RESUMO
Tuberculosis is the most frequent cause of death in humans from a single infectious agent. Due to low numbers of bacteria present in sputum during early infection, diagnosis does not usually occur until >3 to 4 months after symptoms develop. We created a new more sensitive diagnostic that can be carried out in 10 min with no processing or technical expertise. This assay utilizes the Mycobacterium tuberculosis-specific biomarker BlaC in reporter enzyme fluorescence (REF) that has been optimized for clinical samples, designated REFtb, along with a more specific fluorogenic substrate, CDG-3. We report the first evaluation of clinical specimens with REFtb assays in comparison to the gold standards for tuberculosis diagnosis, culture and smear microscopy. REFtb assays allowed diagnosis of 160 patients from 16 different countries with a sensitivity of 89% for smear-positive, culture-positive samples and 88% for smear-negative, culture-positive samples with a specificity of 82%. The negative predictive value of REFtb for tuberculosis infection is 93%, and the positive predictive value is 79%. Overall, these data point toward the need for larger accuracy studies by third parties using a commercially available REFtb kit to determine whether incorporation of REFtb into the clinical toolbox for suspected tuberculosis patients would improve case identification. If results similar to our own can be obtained by all diagnostic laboratories, REFtb would allow proper treatment of more than 85% of patients that would be missed during their initial visit to a clinic using current diagnostic strategies, reducing the potential for further spread of disease.
Assuntos
Testes Diagnósticos de Rotina/métodos , Corantes Fluorescentes/metabolismo , Fluorometria/métodos , Mycobacterium tuberculosis/enzimologia , Tuberculose/diagnóstico , beta-Lactamases/análise , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Fatores de Tempo , Adulto JovemRESUMO
Although tuberculosis (TB) is one of the most common causes of morbidity and mortality in humans worldwide and diagnostic methods have been in place for more than 100 years, diagnosis remains a challenge. The main problems with diagnosis relate to the time needed to obtain a definitive result, difficulty in obtaining sputum, the primary clinical material used, and the ability of the causative agent, Mycobacterium tuberculosis, to cause disease in nearly any tissue within the body. In order to decrease incidence of TB, discovery of a novel interventions will be required, since current technologies have only been able to control numbers of infections, not reduce them. Diagnostic innovation is particularly needed because there are no effective pediatric or extrapulmonary TB diagnostic methods and multiple-drug resistance is only identified in less than 25% of those patients that are thought to have it. The most common diagnostic method worldwide remains acid-fast stain on sputum, with a threshold of â¼10,000 bacteria/ml that is only reached â¼5-6 months after development of symptoms. In order to obtain definitive diagnostic results earlier during the disease process, we have developed a diagnostic method designated reporter enzyme fluorescence (REF) that utilizes BlaC produced by M. tuberculosis and custom substrates to produce a specific fluorescent signal with as few as 10 bacteria/ml in clinical samples. We believe that the unique biology of the REF technique will allow it to contribute new diagnostic information that is complementary to all existing diagnostic tests as well as those currently known to be in development.
Assuntos
Proteínas de Bactérias/metabolismo , Técnicas Bacteriológicas , Corantes Fluorescentes/metabolismo , Mycobacterium tuberculosis/enzimologia , Espectrometria de Fluorescência , Tuberculose Pulmonar/diagnóstico , beta-Lactamases/metabolismo , Carga Bacteriana , Humanos , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Escarro/microbiologia , Especificidade por Substrato , Tuberculose Pulmonar/microbiologiaRESUMO
Antimicrobial drug resistance is a growing threat to global public health. Multidrug resistance among the 'ESKAPE' organisms - encompassing Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. - is of particular concern because they are responsible for many serious infections in hospitals. Although some promising agents are in the pipeline, there is an urgent need for new antibiotic scaffolds. However, antibacterial researchers have struggled to identify new small molecules with meaningful cellular activity, especially those effective against multidrug-resistant Gram-negative pathogens. This difficulty ultimately stems from an incomplete understanding of efflux systems and compound permeation through bacterial membranes. This Opinion article describes findings from target-based and phenotypic screening efforts carried out at AstraZeneca over the past decade, discusses some of the subsequent chemistry challenges and concludes with a description of new approaches comprising a combination of computational modelling and advanced biological tools which may pave the way towards the discovery of new antibacterial agents.
Assuntos
Antibacterianos/farmacologia , Infecções Bacterianas/tratamento farmacológico , Desenho de Fármacos , Animais , Infecções Bacterianas/microbiologia , Biologia Computacional/métodos , Descoberta de Drogas/métodos , Farmacorresistência Bacteriana , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Humanos , Terapia de Alvo MolecularRESUMO
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.
Assuntos
Antibacterianos/farmacologia , Barbitúricos/farmacologia , DNA Girase/metabolismo , DNA Topoisomerase IV/antagonistas & inibidores , DNA Bacteriano/química , Compostos de Espiro/farmacologia , Inibidores da Topoisomerase II/farmacologia , Ciprofloxacina/farmacologia , Ensaios Clínicos como Assunto , DNA/química , DNA/metabolismo , DNA Girase/genética , DNA Topoisomerase IV/genética , DNA Topoisomerase IV/metabolismo , DNA Bacteriano/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Fluoroquinolonas/farmacologia , Expressão Gênica , Humanos , Isoxazóis , Morfolinas , Mutação , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/enzimologia , Neisseria gonorrhoeae/genética , Oxazolidinonas , Especificidade da EspécieRESUMO
With the diminishing effectiveness of current antibacterial therapies, it is critically important to discover agents that operate by a mechanism that circumvents existing resistance. ETX0914, the first of a new class of antibacterial agent targeted for the treatment of gonorrhea, operates by a novel mode-of-inhibition against bacterial type II topoisomerases. Incorporating an oxazolidinone on the scaffold mitigated toxicological issues often seen with topoisomerase inhibitors. Organisms resistant to other topoisomerase inhibitors were not cross-resistant with ETX0914 nor were spontaneous resistant mutants to ETX0914 cross-resistant with other topoisomerase inhibitor classes, including the widely used fluoroquinolone class. Preclinical evaluation of ETX0914 pharmacokinetics and pharmacodynamics showed distribution into vascular tissues and efficacy in a murine Staphylococcus aureus infection model that served as a surrogate for predicting efficacious exposures for the treatment of Neisseria gonorrhoeae infections. A wide safety margin to the efficacious exposure in toxicological evaluations supported progression to Phase 1. Dosing ETX0914 in human volunteers showed sufficient exposure and minimal adverse effects to expect a highly efficacious anti-gonorrhea therapy.
Assuntos
Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Barbitúricos/farmacologia , Barbitúricos/uso terapêutico , Gonorreia/tratamento farmacológico , Compostos de Espiro/farmacologia , Compostos de Espiro/uso terapêutico , Inibidores da Topoisomerase II/farmacologia , Inibidores da Topoisomerase II/uso terapêutico , Adulto , Animais , Antibacterianos/química , Barbitúricos/química , DNA Topoisomerases Tipo II/química , Modelos Animais de Doenças , Cães , Relação Dose-Resposta a Droga , Farmacorresistência Bacteriana , Feminino , Fluoroquinolonas/farmacologia , Gonorreia/microbiologia , Haplorrinos , Humanos , Isoxazóis , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Modelos Moleculares , Conformação Molecular , Morfolinas , Mutação , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética , Oxazolidinonas , Ratos , Compostos de Espiro/química , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Inibidores da Topoisomerase II/química , Adulto JovemRESUMO
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.
Assuntos
Amidoidrolases/antagonistas & inibidores , Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Ácidos Hidroxâmicos/farmacologia , Treonina/análogos & derivados , Animais , Antibacterianos/química , Antibacterianos/farmacocinética , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacocinética , Cinética , Camundongos Endogâmicos , Testes de Sensibilidade Microbiana , Modelos Biológicos , Estrutura Molecular , Ligação Proteica , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Treonina/química , Treonina/farmacocinética , Treonina/farmacologia , Fatores de TempoRESUMO
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.
Assuntos
Antibacterianos/farmacologia , Compostos Azabicíclicos/farmacologia , Proteínas de Bactérias/biossíntese , Ceftazidima/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Seleção Genética , Resistência beta-Lactâmica , beta-Lactamases/biossíntese , Proteínas de Bactérias/genética , Combinação de Medicamentos , Humanos , Testes de Sensibilidade Microbiana , Taxa de Mutação , Pseudomonas aeruginosa/genética , beta-Lactamases/genéticaRESUMO
Measurement of bacterial burden in animal infection models is a key component for both bacterial pathogenesis studies and therapeutic agent research. The traditional quantification means for in vivo bacterial burden requires frequent animal sacrifice and enumerating colony forming units (CFU) recovered from infection loci. To address these issues, researchers have developed a variety of luciferase-expressing bacterial reporter strains to enable bacterial detection in living animals. To date, all such luciferase-based bacterial reporters are in cell-associated form. Production of luciferase-secreting recombinant bacteria could provide the advantage of reporting CFU from both infection loci themselves and remote sampling (eg. body fluid and plasma). Toward this end, we have genetically manipulated a pathogenic Escherichia coli (E. coli) strain, ATCC25922, to secrete the marine copepod Gaussia princeps luciferase (Gluc), and assessed the use of Gluc as both an in situ and ex situ reporter for bacterial burden in mouse tissue cage infections. The E. coli expressing Gluc demonstrates in vivo imaging of bacteria in a tissue cage model of infection. Furthermore, secreted Gluc activity and bacterial CFUs recovered from tissue cage fluid (TCF) are correlated along 18 days of infection. Importantly, secreted Gluc can also be detected in plasma samples and serve as an ex situ indicator for the established tissue cage infection, once high bacterial burdens are achieved. We have demonstrated that Gluc from marine eukaryotes can be stably expressed and secreted by pathogenic E. coli in vivo to enable a facile tool for longitudinal evaluation of persistent bacterial infection.
Assuntos
Infecções por Escherichia coli/microbiologia , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Genes Reporter , Luciferases/metabolismo , Animais , Biocatálise , Cromossomos Bacterianos/genética , Contagem de Colônia Microbiana , Copépodes , Modelos Animais de Doenças , Erwinia/enzimologia , Infecções por Escherichia coli/sangue , Infecções por Escherichia coli/patologia , Loci Gênicos/genética , Imageamento Tridimensional , Luciferases/sangue , Luminescência , Camundongos , Polissacarídeo-Liases/metabolismoRESUMO
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.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Nucleotidiltransferases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Streptococcus pneumoniae/efeitos dos fármacos , Animais , Antibacterianos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ligação Competitiva , Cristalografia por Raios X , Descoberta de Drogas , Inibidores Enzimáticos/química , Feminino , Pulmão/efeitos dos fármacos , Pulmão/microbiologia , Camundongos , Modelos Moleculares , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Panteteína/análogos & derivados , Panteteína/química , Infecções Pneumocócicas/tratamento farmacológico , Infecções Pneumocócicas/microbiologia , Pneumonia Bacteriana/tratamento farmacológico , Pneumonia Bacteriana/microbiologia , Bibliotecas de Moléculas Pequenas/química , Staphylococcus aureus/enzimologia , Staphylococcus aureus/crescimento & desenvolvimento , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/crescimento & desenvolvimento , Coxa da Perna/microbiologiaRESUMO
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.
Assuntos
Compostos Azabicíclicos/química , Escherichia coli/efeitos dos fármacos , beta-Lactamases/química , Antibacterianos/química , Farmacorresistência Bacteriana , Enterobacter cloacae/metabolismo , Inibidores Enzimáticos/química , Escherichia coli/metabolismo , Hidrólise , Cinética , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Plasmídeos/metabolismo , Pseudomonas aeruginosa/metabolismo , Fatores de TempoRESUMO
Avibactam is a ß-lactamase inhibitor that is in clinical development, combined with ß-lactam partners, for the treatment of bacterial infections comprising gram-negative organisms. Avibactam is a structural class of inhibitor that does not contain a ß-lactam core but maintains the capacity to covalently acylate its ß-lactamase targets. Using the TEM-1 enzyme, we characterized avibactam inhibition by measuring the on-rate for acylation and the off-rate for deacylation. The deacylation off-rate was 0.045 min(-1), which allowed investigation of the deacylation route from TEM-1. Using NMR and MS, we showed that deacylation proceeds through regeneration of intact avibactam and not hydrolysis. Other than TEM-1, four additional clinically relevant ß-lactamases were shown to release intact avibactam after being acylated. We showed that avibactam is a covalent, slowly reversible inhibitor, which is a unique mechanism of inhibition among ß-lactamase inhibitors.
Assuntos
Antibacterianos/farmacologia , Compostos Azabicíclicos/farmacologia , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Inibidores de beta-Lactamases , Acilação/efeitos dos fármacos , Antibacterianos/química , Antibacterianos/metabolismo , Compostos Azabicíclicos/química , Compostos Azabicíclicos/metabolismo , Descoberta de Drogas/métodos , Cinética , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Estrutura Molecular , beta-LactamasesRESUMO
NAD(+)-dependent DNA ligases (LigA) are essential bacterial enzymes that catalyze phosphodiester bond formation during DNA replication and repair processes. Phosphodiester bond formation proceeds through a 3-step reaction mechanism. In the first step, the LigA adenylation domain interacts with NAD(+) to form a covalent enzyme-AMP complex. Although it is well established that the specificity for binding of NAD(+) resides within the adenylation domain, the precise recognition elements for the initial binding event remain unclear. We report here the structure of the adenylation domain from Haemophilus influenzae LigA. This structure is a first snapshot of a LigA-AMP intermediate with NAD(+) bound to domain 1a in its open conformation. The binding affinities of NAD(+) for adenylated and nonadenylated forms of the H. influenzae LigA adenylation domain were similar. The combined crystallographic and NAD(+)-binding data suggest that the initial recognition of NAD(+) is via the NMN binding region in domain 1a of LigA.
Assuntos
DNA Ligases/metabolismo , Haemophilus influenzae/enzimologia , NAD/metabolismo , Calorimetria , Clonagem Molecular , Cristalização , DNA Ligases/química , DNA Ligases/isolamento & purificação , Modelos Moleculares , Conformação ProteicaRESUMO
DNA gyrase is an essential enzyme in bacteria, and its inhibition results in the disruption of DNA synthesis and, subsequently, cell death. The pyrrolamides are a novel class of antibacterial agents targeting DNA gyrase. These compounds were identified by a fragment-based lead generation (FBLG) approach using nuclear magnetic resonance (NMR) screening to identify low-molecular-weight compounds that bind to the ATP pocket of DNA gyrase. A pyrrole hit with a binding constant of 1 mM formed the basis of the design and synthesis of a focused library of compounds that resulted in the rapid identification of a lead compound that inhibited DNA gyrase with a 50% inhibitory concentration (IC(50)) of 3 µM. The potency of the lead compound was further optimized by utilizing iterative X-ray crystallography to yield DNA gyrase inhibitors that also displayed antibacterial activity. Spontaneous mutants were isolated in Staphylococcus aureus by plating on agar plates containing pyrrolamide 4 at the MIC. The resistant variants displayed 4- to 8-fold-increased MIC values relative to the parent strain. DNA sequencing revealed two independent point mutations in the pyrrolamide binding region of the gyrB genes from these variants, supporting the hypothesis that the mode of action of these compounds was inhibition of DNA gyrase. Efficacy of a representative pyrrolamide was demonstrated against Streptococcus pneumoniae in a mouse lung infection model. These data demonstrate that the pyrrolamides are a novel class of DNA gyrase inhibitors with the potential to deliver future antibacterial agents targeting multiple clinical indications.
Assuntos
Amidas/farmacologia , Antibacterianos/farmacologia , Pirróis/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Streptococcus pneumoniae/efeitos dos fármacos , Inibidores da Topoisomerase II , Amidas/química , Animais , Antibacterianos/química , Sítios de Ligação , Cristalografia por Raios X , DNA Girase/química , DNA Girase/metabolismo , Farmacorresistência Bacteriana , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Mutação , Ligação Proteica , Pirróis/química , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/crescimento & desenvolvimento , Streptococcus pneumoniae/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Aminopeptidases/metabolismo , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala , Ligases/metabolismo , Metionina/metabolismo , S-Adenosilmetionina/biossíntese , Aminopeptidases/antagonistas & inibidores , Cátions Bivalentes/farmacologia , Ácido Edético/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Ensaios de Triagem em Larga Escala/normas , Cinética , Manganês/farmacologia , Metionil Aminopeptidases , Padrões de Referência , S-Adenosilmetionina/metabolismoRESUMO
DNA ligase is the enzyme that catalyzes the formation of the backbone phosphodiester bond between the 5'-PO(4) and 3'-OH of adjacent DNA nucleotides at single-stranded nicks. These nicks occur between Okazaki fragments during replication of the lagging strand of the DNA as well as during DNA repair and recombination. As essential enzymes for DNA replication, the NAD(+)-dependent DNA ligases of pathogenic bacteria are potential targets for the development of antibacterial drugs. For the purposes of drug discovery, a high-throughput assay for DNA ligase activity is invaluable. This article describes a straightforward, fluorescence resonance energy transfer-based DNA ligase assay that is well suited for high-throughput screening for DNA ligase inhibitors as well as for use in enzyme kinetics studies. Its use is demonstrated for measurement of the steady-state kinetic constants of Haemophilus influenzae NAD(+)-dependent DNA ligase and for measurement of the potency of an inhibitor of this enzyme.
Assuntos
DNA Ligases/metabolismo , Descoberta de Drogas/métodos , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , Antibacterianos/farmacologia , DNA/metabolismo , DNA Ligases/análise , DNA Ligases/antagonistas & inibidores , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Haemophilus influenzae/efeitos dos fármacos , Haemophilus influenzae/enzimologia , Haemophilus influenzae/genética , Cinética , NAD/metabolismo , NAD/farmacologiaRESUMO
DNA ligases are indispensable enzymes playing a critical role in DNA replication, recombination, and repair in all living organisms. Bacterial NAD+-dependent DNA ligase (LigA) was evaluated for its potential as a broad-spectrum antibacterial target. A novel class of substituted adenosine analogs was discovered by target-based high-throughput screening (HTS), and these compounds were optimized to render them more effective and selective inhibitors of LigA. The adenosine analogs inhibited the LigA activities of Escherichia coli, Haemophilus influenzae, Mycoplasma pneumoniae, Streptococcus pneumoniae, and Staphylococcus aureus, with inhibitory activities in the nanomolar range. They were selective for bacterial NAD+-dependent DNA ligases, showing no inhibitory activity against ATP-dependent human DNA ligase 1 or bacteriophage T4 ligase. Enzyme kinetic measurements demonstrated that the compounds bind competitively with NAD+. X-ray crystallography demonstrated that the adenosine analogs bind in the AMP-binding pocket of the LigA adenylation domain. Antibacterial activity was observed against pathogenic Gram-positive and atypical bacteria, such as S. aureus, S. pneumoniae, Streptococcus pyogenes, and M. pneumoniae, as well as against Gram-negative pathogens, such as H. influenzae and Moraxella catarrhalis. The mode of action was verified using recombinant strains with altered LigA expression, an Okazaki fragment accumulation assay, and the isolation of resistant strains with ligA mutations. In vivo efficacy was demonstrated in a murine S. aureus thigh infection model and a murine S. pneumoniae lung infection model. Treatment with the adenosine analogs reduced the bacterial burden (expressed in CFU) in the corresponding infected organ tissue as much as 1,000-fold, thus validating LigA as a target for antibacterial therapy.
Assuntos
Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , DNA Ligases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Animais , Feminino , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/patogenicidadeRESUMO
The pK(a) values of 211 discovery (druglike) compounds were determined experimentally using capillary electrophoresis coupled with ultraviolet spectroscopy and a novel fitting algorithm. These values were compared to those predicted by five different commercially available pK(a) estimation packages: ACDLabs/pK(a), Marvin (ChemAxon), MoKa (Molecular Discovery), Epik (Schrodinger), and Pipeline Pilot (Accelrys). Even though the topological method MoKa was noticeably faster than ACD, the accuracy of those two methods and Marvin was statistically indistinguishable, with a root-mean-squared error of about 1 pK(a) unit compared to experiment. Pipeline Pilot and EpiK both produced pK(a) estimates in significantly worse agreement with the experiment. Interestingly, on a number of compounds, the predictions due to ACD v12 were in poorer agreement with the experiment than ACD v10. Microscopic and "apparent" pK(a) predictions were also compared using ACD v10. Microscopic pK(a)s gave significantly worse agreement with the experiment than the "apparent" values. In all cases, the errors appeared to be randomly distributed across chemical series.
Assuntos
Algoritmos , Fenômenos Químicos , Preparações Farmacêuticas/química , Descoberta de Drogas , SoftwareRESUMO
In high-throughput biochemical assays performed in multiwell plates, the effect of test samples on the activity of the biochemical system is usually measured by optical means such as absorbance, fluorescence, luminescence, or scintillation counting. The test sample often causes detection interference when it remains in the well during the measurement. Interference may be due to light absorption, fluorescence quenching, sample fluorescence, chemical interaction of the sample with a detection reagent, or depression of the meniscus. A simple method is described that corrects for such interference well by well. The interference is measured in a separate artifact assay plate. An appropriate arithmetic correction is then applied to the measurement in the corresponding well of the activity assay plate. The correction procedure can be used for single-point screening or potency measurements on serial dilutions of test samples.
Assuntos
Artefatos , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/normas , Absorção/fisiologia , Algoritmos , Calibragem , Interpretação Estatística de Dados , Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Transferência Ressonante de Energia de Fluorescência/normas , Concentração Inibidora 50 , Luz , Medições Luminescentes/métodos , Medições Luminescentes/normas , Variações Dependentes do Observador , Projetos de Pesquisa/normas , Contagem de Cintilação/métodos , Contagem de Cintilação/normas , Manejo de Espécimes/métodos , Manejo de Espécimes/normasRESUMO
We recently introduced a method (Griffin, B. A.; Adams, S. R.; Tsien, R. Y. Science 1998, 281, 269-272 and Griffin, B. A.; Adams, S. R.; Jones, J.; Tsien, R. Y. Methods Enzymol. 2000, 327, 565-578) for site-specific fluorescent labeling of recombinant proteins in living cells. The sequence Cys-Cys-Xaa-Xaa-Cys-Cys, where Xaa is an noncysteine amino acid, is genetically fused to or inserted within the protein, where it can be specifically recognized by a membrane-permeant fluorescein derivative with two As(III) substituents, FlAsH, which fluoresces only after the arsenics bind to the cysteine thiols. We now report kinetics and dissociation constants ( approximately 10(-11) M) for FlAsH binding to model tetracysteine peptides. Affinities in vitro and detection limits in living cells are optimized with Xaa-Xaa = Pro-Gly, suggesting that the preferred peptide conformation is a hairpin rather than the previously proposed alpha-helix. Many analogues of FlAsH have been synthesized, including ReAsH, a resorufin derivative excitable at 590 nm and fluorescing in the red. Analogous biarsenicals enable affinity chromatography, fluorescence anisotropy measurements, and electron-microscopic localization of tetracysteine-tagged proteins.