RESUMEN
IMPORTANCE: African swine fever virus (ASFV) is a highly fatal swine disease that severely affects the pig industry. Although ASFV has been prevalent for more than 100 years, effective vaccines or antiviral strategies are still lacking. In this study, we identified four Bacillus subtilis strains that inhibited ASFV proliferation in vitro. Pigs fed with liquid biologics or powders derived from four B. subtilis strains mixed with pellet feed showed reduced morbidity and mortality when challenged with ASFV. Further analysis showed that the antiviral activity of B. subtilis was based on its metabolites arctiin and genistein interfering with the function of viral topoisomerase II. Our findings offer a promising new strategy for the prevention and control of ASFV that may significantly alleviate the economic losses in the pig industry.
Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Bacillus subtilis , Animales , Fiebre Porcina Africana/prevención & control , Antivirales/farmacología , ADN-Topoisomerasas de Tipo II/farmacología , Genisteína/farmacología , PorcinosRESUMEN
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.
Asunto(s)
Topoisomerasa de ADN IV , Neisseria gonorrhoeae , Humanos , Girasa de ADN/metabolismo , Roturas del ADN de Doble Cadena , Inhibidores de Topoisomerasa II/farmacología , Inhibidores de Topoisomerasa II/químicaRESUMEN
Type II topoisomerases are essential enzymes that modulate the topological state of DNA supercoiling in all living organisms. These enzymes alter DNA topology by performing double-stranded passage reactions on over- or underwound DNA substrates. This strand passage reaction generates a transient covalent enzyme-cleaved DNA structure known as the cleavage complex. Al-though the cleavage complex is a requisite catalytic intermediate, it is also intrinsically dangerous to genomic stability in biological systems. The potential threat of type II topoisomerase function can also vary based on the nature of the supercoiled DNA substrate. During essential processes such as DNA replication and transcription, cleavage complex formation can be inherently more dangerous on overwound versus underwound DNA substrates. As such, it is important to understand the profound effects that DNA topology can have on the cellular functions of type II topoisomerases. This review will provide a broad assessment of how human and bacterial type II topoisomerases recognize and act on their substrates of various topological states.
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ADN-Topoisomerasas de Tipo II , Lateralidad Funcional , Humanos , ADN-Topoisomerasas de Tipo II/metabolismo , ADN , Isomerasas/genética , Replicación del ADN , ADN-Topoisomerasas de Tipo I/metabolismoRESUMEN
The global increase in multidrug-resistant pathogens has caused severe problems in the treatment of infections. To overcome these difficulties, the advent of a new chemical class of antibacterial drug is eagerly desired. We aimed at creating novel antibacterial agents against bacterial type II topoisomerases, which are well-validated targets. TP0480066 (compound 32) has been identified by using structure-based optimization originated from lead compound 1, which was obtained as a result of our previous lead identification studies. The MIC90 values of TP0480066 against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), and genotype penicillin-resistant Streptococcus pneumoniae (gPRSP) were 0.25, 0.015, and 0.06 µg/mL, respectively. Hence, TP0480066 can be regarded as a promising antibacterial drug candidate of this chemical class.
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Antibacterianos/farmacología , ADN-Topoisomerasas de Tipo II/metabolismo , Quinolinas/farmacología , Inhibidores de Topoisomerasa II/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Farmacorresistencia Bacteriana/efectos de los fármacos , Células Hep G2 , Humanos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/enzimología , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Quinolinas/síntesis química , Quinolinas/química , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/enzimología , Relación Estructura-Actividad , Inhibidores de Topoisomerasa II/síntesis química , Inhibidores de Topoisomerasa II/química , Regulador Transcripcional ERG/antagonistas & inhibidores , Regulador Transcripcional ERG/metabolismo , Enterococos Resistentes a la Vancomicina/efectos de los fármacos , Enterococos Resistentes a la Vancomicina/enzimologíaRESUMEN
Chemical diversification of type II topoisomerase (Topo II) inhibitors remains indispensable to extend their anti-tumor therapeutic values which are limited by their side effects. Herein, we designed and synthesized a novel series of benzimidazole-chalcone hybrids (BCHs). These BCHs showed good inhibitory effect in the Topo II mediated DNA relaxation assay and anti-proliferative effect in 4 tumor cell lines. 4d and 4n were the most potent, with IC50 values less than 5 µM, superior to etoposide. Mechanistic studies indicated that the BCHs functioned as non-intercalative Topo II catalytic inhibitors. Moreover, 4d and 4n demonstrated versatile properties against tumors, including inhibition on the colony formation and cell migration, and promotion of apoptosis of A549 cells. The structure-activity relationship and molecular docking analysis suggested possible contribution of the chalcone motif to the Topo II inhibitory and anti-proliferative potency. These results indicated that 4d and 4n could be promising lead compounds for further anti-tumor drug research.
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Antineoplásicos , Diseño de Fármacos , Neoplasias/tratamiento farmacológico , Inhibidores de Topoisomerasa II , Células A549 , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Bencimidazoles/química , Proliferación Celular/efectos de los fármacos , Chalcona/química , Evaluación Preclínica de Medicamentos , Humanos , Simulación del Acoplamiento Molecular , Relación Estructura-Actividad , Inhibidores de Topoisomerasa II/síntesis química , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
An unrepaired DNA double-strand break (DSB) is lethal to cells. In bacteria, DSBs are usually repaired either via an error-prone pathway, which ligates the ends of the break or an accurate recombination pathway. Due to this lethality, drugs that induce persistent DSBs have been successful in bacterial infection treatment. However, recurrent usage of these drugs has led to emergence of resistant strains. Several articles have thoroughly reviewed the causes, mechanisms and effects of bacterial drug resistance while others have also discussed approaches for facilitating drug discovery and development. Here, we focus on a hypothetical chemotherapeutic strategy that can be explored for minimizing development of resistance to novel DSB-inducing compounds. We also highlight the possibility of utilizing bacterial DSB repair pathways as targets for the discovery and development of novel antibiotics.
RESUMEN
We use genetic assays to suggest that transcription-coupled repair or new origin formation in Escherichia coli involves removal of RNAP to create an RNA primer for DNA synthesis. Transcription factor DksA was shown to play a role in numerous reactions involving RNA polymerase. Some, but not all, of the activities of DksA at promoters or during transcription elongation require (p)ppGpp. In addition to its role during transcription, DksA is also involved in maintaining genome integrity. Cells lacking DksA are sensitive to multiple DNA damaging agents including UV light, ionizing radiation, mitomycin C, and nalidixic acid. Here, we focus on two recent studies addressing the importance of DksA in the repair of double-strand breaks (DSBs), one by Sivaramakrishnan et al. (Nature 550:214-218, 2017) and one originating in our laboratory, Myka et al. (Mol Microbiol 111:1382-1397. https://doi.org/10.1111/mmi.14227 , 2019). It appears that depending on the type and possibly location of DNA damage, DksA can play either a passive or an active role in DSB repair. The passive role relies on exclusion of anti-backtracking factors from the RNAP secondary channel. The exact mechanism of active DksA-mediated DNA repair is unknown. However, DksA was proposed to destabilize transcription complexes, thus clearing the way for recombination and DNA repair. Based on the requirement for DksA, both in repair of DSBs and the R-loop-dependent formation of new origins of DNA replication, we propose that DksA may allow for removal of RNAP without unwinding of the RNA:DNA hybrid, which can then be extended by a DNA polymerase. This mechanism obviates the need for RNAP backtracking to repair damaged DNA.
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Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Roturas del ADN de Doble Cadena/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas de Escherichia coli/genética , Guanosina Pentafosfato/metabolismo , Ácido Nalidíxico/farmacología , Fleomicinas/farmacología , Regiones Promotoras Genéticas , Factores de Transcripción/genéticaRESUMEN
Gyrase is a type IIA topoisomerase that catalyzes negative supercoiling of DNA. The enzyme consists of two GyrA and two GyrB subunits. It is believed to introduce negative supercoils into DNA by converting a positive DNA node into a negative node through strand passage: First, it cleaves both DNA strands of a double-stranded DNA, termed the G-segment, and then it passes a second segment of the same DNA molecule, termed the T-segment, through the gap created. As a two-fold symmetric enzyme, gyrase contains two copies of all elements that are key for the supercoiling reaction: The GyrB subunits provide two active sites for ATP binding and hydrolysis. The GyrA subunits contain two C-terminal domains (CTDs) for DNA binding and wrapping to stabilize the positive DNA node, and two catalytic tyrosines for DNA cleavage. While the presence of two catalytic tyrosines has been ascribed to the necessity of cleaving both strands of the G-segment to enable strand passage, the role of the two ATP hydrolysis events and of the two CTDs has been less clear. This review summarizes recent results on the role of these duplicate elements for individual steps of the supercoiling reaction, and discusses the implications for the mechanism of DNA supercoiling.
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Girasa de ADN/metabolismo , ADN/genética , ADN/metabolismo , Conformación de Ácido Nucleico , Animales , ADN/química , Girasa de ADN/química , ADN-Topoisomerasas de Tipo II/química , ADN-Topoisomerasas de Tipo II/metabolismo , Humanos , Subunidades de Proteína/metabolismo , Relación Estructura-ActividadRESUMEN
High-level fluoroquinolone resistance is conferred by the mutation of conserved serine and acidic amino acids in the quinolone resistance-determining region (QRDR) of the A subunits of the type II topoisomerases, DNA gyrase (GyrA) and topoisomerase IV (ParC). In Japan, fluoroquinolone-resistant Enterococcus faecium continues to emerge in clinical settings. We analyzed 131 Japanese E. faecium clinical isolates for susceptibility to levofloxacin (LVFX), and QRDR mutational status. The bacterial collection had a high percentage of resistance (79%) and showed elevated drug minimal inhibitory concentrations (MICs). Eighty-three isolates had single or combined mutations in gyrA and/or parC; all were resistant to LVFX. A strong correlation was evident between log-transformed MICs and the total number of QRDR mutations (r = 0.7899), confirming the involvement of QRDR mutations in drug resistance, as previously described. Three-dimensional modeling indicated that the amino acid change(s) in QRDR could disrupt the interaction between the enzymes and drugs: the most common cause of quinolone resistance. Interestingly, eight isolates had a single mutation on gyrA and exhibited significantly reduced susceptibility. These data imply that either DNA gyrase or topoisomerase IV can be the primary target of fluoroquinolones, although topoisomerase IV is commonly thought to be the primary target in gram-positive bacteria.
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Antibacterianos/química , Girasa de ADN/genética , Topoisomerasa de ADN IV/genética , Farmacorresistencia Bacteriana/genética , Enterococcus faecium/genética , Mutación , Secuencia de Aminoácidos , Antibacterianos/metabolismo , Antibacterianos/farmacología , Sitios de Unión , Girasa de ADN/química , Girasa de ADN/metabolismo , Topoisomerasa de ADN IV/química , Topoisomerasa de ADN IV/metabolismo , Enterococcus faecium/efectos de los fármacos , Enterococcus faecium/enzimología , Enterococcus faecium/aislamiento & purificación , Fluoroquinolonas/farmacología , Expresión Génica , Infecciones por Bacterias Grampositivas/microbiología , Infecciones por Bacterias Grampositivas/patología , Humanos , Japón , Levofloxacino , Pruebas de Sensibilidad Microbiana , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Alineación de Secuencia , Homología Estructural de ProteínaRESUMEN
We study the elasticity of DNA based on local principal axes of bending identified from over 0.9-µs all-atom molecular dynamics simulations of DNA oligos. The calculated order parameters describe motion of DNA as an elastic rod. In 10 possible dinucleotide steps, bending about the two principal axes is anisotropic yet linearly elastic. Twist about the centroid axis is largely decoupled from bending, but DNA tends to overtwist for unbending beyond the typical range of thermal motion, which is consistent with experimentally observed twist-stretch coupling. The calculated elastic stiffness of dinucleotide steps yield sequence-dependent persistence lengths consistent with previous single-molecule experiments, which is further analyzed by performing coarse-grained simulations of DNA. Flexibility maps of oligos constructed from simulation also match with those from the precalculated stiffness of dinucleotide steps. These support the premise that base pair interaction at the dinucleotide-level is mainly responsible for the elasticity of DNA. Furthermore, we analyze 1381 crystal structures of protein-DNA complexes. In most structures, DNAs are mildly deformed and twist takes the highest portion of the total elastic energy. By contrast, in structures with the elastic energy per dinucleotide step greater than about 4.16 kBT (kBT: thermal energy), the major bending becomes dominant. The extensional energy of dinucleotide steps takes at most 35% of the total elastic energy except for structures containing highly deformed DNAs where linear elasticity breaks down. Such partitioning between different deformational modes provides quantitative insights into the conformational dynamics of DNA as well as its interaction with other molecules and surfaces.
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ADN-Topoisomerasas de Tipo II/química , Proteínas de Unión al ADN/química , ADN/química , Oligodesoxirribonucleótidos/química , Emparejamiento Base , Bases de Datos Factuales , Elasticidad , Humanos , Simulación de Dinámica Molecular , Movimiento (Física) , Conformación de Ácido Nucleico , TermodinámicaRESUMEN
Precise control of supercoiling homeostasis is critical to DNA-dependent processes such as gene expression, replication, and damage response. Topoisomerases are central regulators of DNA supercoiling commonly thought to act independently in the recognition and modulation of chromosome superstructure; however, recent evidence has indicated that cells tightly regulate topoisomerase activity to support chromosome dynamics, transcriptional response, and replicative events. How topoisomerase control is executed and linked to the internal status of a cell is poorly understood. To investigate these connections, we determined the structure of Escherichia coli gyrase, a type IIA topoisomerase bound to YacG, a recently identified chromosomally encoded inhibitor protein. Phylogenetic analyses indicate that YacG is frequently associated with coenzyme A (CoA) production enzymes, linking the protein to metabolism and stress. The structure, along with supporting solution studies, shows that YacG represses gyrase by sterically occluding the principal DNA-binding site of the enzyme. Unexpectedly, YacG acts by both engaging two spatially segregated regions associated with small-molecule inhibitor interactions (fluoroquinolone antibiotics and the newly reported antagonist GSK299423) and remodeling the gyrase holoenzyme into an inactive, ATP-trapped configuration. This study establishes a new mechanism for the protein-based control of topoisomerases, an approach that may be used to alter supercoiling levels for responding to changes in cellular state.
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Girasa de ADN/metabolismo , Escherichia coli/enzimología , Modelos Moleculares , ADN Bacteriano/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Unión Proteica , Estructura Cuaternaria de ProteínaRESUMEN
OBJECTIVES: Francisella tularensis, a CDC class A potential bioterrorism agent, is a Gram-negative bacterium responsible for tularaemia. Understanding the mechanisms of resistance to antibiotics used as first-line treatment is of major security relevance. METHODS: We propagated the three parental reference strains Francisella tularensis subsp. holarctica live vaccine strain, Francisella novicida and Francisella philomiragia with increasing concentrations of ciprofloxacin, a fluoroquinolone used as curative and prophylactic treatment for tularaemia. This evolution procedure provided us with high-level ciprofloxacin-resistant mutants and all evolutionary intermediates towards high-level resistance. We determined the resistance levels to other fluoroquinolones (levofloxacin and moxifloxacin) and other antibiotic families (aminoglycosides, tetracyclines and macrolides) and characterized the genetic changes in the fluoroquinolone target genes encoding DNA gyrase and topoisomerase IV. RESULTS: All high-level resistant mutants shared cross-resistance to the tested fluoroquinolones, while some also revealed striking levels of cross-resistance to other clinically relevant antibiotic classes. High-level resistant mutants carried one to three mutations, including some not previously reported. We mapped all mutations onto known topoisomerase three-dimensional structures. Along the pathways towards high-level resistance, we identified complex evolutionary trajectories including polymorphic states and additional resistance mechanisms likely to be associated with efflux processes. CONCLUSIONS: Our data demonstrated the efficiency and speed of in vitro production of mutants highly resistant to fluoroquinolones in Francisella species. They emphasize the urgent need to identify all antibiotic resistance mechanisms in these species, develop molecular tools for their detection and design new therapeutic alternatives for tularaemia.
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Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Fluoroquinolonas/farmacología , Francisella/efectos de los fármacos , Girasa de ADN/genética , Análisis Mutacional de ADN , Topoisomerasa de ADN IV/genética , Francisella/enzimología , Francisella/genética , Francisella/crecimiento & desarrollo , Humanos , Pruebas de Sensibilidad Microbiana , Selección Genética , Pase SeriadoRESUMEN
Mycobacterium tuberculosis DNA gyrase, a nanomachine involved in the regulation of DNA topology, is the only type II topoisomerase present in this organism and hence is the sole target of fluoroquinolones in the treatment of tuberculosis. The ATPase domain provides the energy required for catalysis by ATP hydrolysis. Two constructs corresponding to this 43 kDa domain, Mtb-GyrB47(C1) and Mtb-GyrB47(C2), have been overproduced, purified and crystallized. Diffraction data were collected from three crystal forms. The crystals belonged to space groups P1 and P21 and diffracted to resolutions of 2.9 and 3.3 Å, respectively.