RESUMEN
BACKGROUND: Latency remains a major obstacle to finding a cure for HIV despite the availability of antiretroviral therapy. Due to virus dormancy, limited biomarkers are available to identify latent HIV-infected cells. Profiling of individual HIV-infected cells is needed to explore potential latency biomarkers and to study the mechanisms of persistence that maintain the HIV reservoir. METHODS: Single cell spatial transcriptomic characterization using the CosMx Spatial Molecular Imager platform was conducted to analyze HIV-infected cells in formalin-fixed paraffin-embedded sections of splenic tissue surgically obtained from an HIV-infected humanized mouse model. Regulation of over a thousand human genes was quantified in both viremic and aviremic specimens. In addition, in situ hybridization and immunohistochemistry were performed in parallel to identify HIV viral RNA- and p24-containing cells, respectively. Finally, initial findings from CosMx gene profiling were confirmed by isolating RNA from CD4 + T cells obtained from a person living with HIV on antiretroviral therapy following either PMA/Ionomycin or DMSO treatment. RNA was quantified using qPCR for a panel of targeted human host genes. RESULTS: Supervised cell typing revealed that most of the HIV-infected cells in the mouse spleen sections were differentiated CD4 + T cells. A significantly higher number of infected cells, 2781 (1.61%) in comparison to 112 (0.06%), and total HIV transcripts per infected cell were observed in viremic samples compared to aviremic samples, respectively, which was consistent with the data obtained from ISH and IHC. Notably, the expression of 55 genes was different in infected cells within tissue from aviremic animals compared to viremic. In particular, both spleen tyrosine kinase (SYK) and CXCL17, were expressed approximately 100-fold higher. This data was further evaluated against bulk RNA isolated from HIV-infected human primary CD4 + T cells. A nearly 6-fold higher expression of SYK mRNA was observed in DMSO-treated CD4 + T cells compared to those stimulated with PMA/Ionomycin. CONCLUSION: This study found that the CosMx SMI platform is valuable for assessing HIV infection and providing insights into host biomarkers associated with HIV reservoirs. Higher relative expression of the SYK gene in aviremic-infected cells from the humanized mouse HIV model was consistent with levels found in CD4 + T cells of aviremic donors.
RESUMEN
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.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Infecciones por Acinetobacter , Acinetobacter baumannii , Antibacterianos , Proteínas Bacterianas , Lipopolisacáridos , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/metabolismo , Antibacterianos/farmacología , Antibacterianos/química , Animales , Lipopolisacáridos/metabolismo , Lipopolisacáridos/antagonistas & inhibidores , Ratones , Humanos , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/metabolismo , Infecciones por Acinetobacter/tratamiento farmacológico , Infecciones por Acinetobacter/microbiología , Pruebas de Sensibilidad MicrobianaRESUMEN
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.
Asunto(s)
Infecciones por Acinetobacter , Acinetobacter baumannii , Antibacterianos , Proteínas Bacterianas , Lipopolisacáridos , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/metabolismo , Lipopolisacáridos/metabolismo , Animales , Infecciones por Acinetobacter/microbiología , Infecciones por Acinetobacter/tratamiento farmacológico , Ratones , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Transporte Biológico , Pruebas de Sensibilidad Microbiana , Humanos , Microscopía por Crioelectrón , Carbapenémicos/farmacología , Carbapenémicos/metabolismo , Modelos Animales de Enfermedad , Femenino , Transportadoras de Casetes de Unión a ATPRESUMEN
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.
Asunto(s)
Infecciones por VIH , VIH-1 , Humanos , Infecciones por VIH/tratamiento farmacológico , Antivirales/uso terapéutico , Apoptosis , Muerte Celular , Linfocitos T CD4-Positivos , Replicación ViralRESUMEN
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.
Asunto(s)
Antibacterianos , Escherichia coli , Antibacterianos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Transporte Biológico , Bacterias Gramnegativas/metabolismo , Permeabilidad , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/metabolismoRESUMEN
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.
Asunto(s)
Infecciones por VIH , VIH-1 , Alquinos , Benzoxazinas , Proteínas Adaptadoras de Señalización CARD/metabolismo , Ciclopropanos , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Infecciones por VIH/tratamiento farmacológico , VIH-1/metabolismo , Humanos , Inflamasomas/metabolismo , Leucocitos Mononucleares , Proteínas de Neoplasias/metabolismoRESUMEN
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.
Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Fluoroquinolonas/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Inhibidores de Topoisomerasa II/farmacología , Antibacterianos/síntesis química , Antibacterianos/metabolismo , Antibacterianos/toxicidad , Sitios de Unión , Línea Celular Tumoral , Girasa de ADN/metabolismo , Topoisomerasa de ADN IV/antagonistas & inhibidores , Topoisomerasa de ADN IV/química , Fluoroquinolonas/síntesis química , Fluoroquinolonas/metabolismo , Fluoroquinolonas/toxicidad , Bacterias Gramnegativas/enzimología , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Relación Estructura-Actividad , Inhibidores de Topoisomerasa II/síntesis química , Inhibidores de Topoisomerasa II/metabolismo , Inhibidores de Topoisomerasa II/toxicidadRESUMEN
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.
Asunto(s)
Aciltransferasas/antagonistas & inhibidores , Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Pirazoles/farmacología , Aciltransferasas/metabolismo , Antibacterianos/metabolismo , Cristalografía por Rayos X , Inhibidores Enzimáticos/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Imidazoles/metabolismo , Pruebas de Sensibilidad Microbiana , Unión Proteica , Pirazoles/metabolismoRESUMEN
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.
Asunto(s)
Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Escherichia coli/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Triazinas/farmacología , Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Proteínas de la Membrana Bacteriana Externa/genética , Transporte Biológico/fisiología , Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/fisiología , Evaluación Preclínica de Medicamentos , Farmacorresistencia Bacteriana/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Pruebas de Sensibilidad MicrobianaRESUMEN
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.
Asunto(s)
Compuestos de Azabiciclo/farmacología , Imipenem/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Antibacterianos/farmacología , Proteínas Bacterianas/efectos de los fármacos , Combinación de Medicamentos , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Humanos , Cinética , Pruebas de Sensibilidad Microbiana , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/enzimología , beta-Lactamasas/efectos de los fármacosRESUMEN
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.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Endopeptidasas , Proteínas de Escherichia coli , Escherichia coli , Proteínas de la Membrana , Desplegamiento Proteico , Factores de Transcripción , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Endopeptidasas/genética , Endopeptidasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Descubrimiento de Drogas , Metabolismo Energético/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/fisiología , Mononucleótido de Flavina/metabolismo , Riboflavina/metabolismo , Riboswitch/efectos de los fármacos , Antibacterianos/farmacología , Clonación Molecular , Genes Reporteros , Ligandos , Mutación , Fenotipo , Plásmidos , Técnica SELEX de Producción de Aptámeros , Bibliotecas de Moléculas PequeñasRESUMEN
The discovery and development of new antibiotics capable of curing infections due to multidrug-resistant and pandrug-resistant Gram-negative bacteria are a major challenge with fundamental importance to our global healthcare system. Part of our broad program at Novartis to address this urgent, unmet need includes the search for new agents that inhibit novel bacterial targets. Here we report the discovery and hit-to-lead optimization of new inhibitors of phosphopantetheine adenylyltransferase (PPAT) from Gram-negative bacteria. Utilizing a fragment-based screening approach, we discovered a number of unique scaffolds capable of interacting with the pantetheine site of E. coli PPAT and inhibiting enzymatic activity, including triazolopyrimidinone 6. Structure-based optimization resulted in the identification of two lead compounds as selective, small molecule inhibitors of bacterial PPAT: triazolopyrimidinone 53 and azabenzimidazole 54 efficiently inhibited E. coli and P. aeruginosa PPAT and displayed modest cellular potency against the efflux-deficient E. coli Δ tolC mutant strain.
Asunto(s)
Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas de Escherichia coli/antagonistas & inhibidores , Compuestos Heterocíclicos con 2 Anillos/farmacología , Nucleotidiltransferasas/antagonistas & inhibidores , Antibacterianos/síntesis química , Antibacterianos/química , Antibacterianos/metabolismo , Bencimidazoles/síntesis química , Bencimidazoles/química , Bencimidazoles/metabolismo , Bencimidazoles/farmacología , Sitios de Unión , Descubrimiento de Drogas , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Compuestos Heterocíclicos con 2 Anillos/síntesis química , Compuestos Heterocíclicos con 2 Anillos/química , Compuestos Heterocíclicos con 2 Anillos/metabolismo , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Nucleotidiltransferasas/química , Nucleotidiltransferasas/metabolismo , Unión Proteica , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/enzimología , Pirimidinonas/síntesis química , Pirimidinonas/química , Pirimidinonas/metabolismo , Pirimidinonas/farmacología , Triazoles/síntesis química , Triazoles/química , Triazoles/metabolismo , Triazoles/farmacologíaRESUMEN
The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current ß-lactam antibiotics and created an urgent need for new treatment options. We report an S. aureus phenotypic screening strategy involving chemical suppression of the growth inhibitory consequences of depleting late-stage wall teichoic acid biosynthesis. This enabled us to identify early-stage pathway-specific inhibitors of wall teichoic acid biosynthesis predicted to be chemically synergistic with ß-lactams. We demonstrated by genetic and biochemical means that each of the new chemical series discovered, herein named tarocin A and tarocin B, inhibited the first step in wall teichoic acid biosynthesis (TarO). Tarocins do not have intrinsic bioactivity but rather demonstrated potent bactericidal synergy in combination with broad-spectrum ß-lactam antibiotics against diverse clinical isolates of methicillin-resistant staphylococci as well as robust efficacy in a murine infection model of MRSA. Tarocins and other inhibitors of wall teichoic acid biosynthesis may provide a rational strategy to develop Gram-positive bactericidal ß-lactam combination agents active against methicillin-resistant staphylococci.
Asunto(s)
Proteínas Bacterianas/metabolismo , Vías Biosintéticas/efectos de los fármacos , Pared Celular/metabolismo , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Ácidos Teicoicos/biosíntesis , beta-Lactamas/farmacología , Animales , Pared Celular/efectos de los fármacos , Dicloxacilina/farmacología , Dicloxacilina/uso terapéutico , Femenino , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Fenotipo , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Resultado del TratamientoRESUMEN
Paramount to any rational discovery of new antibiotics displaying novel mechanisms of action is a deep knowledge of the genetic basis of microbial growth, division and virulence. The bakers' yeast,Saccharomyces cerevisiae, illustrates the highest understanding of the genetic underpinnings of microbial life, and from this framework, a systems biology paradigm has evolved, begging to be emulated in antibacterial discovery. Here, we review landmark events in the history of yeast genomics that provide this new foundation for antibacterial drug discovery.
Asunto(s)
Descubrimiento de Drogas , Genómica , Pruebas de Sensibilidad Microbiana , Saccharomyces cerevisiae/genética , Genoma Bacteriano , Genoma Fúngico , Haploinsuficiencia , Saccharomyces cerevisiae/efectos de los fármacosRESUMEN
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.
Asunto(s)
Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Permeabilidad de la Membrana Celular/genética , Farmacorresistencia Bacteriana Múltiple/genética , Proteínas de Transporte de Membrana/genética , Pseudomonas aeruginosa/genética , Secuencia de Aminoácidos , Transporte Biológico/genética , Membrana Celular/metabolismo , Lipopolisacáridos/metabolismo , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/efectos de los fármacosRESUMEN
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.
Asunto(s)
Pirimidinas/química , Pirimidinas/farmacología , ARN Bacteriano/química , ARN Bacteriano/efectos de los fármacos , Riboswitch/efectos de los fármacos , Animales , Aptámeros de Nucleótidos/química , Bacterias/citología , Bacterias/efectos de los fármacos , Bacterias/crecimiento & desarrollo , Secuencia de Bases , Cristalografía por Rayos X , Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Femenino , Mononucleótido de Flavina/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Proteínas de Choque Térmico/genética , Transferasas Intramoleculares/genética , Ligandos , Ratones , Ratones Endogámicos DBA , Modelos Moleculares , Datos de Secuencia Molecular , Pirimidinas/aislamiento & purificación , Pirimidinas/uso terapéutico , ARN Bacteriano/genética , Reproducibilidad de los Resultados , Riboflavina/biosíntesis , Riboswitch/genética , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Antibacterianos/farmacología , Pirroles/farmacología , Pirrolidinonas/farmacología , Acinetobacter baumannii/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacosRESUMEN
Modern medicine is founded on the discovery of penicillin and subsequent small molecules that inhibit bacterial peptidoglycan (PG) and cell wall synthesis. However, the discovery of new chemically and mechanistically distinct classes of PG inhibitors has become exceedingly rare, prompting speculation that intracellular enzymes involved in PG precursor synthesis are not 'druggable' targets. Here, we describe a ß-lactam potentiation screen to identify small molecules that augment the activity of ß-lactams against methicillin-resistant Staphylococcus aureus (MRSA) and mechanistically characterize a compound resulting from this screen, which we have named murgocil. We provide extensive genetic, biochemical, and structural modeling data demonstrating both in vitro and in whole cells that murgocil specifically inhibits the intracellular membrane-associated glycosyltransferase, MurG, which synthesizes the lipid II PG substrate that penicillin binding proteins (PBPs) polymerize and cross-link into the cell wall. Further, we demonstrate that the chemical synergy and cidality achieved between murgocil and the ß-lactam imipenem is mediated through MurG dependent localization of PBP2 to the division septum. Collectively, these data validate our approach to rationally identify new target-specific bioactive ß-lactam potentiation agents and demonstrate that murgocil now serves as a highly selective and potent chemical probe to assist our understanding of PG biosynthesis and cell wall biogenesis across Staphylococcal species.
Asunto(s)
Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , Peptidoglicano Glicosiltransferasa/metabolismo , Pirazoles/farmacología , Staphylococcus aureus/efectos de los fármacos , Esteroles/farmacología , Simulación por Computador , Farmacorresistencia Bacteriana , Inhibidores Enzimáticos/farmacología , Humanos , Microscopía Fluorescente , Modelos Moleculares , Pirazoles/química , Staphylococcus aureus/enzimología , Esteroles/químicaRESUMEN
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.