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1.
Acta Crystallogr F Struct Biol Commun ; 71(Pt 10): 1242-6, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26457513

RESUMEN

Fluoroquinolone drugs such as moxifloxacin kill bacteria by stabilizing the normally transient double-stranded DNA breaks created by bacterial type IIA topoisomerases. Previous crystal structures of Staphylococcus aureus DNA gyrase with asymmetric DNAs have had static disorder (with the DNA duplex observed in two orientations related by the pseudo-twofold axis of the complex). Here, 20-base-pair DNA homoduplexes were used to obtain crystals of covalent DNA-cleavage complexes of S. aureus DNA gyrase. Crystals with QPT-1, moxifloxacin or etoposide diffracted to between 2.45 and 3.15 Šresolution. A G/T mismatch introduced at the ends of the DNA duplexes facilitated the crystallization of slightly asymmetric complexes of the inherently flexible DNA-cleavage complexes.


Asunto(s)
División del ADN , Girasa de ADN/química , Etopósido/química , Fluoroquinolonas/química , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos de Espiro/química , Staphylococcus aureus/enzimología , Secuencia de Bases , Cristalización , Cristalografía por Rayos X , Datos de Secuencia Molecular , Moxifloxacino
2.
Nat Commun ; 6: 10048, 2015 Dec 07.
Artículo en Inglés | MEDLINE | ID: mdl-26640131

RESUMEN

New antibacterials are needed to tackle antibiotic-resistant bacteria. Type IIA topoisomerases (topo2As), the targets of fluoroquinolones, regulate DNA topology by creating transient double-strand DNA breaks. Here we report the first co-crystal structures of the antibacterial QPT-1 and the anticancer drug etoposide with Staphylococcus aureus DNA gyrase, showing binding at the same sites in the cleaved DNA as the fluoroquinolone moxifloxacin. Unlike moxifloxacin, QPT-1 and etoposide interact with conserved GyrB TOPRIM residues rationalizing why QPT-1 can overcome fluoroquinolone resistance. Our data show etoposide's antibacterial activity is due to DNA gyrase inhibition and suggests other anticancer agents act similarly. Analysis of multiple DNA gyrase co-crystal structures, including asymmetric cleavage complexes, led to a 'pair of swing-doors' hypothesis in which the movement of one DNA segment regulates cleavage and religation of the second DNA duplex. This mechanism can explain QPT-1's bacterial specificity. Structure-based strategies for developing topo2A antibacterials are suggested.


Asunto(s)
Antibacterianos/química , Antineoplásicos/química , Girasa de ADN/química , Etopósido/química , Fluoroquinolonas/química , Staphylococcus aureus/enzimología , Inhibidores de Topoisomerasa II/química , Antibacterianos/farmacología , Antineoplásicos/farmacología , Girasa de ADN/genética , Girasa de ADN/metabolismo , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Farmacorresistencia Bacteriana , Etopósido/farmacología , Fluoroquinolonas/farmacología , Modelos Moleculares , Estructura Molecular , Moxifloxacino , Staphylococcus aureus/química , Staphylococcus aureus/efectos de los fármacos , Inhibidores de Topoisomerasa II/farmacología
3.
J Med Chem ; 45(10): 1959-62, 2002 May 09.
Artículo en Inglés | MEDLINE | ID: mdl-11985462

RESUMEN

Potent nanomolar inhibitors of Staphylococcus aureus methionyl tRNA synthetase have been derived from a file compound high throughput screening hit. Optimized compounds show excellent antibacterial activity against staphylococcal and enterococcal pathogens, including strains resistant to clinical antibiotics. Compound 11 demonstrated in vivo efficacy in an S. aureus rat abscess infection model.


Asunto(s)
Antibacterianos/síntesis química , Enterococcus/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Metionina-ARNt Ligasa/antagonistas & inhibidores , Quinolonas/síntesis química , Staphylococcus/efectos de los fármacos , Absceso/tratamiento farmacológico , Absceso/microbiología , Animales , Antibacterianos/química , Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Quinolonas/química , Quinolonas/farmacología , Ratas , Ratas Sprague-Dawley , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/enzimología , Relación Estructura-Actividad
4.
J Biotechnol ; 109(1-2): 201-11, 2004 Apr 08.
Artículo en Inglés | MEDLINE | ID: mdl-15063628

RESUMEN

Building on the basic design concepts of Randers-Eichhorn [Biotechnol. Bioeng. 55 (1997) 921], an on-line, real-time robust, steam sterilisable optical sensor for monitoring green fluorescent protein (GFP) has been developed. A general cloning vector for fusion expression proteins was constructed, allowing expression of both GFP and the target protein as a fusion. Cultivations were carried out at the 20l scale with the signal from the sensor being relayed directly to the control system of the bioreactors. The production of GFP was then measured on-line, the signal was interfaced directly with other controlling parameters, thereby allowing the microbial process to be controlled directly based on recombinant protein expression. A positive expression correlation between on-line and off-line data was obtained. Protein accretion measured off-line was quantified using both LC-MS and plate reader assays. The potential of such a sensor for many aspects of process development is considerable and we have developed a working system which allows the optimisation of production conditions, for example, linking pH control directly to the fusion protein. Results are also presented that illustrate GFP does not alter the cultivation characteristics of the target protein when compared to the native construct. Whether GFP expressed as a fusion influences the solubility of the target protein is also discussed.


Asunto(s)
Técnicas Biosensibles/instrumentación , Fermentación , Vectores Genéticos/genética , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/biosíntesis
5.
Nat Struct Mol Biol ; 17(9): 1152-3, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20802486

RESUMEN

Quinolone antibacterials have been used to treat bacterial infections for over 40 years. A crystal structure of moxifloxacin in complex with Acinetobacter baumannii topoisomerase IV now shows the wedge-shaped quinolone stacking between base pairs at the DNA cleavage site and binding conserved residues in the DNA cleavage domain through chelation of a noncatalytic magnesium ion. This provides a molecular basis for the quinolone inhibition mechanism, resistance mutations and invariant quinolone antibacterial structural features.


Asunto(s)
Acinetobacter baumannii/enzimología , Topoisomerasa de ADN IV/química , Inhibidores Enzimáticos/química , Quinolonas/química , Topoisomerasa de ADN IV/farmacología , Inhibidores Enzimáticos/farmacología , Modelos Moleculares , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Quinolonas/farmacología
6.
Bioorg Med Chem Lett ; 15(9): 2305-9, 2005 May 02.
Artículo en Inglés | MEDLINE | ID: mdl-15837314

RESUMEN

High throughput screening of Staphylococcus aureus phenylalanyl tRNA synthetase (FRS) identified ethanolamine 1 as a sub-micromolar hit. Optimisation studies led to the enantiospecific lead 64, a single-figure nanomolar inhibitor. The inhibitor series shows selectivity with respect to the mammalian enzyme and the potential for broad spectrum bacterial FRS inhibition.


Asunto(s)
Antibacterianos/síntesis química , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Etanolaminas/síntesis química , Etanolaminas/farmacología , Fenilalanina-ARNt Ligasa/antagonistas & inhibidores , Staphylococcus aureus/enzimología , Animales , Antibacterianos/farmacología , Diseño de Fármacos , Cinética , Mamíferos , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Sensibilidad y Especificidad , Staphylococcus aureus/efectos de los fármacos , Relación Estructura-Actividad
7.
Mol Microbiol ; 49(5): 1191-200, 2003 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-12940980

RESUMEN

Two-component signal transduction (TCST) pathways are regulatory systems that are highly homologous throughout the bacterial kingdom. Their established role in virulence and absence in vertebrates has made TCST an attractive target for therapeutic intervention. However, such systems have yet to yield success in the development of novel antibiotics. CheY serves as a prototype for the analysis of response regulator function. The protein structure exhibits several conformations by both X-ray and nuclear magnetic resonance (NMR) analyses. Knowledge of which structures are relevant in vivo would be valuable in a rational drug design project. Our aim was to probe the in vivo conformation and ligand binding of CheY in Escherichia coli under resting conditions by in-cell NMR methods. CheY was selectively labelled with 15N by the control of growth and expression conditions. NMR spectra obtained in vivo demonstrated that the Mg2+ complex was the predominant form even though cells were resuspended in metal-free buffers and the intracellular free Mg2+ was low. In-cell NMR also confirmed the uptake and in vivo binding mode to CheY of small-molecular-weight compounds identified in vitro. This paper reports the first observation of the structure and interactions with a potential drug of a regulator protein in its native host in vivo using NMR spectroscopy.


Asunto(s)
Proteínas Bacterianas , Escherichia coli/química , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Conformación Proteica , Cationes Bivalentes/metabolismo , Quimiotaxis , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Ligandos , Magnesio/metabolismo , Proteínas Quimiotácticas Aceptoras de Metilo , Modelos Moleculares , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Transducción de Señal
8.
Bioorg Med Chem Lett ; 12(21): 3171-4, 2002 Nov 04.
Artículo en Inglés | MEDLINE | ID: mdl-12372526

RESUMEN

The antimicrobial natural product chuangxinmycin has been found to be a potent and selective inhibitor of bacterial tryptophanyl tRNA synthetase (WRS). A number of analogues have been synthesised. The interaction with WRS appears to be highly constrained, as only sterically smaller analogues afforded significant inhibition. The only analogue to show inhibition comparable to chuangxinmycin also had antibacterial activity. WRS inhibition may contribute to the antibacterial action of chuangxinmycin.


Asunto(s)
Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Staphylococcus aureus/enzimología , Triptófano-ARNt Ligasa/antagonistas & inhibidores , Antibacterianos/síntesis química , Bacterias/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Hidrólisis , Indicadores y Reactivos , Indoles/síntesis química , Pruebas de Sensibilidad Microbiana , Staphylococcus aureus/efectos de los fármacos , Estereoisomerismo , Relación Estructura-Actividad
9.
Antimicrob Agents Chemother ; 46(10): 3118-24, 2002 10.
Artículo en Inglés | MEDLINE | ID: mdl-12234833

RESUMEN

Bacterial enoyl-acyl carrier protein (ACP) reductase (FabI) catalyzes the final step in each elongation cycle of bacterial fatty acid biosynthesis and is an attractive target for the development of new antibacterial agents. High-throughput screening of the Staphylococcus aureus FabI enzyme identified a novel, weak inhibitor with no detectable antibacterial activity against S. aureus. Iterative medicinal chemistry and X-ray crystal structure-based design led to the identification of compound 4 [(E)-N-methyl-N-(2-methyl-1H-indol-3-ylmethyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide], which is 350-fold more potent than the original lead compound obtained by high-throughput screening in the FabI inhibition assay. Compound 4 has exquisite antistaphylococci activity, achieving MICs at which 90% of isolates are inhibited more than 500 times lower than those of nine currently available antibiotics against a panel of multidrug-resistant strains of S. aureus and Staphylococcus epidermidis. Furthermore, compound 4 exhibits excellent in vivo efficacy in an S. aureus infection model in rats. Biochemical and genetic approaches have confirmed that the mode of antibacterial action of compound 4 and related compounds is via inhibition of FabI. Compound 4 also exhibits weak FabK inhibitory activity, which may explain its antibacterial activity against Streptococcus pneumoniae and Enterococcus faecalis, which depend on FabK and both FabK and FabI, respectively, for their enoyl-ACP reductase function. These results show that compound 4 is representative of a new, totally synthetic series of antibacterial agents that has the potential to provide novel alternatives for the treatment of S. aureus infections that are resistant to our present armory of antibiotics.


Asunto(s)
Antibacterianos , Inhibidores Enzimáticos , Oxidorreductasas/antagonistas & inhibidores , Animales , Antibacterianos/química , Antibacterianos/farmacocinética , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Farmacorresistencia Bacteriana Múltiple , Enoil-ACP Reductasa (NADH) , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/enzimología , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Ratas , Ratas Sprague-Dawley , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/enzimología , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/enzimología , Relación Estructura-Actividad
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