Phytochemical Profiling, In Vitro and In Silico Anti-Microbial and Anti-Cancer Activity Evaluations and Staph GyraseB and h-TOP-IIß Receptor-Docking Studies of Major Constituents of Zygophyllum coccineum L. Aqueous-Ethanolic Extract and Its Subsequent Fractions: An Approach to Validate Traditional Phytomedicinal Knowledge.

Zygophyllum coccineum, an edible halophytic plant, is part of the traditional medicine chest in the Mediterranean region for symptomatic relief of diabetes, hypertension, wound healing, burns, infections, and rheumatoid arthritis pain. The current study aimed to characterize Z. coccineum phytoconstituents, and the evaluations of the anti-microbial-biofilm, and anti-cancers bioactivities of the plant's mother liquor, i.e., aqueous-ethanolic extract, and its subsequent fractions. The in silico receptors interaction feasibility of Z. coccineum major constituents with Staph GyraseB, and human topoisomerase-IIß (h-TOP-IIß) were conducted to confirm the plant's anti-microbial and anti-cancer biological activities. Thirty-eight secondary metabolites of flavonoids, stilbene, phenolic acids, alkaloids, and coumarin classes identified by LC-ESI-TOF-MS spectrometric analysis, and tiliroside (kaempferol-3-O-(6''''-p-coumaroyl)-glucoside, 19.8%), zygophyloside-F (12.78%), zygophyloside-G (9.67%), and isorhamnetin-3-O-glucoside (4.75%) were identified as the major constituents. A superior biofilm obliteration activity established the minimum biofilm eradication concentration (MBEC) for the chloroform fraction at 3.9-15.63 µg/mL, as compared to the positive controls (15.63-31.25 µg/mL) against all the microbial strains that produced the biofilm under study, except the Aspergillus fumigatus. The aqueous-ethanolic extract showed cytotoxic effects with IC50 values at 3.47, 3.19, and 2.27 µg/mL against MCF-7, HCT-116, and HepG2 cell-lines, respectively, together with the inhibition of h-TOP-IIß with IC50 value at 45.05 ng/mL in comparison to its standard referral inhibitor (staurosporine, IC50, 135.33 ng/mL). This conclusively established the anti-cancer activity of the aqueous-ethanolic extract that also validated by in silico receptor-binding predicted energy levels and receptor-site docking feasibility of the major constituents of the plant's extract. The study helped to authenticate some of the traditional phytomedicinal properties of the anti-infectious nature of the plant.

Structure-Based Screening of DNA GyraseB Inhibitors for Therapeutic Applications in Tuberculosis: a Pharmacoinformatics Study.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB) and considered as serious public health concern worldwide which kills approximately five thousand people every day. Therefore, TB drug development efforts are in gigantic need for identification of new potential chemical agents to eradicate TB from the society. The bacterial DNA gyrase B (GyrB) protein as an experimentally widely accepted effective drug target for the development of TB chemotherapeutics. In the present study, advanced pharmacoinformatics approaches were used to screen the Mcule database against the GyrB protein. Based on a number of chemometric parameters, five molecules were found to be crucial to inhibit the GyrB. A number of molecular binding interactions between the proposed inhibitors and important active site residues of GyrB were observed. The predicted drug-likeness properties of all molecules were indicated that compounds possess characteristics to be the drug-like candidates. The dynamic nature of each molecule was explored through the molecular dynamics (MD) simulation study. Various analyzing parameters from MD simulation trajectory have suggested rationality of the molecules to be potential GyrB inhibitor. Moreover, the binding free energy was calculated from the entire MD simulation trajectories highlighted greater binding free energy values for all newly identified compounds also substantiated the strong binding affection towards the GyrB in comparison to the novobiocin. Therefore, the proposed molecules might be considered as potential anti-TB chemical agents for future drug discovery purposes subjected to experimental validation. Graphical Abstract.

Discovery of 1,2,3-triazole based quinoxaline-1,4-di-N-oxide derivatives as potential anti-tubercular agents.

A series of thirty one novel 2-(((1-(substituted phenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-3-methylquinoxaline-1,4-dioxide (7a-l), 3-(((1-(substituted phenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-6-chloro-2-methylquinoxaline-1,4-dioxide (8a-l) and 2-(((1-(substituted phenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-6,7-dichloro-3-methylquinoxaline-1,4-dioxide (9a-g) analogues were synthesized, characterized using various analytical techniques and single crystal was developed for the compounds 8 g and 9f. Synthesized compounds were evaluated for in vitro anti-tubercular activity against Mycobacterium tuberculosis H37Rv strain and two clinical isolates Spec. 210 and Spec. 192. The titled compounds exhibited minimum inhibitory concentration (MIC) ranging from 30.35 to 252.00 µM. Among the tested compounds, 8e, 8 l, 9c and 9d exhibited moderate activity (MIC = 47.6 - 52.0 µM) and 8a exhibited significant anti-tubercular activity (MIC = 30.35 µM). Furthermore, 8e, 8 l, and 9d were found to be less toxic against human embryonic kidney, HEK 293 cell lines. Finally, a docking study was also performed using MTB DNA Gyrase (PDB ID: 5BS8) for the significantly active compound 8a to know the exact binding pattern within the active site of the target enzyme.

Identification of new DNA gyrase inhibitors based on bioactive compounds from streptomyces: structure-based virtual screening and molecular dynamics simulations approaches.

DNA gyrase enzyme has vital role in bacterial survival and can be considered as a potential drug target. Owing to the appearance of resistance to gyrase-targeted drugs, especially fluoroquinolone, screening new compounds which bind more efficiently to the mutant binding pocket is essential. Hence, in this work, using Smina Autodock and through structure-based virtual screening of StreptomeDB, several natural products were discovered based on the SimocyclinoneD8 (SD8) binding pocket of GyrA subunit of DNA gyrase. After evaluation of binding affinity, binding modes, critical interactions and physicochemical and pharmaceutical properties, three lead compounds were selected for further analysis. Afterward 60 ns molecular dynamics simulations were performed and binding free energies were calculated by the molecular mechanics/Poisson-Boltzmann surface area method. Also, interaction of the selected lead compounds with the mutated GyrA protein was evaluated. Results indicated that all of the selected compounds could bind to the both wild-type and mutated GyrA with the binding affinities remarkably higher than SimocyclinoneD8. Interestingly, we noticed that the selected compounds comprised angucycline moiety in their structure which could sufficiently interact with GyrA and block the DNA binding pocket of DNA gyrase, in silico. In conclusion, three DNA gyrase inhibitors were identified successfully which were highly capable of impeding DNA gyrase and can be considered as potential drug candidates for treatment of fluoroquinolone-resistant strains.Communicated by Ramaswamy H. Sarma.

Discovery of novel oxoindolin derivatives as atypical dual inhibitors for DNA Gyrase and FabH.

The antibacterial agents and therapies today are facing serious problems such as drug resistance. Introducing dual inhibiting effect is a valid approach to solve this trouble and bring advantages including wide adaptability, favorable safety and superiority of combination. We started from potential DNA Gyrase inhibitory backbone isatin to develop oxoindolin derivatives as atypical dual Gyrase (major) and FabH (assistant) inhibitors via a two-round screening. Aiming at blocking both duplication (Gyrase) and survival (FabH), most of synthesized compounds indicated potency against Gyrase and some of them inferred favorable inhibitory effect on FabH. The top hit I18 suggested comparable Gyrase inhibitory activity (IC50 = 0.025 µM) and antibacterial effect with the positive control Novobiocin (IC50 = 0.040 µM). FabH inhibitory activity (IC50 = 5.20 µM) was also successfully introduced. Docking simulation hinted possible important interacted residues and binding patterns for both target proteins. Adequate Structure-Activity Relation discussions provide the future orientations of modification. With high potency, low initial toxicity and dual inhibiting strategy, advanced compounds with therapeutic methods will be developed for clinical application.

Dynamics of fluoroquinolones induced resistance in DNA gyrase of Mycobacterium tuberculosis.

DNA gyrase is a validated target of fluoroquinolones which are key components of multidrug resistance tuberculosis (TB) treatment. Most frequent occurring mutations associated with high level of resistance to fluoroquinolone in clinical isolates of TB patients are A90V, D94G, and A90V-D94G (double mutant [DM]), present in the larger subunit of DNA Gyrase. In order to explicate the molecular mechanism of drug resistance corresponding to these mutations, molecular dynamics (MD) and mechanics approach was applied. Structure-based molecular docking of complex comprised of DNA bound with Gyrase A (large subunit) and Gyrase C (small subunit) with moxifloxacin (MFX) revealed high binding affinity to wild type with considerably high Glide XP docking score of -7.88 kcal/mol. MFX affinity decreases toward single mutants and was minimum toward the DM with a docking score of -3.82 kcal/mol. Docking studies were also performed against 8-Methyl-moxifloxacin which exhibited higher binding affinity against wild and mutants DNA gyrase when compared to MFX. Molecular Mechanics/Generalized Born Surface Area method predicted the binding free energy of the wild, A90V, D94G, and DM complexes to be -55.81, -25.87, -20.45, and -12.29 kcal/mol, respectively. These complexes were further subjected to 30 ns long MD simulations to examine significant interactions and conformational flexibilities in terms of root mean square deviation, root mean square fluctuation, and strength of hydrogen bond formed. This comparative drug interaction analysis provides systematic insights into the mechanism behind drug resistance and also paves way toward identifying potent lead compounds that could combat drug resistance of DNA gyrase due to mutations.

Drug repurposing: An approach to tackle drug resistance in S. typhimurium.

Drug resistant S. typhimurium pose important public health problem. The development of effective drugs with novel mechanism(s) of action is needed to overcome issues pertaining to drug resistance. Drug repurposing based on computational analyses is considered a viable alternative strategy to circumvent this issue. In this context, 1309 FDA-approved drugs molecules from Mantra 2.0 database were analyzed for this study, against S. typhimurium. Sixteen compounds having similar profiles of gene expression as quinolones were identified from the database, Mantra 2.0. Further, the pharmacophore characteristics of each resultant molecule were identified and compared with the features of nalidixic acid, using the PharamGist program. Subsequently, the activities of these compounds against S. typhimurium DNA gyrase were identified, using molecular docking study. Side effects analysis was also performed for the identified compounds. Molecular dynamics simulation was carried out for the compound to validate its binding efficiency. Further, characterization of screened compound revealed IC50 values in micromolar concentration range, of which flufenamic acid showed comparable in vitro activity alongside ciprofloxacin and nalidixic acid. Thus represent interesting starting points for further optimization against S. typhimurium infections. It may be noted that the results we have obtained are the first experimental evidence of flufenamic acid activity against S. typhimurium.

Identification of Potential Therapeutics to Conquer Drug Resistance in Salmonella typhimurium: Drug Repurposing Strategy.

BACKGROUND: Salmonella typhimurium is the main cause of gastrointestinal illness in humans, and treatment options are decreasing because drug-resistant strains have emerged. OBJECTIVE: The objective of this study was to use computational drug repurposing to identify a novel candidate with an effective mechanism of action to circumvent the drug resistance. METHODS: We used the Mantra 2.0 database to initially screen drug candidates that share similar gene expression profiles to those of quinolones. Data were further reduced using pharmacophore mapping theory. Finally, we employed molecular-simulation studies to calculate the binding affinity of the screened candidates with DNA gyrase, alongside an analysis of side effects. RESULTS: A total of 16 drug candidates from the Mantra 2.0 database were screened. The pharmacophoric features of the screened candidates were examined and nalidixic acid features compared using the PharamGist program. A total of 11 compounds with the highest pharmacophore score were considered for binding energy calculation. Finally, we analysed the side effects of the eight drug candidates that showed significant binding affinity in the simulation study. CONCLUSION: Overall, flufenamic acid and sulconazole may be potential drug candidates that could be studied in vitro to assess their resistance profile against Salmonella enterica Typhimurium.

Molecular docking and inhibition studies on the interactions of Bacopa monnieri's potent phytochemicals against pathogenic Staphylococcus aureus.

BACKGROUND: Bacopa monnieri Linn. (Plantaginaceae), a well-known medicinal plant, is widely used in traditional medicine system. It has long been used in gastrointestinal discomfort, skin diseases, epilepsy and analgesia. This research investigated the in vitro antimicrobial activity of Bacopa monnieri leaf extract against Staphylococcus aureus and the interaction of possible compounds involved in this antimicrobial action. METHODS: Non-edible plant parts were extracted with ethanol and evaporated in vacuo to obtain the crude extract. A zone of inhibition studies and the minimum inhibitory concentration (MIC) of plant extracts were evaluated against clinical isolates by the microbroth dilution method. Docking study was performed to analyze and identify the interactions of possible antimicrobial compounds of Bacopa monnieri in the active site of penicillin binding protein and DNA gyrase through GOLD 4.12 software. RESULTS: A zone of inhibition studies showed significant (p < 0.05) inhibition capacity of different concentrations of Bacopa monnieri's extract against Staphylococcus aureus. The extract also displayed very remarkable minimum inhibitory concentrations (≥16 µg/ml) which was significant compared to that (≥75 µg/ml) of the reference antibiotic against the experimental strain Staphylococcus aureus. Docking studies recommended that luteolin, an existing phytochemical of Bacopa monnieri, has the highest fitness score and more specificity towards the DNA gyrase binding site rather than penicillin binding protein. CONCLUSIONS: Bacopa monnieri extract and its compound luteolin have a significant antimicrobial activity against Staphylococcus aureus. Molecular binding interaction of an in silico data demonstrated that luteolin has more specificity towards the DNA gyrase binding site and could be a potent antimicrobial compound.

Discovery of mono- and disubstituted 1H-pyrazolo[3,4]pyrimidines and 9H-purines as catalytic inhibitors of human DNA topoisomerase†IIα.

Human DNA topoisomerase IIα (htIIα) is a validated target for the development of anticancer agents. Based on structural data regarding the binding mode of AMP-PNP (5'-adenylyl-ß,γ-imidodiphosphate) to htIIα, we designed a two-stage virtual screening campaign that combines structure-based pharmacophores and molecular docking. In the first stage, we identified several monosubstituted 9H-purine compounds and a novel class of 1H-pyrazolo[3,4]pyrimidines as inhibitors of htIIα. In the second stage, disubstituted analogues with improved cellular activities were discovered. Compounds from both classes were shown to inhibit htIIα-mediated DNA decatenation, and surface plasmon resonance (SPR) experiments confirmed binding of these two compounds on the htIIα ATPase domain. Proposed complexes and interaction patterns between both compounds and htIIα were further analyzed in molecular dynamics simulations. Two compounds identified in the second stage showed promising anticancer activities in hepatocellular carcinoma (HepG2) and breast cancer (MCF-7) cell lines. The discovered compounds are suitable starting points for further hit-to-lead development in anticancer drug discovery.

Identification of bacteria pathogenic to or associated with onion (Allium cepa) based on sequence differences in a portion of the conserved gyrase B gene.

We have developed a method for the identification of Gram-negative bacteria, particularly members of the Enterobacteriaceae, based on sequence variation in a portion of the gyrB gene. Thus, we identified, in most cases to species level, over 1000 isolates from onion bulbs and leaves and soil in which onions were grown.

Structure-based discovery of substituted 4,5'-bithiazoles as novel DNA gyrase inhibitors.

Bacterial DNA gyrase is a well-established and validated target for the development of novel antibacterials. Starting from the available structural information about the binding of the natural product inhibitor, clorobiocin, we identified a novel series of 4'-methyl-N(2)-phenyl-[4,5'-bithiazole]-2,2'-diamine inhibitors of gyrase B with a low micromolar inhibitory activity by implementing a two-step structure-based design procedure. This novel class of DNA gyrase inhibitors was extensively investigated by various techniques (differential scanning fluorimetry, surface plasmon resonance, and microscale thermophoresis). The binding mode of the potent inhibitor 18 was revealed by X-ray crystallography, confirming our initial in silico binding model. Furthermore, the high resolution of the complex structure allowed for the placement of the Gly97-Ser108 flexible loop, thus revealing its role in binding of this class of compounds. The crystal structure of the complex protein G24 and inhibitor 18 provides valuable information for further optimization of this novel class of DNA gyrase B inhibitors.

Intein lacking conserved C-terminal motif G retains controllable N-cleavage activity.

A split-intein consists of two complementary fragments (N-intein and C-intein) that can associate to carry out protein trans-splicing. The Ssp GyrB S11 split-intein is an engineered unconventional split-intein consisting of a 150-amino-acid N-intein and an extremely small six-amino-acid C-intein, which comprises the conserved intein motif G. Here, we show that fusion proteins containing the 150-amino-acid N-intein could be triggered to undergo controllable N-cleavage in vitro when the six-amino-acid C-intein or a derivative thereof was added as a synthetic peptide in trans. More importantly, we discovered, unexpectedly, that the 150-amino-acid N-intein could be induced by strong nucleophiles to undergo N-cleavage in vitro, and in Escherichia coli cells, in the absence of the motif G-containing six-amino-acid C-intein. This finding indicated that the first step of the protein splicing mechanism (acyl shift) could occur in the absence of the entire motif G. Extensive kinetic analyses revealed that both the motif G residues and the Ser+1 residue positively influenced N-cleavage rate constants and yields. The 150-amino-acid N-intein could also tolerate various unrelated sequences appended to its C-terminus without disruption of the N-cleavage function, suggesting that the catalytic pocket of the intein has considerable structural flexibility. Our findings reveal interesting insights into intein structure-function relationships, and demonstrate a new and potentially more useful method of controllable, intein-mediated N-cleavage for protein engineering applications.

Anti-tubercular drug designing by structure based screening of combinatorial libraries.

In the current study, the applicability and scope of descriptor based QSAR models to complement virtual screening using molecular docking approach have been applied to identify potential virtual screening hits targeting DNA gyrase A from Mycobacterium tuberculosis, an effective and validated anti-mycobacterial target. Initially QSAR models were developed against M. fortuitum and M. smegmatis using a series of structurally related fluoroquinolone derivatives as DNA gyrase inhibitors. Both the QSAR models yielded significant cross validated Q² values of 0.6715 and 0.6944 and R² values of 0.7250 and 0.7420, respectively. The statistically significant models were validated by a test set of 22 compounds with predictive R² value of 0.7562 and 0.7087 for M. fortuitum and M. smegmatis respectively. To aid the creation of novel antituberculosis compounds, combinatorial library was developed on fluoroquinolone template to derive a data set of 5280 compounds whose activity values have been measured by the above models. Highly active compounds predicted from the models were subjected to molecular docking study to investigate the mechanism of drug binding with the DNA gyrase A protein of M. tuberculosis and the compounds showing similar type of binding patterns with that of the existing drug molecules, like sparfloxacin, were finally reported. It is seen that hydrophobic characteristics of molecular structure together with few hydrogen bond interactions are playing an essential role in antimicrobial activity for the fluoroquinolone derivatives. A representative set of seven compounds with high predicted MIC values were sorted out in the present study.

Site-specific chemical modification of proteins with a prelabelled cysteine tag using the artificially split Mxe GyrA intein.

The selective modification of proteins with a synthetic probe is of central interest for many aspects of protein chemistry. We have recently reported a new approach in which a short cysteine-containing tag (CysTag) fused to one part of a split intein is first modified with a sulfhydryl-reactive probe. In a second step, protein trans-splicing is used to link the labelled CysTag to a target protein that has been expressed in fusion with the complementary split intein fragment. Here, we present the generation and biochemical characterisation of the artificially split Mycobacterium xenopi GyrA intein. We show that this split intein is active without a renaturation step and that it provides a significant improvement for the CysTag protein-labelling approach in terms of product yields and target protein tolerance. Two proteins with multiple cysteine residues, human growth hormone and a multidomain nonribosomal peptide synthetase, were site-specifically modified with high yields. Our approach combines the benefits of the plethora of commercially available cysteine-reactive probes with a straightforward route for their site-specific incorporation even into complex and cysteine-rich proteins.

Discovery of novel DNA gyrase inhibitors by high-throughput virtual screening.

The bacterial type II topoisomerases DNA gyrase and topoisomerase IV are validated targets for clinically useful quinolone antimicrobial drugs. A significant limitation to widely utilized quinolone inhibitors is the emergence of drug-resistant bacteria due to an altered DNA gyrase. To address this problem, we have used structure-based molecular docking to identify novel drug-like small molecules that target sites distinct from those targeted by quinolone inhibitors. A chemical ligand database containing approximately 140,000 small molecules (molecular weight, <500) was molecularly docked onto two sites of Escherichia coli DNA gyrase targeting (i) a previously unexplored structural pocket formed at the dimer interface of subunit A and (ii) a small region of the ATP binding pocket on subunit B overlapping the site targeted by coumarin and cyclothialidine drugs. This approach identified several small-molecule compounds that inhibited the DNA supercoiling activity of purified E. coli DNA gyrase. These compounds are structurally unrelated to previously identified gyrase inhibitors and represent potential scaffolds for the optimization of novel antibacterial agents that act on fluoroquinolone-resistant strains.

Green tea catechins inhibit bacterial DNA gyrase by interaction with its ATP binding site.

Catechins are the main ingredients of green tea extracts and have been shown to possess versatile biological activities, including antimicrobial. We determined that the catechins inhibit bacterial DNA gyrase by binding to the ATP binding site of the gyrase B subunit. In the group of four tested catechins, epigallocatechin gallate (EGCG) had the highest activity, followed by epicatechin gallate (ECG) and epigallocatechin (EGC). Specific binding to the N-terminal 24 kDa fragment of gyrase B was determined by fluorescence spectroscopy and confirmed using heteronuclear two-dimensional NMR spectroscopy of the EGCG-15N-labeled gyrase B fragment complex. Protein residues affected by binding to EGCG were identified through chemical shift perturbation. Molecular docking calculations suggest that the benzopyran ring of EGCG penetrates deeply into the active site while the galloyl moiety anchors it to the cleft through interactions with its hydroxyl groups, which explains the higher activity of EGCG and ECG.

Role of the extended alpha4 domain of Staphylococcus aureus gyrase A protein in determining low sensitivity to quinolones.

Fluoroquinolones target two bacterial type II topoisomerases, DNA gyrase and topoisomerase IV. Acquired resistance to quinolones occurs stepwise, with the first mutation occurring in the more sensitive target enzyme. To limit the emergence of resistance, quinolones should ideally possess dual activities against the two enzymes. For reasons that are as yet unclear, Staphylococcus aureus gyrase is less sensitive to quinolones than topoisomerase IV, counter to its greater sensitivity in Escherichia coli, thereby limiting the use of quinolones for the treatment of staphylococcal infections. Mutations in the alpha4-helix domain of the GyrA subunit of gyrase are important in determining quinolone resistance. We replaced an extended region encompassing the alpha4 domain in the E. coli GyrA protein with its homolog in S. aureus and tested for its ability to complement a thermosensitive gyrase and its catalytic and noncatalytic properties. Purified gyrase reconstituted with chimeric GyrA was more resistant to ciprofloxacin than wild-type gyrase at both inhibition of catalytic activity and stimulation of cleavage complexes, and this difference was more apparent in the presence of K+-glutamate. The chimeric GyrA subunit was able to complement thermosensitive gyrase, similar to wild-type GyrA. Without supplemental K+-glutamate the MICs of ciprofloxacin for thermosensitive E. coli complemented with chimeric DNA gyrase were equal to those for E. coli complemented with wild-type gyrase but were twofold higher in the presence of K+-glutamate. Our findings suggest that the extended alpha4 domain of S. aureus GyrA is responsible, at least in part, for the increased resistance of S. aureus gyrase to quinolones and that this effect is modulated by K+-glutamate.

A 4.2 kDa synthetic peptide as a potential probe to evaluate the antibacterial activity of coumarin drugs.

The coumarin antibiotics are potent inhibitors of DNA replication whose target is the enzyme DNA gyrase, an ATP-dependent bacterial type II topoisomerase. The coumarin drugs inhibit gyrase action by competitive binding to the ATP-binding site of DNA gyrase B protein. The production of new biologically active products has stimulated additional studies on coumarin-gyrase interactions. In this regard, a 4.2 kDa peptide mimic of DNA gyrase B protein from Escherichia coli has been designed and synthesized. The peptide sequence includes the natural fragment 131-146 (coumarin resistance-determining region) and a segment containing the gyrase-DNA interaction region (positions 753-770). The peptide mimic binds to novobiocin (Ka = 1.4+/-0.3 x 10(5) M(-1)), plasmid (Ka = 1.6+/-0.5 x 10(6) M(-1)) and ATP (Ka = 1.9+/-50.4 x 10(3) M(-1)), results previously found with the intact B protein. On the other hand, the binding to novobiocin was reduced when a mutation of Arg-136 to Leu-136 was introduced, a change previously found in the DNA gyrase B protein from several coumarin-resistant clinical isolates of Escherichia coli In contrast, the binding to plasmid and to ATP was not altered. These results suggest that synthetic peptides designed in a similar way to that described here could be used as mimics of DNA gyrase in studies which seek a better understanding of the ATP, as well as coumarin, binding to the gyrase and also the mechanism of action of this class of antibacterial drugs.