RESUMEN
Nitroarenes are less preferred in drug discovery due to their potential to be mutagenic. However, several nitroarenes were shown to be promising antitubercular agents with specific modes of action, namely, nitroimidazoles and benzothiazinones. The nitro group in these compounds is activated through different mechanisms, both enzymatic and non-enzymatic, in mycobacteria prior to binding to the target of interest. From a whole-cell screening program, we identified a novel lead nitrobenzothiazole (BT) series that acts by inhibition of decaprenylphosphoryl-ß-d-ribose 2'-epimerase (DprE1) of Mycobacterium tuberculosis (Mtb). The lead was found to be mutagenic to start with. Our efforts to mitigate mutagenicity resulted in the identification of 6-methyl-7-nitro-5-(trifluoromethyl)-1,3-benzothiazoles (cBTs), a novel class of antitubercular agents that are non-mutagenic and exhibit an improved safety profile. The methyl group ortho to the nitro group decreases the electron affinity of the series, and is hence responsible for the non-mutagenic nature of these compounds. Additionally, the co-crystal structure of cBT in complex with Mtb DprE1 established the mode of binding. This investigation led to a new non-mutagenic antitubercular agent and demonstrates that the mutagenic nature of nitroarenes can be solved by modulation of stereoelectronic properties.
Asunto(s)
Antituberculosos/farmacología , Benzotiazoles/farmacología , Mutágenos/química , Mycobacterium tuberculosis/efectos de los fármacos , Nitrocompuestos/farmacología , Antituberculosos/efectos adversos , Antituberculosos/química , Benzotiazoles/efectos adversos , Benzotiazoles/química , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Nitrocompuestos/efectos adversos , Nitrocompuestos/química , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Discovery of mupirocin, an antibiotic that targets isoleucyl-tRNA synthetase, established aminoacyl-tRNA synthetase as an attractive target for the discovery of novel antibacterial agents. Despite a high degree of similarity between the bacterial and human aminoacyl-tRNA synthetases, the selectivity observed with mupirocin triggered the possibility of targeting other aminoacyl-tRNA synthetases as potential drug targets. These enzymes catalyse the condensation of a specific amino acid to its cognate tRNA in an energy-dependent reaction. Therefore, each organism is expected to encode at least twenty aminoacyl-tRNA synthetases, one for each amino acid. However, a bioinformatics search for genes encoding aminoacyl-tRNA synthetases from Mycobacterium smegmatis returned multiple genes for glutamyl (GluRS), cysteinyl (CysRS), prolyl (ProRS) and lysyl (LysRS) tRNA synthetases. The pathogenic mycobacteria, namely, Mycobacterium tuberculosis and Mycobacterium leprae, were also found to possess two genes each for CysRS and LysRS. A similar search indicated the presence of additional genes for LysRS in gram negative bacteria as well. Herein, we describe sequence and structural analysis of the additional aminoacyl-tRNA synthetase genes found in M. smegmatis. Characterization of conditional expression strains of Cysteinyl and Lysyl-tRNA synthetases generated in M. smegmatis revealed that the canonical aminoacyl-tRNA synthetase are essential, while the additional ones are not essential for the growth of M. smegmatis.
Asunto(s)
Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/metabolismo , Lisina-ARNt Ligasa/química , Lisina-ARNt Ligasa/metabolismo , Mutación/genética , Mycobacterium smegmatis/enzimología , Aminoacil-ARNt Sintetasas/genética , Humanos , Lisina-ARNt Ligasa/genética , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crecimiento & desarrollo , ARN de Transferencia/metabolismoRESUMEN
Conditional expression strains serve as a valuable tool to study the essentiality and to establish the vulnerability of a target under investigation in a drug discovery program. While essentiality implies an absolute requirement of a target function, vulnerability provides valuable information on the extent to which a target function needs to be depleted to achieve bacterial growth inhibition followed by cell death. The critical feature of an ideal conditional expression system is its ability to tightly regulate gene expression to achieve the full spectrum spanning from a high level of expression in order to support growth and near zero level of expression to mimic conditions of gene knockout. A number of bacterial conditional expression systems have been reported for use in mycobacteria. The utility of an isopropylthiogalactoside (IPTG) inducible system in mycobacteria has been reported for protein overexpression and anti-sense gene expression from a replicating multi-copy plasmid. Herein, we report the development of a versatile set of non-replicating IPTG inducible vectors for mycobacteria which can be used for generation of conditional expression strains through homologous recombination. The role of a single lac operator versus a double lac operator to regulate gene expression was evaluated by monitoring the expression levels of ß-galactosidase in Mycobacterium smegmatis. These studies indicated a significant level of leaky expression from the vector with a single lac operator but none from the vector with double lac operator. The significance of the double lac operator vector for target validation was established by monitoring the growth kinetics of an inhA, a rpoB and a ftsZ conditional expression strain grown in the presence of different concentrations of IPTG. The utility of this inducible system in identifying target specific inhibitors was established by screening a focussed library of small molecules using an inhA and a rpoB conditional expression strain.
Asunto(s)
Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Isopropil Tiogalactósido/farmacología , Mycobacterium smegmatis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Operón Lac/genética , Mycobacterium smegmatis/crecimiento & desarrollo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Fenotipo , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
DNA topoisomerases perform the essential function of maintaining DNA topology in prokaryotes. DNA gyrase, an essential enzyme that introduces negative supercoils, is a clinically validated target. However, topoisomerase I (Topo I), an enzyme responsible for DNA relaxation has received less attention as an antibacterial target, probably due to the ambiguity over its essentiality in many organisms. The Mycobacterium tuberculosis genome harbors a single topA gene with no obvious redundancy in its function suggesting an essential role. The topA gene could be inactivated only in the presence of a complementing copy of the gene in M. tuberculosis. Furthermore, down-regulation of topA in a genetically engineered strain of M. tuberculosis resulted in loss of bacterial viability which correlated with a concomitant depletion of intracellular Topo I levels. The topA knockdown strain of M. tuberculosis failed to establish infection in a murine model of TB and was cleared from lungs in two months post infection. Phenotypic screening of a Topo I overexpression strain led to the identification of an inhibitor, thereby providing chemical validation of this target. Thus, our work confirms the attractiveness of Topo I as an anti-mycobacterial target.
Asunto(s)
Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , ADN-Topoisomerasas de Tipo I , Descubrimiento de Drogas , Mycobacterium tuberculosis/efectos de los fármacos , Inhibidores de Topoisomerasa I/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/metabolismo , Regulación Bacteriana de la Expresión Génica , Técnicas de Silenciamiento del Gen , Genotipo , Humanos , Viabilidad Microbiana , Terapia Molecular Dirigida , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/patogenicidad , Fenotipo , Factores de TiempoRESUMEN
The Mur ligases play an essential role in the biosynthesis of bacterial peptidoglycan and hence are attractive antibacterial targets. A screen of the AstraZeneca compound library led to the identification of compound A, a pyrazolopyrimidine, as a potent inhibitor of Escherichia coli and Pseudomonas aeruginosa MurC. However, cellular activity against E. coli or P. aeruginosa was not observed. Compound A was active against efflux pump mutants of both strains. Experiments using an E. coli tolC mutant revealed accumulation of the MurC substrate and a decrease in the level of product upon treatment with compound A ,: indicating inhibition of MurC enzyme in these cells. Such a modulation was not observed in the E. coli wild-type cells. Further, overexpression of MurC in the E. coli tolC mutant led to an increase in the compound A MIC by ≥16-fold, establishing a correlation between MurC inhibition and cellular activity. In addition, estimation of the intracellular compound A level showed an accumulation of the compound over time in the tolC mutant strain. A significant compound A level was not detected in the wild-type E. coli strain even upon treatment with high concentrations of the compound. Therefore, the lack of MIC and absence of MurC inhibition in wild-type E. coli were possibly due to suboptimal compound concentration as a consequence of a high efflux level and/or poor permeativity of compound A.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/citología , Escherichia coli/enzimología , Péptido Sintasas/metabolismo , Alanina/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Mutación , Péptido Sintasas/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Uridina Difosfato Ácido N-Acetilmurámico/metabolismoRESUMEN
The cell envelope of Mycobacterium tuberculosis contains glycans and lipids of peculiar structure that play prominent roles in the biology and pathogenesis of tuberculosis. Consequently, the chemical structure and biosynthesis of the cell wall have been intensively investigated in order to identify novel drug targets. Here, we validate that the function of phosphatidyl-myo-inositol mannosyltransferase PimA is vital for M. tuberculosis in vitro and in vivo. PimA initiates the biosynthesis of phosphatidyl-myo-inositol mannosides by transferring a mannosyl residue from GDP-Man to phosphatidyl-myo-inositol on the cytoplasmic side of the plasma membrane. To prove the essential nature of pimA in M. tuberculosis, we constructed a pimA conditional mutant by using the TetR-Pip off system and showed that downregulation of PimA expression causes bactericidality in batch cultures. Consistent with the biochemical reaction catalyzed by PimA, this phenotype was associated with markedly reduced levels of phosphatidyl-myo-inositol dimannosides, essential structural components of the mycobacterial cell envelope. In addition, the requirement of PimA for viability was clearly demonstrated during macrophage infection and in two different mouse models of infection, where a dramatic decrease in viable counts was observed upon silencing of the gene. Notably, depletion of PimA resulted in complete clearance of the mouse lungs during both the acute and chronic phases of infection. Altogether, the experimental data highlight the importance of the phosphatidyl-myo-inositol mannoside biosynthetic pathway for M. tuberculosis and confirm that PimA is a novel target for future drug discovery programs.
Asunto(s)
Proteínas Bacterianas/metabolismo , Manosiltransferasas/metabolismo , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/crecimiento & desarrollo , Tuberculosis/microbiología , Animales , Proteínas Bacterianas/genética , Femenino , Humanos , Macrófagos/metabolismo , Macrófagos/microbiología , Manosiltransferasas/genética , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/genética , Fosfatidilinositoles/biosíntesisRESUMEN
The bacterial peptidoglycan biosynthesis pathway provides multiple targets for antibacterials, as proven by the clinical success of ß-lactam and glycopeptide classes of antibiotics. The Mur ligases play an essential role in the biosynthesis of the peptidoglycan building block, N-acetyl-muramic acid-pentapeptide. MurC, the first of four Mur ligases, ligates l-alanine to UDP-N-acetylmuramic acid, initiating the synthesis of pentapeptide precursor. Therefore, inhibiting the MurC enzyme should result in bacterial cell death. Herein, we report a novel class of pyrazolopyrimidines with subnanomolar potency against both Escherichia coli and Pseudomonas aeruginosa MurC enzymes, which demonstrates a concomitant bactericidal activity against efflux-deficient strains. Radio-labeled precursor incorporation showed these compounds selectively inhibited peptidoglycan biosynthesis, and genetic studies confirmed the target of pyrazolopyrimidines to be MurC. In the presence of permeability enhancers such as colistin, pyrazolopyrimidines exhibited low micromolar MIC against the wild-type bacteria, thereby, indicating permeability and efflux as major challenges for this chemical series. Our studies provide biochemical and genetic evidence to support the essentiality of MurC and serve to validate the attractiveness of target for antibacterial discovery.
Asunto(s)
Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Escherichia coli/enzimología , Péptido Sintasas/antagonistas & inhibidores , Pseudomonas aeruginosa/enzimología , Pirazoles/farmacología , Pirimidinas/farmacología , Alanina/metabolismo , Antibacterianos/química , Inhibidores Enzimáticos/química , Escherichia coli/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Modelos Químicos , Estructura Molecular , Péptido Sintasas/metabolismo , Proteínas Quinasas/química , Pseudomonas aeruginosa/efectos de los fármacos , Relación Estructura-Actividad , Uridina Difosfato Ácido N-Acetilmurámico/metabolismoRESUMEN
Diarylthiazole (DAT), a hit from diversity screening, was found to have potent antimycobacterial activity against Mycobacterium tuberculosis (Mtb). In a systematic medicinal chemistry exploration, we demonstrated chemical opportunities to optimize the potency and physicochemical properties. The effort led to more than 10 compounds with submicromolar MICs and desirable physicochemical properties. The potent antimycobacterial activity, in conjunction with low molecular weight, made the series an attractive lead (antibacterial ligand efficiency (ALE)>0.4). The series exhibited excellent bactericidal activity and was active against drug-sensitive and resistant Mtb. Mutational analysis showed that mutations in prrB impart resistance to DAT compounds but not to reference drugs tested. The sensor kinase PrrB belongs to the PrrBA two component system and is potentially the target for DAT. PrrBA is a conserved, essential regulatory mechanism in Mtb and has been shown to have a role in virulence and metabolic adaptation to stress. Hence, DATs provide an opportunity to understand a completely new target system for antimycobacterial drug discovery.
Asunto(s)
Antituberculosos/química , Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Proteínas Quinasas/metabolismo , Tiazoles/química , Animales , Antituberculosos/síntesis química , Antituberculosos/farmacología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Polimorfismo de Nucleótido Simple , Proteínas Quinasas/genética , Bibliotecas de Moléculas Pequeñas , Relación Estructura-Actividad , Tiazoles/síntesis química , Tiazoles/farmacologíaRESUMEN
In a previous report, we described the discovery of 1,4-azaindoles, a chemical series with excellent in vitro and in vivo antimycobacterial potency through noncovalent inhibition of decaprenylphosphoryl-ß-d-ribose-2'-epimerase (DprE1). Nevertheless, high mouse metabolic turnover and phosphodiesterase 6 (PDE6) off-target activity limited its advancement. Herein, we report lead optimization of this series, culminating in potent, metabolically stable compounds that have a robust pharmacokinetic profile without any PDE6 liability. Furthermore, we demonstrate efficacy for 1,4-azaindoles in a rat chronic TB infection model. We believe that compounds from the 1,4-azaindole series are suitable for in vivo combination and safety studies.
Asunto(s)
Antituberculosos/síntesis química , Indoles/síntesis química , Oxidorreductasas de Alcohol , Animales , Antituberculosos/farmacocinética , Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/antagonistas & inhibidores , Modelos Animales de Enfermedad , Humanos , Indoles/farmacocinética , Ratones , Mycobacterium tuberculosis/efectos de los fármacos , Oxidorreductasas/antagonistas & inhibidores , Ratas , Relación Estructura-ActividadRESUMEN
4-Aminoquinolone piperidine amides (AQs) were identified as a novel scaffold starting from a whole cell screen, with potent cidality on Mycobacterium tuberculosis (Mtb). Evaluation of the minimum inhibitory concentrations, followed by whole genome sequencing of mutants raised against AQs, identified decaprenylphosphoryl-ß-d-ribose 2'-epimerase (DprE1) as the primary target responsible for the antitubercular activity. Mass spectrometry and enzyme kinetic studies indicated that AQs are noncovalent, reversible inhibitors of DprE1 with slow on rates and long residence times of â¼100 min on the enzyme. In general, AQs have excellent leadlike properties and good in vitro secondary pharmacology profile. Although the scaffold started off as a single active compound with moderate potency from the whole cell screen, structure-activity relationship optimization of the scaffold led to compounds with potent DprE1 inhibition (IC50 < 10 nM) along with potent cellular activity (MIC = 60 nM) against Mtb.
Asunto(s)
Amidas/química , Antituberculosos/química , Proteínas Bacterianas/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Oxidorreductasas/antagonistas & inhibidores , Piperidinas/química , Quinolonas/química , Oxidorreductasas de Alcohol , Amidas/farmacocinética , Amidas/farmacología , Animales , Antituberculosos/farmacocinética , Antituberculosos/farmacología , Dominio Catalítico , Línea Celular Tumoral , Farmacorresistencia Bacteriana , Genoma Bacteriano , Humanos , Cinética , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Mutación , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Piperidinas/farmacocinética , Piperidinas/farmacología , Unión Proteica , Quinolonas/farmacocinética , Quinolonas/farmacología , Ratas Wistar , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Pantothenate kinase (PanK) catalyzes the phosphorylation of pantothenate, the first committed and rate-limiting step toward coenzyme A (CoA) biosynthesis. In our earlier reports, we had established that the type I isoform encoded by the coaA gene is an essential pantothenate kinase in Mycobacterium tuberculosis, and this vital information was then exploited to screen large libraries for identification of mechanistically different classes of PanK inhibitors. The present report summarizes the synthesis and expansion efforts to understand the structure-activity relationships leading to the optimization of enzyme inhibition along with antimycobacterial activity. Additionally, we report the progression of two distinct classes of inhibitors, the triazoles, which are ATP competitors, and the biaryl acetic acids, with a mixed mode of inhibition. Cocrystallization studies provided evidence of these inhibitors binding to the enzyme. This was further substantiated with the biaryl acids having MIC against the wild-type M. tuberculosis strain and the subsequent establishment of a target link with an upshift in MIC in a strain overexpressing PanK. On the other hand, the ATP competitors had cellular activity only in a M. tuberculosis knockdown strain with reduced PanK expression levels. Additionally, in vitro and in vivo survival kinetic studies performed with a M. tuberculosis PanK (MtPanK) knockdown strain indicated that the target levels have to be significantly reduced to bring in growth inhibition. The dual approaches employed here thus established the poor vulnerability of PanK in M. tuberculosis.
Asunto(s)
Antituberculosos/farmacología , Inhibidores Enzimáticos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Western Blotting , Técnicas de Silenciamiento del Gen , Humanos , Pruebas de Sensibilidad Microbiana , Mycobacterium bovis/genética , Mycobacterium tuberculosis/genética , Fenotipo , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Conformación Proteica , Quinolonas/farmacología , Relación Estructura-Actividad , Triazoles/farmacologíaRESUMEN
Pantothenate kinase, an essential enzyme in bacteria and eukaryotes, is involved in catalysing the first step of conversion of pantothenate to coenzyme A (CoA). Three isoforms (type I, II and III) of this enzyme have been reported from various organisms, which can be differentiated from each other on the basis of their biochemical and structural characteristics. Though most bacteria carry only one of the isoforms of pantothenate kinases, some of them possess two isoforms. The physiological relevance of the presence of two types of isozymes in a single organism is not clear. Mycobacterium tuberculosis, an intracellular pathogen, possesses two isoforms of pantothenate kinases (CoaA and CoaX) belonging to type I and III. In order to determine which pantothenate kinase is essential in mycobacteria, we performed gene inactivation of coaA and coaX of M. tuberculosis individually. It was found that coaA could only be inactivated in the presence of an extra copy of the gene, while coaX could be inactivated in the wild-type cells, proving that CoaA is the essential pantothenate kinase in M. tuberculosis. Additionally, the coaA gene of M. tuberculosis was able to complement a temperature-sensitive coaA mutant of Escherichia coli at a non-permissive temperature while coaX could not. The coaX deletion mutant showed no growth defects in vitro, in macrophages or in mice. Taken together, our data suggest that CoaX, which is essential in Bacillus anthracis and thus had been suggested to be a drug target in this organism, might not be a valid target in M. tuberculosis. We have established that the type I isoform, CoaA, is the essential pantothenate kinase in M. tuberculosis and thus can be explored as a drug target.
Asunto(s)
Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Ratones , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Eliminación de Secuencia , Tuberculosis/microbiologíaRESUMEN
Empyema necessitatis is a rare complication of empyema and is more commonly reported in adults. It is characterized by purulence in the pleural space extending into the overlying soft tissues. We report a rare case in an otherwise healthy 3-month-old girl who presented with right chest wall swelling and was subsequently found to have methicillin-resistant Staphylococcus aureus empyema necessitatis requiring thoracotomy with decortication.
Asunto(s)
Empiema Pleural/cirugía , Staphylococcus aureus , Empiema Pleural/diagnóstico por imagen , Empiema Pleural/microbiología , Femenino , Humanos , Lactante , Resistencia a la Meticilina , Procedimientos Quirúrgicos Pulmonares , Radiografía , Infecciones Estafilocócicas/complicacionesRESUMEN
MurG and MraY, essential enzymes involved in the synthesis of bacterial peptidoglycan, are difficult to assay because the substrates are lipidic and hard to prepare in large quantities. Based on the use of Escherichia coli membranes lacking PBP1b, we report a high-throughput method to measure the activity of MurG and, optionally, MraY as well. In these membranes, incubation with the two peptidoglycan sugar precursors results in accumulation of lipid II rather than the peptidoglycan produced by wild-type membranes. MurG was assayed by addition of UDP-[3H]N-acetylglucosamine to membranes in which lipid I was preformed by incubation with UDP-N-acetyl-muramylpentapeptide, and the product was captured by wheat germ agglutinin scintillation proximity assay beads. In a modification of the assay, the activity of MraY was coupled to that of MurG by addition of both sugar precursors together in a single step. This allows simultaneous detection of inhibitors of either enzyme. Both assays could be performed using wild-type membranes by addition of the transglycosylase inhibitor moenomycin. Nisin and vancomycin inhibited the MurG reaction; the MraY-MurG assay was inhibited by tunicamycin as well. Inhibitors of other enzymes of peptidoglycan synthesis--penicillin G, moenomycin, and bacitracin--had no effect. Surprisingly, however, the beta-lactam cephalosporin C inhibited both the MurG and MraY-MurG assays, indicating a secondary mechanism by which this drug inhibits bacterial growth. In addition, it inhibited NADH dehydrogenase in membranes, a hitherto-unreported activity. These assays can be used to screen for novel antibacterial agents.
Asunto(s)
Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Escherichia coli/efectos de los fármacos , N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , Transferasas/antagonistas & inhibidores , Uridina Difosfato Ácido N-Acetilmurámico/análogos & derivados , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cefalosporinas/farmacología , Cromatografía en Papel , Escherichia coli/enzimología , Proteínas de Escherichia coli/genética , Pruebas de Sensibilidad Microbiana/métodos , Mutación , Proteínas de Unión a las Penicilinas/genética , Peptidoglicano/biosíntesis , Peptidoglicano Glicosiltransferasa/genética , Conteo por Cintilación , Sensibilidad y Especificidad , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos) , Uridina Difosfato Ácido N-Acetilmurámico/biosíntesisRESUMEN
Penicillin binding protein (PBP) 1b of Escherichia coli has both transglycosylase and transpeptidase activities, which are attractive targets for the discovery of new antibacterial agents. A high-throughput assay that detects inhibitors of the PBPs was described previously, but it cannot distinguish them from inhibitors of the MraY, MurG, and lipid pyrophosphorylase. We report on a method that distinguishes inhibitors of both activities of the PBPs from those of the other three enzymes. Radioactive peptidoglycan was synthesized by using E. coli membranes. Following termination of the reaction the products were analyzed in three ways. Wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads were added to one set, and the same beads together with a detergent were added to a second set. Type A polyethylenimine-coated WGA-coated SPA beads were added to a third set. By comparison of the results of assays run in parallel under the first two conditions, inhibitors of the transpeptidase and transglycosylase could be distinguished from inhibitors of the other enzymes, as the inhibitors of the other enzymes showed similar inhibitory concentrations (IC(50)s) under both conditions but the inhibitors of the PBPs showed insignificant inhibition in the absence of detergent. Furthermore, comparison of the results of assays run under conditions two and three enabled the distinction of transpeptidase inhibitors. Penicillin and other beta-lactams showed insignificant inhibition with type A beads compared with that shown with WGA-coated SPA beads plus detergent. However, inhibitors of the other four enzymes (tunicamycin, nisin, bacitracin, and moenomycin) showed similar IC(50)s under both conditions. We show that the main PBP being measured under these conditions is PBP 1b. This screen can be used to find novel transglycosylase or transpeptidase inhibitors.