Gyrase and Topoisomerase IV: Recycling Old Targets for New Antibacterials to Combat Fluoroquinolone Resistance.

Beyond their requisite functions in many critical DNA processes, the bacterial type II topoisomerases, gyrase and topoisomerase IV, are the targets of fluoroquinolone antibacterials. These drugs act by stabilizing gyrase/topoisomerase IV-generated DNA strand breaks and by robbing the cell of the catalytic activities of these essential enzymes. Since their clinical approval in the mid-1980s, fluoroquinolones have been used to treat a broad spectrum of infectious diseases and are listed among the five "highest priority" critically important antimicrobial classes by the World Health Organization. Unfortunately, the widespread use of fluoroquinolones has been accompanied by a rise in target-mediated resistance caused by specific mutations in gyrase and topoisomerase IV, which has curtailed the medical efficacy of this drug class. As a result, efforts are underway to identify novel antibacterials that target the bacterial type II topoisomerases. Several new classes of gyrase/topoisomerase IV-targeted antibacterials have emerged, including novel bacterial topoisomerase inhibitors, Mycobacterium tuberculosis gyrase inhibitors, triazaacenaphthylenes, spiropyrimidinetriones, and thiophenes. Phase III clinical trials that utilized two members of these classes, gepotidacin (triazaacenaphthylene) and zoliflodacin (spiropyrimidinetrione), have been completed with positive outcomes, underscoring the potential of these compounds to become the first new classes of antibacterials introduced into the clinic in decades. Because gyrase and topoisomerase IV are validated targets for established and emerging antibacterials, this review will describe the catalytic mechanism and cellular activities of the bacterial type II topoisomerases, their interactions with fluoroquinolones, the mechanism of target-mediated fluoroquinolone resistance, and the actions of novel antibacterials against wild-type and fluoroquinolone-resistant gyrase and topoisomerase IV.

Interactions between Gepotidacin and Escherichia coli Gyrase and Topoisomerase IV: Genetic and Biochemical Evidence for Well-Balanced Dual-Targeting.

Antimicrobial resistance is a global threat to human health. Therefore, efforts have been made to develop new antibacterial agents that address this critical medical issue. Gepotidacin is a novel, bactericidal, first-in-class triazaacenaphthylene antibacterial in clinical development. Recently, phase III clinical trials for gepotidacin treatment of uncomplicated urinary tract infections caused by uropathogens, including Escherichia coli, were stopped for demonstrated efficacy. Because of the clinical promise of gepotidacin, it is important to understand how the compound interacts with its cellular targets, gyrase and topoisomerase IV, from E. coli. Consequently, we determined how gyrase and topoisomerase IV mutations in amino acid residues that are involved in gepotidacin interactions affect the susceptibility of E. coli cells to the compound and characterized the effects of gepotidacin on the activities of purified wild-type and mutant gyrase and topoisomerase IV. Gepotidacin displayed well-balanced dual-targeting of gyrase and topoisomerase IV in E. coli cells, which was reflected in a similar inhibition of the catalytic activities of these enzymes by the compound. Gepotidacin induced gyrase/topoisomerase IV-mediated single-stranded, but not double-stranded, DNA breaks. Mutations in GyrA and ParC amino acid residues that interact with gepotidacin altered the activity of the compound against the enzymes and, when present in both gyrase and topoisomerase IV, reduced the antibacterial activity of gepotidacin against this mutant strain. Our studies provide insights regarding the well-balanced dual-targeting of gyrase and topoisomerase IV by gepotidacin in E. coli.

Target-Mediated Fluoroquinolone Resistance in Neisseria gonorrhoeae: Actions of Ciprofloxacin against Gyrase and Topoisomerase IV.

Fluoroquinolones make up a critically important class of antibacterials administered worldwide to treat human infections. However, their clinical utility has been curtailed by target-mediated resistance, which is caused by mutations in the fluoroquinolone targets, gyrase and topoisomerase IV. An important pathogen that has been affected by this resistance is Neisseria gonorrhoeae, the causative agent of gonorrhea. Over 82 million new cases of this sexually transmitted infection were reported globally in 2020. Despite the impact of fluoroquinolone resistance on gonorrhea treatment, little is known about the interactions of this drug class with its targets in this bacterium. Therefore, we investigated the effects of the fluoroquinolone ciprofloxacin on the catalytic and DNA cleavage activities of wild-type gyrase and topoisomerase IV and the corresponding enzymes that harbor mutations associated with cellular and clinical resistance to fluoroquinolones. Results indicate that ciprofloxacin interacts with both gyrase (its primary target) and topoisomerase IV (its secondary target) through a water-metal ion bridge that has been described in other species. Moreover, mutations in amino acid residues that anchor this bridge diminish the susceptibility of the enzymes for the drug, leading to fluoroquinolone resistance. Results further suggest that ciprofloxacin primarily induces its cytotoxic effects by enhancing gyrase-mediated DNA cleavage as opposed to inhibiting the DNA supercoiling activity of the enzyme. In conclusion, this work links the effects of ciprofloxacin on wild-type and resistant gyrase to results reported for cellular and clinical studies and provides a mechanistic explanation for the targeting and resistance of fluoroquinolones in N. gonorrhoeae.

Discovery of benzopyridone cyanoacetates as new type of potential broad-spectrum antibacterial candidates.

Unique benzopyridone cyanoacetates (BCs) as new type of promising broad-spectrum antibacterial candidates were discovered with large potential to combat the lethal multidrug-resistant bacterial infections. Many prepared BCs showed broad antibacterial spectrum with low MIC values against the tested strains. Some highly active BCs exhibited rapid sterilization capacity, low resistant trend and good predictive pharmacokinetic properties. Furthermore, the highly active sodium BCs (NaBCs) displayed low hemolysis and cytotoxicity, and especially octyl NaBC 5g also showed in vivo potent anti-infective potential and appreciable pharmacokinetic profiles. A series of preliminary mechanistic explorations indicated that these active BCs could effectively eliminate bacterial biofilm and destroy membrane integrity, thus resulting in the leakage of bacterial cytoplasm. Moreover, their unique structures might further bind to intracellular DNA, DNA gyrase and topoisomerase IV through various direct noncovalent interactions to hinder bacterial reproduction. Meanwhile, the active BCs also induced bacterial oxidative stress and metabolic disturbance, thereby accelerating bacterial apoptosis. These results provided a bright hope for benzopyridone cyanoacetates as potential novel multitargeting broad-spectrum antibacterial candidates to conquer drug resistance.

Novel bacterial topoisomerase inhibitors: unique targeting activities of amide enzyme-binding motifs for tricyclic analogs.

Antimicrobial resistance has made a sizeable impact on public health and continues to threaten the effectiveness of antibacterial therapies. Novel bacterial topoisomerase inhibitors (NBTIs) are a promising class of antibacterial agents with a unique binding mode and distinct pharmacology that enables them to evade existing resistance mechanisms. The clinical development of NBTIs has been plagued by several issues, including cardiovascular safety. Herein, we report a sub-series of tricyclic NBTIs bearing an amide linkage that displays promising antibacterial activity, potent dual-target inhibition of DNA gyrase and topoisomerase IV (TopoIV), as well as improved cardiovascular safety and metabolic profiles. These amide NBTIs induced both single- and double-strand breaks in pBR322 DNA mediated by Staphylococcus aureus DNA gyrase, in contrast to prototypical NBTIs that cause only single-strand breaks. Unexpectedly, amides 1a and 1b targeted human topoisomerase IIα (TOP2α) causing both single- and double-strand breaks in pBR322 DNA, and induced DNA strand breaks in intact human leukemia K562 cells. In addition, anticancer drug-resistant K/VP.5 cells containing decreased levels of TOP2α were cross-resistant to amides 1a and 1b. Together, these results demonstrate broad spectrum antibacterial properties of selected tricyclic NBTIs, desirable safety profiles, an unusual ability to induce DNA double-stranded breaks, and activity against human TOP2α. Future work will be directed toward optimization and development of tricyclic NBTIs with potent and selective activity against bacteria. Finally, the current results may provide an additional avenue for development of selective anticancer agents.

Actions of a Novel Bacterial Topoisomerase Inhibitor against Neisseria gonorrhoeae Gyrase and Topoisomerase IV: Enhancement of Double-Stranded DNA Breaks.

Novel bacterial topoisomerase inhibitors (NBTIs) are an emerging class of antibacterials that target gyrase and topoisomerase IV. A hallmark of NBTIs is their ability to induce gyrase/topoisomerase IV-mediated single-stranded DNA breaks and suppress the generation of double-stranded breaks. However, a previous study reported that some dioxane-linked amide NBTIs induced double-stranded DNA breaks mediated by Staphylococcus aureus gyrase. To further explore the ability of this NBTI subclass to increase double-stranded DNA breaks, we examined the effects of OSUAB-185 on DNA cleavage mediated by Neisseria gonorrhoeae gyrase and topoisomerase IV. OSUAB-185 induced single-stranded and suppressed double-stranded DNA breaks mediated by N. gonorrhoeae gyrase. However, the compound stabilized both single- and double-stranded DNA breaks mediated by topoisomerase IV. The induction of double-stranded breaks does not appear to correlate with the binding of a second OSUAB-185 molecule and extends to fluoroquinolone-resistant N. gonorrhoeae topoisomerase IV, as well as type II enzymes from other bacteria and humans. The double-stranded DNA cleavage activity of OSUAB-185 and other dioxane-linked NBTIs represents a paradigm shift in a hallmark characteristic of NBTIs and suggests that some members of this subclass may have alternative binding motifs in the cleavage complex.

Novel fluorobenzothiazole as a dual inhibitor of gyrase B and topoisomerase IV against Gram-positive pathogens.

Aim: The development of a novel inhibitor targeting gyrase B and topoisomerase IV offers an opportunity to combat multidrug resistance. Methods: We investigated the activity of RBx 10080758 against Gram-positive bacteria in vitro and in vivo. Results: RBx 10080758 showed a potent 50% inhibitory concentration of 0.13 µM and 0.25 µM against gyrase B and topoisomerase IV, respectively, and exhibited strong whole-cell in vitro activity with MIC ranges of 0.015-0.06 and 0.015-0.03 µg/ml against Staphylococcus aureus and Streptococcus pneumoniae, respectively. In a rat thigh infection model with methicillin-resistant S. aureus, RBx 10080758 at 45 mg/kg exhibited a >3 log10 CFU reduction in thigh muscles. Conclusion: RBx 10080758 displayed potent activity against multiple multidrug-resistant Gram-positive bacteria with a dual-targeting mechanism of action.

Development of antisense peptide-peptide nucleic acids against fluoroquinolone-resistant Escherichia coli.

BACKGROUND: Fluoroquinolones (FQs) are potent and broad-spectrum antibiotics commonly used to treat MDR bacterial infections, but bacterial resistance to FQs has emerged and spread rapidly around the world. The mechanisms for FQ resistance have been revealed, including one or more mutations in FQ target genes such as DNA gyrase (gyrA) and topoisomerase IV (parC). Because therapeutic treatments for FQ-resistant bacterial infections are limited, it is necessary to develop novel antibiotic alternatives to minimize or inhibit FQ-resistant bacteria. OBJECTIVES: To examine the bactericidal effect of antisense peptide-peptide nucleic acids (P-PNAs) that can block the expression of DNA gyrase or topoisomerase IV in FQ-resistant Escherichia coli (FRE). METHODS: A set of antisense P-PNA conjugates with a bacterial penetration peptide were designed to inhibit the expression of gyrA and parC and were evaluated for their antibacterial activities. RESULTS: Antisense P-PNAs, ASP-gyrA1 and ASP-parC1, targeting the translational initiation sites of their respective target genes significantly inhibited the growth of the FRE isolates. In addition, ASP-gyrA3 and ASP-parC2, which bind to the FRE-specific coding sequence within the gyrA and parC structural genes, respectively, showed selective bactericidal effects against FRE isolates. CONCLUSIONS: Our results demonstrate the potential of targeted antisense P-PNAs as antibiotic alternatives against FQ-resistance bacteria.

Effectiveness of sitafloxacin monotherapy for quinolone-resistant rectal and urogenital Mycoplasma genitalium infections: a prospective cohort study.

BACKGROUND: Mycoplasma genitalium has a tendency to develop macrolide and quinolone resistance. OBJECTIVES: We investigated the microbiological cure rate of a 7 day course of sitafloxacin for the treatment of rectal and urogenital infections in MSM. PATIENTS AND METHODS: This open-label, prospective cohort study was conducted at the National Center for Global Health and Medicine, Tokyo, Japan from January 2019 to August 2022. Patients with M. genitalium urogenital or rectal infections were included. The patients were treated with sitafloxacin 200 mg daily for 7 days. M. genitalium isolates were tested for parC, gyrA and 23S rRNA resistance-associated mutations. RESULTS: In total, 180 patients (median age, 35 years) were included in this study, of whom 77.0% (97/126) harboured parC mutations, including 71.4% (90/126) with G248T(S83I) in parC, and 22.5% (27/120) harboured gyrA mutations. The median time to test of cure was 21 days. The overall microbiological cure rate was 87.8%. The cure rate was 100% for microbes harbouring parC and gyrA WTs, 92.9% for microbes harbouring parC G248T(S83I) and gyrA WT, and 41.7% for microbes harbouring parC G248T(S83I) and gyrA with mutations. The cure rate did not differ significantly between urogenital and rectal infection (P = 0.359). CONCLUSIONS: Sitafloxacin monotherapy was highly effective against infection caused by M. genitalium, except strains with combined parC and gyrA mutations. Sitafloxacin monotherapy can be used as a first-line treatment for M. genitalium infections in settings with a high prevalence of parC mutations and a low prevalence of gyrA mutations.

A CcdB toxin-derived peptide acts as a broad-spectrum antibacterial therapeutic in infected mice.

The bacterial toxin CcdB (Controller of Cell death or division B) targets DNA Gyrase, an essential bacterial topoisomerase, which is also the molecular target for fluoroquinolones. Here, we present a short cell-penetrating 24-mer peptide, CP1-WT, derived from the Gyrase-binding region of CcdB and examine its effect on growth of Escherichia coli, Salmonella Typhimurium, Staphylococcus aureus and a carbapenem- and tigecycline-resistant strain of Acinetobacter baumannii in both axenic cultures and mouse models of infection. The CP1-WT peptide shows significant improvement over ciprofloxacin in terms of its in vivo therapeutic efficacy in treating established infections of S. Typhimurium, S. aureus and A. baumannii. The molecular mechanism likely involves inhibition of Gyrase or Topoisomerase IV, depending on the strain used. The study validates the CcdB binding site on bacterial DNA Gyrase as a viable and alternative target to the fluoroquinolone binding site.

The Formation and Drug Resistance Mechanism of Biofilm for Streptococcus pneumoniae Infection in Severe Respiratory Patients.

This study was to explore whether Streptococcus pneumoniae would form biofilms and the formative factors of biofilms, as well as the drug resistance mechanism of S. pneumoniae. In this study, a total of 150 strains of S. pneumoniae were collected from 5 local hospitals in the past two years, and the minimum inhibitory concentrations (MIC) of levofloxacin, moxifloxacin and penicillin were determined by agar double dilution method to select the drug-resistant strains. The polymerase chain reaction (PCR) amplification and sequencing were performed on specific genes of drug-resistant strains. In addition, 5 strains of S. pneumoniae with penicillin MIC ≤ 0.065 µg/mL, 0.5 µg/mL, 2 µg/mL, ≥ 4µg/mL were randomly selected, and the biofilms were cultured on two kinds of well plates for 24 hours. Finally, whether the biofilms were formed was observed. Experimental results revealed that the resistance rate of S. pneumoniae to erythromycin in this area was as high as 90.3%, and the strains that were resistant to penicillin account for only 1.5%. The amplification and sequencing experiment revealed that one (strain 1) of the strains, which was resistant to both drugs, had a GyrA mutation and ParE mutation, and strain 2 had a parC mutation. All strains generated biofilms, and the optical density (OD) value of penicillin MIC ≤ 0.065 µg/mL group (0.235 ± 0.053) was higher than that of 0.5 µg/mL group (0.192 ± 0.073) (P< 0.05) and higher than the OD value of the 4 µg/mL group (0.200 ± 0.041) (P< 0.05), showing statistically great differences. It was confirmed that the resistance rate of S. pneumoniae to erythromycin remained high, the rate of sensitivity to penicillin was relatively high, and the moxifloxacin and levofloxacin-resistant strains had appeared; S. pneumoniae mainly showed QRDR mutations in gyrA, parE, and parC; and it was confirmed that S. pneumoniae can generate biofilms in vitro.

Contribution of amino acid substitutions in ParE to quinolone resistance in Haemophilus haemolyticus revealed through a horizontal transfer assay using Haemophilus influenzae.

BACKGROUND: In 2019, a high-level quinolone-resistant Haemophilus haemolyticus strain (levofloxacin MIC = 16 mg/L) was isolated from a paediatric patient. In this study, we aimed to determine whether the quinolone resistance of H. haemolyticus could be transferred to Haemophilus influenzae and to identify the mechanism underlying the high-level quinolone resistance of H. haemolyticus. METHODS: A horizontal gene transfer assay to H. influenzae was performed using genomic DNA or PCR-amplified quinolone-targeting genes from the high-level quinolone-resistant H. haemolyticus 2019-19 strain. The amino acids responsible for conferring quinolone resistance were identified through site-directed mutagenesis. RESULTS: By adding the genomic DNA of H. haemolyticus 2019-19, resistant colonies were obtained on agar plates containing quinolones. Notably, H. influenzae grown on levofloxacin agar showed the same level of resistance as H. haemolyticus. Sequencing analysis showed that gyrA, parC and parE of H. influenzae were replaced by those of H. haemolyticus, suggesting that horizontal transfer occurred between the two strains. When the quinolone-targeting gene fragments were added sequentially, the addition of parE, as well as gyrA and parC, contributed to high-level resistance. In particular, amino acid substitutions at both the 439th and 502nd residues of ParE were associated with high-level resistance. CONCLUSIONS: These findings indicate that quinolone resistance can be transferred between species and that amino acid substitutions at the 439th and 502nd residues of ParE, in addition to amino acid substitutions in both GyrA and ParC, contribute to high-level quinolone resistance.

In silico evaluation of usnic acid derivatives to discover potential antibacterial drugs against DNA gyrase B and DNA topoisomerase IV.

Due to the rising increase in infectious diseases brought on by bacteria and anti-bacterial drug resistance, antibacterial therapy has become difficult. The majority of first-line antibiotics are no longer effective against numerous germs, posing a new hazard to global human health in the 21st century. Through the drug-likeness screening, 184 usnic acid derivatives were selected from an in-house database of 340 usnic acid compounds. The pharmacokinetics (ADMET) prediction produced fifteen hit compounds, of which the lead molecule was subsequently obtained through a molecular docking investigation. The lead compounds, labelled compound-277 and compound-276, respectively, with the substantial binding affinity towards the enzymes were obtained through further docking simulation on the DNA gyrase and DNA topoisomerase proteins. Additionally, molecular dynamic (MD) simulation was performed for 300 ns on the lead compounds in order to confirm the stability of the docked complexes and the binding pose discovered during docking tests. Due to their intriguing pharmacological characteristics, these substances may be promising therapeutic candidate for anti-bacterial medication.Communicated by Ramaswamy H. Sarma.

Basis for the discrimination of supercoil handedness during DNA cleavage by human and bacterial type II topoisomerases.

To perform double-stranded DNA passage, type II topoisomerases generate a covalent enzyme-cleaved DNA complex (i.e. cleavage complex). Although this complex is a requisite enzyme intermediate, it is also intrinsically dangerous to genomic stability. Consequently, cleavage complexes are the targets for several clinically relevant anticancer and antibacterial drugs. Human topoisomerase IIα and IIß and bacterial gyrase maintain higher levels of cleavage complexes with negatively supercoiled over positively supercoiled DNA substrates. Conversely, bacterial topoisomerase IV is less able to distinguish DNA supercoil handedness. Despite the importance of supercoil geometry to the activities of type II topoisomerases, the basis for supercoil handedness recognition during DNA cleavage has not been characterized. Based on the results of benchtop and rapid-quench flow kinetics experiments, the forward rate of cleavage is the determining factor of how topoisomerase IIα/IIß, gyrase and topoisomerase IV distinguish supercoil handedness in the absence or presence of anticancer/antibacterial drugs. In the presence of drugs, this ability can be enhanced by the formation of more stable cleavage complexes with negatively supercoiled DNA. Finally, rates of enzyme-mediated DNA ligation do not contribute to the recognition of DNA supercoil geometry during cleavage. Our results provide greater insight into how type II topoisomerases recognize their DNA substrates.

New Dual Inhibitors of Bacterial Topoisomerases with Broad-Spectrum Antibacterial Activity and In Vivo Efficacy against Vancomycin-Intermediate Staphylococcus aureus.

A new series of dual low nanomolar benzothiazole inhibitors of bacterial DNA gyrase and topoisomerase IV were developed. The resulting compounds show excellent broad-spectrum antibacterial activities against Gram-positive Enterococcus faecalis, Enterococcus faecium and multidrug resistant (MDR) Staphylococcus aureus strains [best compound minimal inhibitory concentrations (MICs): range, <0.03125-0.25 µg/mL] and against the Gram-negatives Acinetobacter baumannii and Klebsiella pneumoniae (best compound MICs: range, 1-4 µg/mL). Lead compound 7a was identified with favorable solubility and plasma protein binding, good metabolic stability, selectivity for bacterial topoisomerases, and no toxicity issues. The crystal structure of 7a in complex with Pseudomonas aeruginosa GyrB24 revealed its binding mode at the ATP-binding site. Expanded profiling of 7a and 7h showed potent antibacterial activity against over 100 MDR and non-MDR strains of A. baumannii and several other Gram-positive and Gram-negative strains. Ultimately, in vivo efficacy of 7a in a mouse model of vancomycin-intermediate S. aureus thigh infection was also demonstrated.

Role of the Water-Metal Ion Bridge in Quinolone Interactions with Escherichia coli Gyrase.

Fluoroquinolones are an important class of antibacterials, and rising levels of resistance threaten their clinical efficacy. Gaining a more full understanding of their mechanism of action against their target enzymes-the bacterial type II topoisomerases gyrase and topoisomerase IV-may allow us to rationally design quinolone-based drugs that overcome resistance. As a step toward this goal, we investigated whether the water-metal ion bridge that has been found to mediate the major point of interaction between Escherichia coli topoisomerase IV and Bacillus anthracis topoisomerase IV and gyrase, as well as Mycobacterium tuberculosis gyrase, exists in E. coli gyrase. This is the first investigation of the water-metal ion bridge and its function in a Gram-negative gyrase. Evidence suggests that the water-metal ion bridge does exist in quinolone interactions with this enzyme and, unlike the Gram-positive B. anthracis gyrase, does use both conserved residues (serine and acidic) as bridge anchors. Furthermore, this interaction appears to play a positioning role. These findings raise the possibility that the water-metal ion bridge is a universal point of interaction between quinolones and type II topoisomerases and that it functions primarily as a binding contact in Gram-positive species and primarily as a positioning interaction in Gram-negative species. Future studies will explore this possibility.

A 2.8 Å Structure of Zoliflodacin in a DNA Cleavage Complex with Staphylococcus aureus DNA Gyrase.

Since 2000, some thirteen quinolones and fluoroquinolones have been developed and have come to market. The quinolones, one of the most successful classes of antibacterial drugs, stabilize DNA cleavage complexes with DNA gyrase and topoisomerase IV (topo IV), the two bacterial type IIA topoisomerases. The dual targeting of gyrase and topo IV helps decrease the likelihood of resistance developing. Here, we report on a 2.8 Å X-ray crystal structure, which shows that zoliflodacin, a spiropyrimidinetrione antibiotic, binds in the same DNA cleavage site(s) as quinolones, sterically blocking DNA religation. The structure shows that zoliflodacin interacts with highly conserved residues on GyrB (and does not use the quinolone water-metal ion bridge to GyrA), suggesting it may be more difficult for bacteria to develop target mediated resistance. We show that zoliflodacin has an MIC of 4 µg/mL against Acinetobacter baumannii (A. baumannii), an improvement of four-fold over its progenitor QPT-1. The current phase III clinical trial of zoliflodacin for gonorrhea is due to be read out in 2023. Zoliflodacin, together with the unrelated novel bacterial topoisomerase inhibitor gepotidacin, is likely to become the first entirely novel chemical entities approved against Gram-negative bacteria in the 21st century. Zoliflodacin may also become the progenitor of a new safer class of antibacterial drugs against other problematic Gram-negative bacteria.

Development and radiosterilization of new hydrazono-quinoline hybrids as DNA gyrase and topoisomerase IV inhibitors: Antimicrobial and hemolytic activities against uropathogenic isolates with molecular docking study.

This study aimed to synthesize new potent quinoline derivatives based on hydrazone moieties and evaluate their antimicrobial activity. The newly synthesized hydrazono-quinoline derivatives 2, 5a, 9, and 10b showed the highest antimicrobial activity with MIC values ≤1.0 µg/ml against bacteria and ≤8.0 µg/ml against the fungi. Further, these derivatives exhibited bactericidal and fungicidal effects with MBC/MIC and MFC/MIC ratio ≤4. Surprisingly, the most active compounds displayed good inhibition to biofilm formation with MBEC values ranging between (40.0 ± 10.0 - 230.0 ± 31.0) and (67.0 ± 24.0 - 347.0 ± 15.0) µg/ml against Staphylococcus aureus and Pseudomonas aeruginosa, respectively. The hemolytic assays confirmed that the hydrazono-quinoline derivatives are non-toxic with low % lysis values ranging from 4.62% to 14.4% at a 1.0 mg/ml concentration. Besides, compound 5a exhibited the lowest hemolytic activity value of ~4.62%. Furthermore, the study suggests that the hydrazono-quinoline analogs exert their antibacterial activity as dual inhibitors for DNA gyrase and DNA topoisomerase IV enzymes with IC50 values ranging between (4.56 ± 0.3 - 21.67 ± 0.45) and (6.77 ± 0.4 - 20.41 ± 0.32) µM, respectively. Additionally, the recent work advocated that compound 5a showed the reference SAL at the É£-radiation dose of 10.0 kGy in the sterilization process without affecting its chemical structure. Finally, the in silico drug-likeness, toxicity properties, and molecular docking simulation were performed. Besides, the result exhibited good oral-bioavailability, lower toxicity prediction, and lower binding energy with good binding mode rather than the positive control.

Variable DNA topology is an epigenetic generator of physiological heterogeneity in bacterial populations.

Transcription is a noisy and stochastic process that produces sibling-to-sibling variations in physiology across a population of genetically identical cells. This pattern of diversity reflects, in part, the burst-like nature of transcription. Transcription bursting has many causes and a failure to remove the supercoils that accumulate in DNA during transcription elongation is an important contributor. Positive supercoiling of the DNA ahead of the transcription elongation complex can result in RNA polymerase stalling if this DNA topological roadblock is not removed. The relaxation of these positive supercoils is performed by the ATP-dependent type II topoisomerases DNA gyrase and topoisomerase IV. Interference with the action of these topoisomerases involving, inter alia, topoisomerase poisons, fluctuations in the [ATP]/[ADP] ratio, and/or the intervention of nucleoid-associated proteins with GapR-like or YejK-like activities, may have consequences for the smooth operation of the transcriptional machinery. Antibiotic-tolerant (but not resistant) persister cells are among the phenotypic outliers that may emerge. However, interference with type II topoisomerase activity can have much broader consequences, making it an important epigenetic driver of physiological diversity in the bacterial population.

Synthesis, Structural Characterization and Biological Activity Evaluation of Novel Cu(II) Complexes with 3-(trifluoromethyl)phenylthiourea Derivatives.

Copper complexes with 1,3-disubstituted thiourea derivatives, all containing 3-(trifluoromethyl)phenyl tail and 1-alkyl/halogen-phenyl substituent, were synthesized. The experimental spectroscopic studies and theoretical calculation revealed that two ligands coordinate to Cu(II) in a bidentate fashion via thiocarbonyl S and deprotonated N atoms of thiourea moiety. Such monomers are characteristic of alkylphenylthiourea complexes, whereas the formation of a sandwich-type dimer is observed for halogeno derivatives. For the first time, the structural identifications of CuN2S2-based complexes using experimental and theoretical X-ray absorption near edge structure are demonstrated. The dimeric halogeno derivatives showed higher antimicrobial activity in comparison with alkylphenylthiourea complexes. The Cu(II) complex of 1-(4-chloro-3-nitrophenyl)-3-[3-(trifluoromethyl)phenyl]thiourea was active against 19 strains of methicillin-resistant Staphylococci (MIC = 2 µg/mL). This derivative acted as a dual inhibitor of DNA gyrase and topoisomerase IV isolated from Staphylococcus aureus. Additionally, complexes of halogenphenylthiourea strongly inhibited the growth of mycobacteria isolated from tuberculosis patients, even fourfold stronger than the reference isoniazid. The complexes exerted weak to moderate antitumor activity (towards SW480, SW620, and PC3) being non-toxic towards normal HaCaT cells.