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1.
Nature ; 2020 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-32051588

RESUMO

Addressing the ongoing antibiotic crisis requires the discovery of compounds with novel mechanisms of action that are capable of treating drug-resistant infections1. Many antibiotics are sourced from specialized metabolites produced by bacteria, particularly those of the Actinomycetes family2. Although actinomycete extracts have traditionally been screened using activity-based platforms, this approach has become unfavourable owing to the frequent rediscovery of known compounds. Genome sequencing of actinomycetes reveals an untapped reservoir of biosynthetic gene clusters, but prioritization is required to predict which gene clusters may yield promising new chemical matter2. Here we make use of the phylogeny of biosynthetic genes along with the lack of known resistance determinants to predict divergent members of the glycopeptide family of antibiotics that are likely to possess new biological activities. Using these predictions, we uncovered two members of a new functional class of glycopeptide antibiotics-the known glycopeptide antibiotic complestatin and a newly discovered compound we call corbomycin-that have a novel mode of action. We show that by binding to peptidoglycan, complestatin and corbomycin block the action of autolysins-essential peptidoglycan hydrolases that are required for remodelling of the cell wall during growth. Corbomycin and complestatin have low levels of resistance development and are effective in reducing bacterial burden in a mouse model of skin MRSA infection.

2.
Artigo em Inglês | MEDLINE | ID: mdl-31932375

RESUMO

The rise of Gram-negative pathogens expressing metallo-ß-lactamases (MBLs) is a growing concern, threatening the efficacy of ß-lactam antibiotics, in particular, the carbapenems. There are no inhibitors of MBLs in current clinical use. Aspergillomarasmine A (AMA) is an MBL inhibitor isolated from Aspergillus versicolor with both in vitro and in vivo ability to rescue meropenem activity in MBL-producing bacteria. Here we systematically explored the pairing of AMA with six ß-lactam antibiotic partners against nineteen MBLs from each subclass (B1, B2, B3). Cell-based assays with Escherichia coli and Klebsiella pneumoniae showed that bacteria producing NDM-1 and VIM-2 of subclass B1 were the most susceptible to AMA inhibition, whereas bacteria producing CphA2 and AIM-1 of subclasses B2 and B3, respectively, were the least sensitive. Intracellular antibiotic accumulation assays and in vitro enzyme assays demonstrated that the efficacy of AMA/ß-lactam combinations did not correlate with outer membrane permeability or drug efflux. We determined that the optimal ß-lactam partners for AMA are the carbapenem antibiotics, and the efficacy of AMA is linked to the Zn2+ affinity of specific MBLs.

3.
ACS Infect Dis ; 4(6): 980-987, 2018 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-29634241

RESUMO

Plazomicin is a next-generation, semisynthetic aminoglycoside antibiotic currently under development for the treatment of infections due to multidrug-resistant Enterobacteriaceae. The compound was designed by chemical modification of the natural product sisomicin to provide protection from common aminoglycoside modifying enzymes that chemically alter these drugs via N-acetylation, O-adenylylation, or O-phosphorylation. In this study, plazomicin was profiled against a panel of isogenic strains of Escherichia coli individually expressing twenty-one aminoglycoside resistance enzymes. Plazomicin retained antibacterial activity against 15 of the 17 modifying enzyme-expressing strains tested. Expression of only two of the modifying enzymes, aac(2')-Ia and aph(2″)-IVa, decreased plazomicin potency. On the other hand, expression of 16S rRNA ribosomal methyltransferases results in a complete lack of plazomicin potency. In vitro enzymatic assessment confirmed that AAC(2')-Ia and APH(2'')-IVa (aminoglycoside acetyltransferase, AAC; aminoglycoside phosphotransferase, APH) were able to utilize plazomicin as a substrate. AAC(2')-Ia and APH(2'')-IVa are limited in their distribution to Providencia stuartii and Enterococci, respectively. These data demonstrate that plazomicin is not modified by a broad spectrum of common aminoglycoside modifying enzymes including those commonly found in Enterobacteriaceae. However, plazomicin is inactive in the presence of 16S rRNA ribosomal methyltransferases, which should be monitored in future surveillance programs.


Assuntos
Aminoglicosídeos/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Sisomicina/análogos & derivados , Acetiltransferases/antagonistas & inibidores , Aminoglicosídeos/química , Aminoglicosídeos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Humanos , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Sisomicina/química , Sisomicina/farmacologia , Relação Estrutura-Atividade
4.
Cell Chem Biol ; 25(4): 403-412.e5, 2018 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-29398560

RESUMO

Rifamycin monooxygenases (Rox) are present in a variety of environmental bacteria and are associated with decomposition of the clinically utilized antibiotic rifampin. Here we report the structure and function of a drug-inducible rox gene from Streptomyces venezuelae, which encodes a class A flavoprotein monooxygenase that inactivates a broad range of rifamycin antibiotics. Our findings describe a mechanism of rifamycin inactivation initiated by monooxygenation of the 2-position of the naphthyl group, which subsequently results in ring opening and linearization of the antibiotic. The result is an antibiotic that no longer adopts the basket-like structure essential for binding to the RNA exit tunnel of the target RpoB, thereby providing the molecular logic of resistance. This unique mechanism of enzymatic inactivation underpins the broad spectrum of rifamycin resistance mediated by Rox enzymes and presents a new antibiotic resistance mechanism not yet seen in microbial antibiotic detoxification.


Assuntos
Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana , Oxigenases de Função Mista/metabolismo , Rifamicinas/metabolismo , Streptomyces/enzimologia , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Oxigenases de Função Mista/química , Simulação de Acoplamento Molecular , Conformação Proteica , Rifamicinas/química , Rifamicinas/farmacologia , Streptomyces/química , Streptomyces/efeitos dos fármacos , Streptomyces/metabolismo
5.
Nat Commun ; 9(1): 112, 2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29317655

RESUMO

The production of antibiotics by microbes in the environment and their use in medicine and agriculture select for existing and emerging resistance. To address this inevitability, prudent development of antibiotic drugs requires careful consideration of resistance evolution. Here, we identify the molecular basis for expanded substrate specificity in MphI, a macrolide kinase (Mph) that does not confer resistance to erythromycin, in contrast to other known Mphs. Using a combination of phylogenetics, drug-resistance phenotypes, and in vitro enzyme assays, we find that MphI and MphK phosphorylate erythromycin poorly resulting in an antibiotic-sensitive phenotype. Using likelihood reconstruction of ancestral sequences and site-saturation combinatorial mutagenesis, supported by Mph crystal structures, we determine that two non-obvious mutations in combination expand the substrate range. This approach should be applicable for studying the functional evolution of any antibiotic resistance enzyme and for evaluating the evolvability of resistance enzymes to new generations of antibiotic scaffolds.


Assuntos
Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana , Macrolídeos/metabolismo , Fosfotransferases/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Eritromicina/química , Eritromicina/metabolismo , Eritromicina/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Macrolídeos/química , Macrolídeos/farmacologia , Modelos Moleculares , Estrutura Molecular , Fosfotransferases/classificação , Fosfotransferases/genética , Filogenia , Domínios Proteicos , Especificidade por Substrato
6.
ISME J ; 12(3): 885-897, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29259290

RESUMO

The ecology of antibiotic resistance involves the interplay of a long natural history of antibiotic production in the environment, and the modern selection of resistance in pathogens through human use of these drugs. Important components of the resistome are intrinsic resistance genes of environmental bacteria, evolved and acquired over millennia, and their mobilization, which drives dissemination in pathogens. Understanding the dynamics and evolution of resistance across bacterial taxa is essential to address the current crisis in drug-resistant infections. Here we report the exploration of antibiotic resistance in the Paenibacillaceae prompted by our discovery of an ancient intrinsic resistome in Paenibacillus sp. LC231, recovered from the isolated Lechuguilla cave environment. Using biochemical and gene expression analysis, we have mined the resistome of the second member of the Paenibacillaceae family, Brevibacillus brevis VM4, which produces several antimicrobial secondary metabolites. Using phylogenomics, we show that Paenibacillaceae resistomes are in flux, evolve mostly independent of secondary metabolite biosynthetic diversity, and are characterized by cryptic, redundant, pseudoparalogous, and orthologous genes. We find that in contrast to pathogens, mobile genetic elements are not significantly responsible for resistome remodeling. This offers divergent modes of resistome development in pathogens and environmental bacteria.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Paenibacillus , Brevibacillus/efeitos dos fármacos , Brevibacillus/genética , Cavernas , Ecologia , Perfilação da Expressão Gênica , Humanos , Paenibacillus/efeitos dos fármacos , Paenibacillus/genética
7.
Cell Chem Biol ; 24(1): 98-109, 2017 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-28017602

RESUMO

Solving the antibiotic resistance crisis requires the discovery of new antimicrobial drugs and the preservation of existing ones. The discovery of inhibitors of antibiotic resistance, antibiotic adjuvants, is a proven example of the latter. A major difficulty in identifying new antibiotics is the frequent rediscovery of known compounds, necessitating laborious "dereplication" to identify novel chemical entities. We have developed an antibiotic resistance platform (ARP) that can be used for both the identification of antibiotic adjuvants and for antibiotic dereplication. The ARP is a cell-based array of mechanistically distinct individual resistance elements in an identical genetic background. In dereplication mode, we demonstrate the rapid identification, and thus discrimination, of common antibiotics. In adjuvant discovery mode, we show that the ARP can be harnessed in screens to identify inhibitors of resistance. The ARP is therefore a powerful tool that has broad application in confronting the resistance crisis.


Assuntos
Adjuvantes Farmacêuticos/farmacologia , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Descoberta de Drogas , Farmacorresistência Bacteriana/efeitos dos fármacos , Adjuvantes Farmacêuticos/química , Antibacterianos/química , Ensaios de Triagem em Larga Escala , Estrutura Molecular
8.
Nat Commun ; 7: 13803, 2016 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-27929110

RESUMO

Antibiotic resistance is ancient and widespread in environmental bacteria. These are therefore reservoirs of resistance elements and reflective of the natural history of antibiotics and resistance. In a previous study, we discovered that multi-drug resistance is common in bacteria isolated from Lechuguilla Cave, an underground ecosystem that has been isolated from the surface for over 4 Myr. Here we use whole-genome sequencing, functional genomics and biochemical assays to reveal the intrinsic resistome of Paenibacillus sp. LC231, a cave bacterial isolate that is resistant to most clinically used antibiotics. We systematically link resistance phenotype to genotype and in doing so, identify 18 chromosomal resistance elements, including five determinants without characterized homologues and three mechanisms not previously shown to be involved in antibiotic resistance. A resistome comparison across related surface Paenibacillus affirms the conservation of resistance over millions of years and establishes the longevity of these genes in this genus.


Assuntos
Cavernas/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Paenibacillus/fisiologia , Acetiltransferases/genética , Amidoidrolases/genética , Proteínas de Bactérias/genética , Macrolídeos/metabolismo , Fosfotransferases/genética , Fosfotransferases/metabolismo , Especificidade por Substrato , Sequenciamento Completo do Genoma
9.
Angew Chem Int Ed Engl ; 55(42): 13259-13262, 2016 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-27633338

RESUMO

The fungal secondary metabolite aspergillomarasmine A (AMA) has recently been identified as an inhibitor of metallo-ß-lactamases NDM-1 and VIM-2. Described herein is an efficient and practical route to AMA and its related compounds by a sulfamidate approach. In addition, a series of derivatives has been prepared and tested for biological activity in an effort to explore preliminary structure activity relationships. While it was determined that natural LLL isomer of AMA remains the most effective inactivator of NDM-1 enzyme activity both in vitro and in cells, the structure is highly tolerant of the changes in the stereochemistry at positions 3, 6, and 9.


Assuntos
Amidas/farmacologia , Antibacterianos/farmacologia , Ácido Aspártico/análogos & derivados , Inibidores Enzimáticos/farmacologia , beta-Lactamases/metabolismo , Acinetobacter/efeitos dos fármacos , Acinetobacter/enzimologia , Amidas/química , Antibacterianos/síntese química , Antibacterianos/química , Ácido Aspártico/síntese química , Ácido Aspártico/química , Ácido Aspártico/farmacologia , Relação Dose-Resposta a Droga , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/enzimologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Pseudomonas/efeitos dos fármacos , Pseudomonas/enzimologia , Relação Estrutura-Atividade
10.
Methods Mol Biol ; 1440: 99-108, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27311667

RESUMO

Sortase enzymes have specific endopeptidase activity, cleaving within a defined pentapeptide sequence at the C-terminal end of their protein substrates. Here, we describe how monitoring sortase cleavage activity can be achieved using peptide substrates. Peptide cleavage can be readily analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS), which allows for the precise definition of cleavage sites. This technique could be used to analyze the peptidase activity of any enzyme, and identify sites of cleavage within any peptide.


Assuntos
Cromatografia Líquida/métodos , Cisteína Endopeptidases/metabolismo , Espectrometria de Massas em Tandem/métodos , Peptídeos/química , Proteólise
11.
Nat Commun ; 7: 11343, 2016 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-27103605

RESUMO

Rifampin (RIF) phosphotransferase (RPH) confers antibiotic resistance by conversion of RIF and ATP, to inactive phospho-RIF, AMP and Pi. Here we present the crystal structure of RPH from Listeria monocytogenes (RPH-Lm), which reveals that the enzyme is comprised of three domains: two substrate-binding domains (ATP-grasp and RIF-binding domains); and a smaller phosphate-carrying His swivel domain. Using solution small-angle X-ray scattering and mutagenesis, we reveal a mechanism where the swivel domain transits between the spatially distinct substrate-binding sites during catalysis. RPHs are previously uncharacterized dikinases that are widespread in environmental and pathogenic bacteria. These enzymes are members of a large unexplored group of bacterial enzymes with substrate affinities that have yet to be fully explored. Such an enzymatically complex mechanism of antibiotic resistance augments the spectrum of strategies used by bacteria to evade antimicrobial compounds.


Assuntos
Antibacterianos/metabolismo , Proteínas de Bactérias/química , Listeria monocytogenes/enzimologia , Fosfotransferases/química , Rifampina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biotransformação , Cristalografia por Raios X , Farmacorresistência Bacteriana , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Listeria monocytogenes/classificação , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/genética , Modelos Moleculares , Dados de Sequência Molecular , Fosfotransferases/genética , Fosfotransferases/metabolismo , Filogenia , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rifampina/farmacologia , Alinhamento de Sequência
12.
Angew Chem Int Ed Engl ; 55(6): 2210-2, 2016 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-26709849

RESUMO

Resistance to ß-lactam antibiotics is mediated primarily by enzymes that hydrolytically inactivate the drugs by one of two mechanisms: serine nucleophilic attack or metal-dependent activation of a water molecule. Serine ß-lactamases are countered in the clinic by several codrugs that inhibit these enzymes, thereby rescuing antibiotic action. There are no equivalent inhibitors of metallo-ß-lactamases in clinical use, but the fungal secondary metabolite aspergillomarasmine A has recently been identified as a potential candidate for such a codrug. Herein we report the synthesis of aspergillomarasmine A. The synthesis enabled confirmation of the stereochemical configuration of the compound and offers a route for the synthesis of derivatives in the future.


Assuntos
Ácido Aspártico/análogos & derivados , Inibidores de beta-Lactamases/síntese química , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismo , Ácido Aspártico/síntese química , Ácido Aspártico/química , Ácido Aspártico/farmacologia , Aspergillus/química , Relação Dose-Resposta a Droga , Estrutura Molecular , Relação Estrutura-Atividade , Inibidores de beta-Lactamases/química
13.
J Mol Biol ; 427(12): 2229-43, 2015 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-25900373

RESUMO

One of the main mechanisms of resistance to lincosamide and aminoglycoside antibiotics is their inactivation by O-nucleotidylyltransferases (NTases). Significant sequence variation of lincomycin nucleotidylyltransferase (Lnu) and aminoglycoside nucleotidylyltransferase (ANT) enzymes plus lack of detailed information about the molecular basis for specificity of these enzymes toward chemically distinct antibiotic scaffolds hinders development of a general strategy to curb this resistance mechanism. We conducted an extensive sequence analysis identifying 129 putative antibiotic NTases constituting six distinct subfamilies represented by Lnu(A), Lnu(B), Lnu(C), Lnu(D), Lnu(F)/(G) plus ANT(2") enzymes. Since only the Lnu(B) enzyme has been previously studied in detail, we biochemically characterized the Lnu(A) and Lnu(D) enzymes, with the former representing the most sequence distinct Lnu ortholog. We also determined the crystal structure of the Lnu(A) enzyme in complex with a lincosamide. These data suggested that, while sharing the N-terminal nucleotidylyltransferase domain, the groups of antibiotic NTases feature structurally distinct C-terminal domains (CTDs) adapted to accommodate antibiotics. Comparative structural analysis among antibiotic NTases rationalized their specificity toward lincosamides versus aminoglycosides through active-site plasticity, which allows retention of general catalytic activity while accepting alterations at multiple, specific positions contributed by both domains. Based on this structural analysis, we suggest that antibiotic NTases evolved from an ancestral nucleotidylyltransferase along independent paths according to the identified groups, characterized by structural changes in the active site and recruitment of structurally diverse CTDs. These data show the complexity of enzyme-driven antibiotic resistance and provide a basis for broadly active inhibitors by identifying the key unifying features of antibiotic NTases.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Domínio Catalítico , Análise por Conglomerados , Cristalografia por Raios X , Lincosamidas/química , Lincosamidas/metabolismo , Dados de Sequência Molecular , Nucleotidiltransferases/genética , Filogenia , Ligação Proteica , Conformação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
14.
J Antibiot (Tokyo) ; 68(1): 40-6, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24984798

RESUMO

Rising rates of antibiotic resistance in bacterial pathogens is a medical crisis of global concern that necessitates the development of new treatment strategies. We have isolated a natural product with macrophage-stimulating activity from a screen of microbially produced bioactive molecules. Streptazolin increased bacterial killing and elaboration of immunostimulatory cytokines by macrophages in vitro. Furthermore, we show that streptazolin stimulates the macrophage nuclear factor κB (NF-κB) pathway via phosphatidylinositide 3-kinase (PI3K) signaling, and that the conjugated diene moiety is essential for stimulatory activity. Immunostimulatory molecules like streptazolin represent entries into new treatment paradigms to address the challenge of antibiotic resistance.


Assuntos
Produtos Biológicos/farmacologia , Citocinas/metabolismo , Macrófagos/efeitos dos fármacos , NF-kappa B/metabolismo , Piperidinas/farmacologia , Antibacterianos/farmacologia , Linhagem Celular , Descoberta de Drogas/métodos , Farmacorresistência Bacteriana , Humanos , Macrófagos/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Transdução de Sinais/efeitos dos fármacos
15.
Chembiochem ; 15(17): 2613-23, 2014 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-25255985

RESUMO

In this study, a draft genome sequence of Actinoplanes sp. ATCC 53533 was assembled, and an 81-kb biosynthetic cluster for the unusual sulfated glycopeptide UK-68,597 was identified. Glycopeptide antibiotics are important in the treatment of infections caused by Gram-positive bacteria. Glycopeptides contain heptapeptide backbones that are modified by many tailoring enzymes, including glycosyltransferases, sulfotransferases, methyltransferases, and halogenases, generating extensive chemical and functional diversity. Several tailoring enzymes in the cluster were examined in vitro for their ability to modify glycopeptides, resulting in the synthesis of novel molecules. Tailoring enzymes were also expressed in the producer of the glycopeptide aglycone A47934, generating additional chemical diversity. This work characterizes the biosynthetic program of UK-68,597 and demonstrates the capacity to expand glycopeptide chemical diversity by harnessing the unique chemistry of tailoring enzymes.


Assuntos
Antibacterianos/biossíntese , Vias Biossintéticas/genética , Glicopeptídeos/biossíntese , Micromonosporaceae/enzimologia , Oxirredutases/metabolismo , Transferases/metabolismo , Antibacterianos/química , Glicopeptídeos/química , Micromonosporaceae/genética , Micromonosporaceae/metabolismo , Conformação Molecular , Oxirredutases/genética , Transferases/genética
16.
Proc Natl Acad Sci U S A ; 111(19): 7102-7, 2014 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-24778229

RESUMO

Many environmental bacteria are multidrug-resistant and represent a reservoir of ancient antibiotic resistance determinants, which have been linked to genes found in pathogens. Exploring the environmental antibiotic resistome, therefore, reveals the diversity and evolution of antibiotic resistance and also provides insight into the vulnerability of clinically used antibiotics. In this study, we describe the identification of a highly conserved regulatory motif, the rifampin (RIF) -associated element (RAE), which is found upstream of genes encoding RIF-inactivating enzymes from a diverse collection of actinomycetes. Using gene expression assays, we confirmed that the RAE is involved in RIF-responsive regulation. By using the RAE as a probe for new RIF-associated genes in several actinomycete genomes, we identified a heretofore unknown RIF resistance gene, RIF phosphotransferase (rph). The RPH enzyme is a RIF-inactivating phosphotransferase and represents a new protein family in antibiotic resistance. RPH orthologs are widespread and found in RIF-sensitive bacteria, including Bacillus cereus and the pathogen Listeria monocytogenes. Heterologous expression and in vitro enzyme assays with purified RPHs from diverse bacterial genera show that these enzymes are capable of conferring high-level resistance to a variety of clinically used rifamycin antibiotics. This work identifies a new antibiotic resistance protein family and reinforces the fact that the study of resistance in environmental organisms can serve to identify resistance elements with relevance to pathogens.


Assuntos
Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/genética , Listeria monocytogenes/enzimologia , Fosfotransferases/metabolismo , Rifamicinas/farmacologia , Streptomycetaceae/enzimologia , Actinobacteria/genética , Antibacterianos/química , Antibacterianos/farmacologia , Bacillus cereus/enzimologia , Bacillus cereus/genética , Bacillus cereus/patogenicidade , Proteínas de Bactérias/genética , Sequência de Bases , Sequência Conservada , Desenho de Drogas , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Dados de Sequência Molecular , Fosfotransferases/genética , Rifamicinas/química , Microbiologia do Solo , Streptomycetaceae/genética , Streptomycetaceae/patogenicidade
17.
J Antimicrob Chemother ; 69(7): 1844-55, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24627312

RESUMO

OBJECTIVES: An orthogonal approach taken towards novel antibacterial drug discovery involves the identification of small molecules that potentiate or enhance the activity of existing antibacterial agents. This study aimed to identify natural-product rifampicin adjuvants in the intrinsically resistant organism Escherichia coli. METHODS: E. coli BW25113 was screened against 1120 actinomycete fermentation extracts in the presence of subinhibitory (2 mg/L) concentrations of rifampicin. The active molecule exhibiting the greatest rifampicin potentiation was isolated using activity-guided methods and identified using mass and NMR spectroscopy. Susceptibility testing and biochemical assays were used to determine the mechanism of antibiotic potentiation. RESULTS: The anthracycline Antibiotic 301A(1) was isolated from the fermentation broth of a strain of Streptomyces (WAC450); the molecule was shown to be highly synergistic with rifampicin (fractional inhibitory concentration index = 0.156) and moderately synergistic with linezolid (FIC index = 0.25) in both E. coli and Acinetobacter baumannii. Activity was associated with inhibition of efflux and the synergistic phenotype was lost when tested against E. coli harbouring mutations within the rpoB gene. Structure-activity relationship studies revealed that other anthracyclines do not synergize with rifampicin and removal of the sugar moiety of Antibiotic 301A(1) abolishes activity. CONCLUSIONS: Screening only a subsection of our natural product library identified a small-molecule antibiotic adjuvant capable of sensitizing Gram-negative bacteria to antibiotics to which they are ordinarily intrinsically resistant. This result demonstrates the great potential of this approach in expanding antibiotic effectiveness in the face of the growing challenge of resistance in Gram-negatives.


Assuntos
Antraciclinas/farmacologia , Antibacterianos/farmacologia , Sinergismo Farmacológico , Escherichia coli/efeitos dos fármacos , Rifampina/farmacologia , Acetamidas/farmacologia , Acinetobacter baumannii/efeitos dos fármacos , Antraciclinas/química , Antraciclinas/isolamento & purificação , Antraciclinas/metabolismo , Antibacterianos/química , Antibacterianos/isolamento & purificação , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Linezolida , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Oxazolidinonas/farmacologia , Streptomyces/metabolismo , Relação Estrutura-Atividade
18.
Bioorg Med Chem Lett ; 24(3): 905-10, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24393581

RESUMO

The thienopyridine antiplatelet agent, ticlopidine and its analog, clopidogrel, have been shown to potentiate the action of ß-lactam antibiotics, reversing the methicillin-resistance phenotype of methicillin-resistant Staphylococcus aureus (MRSA), in vitro. Interestingly, these thienopyridines inhibit the action of TarO, the first enzyme in the synthesis of wall teichoic acid, an important cell wall polymer in Gram-positive bacteria. In the human body, both ticlopidine and clopidogrel undergo a rapid P450-dependent oxidation into their respective antiplatelet-active metabolites, resulting in very low plasma concentrations of intact drug. Herein, a series of analogs of ticlopidine and clopidogrel that would avoid oxidative metabolism were designed, prepared and evaluated as inhibitors of TarO. Specifically, we replaced the P450-labile thiophene ring of ticlopidine and clopidogrel to a more stable phenyl group to generate 2-(2-chlorobenzyl)-1,2,3,4-tetrahydro-isoquinoline) (6) and (2-chloro-phenyl)-(3,4-dihydro-1H-isoquinolin-2-yl)-acetic acid methyl ester (22), respectively. The latter molecules displayed inhibitory activity against TarO and formed the basis of a library of analogs. Most synthesized compounds exhibited comparable efficacy to ticlopidine and clopidogrel. So far, it was introduction of a trifluoromethyl group to compound 6, to generate 2-(2-trifluoromethyl-benzyl)-1,2,3,4-tetrahydro-isoquinoline (13) that exhibited enhanced activity against TarO. Compound 13 represents a novel stable inhibitor of TarO with synergistic impact on ß-lactam antibiotics against MRSA and low potential for P-450 metabolism.


Assuntos
Desenho de Drogas , Ácidos Teicoicos/antagonistas & inibidores , Ticlopidina/análogos & derivados , Ticlopidina/química , Clopidogrel , Concentração Inibidora 50 , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Estrutura Molecular , Oxirredução/efeitos dos fármacos , Ácidos Teicoicos/química , Ticlopidina/farmacologia
19.
J Antibiot (Tokyo) ; 67(1): 31-41, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24220108

RESUMO

Glycopeptides such as vancomycin, teicoplanin and telavancin are essential for treating infections caused by Gram-positive bacteria. Unfortunately, the dwindled pipeline of new antibiotics into the market and the emergence of glycopeptide-resistant enterococci and other resistant bacteria are increasingly making effective antibiotic treatment difficult. We have now learned a great deal about how bacteria produce antibiotics. This information can be exploited to develop the next generation of antimicrobials. The biosynthesis of glycopeptides via nonribosomal peptide assembly and unusual amino acid synthesis, crosslinking and tailoring enzymes gives rise to intricate chemical structures that target the bacterial cell wall. This review seeks to describe recent advances in our understanding of both biosynthesis and resistance of these important antibiotics.


Assuntos
Antibacterianos/biossíntese , Desenho de Drogas , Glicopeptídeos/biossíntese , Antibacterianos/farmacologia , Parede Celular/metabolismo , Farmacorresistência Bacteriana , Glicopeptídeos/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Terapia de Alvo Molecular
20.
Chem Biol ; 20(8): 983-90, 2013 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-23891151

RESUMO

Edeines are atypical cationic peptides produced by Brevibacillus brevis Vm4 with broad-spectrum antimicrobial activity. These linear nonribosomal peptides bind to the 30S ribosomal subunit and block t-RNA binding to the P-site. To identify the mechanism of high-level self-resistance in the producing organism, the B. brevis Vm4 genome was sequenced and the edeine biosynthetic cluster discovered. A potential edeine-modifying enzyme, EdeQ, showed similarity to spermidine N-acetyltransferases. EdeQ was purified and shown to convert edeine to N-acetyledeine, which is inactive against cells in vivo and against cell-free extracts. Unexpectedly, tandem mass spectroscopy and nuclear magnetic resonance demonstrate that N-acylation occurs on the free amine of the internal diaminopropionic acid rather than the N-terminal spermidine polyamine. Acetylation of edeine by EdeQ abolishes its ability to inhibit translation, thus conferring resistance to the antibiotic in the producing organism.


Assuntos
Acetiltransferases/metabolismo , Antibacterianos/metabolismo , Brevibacillus/enzimologia , Edeína/metabolismo , Acetilação , Acetiltransferases/genética , Antibacterianos/química , Brevibacillus/genética , Brevibacillus/metabolismo , Edeína/química , Genes Bacterianos , Modelos Moleculares , Família Multigênica , Inibidores da Síntese de Ácido Nucleico/química , Inibidores da Síntese de Ácido Nucleico/metabolismo
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