ABSTRACT
Acinetobacter baumannii-calcoaceticus complex (ABC) causes severe, difficult-to-treat infections that are frequently antibiotic resistant. Sulbactam-durlobactam (SUL-DUR) is a targeted ß-lactam/ß-lactamase inhibitor combination antibiotic designed to treat ABC infections, including those caused by multidrug-resistant strains. In a global, pathogen-specific, randomized, controlled phase 3 trial (ATTACK), the efficacy and safety of SUL-DUR were compared to colistin, both dosed with imipenem-cilastatin as background therapy, in patients with serious infections caused by carbapenem-resistant ABC. Results from ATTACK showed that SUL-DUR met the criteria for non-inferiority to colistin for the primary efficacy endpoint of 28-day all-cause mortality with improved clinical and microbiological outcomes compared to colistin. This report describes the characterization of the baseline ABC isolates from patients enrolled in ATTACK, including an analysis of the correlation of microbiological outcomes with SUL-DUR MIC values and the molecular drivers of SUL-DUR resistance.
Subject(s)
Acinetobacter Infections , Acinetobacter baumannii , Anti-Bacterial Agents , Colistin , Microbial Sensitivity Tests , Sulbactam , Humans , Male , Acinetobacter baumannii/drug effects , Acinetobacter calcoaceticus/drug effects , Acinetobacter calcoaceticus/genetics , Acinetobacter Infections/drug therapy , Acinetobacter Infections/microbiology , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Azabicyclo Compounds/pharmacology , Azabicyclo Compounds/therapeutic use , Cilastatin, Imipenem Drug Combination/therapeutic use , Colistin/pharmacology , Colistin/therapeutic use , Drug Resistance, Multiple, Bacterial , Sulbactam/therapeutic use , Sulbactam/pharmacologyABSTRACT
Background: In a previous study, the efficacy and safety of sulbactam-durlobactam vs colistin for the treatment of patients with carbapenem-resistant Acinetobacter baumannii-calcoaceticus complex (CRABC) infections were evaluated in a randomized controlled phase 3 trial. Both arms were dosed on a background of imipenem-cilastatin to treat coinfecting gram-negative pathogens. Thirty-six percent of infections in the primary efficacy population were polymicrobial. Methods: A subset analysis was performed to compare clinical and microbiological outcomes at test of cure (7 ± 2 days after the last dose) for patients with monomicrobial and polymicrobial CRABC infections. Minimal inhibitory concentrations of antibiotics against baseline isolates were determined by broth microdilution according to Clinical and Laboratory Standards Institute methodology. Results: Clinical cure, 28-day all-cause mortality, and microbiological outcomes were similar for patients in the sulbactam-durlobactam treatment arm with monomicrobial or polymicrobial A baumannii-calcoaceticus infections. Patients in the colistin arm with monomicrobial CRABC infections had higher mortality rates with worse clinical and microbiological outcomes as compared with those with polymicrobial infections. For patients who received sulbactam-durlobactam, imipenem susceptibility of coinfecting gram-negative pathogens trended with clinical benefit for patients with polymicrobial A baumannii-calcoaceticus infections. When tested in vitro, durlobactam restored imipenem susceptibility to the majority of coinfecting gram-negative pathogens from the sulbactam-durlobactam arm. This phenotype appeared to be related to the clinical outcome in 13 of 15 evaluable cases. Conclusions: These results suggest that the use of sulbactam-durlobactam plus a carbapenem could be an effective approach to treat polymicrobial infections that include CRABC, but additional clinical data are needed to demonstrate efficacy.
ABSTRACT
The treatment of patients with infection secondary to carbapenem-resistant Acinetobacter baumannii with emerging cefiderocol resistance remains challenging and unclear. We present a case of in vivo emergence of pandrug-resistant A baumannii that was successfully treated with the compassionate use of investigational sulbactam-durlobactam-based antibiotic regimen. We also performed a longitudinal genomic analysis of the bacterial isolates and showed the development of resistance and genetic mutations over time.
ABSTRACT
Acinetobacter baumannii-calcoaceticus complex (ABC) causes severe infections that are difficult to treat due to pre-existing antibiotic resistance. Sulbactam-durlobactam (SUL-DUR) is a targeted ß-lactam/ß-lactamase inhibitor combination antibiotic designed to treat serious infections caused by Acinetobacter, including multidrug- and carbapenem-resistant strains. In a recent global surveillance study of 5,032 ABC clinical isolates collected from 2016 to 2021, less than 2% of ABC isolates had SUL-DUR MIC values >4 µg/mL. Molecular characterization of these isolates confirmed the primary drivers of resistance are metallo-ß-lactamases or penicillin-binding protein 3 (PBP3) mutations, as previously described. In addition, this study shows that certain common PBP3 variants, such as A515V, are insufficient to confer sulbactam resistance and that the efflux of durlobactam by AdeIJK is likely to play a role in a subset of strains.
Subject(s)
Acinetobacter baumannii , Sulbactam , Sulbactam/pharmacology , Sulbactam/therapeutic use , Anti-Bacterial Agents/therapeutic use , Azabicyclo Compounds/pharmacology , beta-Lactamase Inhibitors/pharmacology , beta-Lactamase Inhibitors/therapeutic use , Monobactams , Microbial Sensitivity TestsABSTRACT
The development of new antibiotics to treat infections caused by drug-resistant Gram-negative pathogens is of paramount importance as antibiotic resistance continues to increase worldwide1. Here we describe a strategy for the rational design of diazabicyclooctane inhibitors of penicillin-binding proteins from Gram-negative bacteria to overcome multiple mechanisms of resistance, including ß-lactamase enzymes, stringent response and outer membrane permeation. Diazabicyclooctane inhibitors retain activity in the presence of ß-lactamases, the primary resistance mechanism associated with ß-lactam therapy in Gram-negative bacteria2,3. Although the target spectrum of an initial lead was successfully re-engineered to gain in vivo efficacy, its ability to permeate across bacterial outer membranes was insufficient for further development. Notably, the features that enhanced target potency were found to preclude compound uptake. An improved optimization strategy leveraged porin permeation properties concomitant with biochemical potency in the lead-optimization stage. This resulted in ETX0462, which has potent in vitro and in vivo activity against Pseudomonas aeruginosa plus all other Gram-negative ESKAPE pathogens, Stenotrophomonas maltophilia and biothreat pathogens. These attributes, along with a favourable preclinical safety profile, hold promise for the successful clinical development of the first novel Gram-negative chemotype to treat life-threatening antibiotic-resistant infections in more than 25 years.
Subject(s)
Anti-Bacterial Agents/pharmacology , Drug Design , Drug Resistance, Multiple, Bacterial , Gram-Negative Bacteria/drug effects , Animals , Anti-Bacterial Agents/chemistry , Aza Compounds/chemistry , Aza Compounds/pharmacology , Cyclooctanes/chemistry , Cyclooctanes/pharmacology , Female , Mice , Mice, Inbred BALB C , Molecular Structure , Penicillin-Binding Proteins/antagonists & inhibitors , Pseudomonas aeruginosa/drug effects , beta-LactamasesABSTRACT
Durlobactam is a new member of the diazabicyclooctane class of ß-lactamase inhibitors with broad spectrum activity against Ambler class A, C, and D serine ß-lactamases. Sulbactam is a first generation ß-lactamase inhibitor with activity limited to a subset of class A enzymes that also has direct-acting antibacterial activity against Acinetobacter spp. The latter feature is due to sulbactam's ability to inhibit certain penicillin-binding proteins, essential enzymes involved in bacterial cell wall synthesis in this pathogen. Because sulbactam is also susceptible to cleavage by numerous ß-lactamases, its clinical utility for the treatment of contemporary Acinetobacter infections is quite limited. However, when combined with durlobactam, the activity of sulbactam is effectively restored against these notoriously multidrug-resistant strains. This sulbactam-durlobactam combination is currently in late-stage development for the treatment of Acinectobacter infections, including those caused by carbapenem-resistant isolates, for which there is a high unmet medical need. The following mini-review summarizes the molecular drivers of efficacy of this combination against this troublesome pathogen, with an emphasis on the biochemical features of each partner.
ABSTRACT
Mutations in KPC-2 and KPC-3 ß-lactamase can confer resistance to the ß-lactam/ß-lactamase inhibitor antibacterial intravenous drug combination ceftazidime-avibactam, introduced in 2015. Avibactam was the first of the diazabicyclooctane class of non-ß-lactam ß-lactamase inhibitors to be approved for clinical use. The orally bioavailable prodrug ETX0282 of the diazabicyclooctane ß-lactamase inhibitor ETX1317 is in clinical development in combination with the oral ß-lactam prodrug cefpodoxime proxetil for use against complicated urinary tract infections. We investigated the effects of 3 ceftazidime-avibactam resistance mutations in KPC-3 (V240G, D179Y, and D179Y/T243M) on the ability of ETX1317 to overcome KPC-3-induced cefpodoxime resistance. Isogenic Escherichia coli strains, each expressing the wild-type or a mutant KPC-3 at similar levels, retained susceptibility to cefpodoxime-ETX1317 (1:2) with essentially identical minimal inhibitory concentrations of 0.125-0.25 µg/mL cefpodoxime. The KPC-3 mutations had little or no effect on the kinact/Ki values for inhibition by each of 3 diazabicyclooctanes: avibactam, durlobactam (ETX2514), and ETX1317. The KM values for hydrolysis of cefpodoxime were similar for all 4 variants, but the kcat values of the D179Y and D179Y/T243M variants were much lower than those of the wild-type and V240G mutant enzymes. All 4 KPC-3 variants formed stable, reversibly covalent complexes with ETX1317, but dissociation of ETX1317 was much slower from the D179Y and D179Y/T243M mutants than from the wild-type and V240G mutant enzymes. Thus, the KPC-3 variants examined here that cause resistance to ceftazidime-avibactam do not cause resistance to cefpodoxime-ETX1317.
Subject(s)
Azabicyclo Compounds , beta-Lactamases , Azabicyclo Compounds/pharmacology , Ceftazidime , Ceftizoxime/analogs & derivatives , Drug Combinations , Mutation , beta-Lactamases/genetics , CefpodoximeABSTRACT
UDP-3-O-(R-3-hydroxyacyl)-N-acetylglucosamine deacetylase (LpxC), the zinc metalloenzyme catalyzing the first committed step of lipid A biosynthesis in Gram-negative bacteria, has been a target for antibacterial drug discovery for many years. All inhibitor chemotypes reaching an advanced preclinical stage and clinical phase 1 have contained terminal hydroxamic acid, and none have been successfully advanced due, in part, to safety concerns, including hemodynamic effects. We hypothesized that the safety of LpxC inhibitors could be improved by replacing the terminal hydroxamic acid with a different zinc-binding group. After choosing an N-hydroxyformamide zinc-binding group, we investigated the structure-activity relationship of each part of the inhibitor scaffold with respect to Pseudomonas aeruginosa and Escherichia coli LpxC binding affinity, in vitro antibacterial potency and pharmacological properties. We identified a novel, potency-enhancing hydrophobic binding interaction for an LpxC inhibitor. We demonstrated in vivo efficacy of one compound in a neutropenic mouse E. coli infection model. Another compound was tested in a rat hemodynamic assay and was found to have a hypotensive effect. This result demonstrated that replacing the terminal hydroxamic acid with a different zinc-binding group was insufficient to avoid this previously recognized safety issue with LpxC inhibitors.
Subject(s)
Amidohydrolases/antagonists & inhibitors , Anti-Bacterial Agents/pharmacology , Enzyme Inhibitors/chemistry , Formamides/chemistry , Hemodynamics/drug effects , Amidohydrolases/metabolism , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/therapeutic use , Binding Sites , Crystallography, X-Ray , Disease Models, Animal , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Escherichia coli/drug effects , Escherichia coli Infections/drug therapy , Escherichia coli Infections/pathology , Female , Formamides/metabolism , Formamides/pharmacology , Formamides/therapeutic use , Half-Life , Male , Mice , Molecular Dynamics Simulation , Rats , Rats, Sprague-Dawley , Structure-Activity RelationshipABSTRACT
Optimal phage propagation depends on the regulation of the lysis of the infected host cell. In T4 phage infection, lysis occurs when the holin protein (T) forms lesions in the host membrane. However, the lethal function of T can be blocked by an antiholin (RI) during lysis inhibition (LIN). LIN sets if the infected cell undergoes superinfection, then the lysis is delayed until host/phage ratio becomes more favorable for the release of progeny. It has been thought that a signal derived from the superinfection is required to activate RI. Here we report structures that suggest a radically different model in which RI binds to T irrespective of superinfection, causing it to accumulate in a membrane as heterotetrameric 2RI-2T complex. Moreover, we show the complex binds non-specifically to DNA, suggesting that the gDNA from the superinfecting phage serves as the LIN signal and that stabilization of the complex by DNA binding is what defines LIN. Finally, we show that soluble domain of free RI crystallizes in a domain-swapped homotetramer, which likely works as a sink for RI molecules released from the RI-T complex to ensure efficient lysis. These results constitute the first structural basis and a new model not only for the historic LIN phenomenon but also for the temporal regulation of phage lysis in general.
Subject(s)
Bacteriophage T4/physiology , DNA, Viral/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolism , Bacterial Physiological Phenomena , Bacteriolysis , Cell Membrane/metabolism , Cryoelectron Microscopy , Crystallography, X-Ray , Models, Molecular , Protein Conformation , Protein DomainsABSTRACT
Multi-drug-resistant Enterobacteriales expressing a wide array of ß-lactamases are emerging as a global health threat in both hospitals and communities. Although several intravenous drugs have recently been approved to address this need, there are no oral Gram-negative agents that are both safe and broadly effective against such pathogens. The lack of an effective oral agent is of concern for common infections which could otherwise be treated in the community but, due to antibiotic resistance, require hospitalization to allow for intravenous therapy. ETX1317 is a novel, broad spectrum, serine ß-lactamase inhibitor of the diazabicyclooctane class that restores the antibacterial activity of multiple ß-lactams against multiple species of multi-drug-resistant Enterobacteriales, including carbapenem-resistant strains. A combination of its oral prodrug, ETX0282, and the oral prodrug of a third-generation cephalosporin, cefpodoxime proxetil, is currently in clinical development. This report describes the biochemical and microbiological properties of ETX1317, which is more potent and demonstrates a greater breadth of inhibition than avibactam, the parenteral prototype of this class of ß-lactamase inhibitors.
Subject(s)
Pharmaceutical Preparations , beta-Lactamase Inhibitors , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Carbapenems , beta-Lactamase Inhibitors/pharmacology , beta-LactamsABSTRACT
Acinetobacter baumannii-calcoaceticus complex (ABC) organisms cause severe infections that are difficult to treat due to preexisting antibiotic resistance. Sulbactam-durlobactam (formerly sulbactam-ETX2514) (SUL-DUR) is a ß-lactam-ß-lactamase inhibitor combination antibiotic designed to treat serious infections caused by ABC organisms, including multidrug-resistant (MDR) strains. The in vitro antibacterial activities of SUL-DUR and comparator agents were determined by broth microdilution against 1,722 clinical isolates of ABC organisms collected in 2016 and 2017 from 31 countries across Asia/South Pacific, Europe, Latin America, the Middle East, and North America. Over 50% of these isolates were resistant to carbapenems. Against this collection of global isolates, SUL-DUR had a MIC50/MIC90 of 1/2 µg/ml compared to a MIC50/MIC90 of 8/64 µg/ml for sulbactam alone. This level of activity was found to be consistent across organisms, regions, sources of infection, and subsets of resistance phenotypes, including MDR and extensively drug-resistant isolates. The SUL-DUR activity was superior to those of the tested comparators, with only colistin having similar potency. Whole-genome sequencing of the 39 isolates (2.3%) with a SUL-DUR MIC of >4 µg/ml revealed that these strains encoded either the metallo-ß-lactamase NDM-1, which durlobactam does not inhibit, or single amino acid substitutions near the active site of penicillin binding protein 3 (PBP3), the primary target of sulbactam. In summary, SUL-DUR demonstrated potent antibacterial activity against recent, geographically diverse clinical isolates of ABC organisms, including MDR isolates.
Subject(s)
Acinetobacter Infections/microbiology , Acinetobacter/drug effects , Anti-Bacterial Agents/pharmacology , Azabicyclo Compounds/pharmacology , Sulbactam/pharmacology , Acinetobacter/genetics , Acinetobacter baumannii/drug effects , Acinetobacter baumannii/isolation & purification , Community-Acquired Infections/microbiology , Drug Combinations , Drug Resistance, Bacterial/drug effects , Drug Resistance, Bacterial/genetics , Humans , Microbial Sensitivity Tests , Whole Genome SequencingABSTRACT
Multidrug-resistant (MDR) Acinetobacter spp. poses a significant therapeutic challenge in part due to the presence of chromosomally encoded ß-lactamases, including class C Acinetobacter-derived cephalosporinases (ADC) and class D oxacillinases (OXA), as well as plasmid-mediated class A ß-lactamases. Importantly, OXA-like ß-lactamases represent a gap in the spectrum of inhibition by recently approved ß-lactamase inhibitors such as avibactam and vaborbactam. ETX2514 is a novel, rationally designed, diazabicyclooctenone inhibitor that effectively targets class A, C, and D ß-lactamases. We show that addition of ETX2514 significantly increased the susceptibility of clinical Acinetobacterbaumannii isolates to sulbactam. AdeB and AdeJ were identified to be key efflux constituents for ETX2514 in A. baumannii The combination of sulbactam and ETX2514 was efficacious against A. baumannii carrying blaTEM-1, blaADC-82, blaOXA-23, and blaOXA-66 in a neutropenic murine thigh infection model. We also show that, in vitro, ETX2514 inhibited ADC-7 (k2/Ki 1.0 ± 0.1 × 106 M-1 s-1) and OXA-58 (k2/Ki 2.5 ± 0.3 × 105 M-1 s-1). Cocrystallization of ETX2514 with OXA-24/40 revealed hydrogen bonding interactions between ETX2514 and residues R261, S219, and S128 of OXA-24/40 in addition to a chloride ion occupied in the active site. Further, the C3 methyl group of ETX2514 shifts the position of M223. In conclusion, the sulbactam-ETX2514 combination possesses a broadened inhibitory range to include class D ß-lactamases as well as class A and C ß-lactamases and is a promising therapeutic candidate for infections caused by MDR Acinetobacter spp.IMPORTANCE The number and diversity of ß-lactamases are steadily increasing. The emergence of ß-lactamases that hydrolyze carbapenems poses a significant threat to our antibiotic armamentarium. The explosion of OXA enzymes that are carbapenem hydrolyzers is a major challenge (carbapenem-hydrolyzing class D [CHD]). An urgent need exists to discover ß-lactamase inhibitors with class D activity. The sulbactam-ETX2514 combination demonstrates the potential to become a treatment regimen of choice for Acinetobacter spp. producing class D ß-lactamases.
Subject(s)
Acinetobacter Infections/drug therapy , Acinetobacter baumannii/drug effects , Anti-Bacterial Agents/administration & dosage , Azabicyclo Compounds/administration & dosage , Sulbactam/administration & dosage , beta-Lactamase Inhibitors/administration & dosage , Acinetobacter Infections/microbiology , Animals , Anti-Bacterial Agents/pharmacology , Azabicyclo Compounds/pharmacology , Crystallography, X-Ray , Disease Models, Animal , Mice , Protein Binding , Protein Conformation , Sulbactam/pharmacology , Treatment Outcome , beta-Lactamase Inhibitors/pharmacology , beta-Lactamases/chemistry , beta-Lactamases/metabolismABSTRACT
The susceptibility of small molecules to Gram-negative bacterial efflux is typically evaluated using an antibacterial activity-based efflux ratio, which is computed as the ratio of the antibacterial activity for a wild-type strain and its isogenic efflux mutant (typically lacking genes encoding major efflux pumps). The magnitude of the ratio is often used as an efflux index. However, early in drug discovery, hits with suboptimal physicochemical properties often lack whole cell inhibition against wild-type strains, which makes efflux ratios indeterminable. To address this gap, we developed an assay to titrate levels of total efflux by varying the TolC expression using an arabinose-inducible promoter (pBAD) in an Escherichia coli Δ tolC strain. We provide a proof of concept for the assay using sets of related compounds from two antibiotic classes and show that the TolC titration provides a sensitive method for rank ordering compounds with respect to their efflux susceptibility.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Outer Membrane Proteins/analysis , Escherichia coli Proteins/analysis , Escherichia coli/chemistry , Membrane Transport Proteins/analysis , Arabinose/chemistry , Bacterial Outer Membrane Proteins/genetics , Drug Discovery , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Membrane Transport Proteins/genetics , Microbial Sensitivity Tests , Promoter Regions, Genetic , Proof of Concept StudyABSTRACT
The major Gram-negative gated efflux channel TolC has been extensively characterized in Escherichia coli but there is minimal information about Klebsiella pneumoniae TolC. Using an arabinose-inducible plasmid-based expression system, we show that the K. pneumoniae TolC complements the efflux defect in an E. coli K-12 ΔtolC strain, restoring wild-type levels of resistance towards most antibiotics suggesting that it can interact with the E. coli AcrB efflux pump. We characterize the efflux properties of K. pneumoniae TolC using an orthogonal whole cell-based assay and quantify the extrusion of environment-sensitive fluorescent probes and contrast the findings with the E. coli ortholog.
Subject(s)
Anti-Bacterial Agents/metabolism , Bacterial Outer Membrane Proteins/metabolism , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/metabolism , Membrane Transport Proteins/metabolism , Bacterial Outer Membrane Proteins/genetics , Escherichia coli Proteins , Membrane Transport Proteins/geneticsABSTRACT
The novel diazabicyclooctenone ETX2514 is a potent, broad-spectrum serine ß-lactamase inhibitor that restores sulbactam activity against resistant Acinetobacter baumannii The frequency of spontaneous resistance to sulbactam-ETX2514 in clinical isolates was found to be 7.6 × 10-10 to <9.0 × 10-10 at 4× MIC and mapped to residues near the active site of penicillin binding protein 3 (PBP3). Purified mutant PBP3 proteins demonstrated reduced affinity for sulbactam. In a sulbactam-sensitive isolate, resistance also mapped to stringent response genes associated with resistance to PBP2 inhibitors, suggesting that in addition to ß-lactamase inhibition, ETX2514 may enhance sulbactam activity in A. baumannii via inhibition of PBP2.
Subject(s)
Acinetobacter baumannii/drug effects , Anti-Bacterial Agents/pharmacology , Azabicyclo Compounds/pharmacology , Drug Resistance, Multiple, Bacterial/drug effects , Gene Expression Regulation, Bacterial , Sulbactam/pharmacology , beta-Lactamase Inhibitors/pharmacology , Acinetobacter Infections/microbiology , Acinetobacter baumannii/genetics , Acinetobacter baumannii/isolation & purification , Acinetobacter baumannii/metabolism , Binding Sites , Drug Resistance, Multiple, Bacterial/genetics , Drug Therapy, Combination , Humans , Microbial Sensitivity Tests , Models, Molecular , Mutation , Penicillin-Binding Proteins/antagonists & inhibitors , Penicillin-Binding Proteins/chemistry , Penicillin-Binding Proteins/genetics , Penicillin-Binding Proteins/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/genetics , Protein Isoforms/metabolism , beta-Lactamases/genetics , beta-Lactamases/metabolismABSTRACT
OmpAAb is a conserved, abundantly expressed outer membrane porin in Acinetobacter baumannii whose presumed role in antibiotic permeation has not been clearly demonstrated. In this report, we use a titratable heterologous expression system to express OmpAAb in isolation and demonstrate selective passage of small molecule antibiotics through OmpAAb. ETX2514, a recently discovered broad-spectrum ß-lactamase inhibitor, in combination with sulbactam, is currently in clinical testing for the treatment of drug-resistant A. baumannii infections. We demonstrate that ETX2514 permeates OmpAAb and potentiates the activity of sulbactam in an OmpAAb-dependent manner. In addition, we show that small modifications in the structure of ETX2514 differentially affect its passage through OmpAAb, revealing unique structure-porin-permeation relationships. Finally, we confirm the contribution of OmpAAb to bacterial fitness using a murine thigh model of A. baumannii infection. These results, combined with the high sequence homology of OmpA across Acinetobacter spp., suggest that optimization of antibiotic entry through OmpAAb may prove to be a feasible medicinal chemistry design strategy for future antibacterial discovery efforts.
Subject(s)
Acinetobacter baumannii/enzymology , Acinetobacter baumannii/metabolism , Anti-Bacterial Agents/metabolism , Bacterial Outer Membrane Proteins/metabolism , Porins/metabolism , Acinetobacter Infections/microbiology , Acinetobacter baumannii/physiology , Animals , Azabicyclo Compounds/metabolism , Biological Transport , Disease Models, Animal , Genetic Fitness , Mice , Structure-Activity Relationship , Sulbactam/metabolismABSTRACT
Multidrug-resistant (MDR) bacterial infections are a serious threat to public health. Among the most alarming resistance trends is the rapid rise in the number and diversity of ß-lactamases, enzymes that inactivate ß-lactams, a class of antibiotics that has been a therapeutic mainstay for decades. Although several new ß-lactamase inhibitors have been approved or are in clinical trials, their spectra of activity do not address MDR pathogens such as Acinetobacter baumannii. This report describes the rational design and characterization of expanded-spectrum serine ß-lactamase inhibitors that potently inhibit clinically relevant class A, C and D ß-lactamases and penicillin-binding proteins, resulting in intrinsic antibacterial activity against Enterobacteriaceae and restoration of ß-lactam activity in a broad range of MDR Gram-negative pathogens. One of the most promising combinations is sulbactam-ETX2514, whose potent antibacterial activity, in vivo efficacy against MDR A. baumannii infections and promising preclinical safety demonstrate its potential to address this significant unmet medical need.
Subject(s)
Acinetobacter baumannii/drug effects , Azabicyclo Compounds/chemistry , Azabicyclo Compounds/pharmacology , Gram-Negative Bacteria/drug effects , beta-Lactamase Inhibitors/chemistry , beta-Lactamase Inhibitors/pharmacology , Acinetobacter Infections/drug therapy , Acinetobacter Infections/microbiology , Animals , Azabicyclo Compounds/therapeutic use , Azabicyclo Compounds/toxicity , Carbapenems/pharmacology , Dogs , Drug Design , Drug Evaluation, Preclinical , Drug Resistance, Multiple, Bacterial , Enterobacteriaceae/drug effects , Gram-Negative Bacterial Infections/drug therapy , Humans , Mice , Models, Molecular , Penicillin-Binding Proteins/antagonists & inhibitors , Rats , Sulbactam/chemistry , Sulbactam/pharmacology , beta-Lactamase Inhibitors/therapeutic use , beta-Lactamase Inhibitors/toxicity , beta-Lactamases/metabolism , beta-Lactams/pharmacologyABSTRACT
The majority of bacterial proteins are dispensable for growth in the laboratory but nevertheless have important physiological roles. There are no systematic approaches to identify cell-permeable small-molecule inhibitors of these proteins. We demonstrate a strategy to identify such inhibitors that exploits synthetic lethal relationships both for small-molecule discovery and for target identification. Applying this strategy in Staphylococcus aureus, we have identified a compound that inhibits DltB, a component of the teichoic acid D-alanylation machinery that has been implicated in virulence. This D-alanylation inhibitor sensitizes S. aureus to aminoglycosides and cationic peptides and is lethal in combination with a wall teichoic acid inhibitor. We conclude that DltB is a druggable target in the D-alanylation pathway. More broadly, the work described demonstrates a systematic method to identify biologically active inhibitors of major bacterial processes that can be adapted to numerous organisms.
Subject(s)
Amsacrine/pharmacology , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Drug Evaluation, Preclinical/methods , Staphylococcus aureus/drug effects , Aminoglycosides/pharmacology , Amsacrine/chemistry , Anti-Bacterial Agents/chemistry , Antimicrobial Cationic Peptides/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Wall/metabolism , High-Throughput Screening Assays/methods , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Microbial Sensitivity Tests , Mutation , Small Molecule Libraries/pharmacology , Staphylococcus aureus/genetics , Staphylococcus aureus/metabolism , Staphylococcus aureus/pathogenicity , Teichoic Acids/metabolismABSTRACT
BACKGROUND: Staphylococcus aureus readily develops resistance to antibiotics and achieving effective therapies to overcome resistance requires in-depth understanding of S. aureus biology. High throughput, parallel-sequencing methods for analyzing transposon mutant libraries have the potential to revolutionize studies of S. aureus, but the genetic tools to take advantage of the power of next generation sequencing have not been fully developed. RESULTS: Here we report a phage-based transposition system to make ultra-high density transposon libraries for genome-wide analysis of mutant fitness in any Φ11-transducible S. aureus strain. The high efficiency of the delivery system has made it possible to multiplex transposon cassettes containing different regulatory elements in order to make libraries in which genes are over- or under-expressed as well as deleted. By incorporating transposon-specific barcodes into the cassettes, we can evaluate how null mutations and changes in gene expression levels affect fitness in a single sequencing data set. Demonstrating the power of the system, we have prepared a library containing more than 690,000 unique insertions. Because one unique feature of the phage-based approach is that temperature-sensitive mutants are retained, we have carried out a genome-wide study of S. aureus genes involved in withstanding temperature stress. We find that many genes previously identified as essential are temperature sensitive and also identify a number of genes that, when disrupted, confer a growth advantage at elevated temperatures. CONCLUSIONS: The platform described here reliably provides mutant collections of unparalleled genotypic diversity and will enable a wide range of functional genomic studies in S. aureus.
Subject(s)
Bacteriophages/genetics , DNA Transposable Elements , Gene Library , Genetic Vectors/metabolism , Staphylococcus aureus/genetics , Gene Expression , Genes, Essential , High-Throughput Nucleotide Sequencing , Mutation , Sequence Analysis, DNA , Staphylococcus aureus/growth & development , Staphylococcus aureus/metabolism , TemperatureABSTRACT
Staphylococcus aureus contains two distinct teichoic acid (TA) polymers, lipoteichoic acid (LTA) and wall teichoic acid (WTA), which are proposed to play redundant roles in regulating cell division. To gain insight into the underlying biology of S. aureus TAs, we used a small molecule inhibitor to screen a highly saturated transposon library for cellular factors that become essential when WTA is depleted. We constructed an interaction network connecting WTAs with genes involved in LTA synthesis, peptidoglycan synthesis, surface protein display, and D-alanine cell envelope modifications. Although LTAs and WTAs are synthetically lethal, we report that they do not have the same synthetic interactions with other cell envelope genes. For example, D-alanylation, a tailoring modification of both WTAs and LTAs, becomes essential when the former, but not the latter, are removed. Therefore, D-alanine-tailored LTAs are required for survival when WTAs are absent. Examination of terminal phenotoypes led to the unexpected discovery that cells lacking both LTAs and WTAs lose their ability to form Z rings and can no longer divide. We have concluded that the presence of either LTAs or WTAs on the cell surface is required for initiation of S. aureus cell division, but these polymers act as part of distinct cellular networks.