RESUMEN
Many drug candidates fail in clinical trials owing to a lack of efficacy from limited target engagement or an insufficient therapeutic index. Minimizing off-target effects while retaining the desired pharmacodynamic (PD) response can be achieved by reduced exposure for drugs that display kinetic selectivity in which the drug-target complex has a longer half-life than off-target-drug complexes. However, though slow-binding inhibition kinetics are a key feature of many marketed drugs, prospective tools that integrate drug-target residence time into predictions of drug efficacy are lacking, hindering the integration of drug-target kinetics into the drug discovery cascade. Here we describe a mechanistic PD model that includes drug-target kinetic parameters, including the on- and off-rates for the formation and breakdown of the drug-target complex. We demonstrate the utility of this model by using it to predict dose response curves for inhibitors of the LpxC enzyme from Pseudomonas aeruginosa in an animal model of infection.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Ácidos Hidroxámicos/farmacología , Treonina/análogos & derivados , Animales , Antibacterianos/química , Antibacterianos/farmacocinética , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/farmacocinética , Cinética , Ratones Endogámicos , Pruebas de Sensibilidad Microbiana , Modelos Biológicos , Estructura Molecular , Unión Proteica , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/enzimología , Treonina/química , Treonina/farmacocinética , Treonina/farmacología , Factores de TiempoRESUMEN
In Gram-negative bacteria, the cell wall is surrounded by an outer membrane, the outer leaflet of which is comprised of charged lipopolysaccharide (LPS) molecules. Lipid A, a component of LPS, anchors this molecule to the outer membrane. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a zinc-dependent metalloamidase that catalyzes the first committed step of biosynthesis of Lipid A, making it a promising target for antibiotic therapy. Formation of soluble aggregates of Pseudomonas aeruginosa LpxC protein when overexpressed in Escherichia coli has limited the availability of high quality protein for X-ray crystallography. Expression of LpxC in the presence of an inhibitor dramatically increased protein solubility, shortened crystallization time and led to a high-resolution crystal structure of LpxC bound to the inhibitor. However, this approach required large amounts of compound, restricting its use. To reduce the amount of compound needed, an overexpression strain of E. coli was created lacking acrB, a critical component of the major efflux pump. By overexpressing LpxC in the efflux deficient strain in the presence of LpxC inhibitors, several structures of P. aeruginosa LpxC in complex with different compounds were solved to accelerate structure-based drug design.
Asunto(s)
Amidohidrolasas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Pseudomonas aeruginosa/enzimología , Amidohidrolasas/antagonistas & inhibidores , Amidohidrolasas/genética , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Catálisis , Cromatografía Liquida , Cristalografía por Rayos X , Escherichia coli , Expresión Génica , Espectrometría de Masas , Conformación Proteica , Zinc/química , Zinc/metabolismoRESUMEN
The antimicrobial activity of phenyl-thiazolylurea-sulfonamides against Staphylococcus aureus PheRS are dependent upon phenylalanine levels in the extracellular fluids. Inhibitor efficacy in animal models of infection is substantially diminished by dietary phenylalanine intake, thereby reducing the perceived clinical utility of this inhibitor class. The search for novel antibacterial compounds against Gram-negative pathogens led to a re-evaluation of this phenomenon, which is shown here to be unique to S. aureus. Inhibition of macromolecular syntheses and characterization of novel resistance mutations in Escherichia coli demonstrate that antimicrobial activity of phenyl-thiazolylurea-sulfonamides is mediated by PheRS inhibition, validating this enzyme as a viable drug discovery target for Gram-negative pathogens. A search for novel inhibitors of PheRS yielded three novel chemical starting points. NMR studies were used to confirm direct target engagement for phenylalanine-competitive hits. The crystallographic structure of Pseudomonas aeruginosa PheRS defined the binding modes of these hits and revealed an auxiliary hydrophobic pocket that is positioned adjacent to the phenylalanine binding site. Three viable inhibitor-resistant mutants were mapped to this pocket, suggesting that this region is a potential liability for drug discovery.
Asunto(s)
Antibacterianos/farmacología , Bacterias Gramnegativas/enzimología , Fenilalanina-ARNt Ligasa/metabolismo , Sitios de Unión , Farmacorresistencia Bacteriana , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/genética , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Fenilalanina-ARNt Ligasa/química , Sulfonamidas/farmacologíaRESUMEN
Inhibitors of 4'-phosphopantetheine adenylyltransferase (PPAT) were identified through high-throughput screening of the AstraZeneca compound library. One series, cycloalkyl pyrimidines, showed inhibition of PPAT isozymes from several species, with the most potent inhibition of enzymes from Gram-positive species. Mode-of-inhibition studies with Streptococcus pneumoniae and Staphylococcus aureus PPAT demonstrated representatives of this series to be reversible inhibitors competitive with phosphopantetheine and uncompetitive with ATP, binding to the enzyme-ATP complex. The potency of this series was optimized using structure-based design, and inhibition of cell growth of Gram-positive species was achieved. Mode-of-action studies, using generation of resistant mutants with targeted sequencing as well as constructs that overexpress PPAT, demonstrated that growth suppression was due to inhibition of PPAT. An effect on bacterial burden was demonstrated in mouse lung and thigh infection models, but further optimization of dosing requirements and compound properties is needed before these compounds can be considered for progress into clinical development. These studies validated PPAT as a novel target for antibacterial therapy.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Nucleotidiltransferasas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Staphylococcus aureus/efectos de los fármacos , Streptococcus pneumoniae/efectos de los fármacos , Animales , Antibacterianos/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Unión Competitiva , Cristalografía por Rayos X , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Femenino , Pulmón/efectos de los fármacos , Pulmón/microbiología , Ratones , Modelos Moleculares , Nucleotidiltransferasas/química , Nucleotidiltransferasas/metabolismo , Panteteína/análogos & derivados , Panteteína/química , Infecciones Neumocócicas/tratamiento farmacológico , Infecciones Neumocócicas/microbiología , Neumonía Bacteriana/tratamiento farmacológico , Neumonía Bacteriana/microbiología , Bibliotecas de Moléculas Pequeñas/química , Staphylococcus aureus/enzimología , Staphylococcus aureus/crecimiento & desarrollo , Streptococcus pneumoniae/enzimología , Streptococcus pneumoniae/crecimiento & desarrollo , Muslo/microbiologíaRESUMEN
The tRNA-(N(1)G37) methyltransferase (TrmD) is essential for growth and highly conserved in both Gram-positive and Gram-negative bacterial pathogens. Additionally, TrmD is very distinct from its human orthologue TRM5 and thus is a suitable target for the design of novel antibacterials. Screening of a collection of compound fragments using Haemophilus influenzae TrmD identified inhibitory, fused thieno-pyrimidones that were competitive with S-adenosylmethionine (SAM), the physiological methyl donor substrate. Guided by X-ray cocrystal structures, fragment 1 was elaborated into a nanomolar inhibitor of a broad range of Gram-negative TrmD isozymes. These compounds demonstrated no activity against representative human SAM utilizing enzymes, PRMT1 and SET7/9. This is the first report of selective, nanomolar inhibitors of TrmD with demonstrated ability to order the TrmD lid in the absence of tRNA.
Asunto(s)
Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Haemophilus influenzae/enzimología , ARNt Metiltransferasas/antagonistas & inhibidores , Adenosina/metabolismo , Aminas/síntesis química , Aminas/química , Aminas/metabolismo , Aminas/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Antibacterianos/metabolismo , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Haemophilus influenzae/efectos de los fármacos , Humanos , Metionina/metabolismo , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Terciaria de Proteína , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , ARNt Metiltransferasas/química , ARNt Metiltransferasas/metabolismoRESUMEN
Enzyme assays for the catalytic activity of aminoacyl-tRNA synthetases generally measure the incorporation of radioactive amino acids into tRNA. Such assays are necessarily discontinuous. Leucyl-tRNA synthetase has recently gained attention as the target of novel antimicrobial compounds based on the oxaborole scaffold, examples of which have been shown to have slow binding and dissociation kinetics. Investigations of the kinetics of inhibition by these compounds would be facilitated by a continuous assay of leucyl-tRNA synthetase catalysis. Here we report a continuous fluorescence intensity-based assay for leucyl-tRNA synthetase in which the pyrophosphate product is converted to phosphate, which is detected with nanomolar sensitivity by a phosphate sensor protein. This assay was used to measure the time constants for the slow onset of inhibition and long residence time of an oxaborole-based inhibitor.
Asunto(s)
Compuestos de Boro/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/metabolismo , Escherichia coli/enzimología , Leucina-ARNt Ligasa/antagonistas & inhibidores , Espectrometría de Fluorescencia , Compuestos de Boro/química , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Colorantes Fluorescentes/química , Cinética , Leucina-ARNt Ligasa/genética , Leucina-ARNt Ligasa/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de TiempoRESUMEN
Peptidoglycan biosynthesis is an essential process in bacteria and is therefore a suitable target for the discovery of new antibacterial drugs. One of the last cytoplasmic steps of peptidoglycan biosynthesis is catalyzed by the integral membrane protein MraY, which attaches soluble UDP-N-acetylmuramoyl-pentapeptide to the membrane-bound acceptor undecaprenyl phosphate. Although several natural product-derived inhibitors of MraY are known, none have the properties necessary to be of clinical use as antibacterial drugs. Here we describe a novel, homogeneous, fluorescence resonance energy transfer-based MraY assay that is suitable for high-throughput screening for novel MraY inhibitors. The assay allows for continuous measurement, or it can be quenched prior to measurement.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Transferasas (Grupos de Otros Fosfatos Sustitutos)/antagonistas & inhibidores , Descubrimiento de Drogas , Pruebas de Enzimas/métodos , Inhibidores Enzimáticos/farmacología , Humanos , Concentración 50 Inhibidora , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Bibliotecas de Moléculas Pequeñas/farmacología , Transferasas (Grupos de Otros Fosfatos Sustitutos)/químicaRESUMEN
A new, homogeneous, high-throughput-compatible assay method is described for the fluorescence-based quantitation of nanomolar concentrations of ribonucleoside diphosphates (rNDPs). The principle of the method is the conversion of the rNDPs to RNA by the enzyme polynucleotide phosphorylase (EC 2.7.7.8) and detection of the RNA by the increased fluorescence of a commercial nucleic acid detection dye. A commercial RNA homopolymer complementary to the RNA product is included to increase the sensitivity for ADP and UDP. Standard curves for nanomolar concentrations of ADP, UDP, GDP, and CDP are shown. The assay detected 75 nM ADP produced by the pyruvate kinase-catalyzed phosphorylation of pyruvate with a signal-to-baseline ratio of 2.8. The assay may be used in either a continuous or a discontinuous mode.
Asunto(s)
Adenosina Difosfato/análisis , Espectrometría de Fluorescencia/métodos , Citidina Difosfato/análisis , Colorantes Fluorescentes/química , Guanosina Difosfato/análisis , Ensayos Analíticos de Alto Rendimiento/métodos , Cinética , Fosforilación , Piruvato Quinasa/metabolismo , Piruvatos/metabolismo , ARN/química , Uridina Difosfato/análisisRESUMEN
The degree of supercoiling of DNA is vital for cellular processes, such as replication and transcription. DNA topology is controlled by the action of DNA topoisomerase enzymes. Topoisomerases, because of their importance in cellular replication, are the targets of several anticancer and antibacterial drugs. In the search for new drugs targeting topoisomerases, a biochemical assay compatible with automated high-throughput screening (HTS) would be valuable. Gel electrophoresis is the standard method for measuring changes in the extent of supercoiling of plasmid DNA when acted upon by topoisomerases, but this is a low-throughput and laborious method. A medium-throughput method was described previously that quantitatively distinguishes relaxed and supercoiled plasmids by the difference in their abilities to form triplex structures with an immobilized oligonucleotide. In this article, the authors describe a homogeneous supercoiling assay based on triplex formation in which the oligonucleotide strand is labeled with a fluorescent dye and the readout is fluorescence anisotropy. The new assay requires no immobilization, filtration, or plate washing steps and is therefore well suited to HTS for inhibitors of topoisomerases. The utility of this assay is demonstrated with relaxation of supercoiled plasmid by Escherichia coli topoisomerase I, supercoiling of relaxed plasmid by E. coli DNA gyrase, and inhibition of gyrase by fluoroquinolones and nalidixic acid.
Asunto(s)
ADN Superhelicoidal/química , Polarización de Fluorescencia/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Biocatálisis/efectos de los fármacos , ADN-Topoisomerasas de Tipo I/metabolismo , Electroforesis en Gel de Agar , Concentración de Iones de Hidrógeno/efectos de los fármacos , Magnesio/farmacología , Conformación de Ácido Nucleico/efectos de los fármacos , Oligonucleótidos/química , Plásmidos/química , Inhibidores de Topoisomerasa IIRESUMEN
A homogeneous, fluorescence resonance energy transfer (FRET)-based DNA polymerase assay that is suitable for high-throughput screening for inhibitors, and can also be used for steady-state kinetic investigations, is described. The activity, kinetic mechanism, and processivity of the isolated alpha subunit of DNA polymerase III, the product of the dnaE gene, from the gram-negative pathogen Haemophilus influenzae were investigated using the FRET assay.