Design, synthesis, molecular modeling, and antimicrobial potential of novel 3-[(1H-pyrazol-3-yl)imino]indolin-2-one derivatives as DNA gyrase inhibitors.

A series of 3-[(1H-pyrazol-3-yl)imino]indolin-2-one derivatives were designed using the molecular hybridization method, characterized using different spectroscopic techniques, and evaluated for their in vitro antimicrobial activity. Most of the target compounds demonstrated good to moderate antimicrobial activity compared with ciprofloxacin and fluconazole. Four compounds (8b, 9a, 9c, and 10a) showed encouraging results, with minimal inhibitory concentration (MIC) values (53.45-258.32 µM) comparable to those of norfloxacin (100.31-200.63 µM) and ciprofloxacin (48.33-96.68 µM). Noticeably, the four derivatives revealed excellent bactericidal and fungicidal activities, except for the bacteriostatic potential of compounds 8b and 9a against Escherichia coli and Staphylococcus aureus, respectively. The time-killing kinetic study against S. aureus confirmed the efficacy of these derivatives. Furthermore, two of the four promising derivatives, 9a and 10a, could prevent the formation of biofilms of S. aureus without affecting the bacterial growth at low concentrations. A combination study with seven commercial antibiotics against the multidrug-resistant bacterium P. aeruginosa showed a notable reduction in the antibiotic MIC values, represented mainly through a synergistic or additive effect. The enzymatic assay implied that the most active derivatives had inhibition potency against DNA gyrase comparable to that of ciprofloxacin. Molecular docking and density functional theory calculations were performed to explore the binding mode and study the reactivity of the promising compounds.

Synthesis, Identification, Computer-Aided Docking Studies, and ADMET Prediction of Novel Benzimidazo-1,2,3-triazole Based Molecules as Potential Antimicrobial Agents.

2-azido-1H-benzo[d]imidazole derivatives 1a,b were reacted with a ß-ketoester such as acetylacetone in the presence of sodium ethoxide to obtain the desired molecules 2a,b. The latter acted as a key molecule for the synthesis of new carbazone derivatives 4a,b that were submitted to react with 2-oxo-N-phenyl-2-(phenylamino)acetohydrazonoyl chloride to obtain the target thiadiazole derivatives 6a,b. The structures of the newly synthesized compounds were inferred from correct spectral and microanalytical data. Moreover, the newly prepared compounds were subjected to molecular docking studies with DNA gyrase B and exhibited binding energy that extended from -9.8 to -6.4 kcal/mol, which confirmed their excellent potency. The compounds 6a,b were found to be with the minimum binding energy (-9.7 and -9.8 kcal/mol) as compared to the standard drug ciprofloxacin (-7.4 kcal/mol) against the target enzyme DNA gyrase B. In addition, the newly synthesized compounds were also examined and screened for their in vitro antimicrobial activity against pathogenic microorganisms Staphylococcus aureus, E. coli, Pseudomonas aeruginosa, Aspergillus niger, and Candida albicans. Among the newly synthesized molecules, significant antimicrobial activity against all the tested microorganisms was obtained for the compounds 6a,b. The in silico and in vitro findings showed that compounds 6a,b were the most active against bacterial strains, and could serve as potential antimicrobial agents.

Biological Effects of Quinolones: A Family of Broad-Spectrum Antimicrobial Agents.

Broad antibacterial spectrum, high oral bioavailability and excellent tissue penetration combined with safety and few, yet rare, unwanted effects, have made the quinolones class of antimicrobials one of the most used in inpatients and outpatients. Initially discovered during the search for improved chloroquine-derivative molecules with increased anti-malarial activity, today the quinolones, intended as antimicrobials, comprehend four generations that progressively have been extending antimicrobial spectrum and clinical use. The quinolone class of antimicrobials exerts its antimicrobial actions through inhibiting DNA gyrase and Topoisomerase IV that in turn inhibits synthesis of DNA and RNA. Good distribution through different tissues and organs to treat Gram-positive and Gram-negative bacteria have made quinolones a good choice to treat disease in both humans and animals. The extensive use of quinolones, in both human health and in the veterinary field, has induced a rise of resistance and menace with leaving the quinolones family ineffective to treat infections. This review revises the evolution of quinolones structures, biological activity, and the clinical importance of this evolving family. Next, updated information regarding the mechanism of antimicrobial activity is revised. The veterinary use of quinolones in animal productions is also considered for its environmental role in spreading resistance. Finally, considerations for the use of quinolones in human and veterinary medicine are discussed.

Effectiveness of Fluoroquinolones with Difluoropyridine Derivatives as R1 Groups on the Salmonella DNA Gyrase in the Presence and Absence of Plasmid-Encoded Quinolone Resistance Protein QnrB19.

Aims: WQ-3810 has strong inhibitory activity against Salmonella and other fluoroquinolone-resistant pathogens. The unique potentiality of this is attributed to 6-amino-3,5-difluoropyridine-2-yl at R1 group. The aim of this study was to examine WQ-3810 and its derivatives WQ-3334 and WQ-4065 as the new drug candidate for wild-type Salmonella and that carrying QnrB19. Materials and Methods: The half maximal inhibitory concentrations (IC50s) of WQ-3810, WQ-3334 (Br atom in place of methyl group at R8), and WQ-4065 (6-ethylamino-3,5-difluoropyridine-2-yl in place of 6-amino-3,5-difluoropyridine-2-yl group at R1) in the presence or absence of QnrB19 were assessed by in vitro DNA supercoiling assay utilizing recombinant DNA gyrase and QnrB19. Results: IC50s of WQ-3810, WQ-3334, and WQ-4065 against Salmonella DNA gyrase were 0.031 ± 0.003, 0.068 ± 0.016, and 0.72 ± 0.39 µg/mL, respectively, while QnrB19 increased IC50s of WQ-3810, WQ-3334, and WQ-4065 to 0.44 ± 0.05, 0.92 ± 0.34, and 9.16 ± 2.21 µg/mL, respectively. Conclusion: WQ-3810 and WQ-3334 showed stronger inhibitory activity against Salmonella Typhimurium DNA gyrases than WQ-4065 even in the presence of QnrB19. The results suggest that 6-amino-3,5-difluoropyridine-2-yl group at R1 is playing an important role and WQ-3810 and WQ-3334 to be good candidates for Salmonella carrying QnrB19.

DNA supercoiling differences in bacteria result from disparate DNA gyrase activation by polyamines.

DNA supercoiling is essential for all living cells because it controls all processes involving DNA. In bacteria, global DNA supercoiling results from the opposing activities of topoisomerase I, which relaxes DNA, and DNA gyrase, which compacts DNA. These enzymes are widely conserved, sharing >91% amino acid identity between the closely related species Escherichia coli and Salmonella enterica serovar Typhimurium. Why, then, do E. coli and Salmonella exhibit different DNA supercoiling when experiencing the same conditions? We now report that this surprising difference reflects disparate activation of their DNA gyrases by the polyamine spermidine and its precursor putrescine. In vitro, Salmonella DNA gyrase activity was sensitive to changes in putrescine concentration within the physiological range, whereas activity of the E. coli enzyme was not. In vivo, putrescine activated the Salmonella DNA gyrase and spermidine the E. coli enzyme. High extracellular Mg2+ decreased DNA supercoiling exclusively in Salmonella by reducing the putrescine concentration. Our results establish the basis for the differences in global DNA supercoiling between E. coli and Salmonella, define a signal transduction pathway regulating DNA supercoiling, and identify potential targets for antibacterial agents.

Antibacterial activity of novel dual bacterial DNA type II topoisomerase inhibitors.

In this study, a drug discovery programme that sought to identify novel dual bacterial topoisomerase II inhibitors (NBTIs) led to the selection of six optimized compounds. In enzymatic assays, the molecules showed equivalent dual-targeting activity against the DNA gyrase and topoisomerase IV enzymes of Staphylococcus aureus and Escherichia coli. Consistently, the compounds demonstrated potent activity in susceptibility tests against various Gram-positive and Gram-negative reference species, including ciprofloxacin-resistant strains. The activity of the compounds against clinical multidrug-resistant isolates of S. aureus, Clostridium difficile, Acinetobacter baumannii, Neisseria gonorrhoeae, E. coli and vancomycin-resistant Enterococcus spp. was also confirmed. Two compounds (1 and 2) were tested in time-kill and post-antibiotic effect (PAE) assays. Compound 1 was bactericidal against all tested reference strains and showed higher activity than ciprofloxacin, and compound 2 showed a prolonged PAE, even against the ciprofloxacin-resistant S. aureus BAA-1720 strain. Spontaneous development of resistance to both compounds was selected for in S. aureus at frequencies comparable to those obtained for quinolones and other NBTIs. S. aureus BAA-1720 mutants resistant to compounds 1 and 2 had single point mutations in gyrA or gyrB outside of the quinolone resistance-determining region (QRDR), confirming the distinct site of action of these NBTIs compared to that of quinolones. Overall, the very good antibacterial activity of the compounds and their optimizable in vitro safety and physicochemical profile may have relevant implications for the development of new broad-spectrum antibiotics.

WQ-3810 inhibits DNA gyrase activity in ofloxacin-resistant Mycobacterium leprae.

BACKGROUND: Mycobacterium leprae causes leprosy and ofloxacin is used to control this bacterium. However, specific amino acid substitutions in DNA gyrases of M. leprae interferes with the effect of ofloxacin. METHODOLOGY/PRINCIPAL FINDINGS: Here we tested the inhibitory effect of WQ-3810 on DNA gyrases in M. leprae, using recombinant gyrases. We theorized that WQ-3810 and DNA gyrases interacted, which was tested in silico. Compared with control drugs like ofloxacin, WQ-3810 showed a better inhibitory effect on ofloxacin-resistant DNA gyrases. The in-silico study showed that, unlike control drugs, a specific linkage between a R1 group in WQ-3810 and aspartic acid at position 464 in the subunit B of DNA gyrases existed, which would enhance the inhibitory effect of WQ-3810. This linkage was confirmed in a further experiment, using recombinant DNA gyrases with amino acid substitutions in subunits B instead. CONCLUSIONS/SIGNIFICANCE: The inhibitory effect of WQ-3810 was likely enhanced by the specific linkage between a R1 group residue in its structure and DNA gyrases. Using interactions like the one found in the present work may help design new fluoroquinolones that contribute to halt the emergence of antibiotic-resistant pathogens.

Novel 2-arylbenzothiazole DNA gyrase inhibitors: Synthesis, antimicrobial evaluation, QSAR and molecular docking studies.

A series of new 2-arylbenzothiazole derivatives (4, 5, 6a-j, 7a-i and 8a,b) was synthesized and tested for their antimicrobial activity against different Gram-positive, Gram-negative bacteria and yeast using ciprofloxacin and fluconazole as positive controls for the antibacterial and antifungal activities, respectively. The target compounds showed stronger inhibitory activity against Gram-negative than Gram-positive bacteria. The minimum inhibitory concentration (MIC) values were determined for those compounds showed zone of inhibition ≥ 13 mm. Based on the MIC values for the tested compounds against E. coli, compounds (4, 5, 6c, 6d, 6g, 6i, 6j, 7b, 7c, 7g and 8a) were selected and tested for their E. coli gyrase inhibitory activity. The tested compounds showed moderate inhibitory activity against E. coli gyrase. Compounds 5, 6c, 6i, 6j and 7b displayed high inhibitory activity against E. coli gyrase with IC50 values below 10 µM, however, they were less active than ciprofloxacin (E. coli gyrase IC50 = 1.14 µM). The p-hydroxy-m-methoxy benzothiazole analogue 6c was the most active tested compound (E. coli gyrase IC50 = 4.85 µM). Quantitative structure-activity relationship (QSAR) study was also implemented for the newly synthesized compounds. The QSAR study indicated that the structural feature that governs the anti-microbial activity for the newly synthesized benzothiazole derivatives is their structural hydrophilic-lipophilic balance what agrees with the chemical intuition where this balance governs their cellular absorption and so their antimicrobial activity. Molecular docking showed that the newly synthesized compounds possess the required structural feature for E. coli gyrase B inhibition through interaction with the key amino acids Asp73 and Gly77.

WQ-3810 exerts high inhibitory effect on quinolone-resistant DNA gyrase of Salmonella Typhimurium.

The inhibitory effect of WQ-3810 on DNA gyrase was assayed to evaluate the potential of WQ-3810 as a candidate drug for the treatment of quinolone resistant Salmonella Typhymurium infection. The inhibitory effect of WQ-3810, ciprofloxacin and nalidixic acid was compared by accessing the drug concentration that halves the enzyme activity (IC50) of purified S. Typhimurium wildtype and mutant DNA gyrase with amino acid substitution at position 83 or/and 87 in subunit A (GyrA) causing quinolone resistance. As a result, WQ-3810 reduced the enzyme activity of both wildtype and mutant DNA gyrase at a lower concentration than ciprofloxacin and nalidixic acid. Remarkably, WQ-3810 showed a higher inhibitory effect on DNA gyrase with amino acid substitutions at position 87 than with that at position 83 in GyrA. This study revealed that WQ-3810 could be an effective therapeutic agent, especially against quinolone resistant Salmonella enterica having amino acid substitution at position 87.

Discovery of a Novel DNA Gyrase-Targeting Antibiotic through the Chemical Perturbation of Streptomyces venezuelae Sporulation.

Common approaches to antibiotic discovery include small-molecule screens for growth inhibition in target pathogens and screens for inhibitors of purified enzymes. These approaches have a shared intent of seeking to directly target a vital Achilles heel in a pathogen of interest. Here, we report the first screen against a sporulation pathway in a non-pathogenic bacterium as a means of discovering novel antibiotics-this effort has resulted in two important discoveries. First, we show that the sporulation program of Streptomyces venezuelae is exquisitely sensitive to numerous forms of DNA damage. Second, we have identified a DNA gyrase inhibitor. This molecule, EN-7, is active against pathogenic species that are resistant to ciprofloxacin and other clinically important antibiotics. We suggest that this strategy could be applied to other morphogenetic pathways in prokaryotes or eukaryotes as a means of identifying novel chemical matter having scientific and clinical utility.

The Microbial Toxin Microcin B17: Prospects for the Development of New Antibacterial Agents.

Microcin B17 (MccB17) is an antibacterial peptide produced by strains of Escherichia coli harboring the plasmid-borne mccB17 operon. MccB17 possesses many notable features. It is able to stabilize the transient DNA gyrase-DNA cleavage complex, a very efficient mode of action shared with the highly successful fluoroquinolone drugs. MccB17 stabilizes this complex by a distinct mechanism making it potentially valuable in the fight against bacterial antibiotic resistance. MccB17 was the first compound discovered from the thiazole/oxazole-modified microcins family and the linear azole-containing peptides; these ribosomal peptides are post-translationally modified to convert serine and cysteine residues into oxazole and thiazole rings. These chemical moieties are found in many other bioactive compounds like the vitamin thiamine, the anti-cancer drug bleomycin, the antibacterial sulfathiazole and the antiviral nitazoxanide. Therefore, the biosynthetic machinery that produces these azole rings is noteworthy as a general method to create bioactive compounds. Our knowledge of MccB17 now extends to many aspects of antibacterial-bacteria interactions: production, transport, interaction with its target, and resistance mechanisms; this knowledge has wide potential applicability. After a long time with limited progress on MccB17, recent publications have addressed critical aspects of MccB17 biosynthesis as well as an explosion in the discovery of new related compounds in the thiazole/oxazole-modified microcins/linear azole-containing peptides family. It is therefore timely to summarize the evidence gathered over more than 40 years about this still enigmatic molecule and place it in the wider context of antibacterials.

In Vitro and In Vivo Activity of Zabofloxacin and Other Fluoroquinolones Against MRSA Isolates from A University Hospital in Egypt.

The widespread of infections caused by methicillin-resistant Staphylococcus aureus (MRSA), has necessitated the search for alternative therapies; introduction of new agents being a suggestion. This study compares the in vitro and in vivo activities of zabofloxacin, a novel fluoroquinolone, with moxifloxacin, levofloxacin and ciprofloxacin against clinical isolates of MRSA from patients hospitalized in the Alexandria Main University hospital; a tertiary hospital in Alexandria, Egypt, where zabofloxacin has not been yet introduced. The strains tested showed the highest percentage of susceptibility to zabofloxacin (61.2%) among the tested fluoroquinolones with the most effective MIC50 and MIC90 (0.25 and 2 µg/ml, respectively). Time-kill curve analysis revealed a rapid bactericidal activity of zabofloxacin after 6 h of incubation with a quinolone-resistant isolate and complete killing when tested against a quinolone-sensitive isolate with inhibition of regrowth in both cases. PCR amplification and sequencing of QRDRs in selected strains revealed the following amino acid substitutions: Ser-84→Leu in GyrA, Ser-80→Phe in GrlA and Pro-451→Ser in GrlB. The in vivo studies demonstrated that zabofloxacin possessed the most potent protective effect against systemic infection in mice (ED50: 29.05 mg/kg) with lowest count in the dissected lungs (3.66 log10 CFU/ml). The histopathological examination of lung specimens of mice treated with zabofloxacin displayed least congestion, inflammation, oedema and necrosis with clear alveolar spaces and normal vessels. In conclusion, zabofloxacin was proved to possess high in vitro and in vivo efficacy encompassing its comparators and could be considered as a possible candidate for the treatment of infections caused by MRSA. To our knowledge, this is the first study evaluating the in vitro and in vivo activity of zabofloxacin against Egyptian MRSA clinical isolates.The widespread of infections caused by methicillin-resistant Staphylococcus aureus (MRSA), has necessitated the search for alternative therapies; introduction of new agents being a suggestion. This study compares the in vitro and in vivo activities of zabofloxacin, a novel fluoroquinolone, with moxifloxacin, levofloxacin and ciprofloxacin against clinical isolates of MRSA from patients hospitalized in the Alexandria Main University hospital; a tertiary hospital in Alexandria, Egypt, where zabofloxacin has not been yet introduced. The strains tested showed the highest percentage of susceptibility to zabofloxacin (61.2%) among the tested fluoroquinolones with the most effective MIC50 and MIC90 (0.25 and 2 µg/ml, respectively). Time-kill curve analysis revealed a rapid bactericidal activity of zabofloxacin after 6 h of incubation with a quinolone-resistant isolate and complete killing when tested against a quinolone-sensitive isolate with inhibition of regrowth in both cases. PCR amplification and sequencing of QRDRs in selected strains revealed the following amino acid substitutions: Ser-84→Leu in GyrA, Ser-80→Phe in GrlA and Pro-451→Ser in GrlB. The in vivo studies demonstrated that zabofloxacin possessed the most potent protective effect against systemic infection in mice (ED50: 29.05 mg/kg) with lowest count in the dissected lungs (3.66 log10 CFU/ml). The histopathological examination of lung specimens of mice treated with zabofloxacin displayed least congestion, inflammation, oedema and necrosis with clear alveolar spaces and normal vessels. In conclusion, zabofloxacin was proved to possess high in vitro and in vivo efficacy encompassing its comparators and could be considered as a possible candidate for the treatment of infections caused by MRSA. To our knowledge, this is the first study evaluating the in vitro and in vivo activity of zabofloxacin against Egyptian MRSA clinical isolates.

Targeting DNA Gyrase to Combat Mycobacterium tuberculosis: An Update.

DNA gyrase is a clinically validated drug target, currently targeted only by fluoroquinolone class of antibacterials. However, owing to increasing drug resistance as well as a concomitant reduction in the availability of newer classes of antibiotics, fluoroquinolones are increasingly being over-utilized in order to treat serious infections, including multi-drug resistant tuberculosis. This, in turn, increases the probability of resistance to fluoroquinolones, which is mediated by a single amino acid change in gyrA, leading to class-wide resistance. In this review, we provide an overview of the recent progress in identifying novel scaffolds which target DNA gyrase and provide an update on their discovery and development status.

An optimised series of substituted N-phenylpyrrolamides as DNA gyrase B inhibitors.

ATP competitive inhibitors of DNA gyrase and topoisomerase IV have great therapeutic potential, but none of the described synthetic compounds has so far reached the market. To optimise the activities and physicochemical properties of our previously reported N-phenylpyrrolamide inhibitors, we have synthesized an improved, chemically variegated selection of compounds and evaluated them against DNA gyrase and topoisomerase IV enzymes, and against selected Gram-positive and Gram-negative bacteria. The most potent compound displayed IC50 values of 6.9 nM against Escherichia coli DNA gyrase and 960 nM against Staphylococcus aureus topoisomerase IV. Several compounds displayed minimum inhibitory concentrations (MICs) against Gram-positive strains in the 1-50 µM range, one of which inhibited the growth of Enterococcus faecalis, Enterococcus faecium, S. aureus and Streptococcus pyogenes with MIC values of 1.56 µM, 1.56 µM, 0.78 µM and 0.72 µM, respectively. This compound has been investigated further on methicillin-resistant S. aureus (MRSA) and on ciprofloxacin non-susceptible and extremely drug resistant strain of S. aureus (MRSA VISA). It exhibited the MIC value of 2.5 µM on both strains, and MIC value of 32 µM against MRSA in the presence of inactivated human blood serum. Further studies are needed to confirm its mode of action.

Design and synthesis of aminothiazolyl norfloxacin analogues as potential antimicrobial agents and their biological evaluation.

A series of aminothiazolyl norfloxacin analogues as a new type of potential antimicrobial agents were synthesized and screened for their antimicrobial activities. Most of the prepared compounds exhibited excellent inhibitory efficiencies. Especially, norfloxacin analogue II-c displayed superior antimicrobial activities against K. pneumoniae and C. albicans with MIC values of 0.005 and 0.010 mM to reference drugs, respectively. This compound not only showed broad antimicrobial spectrum, rapid bactericidal efficacy and strong enzymes inhibitory potency including DNA gyrase and chitin synthase (CHS), low toxicity against mammalian cells and no obvious propensity to trigger the development of bacterial resistance, but also exerted efficient membrane permeability, and could effectively intercalate into K. pneumoniae DNA to form a steady supramolecular complex, which might block DNA replication to exhibit their powerful antimicrobial activity. Quantum chemical studies were also performed to explain the high antimicrobial activities. Molecular docking showed that compound II-c could bind with gyrase-DNA and topoisomerase IV-DNA through hydrogen bonds and π-π stacking.

ATP-competitive DNA gyrase and topoisomerase IV inhibitors as antibacterial agents.

INTRODUCTION: The bacterial topoisomerases DNA gyrase and topoisomerase IV are validated targets for development of novel antibacterial agents. Fluoroquinolones inhibit the catalytic GyrA and/or ParC(GrlA) subunit and have been commonly used, although these have toxicity liabilities that restrict their use. The ATPase GyrB and ParE(GrlB) subunits have been much less explored and after withdrawal of novobiocin, there are no further marketed inhibitors . ATP-competitive inhibitors of GyrB and/or ParE(GrlB) are of special interest, as this target has been validated, and it is expected that many of the problems associated with fluoroquinolones can be avoided. AREAS COVERED: This review summarises the development of ATP-competitive inhibitors of GyrB and/or ParE(GrlB) as novel antibacterial agents over the last 10 years. Structural features of the new inhibitors and their optimisation strategies are highlighted. EXPERT OPINION: The development of novel ATP-competitive inhibitors of GyrB and/or ParE(GrlB) is ongoing in industrial and academical research. Development of resistance is one of the most problematic issues, but GyrB/ParE(GrlB) inhibitors do not show cross-resistance with fluoroquinolones. Other common issues, such as low solubility, high protein binding, development of off-target resistance, are related to the structures of the inhibitors themselves, which is thus a main focus of design strategies. With some now in early clinical development, there is reasonable expectation that novel ATP-competitive inhibitors of GyrB/ParE(GrlB) will reach the market in the near future.

Design, Synthesis, and Biological Evaluation of Novel DNA Gyrase-Inhibiting Spiropyrimidinetriones as Potent Antibiotics for Treatment of Infections Caused by Multidrug-Resistant Gram-Positive Bacteria.

Spiropyrimidinetriones are a novel class of antibacterial agents that target the bacterial type II topoisomerase via a new mode of action. Compound ETX0914 is thus far the only drug from this class that is being evaluated in clinical trials. To improve the antibacterial activity and pharmacokinetic properties of ETX0914, we carried out systematic structural modification of this compound, and a number of compounds with increased potency were obtained. The most promising compound 33e, with incorporation of a spirocyclopropane at the oxazolidinone 5 position reduced metabolism, exhibited excellent antibacterial activity against Gram-positive pathogens and a good pharmacokinetic profile combined with high aqueous solubility. In addition, compound 33e exhibited good selectivity for Staphylococcus aureus gyrase over human Topo IIα. In a murine model of systemic methicillin-resistant S. aureus infection, 33e exhibited superior in vivo efficacy (ED50 = 3.87 mg/kg) compared to ETX0914 (ED50 = 11.51 mg/kg).

Synthesis and Biological Evaluation of Novel Indole-Derived Thioureas.

A series of 2-(1H-indol-3-yl)ethylthiourea derivatives were prepared by condensation of 2-(1H-indol-3-yl)ethanamine with appropriate aryl/alkylisothiocyanates in anhydrous media. The structures of the newly synthesized compounds were confirmed by spectroscopic analysis and the molecular structures of 8 and 28 were confirmed by X-ray crystallography. All obtained compounds were tested for antimicrobial activity against Gram-positive cocci, Gram-negative rods and for antifungal activity. Microbiological evaluation was carried out over 20 standard strains and 30 hospital strains. Compound 6 showed significant inhibition against Gram-positive cocci and had inhibitory effect on the S. aureus topoisomerase IV decatenation activity and S. aureus DNA gyrase supercoiling activity. Compounds were tested for cytotoxicity and antiviral activity against a large panel of DNA and RNA viruses, including HIV-1 and other several important human pathogens. Interestingly, derivative 8 showed potent activity against HIV-1 wild type and variants bearing clinically relevant mutations. Newly synthesized tryptamine derivatives showed also a wide spectrum activity, proving to be active against positive- and negative-sense RNA viruses.

Genome Editing Reveals Novel Thiotemplated Assembly of Polythioamide Antibiotics in Anaerobic Bacteria.

Closthioamide (CTA) is a unique symmetric nonribosomal peptide with six thioamide moieties that is produced by the Gram-positive obligate anaerobe Ruminiclostridium cellulolyticum. CTA displays potent inhibitory activity against important clinical pathogens, making it a promising drug candidate. Yet, the biosynthesis of this DNA gyrase-targeting antibiotic has remained enigmatic. Using a combination of genome mining, genome editing (targeted group II intron, CRISPR/Cas9), and heterologous expression, we show that CTA biosynthesis involves specialized enzymes for starter unit biosynthesis, amide bond formation, thionation, and dimerization. Surprisingly, CTA biosynthesis involves a novel thiotemplated peptide assembly line that markedly differs from known nonribosomal peptide synthetases. These findings provide the first insights into the biosynthesis of thioamide-containing nonribosomal peptides and offer a starting point for the discovery of related natural products.

Antimicrobial Resistance in Mycoplasma spp.

Mycoplasmas are intrinsically resistant to antimicrobials targeting the cell wall (fosfomycin, glycopeptides, or ß-lactam antibiotics) and to sulfonamides, first-generation quinolones, trimethoprim, polymixins, and rifampicin. The antibiotics most frequently used to control mycoplasmal infections in animals are macrolides and tetracyclines. Lincosamides, fluoroquinolones, pleuromutilins, phenicols, and aminoglycosides can also be active. Standardization of methods used for determination of susceptibility levels is difficult since no quality control strains are available and because of species-specific growth requirements. Reduced susceptibility levels or resistances to several families of antimicrobials have been reported in field isolates of pathogenic Mycoplasma species of major veterinary interest: M. gallisepticum and M. synoviae in poultry; M. hyopneumoniae, M. hyorhinis, and M. hyosynoviae in swine; M. bovis in cattle; and M. agalactiae in small ruminants. The highest resistances are observed for macrolides, followed by tetracyclines. Most strains remain susceptible to fluoroquinolones. Pleuromutilins are the most effective antibiotics in vitro. Resistance frequencies vary according to the Mycoplasma species but also according to the countries or groups of animals from which the samples were taken. Point mutations in the target genes of different antimicrobials have been identified in resistant field isolates, in vitro-selected mutants, or strains reisolated after an experimental infection followed by one or several treatments: DNA-gyrase and topoisomerase IV for fluoroquinolones; 23S rRNA for macrolides, lincosamides, pleuromutilins, and amphenicols; 16S rRNAs for tetracyclines and aminoglycosides. Further work should be carried out to determine and harmonize specific breakpoints for animal mycoplasmas so that in vitro information can be used to provide advice on selection of in vivo treatments.