In Silico Modeling and Docking Analysis of CTX-M-5, Cefotaxime-Hydrolyzing ?-Lactamase from Human-Associated Salmonella Typhimurium

Objective: To computationally model the CTX-M-5 ?-lactamase and establish its structure, which is exclusively present in human-associated Salmonella. Methods: The CTX-M-5 aminoacid sequence (Uniprot ID:O65975) of Salmonella enterica subsp. enterica serovar typhimurium was retrieved from UniProt database and subjected to homology modeling using MODELLER 9v7. The homology models were duly validated using RAMPAGE tool by generating Ramachandran plots, ERRAT graphs, and ProSA score. DoGSiteScorer server and ConSurf server were used to detect the cavities, pockets, and clefts to identify conserved amino acid sites in the predicted model. Subsequently, the modeled structure was docked using CLC Drug Discovery Workbench against proven drugs and known inhibitors. Results: Obtained high-quality homology model with 91.7% of the residues in favorable regions in Ramachandran plot and qualified in other quality parameters. Docking studies resulted in a higher dock score for PNK (D-benzylpenicilloic acid) molecule when compared to other reported inhibitors. Conclusion: This in silico study suggests that the compound PNK could be an efficient ligand for CTX-M-5 ?-lactamase and serve as a potent inhibitor of CTX-M-5.

Application of homology modeling technique in congenital cardiovascular disease / 国际儿科学杂志

Congenital cardiovascular disease is a kind of congenital disease caused by the interaction of multiple factors such as heredity, environment and pregnancy infection, but its exact etiology is still unclear.With the deepening of research, more and more evidences show that genetic factors play a significant role in its pathogenesis.Many genes are involved in the development of heart embryos, and the mutation of these genes often leads to congenital cardiovascular diseases.Homology modeling is a technique to predict the structural and functional changes of proteins caused by gene mutation through computer algorithm, and its application in the pathogenesis of congenital diseases has become a future trend.The purpose of this paper is to review homology modeling and its application in pathogenesis of congenital cardiovascular diseases briefly.

Effect of amino acid site modification on stability of foot-and-mouth disease virus-like particles / 生物工程学报

The stability of virus-like particles (VLPs) is currently the main factor affecting the quality of foot-and-mouth disease VLPs vaccines. In order to further improve the quality of the VLPs vaccine of foot-and-mouth disease (FMD), three amino acid modification sites were designed and screened through kinetic analysis software, based on the three-dimensional structure of FMDV. The three mutant recombinant plasmids were successfully prepared by the point mutation kit, transformed into Escherichia coli strain BL21 and expressed in vitro. After purification by Ni ion chromatography column, SDS-PAGE proved that the three amino acid mutations did not affect the expression of the target protein. The results of the stability study of three FMD mutant VLPs obtained by in vitro assembly show that the introduction of internal hydrophobic side chain amino acids made the morphology of VLPs more uniform (N4017W), and their stability was significantly improved compared to the other two VLPs. The internal hydrophobic force of the capsid contributes to the formation of VLPs and helps to maintain the stability of the capsid, providing new experimental ideas for improving the quality of VLPs vaccines, and helping to promote the development of VLPs vaccines.

In Silico Study of Protein-Protein Interactions in Mapks and pp2cs of Medicago sativa Discloses its Docking Sites Variations

Abstract A wide variety of cellular mechanisms such as cell division and metabolic processes are maintained by protein-protein interactions (PPIs). The identification of PPI through laboratory techniques is costly, time-consuming, difficult, and challenging. However, computational techniques were generated for PPIs prediction. In alfalfa (Madicago sativa), PPI was predicted among 12 MsMAPKs and 4 MsPP2Cs using a docking approach. For homology modelling, the Swiss model was employed while PROCHECK, ERRAT, and Verify3D were used to validate 3D models. The Ramachandran plots were obtained from PROCHECK which showed value more than 90% (nPP2C1, PP2C1, PP2C, and MSK-3 revealed 92.9%, 94.2%, 92.4%, and 91.1% respectively) for high-quality structures. The HawkDock server and the BIPSPI server were used to analyse protein docking and predict interaction sites, respectively. Our findings demonstrated that MsPP2C docking sites play an important role in the identification and docking of MsMAPKs. The binding free energy ranged from -0.16Kcal/mol to -49.15Kcal/mol for all MsMAPKs and MsPP2Cs, indicating that they interact. Docking site analysis showed that there were 48 pairs of PPIs which indicated that MsPP2Cs can perform a vital role in other signaling pathways. This study found that all MsPP2Cs have docking sites for MsMAPKs, indicating that this method can accurately determine protein-protein interactions.

Comparative modeling of sodium- and chloride-dependent GABA transporter 1 and docking studies with natural compounds

Epilepsy is a chronic neurological disorder that causes uncontrolled seizures which can affect the bodyphysically and psychologically. When a person experiences seizure, it is very difficult for them to breathe andthey bite their tongue as a reflex. Glutamatergic and gamma-aminobutyric acid ergic (GABAergic) transmissionin the brain causes seizures. The immature brain is more prone to seizures than the adult brain. Gene SLC6A1produces GABA1 protein which helps in reuptake of GABA from the synapse. Presently in this study, proteinmodeling and molecular docking were performed on protein sequence sodium- and chloride-dependent GABAtransporter 1 that was retrieved from uniport. MODELLER 9.21 versions were used to develop a homologymodel. X-ray structure of Drosophila dopamine transporter in complex with cocaine (4XP4) from speciesDrosophila melanogaster was used as a template. Autodock4.2, a docking software, was used for moleculardocking studies. Against the modeled protein, 22 natural compounds were docked. According to the results,natural compounds like Morusin showed high binding energy against modeled protein than standard drugs.

Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods

SARS-CoV-2 has caused tens of thousands of infections and more than one thousand deaths. There are currently no registered therapies for treating coronavirus infections. Because of time consuming process of new drug development, drug repositioning may be the only solution to the epidemic of sudden infectious diseases. We systematically analyzed all the proteins encoded by SARS-CoV-2 genes, compared them with proteins from other coronaviruses, predicted their structures, and built 19 structures that could be done by homology modeling. By performing target-based virtual ligand screening, a total of 21 targets (including two human targets) were screened against compound libraries including ZINC drug database and our own database of natural products. Structure and screening results of important targets such as 3-chymotrypsin-like protease (3CLpro), Spike, RNA-dependent RNA polymerase (RdRp), and papain like protease (PLpro) were discussed in detail. In addition, a database of 78 commonly used anti-viral drugs including those currently on the market and undergoing clinical trials for SARS-CoV-2 was constructed. Possible targets of these compounds and potential drugs acting on a certain target were predicted. This study will provide new lead compounds and targets for further and studies of SARS-CoV-2, new insights for those drugs currently ongoing clinical studies, and also possible new strategies for drug repositioning to treat SARS-CoV-2 infections.

Evolution of VP1 gene of coxsackievirus A16 in hand, foot, and mouth disease / 中华传染病杂志

Objective To investigate the genetic evolution of VP1 gene of pathogenic coxsackievirus A16 (CV-A16) strain isolated from clinical hand,foot,and mouth disease (HFMD) patients.Methods A total of 160 HFMD cases with CV-A16-positive results were collected from hospitals in Kunming during January 2015 to June 2017.Fecal samples were collected.Real-time polymerase chain reaction (PCR) was used to detect the CV-A16 virus nucleic acid.The VP1 genes of CV-A16-positive samples were amplified by reverse transcription-PCR.The amplified positive products were sequenced and aligned.The homologies were identified and their subgenotypes were determined.The phylogenetic tree was constructed and homology modeling was conducted.Results All the 160 CV-A16 isolates were B2 subtypes.The genetic distance between detected strains of CV-A16 and the strains in Fujian,Beijing,Nanjing was 0.76.The genetic distance to the strains in Malaysia was 0.78,and to the strains in Australia was 1.86.Homologous modeling revealed that the amino acid sequence of the VP1 gene of the strain had a G227R mutation.Conclusions There is no major genetic variation in the CV-A16 strains during 3 years.CV-A16 isolates are close to those of epidemic strains in Beijing,Fujian and Malaysia,but are far fram the strains from Australia.

Functional characterization of SsNES responsible for nerolidol biosynthesis in Senecio scandens / 中国中药杂志

A short terpene synthase gene was obtained by screening the transcriptome data of Senecio scandens. The phylogenetic tree and sequence alignment putatively identified this gene as a nerolidol synthase gene, named SsNES(GenBank MH518312). Protein homology modeling indicated that SsNES contained a complete conserved domain and folded correctly. SsNES was cloned and successfully expressed in Escherichia coli as soluble protein. The biochemical function of SsNES was characterized by E. coli metabolic engineering, which showed that SsNES catalyzed formation of trans-nerolidol with(E, E)-farnesyl diphosphate as the substrate. Nerolidol was also detected in stems and leaves of S. scandens, indicating that SsNES might act as the nerolidol synthase in plant. RT-PCR analysis indicated that SsNES was mainly expressed in stem, flowers and leaves, and no expression was observed in roots. After the treatment of SA, MeJA or Ala, SsNES was induced significantly at 6 h, indicating involvement in the defense response of S. scandens. The identification of SsNES not only clarified biosynthesis of nerolidol in S. scandens, but also provided diversity of sesquiterpene synthase, as well as theoretical basis for disease and pest defense mediated by the terpene metabolites.

Correlation study on Tibetan medicine Pterocephalus hookeri of bitter taste receptors based on molecular docking technology / 中国中药杂志

In order to study the interaction between Pterocephalus hookeri and bitter taste receptors,three-dimensional structural models of bitter taste receptors TAS2 R16,TAS2 R14 and TAS2 R13 were established by homology modeling in this paper. Maestro software was used for docking the chemical constituents of P. hookeri with bitter taste receptors. The results showed that 25 chemical components of P. hookeri can regulate three bitter taste receptors. And these components were mainly iridoid glycosides and phenolic acids.This research focused on the comprehensive application of homology modeling and molecular docking technology to explore the interaction between bitter chemical constituents of P. hookeri and bitter taste receptors. This study provided assistance in revealing pharmacodynamic basis of bitter Tibetan medicine at molecular level. It also provided new ideas and methods for the study of Tibetan medicine.

Study on Homology Modeling of eEF 2K Protein and Virtual Screening of Its Inhibitors Molecules / 中国药房

OBJECTIVE： To screen potential eEF2K inhibitor molecules， and to provide reference for the design and R&D of eEF2K inhibitor. METHODS： The eEF2K crystal structure model was constructed by homology modeling technique. The model was optimized by Loop optimization and molecular dynamics. With the help of SAVES online server， the above models were evaluated from three aspects such as Verify_3D， EERAT and Laplace diagram. Totally 55 eEF2K inhibitor molecules were collected. Hypogen pharmacophore model with activity prediction ability was constructed based on 28 of them （odd number， as training set） by Insight Ⅱ software and validated by other 27 （even number， as test set）. The optimal pharmacophore model was screened by fitting the predicted and experimental values of activity [i.e. negative logarithm of half inhibitory concentration （pIC50）] and using Ligand profiler thermogram. The virtual screening of small molecules of eEF2K inhibitors was carried out by combining the above pharmacophore model， Lipinski’s five rules and molecular docking method. RESULTS & CONCLUSIONS： The overall quality factor score of the crystal structure model of eEF2K protein was 93.697. Among them， 83.33％ of the amino acid Verify_3D score was more than or equal to 0.2， and 1.7％ of the total amino acids were located in the non-permissible region. The amino acid conformation and skeleton structure of the model were reasonable and the reliability of the model was high. Totally 9 Hypogen pharmacophore models （No. 02-10） with active predictive function were constructed， among which No. 03 pharmacophore model included 2 hydrogen bond receptors and 2 conjugated aromatic rings， which could better distinguish active and inactive molecules. The predicted value of pIC50 fitted the experimental value best （the correlation coefficient was 0.665 3）， and it had good predictive ability and high reliability. Finally， 9 potential eEF2K inhibitor molecules were obtained through virtual screening （pIC50 ranged from 1.074 to 1.185， and Dcoking-score of protein-molecule interaction ranged from －9.730 to －7.467）. Pro268， Asp267， Gln171， Phe121 and Glu212 may be the key amino acids for the interaction between eEF2K inhibitors and target proteins， including hydrogen bonds， salt bridges and hydrophobicity. These 9 molecules are expected to be the lead compounds for the development of eEF2K inhibitors.

In Silico Characterization of Meloidogyne Genus Nematode Cellulose Binding Proteins

Abstract Root-knot nematodes are a group of endoparasites species that induce the formation of giant cells in the hosts, by which they guarantee their feeding and development. Meloidogyne species infect over 2000 plant species, and are highly destructive, causing damage to many crops around the world. M. enterolobii is considered the most aggressive species in tropical regions, such as Africa and South America. Phytonematodes are able to penetrate and migrate within plant tissues, establishing a sophisticated interaction with their hosts through parasitism factors, which include a series of cell wall degradation enzymes and plant cell modification. Among the parasitism factors documented in the M. enterolobii species, cellulose binding protein (CBP), a nematode excretion protein that appears to be associated with the breakdown of cellulose present in the plant cell wall. In silico analysis can be of great importance for the identification, structural and functional characterization of genomic sequences, besides making possible the prediction of structures and functions of proteins. The present work characterized 12 sequences of the CBP protein of nematodes of the genus Meloidogyne present in genomic databases. The results showed that all CBP sequences had signal peptide and that, after their removal, they had an isoelectric point that characterized them as unstable in an acid medium. The values of the average hydrophilicity demonstrated the hydrophilic character of the analyzed sequences. Phylogenetic analyzes were also consistent with the taxonomic classification of the nematode species of this study. Five motifs were identified, which are present in all sequences analyzed. These results may provide theoretical grounds for future studies of plant resistance to nematode infection.

Homology modeling and epitope prediction of Der f 33

Dermatophagoides farinae (Der f), one of the main species of house dust mites, produces more than 30 allergens. A recently identified allergen belonging to the alpha-tubulin protein family, Der f 33, has not been characterized in detail. In this study, we used bioinformatics tools to construct the secondary and tertiary structures and predict the B and T cell epitopes of Der f 33. First, protein attribution, protein patterns, and physicochemical properties were predicted. Then, a reasonable tertiary structure was constructed by homology modeling. In addition, six B cell epitopes (amino acid positions 34-45, 63-67, 103-108, 224-230, 308-316, and 365-377) and four T cell epitopes (positions 178-186, 241-249, 335-343, and 402-410) were predicted. These results established a theoretical basis for further studies and eventual epitope-based vaccine design against Der f 33.

Study on the Molecular Docking of Penehyclidine Optical Isomers and Muscarinic Receptor Subtypes / 中国药房

OBJECTIVE:To study the affinity of penehyclidine optical isomers to muscarinic(M)receptor subtypes,and pro-vide reference for revealing the action targets and efficacy selectivity of penehyclidine. METHODS:Homology modeling,molecu-lar docking and other molecular simulation technologies were used to analyze and predict the binding energy of 4 optical isomers to M receptor subtypes and judge its affinity by comparing the binding energy of different optical isomers R1 (3R,2′R),R2 (3R, 2′S),S1(3S,2′R),S2(3S,2′S)with M receptor subtypes M1-M5. RESULTS:All the 4 optical isomers can dock into the ac-tive sites of M receptor subtypes,and different optical isomers showed great differences in the molecular docking with different M receptor subtypes. Penehyclidine isomers showed larger binding energy to M3,the binding energy of 4 optical isomers ranged in 5736.519-5907.143 kcal/mol. The binding energy of R1 to M1 was 1190.041 kcal/mol;while those of other optical isomers to each receptor subtype were lower or negative. CONCLUSIONS:R1 shows the affinity to M1 receptor. And all the 4 optical isomer show the affinity to M3.

Homology modeling and application of 3 D structure of Cav1.2 channel / 中国药理学通报

Aim To construct 3 D structure model of cardiac Cav1.2 channel and check its accuracy and re-liability.Methods Homology model of Cav1.2 chan-nel α1 subunit was constructed using SWISS-MODEL server.The model was submitted to an online testing server built by University of California and scored by it.The binding of Cav1.2 channel with blocker or drug was simulated by MOE software molecular docking pro-gram to check the model′s accuracy and reliability.Re-sults Both the target sequence Cav1.2 α1 C and the template sequence Cav1.1 α1 S searched by SWISS-MODEL server belonged to L-type Ca2+channel.Since the homology was 7 1.5% revealed by sequence align-ment,homology modeling was performed using automa-ted mode.L-type Ca2+ channel blockers Verapamil, Nifedipine and Diltiazem could bind to the 3 D structure model of Cav1.2 channel,while sodium channel bloc-ker TTX could not.Furthermore,active ingredient of traditional Chinese drug Praeruptorin A and Berberine could also bind to the 3D structure model of Cav1.2 channel.Conclusion The 3 D structure model of Cav1.2 channel was constructed successfully,which provides reliable materials for further studies and estab-lishes the foundation for the application of homology modeling in the study of 3 D structure prediction of ion channels.

Prediction of ETA oligopeptides antagonists from Glycine max based on in silico proteolysis / 中国中药杂志

Oligopeptides are one of the the key pharmaceutical effective constituents of traditional Chinese medicine(TCM). Systematic study on composition and efficacy of TCM oligopeptides is essential for the analysis of material basis and mechanism of TCM. In this study, the potential anti-hypertensive oligopeptides from Glycine max and their endothelin receptor A (ETA) antagonistic activity were discovered and predicted based on in silico technologies.Main protein sequences of G. max were collected and oligopeptides were obtained using in silico gastrointestinal tract proteolysis. Then, the pharmacophore of ETA antagonistic peptides was constructed and included one hydrophobic feature, one ionizable negative feature, one ring aromatic feature and five excluded volumes. Meanwhile, three-dimensional structure of ETA was developed by homology modeling methods for further docking studies. According to docking analysis and consensus score, the key amino acid of GLN165 was identified for ETA antagonistic activity. And 27 oligopeptides from G. max were predicted as the potential ETA antagonists by pharmacophore and docking studies.In silico proteolysis could be used to analyze the protein sequences from TCM. According to combination of in silico proteolysis and molecular simulation, the biological activities of oligopeptides could be predicted rapidly based on the known TCM protein sequence. It might provide the methodology basis for rapidly and efficiently implementing the mechanism analysis of TCM oligopeptides.

Homology modeling and site-directed mutagenesis of caffeic acid-3-O-methyltransferase from Ligusticum chuanxiong / 中草药

Objective: To construct the three dimensional models of L. chuanxiong caffeic acid-3-O-methyltransferase (LCCOMT) and verify the model using site-directed mutagenesis technology. Methods: The three-dimensional model was constructed by homology modeling using the crystal structure of COMT from Medicago sativa as a temple. Caffeic acid was docked into the optimized model of LCCOMT to predict the active site. The predicted site was mutated using site-directed mutagenesis technology. Then, the activity of mutant enzyme was detected. Results: The molecular docking, which showed there were hydrogen bonds between His268 and 3-OH of caffeic acid, was successful. Two mutant enzymes, H268N and H268Q, lost 94.85% and 95.28% of their activity respectively. Conclusion: The His268 is confirmed as one of the key residues of LCCOMT. It may play a role as a base in the deprotonation reaction of the 3-OH of caffeic acid.

Virtual screening for components in Chicory combined with CNT2 target based on molecular docking / 中国中药杂志

To virtual screen the compound of Chicory combined with the concentrative nucleoside transporter 2 (CNT2) in molecular docking technology.The homology model of hCNT2 was produced, and then the Vina software was employed to virtual screen the Chicory compound combined with CNT2. Compared with 7,8,3'-trihydroxyflavone, a CNT2 inhibitors, 23 score higher chicory compounds were hit.Meanwhile, the ten top compounds have been revealed that play important role in decrease the uric level. The bioactivity to CNT2 needs to be investigatedin experiment. CNT2 may be a potential target of chicory, which decreases the absorption of purine nucleoside in intestinal tract.

Homology Modeling of DNA polymerases of Herpesviridae family and structure-based virtual screening for inhibitor identification.

Human herpes viruses are responsible for the several transmitted infections in human. It is known that the DNA polymerase enzyme is one of the putative targets for herpes. Therefore, it is of interest to model all known DNA polymerases of Herpesviridae family. Here, all the DNA polymerases of Herpesviridae without any crystal structure were modeled using HHV-1 DNA polymerase as a template. Modeled structures were screened by ramachandran plot and Descrete Optimization of Protein Energy (DOPE) score. To find out multi-target inhibitor for Herpesviridae, 21 natural antiviral compounds were selected from literature and screened using Lipinski’s rule of five. Binding pose of acyclovir with HHV-1 DNA polymerase was taken for the comparative docking study. Comparative binding analysis was done after settling of 120 and eight partial mono flexible protein-ligand docking sets for natural compounds and acyclovir, respectively. From the study it is found that alliin and gallic acid exhibit good binding affinity than acyclovir and other natural compounds. So, here we purpose that these two compounds can be potential candidates to inhibit Herpesviridae family.

Construction of a Single Chain Variable Fragment Antibody Against Sulfonamides and Its Interaction with Sulfathiazole / 分析化学

The interaction between the antibody and the corresponding target molecule determines the characteristics of immunoassay. In this study, a single chain variable fragment antibody (scFv4C7) derived from the hybridoma strain 4C7 were prepared via genetic engineering technique. The recognition properties of scFv4 C7 was determined and compared to those of the parent monoclonal antibody by indirect competitive enzyme-linked immunosorbent assay(ic-ELISA). Three dimensional structure of the scFv4C7 was presented by Swiss-Model, and sulfathiazole ( STZ) was docked to the scFv4C7 model to obtain the structure of the binding complex. The results from the ic-ELISA showed that the binding properties of scFv4C7 were comparable with the parent monoclonal antibody and STZ was almost completely buried in a deep binding pocket formed by the heavy chain and light chain of the antibody. The interaction between STZ and scFv4C7 was more closely related to the heavy chain and the complementarity-determining region ( CDR ) H3 loop played more important role than other CDR loops. The study preliminary provided the necessary structural information for the preparation of antibody with broader specificity and higher affinity.

Effect of mutation on aggregation propensity in homology model structures of syntaxin-3 from Homo sapiens.

Perception of molecular mechanism would provide potent additional knowledge on mammalian membrane proteins involved in causing diseases. In human, syntaxin-3 (STX3) is a significant apical targeting protein in the epithelial membrane and in exocytosis process; it also acts as a vesicle transporter by cellular receptor in neutrophils, which is crucial for protein trafficking event. Structurally, syntaxin-3 has hydrophobic domain at carboxyl terminus that directs itself to intra-cellular compartments. In addition, the experimental structure of STX3 is not available and no mutational study has been carried out with natural variants of proteins. Moreover, there is no evidence so far for the natural variant Val286 of STX3 causing any diseases. Hence, in the present study, analyses of residue-based properties of the homology model STX3 were carried out along with mutations at carboxyl terminus of STX3 by implementing protein engineering and in silico approaches. The model structure of STX3 was constructed adopting Modeller v9.11 and the aggregation propensity was analyzed with BioLuminate tool. The results showed that there was reduction in aggregation propensity with point mutation at Val286, instead of Ile, resulting into increasing the structural stability of STX3. In conclusion, the Ccap exposed residue would be a suitable position for further mutational studies, particularly with Val286 of STX3 in human. This approach could gainfully be applied to STX3 for efficient drug designing which would be a valuable target in the cancer treatment.