Infected foot ulcers in male and female diabetic patients: a clinico-bioinformative study

<p>Abstract</p> <p>Background</p> <p>The study aimed at (i) characterizing the mode of transmission of <it>bla</it>_CTX-Mand <it>bla</it>_TEM-1among extended-spectrum-β-lactamase (ESBL)-producing <it>Escherichia coli </it>strains isolated from infected diabetic foot ulcers, and (ii) identifying the risk factors for "sex-associated multidrug resistant Gram-negative bacterial (MDRGNB)-infection status" of the ulcers.</p> <p>Methods</p> <p>Seventy-seven diabetic patients having clinically infected foot ulcers were studied in a consecutive series. The <it>E. coli </it>strains isolated from the ulcers were screened for <it>bla</it>_CTX-M, <it>bla</it>_TEM-1, <it>armA</it>, <it>rmtA </it>and <it>rmtB </it>during the 2-year study-period. PCR amplified <it>bla</it>_CTX-Mgenes were cloned and sequenced. Enterobacterial repetitive intergenic consensus (ERIC)-PCR was used for the analysis of genetic relatedness of the ESBL-producers. Risk factors for "sex-associated MDRGNB-infection status" of ulcers were assessed. Modeling was performed using Swiss-Model-Server and verified by Procheck and verify3D programmes. Discovery Studio2.0 (Accelrys) was used to prepare Ramachandran plots. Z-scores were calculated using 'WHAT IF'-package. Docking of cefotaxime with modeled CTX-M-15 enzyme was performed using Hex5.1.</p> <p>Results</p> <p>Among 51 <it>E. coli </it>isolates, 14 (27.5%) ESBL-producers were identified. Only 7 Class1 integrons, 2 <it>bla</it>_CTX-M-15, and 1 <it>bla</it>_TEM-1were detected. Ceftazidime and cefotaxime resistance markers were present on the plasmidic DNA of both the <it>bla</it>_CTX-M-15positive strains and were transmissible through conjugation. The residues Asn132, Glu166, Pro167, Val172, Lys234 and Thr235 of CTX-M-15 were found to make important contacts with cefotaxime in the docked-complex. Multivariate analysis proved 'Glycemic control at discharge' as the single independent risk factor.</p> <p>Conclusions</p> <p>Male diabetic patients with MDRGNB-infected foot ulcers have poor glycemic control and hence they might have higher mortality rates compared to their female counterparts. Plasmid-mediated conjugal transfer, albeit at a low frequency might be the possible mechanism of transfer of <it>bla</it>_CTX-M-15resistance marker in the present setting. Since the docking results proved that the amino acid residues Asn132, Glu166, Pro167, Val172, Lys234 and Thr235 of CTX-M-15 (enzyme) make important contacts with cefotaxime (drug) in the 'enzyme-drug complex', researchers are expected to duly utilize this information for designing more potent and versatile CTX-M-inhibitors.</p

A simple click by click protocol to perform docking

Recently, bioinformatics has advanced to the level that it allows almost accurate prediction of molecular interactions that hold together a protein and a ligand in the bound state. For instance, the program AutoDock has been developed to provide a procedure for predicting the interaction of small molecules with macromolecular targets which can easily separate compounds with micromolar and nanomolar binding constants from those with millimolar binding constants and can often rank molecules with finer differences in affinity. AutoDock can be used to screen a variety of possible compounds, searching for new compounds with specific binding properties or testing a range of modifications of an existing compound. The present work is a detailed outline of the protocol to use AutoDock in a more user-friendly manner. The first step is to retrieve required Ligand and Target.pdb files from major databases. The second step is preparing PDBQT format files for Target and Ligand (Target.pdbqt, Ligand.pdbqt) and Grid and Docking Parameter file (a.gpf and a.dpf) using AutoDock 4.2. The third step is to perform molecular docking using Cygwin and finally the results are analyzed. With due confidence, this is our humble claim that a researcher with no previous background in bioinformatics research would be able to perform molecular docking using AutoDock 4.2 program by following stepwise guide lines given in this article

Molecular interaction of 4-amino-N’-(benzoyloxy)-N-(2,4- dimethylphenyl)-1,2,5-oxadiazole-3-carboximidamide with the methotrexate binding site of human DHFR, and its implication in rheumatoid arthritis

Purpose: To identify an improved lead molecule for the human dihydrofolate reductase (DHFR) inhibition that ‘sits’ in the same binding cavity as methotrexate by high throughput computationalscreening.Methods: The 3-D structure of the DHFR binding site was examined using ‘CASTp3.0’. Structure based in silico screening of about 5 million drug candidates housed in the MCULE database was performed. The obtained molecule-hits were ranked in accordance with their VINA scores, made to pass through drug-likeness filters, ΔG cut-off criterion, toxicity-checker and finally ‘zero RO5 criterion’.Results: The ‘top molecule’, namely, 4-amino-N'-(benzoyloxy)-N-(2,4-dimethylphenyl)-1,2,5-oxadiazole- 3-carboximidamide, displayed robust binding with human DHFR through 21 amino acid residues (ΔG = - 9.6 kcal/mol) while 10 of these residues were the same as those displayed by ‘methotrexate binding interactions’. It passed through relevant drug screening filters including the ‘Toxicity Checker’.Conclusion: This research work describes the molecular interaction of human DHFR with an improved lead molecule named, 4-amino- N’-(benzoyloxy)-N-(2,4-dimethylphenyl)-1,2,5-oxadiazole-3- carboximidamide, with a ΔG of -9.6 kcal/mol, thus satisfying adequate ADME features for further in vitro and in vivo validation in the context of rheumatoid arthritis. Keywords: Dihydrofolate reductase, In silico screening, Methotrexate, Rheumatoid arthritis, DHF

Identification of a putative anti-rheumatoid arthritis molecule by virtual screening

Purpose: To propose an improved chemical skeleton whose scaffolds could be used for the design of future thymidylate synthase (TS)-inhibitors against rheumatoid arthritis. Methods: The drug discovery platform, ‘MCULE’, was employed for inhibitor-screening. The ‘methotrexate-interaction site’ in the crystal (PDB ID 5X66) was used as a target. One ‘RO5 violation’ was permitted. A maximum of ‘10 rotatable bonds’ and ‘100 diverse molecules’ were also allowed in the protocol. The ‘threshold similarity cut off’ was 0.7. The input values describing the remaining parameters were kept as ‘default’. The ‘Open Babel Linear Fingerprint’ was used for the analyses of molecular descriptors, followed by ADME-check. Results: 4-(4-Methyl-1-piperazinyl)-2-phenyl[1]benzofuro[3,2-d]pyrimidine corresponding to the MCULE ID-7590816301-0-93 exhibited the overall best binding with TS. The free energy of binding was -8.6 kcal/mol. A total of 17 amino acid residues were significant for the binding interactions. Importantly, 9 residues were common to methotrexate binding. It satisfied pertinent ADME conditions. Conclusion: 4-(4-Methyl-1-piperazinyl)-2-phenyl[1]benzofuro[3,2-d]pyrimidinemay emerge as a potent seed molecule for TS-inhibitor design in the context of rheumatoid arthritis. It has satisfied pertinent ADME features. However, there is need for further wet laboratory validation. Keywords: Anti-rheumatoid arthritis, Inhibitor design, Methotrexate, Seed molecule, Thymidylate synthase, Virtual screenin

Interaction of CTXM15 enzyme with cefotaxime: a molecular modelling and docking study. Bioinformation 4: 468–472. Detection of E. coli and b-Lactamases

Abstract: Extended-spectrum β-lactamases (ESBLs) are the bacterial enzymes that make them resistant to advanced-generation cephalosporins. CTX-M enzymes (the most prevalent ESBL-type) target cefotaxime. Aims of the study were: (i) Modelling of CTX-M enzyme from blaCTX-M sequences of clinical Escherichia coli isolates (ii) Docking of cefotaxime with modelled CTX-M enzymes to identify amino acid residues crucial to their interaction (iii) To hypothesize a possible relationship between &apos;interaction energy of the docked enzyme-antibiotic complex&apos; and &apos;minimum inhibitory concentration (MIC) of the antibiotic against the bacteria producing that enzyme&apos;. Seven E. coli strains of clinical origin which were confirmed as PCR-positive for blaCTX-M were selected for the study. C600 cells harboring cloned blaCTX-M were tested for ESBL-production by double-disk-synergy test. BLAST analysis confirmed all the blaCTX-M genes as blaCTX-M-15. Four of the 7 strains were found to be clonally related. Modelling was performed using Swiss Model Server. Discovery Studio 2.0 (Accelrys) was used to prepare Ramachandran plots for the modelled structures. Ramachandran Z-scores for modelled CTX-M enzymes from E. coli strains D8, D183, D253, D281, D282, D295 and D296 were found to be -0.449, 0.096, 0.027, 0.043, 0.032, -1.249 and -1.107, respectively. Docking was performed using Hex 5.1 and the results were further confirmed by Autodock 4.0. The amino acid residues Asn 104, Asn132, Gly 227, Thr 235, Gly 236, and Ser237 were found to be responsible for positioning cefotaxime into the active site of the CTX-M-15 enzyme. It was found that cefotaxime MICs for the CTX-M-15-producers increased with the increasing negative interaction energy of the enzyme-antibiotic complex

High Throughput Virtual Screening and Molecular Dynamics Simulation for Identifying a Putative Inhibitor of Bacterial CTX-M-15

Background: Multidrug resistant bacteria are a major therapeutic challenge. CTX-M-type enzymes are an important group of class A extended-spectrum β-lactamases (ESBLs). ESBLs are the enzymes that arm bacterial pathogens with drug resistance to an array of antibiotics, notably the advanced-generation cephalosporins. The current need for an effective CTX-M-inhibitor is high. Objective: The aim of the current study was to identify a promising anti-CTX-M-15 ligand whose chemical skeleton could be used as a ‘seed-molecule’ for future drug design against resistant bacteria. Methods: Virtual screening of 5,000,000 test molecules was performed by ‘MCULE Drug Discovery Platform’. ‘ADME analyses’ was performed by ‘SWISS ADME’. TOXICITY CHECKER of MCULE was employed to predict the safety profile of the test molecules. The complex of the ‘Top inhibitor’ with the ‘bacterial CTX-M-15 enzyme’ was subjected to 102.25 ns molecular dynamics simulation. This simulation was run for 3 days on a HP ZR30w workstation. Trajectory analyses were performed by employing the macro ‘md_analyze.mcr’ of YASARA STRUCTURE version 20.12.24.W.64 using AMBER14 force field. YANACONDA macro language was used for complex tasks. Figures, including RMSD and RMSF plots, were generated. Snapshots were acquired after every 250 ps. Finally, two short videos of ‘41 s’ and ‘1 min and 22 s’ duration were recorded. Results: 5-Amino-1-(2H-[1,2,4]triazino[5,6-b]indol-3-yl)-1H-pyrazole-4-carbonitrile, denoted by the MCULE-1352214421-0-56, displayed the most efficient binding with bacterial CTX-M-15 enzyme. This screened molecule significantly interacted with CTX-M-15 via 13 amino acid residues. Notably, nine amino acid residues were found common to avibactam binding (the reference ligand). Trajectory analysis yielded 410 snapshots. The RMSD plot revealed that around 26 ns, equilibrium was achieved and, thereafter, the complex remained reasonably stable. After a duration of 26 ns and onwards until 102.25 ns, the backbone RMSD fluctuations were found to be confined within a range of 0.8–1.4 Å. Conclusion: 5-Amino-1-(2H-[1,2,4]triazino[5,6-b]indol-3-yl)-1H-pyrazole-4-carbonitrile could emerge as a promising seed molecule for CTX-M-15-inhibitor design. It satisfied ADMET features and displayed encouraging ‘simulation results’. Advanced plots obtained by trajectory analyses predicted the stability of the proposed protein-ligand complex. ‘Hands on’ wet laboratory validation is warranted