BACKGROUND: The current standard treatment for Helicobacter pylori infection, which involves a combination of two broad-spectrum antibiotics, faces significant challenges due to its detrimental impact on the gut microbiota and the emergence of drug-resistant strains. This underscores the urgent requirement for the development of novel anti-H. pylori drugs. Zoliflodacin, a novel bacterial gyrase inhibitor, is currently undergoing global phase III clinical trials for treating uncomplicated Neisseria gonorrhoeae. However, there is no available data regarding its activity against H. pylori.
METHODS: We evaluated the in vitro activity of zoliflodacin against H. pylori clinical isolates (n = 123) with diverse multidrug resistance. We performed DNA gyrase supercoiling and microscale thermophoresis assays to identify the target of zoliflodacin in H. pylori. We analyzed 2262 H. pylori whole genome sequences to identify Asp424Asn and Lys445Asn mutations in DNA gyrase subunit B (GyrB) that are associated with zoliflodacin resistance.
RESULTS: Zoliflodacin exhibits potent activity against all tested isolates, with minimal inhibitory concentration (MIC) values ranging from 0.008 to 1 μg/mL (MIC50: 0.125 μg/mL; MIC90: 0.25 μg/mL). Importantly, there was no evidence of cross-resistance to any of the four first-line antibiotics commonly used against H. pylori. We identified GyrB as the primary target of zoliflodacin, with Asp424Asn or Lys445Asn substitutions conferring resistance. Screening of 2262 available H. pylori genomes for the two mutations revealed only one clinical isolate carrying Asp424Asn substitution.
CONCLUSIONS: These findings support the potential of zoliflodacin as a promising candidate for H. pylori treatment, warranting further development and evaluation.

BACKGROUND: It is well known that antibiotic resistance is a major health hazard. To eradicate antibiotic-resistant bacterial infections, it is essential to find a novel antibacterial agent. Hence, in this study, a quantitative structure-activity relationship (QSAR) model was developed using 43 DNA gyrase inhibitors, and 700 natural compounds were screened for their antibacterial properties. Based on molecular docking and absorption, distribution, metabolism, excretion, and toxicity (ADMET) studies, the top three leads viz., apigenin-4\'-glucoside, 8-deoxygartanin, and cryptodorine were selected and structurally optimized using density functional theory (DFT) studies. The optimized structures were redocked, and molecular dynamic (MD) simulations were performed. Binding energies were calculated by molecular mechanics/Poisson-Boltzmann surface area solvation (MM-PBSA). Based on the above studies, apigenin-4\'-glucoside was identified as a potent antibacterial lead. Further in vitro confirmation studies were performed using the plant Lawsonia inermis containing apigenin-4\'-glucoside to confirm the antibacterial activity.
METHODS: For QSAR modeling, 2D descriptors were calculated by PaDEL-Descriptors v2.21 software, and the model was developed using the DTClab QSAR tool. Docking was performed using PyRx v0.8 software. ORCA v5.0.1 computational package was used to optimize the structures. The job type used in optimization was equilibrium structure search using the DFT hybrid functional ORCA method B3LYP. The basis set was 6-311G (3df, 3pd) plus four polarization functions for all atoms. Accurate docking was performed for optimized leads using the iGEMDOCK v2.1 tool with a genetic algorithm by 10 solutions each of 80 generations. Molecular dynamic simulations were performed using GROMACS 2020.04 software with CHARMM36 all-atom force field.

Cells have evolved strategies to safeguard their genome integrity. We describe a mechanism to counter double strand breaks in the chromosome that involves the protection of an essential housekeeping enzyme from external agents. YacG is a DNA gyrase inhibitory protein from Escherichia coli that protects the bacterium from the cytotoxic effects of catalytic inhibitors as well as cleavage-complex stabilizers of DNA gyrase. By virtue of blocking the primary DNA binding site of the enzyme, YacG prevents the accumulation of double strand breaks induced by gyrase poisons. It also enables the bacterium to resist the growth-inhibitory property of novobiocin. Gyrase poison-induced oxidative stress upregulates YacG production, probably as a cellular response to counter DNA damage. YacG-mediated protection of the genome is specific for gyrase targeting agents as the protection is not observed from the action of general DNA damaging agents. YacG also intensifies the transcription stress induced by rifampicin substantiating the importance of gyrase activity during transcription. Although essential for bacterial survival, DNA gyrase often gets entrapped by external inhibitors and poisons, resulting in cell death. The existence of YacG to specifically protect an essential housekeeping enzyme might be a strategy adopted by bacteria for competitive fitness advantage.

Toxin-antitoxin (TA) systems consist of a toxin inhibiting essential cellular functions (such as DNA, RNA and protein synthesis), and its cognate antitoxin neutralizing the toxicity. Recently, we identified a TA system termed TsbA/TsbT in the Staphylococcus aureus genome. The induction of the tsbT gene in Escherichia coli halted both DNA and RNA synthesis, reduced supercoiled plasmid and resulted in increasingly relaxed DNA. These results suggested that DNA gyrase was the target of TsbT. In addition, TsbT inhibited both E. coli and S. aureus DNA gyrase activity and induced linearization of plasmid DNA in vitro. Taken together, these results demonstrate that the TsbT toxin targets DNA gyrase in vivo. Site-directed mutagenesis experiments showed that the E27 and D37 residues in TsbT are critical for toxicity. Secondary structure prediction combining the analysis of vacuum-ultraviolet circular-dichroism spectroscopy and neural network method demonstrated that the 22nd-32nd residues of TsbT form an α-helix structure, and that the E27 residue is located around the centre of the α-helix segment. These findings give new insights not only into S. aureus TA systems, but also into bacterial toxins targeting DNA topoisomerases.

DNA gyrase and topoisomerase IV are proven to be validated targets in the design of novel antibacterial drugs. In this study, we report the antibacterial evaluation and molecular docking studies of previously synthesized two series of cyclic diphenylphosphonates (1a-e and 2a-e) as DNA gyrase inhibitors. The synthesized compounds were screened for their activity (antibacterial and DNA gyrase inhibition) against ciprofloxacin-resistant E.coli and Klebsiella pneumoniae clinical isolates having mutations (deletion and substitution) in QRDR region of DNA gyrase. The target compound (2a) that exhibited the most potent activity against ciprofloxacin Gram-negative clinical isolates was selected to screen its inhibitory activity against DNA gyrase displayed IC50 of 12.03 µM. In addition, a docking study was performed with inhibitor (2a), to illustrate its binding mode in the active site of DNA gyrase and the results were compatible with the observed inhibitory potency. Furthermore, the docking study revealed that the binding of inhibitor (2a) to DNA gyrase is mediated and modulated by divalent Mg2+ at good binding energy (-9.08 Kcal/mol). Moreover, structure-activity relationships (SARs) demonstrated that the combination of hydrazinyl moiety in conjunction with the cyclic diphenylphosphonate based scaffold resulted in an optimized molecule that inhibited the bacterial DNA gyrase by its detectable effect in vitro on gyrase-catalyzed DNA supercoiling activity.

SPR720 (phosphate prodrug of SPR719) is a novel aminobenzimidazole bacterial DNA gyrase (GyrB) inhibitor in development for nontuberculous mycobacterial pulmonary disease (NTM-PD) and pulmonary tuberculosis. SPR719 has demonstrated activity against clinically relevant mycobacteria in vitro and in murine and hollow-fiber infection models. This phase 1 randomized, double-blind, placebo-controlled, single ascending dose (SAD)/multiple ascending dose (MAD) trial evaluated the safety, tolerability, and pharmacokinetics of SPR720/SPR719. A total of 96 healthy volunteers (n = 8/cohort, 3:1 randomization) received SPR720 (or placebo) as single oral doses ranging from 100 to 2,000 mg or repeat total daily doses ranging from 500 to 1,500 mg for 7 or 14 days. SPR720 was well tolerated at daily doses of up to 1,000 mg for up to 14 days. Across SAD/MAD cohorts, the most common adverse events (AEs) were gastrointestinal (nausea, vomiting, and diarrhea) and headache, all of mild or moderate severity and dose dependent. No serious AEs were reported. The median SPR719 Tmax ranged from 2.8 to 8.0 h across cohorts, and the t1/2 ranged from 2.9 to 4.5 h and was shown to be dose independent. Dosing with food decreased SPR719 plasma exposure by approximately 20%. In the MAD cohorts, SPR719 plasma exposure declined approximately 40% between days 1 and 7, suggesting induction of an elimination pathway. However, plasma AUC0-24 was comparable between days 7 and 14. The results of this first-in-human study suggest that predicted therapeutic exposures of SPR719 can be attained with a once-daily oral administration of SPR720. (This study has been registered at ClinicalTrials.gov under registration no. NCT03796910.).

The design and synthesis of novel multi-substituted benzo-indole pyrazole Schiff base derivatives of potent DNA gyrase inhibitory activity were the main aims of this study. All the novel synthesized compounds were examined for their antibacterial activities against Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, and Salmonella. In addition, we selected 20 compounds for the in vitro antibacterial activities assay of 6 drug-resistant bacteria strains. The result revealed compound 8I-w exhibited excellent antibacterial activity against 4 drug-resistant E. coli bacteria strains with IC50 values of 7.0, 17.0, 13.5, and 1.0 μM, respectively. In vitro enzyme inhibitory assay showed that compound 8I-w displayed potent inhibition against DNA gyrase with IC50 values of 0.10 μM. The molecular docking model indicated that compounds 8I-w can bind well to the DNA gyrase by interacting with various amino acid residues. This study demonstrated that the compound 8I-w can act as the most potent DNA gyrase inhibitor in the reported series of compounds and provide valuable information for the commercial DNA gyrase inhibiting bactericides.

The aim of this study was to screen lead compounds exhibiting potent in vitro antimicrobial activity against multidrug-resistant (MDR) Acinetobacter baumannii strains from a library of chemical compounds. In a high-throughput screening analysis of 7520 compounds representative of 340,000 small molecules, two 4H-4-oxoquinolizine compounds were the most active against A. baumannii ATCC 17978. Subsequent selection and analysis of 70 4H-4-oxoquinolizine compounds revealed that the top 7 compounds were extremely active against extensively drug-resistant (XDR) A. baumannii isolates. These compounds commonly carried a 1-cyclopropyl-7-fluoro-4-oxo-4H-quinolizine-3-carboxylic acid core structure but had different C-8 and/or C-9 moieties. Minimum inhibitory concentrations (MICs) of the seven compounds against fluoroquinolone-resistant A. baumannii isolates were found to be in the range of 0.02-1.70 µg/mL regardless of the mutation types in the quinolone resistance-determining region (QRDR) of GyrA and ParC. Cytotoxicity of the seven compounds was observed in HeLa and U937 cells at a concentration of 50 µg/mL, which was >32.5- to 119-fold higher than the MIC90 for A. baumannii isolates. In conclusion, novel 4H-4-oxoquinolizine compounds represent a promising scaffold on which to develop antimicrobial agents against drug-resistant A. baumannii strains.

Owing to essential role in bacterial survival, DNA gyrase has been exploited as a validated drug target. However, rapidly emerging resistance to gyrase-targeted drugs such as widely utilized fluoroquinolones reveals the necessity to develop novel compounds with new mechanism of actions against this enzyme. Here, an attempt has been made to identify new drug-like molecules for Shigella flexneri DNA gyrase inhibition through in silico approaches. The structural similarity search was carried out using the natural product simocyclinone D8, a unique gyrase inhibitor, to virtually screen ZINC database. A total of 11830 retrieved hits were further screened for selection of high-affinity compounds by implementing molecular docking followed by investigation of druggability according to Lipinski\'s rule, biological activity and physiochemical properties. Among the hits initially identified, three molecules were then confirmed to have reasonable gyrase-binding affinity and to follow Lipinski\'s rule. Based on these in silico findings, three compounds with different chemical structures from previously identified gyrase inhibitors were proposed as potential candidates for the treatment of fluoroquinolone-resistant strains and deserve further investigations.

Mycobacterium tuberculosis H37Rv escapes host-generated stresses by entering a dormant persistent state. Activation of toxin-antitoxin modules is one of the mechanisms known to trigger such a state with low metabolic activity. M. tuberculosis harbors a large number of TA systems mostly located within discernible genomic islands. We have investigated the parDE2 operon of M. tuberculosis H37Rv encoding MParE2 toxin and MParD2 antitoxin proteins. The parDE2 locus was transcriptionally active from growth phase till late stationary phase in M. tuberculosis. A functional promoter located upstream of parD2 GTG start-site was identified by 5\'-RACE and lacZ reporter assay. The MParD2 protein transcriptionally regulated the P parDE2 promoter by interacting through Arg16 and Ser15 residues located in the N-terminus. In Escherichia coli, ectopic expression of MParE2 inhibited growth in early stages, with a drastic reduction in colony forming units. Live-dead analysis revealed that the reduction was not due to cell death alone but due to formation of viable but non-culturable cells (VBNCs) also. The toxic activity of the protein, identified in the C-terminal residues Glu98 and Arg102, was neutralized by the antitoxin MParD2, both in vivo and in vitro. MParE2 inhibited mycobacterial DNA gyrase and interacted with the GyrB subunit without affecting its ATPase activity. Introduction of parE2 gene in the heterologous M. smegmatis host prevented growth and colony formation by the transformed cells. An M. smegmatis strain containing the parDE2 operon also switched to a non-culturable phenotype in response to oxidative stress. Loss in colony-forming ability of a major part of the MParE2 expressing cells suggests its potential role in dormancy, a cellular strategy for adaptation to environmental stresses. Our study has laid the foundation for future investigations to explore the physiological significance of parDE2 operon in mycobacterial pathogenesis.