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.

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.

Structural basis of DNA crossover capture by Escherichia coli DNA gyrase.

DNA supercoiling must be precisely regulated by topoisomerases to prevent DNA entanglement. The interaction of type IIA DNA topoisomerases with two DNA molecules, enabling the transport of one duplex through the transient double-stranded break of the other, remains elusive owing to structures derived solely from single linear duplex DNAs lacking topological constraints. Using cryo-electron microscopy, we solved the structure of Escherichia coli DNA gyrase bound to a negatively supercoiled minicircle DNA. We show how DNA gyrase captures a DNA crossover, revealing both conserved molecular grooves that accommodate the DNA helices. Together with molecular tweezer experiments, the structure shows that the DNA crossover is of positive chirality, reconciling the binding step of gyrase-mediated DNA relaxation and supercoiling in a single structure.

TisB protein is the single molecular determinant underlying multiple downstream effects of ofloxacin in Escherichia coli.

Bactericidal antibiotics can cause metabolic perturbations that contribute to antibiotic-induced lethality. The molecular mechanism underlying these downstream effects remains unknown. Here, we show that ofloxacin, a fluoroquinolone that poisons DNA gyrase, induces a cascade of metabolic changes that are dependent on an active SOS response. We identified the SOS-regulated TisB protein as the unique molecular determinant responsible for cytoplasmic condensation, proton motive force dissipation, loss of pH homeostasis, and H2O2 accumulation in Escherichia coli cells treated with high doses of ofloxacin. However, TisB is not required for high doses of ofloxacin to interfere with the function of DNA gyrase or the resulting rapid inhibition of DNA replication and lethal DNA damage. Overall, the study sheds light on the molecular mechanisms by which ofloxacin affects bacterial cells and highlights the role of the TisB protein in mediating these effects.

Novel terpyridines as Staphylococcus aureus gyrase inhibitors: efficient synthesis and antibacterial assessment via solvent-drop grinding.

Aim: This study was designed to synthesize a novel series of terpyridines with potential antibacterial properties, targeting multidrug resistance. Materials & methods: Terpyridines (4a-h and 6a-c) were synthesized via a one-pot multicomponent reaction using 2,6-diacetylpyridines, benzaldehyde derivatives and malononitrile or ethyl 2-cyanoacetate. The reactions, conducted under grinding conditions with glacial acetic acid, produced high-yield compounds, confirmed by spectroscopic data. Results: The synthesized terpyridines exhibited potent antibacterial activity. Notably, compounds 4d and 4h demonstrated significant inhibition zones against Staphylococcus aureus and Bacillus subtilis, outperforming ciprofloxacin. Conclusion: Molecular docking studies highlighted compounds 4d, 4h and 6c as having strong binding affinity to DNA gyrase B, correlating with their robust antibacterial activity, suggesting their potential as effective agents against multidrug-resistant bacterial strains.

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.

Investigation of new 1,2,3-triazolyl-quinolinyl-propan-2-ol derivatives as potential antimicrobial agents: in vitro and in silico approach.

A new series of 1-((1-(4-substituted benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2-(2-substituted quinolin-4-yl)propan-2-ol (9a-x) have been synthesized. The newly synthesized 1,2,3-triazolyl-quinolinyl-propan-2-ol (9a-x) derivatives were screened for in vitro antimicrobial activity against M. tuberculosis H37Rv, E. coli, P. mirabilis, B. subtilis, and S. albus. Most of the compounds showed good to moderate antibacterial activity and all derivatives have shown excellent to good antitubercular activity with MIC 0.8-12.5 µg/mL. To know the plausible mode of action for antibacterial activity the docking study against DNA gyrase from M. tuberculosis and S. aureus was investigated. The compounds have shown significant docking scores in the range of -9.532 to -7.087 and -9.543 to -6.621 Kcal/mol with the DNA gyrase enzyme of S. aureus (PDB ID: 2XCT) and M. tuberculosis (PDB ID: 5BS8), respectively. Against the S. aureus and M. tuberculosis H37Rv strains, the compound 9 l showed good activity with MIC values of 62.5 and 3.33 µM. It also showed significant docking scores in both targets with -8.291 and -8.885 Kcal/mol, respectively. Molecular dynamics was studied to investigate the structural and dynamics transitions at the atomistic level in S. aureus DNA gyrase (2XCT) and M. tuberculosis DNA gyrase (5BS8). The results revealed that the residues in the active binding pockets of the S. aureus and M. tuberculosis DNA gyrase proteins that interacted with compound 9 l remained relatively consistent throughout the MD simulations and thus, reflected the conformation stability of the respective complexes. Thus, the significant antimicrobial activity of derivatives 9a-x recommended that these compounds could assist in the development of lead compounds to treat for bacterial infections.Communicated by Ramaswamy H. Sarma.

Differential cellular localization of DNA gyrase and topoisomerase IB in response to DNA damage in Deinococcus radiodurans.

Topoisomerases are crucial enzymes in genome maintenance that modulate the topological changes during DNA metabolism. Deinococcus radiodurans, a Gram-positive bacterium is characterized by its resistance to many abiotic stresses including gamma radiation. Its multipartite genome encodes both type I and type II topoisomerases. Time-lapse studies using fluorescently tagged topoisomerase IB (drTopoIB-RFP) and DNA gyrase (GyrA-RFP) were performed to check the dynamics and localization with respect to DNA repair and cell division under normal and post-irradiation growth conditions. Results suggested that TopoIB and DNA gyrase are mostly found on nucleoid, highly dynamic, and show growth phase-dependent subcellular localization. The drTopoIB-RFP was also present at peripheral and septum regions but does not co-localize with the cell division protein, drFtsZ. On the other hand, DNA gyrase co-localizes with PprA a pleiotropic protein involved in radioresistance, on the nucleoid during the post-irradiation recovery (PIR). The topoIB mutant was found to be sensitive to hydroxyurea treatment, and showed more accumulation of single-stranded DNA during the PIR, compared to the wild type suggesting its role in DNA replication stress. Together, these results suggest differential localization of drTopoIB-RFP and GyrA-RFP in D. radiodurans and their interaction with PprA protein, emphasizing the functional significance and role in radioresistance.

Physiologic and Transcriptomic Effects Triggered by Overexpression of Wild Type and Mutant DNA Topoisomerase I in Streptococcus pneumoniae.

Topoisomerase I (TopoI) in Streptococcus pneumoniae, encoded by topA, is a suitable target for drug development. Seconeolitsine (SCN) is a new antibiotic that specifically blocks this enzyme. We obtained the topARA mutant, which encodes an enzyme less active than the wild type (topAWT) and more resistant to SCN inhibition. Likely due to the essentiality of TopoI, we were unable to replace the topAWT allele by the mutant topARA version. We compared the in vivo activity of TopoIRA and TopoIWT using regulated overexpression strains, whose genes were either under the control of a moderately (PZn) or a highly active promoter (PMal). Overproduction of TopoIRA impaired growth, increased SCN resistance and, in the presence of the gyrase inhibitor novobiocin (NOV), caused lower relaxation than TopoIWT. Differential transcriptomes were observed when the topAWT and topARA expression levels were increased about 5-fold. However, higher increases (10-15 times), produced a similar transcriptome, affecting about 52% of the genome, and correlating with a high DNA relaxation level with most responsive genes locating in topological domains. These results confirmed that TopoI is indeed the target of SCN in S. pneumoniae and show the important role of TopoI in global transcription, supporting its suitability as an antibiotic target.

Norfloxacin derivatives as DNA gyrase and urease inhibitors: synthesis, biological evaluation and molecular docking.

Background: DNA gyrase and urease enzymes are important targets for the treatment of gastroenteritis, appendicitis, tuberculosis, urinary tract infections and Crohn's disease. Materials & methods: Esterification of norfloxacin was performed to enhance DNA gyrase and urease enzyme inhibition potential. Structure elucidation and chemical characterization were done through spectral (1H NMR, Fourier transform IR, 13C NMR) and carbon, hydrogen, nitrogen and sulfur analysis along with molecular docking. Results & conclusion: The majority of derivatives exhibited significant results but the 3e derivative showed maximum bactericidal, DPPH scavenging (96%), DNA gyrase and urease enzyme inhibitory activity with IC50 of 0.15 ± 0.24 and 1.14 ± 0.11 µM respectively which was further supported by molecular docking studies. So, the active derivatives can serve as a lead compound for the treatment of various pathological conditions.

Macromolecular crowding potently stimulates DNA supercoiling activity of Mycobacterium tuberculosis DNA gyrase.

Macromolecular crowding, manifested by high concentrations of proteins and nucleic acids in living cells, significantly influences biological processes such as enzymatic reactions. Studying these reactions in vitro, using agents such as polyetthylene glycols (PEGs) and polyvinyl alcohols (PVAs) to mimic intracellular crowding conditions, is essential due to the notable differences from enzyme behaviors observed in diluted aqueous solutions. In this article, we studied Mycobacterium tuberculosis (Mtb) DNA gyrase under macromolecular crowding conditions by incorporating PEGs and PVAs into the DNA supercoiling reactions. We discovered that high concentrations of potassium glutamate, glycine betaine, PEGs, and PVA substantially stimulated the DNA supercoiling activity of Mtb DNA gyrase. Steady-state kinetic studies showed that glycine betaine and PEG400 significantly reduced the KM of Mtb DNA gyrase and simultaneously increased the Vmax or kcat of Mtb DNA gyrase for ATP and the plasmid DNA molecule. Molecular dynamics simulation studies demonstrated that PEG molecules kept the ATP lid of DNA gyrase subunit B in a closed or semiclosed conformation, which prevented ATP molecules from leaving the ATP-binding pocket of DNA gyrase subunit B. The stimulation of the DNA supercoiling activity of Mtb DNA gyrase by these molecular crowding agents likely results from a decrease in water activity and an increase in excluded volume.

The Impact of Substitutions at Positions 1 and 8 of Fluoroquinolones on the Activity Against Mutant DNA Gyrases of Salmonella Typhimurium.

Although many drug-resistant nontyphoidal Salmonella (NTS) infections are reported globally, their treatment is challenging owing to the ineffectiveness of the currently available antimicrobial drugs against resistant bacteria. It is therefore essential to discover novel antimicrobial drugs for the management of these infections. In this study, we report high inhibitory activities of the novel fluoroquinolones (FQs; WQ-3810 and WQ-3334) with substitutions at positions R-1 by 6-amino-3,5-difluoropyridine-2-yl and R-8 by methyl group or bromine, respectively, against wild-type and mutant DNA gyrases of Salmonella Typhimurium. The inhibitory activities of these FQs were assessed against seven amino acid substitutions in DNA gyrases conferring FQ resistance to S. Typhimurium, including high-level resistant mutants, Ser83Ile and Ser83Phe-Asp87Asn by in vitro DNA supercoiling assay. Drug concentrations of WQ compounds with 6-amino-3,5-difluoropyridine-2-yl that suppressed DNA supercoiling by 50% (IC50) were found to be ∼150-fold lower than ciprofloxacin against DNA gyrase with double amino acid substitutions. Our findings highlight the importance of the chemical structure of an FQ drug on its antimicrobial activity. Particularly, the presence of 6-amino-3,5-difluoropyridine-2-yl at R-1 and either methyl group or bromine at R-8 of WQ-3810 and WQ-3334, respectively, was associated with improved antimicrobial activity. Therefore, WQ-3810 and WQ-3334 are promising candidates for use in the treatment of patients infected by FQ-resistant Salmonella spp.

Benzopyrone-mediated quinolones as potential multitargeting antibacterial agents.

A new type of benzopyrone-mediated quinolones (BMQs) was rationally designed and efficiently synthesized as novel potential antibacterial molecules to overcome the global increasingly serious drug resistance. Some synthesized BMQs effectively suppressed the growth of the tested strains, outperforming clinical drugs. Notably, ethylidene-derived BMQ 17a exhibited superior antibacterial potential with low MICs of 0.5-2 µg/mL to clinical drugs norfloxacin, it not only displayed rapid bactericidal performance and inhibited bacterial biofilm formation, but also showed low toxicity toward human red blood cells and normal MDA-kb2 cells. Mechanistic investigation demonstrated that BMQ 17a could effectually induce bacterial metabolic disorders and promote the enhancement of reactive oxygen species to disrupt the bacterial antioxidant defense system. It was found that the active molecule BMQ 17a could not only form supramolecular complex with lactate dehydrogenase, which disturbed the biological functions, but also effectively embed into calf thymus DNA, thus affecting the normal function of DNA and achieving cell death. This work would provide an insight into developing new molecules to reduce drug resistance and expand antibacterial spectrum.

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.

Mycobacterium tuberculosis Inhibitors Based on Arylated Quinoline Carboxylic Acid Backbones with Anti-Mtb Gyrase Activity.

New antitubercular agents with either a novel mode of action or novel mode of inhibition are urgently needed to overcome the threat of drug-resistant tuberculosis (TB). The present study profiles new arylated quinoline carboxylic acids (QCAs) having activity against replicating and non-replicating Mycobacterium tuberculosis (Mtb), the causative agent of TB. Thus, the synthesis, characterization, and in vitro screening (MABA and LORA) of 48 QCAs modified with alkyl, aryl, alkoxy, halogens, and nitro groups in the quinoline ring led to the discovery of two QCA derivatives, 7i and 7m, adorned with C-2 2-(naphthalen-2-yl)/C-6 1-butyl and C-2 22-(phenanthren-3-yl)/C-6 isopropyl, respectively, as the best Mtb inhibitors. DNA gyrase inhibition was shown to be exhibited by both, with QCA 7m illustrating better activity up to a 1 µM test concentration. Finally, a docking model for both compounds with Mtb DNA gyrase was developed, and it showed a good correlation with in vitro results.

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.

New pyrrole derivatives as DNA gyrase and 14α-demethylase inhibitors: Design, synthesis, antimicrobial evaluation, and molecular docking.

An efficient one-pot reaction utilizing readily available chemical reagents was used to prepare novel 2-amino-1,5-diaryl-1H-pyrrole-3-carbonitrile derivatives and the structures of these compounds were validated by spectroscopic data and elemental analyses. All the synthetic compounds were evaluated for their antimicrobial activities (MZI assay). The tested compounds proved high activities on Staphylococcus aureus (Gram-positive bacteria) and Candida albicans (Pathogenic fungi). However, they did not show any activity on Escherichia coli (Gram-negative bacteria). The most effective compounds in MZI assay 7c, 9a, 9b, 11a, and 11b were selected to determine their MIC on S. aureus and C. albicans. Furthermore, DNA gyrase and 14-α demethylase inhibitory assays were performed to study the inhibitory activities of 7c, 9a, 9b, 11a, and 11b. The results illustrated that compound 9b was the most DNA gyrase inhibitor (IC50 of 0.0236 ± 0.45 µM, which was 1.3- fold higher than gentamicin reference IC50 values of 0.0323 ± 0.81 µM). In addition, compound 9b demonstrated the highest 14-α demethylase inhibitory effect with IC50 of 0.0013 ± 0.02 µM, compared to ketoconazole (IC50 of 0.0008 ± 0.03 µM) and fluconazole (IC50 of 0.00073 ± 0.01 µM), as antifungal reference drugs. Lastly, docking studies were performed to rationalize the dual inhibitory activities of the highly active compounds on both DNA gyrase and 14-α demethylase enzymes.

Distinct subunit architecture and assembly pattern of DNA gyrase from mycobacteria.

DNA gyrase, the sole negative supercoiling type II topoisomerase, is composed of two subunits, GyrA and GyrB, encoded by the gyrA and gyrB genes, respectively, that form a quaternary complex of A2 B2 . In this study, we have investigated the assembly of mycobacterial DNA gyrase from its individual subunits, a step prerequisite for its activity. Using analytical size-exclusion chromatography, we show that GyrA from Mycobacterium tuberculosis and Mycobacterium smegmatis forms tetramers (A4 ) in solution unlike in Escherichia coli and other bacteria where GyrA exists as a dimer. GyrB, however, persists as a monomer, resembling the pattern found in E. coli. GyrB in both mycobacterial species interacts with GyrA and triggers the dissociation of the GyrA tetramer to facilitate the formation of catalytically active A2 B2 . Despite oligomerisation, the GyrA tetramer retained its DNA binding ability, and DNA binding had no effect on GyrA's oligomeric state in both species. Moreover, the presence of DNA facilitated the assembly of holoenzyme in the case of M. smegmatis by stabilising the GyrA2 B2 tetramer but with little effect in M. tuberculosis. Thus, in addition to the distinct organisation and regulation of the gyr locus in mycobacteria, the enzyme assembly also follows a different pattern.