ABSTRACT
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
Many drug candidates fail in clinical trials owing to a lack of efficacy from limited target engagement or an insufficient therapeutic index. Minimizing off-target effects while retaining the desired pharmacodynamic (PD) response can be achieved by reduced exposure for drugs that display kinetic selectivity in which the drug-target complex has a longer half-life than off-target-drug complexes. However, though slow-binding inhibition kinetics are a key feature of many marketed drugs, prospective tools that integrate drug-target residence time into predictions of drug efficacy are lacking, hindering the integration of drug-target kinetics into the drug discovery cascade. Here we describe a mechanistic PD model that includes drug-target kinetic parameters, including the on- and off-rates for the formation and breakdown of the drug-target complex. We demonstrate the utility of this model by using it to predict dose response curves for inhibitors of the LpxC enzyme from Pseudomonas aeruginosa in an animal model of infection.
Subject(s)
Amidohydrolases/antagonists & inhibitors , Anti-Bacterial Agents/pharmacology , Enzyme Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Threonine/analogs & derivatives , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacokinetics , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacokinetics , Kinetics , Mice, Inbred Strains , Microbial Sensitivity Tests , Models, Biological , Molecular Structure , Protein Binding , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/enzymology , Threonine/chemistry , Threonine/pharmacokinetics , Threonine/pharmacology , Time FactorsABSTRACT
AZD0914 is a new spiropyrimidinetrione bacterial DNA gyrase/topoisomerase inhibitor with potent in vitro antibacterial activity against key Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus agalactiae), fastidious Gram-negative (Haemophilus influenzae and Neisseria gonorrhoeae), atypical (Legionella pneumophila), and anaerobic (Clostridium difficile) bacterial species, including isolates with known resistance to fluoroquinolones. AZD0914 works via inhibition of DNA biosynthesis and accumulation of double-strand cleavages; this mechanism of inhibition differs from those of other marketed antibacterial compounds. AZD0914 stabilizes and arrests the cleaved covalent complex of gyrase with double-strand broken DNA under permissive conditions and thus blocks religation of the double-strand cleaved DNA to form fused circular DNA. Whereas this mechanism is similar to that seen with fluoroquinolones, it is mechanistically distinct. AZD0914 exhibited low frequencies of spontaneous resistance in S. aureus, and if mutants were obtained, the mutations mapped to gyrB. Additionally, no cross-resistance was observed for AZD0914 against recent bacterial clinical isolates demonstrating resistance to fluoroquinolones or other drug classes, including macrolides, ß-lactams, glycopeptides, and oxazolidinones. AZD0914 was bactericidal in both minimum bactericidal concentration and in vitro time-kill studies. In in vitro checkerboard/synergy testing with 17 comparator antibacterials, only additivity/indifference was observed. The potent in vitro antibacterial activity (including activity against fluoroquinolone-resistant isolates), low frequency of resistance, lack of cross-resistance, and bactericidal activity of AZD0914 support its continued development.
Subject(s)
Anti-Bacterial Agents/pharmacology , Barbiturates/pharmacology , DNA Gyrase/drug effects , Nucleic Acid Synthesis Inhibitors/pharmacology , Spiro Compounds/pharmacology , Topoisomerase II Inhibitors/pharmacology , Atypical Bacterial Forms/drug effects , Drug Resistance, Bacterial , Fluoroquinolones/pharmacology , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Isoxazoles , Microbial Sensitivity Tests , Morpholines , OxazolidinonesABSTRACT
OBJECTIVES: The spread of NDM-1 amongst Enterobacteriaceae has highlighted a significant threat to the clinical management of serious infections. The combination of aztreonam and avibactam, a non-ß-lactam ß-lactamase inhibitor, may provide a much-needed therapeutic alternative. This combination was potent against most NDM-containing Enterobacteriaceae, although activity was diminished against many Escherichia coli isolates. These E. coli isolates were characterized to elucidate the mechanism of decreased susceptibility to aztreonam/avibactam. METHODS: MIC determinations were performed using broth microdilution, and whole-genome sequencing was performed to enable sequence-based analyses. RESULTS: The decreased susceptibility was not due to avibactam being unable to inhibit the serine ß-lactamases found in the E. coli isolates. Rather, it was manifested by a four-amino-acid insertion in PBP3. This same insertion was also found in non-NDM-containing E. coli that had reduced susceptibility to aztreonam/avibactam. Construction of an isogenic mutant confirmed that this insertion resulted in decreased susceptibility to aztreonam and several cephalosporins, but had no impact on carbapenem potency. Structural analysis suggests that this insertion will impact the accessibility of the ß-lactam drugs to the transpeptidase pocket of PBP3. CONCLUSIONS: The acquisition of ß-lactamases is the predominant mechanism of ß-lactam resistance in Enterobacteriaceae. We have demonstrated that small PBP3 changes will affect the susceptibility to a broad range of ß-lactams. These changes were identified in multiple MLST lineages of E. coli, and were enriched in NDM-containing isolates. However, they were not present in other key species of Enterobacteriaceae despite significant conservation among the PBP3 proteins.
Subject(s)
Anti-Bacterial Agents/pharmacology , Azabicyclo Compounds/pharmacology , Aztreonam/pharmacology , Escherichia coli/drug effects , Penicillin-Binding Proteins/genetics , beta-Lactam Resistance , beta-Lactamases/metabolism , Computational Biology , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli/isolation & purification , Escherichia coli Infections/microbiology , Genome, Bacterial , Humans , Microbial Sensitivity Tests , Mutagenesis, Insertional , Sequence Analysis, DNAABSTRACT
OBJECTIVES: Pseudomonas aeruginosa is an important nosocomial pathogen that can cause a wide range of infections resulting in significant morbidity and mortality. Avibactam, a novel non-ß-lactam ß-lactamase inhibitor, is being developed in combination with ceftazidime and has the potential to be a valuable addition to the treatment options for the infectious diseases practitioner. We compared the frequency of resistance development to ceftazidime/avibactam in three P. aeruginosa strains that carried derepressed ampC alleles. METHODS: The strains were incubated in the presence of increasing concentrations of ceftazidime with a fixed concentration (4 mg/L) of avibactam to calculate the frequency of spontaneous resistance. The mutants were characterized by WGS to identify the underlying mechanism of resistance. A representative mutant protein was characterized biochemically. RESULTS: The resistance frequency was very low in all strains. The resistant variants isolated exhibited ceftazidime/avibactam MIC values that ranged from 64 to 256 mg/L. All of the mutants exhibited changes in the chromosomal ampC gene, the majority of which were deletions of various sizes in the Ω-loop region of AmpC. The mutant enzyme that carried the smallest Ω-loop deletion, which formed a part of the avibactam-binding pocket, was characterized biochemically and found to be less effectively inhibited by avibactam as well as exhibiting increased hydrolysis of ceftazidime. CONCLUSIONS: The development of high-level resistance to ceftazidime/avibactam appears to occur at low frequency, but structural modifications in AmpC can occur that impact the ability of avibactam to inhibit the enzyme and thereby protect ceftazidime from hydrolysis.
Subject(s)
Anti-Bacterial Agents/pharmacology , Azabicyclo Compounds/pharmacology , Bacterial Proteins/biosynthesis , Ceftazidime/pharmacology , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/enzymology , Selection, Genetic , beta-Lactam Resistance , beta-Lactamases/biosynthesis , Bacterial Proteins/genetics , Drug Combinations , Humans , Microbial Sensitivity Tests , Mutation Rate , Pseudomonas aeruginosa/genetics , beta-Lactamases/geneticsABSTRACT
Two novel compounds, pyridopyrimidines (1) and naphthyridines (2) were identified as potent inhibitors of bacterial NAD(+)-dependent DNA ligase (Lig) A in a fragment screening. SAR was guided by molecular modeling and X-ray crystallography. It was observed that the diaminonitrile pharmacophore made a key interaction with the ligase enzyme, specifically residues Glu114, Lys291, and Leu117. Synthetic challenges limited opportunities for diversification of the naphthyridine core, therefore most of the SAR was focused on a pyridopyrimidine scaffold. The initial diversification at R(1) improved both enzyme and cell potency. Further SAR developed at the R(2) position using the Negishi cross-coupling reaction provided several compounds, among these compounds 22g showed good enzyme potency and cellular potency.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , DNA Ligases/antagonists & inhibitors , NAD/metabolism , Naphthyridines/pharmacology , Pyrimidines/pharmacology , Anti-Bacterial Agents/chemical synthesis , Bacterial Proteins/chemistry , DNA Ligases/chemistry , Haemophilus influenzae/drug effects , Microbial Sensitivity Tests , Naphthyridines/chemical synthesis , Pyrimidines/chemical synthesis , Staphylococcus aureus/drug effects , Streptococcus pneumoniae/drug effects , Structure-Activity RelationshipABSTRACT
The need for new antibiotics that address serious Gram-negative infections is well recognized. Our efforts with a series of novel bacterial type II topoisomerase inhibitors (NBTIs) led to the discovery of NBTI 5463, an agent with improved activity over other NBTIs against Gram-negative bacteria, in particular against Pseudomonas aeruginosa (F. Reck, D. E. Ehmann, T. J. Dougherty, J. V. Newman, S. Hopkins, G. Stone, N. Agrawal, P. Ciaccio, J. McNulty, H. Barthlow, J. O'Donnell, K. Goteti, J. Breen, J. Comita-Prevoir, M. Cornebise, M. Cronin, C. J. Eyermann, B. Geng, G. R. Carr, L. Pandarinathan, X. Tang, A. Cottone, L. Zhao, N. Bezdenejnih-Snyder, submitted for publication). In the present work, NBTI 5463 demonstrated promising activity against a broad range of Gram-negative pathogens. In contrast to fluoroquinolones, the compound did not form a double-strand DNA cleavable complex with Escherichia coli DNA gyrase and DNA, but it was a potent inhibitor of both DNA gyrase and E. coli topoisomerase IV catalytic activities. In studies with P. aeruginosa, NBTI 5463 was bactericidal. Resistant mutants arose at a low rate, and the mutations were found exclusively in the nfxB gene, a regulator of the MexCD-OprJ efflux system. Levofloxacin-selected resistance mutations in GyrA did not result in decreased susceptibility to NBTI 5463. Animal infection studies demonstrated that NBTI 5463 was efficacious in mouse models of lung, thigh, and ascending urinary tract infections.
Subject(s)
Anti-Bacterial Agents/pharmacology , Morpholines/pharmacology , Naphthyridines/pharmacology , Topoisomerase II Inhibitors/pharmacology , DNA Gyrase/metabolism , DNA Topoisomerase IV/metabolism , Escherichia coli/drug effects , Escherichia coli/enzymology , Fluoroquinolones/pharmacology , Gram-Negative Bacteria/drug effects , Microbial Sensitivity TestsABSTRACT
BACKGROUND: Extended-spectrum AmpC (ESAC) ß-lactamase enzymes, which are either chromosomally encoded or plasmid encoded, have minor structural changes that broaden their substrate hydrolysis profile. The derepressed AmpC enzyme found once in Enterobacter cloacae CHE was shown to contain a six residue deletion in the H-10 helix in close proximity to the active site. Avibactam is a non-ß-lactam inhibitor of Ambler class A, class C and some class D ß-lactamases that is in clinical development with several ß-lactam agents. It has been shown to inhibit AmpC enzymes, but its microbiological activity against isolates carrying different ESAC enzymes is less well understood. METHODS: MICs were determined using the broth microdilution technique. RT-PCR analyses were performed to measure the level of ampC expression and whole genome sequencing was performed to enable sequence-based analyses. RESULTS: Structural analyses of avibactam bound to a representative AmpC ß-lactamase suggested that the H-10 helix deletion would impact the potency of the inhibitor. Under standard conditions, the ceftazidime/avibactam and ceftaroline/avibactam MIC values for E. cloacae CHE were 64 and 4 mg/L, respectively, representing a significant decrease in susceptibility over control E. cloacae isolates. However, use of higher avibactam concentrations restored the susceptibility of E. cloacae CHE in a dose-dependent manner. Comparison with other E. cloacae isolates carrying derepressed AmpC enzymes suggested that this difference in inhibition by avibactam was unrelated to the level of AmpC being produced. CONCLUSIONS: The E. cloacae CHE ESAC enzyme is inhibited less efficiently by avibactam than other E. cloacae AmpC proteins due to a subtle rearrangement of the binding site. Although the variants are not commonly observed, the different ESAC enzymes may be inhibited to varied extents by avibactam.
Subject(s)
Azabicyclo Compounds/pharmacology , Bacterial Proteins/antagonists & inhibitors , Enterobacter cloacae/drug effects , beta-Lactamase Inhibitors/pharmacology , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Enterobacter cloacae/enzymology , Enterobacter cloacae/genetics , Humans , Molecular Sequence Data , Protein Structure, Secondary , Protein Structure, Tertiary , beta-Lactamases/genetics , beta-Lactamases/metabolismABSTRACT
Inhibitors of 4'-phosphopantetheine adenylyltransferase (PPAT) were identified through high-throughput screening of the AstraZeneca compound library. One series, cycloalkyl pyrimidines, showed inhibition of PPAT isozymes from several species, with the most potent inhibition of enzymes from Gram-positive species. Mode-of-inhibition studies with Streptococcus pneumoniae and Staphylococcus aureus PPAT demonstrated representatives of this series to be reversible inhibitors competitive with phosphopantetheine and uncompetitive with ATP, binding to the enzyme-ATP complex. The potency of this series was optimized using structure-based design, and inhibition of cell growth of Gram-positive species was achieved. Mode-of-action studies, using generation of resistant mutants with targeted sequencing as well as constructs that overexpress PPAT, demonstrated that growth suppression was due to inhibition of PPAT. An effect on bacterial burden was demonstrated in mouse lung and thigh infection models, but further optimization of dosing requirements and compound properties is needed before these compounds can be considered for progress into clinical development. These studies validated PPAT as a novel target for antibacterial therapy.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Nucleotidyltransferases/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Staphylococcus aureus/drug effects , Streptococcus pneumoniae/drug effects , Animals , Anti-Bacterial Agents/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Binding, Competitive , Crystallography, X-Ray , Drug Discovery , Enzyme Inhibitors/chemistry , Female , Lung/drug effects , Lung/microbiology , Mice , Models, Molecular , Nucleotidyltransferases/chemistry , Nucleotidyltransferases/metabolism , Pantetheine/analogs & derivatives , Pantetheine/chemistry , Pneumococcal Infections/drug therapy , Pneumococcal Infections/microbiology , Pneumonia, Bacterial/drug therapy , Pneumonia, Bacterial/microbiology , Small Molecule Libraries/chemistry , Staphylococcus aureus/enzymology , Staphylococcus aureus/growth & development , Streptococcus pneumoniae/enzymology , Streptococcus pneumoniae/growth & development , Thigh/microbiologyABSTRACT
Lipopolysaccharide (LPS) biosynthesis is an attractive antibacterial target as it is both conserved and essential for the survival of key pathogenic bacteria. Lipid A is the hydrophobic anchor for LPS and a key structural component of the outer membrane of Gram-negative bacteria. Lipid A biosynthesis is performed in part by a unique zinc dependent metalloamidase, LpxC (UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase), which catalyzes the first non-reversible step in lipid A biosynthesis. The UDP portion of the LpxC substrate-binding pocket has been relatively unexplored. We have designed and evaluated a series of hydroxamate based inhibitors which explore the SAR of substitutions directed into the UDP pocket with a range of substituted α-amino acid based linkers. We also provide the first wild type structure of Pseudomonas aeruginosa LpxC which was utilized in the design of many of these analogs.
Subject(s)
Amidohydrolases/antagonists & inhibitors , Amidohydrolases/chemistry , Amino Acids, Cyclic/chemistry , Uridine Diphosphate/chemistry , Amidohydrolases/metabolism , Amino Acid Sequence , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Binding Sites , Drug Design , Hydrophobic and Hydrophilic Interactions , Hydroxamic Acids/chemical synthesis , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacology , Lipid A/antagonists & inhibitors , Lipid A/biosynthesis , Lipopolysaccharides/antagonists & inhibitors , Lipopolysaccharides/biosynthesis , Models, Molecular , Protein Conformation , Structure-Activity Relationship , Uridine Diphosphate/metabolismABSTRACT
While numerous insect studies have demonstrated the effects environmental conditions, genetic variation and other factors have on thermal resistance, often showing patterns consistent with adaptive plasticity and local adaptation, few experiments have considered the effects of multiple factors simultaneously. Here however, we have investigated the impact of sex, rearing conditions, hardening, population, and laboratory rearing period on adult heat resistance in stocks of Drosophila hydei, a cosmopolitan species that occurs across a range of climatic zones. We show that population and putative laboratory adaptation effects are larger than those associated with rearing temperature and hardening, although there was also a notable interaction between hardening and sex, in that females showed a cost of hardening that was not present in males. In separate experiments, we found that environmental effects across a generation were small and similar in magnitude to those within a generation. These findings suggest multiple sources of variation on heat resistance and place potential genetic versus environmental sources in context.
Subject(s)
Drosophila/physiology , Environment , Genetic Variation , Thermotolerance , Acclimatization , Animals , Drosophila/genetics , Drosophila/growth & development , Female , Hot Temperature , Male , Population Dynamics , Sex FactorsABSTRACT
Wild animals in urban environments are exposed to a broad range of human activities that have the potential to disturb their life history and behaviour. Wildlife responses to disturbance can range from emigration to modified behaviour, or elevated stress, but these responses are rarely evaluated in concert. We simultaneously examined population, behavioural and hormonal responses of an urban population of black swans Cygnus atratus before, during and after an annual disturbance event involving large crowds and intense noise, the Australian Formula One Grand Prix. Black swan population numbers were lowest one week before the event and rose gradually over the course of the study, peaking after the event, suggesting that the disturbance does not trigger mass emigration. We also found no difference in the proportion of time spent on key behaviours such as locomotion, foraging, resting or self-maintenance over the course of the study. However, basal and capture stress-induced corticosterone levels showed significant variation, consistent with a modest physiological response. Basal plasma corticosterone levels were highest before the event and decreased over the course of the study. Capture-induced stress levels peaked during the Grand Prix and then also declined over the remainder of the study. Our results suggest that even intensely noisy and apparently disruptive events may have relatively low measurable short-term impact on population numbers, behaviour or physiology in urban populations with apparently high tolerance to anthropogenic disturbance. Nevertheless, the potential long-term impact of such disturbance on reproductive success, individual fitness and population health will need to be carefully evaluated.