RESUMEN
Over recent decades, the pipeline of antibiotics acting against Gram-negative bacteria is running dry, as most discovered candidate antibiotics suffer from insufficient potency, pharmacokinetic properties, or toxicity. The darobactins, a promising new small peptide class of drug candidates, bind to novel antibiotic target BamA, an outer membrane protein. Previously, we reported that biosynthetic engineering in a heterologous host generated novel darobactins with enhanced antibacterial activity. Here we utilize an optimized purification method and present cryo-EM structures of the Bam complex with darobactin 9 (D9), which served as a blueprint for the biotechnological generation of twenty new darobactins including halogenated analogs. The newly engineered darobactin 22 binds more tightly to BamA and outperforms the favorable activity profile of D9 against clinically relevant pathogens such as carbapenem-resistant Acinetobacter baumannii up to 32-fold, without observing toxic effects.
Asunto(s)
Acinetobacter baumannii , Fenilpropionatos , Antibacterianos/farmacología , Antibacterianos/química , Microscopía por Crioelectrón , Fenilpropionatos/farmacología , Bacterias Gramnegativas , Pruebas de Sensibilidad MicrobianaRESUMEN
Class B metallo-ß-lactamases (MBLs) are Zn2+-dependent enzymes that catalyze the hydrolysis of ß-lactam antibiotics to confer resistance in bacteria. Several problematic groups of MBLs belong to subclass B1, including the binuclear New Delhi MBL (NDM), Verona integrin-encoded MBL, and imipenemase-type enzymes, which are responsible for widespread antibiotic resistance. Aspergillomarasmine A (AMA) is a natural aminopolycarboxylic acid that functions as an effective inhibitor of class B1 MBLs. The precise mechanism of action of AMA is not thoroughly understood, but it is known to inactivate MBLs by removing one catalytic Zn2+ cofactor. We investigated the kinetics of MBL inactivation in detail and report that AMA is a selective Zn2+ scavenger that indirectly inactivates NDM-1 by encouraging the dissociation of a metal cofactor. To further investigate the mechanism in living bacteria, we used an active site probe and showed that AMA causes the loss of a Zn2+ ion from a low-affinity binding site of NDM-1. Zn2+-depleted NDM-1 is rapidly degraded, contributing to the efficacy of AMA as a ß-lactam potentiator. However, MBLs with higher metal affinity and stability such as NDM-6 and imipenemase-7 exhibit greater tolerance to AMA. These results indicate that the mechanism of AMA is broadly applicable to diverse Zn2+ chelators and highlight that leveraging Zn2+ availability can influence the survival of MBL-producing bacteria when they are exposed to ß-lactam antibiotics.
Asunto(s)
Antibacterianos/farmacología , Ácido Aspártico/análogos & derivados , Bacterias/efectos de los fármacos , Zinc/química , Inhibidores de beta-Lactamasas/farmacología , beta-Lactamasas/química , Ácido Aspártico/farmacología , Bacterias/enzimología , Quelantes/farmacología , Farmacorresistencia Bacteriana , Pruebas de Sensibilidad Microbiana/métodos , beta-Lactamasas/metabolismoRESUMEN
The increasing prevalence of metallo-ß-lactamase (MBL)-expressing bacteria presents a worrying trend in antibiotic resistance. MBLs rely on active site zinc ions for their hydrolytic activity and the pursuit of MBL-inhibitors has therefore involved the investigation of zinc chelators. To ensure that such chelators specifically target MBLs, a series of cephalosporin prodrugs of two potent zinc-binders: dipicolinic acid (DPA) and 8-thioquinoline (8-TQ) was prepared. Although both DPA and 8-TQ bind free zinc very tightly (Kd values in the low nm range), the corresponding cephalosporin conjugates do not. The cephalosporin conjugates are efficiently hydrolyzed by MBLs to release DPA or 8-TQ, as confirmed by using both NMR and LC-MS studies. Notably, the cephalosporin prodrugs of DPA and 8-TQ show potent inhibitory activity against NDM, VIM, and IMP classes of MBLs and display potent synergy with meropenem against MBL-expressing clinical isolates of K. pneumoniae and E. coli.
Asunto(s)
Cefalosporinas/farmacología , Farmacorresistencia Microbiana/efectos de los fármacos , Profármacos/farmacología , Inhibidores de beta-Lactamasas/farmacología , beta-Lactamasas/metabolismo , Escherichia coli/efectos de los fármacos , Klebsiella pneumoniae/efectos de los fármacos , Pruebas de Sensibilidad MicrobianaRESUMEN
The rapid emergence of antimicrobial resistance is a major threat to human health. Antibiotics modulate a wide range of biological processes in bacteria and as such, the study of bacterial cellular signaling could aid the development of urgently needed new antibiotic agents. Due to the advances in bacterial phosphoproteomics, such a systemwide analysis of bacterial signaling in response to antibiotics has recently become feasible. Here we present a dynamic view of differential protein phosphorylation upon antibiotic treatment and antibiotic resistance. Most strikingly, differential phosphorylation was observed on highly conserved residues of resistance regulating transcription factors, implying a previously unanticipated role of phosphorylation mediated regulation. Using the comprehensive phosphoproteomics data presented here as a resource, future research can now focus on deciphering the precise signaling mechanisms contributing to resistance, eventually leading to alternative strategies to combat antimicrobial resistance.
Asunto(s)
Antibacterianos/metabolismo , Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Cromatografía Liquida , Proteínas de Escherichia coli , Humanos , Fosforilación , Proteómica/métodos , Espectrometría de Masas en Tándem , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Food for human consumption is screened widely for the presence of antibiotic-resistant bacteria to assess the potential for transfer of resistant bacteria to the general population. Here, we describe an Enterobacter cloacae complex isolated from imported seafood that encodes two carbapenemases on two distinct plasmids. Both enzymes belong to Ambler class A ß-lactamases, the previously described IMI-2 and a novel family designated FLC-1. The hydrolytic activity of the novel enzyme against aminopenicillins, cephalosporins, and carbapenems was determined.
Asunto(s)
Proteínas Bacterianas/metabolismo , Enterobacter cloacae/enzimología , beta-Lactamasas/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Cefalosporinas/metabolismo , Enterobacter/efectos de los fármacos , Enterobacter cloacae/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , beta-Lactamasas/genéticaRESUMEN
Antimicrobial resistance is a leading cause of mortality, calling for the development of new antibiotics. The fungal antibiotic plectasin is a eukaryotic host defence peptide that blocks bacterial cell wall synthesis. Here, using a combination of solid-state nuclear magnetic resonance, atomic force microscopy and activity assays, we show that plectasin uses a calcium-sensitive supramolecular killing mechanism. Efficient and selective binding of the target lipid II, a cell wall precursor with an irreplaceable pyrophosphate, is achieved by the oligomerization of plectasin into dense supra-structures that only form on bacterial membranes that comprise lipid II. Oligomerization and target binding of plectasin are interdependent and are enhanced by the coordination of calcium ions to plectasin's prominent anionic patch, causing allosteric changes that markedly improve the activity of the antibiotic. Structural knowledge of how host defence peptides impair cell wall synthesis will likely enable the development of superior drug candidates.
Asunto(s)
Calcio , Pared Celular , Péptidos , Uridina Difosfato Ácido N-Acetilmurámico , Pared Celular/metabolismo , Pared Celular/efectos de los fármacos , Pared Celular/química , Calcio/metabolismo , Péptidos/farmacología , Péptidos/metabolismo , Péptidos/química , Uridina Difosfato Ácido N-Acetilmurámico/análogos & derivados , Uridina Difosfato Ácido N-Acetilmurámico/metabolismo , Uridina Difosfato Ácido N-Acetilmurámico/química , Microscopía de Fuerza Atómica , Antibacterianos/farmacología , Antibacterianos/química , Espectroscopía de Resonancia Magnética , Unión ProteicaRESUMEN
Gram-positive bacterial infections present a major clinical challenge, with methicillin- and vancomycin-resistant strains continuing to be a cause for concern. In recent years, semisynthetic vancomycin derivatives have been developed to overcome this problem as exemplified by the clinically used telavancin, which exhibits increased antibacterial potency but has also raised toxicity concerns. Thus, glycopeptide antibiotics with enhanced antibacterial activities and improved safety profiles are still necessary. We describe the development of a class of highly potent semisynthetic glycopeptide antibiotics, the guanidino lipoglycopeptides, which contain a positively charged guanidino moiety bearing a variable lipid group. These glycopeptides exhibited enhanced in vitro activity against a panel of Gram-positive bacteria including clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant strains, showed minimal toxicity toward eukaryotic cells, and had a low propensity for resistance selection. Mechanistically, guanidino lipoglycopeptides engaged with bacterial cell wall precursor lipid II with a higher binding affinity than vancomycin. Binding to both wild-type d-Ala-d-Ala lipid II and the vancomycin-resistant d-Ala-d-Lac variant was confirmed, providing insight into the enhanced activity of guanidino lipoglycopeptides against vancomycin-resistant isolates. The in vivo efficacy of guanidino lipoglycopeptide EVG7 was evaluated in a S. aureus murine thigh infection model and a 7-day sepsis survival study, both of which demonstrated superiority to vancomycin. Moreover, the minimal to mild kidney effects at supratherapeutic doses of EVG7 indicate an improved therapeutic safety profile compared with vancomycin. These findings position guanidino lipoglycopeptides as candidates for further development as antibacterial agents for the treatment of clinically relevant multidrug-resistant Gram-positive infections.
Asunto(s)
Antibacterianos , Lipoglucopéptidos , Pruebas de Sensibilidad Microbiana , Animales , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/uso terapéutico , Lipoglucopéptidos/farmacología , Lipoglucopéptidos/uso terapéutico , Ratones , Humanos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Glicopéptidos/farmacología , Glicopéptidos/química , Glicopéptidos/uso terapéutico , Bacterias Grampositivas/efectos de los fármacos , FemeninoRESUMEN
Metallo-ß-lactamases (MBLs) are zinc-dependent bacterial enzymes that inactivate essentially all classes of ß-lactam antibiotics including last-resort carbapenems. At present there are no clinically approved MBL inhibitors, and in order to develop such agents it is essential to understand their inhibitory mechanisms. Herein, we describe a comprehensive mechanistic study of a panel of structurally distinct MBL inhibitors reported in both the scientific and patent literature. Specifically, we determined the half-maximal inhibitory concentration (IC50 ) for each inhibitor against MBLs belonging to the NDM and IMP families. In addition, the binding affinities of the inhibitors for Zn2+ , Ca2+ and Mg2+ were assessed by using isothermal titration calorimetry (ITC). We also compared the ability of the different inhibitors to resensitize a highly resistant MBL-expressing Escherichia coli strain to meropenem. These investigations reveal clear differences between the MBL inhibitors studied in terms of their IC50 value, metal binding ability, and capacity to synergize with meropenem. Notably, our studies demonstrate that potent MBL inhibition and synergy with meropenem are not explicitly dependent on the capacity of an inhibitor to strongly chelate zinc.
Asunto(s)
Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Inhibidores de beta-Lactamasas/farmacología , beta-Lactamasas/metabolismo , Antibacterianos/síntesis química , Antibacterianos/química , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Piridinas/química , Piridinas/farmacología , Relación Estructura-Actividad , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/farmacología , Zinc/química , Zinc/farmacología , Inhibidores de beta-Lactamasas/síntesis química , Inhibidores de beta-Lactamasas/químicaRESUMEN
In an attempt to exploit the hydrolytic mechanism by which ß-lactamases degrade cephalosporins, we designed and synthesized a series of novel cephalosporin prodrugs aimed at delivering thiol-based inhibitors of metallo-ß-lactamases (MBLs) in a spatiotemporally controlled fashion. While enzymatic hydrolysis of the ß-lactam ring was observed, it was not accompanied by inhibitor release. Nonetheless, the cephalosporin prodrugs, especially thiomandelic acid conjugate (8), demonstrated potent inhibition of IMP-type MBLs. In addition, conjugate 8 was also found to greatly reduce the minimum inhibitory concentration of meropenem against IMP-producing bacteria. The results of kinetic experiments indicate that these prodrugs inhibit IMP-type MBLs by acting as slowly turned-over substrates. Structure-activity relationship studies revealed that both phenyl and carboxyl moieties of 8 are crucial for its potency. Furthermore, modeling studies indicate that productive interactions of the thiomandelic acid moiety of 8 with Trp28 within the IMP active site may contribute to its potency and selectivity.
Asunto(s)
Antibacterianos/farmacología , Cefalosporinas/farmacología , Inhibidores de beta-Lactamasas/farmacología , beta-Lactamasas/metabolismo , Antibacterianos/síntesis química , Antibacterianos/química , Cefalosporinas/síntesis química , Cefalosporinas/química , Relación Dosis-Respuesta a Droga , Estructura Molecular , Relación Estructura-Actividad , Inhibidores de beta-Lactamasas/síntesis química , Inhibidores de beta-Lactamasas/químicaRESUMEN
In the search for new inhibitors of bacterial metallo-ß-lactamases (MBLs), a series of commonly used small molecule carboxylic acid derivatives were evaluated for their ability to inhibit New Delhi metallo-ß-lactamase (NDM)-, Verona integron-encoded metallo-ß-lactamase (VIM)-, and imipenemase (IMP)-type enzymes. Nitrilotriacetic acid (3) and N-(phosphonomethyl)iminodiacetic acid (5) showed promising activity especially against NDM-1 and VIM-2 with IC50 values in the low-to-sub µM range. Binding assays using isothermal titration calorimetry reveal that 3 and 5 bind zinc with high affinity with dissociation constant (Kd) values of 121 and 56 nM, respectively. The in vitro biological activity of 3 and 5 against E. coli expressing NDM-1 was evaluated in checkerboard format, demonstrating a strong synergistic relationship for both compounds when combined with Meropenem. Compounds 3 and 5 were then tested against 35 pathogenic strains expressing MBLs of the NDM, VIM, or IMP classes. Notably, when combined with Meropenem, compounds 3 and 5 were found to lower the minimum inhibitory concentration (MIC) of Meropenem up to 128-fold against strains producing NDM- and VIM-type enzymes.
Asunto(s)
Escherichia coli , Inhibidores de beta-Lactamasas , Antibacterianos/farmacología , Ácidos Carboxílicos , Escherichia coli/genética , Meropenem/farmacología , Inhibidores de beta-Lactamasas/farmacología , beta-Lactamasas/genéticaRESUMEN
A series of aminocarboxylic acid analogues of aspergillomarasmine A (AMA) and ethylenediamine-N,N'-disuccinic acid (EDDS) were chemoenzymatically synthesized via the addition of various mono- and diamine substrates to fumaric acid catalyzed by the enzyme EDDS lyase. Many of these novel AMA and EDDS analogues demonstrate potent inhibition of the bacterial metallo-ß-lactamase NDM-1. Isothermal titration calorimetry assays revealed a strong correlation between the inhibitory potency of the compounds and their ability to bind zinc. Compounds 1a (AMA), 1b (AMB), 5 (EDDS), followed by 1d and 8a, demonstrate the highest synergy with meropenem resensitizing an NDM-1 producing strain of E. coli to this important carbapenem of last resort.
Asunto(s)
Ácido Aspártico/análogos & derivados , Complejos de Coordinación/farmacología , Proteínas de Escherichia coli/antagonistas & inhibidores , Etilenodiaminas/farmacología , Succinatos/farmacología , Zinc/farmacología , Inhibidores de beta-Lactamasas/farmacología , Aminoácidos/química , Aminoácidos/farmacología , Ácido Aspártico/química , Ácido Aspártico/farmacología , Complejos de Coordinación/síntesis química , Complejos de Coordinación/química , Relación Dosis-Respuesta a Droga , Proteínas de Escherichia coli/metabolismo , Etilenodiaminas/química , Estructura Molecular , Relación Estructura-Actividad , Succinatos/química , Zinc/química , Inhibidores de beta-Lactamasas/síntesis química , Inhibidores de beta-Lactamasas/química , beta-Lactamasas/metabolismoRESUMEN
Antibiotic resistance caused by ß-lactamase production continues to present a growing challenge to the efficacy of ß-lactams and their role as the most important class of clinically used antibiotics. In response to this threat however, only a handful of ß-lactamase inhibitors have been introduced to the market over the past thirty years. The first-generation ß-lactamase inhibitors (clavulanic acid, sulbactam and tazobactam) are all ß-lactam derivatives and work primarily by inactivating class A and some class C serine ß-lactamases. The newer generations of ß-lactamase inhibitors including avibactam and vaborbactam are based on non-ß-lactam structures and their spectrum of inhibition is extended to KPC as an important class A carbapenemase. Despite these advances several class D and virtually all important class B ß-lactamases are resistant to existing inhibitors. The present review provides an overview of recent FDA-approved ß-lactam/ß-lactamase inhibitor combinations as well as an update on research efforts aimed at the discovery and development of novel ß-lactamase inhibitors.
RESUMEN
Metallo-ß-lactamases (MBLs) are a growing threat to the continued efficacy of ß-lactam antibiotics. Recently, aspergillomarasmine A (AMA) was identified as an MBL inhibitor, but the mode of inhibition was not fully characterized. Equilibrium dialysis and metal analysis studies revealed that 2 equiv of AMA effectively removes 1 equiv of Zn(II) from MBLs NDM-1, VIM-2, and IMP-7 when the MBL is at micromolar concentrations. Conversely, 1H NMR studies revealed that 2 equiv of AMA remove 2 equiv of Co(II) from Co(II)-substituted NDM-1, VIM-2, and IMP-7 when the MBL/AMA are at millimolar concentrations. Our findings reveal that AMA inhibits the MBLs by removal of the active site metal ions required for ß-lactam hydrolysis among the most clinically significant MBLs.