RESUMEN
Multidrug-resistant (MDR) bacterial infections are a serious threat to public health. Among the most alarming resistance trends is the rapid rise in the number and diversity of ß-lactamases, enzymes that inactivate ß-lactams, a class of antibiotics that has been a therapeutic mainstay for decades. Although several new ß-lactamase inhibitors have been approved or are in clinical trials, their spectra of activity do not address MDR pathogens such as Acinetobacter baumannii. This report describes the rational design and characterization of expanded-spectrum serine ß-lactamase inhibitors that potently inhibit clinically relevant class A, C and D ß-lactamases and penicillin-binding proteins, resulting in intrinsic antibacterial activity against Enterobacteriaceae and restoration of ß-lactam activity in a broad range of MDR Gram-negative pathogens. One of the most promising combinations is sulbactam-ETX2514, whose potent antibacterial activity, in vivo efficacy against MDR A. baumannii infections and promising preclinical safety demonstrate its potential to address this significant unmet medical need.
Asunto(s)
Acinetobacter baumannii/efectos de los fármacos , Compuestos de Azabiciclo/química , Compuestos de Azabiciclo/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Inhibidores de beta-Lactamasas/química , Inhibidores de beta-Lactamasas/farmacología , Infecciones por Acinetobacter/tratamiento farmacológico , Infecciones por Acinetobacter/microbiología , Animales , Compuestos de Azabiciclo/uso terapéutico , Compuestos de Azabiciclo/toxicidad , Carbapenémicos/farmacología , Perros , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Farmacorresistencia Bacteriana Múltiple , Enterobacteriaceae/efectos de los fármacos , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Humanos , Ratones , Modelos Moleculares , Proteínas de Unión a las Penicilinas/antagonistas & inhibidores , Ratas , Sulbactam/química , Sulbactam/farmacología , Inhibidores de beta-Lactamasas/uso terapéutico , Inhibidores de beta-Lactamasas/toxicidad , beta-Lactamasas/metabolismo , beta-Lactamas/farmacologíaRESUMEN
We characterized the inhibition of Neisseria gonorrhoeae type II topoisomerases gyrase and topoisomerase IV by AZD0914 (AZD0914 will be henceforth known as ETX0914 (Entasis Therapeutics)), a novel spiropyrimidinetrione antibacterial compound that is currently in clinical trials for treatment of drug-resistant gonorrhea. AZD0914 has potent bactericidal activity against N. gonorrhoeae, including multidrug-resistant strains and key Gram-positive, fastidious Gram-negative, atypical, and anaerobic bacterial species (Huband, M. D., Bradford, P. A., Otterson, L. G., Basrab, G. S., Giacobe, R. A., Patey, S. A., Kutschke, A. C., Johnstone, M. R., Potter, M. E., Miller, P. F., and Mueller, J. P. (2014) In Vitro Antibacterial Activity of AZD0914: A New Spiropyrimidinetrione DNA Gyrase/Topoisomerase Inhibitor with Potent Activity against Gram-positive, Fastidious Gram-negative, and Atypical Bacteria. Antimicrob. Agents Chemother. 59, 467-474). AZD0914 inhibited DNA biosynthesis preferentially to other macromolecules in Escherichia coli and induced the SOS response to DNA damage in E. coli. AZD0914 stabilized the enzyme-DNA cleaved complex for N. gonorrhoeae gyrase and topoisomerase IV. The potency of AZD0914 for inhibition of supercoiling and the stabilization of cleaved complex by N. gonorrhoeae gyrase increased in a fluoroquinolone-resistant mutant enzyme. When a mutation, conferring mild resistance to AZD0914, was present in the fluoroquinolone-resistant mutant, the potency of ciprofloxacin for inhibition of supercoiling and stabilization of cleaved complex was increased greater than 20-fold. In contrast to ciprofloxacin, religation of the cleaved DNA did not occur in the presence of AZD0914 upon removal of magnesium from the DNA-gyrase-inhibitor complex. AZD0914 had relatively low potency for inhibition of human type II topoisomerases α and ß.
Asunto(s)
Antibacterianos/farmacología , Barbitúricos/farmacología , Girasa de ADN/metabolismo , Topoisomerasa de ADN IV/antagonistas & inhibidores , ADN Bacteriano/química , Compuestos de Espiro/farmacología , Inhibidores de Topoisomerasa II/farmacología , Ciprofloxacina/farmacología , Ensayos Clínicos como Asunto , ADN/química , ADN/metabolismo , Girasa de ADN/genética , Topoisomerasa de ADN IV/genética , Topoisomerasa de ADN IV/metabolismo , ADN Bacteriano/metabolismo , Farmacorresistencia Bacteriana/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Escherichia coli/genética , Fluoroquinolonas/farmacología , Expresión Génica , Humanos , Isoxazoles , Morfolinas , Mutación , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/enzimología , Neisseria gonorrhoeae/genética , Oxazolidinonas , Especificidad de la EspecieRESUMEN
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
Sulbactam is a class A ß-lactamase inhibitor with intrinsic whole-cell activity against certain bacterial species, including Acinetobacter baumannii. The clinical use of sulbactam for A. baumannii infections is of interest due to increasing multidrug resistance in this pathogen. However, the molecular drivers of its antibacterial activity and resistance determinants have yet to be precisely defined. Here we show that the antibacterial activities of sulbactam vary widely across contemporary A. baumannii clinical isolates and are mediated through inhibition of the penicillin-binding proteins (PBPs) PBP1 and PBP3, with very low frequency of resistance; the rare pbp3 mutants with high levels of resistance to sulbactam are attenuated in fitness. These results support further investigation of the potential clinical utility of sulbactam.
Asunto(s)
Acinetobacter baumannii/efectos de los fármacos , Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/fisiología , Sulbactam/farmacología , Proteínas de Unión a las Penicilinas/antagonistas & inhibidoresRESUMEN
Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in young children and other high-risk populations. RSV nucleoprotein (N) is essential for virus assembly and replication as part of the viral ribonucleoprotein (RNP) complex. RSV604 was a putative N inhibitor in phase 2 clinical trials whose molecular mechanism of action (MoA) was not well understood. This study investigated the cell line-dependent potency of RSV604 and demonstrated its direct binding to the N protein in vitro, providing the first evidence of direct target engagement for this class of inhibitors reported to date. The affinity of RSV604 N binding was not affected by RSV604 resistance mutations in the N protein. RSV604 engaged in two different MoAs in HeLa cells, inhibiting both RSV RNA synthesis and the infectivity of released virus. The lack of inhibition of viral RNA synthesis in some cell lines explained the cell-type-dependent potency of the inhibitor. RSV604 did not inhibit viral RNA synthesis in the RSV subgenomic replicon cells or in the cell-free RNP assay, suggesting that it might act prior to viral replication complex formation. RSV604 did not alter N protein localization in the infected cells. Taken together, these results provide new insights leading to an understanding of the MoAs of RSV604 and other similar N inhibitors.
Asunto(s)
Benzodiazepinonas/farmacología , Compuestos de Fenilurea/farmacología , ARN Viral/efectos de los fármacos , Virus Sincitiales Respiratorios/efectos de los fármacos , Línea Celular , Humanos , Replicación Viral/efectos de los fármacosRESUMEN
In a previous report (T. J. Dougherty, A. Nayar, J. V. Newman, S. Hopkins, G. G. Stone, M. Johnstone, A. B. Shapiro, M. Cronin, F. Reck, and D. E. Ehmann, Antimicrob Agents Chemother 58:2657-2664, 2014), a novel bacterial type II topoisomerase inhibitor, NBTI 5463, with activity against Gram-negative pathogens was described. First-step resistance mutations in Pseudomonas aeruginosa arose exclusively in the nfxB gene, a regulator of the MexCD-OprJ efflux pump system. The present report describes further resistance studies with NBTI 5463 in both Pseudomonas aeruginosa and Escherichia coli. Second-step mutations in P. aeruginosa arose at aspartate 82 of the gyrase A subunit and led to 4- to 8-fold increases in the MIC over those seen in the parental strain with a first-step nfxB efflux mutation. A third-step mutant showed additional GyrA changes, with no changes in topoisomerase IV. Despite repeated efforts, resistance mutations could not be selected in E. coli. Genetic introduction of the Asp82 mutations observed in P. aeruginosa did not significantly increase the NBTI MIC in E. coli. However, with the aspartate 82 mutation present, it was possible to select second-step mutations in topoisomerase IV that did lead to MIC increases of 16- and 128-fold. As with the gyrase aspartate 82 mutation, the mutations in topoisomerase IV did not by themselves raise the NBTI MIC in E. coli. Only the presence of mutations in both targets of E. coli led to an increase in NBTI MIC values. This represents a demonstration of the value of balanced dual-target activity in mitigating resistance development.
Asunto(s)
Farmacorresistencia Bacteriana/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Morfolinas/farmacología , Naftiridinas/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Inhibidores de Topoisomerasa II/farmacología , Proteínas Bacterianas/genética , Girasa de ADN/genética , Farmacorresistencia Bacteriana/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Pruebas de Sensibilidad Microbiana , Mutación , Pseudomonas aeruginosa/genéticaRESUMEN
BACKGROUND: There are no approved small molecule drug therapies for human respiratory syncytial virus (hRSV), a cause of morbidity and mortality in at-risk newborns, the immunocompromised, and the elderly. We have investigated as a potential novel hRSV drug target the protein-protein interaction between the C-terminus of the viral phosphoprotein (P) and the viral nucleocapsid protein (N), components of the ribonucleoprotein complex that contains, replicates, and transcribes the viral RNA genome. Earlier work by others established that the 9 C-terminal residues of P are necessary and sufficient for binding to N. METHODS: We used a fluorescence anisotropy assay, surface plasmon resonance and 2-D NMR to quantify the affinities of peptides based on the C terminus of P for RNA-free, monomeric N-terminal-truncated N(13-391). We calculated the contributions to the free energies of binding of P to N(13-391) attributable to the C-terminal 11 residues, phosphorylation of the C-terminal 2 serine residues, the C-terminal Asp-Phe, and the phenyl ring of the C-terminal Phe. RESULTS: Binding studies confirmed the crucial role of the phosphorylated C-terminal peptide D(pS)DNDL(pS)LEDF for binding of P to RNA-free, monomeric N(13-391), contributing over 90% of the binding free energy at low ionic strength. The phenyl ring of the C-terminal Phe residue contributed an estimated -2.7 kcal/mole of the free energy of binding, the C-terminal Asp-Phe residues contributed -3.8 kcal/mole, the sequence DSDNDLSLE contributed -3.1 kcal/mole, and phosphorylation of the 2 Ser residues contributed -1.8 kcal/mole. Due to the high negative charge of the C-terminal peptide, the affinity of the P C-terminus for N(13-391) decreased as the ionic strength increased. CONCLUSIONS: The results support the idea that the interaction of the C-terminal residues of P with N constitutes a protein-protein interaction hotspot that may be a suitable target for small-molecule drugs that inhibit viral genome replication and transcription.
Asunto(s)
Nucleoproteínas/química , Nucleoproteínas/metabolismo , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/metabolismo , Proteínas Estructurales Virales/química , Proteínas Estructurales Virales/metabolismo , Secuencias de Aminoácidos , Sitios de Unión , Humanos , Cinética , Nucleoproteínas/genética , Fosforilación , Unión Proteica , Virus Sincitial Respiratorio Humano/química , Virus Sincitial Respiratorio Humano/genética , Proteínas Estructurales Virales/genéticaRESUMEN
In the search for novel Gram-negative agents, we performed a comprehensive search of the AstraZeneca collection and identified a tetrahydropyran-based matrix metalloprotease (MMP) inhibitor that demonstrated nanomolar inhibition of UDP-3-O-(acyl)-N-acetylglucosamine deacetylase (LpxC). Crystallographic studies in Aquifex aeolicus LpxC indicated the tetrahydropyran engaged in the same hydrogen bonds and van der Waals interactions as other known inhibitors. Systematic optimization of three locales on the scaffold provided compounds with improved Gram-negative activity. However, the optimization of LpxC activity was not accompanied by reduced inhibition of MMPs. Comparison of the crystal structure of the native product, UDP-3-O-(acyl)-glucosamine, in Aquifex aeolicus to the structure of a tetrahydropyran-based inhibitor indicates pathways for future optimization.
RESUMEN
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
High-molecular-weight penicillin-binding proteins (PBPs) are essential integral membrane proteins of the bacterial cytoplasmic membrane responsible for biosynthesis of peptidoglycan. They are the targets of antibacterial ß-lactam drugs, including penicillins, cephalosporins, and carbapenems. ß-Lactams covalently acylate the active sites of the PBP transpeptidase domains. Because ß-lactams are time-dependent inhibitors, quantitative assessment of the inhibitory activity of these compounds ideally involves measurement of their second-order acylation rate constants. We previously described a fluorescence anisotropy-based assay to measure these rate constants for soluble constructs of PBP3 (Anal. Biochem. 439 (2013) 37-43). Here we report the expression and purification of a soluble construct of Pseudomonas aeruginosa PBP2 as a fusion protein with NusA. This soluble PBP2 was used to measure second-order acylation rate constants with the fluorescence anisotropy assay. Measurements were obtained for mecillinam, which reacts specifically with PBP2, and for several carbapenems. The assay also revealed that PBP2 slowly hydrolyzed mecillinam and was used to measure the rate constant for this deacylation reaction.
Asunto(s)
Polarización de Fluorescencia , Pseudomonas aeruginosa/metabolismo , Acilación , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Compuestos de Boro/metabolismo , Electroforesis en Gel de Poliacrilamida , Cinética , Proteínas de Unión a las Penicilinas/química , Proteínas de Unión a las Penicilinas/genética , Proteínas de Unión a las Penicilinas/metabolismo , Penicilinas/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
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
Lipopolysaccharide (LPS) biosynthesis is an attractive antibacterial target as it is both conserved and essential for the survival of key pathogenic bacteria. Lipid A is the hydrophobic anchor for LPS and a key structural component of the outer membrane of Gram-negative bacteria. Lipid A biosynthesis is performed in part by a unique zinc dependent metalloamidase, LpxC (UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase), which catalyzes the first non-reversible step in lipid A biosynthesis. The UDP portion of the LpxC substrate-binding pocket has been relatively unexplored. We have designed and evaluated a series of hydroxamate based inhibitors which explore the SAR of substitutions directed into the UDP pocket with a range of substituted α-amino acid based linkers. We also provide the first wild type structure of Pseudomonas aeruginosa LpxC which was utilized in the design of many of these analogs.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Amidohidrolasas/química , Aminoácidos Cíclicos/química , Uridina Difosfato/química , Amidohidrolasas/metabolismo , Secuencia de Aminoácidos , Antibacterianos/síntesis química , Antibacterianos/química , Sitios de Unión , Diseño de Fármacos , Interacciones Hidrofóbicas e Hidrofílicas , Ácidos Hidroxámicos/síntesis química , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/farmacología , Lípido A/antagonistas & inhibidores , Lípido A/biosíntesis , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/biosíntesis , Modelos Moleculares , Conformación Proteica , Relación Estructura-Actividad , Uridina Difosfato/metabolismoRESUMEN
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.
Asunto(s)
Deshidrogenasas de Carbohidratos/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Adenosina Difosfato/metabolismo , Antibacterianos/química , Deshidrogenasas de Carbohidratos/análisis , Pared Celular , Descubrimiento de Drogas/métodos , Escherichia coli/metabolismo , Fluorescencia , Peptidoglicano/biosíntesis , Polirribonucleótido Nucleotidiltransferasa/metabolismoRESUMEN
Methionine aminopeptidase (MAP) (E.C. 3.4.11.18) is a metallopeptidase that cleaves the N-terminal methionine (Met) residue from some proteins. MAP is essential for growth of several bacterial pathogens, making it a target for antibacterial drug discovery. MAP enzymes are also present in eukaryotic cells, and one is a target for antiangiogenic cancer therapy. To screen large compound libraries for MAP inhibitors as the starting point for drug discovery, a high-throughput-compatible assay is valuable. Here the authors describe a novel assay, which detects the Met product of MAP-catalyzed peptide cleavage by coupling it to adenosine triphosphate (ATP)-dependent production of S-adenosyl-L-methionine (SAM) and inorganic phosphate (P(i)) by SAM synthetase (MetK) combined with inorganic pyrophosphatase. The three P(i) ions produced for each Met consumed are detected using Malachite Green/molybdate reagent. This assay can use any unmodified peptide MAP substrate with an N-terminal Met. The assay was used to measure kinetic constants for Escherichia coli MAP using Mn(2+) as the activator and the peptide Met-Gly-Met-Met as the substrate, as well as to measure the potency of a MAP inhibitor. A Mn(2+) buffer is described that can be used to prevent free Mn(2+) depletion by chelating compounds from interfering in screens for MAP inhibitors.