homology-modeling approaches. To investigate deeper into the predicted structure, we further performed a molecular dynamics simulation for 10 ns and calculated several supporting analysis for structural properties such as RMSF, radius of gyration, and the total energy calculation to support the predicted structured model of cysteine synthase. The present findings led us to conclude that the proposed model is stereochemically stable. The overall PROCHECK G factor for the homology-modeled structure was −0.04. On the basis of the virtual screening for cysteine synthase against the NCI subset II molecule, we present the molecule 1-N, 4-N-bis [3-(1H-benzimidazol-2-yl) phenyl] benzene-1,4-dicarboxamide (ZINC01690699) having the minimum energy score (−13.0 Kcal/Mol) and a log P value of 6 as a potential inhibitory molecule used to inhibit the growth of T. vaginalis infection.

Genome-wide identification and comprehensive analysis of excretory/secretory proteins in nematodes provide potential drug targets for parasite control

Nematodes are responsible for causing severe diseases in plants, humans and other animals. Infection is associated with the release of Excretory/Secretory (ES) proteins into host cytoplasm and interference with the host immune system which make them attractive targets for therapeutic use. The identification of ES proteins through bioinformatics approaches is cost- and time-effective and could be used for screening of potential targets for parasitic diseases for further experimental studies. Here, we identified and functionally annotated 93,949 ES proteins, in the genome of 73 nematodes using integration of various bioinformatics tools. 30.6% of ES proteins were found to be supported at RNA level. The predicted ES proteins, annotated by Gene Ontology terms, domains, metabolic pathways, proteases and enzyme class analysis were enriched in molecular functions of proteases, protease inhibitors, c-type lectin and hydrolases which are strongly associated with typical functions of ES proteins. We identified a total of 452 ES proteins from human and plant parasitic nematodes, homologues to DrugBank-approved targets and C. elegans RNA interference phenotype genes which could represent potential targets for parasite control and provide valuable resource for further experimental studies to understand host-pathogen interactions.

Identification and analysis of insulin like peptides in nematode secretomes provide targets for parasite control

Insulin-like (ins) peptides play an important role in development and metabolism across the metazoa. In nematodes, these are also required for dauer formation and longevity and are expressed in different types of neurons across various life stages which demonstrate their role in parasites and could become possible targets for parasite control. To date, many nematode genomes are publically available. However, a systematic screening of ins peptides across different nematode group has not been reported. In the present study, we systematically identified ins peptides in the secretomes of 73 nematodes with fully sequenced genomes covering five different groups viz. plant parasitic, animal parasitic, human parasitic, entomopathogenic and free living nematodes. From the total of 93,949 secretory proteins, 176 proteins were uniquely mapped to 40 identified C. elegans ins families. The obtained result showed that 74.15% of the identified ins proteins were represented in free living nematodes only and remaining 25.84% were combinedly identified in all other nematode groups. The ins-1, ins-17 and ins-18 were the only ins families which were detected in all the studied nematode groups. Out of 176 proteins, 96 of ins proteins were predicted as hydrophilic in nature and 39 proteins were found stable using ProtParam analysis. Our study provides insight into the distribution of ins peptides across different group of nematodes and this information could be useful for further experimental study.