RESUMEN
OBJECTIVES: Pseudomonas aeruginosa is an important nosocomial pathogen that can cause a wide range of infections resulting in significant morbidity and mortality. Avibactam, a novel non-ß-lactam ß-lactamase inhibitor, is being developed in combination with ceftazidime and has the potential to be a valuable addition to the treatment options for the infectious diseases practitioner. We compared the frequency of resistance development to ceftazidime/avibactam in three P. aeruginosa strains that carried derepressed ampC alleles. METHODS: The strains were incubated in the presence of increasing concentrations of ceftazidime with a fixed concentration (4 mg/L) of avibactam to calculate the frequency of spontaneous resistance. The mutants were characterized by WGS to identify the underlying mechanism of resistance. A representative mutant protein was characterized biochemically. RESULTS: The resistance frequency was very low in all strains. The resistant variants isolated exhibited ceftazidime/avibactam MIC values that ranged from 64 to 256 mg/L. All of the mutants exhibited changes in the chromosomal ampC gene, the majority of which were deletions of various sizes in the Ω-loop region of AmpC. The mutant enzyme that carried the smallest Ω-loop deletion, which formed a part of the avibactam-binding pocket, was characterized biochemically and found to be less effectively inhibited by avibactam as well as exhibiting increased hydrolysis of ceftazidime. CONCLUSIONS: The development of high-level resistance to ceftazidime/avibactam appears to occur at low frequency, but structural modifications in AmpC can occur that impact the ability of avibactam to inhibit the enzyme and thereby protect ceftazidime from hydrolysis.
Asunto(s)
Antibacterianos/farmacología , Compuestos de Azabiciclo/farmacología , Proteínas Bacterianas/biosíntesis , Ceftazidima/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/enzimología , Selección Genética , Resistencia betalactámica , beta-Lactamasas/biosíntesis , Proteínas Bacterianas/genética , Combinación de Medicamentos , Humanos , Pruebas de Sensibilidad Microbiana , Tasa de Mutación , Pseudomonas aeruginosa/genética , beta-Lactamasas/genéticaRESUMEN
Negamycin is a natural product with broad-spectrum antibacterial activity and efficacy in animal models of infection. Although its precise mechanism of action has yet to be delineated, negamycin inhibits cellular protein synthesis and causes cell death. Here, we show that single point mutations within 16S rRNA that confer resistance to negamycin are in close proximity of the tetracycline binding site within helix 34 of the small subunit head domain. As expected from its direct interaction with this region of the ribosome, negamycin was shown to displace tetracycline. However, in contrast to tetracycline-class antibiotics, which serve to prevent cognate tRNA from entering the translating ribosome, single-molecule fluorescence resonance energy transfer investigations revealed that negamycin specifically stabilizes near-cognate ternary complexes within the A site during the normally transient initial selection process to promote miscoding. The crystal structure of the 70S ribosome in complex with negamycin, determined at 3.1 Å resolution, sheds light on this finding by showing that negamycin occupies a site that partially overlaps that of tetracycline-class antibiotics. Collectively, these data suggest that the small subunit head domain contributes to the decoding mechanism and that small-molecule binding to this domain may either prevent or promote tRNA entry by altering the initial selection mechanism after codon recognition and before GTPase activation.
Asunto(s)
Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , ARN Bacteriano/efectos de los fármacos , ARN Ribosómico 16S/efectos de los fármacos , Ribosomas/efectos de los fármacos , Aminoácidos Diaminos/farmacología , Antibacterianos/metabolismo , Emparejamiento Base , Sitios de Unión , Unión Competitiva , Cristalografía por Rayos X , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli/genética , Minociclina/análogos & derivados , Minociclina/farmacología , Modelos Moleculares , Conformación de Ácido Nucleico , Mutación Puntual , Biosíntesis de Proteínas/efectos de los fármacos , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/fisiología , ARN Ribosómico 16S/química , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/fisiología , ARN de Transferencia/metabolismo , Ribosomas/ultraestructura , Resistencia a la Tetraciclina/genética , Tetraciclinas/metabolismo , Tetraciclinas/farmacología , TigeciclinaRESUMEN
A novel, ultrahigh-throughput, fluorescence anisotropy-based assay was developed and used to screen a 1.4-million-sample library for compounds that compete with adenosine triphosphate (ATP) for binding to Escherichia coli tRNA(Ile) lysidine synthetase (TilS), an essential, conserved, ATP-dependent, tRNA-modifying enzyme of bacterial pathogens. TilS modifies a cytidine base in the anticodon loop of Ile2 tRNA by attaching lysine, thereby altering codon recognition of the CAU anticodon from AUG (methionine) to AUA (isoleucine). A scintillation proximity assay for the incorporation of lysine into Ile2 tRNA was used to eliminate false positives in the initial screen resulting from detection artifacts as well as compounds competitive with the fluorescent label instead of ATP, and to measure inhibitor potencies against E. coli and Pseudomonas aeruginosa TilS isozymes. The tRNA(Ile) substrate for P. aeruginosa TilS was identified for the first time to enable these measurements. ATP-competitive binding of inhibitors was confirmed by one-dimensional ligand-observe nuclear magnetic resonance. A preliminary structure-activity relationship is shown for two inhibitor series.
Asunto(s)
Aminoacil-ARNt Sintetasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Proteínas de Escherichia coli/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento/métodos , Adenosina Trifosfato/metabolismo , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Anticodón , Unión Competitiva , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Polarización de Fluorescencia/métodos , Lisina/metabolismo , Pseudomonas aeruginosa/enzimología , Relación Estructura-ActividadRESUMEN
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
Avibactam is a non-ß-lactam ß-lactamase inhibitor with a spectrum of activity that includes ß-lactamase enzymes of classes A, C, and selected D examples. In this work acylation and deacylation rates were measured against the clinically important enzymes CTX-M-15, KPC-2, Enterobacter cloacae AmpC, Pseudomonas aeruginosa AmpC, OXA-10, and OXA-48. The efficiency of acylation (k2/Ki) varied across the enzyme spectrum, from 1.1 × 10(1) m(-1)s(-1) for OXA-10 to 1.0 × 10(5) for CTX-M-15. Inhibition of OXA-10 was shown to follow the covalent reversible mechanism, and the acylated OXA-10 displayed the longest residence time for deacylation, with a half-life of greater than 5 days. Across multiple enzymes, acyl enzyme stability was assessed by mass spectrometry. These inhibited enzyme forms were stable to rearrangement or hydrolysis, with the exception of KPC-2. KPC-2 displayed a slow hydrolytic route that involved fragmentation of the acyl-avibactam complex. The identity of released degradation products was investigated, and a possible mechanism for the slow deacylation from KPC-2 is proposed.
Asunto(s)
Compuestos de Azabiciclo/química , Escherichia coli/efectos de los fármacos , beta-Lactamasas/química , Antibacterianos/química , Farmacorresistencia Bacteriana , Enterobacter cloacae/metabolismo , Inhibidores Enzimáticos/química , Escherichia coli/metabolismo , Hidrólisis , Cinética , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Pruebas de Sensibilidad Microbiana , Plásmidos/metabolismo , Pseudomonas aeruginosa/metabolismo , Factores de TiempoRESUMEN
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
We report a simple, rapid, and reproducible fluorescence anisotropy-based method for measuring rate constants for acylation and deacylation of soluble penicillin binding protein (PBP) constructs by compounds in microtiter plates by means of competition with time-dependent acylation by BOCILLIN FL. The method is demonstrated by measuring the acylation rate constants of the PBP3 periplasmic domains from Pseudomonas aeruginosa and Acinetobacter baumannii by BOCILLIN FL, aztreonam, meropenem, and ceftazidime. The new method requires very little protein and can be completed in approximately 1h per compound. A set of BOCILLIN FL acylation progress curves collected over a range of competitor concentrations is fit globally to a kinetic model by numerical integration. First-order deacylation rate constants could also be measured, as demonstrated with a catalytically impaired mutant OXA-10 ß-lactamase.
Asunto(s)
Compuestos de Boro/metabolismo , Polarización de Fluorescencia/métodos , Proteínas de Unión a las Penicilinas/metabolismo , Penicilinas/metabolismo , Acinetobacter baumannii/citología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Biocatálisis , Cinética , Proteínas de Unión a las Penicilinas/química , Periplasma/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Pseudomonas aeruginosa/citologíaRESUMEN
LpxA, the first enzyme in the biosynthetic pathway for the Lipid A component of the outer membrane lipopolysaccharide in Gram-negative bacteria, is a potential target for novel antibacterial drug discovery. A fluorescence polarization assay was developed to facilitate high-throughput screening for competitive inhibitors of LpxA. The assay detects displacement of a fluorescently labeled peptide inhibitor, based on the previously reported inhibitor peptide 920, by active site ligands. The affinity of the fluorescent ligand was increased ~10-fold by acyl carrier protein (ACP). Competition with peptide binding was observed with UDP-N-acetylglucosamine (IC(50) ~6 mM), UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine (IC(50) ~200 nM), and DL-3-hydroxymyristic acid (IC(50) ~50 µM) and peptide 920 (IC(50) ~600 nM). The IC(50)s were not significantly affected by the presence of ACP.
Asunto(s)
Aciltransferasas/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/farmacología , Escherichia coli/enzimología , Polarización de Fluorescencia/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Proteína Transportadora de Acilo/metabolismo , Aciltransferasas/química , Unión Competitiva , Dominio Catalítico , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Concentración 50 Inhibidora , Ligandos , Lípido A/metabolismo , Ácidos Mirísticos/química , Ácidos Mirísticos/metabolismo , Péptidos/química , Péptidos/metabolismo , Uridina Difosfato N-Acetilglucosamina/análogos & derivados , Uridina Difosfato N-Acetilglucosamina/química , Uridina Difosfato N-Acetilglucosamina/metabolismoRESUMEN
The NAD-dependent DNA ligase is an excellent target for the discovery of antibacterial agents with a novel mode of action. In this work the DNA ligase from Streptococcus pneumoniae was investigated for its steady-state kinetic parameters and inhibition by compounds with an adenosine substructure. Inhibition by substrate DNA that was observed in the enzyme turnover experiments was verified by direct binding measurements using isothermal titration calorimetry (ITC). The substrate-inhibited enzyme form was identified as deadenylated DNA ligase. The binding potencies of 2-(butylsulfanyl) adenosine and 2-(cyclopentyloxy) adenosine were not significantly affected by the presence of the enzyme-bound DNA substrate. Finally, a mutant protein was prepared that was known to confer resistance to the adenosine compounds' antibacterial activity. The mutant protein was shown to have little catalytic impairment yet it was less susceptible to adenosine compound inhibition.
Asunto(s)
Adenosina/farmacología , Antibacterianos/farmacología , ADN Ligasas/metabolismo , Inhibidores Enzimáticos/farmacología , Streptococcus pneumoniae/enzimología , Adenosina/química , Antibacterianos/química , Secuencia de Bases , Calorimetría , ADN Ligasas/antagonistas & inhibidores , ADN Ligasas/genética , Cartilla de ADN , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Cinética , Mutagénesis Sitio-DirigidaRESUMEN
A novel assay for the NADPH-dependent bacterial enzyme UDP-N-acetylenolpyruvylglucosamine reductase (MurB) is described that has nanomolar sensitivity for product formation and is suitable for high-throughput applications. MurB catalyzes an essential cytoplasmic step in the synthesis of peptidoglycan for the bacterial cell wall, reduction of UDP-N-acetylenolpyruvylglucosamine to UDP-N-acetylmuramic acid (UNAM). Interruption of this biosynthetic pathway leads to cell death, making MurB an attractive target for antibacterial drug discovery. In the new assay, the UNAM product of the MurB reaction is ligated to L-alanine by the next enzyme in the peptidoglycan biosynthesis pathway, MurC, resulting in hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). The ADP is detected with nanomolar sensitivity by converting it to oligomeric RNA with polynucleotide phosphorylase and detecting the oligomeric RNA with a fluorescent dye. The product sensitivity of the new assay is 1000-fold greater than that of the standard assay that follows the absorbance decrease resulting from the conversion of NADPH to NADP(+). This sensitivity allows inhibitor screening to be performed at the low substrate concentrations needed to make the assay sensitive to competitive inhibition of MurB.