RESUMEN
The development of antibacterial drugs based on novel chemotypes is essential to the future management of serious drug resistant infections. We herein report the design, synthesis and SAR of a novel series of N-ethylurea inhibitors based on a pyridine-3-carboxamide scaffold targeting the ATPase sub-unit of DNA gyrase. Consideration of structural aspects of the GyrB ATPase site has aided the development of this series resulting in derivatives that demonstrate excellent enzyme inhibitory activity coupled to potent Gram positive antibacterial efficacy.
Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Girasa de ADN/metabolismo , Diseño de Fármacos , Inhibidores de Topoisomerasa II/farmacología , Urea/análogos & derivados , Urea/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Bacterias/enzimología , Bacterias/metabolismo , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Inhibidores de Topoisomerasa II/síntesis química , Inhibidores de Topoisomerasa II/química , Urea/síntesis química , Urea/químicaRESUMEN
The discovery and optimisation of a new class of benzothiazole small molecules that inhibit bacterial DNA gyrase and topoisomerase IV are described. Antibacterial properties have been demonstrated by activity against DNA gyrase ATPase and potent activity against Staphylococcus aureus, Enterococcus faecalis, Streptococcus pyogenes and Haemophilus influenzae. Further refinements to the scaffold designed to enhance drug-likeness included analogues bearing an α-substituent to the carboxylic acid group, resulting in excellent solubility and favourable pharmacokinetic properties.
Asunto(s)
Benzotiazoles/química , Benzotiazoles/farmacología , Topoisomerasa de ADN IV/antagonistas & inhibidores , Diseño de Fármacos , Ácidos Isonipecóticos/química , Inhibidores de Topoisomerasa II/síntesis química , Inhibidores de Topoisomerasa II/farmacología , Animales , Antibacterianos/síntesis química , Antibacterianos/química , Antibacterianos/farmacocinética , Antibacterianos/farmacología , Benzotiazoles/síntesis química , Girasa de ADN/química , Girasa de ADN/metabolismo , Topoisomerasa de ADN IV/metabolismo , Enterococcus faecalis/efectos de los fármacos , Enterococcus faecalis/enzimología , Activación Enzimática/efectos de los fármacos , Haemophilus influenzae/efectos de los fármacos , Haemophilus influenzae/enzimología , Semivida , Ratones , Pruebas de Sensibilidad Microbiana , Ratas , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/enzimología , Streptococcus pyogenes/efectos de los fármacos , Streptococcus pyogenes/enzimología , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/farmacocinéticaRESUMEN
The type II topoisomerases DNA gyrase (GyrA/GyrB) and topoisomerase IV (ParC/ParE) are well-validated targets for antibacterial drug discovery. Because of their structural and functional homology, these enzymes are amenable to dual targeting by a single ligand. In this study, two novel benzothiazole ethyl urea-based small molecules, designated compound A and compound B, were evaluated for their biochemical, antibacterial, and pharmacokinetic properties. The two compounds inhibited the ATPase activity of GyrB and ParE with 50% inhibitory concentrations of <0.1 µg/ml. Prevention of DNA supercoiling by DNA gyrase was also observed. Both compounds potently inhibited the growth of a range of bacterial organisms, including staphylococci, streptococci, enterococci, Clostridium difficile, and selected Gram-negative respiratory pathogens. MIC90s against clinical isolates ranged from 0.015 µg/ml for Streptococcus pneumoniae to 0.25 µg/ml for Staphylococcus aureus. No cross-resistance with common drug resistance phenotypes was observed. In addition, no synergistic or antagonistic interactions between compound A or compound B and other antibiotics, including the topoisomerase inhibitors novobiocin and levofloxacin, were detected in checkerboard experiments. The frequencies of spontaneous resistance for S. aureus were <2.3 × 10(-10) with compound A and <5.8 × 10(-11) with compound B at concentrations equivalent to 8× the MICs. These values indicate a multitargeting mechanism of action. The pharmacokinetic properties of both compounds were profiled in rats. Following intravenous administration, compound B showed approximately 3-fold improvement over compound A in terms of both clearance and the area under the concentration-time curve. The measured oral bioavailability of compound B was 47.7%.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Benzotiazoles/farmacología , Topoisomerasa de ADN IV/antagonistas & inhibidores , ADN-Topoisomerasas de Tipo II/metabolismo , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Inhibidores de Topoisomerasa/farmacología , Urea/análogos & derivados , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Antibacterianos/química , Antibacterianos/farmacocinética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Benzotiazoles/química , Benzotiazoles/farmacocinética , Supervivencia Celular/efectos de los fármacos , Topoisomerasa de ADN IV/genética , Topoisomerasa de ADN IV/metabolismo , ADN-Topoisomerasas de Tipo II/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Bacterias Gramnegativas/enzimología , Bacterias Gramnegativas/crecimiento & desarrollo , Bacterias Grampositivas/enzimología , Bacterias Grampositivas/crecimiento & desarrollo , Células Hep G2 , Humanos , Interleucina-33 , Interleucinas , Levofloxacino/farmacología , Masculino , Pruebas de Sensibilidad Microbiana , Novobiocina/farmacología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Inhibidores de Topoisomerasa/química , Inhibidores de Topoisomerasa/farmacocinética , Urea/química , Urea/farmacocinética , Urea/farmacologíaRESUMEN
Bacteria use molecular machines or weapons to colonize, invade or fight other bacteria and eukaryotic cells. In addition to these various secretion systems, two different systems that release bacterial compounds have also been described. The first one corresponds to membrane vesicle formation and to long distance delivery of membrane or soluble components. The second system is dependent of the expression of the colicin lysis genes known for releasing cytoplasmic colicins as well as other soluble proteins. Both systems will be described thereafter.
Asunto(s)
Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos , Animales , Bacterias/genética , Infecciones Bacterianas/microbiología , Proteínas Bacterianas/genética , Colicinas/genética , Colicinas/metabolismo , Humanos , Transporte de ProteínasRESUMEN
The opportunistic human pathogen, Pseudomonas aeruginosa, is a major cause of infections in chronic wounds, burns and the lungs of cystic fibrosis patients. The P. aeruginosa genome encodes at least three proteins exhibiting the characteristic three domain structure of autotransporters, but much remains to be understood about the functions of these three proteins and their role in pathogenicity. Autotransporters are the largest family of secreted proteins in Gram-negative bacteria, and those characterised are virulence factors. Here, we demonstrate that the PA0328 autotransporter is a cell-surface tethered, arginine-specific aminopeptidase, and have defined its active site by site directed mutagenesis. Hence, we have assigned PA0328 with the name AaaA, for arginine-specific autotransporter of P. aeruginosa. We show that AaaA provides a fitness advantage in environments where the sole source of nitrogen is peptides with an aminoterminal arginine, and that this could be important for establishing an infection, as the lack of AaaA led to attenuation in a mouse chronic wound infection which correlated with lower levels of the cytokines TNFα, IL-1α, KC and COX-2. Consequently AaaA is an important virulence factor playing a significant role in the successful establishment of P. aeruginosa infections.
Asunto(s)
Aminopeptidasas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Infecciones por Pseudomonas/enzimología , Pseudomonas aeruginosa/patogenicidad , Factores de Virulencia/metabolismo , Infección de Heridas/enzimología , Aminopeptidasas/genética , Animales , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Enfermedad Crónica , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Péptidos/metabolismo , Infecciones por Pseudomonas/genética , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genética , Factores de Virulencia/genética , Infección de Heridas/genética , Infección de Heridas/microbiologíaRESUMEN
The Tol-Pal proteins of the cell envelope of Escherichia coli are required for maintaining outer membrane integrity. This system forms protein complexes in which TolA plays a central role by providing a bridge between the inner and outer membranes via its interaction with the Pal lipoprotein. The Tol proteins are parasitized by filamentous bacteriophages and group A colicins. The N-terminal domain of the Ff phage g3p protein and the translocation domains of colicins interact directly with TolA during the processes of import through the cell envelope. Recently, a four-amino-acid sequence in Pal has been shown to be involved in Pal's interaction with TolA. A similar motif is also present in the sequence of two TolA partners, g3p and colicin A. Here, a mutational study was conducted to define the function of these motifs in the binding activity and import process of TolA. The various domains were produced and exported to the bacterial periplasm, and their cellular effects were analyzed. Cells producing the g3p domain were tolerant to colicins and filamentous phages and had destabilized outer membranes, while g3p deleted of three residues in the motif was affected in TolA binding and had no effect on cell integrity or colicin or phage import. A conserved Tyr residue in the colicin A translocation domain was involved in TolA binding and colicin A import. Furthermore, in vivo and in vitro coprecipitation analyses demonstrated that colicin A and g3p N-terminal domains compete for binding to TolA.
Asunto(s)
Secuencias de Aminoácidos , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiología , Transporte de Proteínas , Secuencia de Aminoácidos , Transporte Biológico , Colicinas/genética , Colicinas/metabolismo , Secuencia Conservada , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/aislamiento & purificación , Proteínas de Escherichia coli/fisiología , Inovirus/genética , Inovirus/fisiología , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Unión Proteica , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Outer membrane vesicle formation occurs during Gram-negative bacterial growth. However, natural production of large amounts of outer membrane vesicles has only been described in a few bacterial genera. The purified vesicles of some bacterial pathogens have shown potential applications in vaccinology and in antibiotic therapy. This study focused on the development of a gene expression system able to induce production of large amounts of outer membrane vesicles. The Tol-Pal system of Escherichia coli, required to maintain outer membrane integrity, is composed of five cell envelope proteins, TolA, TolB, TolQ, TolR and Pal. Tol proteins are parasitized by filamentous bacteriophages and by colicins. The phage infection process and colicin import require, respectively, the N-terminal domain of the minor coat g3p protein and the translocation domain of colicins, with both domains interacting with Tol proteins. In this study, we show that the periplasmic production of either Tol, g3p or colicin domains was able to specifically destabilize the E. coli or Shigella flexneri cell envelope and to induce production of high amounts of vesicles. This technique was further found to work efficiently in Salmonella enterica serovar Typhimurium.