RESUMO
Gram-negative bacteria are notoriously more resistant to antibiotics than Gram-positive bacteria, primarily due to the presence of the outer membrane and a plethora of active efflux pumps. However, the potency of antibiotics also varies dramatically between different Gram-negative pathogens, suggesting major mechanistic differences in how antibiotics penetrate permeability barriers. Two approaches are used broadly to analyze how permeability barriers affect intracellular accumulation of antibiotics. One compares the antibacterial activities of compounds, while the other measures the total intracellular concentrations of compounds in nongrowing cells, with both approaches using strains harboring wild-type or genetically modified efflux systems and permeability barriers. Whether the two assays provide similar mechanistic insights remains unclear. In this study, we analyzed the intracellular accumulation and antibacterial activities of antibiotics representative of major clinical classes in three Gram-negative pathogens of high clinical importance, Pseudomonas aeruginosa, Escherichia coli, and Acinetobacter baumannii. We found that both assays are informative about properties of permeability barriers, but there is no quantitative agreement between the assays. Our results show that the three pathogens differ dramatically in their permeability barriers, with the outer membrane playing the dominant role in E. coli and P. aeruginosa but efflux dominating in A. baumannii. However, even compounds of the same chemotype may use different permeation pathways depending on small chemical modifications. Accordingly, a classification analysis revealed limited conservation of molecular properties that define compound penetration into the three bacteria.
Assuntos
Antibacterianos , Escherichia coli , Antibacterianos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Transporte Biológico , Bactérias Gram-Negativas/metabolismo , Permeabilidade , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/metabolismoRESUMO
The development of new antimicrobial drugs is a priority to combat the increasing spread of multidrug-resistant bacteria. This development is especially problematic in gram-negative bacteria due to the outer membrane (OM) permeability barrier and multidrug efflux pumps. Therefore, we screened for compounds that target essential, nonredundant, surface-exposed processes in gram-negative bacteria. We identified a compound, MRL-494, that inhibits assembly of OM proteins (OMPs) by the ß-barrel assembly machine (BAM complex). The BAM complex contains one essential surface-exposed protein, BamA. We constructed a bamA mutagenesis library, screened for resistance to MRL-494, and identified the mutation bamAE470K BamAE470K restores OMP biogenesis in the presence of MRL-494. The mutant protein has both altered conformation and activity, suggesting it could either inhibit MRL-494 binding or allow BamA to function in the presence of MRL-494. By cellular thermal shift assay (CETSA), we determined that MRL-494 stabilizes BamA and BamAE470K from thermally induced aggregation, indicating direct or proximal binding to both BamA and BamAE470K Thus, it is the altered activity of BamAE470K responsible for resistance to MRL-494. Strikingly, MRL-494 possesses a second mechanism of action that kills gram-positive organisms. In microbes lacking an OM, MRL-494 lethally disrupts the cytoplasmic membrane. We suggest that the compound cannot disrupt the cytoplasmic membrane of gram-negative bacteria because it cannot penetrate the OM. Instead, MRL-494 inhibits OMP biogenesis from outside the OM by targeting BamA. The identification of a small molecule that inhibits OMP biogenesis at the cell surface represents a distinct class of antibacterial agents.
Assuntos
Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/antagonistas & inibidores , Escherichia coli/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Triazinas/farmacologia , Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Proteínas da Membrana Bacteriana Externa/genética , Transporte Biológico/fisiologia , Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/fisiologia , Avaliação Pré-Clínica de Medicamentos , Farmacorresistência Bacteriana/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Testes de Sensibilidade MicrobianaRESUMO
Riboswitches are non-coding RNA structures located in messenger RNAs that bind endogenous ligands, such as a specific metabolite or ion, to regulate gene expression. As such, riboswitches serve as a novel, yet largely unexploited, class of emerging drug targets. Demonstrating this potential, however, has proven difficult and is restricted to structurally similar antimetabolites and semi-synthetic analogues of their cognate ligand, thus greatly restricting the chemical space and selectivity sought for such inhibitors. Here we report the discovery and characterization of ribocil, a highly selective chemical modulator of bacterial riboflavin riboswitches, which was identified in a phenotypic screen and acts as a structurally distinct synthetic mimic of the natural ligand, flavin mononucleotide, to repress riboswitch-mediated ribB gene expression and inhibit bacterial cell growth. Our findings indicate that non-coding RNA structural elements may be more broadly targeted by synthetic small molecules than previously expected.
Assuntos
Pirimidinas/química , Pirimidinas/farmacologia , RNA Bacteriano/química , RNA Bacteriano/efeitos dos fármacos , Riboswitch/efeitos dos fármacos , Animais , Aptâmeros de Nucleotídeos/química , Bactérias/citologia , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Sequência de Bases , Cristalografia por Raios X , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Feminino , Mononucleotídeo de Flavina/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Transferases Intramoleculares/genética , Ligantes , Camundongos , Camundongos Endogâmicos DBA , Modelos Moleculares , Dados de Sequência Molecular , Pirimidinas/isolamento & purificação , Pirimidinas/uso terapêutico , RNA Bacteriano/genética , Reprodutibilidade dos Testes , Riboflavina/biossíntese , Riboswitch/genética , Especificidade por SubstratoRESUMO
The outer membrane (OM) of Gram-negative bacteria forms a robust permeability barrier that blocks entry of toxins and antibiotics. Most OM proteins (OMPs) assume a ß-barrel fold, and some form aqueous channels for nutrient uptake and efflux of intracellular toxins. The Bam machine catalyzes rapid folding and assembly of OMPs. Fidelity of OMP biogenesis is monitored by the σE stress response. When OMP folding defects arise, the proteases DegS and RseP act sequentially to liberate σE into the cytosol, enabling it to activate transcription of the stress regulon. Here, we identify batimastat as a selective inhibitor of RseP that causes a lethal decrease in σE activity in Escherichia coli, and we further identify RseP mutants that are insensitive to inhibition and confer resistance. Remarkably, batimastat treatment allows the capture of elusive intermediates in the OMP biogenesis pathway and offers opportunities to better understand the underlying basis for σE essentiality.
Assuntos
Proteínas da Membrana Bacteriana Externa , Endopeptidases , Proteínas de Escherichia coli , Escherichia coli , Proteínas de Membrana , Desdobramento de Proteína , Fatores de Transcrição , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The lipopolysaccharide biosynthesis pathway is considered an attractive drug target against the rising threat of multi-drug-resistant Gram-negative bacteria. Here, we report two novel small-molecule inhibitors (compounds 1 and 2) of the acyltransferase LpxA, the first enzyme in the lipopolysaccharide biosynthesis pathway. We show genetically that the antibacterial activities of the compounds against efflux-deficient Escherichia coli are mediated by LpxA inhibition. Consistently, the compounds inhibited the LpxA enzymatic reaction in vitro. Intriguingly, using biochemical, biophysical, and structural characterization, we reveal two distinct mechanisms of LpxA inhibition; compound 1 is a substrate-competitive inhibitor targeting apo LpxA, and compound 2 is an uncompetitive inhibitor targeting the LpxA/product complex. Compound 2 exhibited more favorable biological and physicochemical properties than compound 1 and was optimized using structural information to achieve improved antibacterial activity against wild-type E. coli. These results show that LpxA is a promising antibacterial target and imply the advantages of targeting enzyme/product complexes in drug discovery.
Assuntos
Aciltransferases/antagonistas & inibidores , Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Pirazóis/farmacologia , Aciltransferases/metabolismo , Antibacterianos/metabolismo , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Imidazóis/metabolismo , Testes de Sensibilidade Microbiana , Ligação Proteica , Pirazóis/metabolismoRESUMO
BACKGROUND: The prevalence of antibiotic resistance is increasing, and multidrug-resistant Pseudomonas aeruginosa has been identified as a serious threat to human health. The production of ß-lactamase is a key mechanism contributing to imipenem resistance in P. aeruginosa. Relebactam is a novel ß-lactamase inhibitor, active against class A and C ß-lactamases, that has been shown to restore imipenem susceptibility. In a series of studies, we assessed the interaction of relebactam with key mechanisms involved in carbapenem resistance in P. aeruginosa and to what extent relebactam might overcome imipenem non-susceptibility. RESULTS: Relebactam demonstrated no intrinsic antibacterial activity against P. aeruginosa, had no inoculum effect, and was not subject to efflux. Enzymology studies showed relebactam is a potent (overall inhibition constant: 27 nM), practically irreversible inhibitor of P. aeruginosa AmpC. Among P. aeruginosa clinical isolates from the SMART global surveillance program (2009, n = 993; 2011, n = 1702; 2015, n = 5953; 2016, n = 6165), imipenem susceptibility rates were 68.4% in 2009, 67.4% in 2011, 70.4% in 2015, and 67.3% in 2016. With the addition of 4 µg/mL relebactam, imipenem susceptibility rates increased to 87.6, 86.0, 91.7, and 89.8%, respectively. When all imipenem-non-susceptible isolates were pooled, the addition of 4 µg/mL relebactam reduced the mode imipenem minimum inhibitory concentration (MIC) 8-fold (from 16 µg/mL to 2 µg/mL) among all imipenem-non-susceptible isolates. Of 3747 imipenem-non-susceptible isolates that underwent molecular profiling, 1200 (32%) remained non-susceptible to the combination imipenem/relebactam (IMI/REL); 42% of these encoded class B metallo-ß-lactamases, 11% encoded a class A GES enzyme, and no class D enzymes were detected. No relationship was observed between alleles of the chromosomally-encoded P. aeruginosa AmpC and IMI/REL MIC. CONCLUSIONS: IMI/REL exhibited potential in the treatment of carbapenem-resistant P. aeruginosa infections, with the exception of isolates encoding class B, some GES alleles, and class D carbapenemases.
Assuntos
Compostos Azabicíclicos/farmacologia , Imipenem/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Antibacterianos/farmacologia , Proteínas de Bactérias/efeitos dos fármacos , Combinação de Medicamentos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Humanos , Cinética , Testes de Sensibilidade Microbiana , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/enzimologia , beta-Lactamases/efeitos dos fármacosRESUMO
Gram-negative bacteria provide a particular challenge to antibacterial drug discovery due to their cell envelope structure. Compound entry is impeded by the lipopolysaccharide (LPS) of the outer membrane (OM), and those molecules that overcome this barrier are often expelled by multidrug efflux pumps. Understanding how efflux and permeability affect the ability of a compound to reach its target is paramount to translating in vitro biochemical potency to cellular bioactivity. Herein, a suite of Pseudomonas aeruginosa strains were constructed in either a wild-type or efflux-null background in which mutations were engineered in LptD, the final protein involved in LPS transport to the OM. These mutants were demonstrated to be defective in LPS transport, resulting in compromised barrier function. Using isogenic strain sets harboring these newly created alleles, we were able to define the contributions of permeability and efflux to the intrinsic resistance of P. aeruginosa to a variety of antibiotics. These strains will be useful in the design and optimization of future antibiotics against Gram-negative pathogens.
Assuntos
Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Permeabilidade da Membrana Celular/genética , Farmacorresistência Bacteriana Múltipla/genética , Proteínas de Membrana Transportadoras/genética , Pseudomonas aeruginosa/genética , Sequência de Aminoácidos , Transporte Biológico/genética , Membrana Celular/metabolismo , Lipopolissacarídeos/metabolismo , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacosRESUMO
Bacterial resistance to antibiotics continues to grow and pose serious challenges, while the discovery rate for new antibiotics declines. Kibdelomycin is a recently discovered natural-product antibiotic that inhibits bacterial growth by inhibiting the bacterial DNA replication enzymes DNA gyrase and topoisomerase IV. It was reported to be a broad-spectrum aerobic Gram-positive agent with selective inhibition of the anaerobic bacterium Clostridium difficile. We have extended the profiling of kibdelomycin by using over 196 strains of Gram-positive and Gram-negative aerobic pathogens recovered from worldwide patient populations. We report the MIC50s, MIC90s, and bactericidal activities of kibdelomycin. We confirm the Gram-positive spectrum and report for the first time that kibdelomycin shows strong activity (MIC90, 0.125 µg/ml) against clinical strains of the Gram-negative nonfermenter Acinetobacter baumannii but only weak activity against Pseudomonas aeruginosa. We confirm that well-characterized resistant strains of Staphylococcus aureus and Streptococcus pneumoniae show no cross-resistance to kibdelomycin and quinolones and coumarin antibiotics. We also show that kibdelomycin is not subject to efflux in Pseudomonas, though it is in Escherichia coli, and it is generally affected by the outer membrane permeability entry barrier in the nonfermenters P. aeruginosa and A. baumannii, which may be addressable by structure-based chemical modification.
Assuntos
Antibacterianos/farmacologia , Pirróis/farmacologia , Pirrolidinonas/farmacologia , Acinetobacter baumannii/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacosRESUMO
Carbapenem-resistant Acinetobacter baumannii infections have limited treatment options. Synthesis, transport and placement of lipopolysaccharide or lipooligosaccharide (LOS) in the outer membrane of Gram-negative bacteria are important for bacterial virulence and survival. Here we describe the cerastecins, inhibitors of the A. baumannii transporter MsbA, an LOS flippase. These molecules are potent and bactericidal against A. baumannii, including clinical carbapenem-resistant Acinetobacter baumannii isolates. Using cryo-electron microscopy and biochemical analysis, we show that the cerastecins adopt a serpentine configuration in the central vault of the MsbA dimer, stalling the enzyme and uncoupling ATP hydrolysis from substrate flipping. A derivative with optimized potency and pharmacokinetic properties showed efficacy in murine models of bloodstream or pulmonary A. baumannii infection. While resistance development is inevitable, targeting a clinically unexploited mechanism avoids existing antibiotic resistance mechanisms. Although clinical validation of LOS transport remains undetermined, the cerastecins may open a path to narrow-spectrum treatment modalities for important nosocomial infections.
Assuntos
Infecções por Acinetobacter , Acinetobacter baumannii , Antibacterianos , Proteínas de Bactérias , Lipopolissacarídeos , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/metabolismo , Lipopolissacarídeos/metabolismo , Animais , Infecções por Acinetobacter/microbiologia , Infecções por Acinetobacter/tratamento farmacológico , Camundongos , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Transporte Biológico , Testes de Sensibilidade Microbiana , Humanos , Microscopia Crioeletrônica , Carbapenêmicos/farmacologia , Carbapenêmicos/metabolismo , Modelos Animais de Doenças , Feminino , Transportadores de Cassetes de Ligação de ATPRESUMO
Acinetobacter baumannii, a commonly multidrug-resistant Gram-negative bacterium responsible for large numbers of bloodstream and lung infections worldwide, is increasingly difficult to treat and constitutes a growing threat to human health. Structurally novel antibacterial chemical matter that can evade existing resistance mechanisms is essential for addressing this critical medical need. Herein, we describe our efforts to inhibit the essential A. baumannii lipooligosaccharide (LOS) ATP-binding cassette (ABC) transporter MsbA. An unexpected impurity from a phenotypic screening was optimized as a series of dimeric compounds, culminating with 1 (cerastecin D), which exhibited antibacterial activity in the presence of human serum and a pharmacokinetic profile sufficient to achieve efficacy against A. baumannii in murine septicemia and lung infection models.
RESUMO
Elongation factor P (EF-P) is a highly conserved ribosomal initiation factor responsible for stimulating formation of the first peptide bond. Its essentiality has been debated and may differ depending on the organism. Here, we demonstrate that EF-P is dispensable in Escherichia coli and Pseudomonas aeruginosa under laboratory growth conditions. Although knockouts are viable, growth rates are diminished compared with wild-type strains. Despite this cost in fitness, these mutants are not more susceptible to a wide range of antibiotics; including ribosome targeting antibiotics, such as lincomycin, chloramphenicol, and streptomycin, which have been shown previously to disrupt EF-P function in vitro. In Pseudomonas, knockout of efp leads to an upregulation of mexX, a phenotype previously observed with other genetic lesions affecting ribosome function and that can be induced by the treatment with antibiotics affecting protein synthesis.
Assuntos
Escherichia coli/enzimologia , Escherichia coli/genética , Deleção de Genes , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Escherichia coli/crescimento & desenvolvimento , Genes Essenciais , Viabilidade Microbiana , Pseudomonas aeruginosa/crescimento & desenvolvimentoRESUMO
Antiretroviral therapy inhibits HIV-1 replication but is not curative due to establishment of a persistent reservoir after virus integration into the host genome. Reservoir reduction is therefore an important HIV-1 cure strategy. Some HIV-1 nonnucleoside reverse transcriptase inhibitors induce HIV-1 selective cytotoxicity in vitro but require concentrations far exceeding approved dosages. Focusing on this secondary activity, we found bifunctional compounds with HIV-1-infected cell kill potency at clinically achievable concentrations. These targeted activator of cell kill (TACK) molecules bind the reverse transcriptase-p66 domain of monomeric Gag-Pol and act as allosteric modulators to accelerate dimerization, resulting in HIV-1+ cell death through premature intracellular viral protease activation. TACK molecules retain potent antiviral activity and selectively eliminate infected CD4+ T cells isolated from people living with HIV-1, supporting an immune-independent clearance strategy.
Assuntos
Infecções por HIV , HIV-1 , Humanos , Infecções por HIV/tratamento farmacológico , Antivirais/uso terapêutico , Apoptose , Morte Celular , Linfócitos T CD4-Positivos , Replicação ViralRESUMO
Although current antiretroviral therapy can control HIV-1 replication and prevent disease progression, it is not curative. Identifying mechanisms that can lead to eradication of persistent viral reservoirs in people living with HIV-1 (PLWH) remains an outstanding challenge to achieving cure. Utilizing a phenotypic screen, we identified a novel chemical class capable of killing HIV-1 infected peripheral blood mononuclear cells. Tool compounds ICeD-1 and ICeD-2 ("inducer of cell death-1 and 2"), optimized for potency and selectivity from screening hits, were used to deconvolute the mechanism of action using a combination of chemoproteomic, biochemical, pharmacological, and genetic approaches. We determined that these compounds function by modulating dipeptidyl peptidase 9 (DPP9) and activating the caspase recruitment domain family member 8 (CARD8) inflammasome. Efficacy of ICeD-1 and ICeD-2 was dependent on HIV-1 protease activity and synergistic with efavirenz, which promotes premature activation of HIV-1 protease at high concentrations in infected cells. This in vitro synergy lowers the efficacious cell kill concentration of efavirenz to a clinically relevant dose at concentrations of ICeD-1 or ICeD-2 that do not result in complete DPP9 inhibition. These results suggest engagement of the pyroptotic pathway as a potential approach to eliminate HIV-1 infected cells.
Assuntos
Infecções por HIV , HIV-1 , Alcinos , Benzoxazinas , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Ciclopropanos , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Infecções por HIV/tratamento farmacológico , HIV-1/metabolismo , Humanos , Inflamassomos/metabolismo , Leucócitos Mononucleares , Proteínas de Neoplasias/metabolismoRESUMO
Coenzyme A (CoA) plays a central and essential role in all living organisms. The pathway leading to CoA biosynthesis has been considered an attractive target for developing new antimicrobial agents with novel mechanisms of action. By using an arabinose-regulated expression system, the essentiality of coaBC, a single gene encoding a bifunctional protein catalyzing two consecutive steps in the CoA pathway converting 4'-phosphopantothenate to 4'-phosphopantetheine, was confirmed in Escherichia coli. Utilizing this regulated coaBC strain, it was further demonstrated that E. coli can effectively metabolize pantethine to bypass the requirement for coaBC. Interestingly, pantethine cannot be used by Pseudomonas aeruginosa to obviate coaBC. Through reciprocal complementation studies in combination with biochemical characterization, it was demonstrated that the differential characteristics of pantethine utilization in these two microorganisms are due to the different substrate specificities associated with endogenous pantothenate kinase, the first enzyme in the CoA biosynthetic pathway encoded by coaA in E. coli and coaX in P. aeruginosa.
Assuntos
Carboxiliases/deficiência , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Panteteína/análogos & derivados , Peptídeo Sintases/deficiência , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/metabolismo , Vias Biossintéticas/genética , Proteínas de Escherichia coli , Deleção de Genes , Genes Essenciais , Teste de Complementação Genética , Complexos Multienzimáticos/deficiência , Panteteína/metabolismoRESUMO
Transcriptional profiling data accumulated in recent years for the clinically relevant pathogen Staphylococcus aureus have established a cell wall stress stimulon, which comprises a coordinately regulated set of genes that are upregulated in response to blockage of cell wall biogenesis. In particular, the expression of cwrA (SA2343, N315 notation), which encodes a putative 63 amino acid polypeptide of unknown biological function, increases over 100-fold in response to cell wall inhibition. Herein, we seek to understand the biological role that this gene plays in S. aureus. cwrA was found to be robustly induced by all cell wall-targeting antibiotics tested - vancomycin, oxacillin, penicillin G, phosphomycin, imipenem, hymeglusin and bacitracin - but not by antibiotics with other mechanisms of action, including ciprofloxacin, erythromycin, chloramphenicol, triclosan, rifampicin, novobiocin and carbonyl cyanide 3-chlorophenylhydrazone. Although a DeltacwrA S. aureus strain had no appreciable shift in MICs for cell wall-targeting antibiotics, the knockout was shown to have reduced cell wall integrity in a variety of other assays. Additionally, the gene was shown to be important for virulence in a mouse sepsis model of infection.
Assuntos
Proteínas de Bactérias/fisiologia , Parede Celular/fisiologia , Staphylococcus aureus/genética , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Bacteriólise , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Genes Reporter , Lisostafina/farmacologia , Camundongos , Testes de Sensibilidade Microbiana , Sepse/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Staphylococcus aureus/ultraestrutura , VirulênciaRESUMO
Since their discovery over 5 decades ago, quinolone antibiotics have found enormous success as broad spectrum agents that exert their activity through dual inhibition of bacterial DNA gyrase and topoisomerase IV. Increasing rates of resistance, driven largely by target-based mutations in the GyrA/ParC quinolone resistance determining region, have eroded the utility and threaten the future use of this vital class of antibiotics. Herein we describe the discovery and optimization of a series of 4-(aminomethyl)quinolin-2(1H)-ones, exemplified by 34, that inhibit bacterial DNA gyrase and topoisomerase IV and display potent activity against ciprofloxacin-resistant Gram-negative pathogens. X-ray crystallography reveals that 34 occupies the classical quinolone binding site in the topoisomerase IV-DNA cleavage complex but does not form significant contacts with residues in the quinolone resistance determining region.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Fluoroquinolonas/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Inibidores da Topoisomerase II/farmacologia , Antibacterianos/síntese química , Antibacterianos/metabolismo , Antibacterianos/toxicidade , Sítios de Ligação , Linhagem Celular Tumoral , DNA Girase/metabolismo , DNA Topoisomerase IV/antagonistas & inibidores , DNA Topoisomerase IV/química , Fluoroquinolonas/síntese química , Fluoroquinolonas/metabolismo , Fluoroquinolonas/toxicidade , Bactérias Gram-Negativas/enzimologia , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/síntese química , Inibidores da Topoisomerase II/metabolismo , Inibidores da Topoisomerase II/toxicidadeRESUMO
Isoprenoids are a class of ubiquitous organic molecules synthesized from the five-carbon starter unit isopentenyl pyrophosphate (IPP). Comprising more than 30,000 known natural products, isoprenoids serve various important biological functions in many organisms. In bacteria, undecaprenyl pyrophosphate is absolutely required for the formation of cell wall peptidoglycan and other cell surface structures, while ubiquinones and menaquinones, both containing an essential prenyl moiety, are key electron carriers in respiratory energy generation. There is scant knowledge on the nature and regulation of bacterial isoprenoid pathways. In order to explore the cellular responses to perturbations in the mevalonate pathway, responsible for producing the isoprenoid precursor IPP in many gram-positive bacteria and eukaryotes, we constructed three strains of Staphylococcus aureus in which each of the mevalonate pathway genes is regulated by an IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible promoter. We used DNA microarrays to profile the transcriptional effects of downregulating the components of the mevalonate pathway in S. aureus and demonstrate that decreased expression of the mevalonate pathway leads to widespread downregulation of primary metabolism genes, an upregulation in virulence factors and cell wall biosynthetic determinants, and surprisingly little compensatory expression in other isoprenoid biosynthetic genes. We subsequently correlate these transcriptional changes with downstream metabolic consequences.
Assuntos
Ácido Mevalônico/metabolismo , Transdução de Sinais/fisiologia , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Glicólise/genética , Hemiterpenos/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Compostos Organofosforados/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transcrição GênicaRESUMO
Siderophores are key virulence factors that allow bacteria to grow in iron-restricted environments. The Gram-positive pathogen Staphylococcus aureus is known to produce four siderophores for which genetic and/or structural data are unknown. Here we characterize the gene cluster responsible for producing the prevalent siderophore staphyloferrin A. In addition to expressing the cluster in the heterologous host Escherichia coli, which confers the ability to synthesize the siderophore, we reconstituted staphyloferrin A biosynthesis in vitro by expressing and purifying two key enzymes in the pathway. As with other polycarboxylate siderophores, staphyloferrin A is biosynthesized using the recently described nonribosomal peptide synthetase independent siderophore (NIS) biosynthetic pathway. Two NIS synthetases condense two molecules of citric acid to d-ornithine in a stepwise ordered process with SfnaD using the delta-amine as a nucleophile to form the first amide followed by SfnaB utilizing the alpha-amine to complete staphyloferrin A synthesis.
Assuntos
Proteínas de Bactérias/genética , Família Multigênica , Ornitina/análogos & derivados , Peptídeo Sintases/genética , Sideróforos/genética , Staphylococcus aureus/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Citratos/química , Biologia Computacional/métodos , Escherichia coli/enzimologia , Escherichia coli/genética , Código Genético , Ornitina/química , Ornitina/genética , Peptídeo Sintases/química , Sideróforos/biossíntese , Sideróforos/química , Staphylococcus aureus/químicaRESUMO
The last stages of assembly of the aminocoumarin antibiotics, clorobiocin and coumermycin A(1), which target the GyrB subunits of bacterial DNA gyrase, involve enzymatic transfer of the pyrrolyl-2-carbonyl acyl group from a carrier protein (CloN1/CouN1) to the 3'-OH of the noviosyl moiety of the antibiotic scaffold. The enzyme, CouN7, will catalyze both the forward and back reaction on both arms of the coumermycin scaffold. This occurs via an O-acyl-Ser(101)-CouN7 intermediate, as shown by transient labeling of the enzyme with [(14)C]acetyl-S-CouN1 as donor and by inactivating mutation of the active site, Ser(101), to Ala. The intermediacy of the pyrrolyl-2-carbonyl-O-CouN7 allows net pyrrole transfer between distinct aminocoumarin scaffolds, for example, between the descarbamoylnovobiocin scaffold and coumermycin A(1) and vice versa. CouN7 also allows shuttling of surrogate acyl groups between noviosyl-aminocoumarin scaffolds to generate new antibiotic variants.
Assuntos
Aciltransferases , Antibacterianos/biossíntese , Antibacterianos/química , Acil Coenzima A/química , Acilação , Aciltransferases/biossíntese , Aciltransferases/química , Aciltransferases/genética , Aminocumarinas/química , Catálise , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Escherichia coli/metabolismo , Cinética , Streptomyces/enzimologia , Streptomyces/genéticaRESUMO
A riboflavin biosynthesis pathway-specific phenotypic screen using a library of compounds, all with unspecified antibiotic activity, identified one small molecule later named ribocil, for which intrinsic antibacterial activity against Escherichia coli was completely suppressed by addition of exogenous riboflavin to the bacterial growth medium. The ability of riboflavin to suppress the activity of ribocil, and further demonstration that ribocil inhibited riboflavin synthesis (IC50 = 0.3 µM), supported that a component of the riboflavin synthesis pathway was the molecular target. Remarkably, resistance mutation selection and whole-genome sequencing showed that the target of ribocil was not an enzyme in the riboflavin biosynthesis pathway, but instead the flavin mononucleotide riboswitch, a noncoding structural RNA element in the ribB gene that encodes a key riboflavin synthesis enzyme. Although ribocil is structurally distinct from the natural riboswitch regulatory ligand flavin mononucleotide, ribocil binding to the riboswitch results in efficient repression of ribB expression and inhibition of riboflavin biosynthesis and bacterial growth. A cell-based riboswitch regulated gene reporter assay as well as an in vitro riboswitch RNA aptamer-binding assay, both of which are described in detail here along with the riboflavin pathway-specific screen, were developed to further validate the mechanism of action of ribocil and to facilitate the discovery of more potent analogues.