RESUMEN
In this work, we describe an improved series of N-phenylpyrrolamide inhibitors that exhibit potent activity against DNA gyrase and are highly effective against high-priority gram-positive bacteria. The most potent compounds show low nanomolar IC50 values against Escherichia coli DNA gyrase, and in addition, compound 7c also inhibits E. coli topoisomerase IV in the nanomolar concentration range, making it a promising candidate for the development of potent dual inhibitors for these enzymes. All tested compounds show high selectivity towards the human isoform DNA topoisomerase IIα. Compounds 6a, 6d, 6e and 6f show MIC values between 0.031 and 0.0625 µg/mL against vancomycin-intermediate S. aureus (VISA) and Enterococcus faecalis strains. Compound 6g shows an inhibitory effect against the methicillin-resistant S. aureus strain (MRSA) with a MIC of 0.0625 µg/mL and against the E. faecalis strain with a MIC of 0.125 µg/mL. In a time-kill assay, compound 6d showed a dose-dependent bactericidal effect on the MRSA strain and achieved bactericidal activity at 8 × MIC after 8 h. The duration of the post-antibiotic effect (PAE) on the MRSA strain for compound 6d was 2 h, which corresponds to the PAE duration for ciprofloxacin. The compounds were not cytotoxic at effective concentrations, as determined in an MTS assay on the MCF-7 breast cancer cell line.
Asunto(s)
Antibacterianos , Girasa de ADN , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Inhibidores de Topoisomerasa II , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Humanos , Inhibidores de Topoisomerasa II/farmacología , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/síntesis química , Girasa de ADN/metabolismo , Relación Estructura-Actividad , Estructura Molecular , Enterococcus faecalis/efectos de los fármacos , Pirroles/farmacología , Pirroles/química , Pirroles/síntesis química , Amidas/farmacología , Amidas/química , Amidas/síntesis química , Escherichia coli/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacosRESUMEN
Type II topoisomerases (topos) are a ubiquitous and essential class of enzymes that form transient enzyme-bound double-stranded breaks on DNA called cleavage complexes. The location and frequency of these cleavage complexes on DNA is important for cellular function, genomic stability and a number of clinically important anticancer and antibacterial drugs, e.g. quinolones. We developed a simple high-accuracy end-sequencing (SHAN-seq) method to sensitively map type II topo cleavage complexes on DNA in vitro. Using SHAN-seq, we detected Escherichia coli gyrase and topoisomerase IV cleavage complexes at hundreds of sites on supercoiled pBR322 DNA, approximately one site every ten bp, with frequencies that varied by two-to-three orders of magnitude. These sites included previously identified sites and 20-50-fold more new sites. We show that the location and frequency of cleavage complexes at these sites are enzyme-specific and vary substantially in the presence of the quinolone, ciprofloxacin, but not with DNA supercoil chirality, i.e. negative versus positive supercoiling. SHAN-seq's exquisite sensitivity provides an unprecedented single-nucleotide resolution view of the distribution of gyrase and topoisomerase IV cleavage complexes on DNA. Moreover, the discovery that these enzymes can cleave DNA at orders of magnitude more sites than the relatively few previously known sites resolves the apparent paradox of how these enzymes resolve topological problems throughout the genome.
Asunto(s)
División del ADN , Girasa de ADN , Topoisomerasa de ADN IV , ADN-Topoisomerasas de Tipo II , Escherichia coli , Escherichia coli/genética , Escherichia coli/enzimología , Girasa de ADN/metabolismo , Girasa de ADN/genética , Girasa de ADN/química , Topoisomerasa de ADN IV/metabolismo , Topoisomerasa de ADN IV/genética , Topoisomerasa de ADN IV/química , ADN-Topoisomerasas de Tipo II/metabolismo , ADN-Topoisomerasas de Tipo II/genética , Análisis de Secuencia de ADN/métodos , ADN Superhelicoidal/metabolismo , ADN Superhelicoidal/química , Ciprofloxacina/farmacología , Secuenciación de Nucleótidos de Alto Rendimiento , ADN/metabolismo , ADN/químicaRESUMEN
Two series of C-Mannich base derivatives were synthesized and evaluated through the reaction of formaldehyde, two thiazolo-pyrimidine compounds, and various 2°-amines. The chemical structures and inherent properties of the synthesized compounds were authenticated using a variety of spectroscopic techniques. The aseptic bactericidal potential of the compounds was assessed alongside five common bacterial microbes, with Ampicillin employed as the reference drug. Compounds 9b and 9d demonstrated comparable antibacterial activity to ampicillin against Bacillus subtilis and Bacillus megaterium, respectively, at 100 µg/mL. Furthermore, compounds 9f and 10f exhibited noteworthy action against Staphylococcus aureus (MIC: 250 µg/mL). Compounds 10b and 10f displayed excellent efficacy versus Escherichia coli, boasting (MIC: 50 µg/mL). Molecular docking studies elucidated the necessary connections and energies of molecular entities with the E. coli DNA gyrase B enzyme, a pivotal target in bacterial DNA replication. Further thermodynamic stability of the ligand-receptor complex of 10b and 10f were further validated though 200 ns molecular dynamics simulation. The findings highlight the potential of these synthesized derivatives as effective antibacterial agents and provide valuable insights into their mechanism of action.
Asunto(s)
Antibacterianos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Pirimidinas , Antibacterianos/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Pirimidinas/química , Pirimidinas/farmacología , Pirimidinas/síntesis química , Bacillus subtilis/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Tiazoles/química , Tiazoles/farmacología , Tiazoles/síntesis química , Pruebas de Sensibilidad Microbiana , Escherichia coli/efectos de los fármacos , Girasa de ADN/metabolismo , Girasa de ADN/químicaRESUMEN
A series of new 1,2,3-triazole fused chromene based glucose triazole conjugates were synthesized from chromene fused 1,2,3-triazolyl extended alkyne and 2,3,4,6-tetra-O-acetyl-ß-d-glucopyranosyl azide in good to excellent yield by a copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction. The major advantages include mild reaction conditions, high yield, good substrate scope, and shorter reaction time. The antibacterial efficacy of the compounds were assessed in vitro against human pathogenic Gram-negative E. coli and Gram-positive S. aureus bacteria. Compound 24j was found to be the most potent molecule with zone of inhibition (ZI) of 17 mm and minimum inhibitory concentration (MIC) of 25 µg mL-1 in E. coli and ZI of 16 mm and MIC of 25 µg mL-1 in S. aureus. Also, it significantly inhibited E. coli DNA-gyrase in silico with a binding affinity of -9.4 kcal/mol. Among all the synthesized compounds, 24i, 24d, 24e and 24f showed significant antibacterial activity against both strains and inhibited DNA-gyrase in silico with good binding affinities. Hence, these 1,2,3-triazole fused chromene based glucose triazole conjugates may evolve to be powerful antibacterial agents in recent future, according to structure-activity relationships based on strong antibacterial properties and molecular docking studies.
Asunto(s)
Antibacterianos , Benzopiranos , Química Clic , Escherichia coli , Glucosa , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Staphylococcus aureus , Triazoles , Triazoles/química , Triazoles/farmacología , Triazoles/síntesis química , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Escherichia coli/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Benzopiranos/química , Benzopiranos/farmacología , Benzopiranos/síntesis química , Glucosa/química , Glucosa/análogos & derivados , Relación Estructura-Actividad , Estructura Molecular , Girasa de ADN/metabolismo , Girasa de ADN/química , HumanosRESUMEN
New 2-pyrrolamidobenzothiazole-based inhibitors of mycobacterial DNA gyrase were discovered. Among these, compounds 49 and 51, show excellent antibacterial activity against Mycobacterium tuberculosis and Mycobacterium abscessus with a notable preference for mycobacteria. Both compounds can penetrate infected macrophages and reduce intracellular M. tuberculosis load. Compound 51 is a potent inhibitor of DNA gyrase (M. tuberculosis DNA gyrase IC50 = 4.1 nM, Escherichia coli DNA gyrase IC50 of <10 nM), selective for bacterial topoisomerases. It displays low MIC90 values (M. tuberculosis: 0.63 µM; M. abscessus: 2.5 µM), showing specificity for mycobacteria, and no apparent toxicity. Compound 49 not only displays potent antimycobacterial activity (MIC90 values of 2.5 µM for M. tuberculosis and 0.63 µM for M. abscessus) and selectivity for mycobacteria but also exhibits favorable solubility (kinetic solubility = 55 µM) and plasma protein binding (with a fraction unbound of 2.9 % for human and 4.7 % for mouse). These findings underscore the potential of fine-tuning molecular properties to develop DNA gyrase B inhibitors that specifically target the mycobacterial chemical space, mitigating the risk of resistance development in non-target pathogens and minimizing harm to the microbiome.
Asunto(s)
Antibacterianos , Girasa de ADN , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis , Inhibidores de Topoisomerasa II , Girasa de ADN/metabolismo , Inhibidores de Topoisomerasa II/farmacología , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/síntesis química , Humanos , Mycobacterium tuberculosis/efectos de los fármacos , Relación Estructura-Actividad , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Estructura Molecular , Ratones , Animales , Relación Dosis-Respuesta a Droga , Antituberculosos/farmacología , Antituberculosos/química , Antituberculosos/síntesis química , Desarrollo de Medicamentos , Mycobacterium/efectos de los fármacosRESUMEN
Mycobacterium tuberculosis is the single most important global infectious disease killer and a World Health Organization critical priority pathogen for development of new antimicrobials. M. tuberculosis DNA gyrase is a validated target for anti-TB agents, but those in current use target DNA breakage-reunion, rather than the ATPase activity of the GyrB subunit. Here, virtual screening, subsequently validated by whole-cell and enzyme inhibition assays, was applied to identify candidate compounds that inhibit M. tuberculosis GyrB ATPase activity from the Specs compound library. This approach yielded six compounds: four carbazole derivatives (1, 2, 3, and 8), the benzoindole derivative 11, and the indole derivative 14. Carbazole derivatives can be considered a new scaffold for M. tuberculosis DNA gyrase ATPase inhibitors. IC50 values of compounds 8, 11, and 14 (0.26, 0.56, and 0.08 µM, respectively) for inhibition of M. tuberculosis DNA gyrase ATPase activity are 5-fold, 2-fold, and 16-fold better than the known DNA gyrase ATPase inhibitor novobiocin. MIC values of these compounds against growth of M. tuberculosis H37Ra are 25.0, 3.1, and 6.2 µg/mL, respectively, superior to novobiocin (MIC > 100.0 µg/mL). Molecular dynamics simulations of models of docked GyrB:inhibitor complexes suggest that hydrogen bond interactions with GyrB Asp79 are crucial for high-affinity binding of compounds 8, 11, and 14 to M. tuberculosis GyrB for inhibition of ATPase activity. These data demonstrate that virtual screening can identify known and new scaffolds that inhibit both M. tuberculosis DNA gyrase ATPase activity in vitro and growth of M. tuberculosis bacteria.
Asunto(s)
Antituberculosos , Girasa de ADN , Indoles , Mycobacterium tuberculosis , Inhibidores de Topoisomerasa II , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/metabolismo , Antituberculosos/farmacología , Antituberculosos/química , Girasa de ADN/metabolismo , Girasa de ADN/química , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Indoles/farmacología , Indoles/química , Ligandos , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/efectos de los fármacos , Inhibidores de Topoisomerasa II/farmacología , Inhibidores de Topoisomerasa II/químicaRESUMEN
Cumulative escalation in antibiotic-resistant pathogens necessitates the quest for novel antimicrobial agents, as current options continue to diminish bacterial resistance. Herein, we report the synthesis of di-heterocyclic benzazole structures (12-19) and their in vitro evaluation for some biological activities. Compounds 16 and 17 demonstrated potent antibacterial activity (MIC = 7.81 µg/mL) against Staphylococcus aureus, along with significant anti-biofilm activity. Noteworthy is the capability of Compound 17 to inhibit biofilm formation by at least 50% at sub-MIC (3.90 µg/mL) concentration. Furthermore, both compounds exhibited the potential to inhibit preformed biofilm by at least 50% at the MIC concentration (7.81 µg/mL). Additionally, Compounds 16 and 17 were examined for cytotoxic effects in HFF-1 cells, using the MTT method, and screened for binding interactions within the active site of S. aureus DNA gyrase using in silico molecular docking technique, employing AutoDock 4.2.6 and Schrödinger Glidse programs. Overall, our findings highlight Compounds 16 and 17 as promising scaffolds warranting further optimization for the development of effective antibacterial and anti-biofilm agents.
Asunto(s)
Antibacterianos , Biopelículas , Girasa de ADN , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Staphylococcus aureus , Biopelículas/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Girasa de ADN/metabolismo , Humanos , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/farmacología , Compuestos Heterocíclicos/síntesis química , Relación Estructura-Actividad , Línea Celular , Dominio CatalíticoRESUMEN
Efflux pumps play a crucial role in the development of antibiotic resistance. The aim of this study was to investigate the relationship between efflux pump gene expression and resistance gene mutations in Helicobacter pylori. Twenty-six clinical strains with varying resistance characteristics were selected for further experiment. Seven susceptible strains were induced to become resistant, and the expression of efflux pump genes and point mutations were recorded. Four susceptible strains were selected to undergo candidate mutation construction, and changes in efflux pump gene expression were detected. Efflux pump knockout strains were constructed, and their effects on preventing and reversing antibiotic resistance gene mutations were assessed. Results showed that the expression of efflux pump genes hefA and hefD was significantly higher in the multidrug-resistant group compared to other groups. During the process of antibiotic-induced resistance, efflux pump gene expression did not exhibit a steady increase or decrease. Strains with the A2143G or A2142G point mutations in 23S rRNA exhibited lower hefA gene expression. Strains with mutations at 87K/91N, 87N/91 G, 87K/91D, or 87N/91Y in gyrA and the 194insertA mutation in rdxA showed higher hefA gene expression compared to the wild-type strain. During the process of antibiotic-induced resistance, the strain with the knockout of the efflux pump gene hefA developed mutations in the 23S rRNA, gyrA, or rdxA genes later compared to the wild-type strain. Knockout of the efflux pump gene could reverse the phenotypic resistance to clarithromycin or metronidazole in some strains but had no effect on reverse resistance gene mutation. This study suggested that different resistance gene point mutations may have varying effects on efflux pump gene expression. Knockout of the efflux pump gene can delay or prevent antibiotic resistance gene mutations to some extent and can reverse phenotypic resistance to clarithromycin and metronidazole in certain strains.
Asunto(s)
Antibacterianos , Proteínas Bacterianas , Infecciones por Helicobacter , Helicobacter pylori , Proteínas de Transporte de Membrana , Helicobacter pylori/genética , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/metabolismo , Antibacterianos/farmacología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Infecciones por Helicobacter/microbiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Humanos , Farmacorresistencia Bacteriana/genética , Mutación Puntual , Mutación , Pruebas de Sensibilidad Microbiana , Regulación Bacteriana de la Expresión Génica , Farmacorresistencia Bacteriana Múltiple/genética , ARN Ribosómico 23S/genética , Girasa de ADN/genética , Girasa de ADN/metabolismoRESUMEN
Bacterial DNA gyrase and topoisomerase IV inhibition has emerged as a promising strategy for the cure of infections caused by antibiotic-resistant bacteria. The Novel Bacterial Topoisomerase Inhibitors (NBTIs) bind to a different site from that of the quinolones with novel mechanism of action. This evades the existing target-mediated bacterial resistance associated with quinolones. This article presents our efforts to identify in vitro potent and broad-spectrum antibacterial agent 4l.
Asunto(s)
Antibacterianos , Pruebas de Sensibilidad Microbiana , Piperidinas , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Piperidinas/química , Piperidinas/farmacología , Piperidinas/síntesis química , Relación Estructura-Actividad , Inhibidores de Topoisomerasa/farmacología , Inhibidores de Topoisomerasa/química , Inhibidores de Topoisomerasa/síntesis química , Girasa de ADN/metabolismo , Inhibidores de Topoisomerasa II/farmacología , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/síntesis química , Topoisomerasa de ADN IV/antagonistas & inhibidores , Topoisomerasa de ADN IV/metabolismo , Estructura Molecular , Descubrimiento de Drogas , Relación Dosis-Respuesta a Droga , HumanosRESUMEN
Gyrase and topoisomerase IV are the cellular targets for fluoroquinolones, a critically important class of antibacterial agents used to treat a broad spectrum of human infections. Unfortunately, the clinical efficacy of the fluoroquinolones has been curtailed by the emergence of target-mediated resistance. This is especially true for Neisseria gonorrhoeae, the causative pathogen of the sexually transmitted infection gonorrhea. Spiropyrimidinetriones (SPTs), a new class of antibacterials, were developed to combat the growing antibacterial resistance crisis. Zoliflodacin is the most clinically advanced SPT and displays efficacy against uncomplicated urogenital gonorrhea in human trials. Like fluoroquinolones, the primary target of zoliflodacin in N. gonorrhoeae is gyrase, and topoisomerase IV is a secondary target. Because unbalanced gyrase/topoisomerase IV targeting has facilitated the evolution of fluoroquinolone-resistant bacteria, it is important to understand the underlying basis for the differential targeting of zoliflodacin in N. gonorrhoeae. Therefore, we assessed the effects of this SPT on the catalytic and DNA cleavage activities of N. gonorrhoeae gyrase and topoisomerase IV. In all reactions examined, zoliflodacin displayed higher potency against gyrase than topoisomerase IV. Moreover, zoliflodacin generated more DNA cleavage and formed more stable enzyme-cleaved DNA-SPT complexes with gyrase. The SPT also maintained higher activity against fluoroquinolone-resistant gyrase than topoisomerase IV. Finally, when compared to zoliflodacin, the novel SPT H3D-005722 induced more balanced double-stranded DNA cleavage with gyrase and topoisomerase IV from N. gonorrhoeae, Escherichia coli, and Bacillus anthracis. This finding suggests that further development of the SPT class could yield compounds with a more balanced targeting against clinically important bacterial infections.
Asunto(s)
Antibacterianos , Girasa de ADN , Topoisomerasa de ADN IV , Neisseria gonorrhoeae , Inhibidores de Topoisomerasa II , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/enzimología , Topoisomerasa de ADN IV/metabolismo , Topoisomerasa de ADN IV/antagonistas & inhibidores , Topoisomerasa de ADN IV/genética , Girasa de ADN/metabolismo , Girasa de ADN/genética , Girasa de ADN/química , Antibacterianos/farmacología , Antibacterianos/química , Inhibidores de Topoisomerasa II/farmacología , Inhibidores de Topoisomerasa II/química , Humanos , Oxazolidinonas/farmacología , Oxazolidinonas/química , Barbitúricos/farmacología , Barbitúricos/química , Pruebas de Sensibilidad Microbiana , Farmacorresistencia Bacteriana , Isoxazoles , Morfolinas , Compuestos de EspiroRESUMEN
Globally, there have been increasing reports of antimicrobial resistance in nontyphoidal Salmonella (NTS), which can develop into severe and potentially life-threatening diarrhea. This study focuses on the synergistic effects of DNA gyrase mutations and plasmid-mediated quinolone resistance (PMQR) genes, specifically qnrB19, on fluoroquinolone (FQ) resistance in Salmonella Typhimurium. By utilizing recombinant mutants, GyrAS83F and GyrAD87N, and QnrB19's, we discovered a significant increase in fluoroquinolones resistance when QnrB19 is present. Specifically, ciprofloxacin and moxifloxacin's inhibitory concentrations rose 10- and 8-fold, respectively. QnrB19 was found to enhance the resistance capacity of mutant DNA gyrases, leading to high-level FQ resistance. Additionally, we observed that the ratio of QnrB19 to DNA gyrase played a critical role in determining whether QnrB19 could protect DNA gyrase against FQ inhibition. Our findings underscore the critical need to understand these resistance mechanisms, as their coexistence enables bacteria to withstand therapeutic FQ levels, posing a significant challenge to treatment efficacy.
Asunto(s)
Sustitución de Aminoácidos , Antibacterianos , Girasa de ADN , Farmacorresistencia Bacteriana , Fluoroquinolonas , Pruebas de Sensibilidad Microbiana , Salmonella typhimurium , Girasa de ADN/genética , Girasa de ADN/metabolismo , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Fluoroquinolonas/farmacología , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Ciprofloxacina/farmacología , Mutación , Plásmidos/genéticaRESUMEN
Bacteria have evolved resistance to nearly all known antibacterials, emphasizing the need to identify antibiotics that operate via novel mechanisms. Here we report a class of allosteric inhibitors of DNA gyrase with antibacterial activity against fluoroquinolone-resistant clinical isolates of Escherichia coli. Screening of a small-molecule library revealed an initial isoquinoline sulfonamide hit, which was optimized via medicinal chemistry efforts to afford the more potent antibacterial LEI-800. Target identification studies, including whole-genome sequencing of in vitro selected mutants with resistance to isoquinoline sulfonamides, unanimously pointed to the DNA gyrase complex, an essential bacterial topoisomerase and an established antibacterial target. Using single-particle cryogenic electron microscopy, we determined the structure of the gyrase-LEI-800-DNA complex. The compound occupies an allosteric, hydrophobic pocket in the GyrA subunit and has a mode of action that is distinct from the clinically used fluoroquinolones or any other gyrase inhibitor reported to date. LEI-800 provides a chemotype suitable for development to counter the increasingly widespread bacterial resistance to fluoroquinolones.
Asunto(s)
Antibacterianos , Girasa de ADN , Farmacorresistencia Bacteriana , Escherichia coli , Fluoroquinolonas , Isoquinolinas , Sulfonamidas , Inhibidores de Topoisomerasa II , Inhibidores de Topoisomerasa II/farmacología , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/síntesis química , Isoquinolinas/química , Isoquinolinas/farmacología , Isoquinolinas/síntesis química , Sulfonamidas/farmacología , Sulfonamidas/química , Sulfonamidas/síntesis química , Fluoroquinolonas/farmacología , Fluoroquinolonas/química , Fluoroquinolonas/síntesis química , Girasa de ADN/metabolismo , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Farmacorresistencia Bacteriana/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Pruebas de Sensibilidad Microbiana , Relación Estructura-Actividad , Descubrimiento de Drogas , Regulación Alostérica/efectos de los fármacosRESUMEN
DNA gyrase, a ubiquitous bacterial enzyme, is a type IIA topoisomerase formed by heterotetramerisation of 2 GyrA subunits and 2 GyrB subunits, to form the active complex. DNA gyrase can loop DNA around the C-terminal domains (CTDs) of GyrA and pass one DNA duplex through a transient double-strand break (DSB) established in another duplex. This results in the conversion from a positive (+1) to a negative (-1) supercoil, thereby introducing negative supercoiling into the bacterial genome by steps of 2, an activity essential for DNA replication and transcription. The strong protein interface in the GyrA dimer must be broken to allow passage of the transported DNA segment and it is generally assumed that the interface is usually stable and only opens when DNA is transported, to prevent the introduction of deleterious DSBs in the genome. In this paper, we show that DNA gyrase can exchange its DNA-cleaving interfaces between two active heterotetramers. This so-called interface 'swapping' (IS) can occur within a few minutes in solution. We also show that bending of DNA by gyrase is essential for cleavage but not for DNA binding per se and favors IS. Interface swapping is also favored by DNA wrapping and an excess of GyrB. We suggest that proximity, promoted by GyrB oligomerization and binding and wrapping along a length of DNA, between two heterotetramers favors rapid interface swapping. This swapping does not require ATP, occurs in the presence of fluoroquinolones, and raises the possibility of non-homologous recombination solely through gyrase activity. The ability of gyrase to undergo interface swapping explains how gyrase heterodimers, containing a single active-site tyrosine, can carry out double-strand passage reactions and therefore suggests an alternative explanation to the recently proposed 'swivelling' mechanism for DNA gyrase (Gubaev et al., 2016).
Asunto(s)
Girasa de ADN , Girasa de ADN/metabolismo , Girasa de ADN/química , Girasa de ADN/genética , Multimerización de Proteína , ADN Bacteriano/metabolismo , ADN Bacteriano/genética , Escherichia coli/genética , Escherichia coli/enzimología , Escherichia coli/metabolismo , ADN/metabolismo , ADN/químicaRESUMEN
N-(Benzothiazole-2-yl)pyrrolamide DNA gyrase inhibitors with benzyl or phenethyl substituents attached to position 3 of the benzothiazole ring or to the carboxamide nitrogen atom were prepared and studied for their inhibition of Escherichia coli DNA gyrase by supercoiling assay. Compared to inhibitors bearing the substituents at position 4 of the benzothiazole ring, the inhibition was attenuated by moving the substituent to position 3 and further to the carboxamide nitrogen atom. A co-crystal structure of (Z)-3-benzyl-2-((4,5-dibromo-1H-pyrrole-2-carbonyl)imino)-2,3-dihydrobenzo[d]-thiazole-6-carboxylic acid (I) in complex with E. coli GyrB24 (ATPase subdomain) was solved, revealing the binding mode of this type of inhibitor to the ATP-binding pocket of the E. coli GyrB subunit. The key binding interactions were identified and their contribution to binding was rationalised by quantum theory of atoms in molecules (QTAIM) analysis. Our study shows that the benzyl or phenethyl substituents bound to the benzothiazole core interact with the lipophilic floor of the active site, which consists mainly of residues Gly101, Gly102, Lys103 and Ser108. Compounds with substituents at position 3 of the benzothiazole core were up to two orders of magnitude more effective than compounds with substituents at the carboxamide nitrogen. In addition, the 6-oxalylamino compounds were more potent inhibitors of E. coli DNA gyrase than the corresponding 6-acetamido analogues.
Asunto(s)
Girasa de ADN , Escherichia coli , Inhibidores de Topoisomerasa II , Inhibidores de Topoisomerasa II/farmacología , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/síntesis química , Girasa de ADN/metabolismo , Girasa de ADN/química , Sitios de Unión , Escherichia coli/enzimología , Escherichia coli/efectos de los fármacos , Relación Estructura-Actividad , Benzotiazoles/química , Benzotiazoles/farmacología , Benzotiazoles/síntesis química , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/química , Estructura Molecular , Teoría Cuántica , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Modelos MolecularesRESUMEN
INTRODUCTION: Neisseria gonorrhoeae is a common sexually transmitted disease connected with extensive drug resistance to many antibiotics. Presently, only expanded spectrum cephalosporins (ceftriaxone and cefixime) and azithromycin remain useful for its management. AREAS COVERED: New chemotypes for the classical antibiotic drug target gyrase/topoisomerase IV afforded inhibitors with potent binding to these enzymes, with an inhibition mechanism distinct from that of fluoroquinolones, and thus less prone to mutations. The α-carbonic anhydrase from the genome of this bacterium (NgCAα) was also validated as an antibacterial target. EXPERT OPINION: By exploiting different subunits from the gyrase/topoisomerase IV as well as new chemotypes, two new antibiotics reached Phase II/III clinical trials, zoliflodacin and gepotidacin. They possess a novel inhibition mechanism, binding in distinct parts of the enzyme compared to the fluoroquinolones. Other chemotypes with inhibitory activity in these enzymes were also reported. NgCAα inhibitors belonging to a variety of classes were obtained, with several sulfonamides showing MIC values in the range of 0.25-4 µg/mL and significant activity in animal models of this infection. Acetazolamide and similar CA inhibitors might thus be repurposed as antiinfectives. The scientific/patent literature has been searched for on PubMed, ScienceDirect, Espacenet, and PatentGuru, from 2016 to 2024.
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Antibacterianos , Reposicionamiento de Medicamentos , Farmacorresistencia Bacteriana , Gonorrea , Neisseria gonorrhoeae , Patentes como Asunto , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/enzimología , Antibacterianos/farmacología , Humanos , Animales , Gonorrea/tratamiento farmacológico , Gonorrea/microbiología , Inhibidores de Topoisomerasa II/farmacología , Oxazolidinonas/farmacología , Pruebas de Sensibilidad Microbiana , Topoisomerasa de ADN IV/antagonistas & inhibidores , Topoisomerasa de ADN IV/metabolismo , Girasa de ADN/metabolismo , Morfolinas , Isoxazoles , Compuestos de Espiro , Compuestos Heterocíclicos con 3 Anillos , Barbitúricos , AcenaftenosRESUMEN
DNA gyrases catalyze negative supercoiling of DNA, are essential for bacterial DNA replication, transcription, and recombination, and are important antibacterial targets in multiple pathogens, including Mycobacterium tuberculosis, which in 2021 caused >1.5 million deaths worldwide. DNA gyrase is a tetrameric (A2B2) protein formed from two subunit types: gyrase A (GyrA) carries the breakage-reunion active site, whereas gyrase B (GyrB) catalyzes ATP hydrolysis required for energy transduction and DNA translocation. The GyrB ATPase domains dimerize in the presence of ATP to trap the translocated DNA (T-DNA) segment as a first step in strand passage, for which hydrolysis of one of the two ATPs and release of the resulting inorganic phosphate is rate-limiting. Here, dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations of the dimeric 43 kDa N-terminal fragment of M. tuberculosis GyrB show how events at the ATPase site (dissociation/hydrolysis of bound nucleotides) are propagated through communication pathways to other functionally important regions of the GyrB ATPase domain. Specifically, our simulations identify two distinct pathways that respectively connect the GyrB ATPase site to the corynebacteria-specific C-loop, thought to interact with GyrA prior to DNA capture, and to the C-terminus of the GyrB transduction domain, which in turn contacts the C-terminal GyrB topoisomerase-primase (TOPRIM) domain responsible for interactions with GyrA and the centrally bound G-segment DNA. The connection between the ATPase site and the C-loop of dimeric GyrB is consistent with the unusual properties of M. tuberculosis DNA gyrase relative to those from other bacterial species.
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Adenosina Trifosfatasas , Girasa de ADN , Simulación de Dinámica Molecular , Mycobacterium tuberculosis , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Girasa de ADN/metabolismo , Girasa de ADN/química , Girasa de ADN/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Dominios Proteicos , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Transducción de SeñalRESUMEN
Launaea sarmentosa, also known as Sa Sam Nam, is a widely used remedy in Vietnamese traditional medicine and cuisine. However, the chemical composition and bioactivity of its essential oil have not been elucidated yet. In this study, we identified 40 compounds (98.6% of total peak area) in the essential oil via GC-MS analysis at the first time. Among them, five main compounds including Thymohydroquinone dimethyl ether (52.4%), (E)-α-Atlantone (9.0%), Neryl isovalerate (6.6%), Davanol D2 (isomer 2) (3.9%), and trans-Sesquisabinene hydrate (3.9%) have accounted for 75.8% of total peak area. The anti-bacterial activity of the essential oil against 4 microorganisms including Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa has also investigated via agar well diffusion assay. The results showed that the essential oil exhibited a strong antibacterial activity against Bacillus subtilis with the inhibition zones ranging from 8.2 to 18.7 mm. To elucidate the anti-bacterial effect mechanism of the essential oil, docking study of five main compounds of the essential oil (Thymohydroquinone dimethyl ether, (E)-α-Atlantone, Neryl isovalerate, Davanol D2 (isomer 2), and trans-Sesquisabinene hydrate) against some key proteins for bacterial growth such as DNA gyrase B, penicillin binding protein 2A, tyrosyl-tRNA synthetase, and dihydrofolate reductase were performed. The results showed that the main constituents of essential oil were highly bound with penicillin binding protein 2A with the free energies ranging -27.7 to -44.8 kcal/mol, which suggests the relationship between the antibacterial effect of essential oil and the affinity of main compounds with penicillin binding protein. In addition, the free energies of main compounds of the essential oil with human cyclooxygenase 1, cyclooxygenase 2, and phospholipase A2, the crucial proteins related with inflammatory response were less than diclofenac, a non-steroidal antiinflammatory drug. These findings propose the essential oil as a novel and promising anti-bacterial and anti-inflammatory medicine or cosmetic products.
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Antibacterianos , Bacillus subtilis , Hemiterpenos , Simulación del Acoplamiento Molecular , Aceites Volátiles , Ácidos Pentanoicos , Antibacterianos/farmacología , Antibacterianos/aislamiento & purificación , Antibacterianos/química , Aceites Volátiles/farmacología , Aceites Volátiles/química , Aceites Volátiles/aislamiento & purificación , Bacillus subtilis/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Tetrahidrofolato Deshidrogenasa/metabolismo , Girasa de ADN/metabolismo , Sesquiterpenos/aislamiento & purificación , Sesquiterpenos/farmacología , Pruebas de Sensibilidad Microbiana , Cromatografía de Gases y Espectrometría de MasasRESUMEN
DNA gyrase and topoisomerase IV play significant role in maintaining the correct structure of DNA during replication and they have been identified as validated targets in antibacterial drug discovery. Inadequate pharmacokinetic properties are responsible for many failures during drug discovery and their estimation in the early phase of this process maximizes the chance of getting useful drug candidates. Passive gastrointestinal absorption of a selected group of thirteen dual DNA gyrase and topoisomerase IV inhibitors was estimated using two in vitro tests - parallel artificial membrane permeability assay (PAMPA) and biopartitioning micellar chromatography (BMC). Due to good correlation between obtained results, passive gastrointestinal absorption of remaining ten compounds was estimated using only BMC. With this experimental setup, it was possible to identify compounds with high values of retention factors (k) and highest expected passive gastrointestinal absorption, and compounds with low values of k for which low passive gastrointestinal absorption is predicted. Quantitative structure-retention relationship (QSRR) modelling was performed by creating multiple linear regression (MLR), partial least squares (PLS) and support vector machines (SVM) models. Descriptors with the highest influence on retention factor were identified and their interpretation can be used for the design of new compounds with improved passive gastrointestinal absorption.
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Absorción Gastrointestinal , Relación Estructura-Actividad Cuantitativa , Inhibidores de Topoisomerasa II , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/farmacocinética , Micelas , Modelos Lineales , Membranas Artificiales , Girasa de ADN/metabolismo , Girasa de ADN/química , Humanos , Topoisomerasa de ADN IV/metabolismo , Topoisomerasa de ADN IV/antagonistas & inhibidores , Topoisomerasa de ADN IV/químicaRESUMEN
Cervimycins A-D are bis-glycosylated polyketide antibiotics produced by Streptomyces tendae HKI 0179 with bactericidal activity against Gram-positive bacteria. In this study, cervimycin C (CmC) treatment caused a spaghetti-like phenotype in Bacillus subtilis 168, with elongated curved cells, which stayed joined after cell division, and exhibited a chromosome segregation defect, resulting in ghost cells without DNA. Electron microscopy of CmC-treated Staphylococcus aureus (3 × MIC) revealed swollen cells, misshapen septa, cell wall thickening, and a rough cell wall surface. Incorporation tests in B. subtilis indicated an effect on DNA biosynthesis at high cervimycin concentrations. Indeed, artificial downregulation of the DNA gyrase subunit B gene (gyrB) increased the activity of cervimycin in agar diffusion tests, and, in high concentrations (starting at 62.5 × MIC), the antibiotic inhibited S. aureus DNA gyrase supercoiling activity in vitro. To obtain a more global view on the mode of action of CmC, transcriptomics and proteomics of cervimycin treated versus untreated S. aureus cells were performed. Interestingly, 3 × MIC of cervimycin did not induce characteristic responses, which would indicate disturbance of the DNA gyrase activity in vivo. Instead, cervimycin induced the expression of the CtsR/HrcA heat shock operon and the expression of autolysins, exhibiting similarity to the ribosome-targeting antibiotic gentamicin. In summary, we identified the DNA gyrase as a target, but at low concentrations, electron microscopy and omics data revealed a more complex mode of action of cervimycin, which comprised induction of the heat shock response, indicating protein stress in the cell.IMPORTANCEAntibiotic resistance of Gram-positive bacteria is an emerging problem in modern medicine, and new antibiotics with novel modes of action are urgently needed. Secondary metabolites from Streptomyces species are an important source of antibiotics, like the cervimycin complex produced by Streptomyces tendae HKI 0179. The phenotypic response of Bacillus subtilis and Staphylococcus aureus toward cervimycin C indicated a chromosome segregation and septum formation defect. This effect was at first attributed to an interaction between cervimycin C and the DNA gyrase. However, omics data of cervimycin treated versus untreated S. aureus cells indicated a different mode of action, because the stress response did not include the SOS response but resembled the response toward antibiotics that induce mistranslation or premature chain termination and cause protein stress. In summary, these results point toward a possibly novel mechanism that generates protein stress in the cells and subsequently leads to defects in cell and chromosome segregation.
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Antibacterianos , Bacillus subtilis , Pruebas de Sensibilidad Microbiana , Staphylococcus aureus , Streptomyces , Antibacterianos/farmacología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Streptomyces/genética , Streptomyces/metabolismo , Streptomyces/efectos de los fármacos , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Policétidos/farmacología , Policétidos/metabolismo , Glicósidos/farmacología , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Proteómica , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Girasa de ADN/genética , Girasa de ADN/metabolismoRESUMEN
INTRODUCTION: Campylobacteriosis stands as one of the most frequent bacterial gastroenteritis worldwide necessitating antibiotic treatment in severe cases and the rise of quinolones-resistant Campylobacter jejuni poses a significant challenge. The predominant mechanism of quinolones-resistance in this bacterium involves point mutations in the gyrA, resulting in amino acid substitution from threonine to isoleucine at 86th position, representing more than 90% of mutant DNA gyrase, and aspartic acid to asparagine at 90th position. WQ-3334, a novel quinolone, has demonstrated strong inhibitory activity against various bacteria. This study aims to investigate the effectiveness of WQ-3334, and its analogues, WQ-4064 and WQ-4065, with a unique modification in R1 against quinolones-resistant C. jejuni. METHODS: The structure-activity relationship of the examined drugs was investigated by measuring IC50 and their antimicrobial activities were accessed by MIC against C. jejuni strains. Additionally, in silico docking simulations were carried out using the crystal structure of the Escherichia coli DNA gyrase. RESULT: WQ-3334 exhibited the lowest IC50 against WT (0.188 ± 0.039 mg/L), T86I (11.0 ± 0.7 mg/L) and D90 N (1.60 ± 0.28 mg/L). Notably, DNA gyrases with T86I substitutions displayed the highest IC50 values among the examined WQ compounds. Moreover, WQ-3334 demonstrated the lowest MICs against wild-type and mutant strains. The docking simulations further confirmed the interactions between WQ-3334 and DNA gyrases. CONCLUSION: WQ-3334 with 6-amino-3,5-difluoropyridine-2-yl at R1 severed as a remarkable candidate for the treatment of foodborne diseases caused by quinolones-resistant C. jejuni as shown by the high inhibitory activity against both wild-type and the predominant quinolones-resistant strains.