ABSTRACT
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.
Subject(s)
ATP-Binding Cassette Transporters , Acinetobacter Infections , Acinetobacter baumannii , Anti-Bacterial Agents , Bacterial Proteins , Lipopolysaccharides , Acinetobacter baumannii/drug effects , Acinetobacter baumannii/metabolism , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Animals , Lipopolysaccharides/metabolism , Lipopolysaccharides/antagonists & inhibitors , Mice , Humans , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/metabolism , ATP-Binding Cassette Transporters/antagonists & inhibitors , ATP-Binding Cassette Transporters/metabolism , Acinetobacter Infections/drug therapy , Acinetobacter Infections/microbiology , Microbial Sensitivity TestsABSTRACT
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.
Subject(s)
Acinetobacter Infections , Acinetobacter baumannii , Anti-Bacterial Agents , Bacterial Proteins , Lipopolysaccharides , Acinetobacter baumannii/drug effects , Acinetobacter baumannii/metabolism , Lipopolysaccharides/metabolism , Animals , Acinetobacter Infections/microbiology , Acinetobacter Infections/drug therapy , Mice , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/metabolism , Biological Transport , Microbial Sensitivity Tests , Humans , Cryoelectron Microscopy , Carbapenems/pharmacology , Carbapenems/metabolism , Disease Models, Animal , Female , ATP-Binding Cassette TransportersABSTRACT
The use of ß-lactam (BL) and ß-lactamase inhibitor combination to overcome BL antibiotic resistance has been validated through clinically approved drug products. However, unmet medical needs still exist for the treatment of infections caused by Gram-negative (GN) bacteria expressing metallo-ß-lactamases. Previously, we reported our effort to discover pan inhibitors of three main families in this class: IMP, VIM, and NDM. Herein, we describe our work to improve the GN coverage spectrum in combination with imipenem and relebactam. This was achieved through structure- and property-based optimization to tackle the GN cell penetration and efflux challenges. A significant discovery was made that inhibition of both VIM alleles, VIM-1 and VIM-2, is essential for broad GN coverage, especially against VIM-producing P. aeruginosa. In addition, pharmacokinetics and nonclinical safety profiles were investigated for select compounds. Key findings from this drug discovery campaign laid the foundation for further lead optimization toward identification of preclinical candidates.
Subject(s)
Anti-Bacterial Agents , beta-Lactamase Inhibitors , Humans , beta-Lactamase Inhibitors/pharmacology , beta-Lactamase Inhibitors/therapeutic use , beta-Lactamase Inhibitors/chemistry , Anti-Bacterial Agents/chemistry , Imipenem/pharmacology , beta-Lactamases , Gram-Negative Bacteria , Microbial Sensitivity TestsABSTRACT
Multidrug-resistant (MDR) Pseudomonas aeruginosa infections are a major clinical challenge. Many isolates are carbapenem resistant, which severely limits treatment options; thus, novel therapeutic combinations, such as imipenem-relebactam (IMI/REL), ceftazidime-avibactam (CAZ/AVI), ceftolozane-tazobactam (TOL/TAZO), and meropenem-vaborbactam (MEM/VAB) were developed. Here, we studied two extensively drug-resistant (XDR) P. aeruginosa isolates, collected in the United States and Mexico, that demonstrated resistance to IMI/REL. Whole-genome sequencing (WGS) showed that both isolates contained acquired GES ß-lactamases, intrinsic PDC and OXA ß-lactamases, and disruptions in the genes encoding the OprD porin, thereby inhibiting uptake of carbapenems. In one isolate (ST17), the entire C terminus of OprD deviated from the expected amino acid sequence after amino acid G388. In the other (ST309), the entire oprD gene was interrupted by an ISPa1328 insertion element after amino acid D43, rendering this porin nonfunctional. The poor inhibition by REL of the GES ß-lactamases (GES-2, -19, and -20; apparent Ki of 19 ± 2 µM, 23 ± 2 µM, and 21 ± 2 µM, respectively) within the isolates also contributed to the observed IMI/REL-resistant phenotype. Modeling of REL binding to the active site of GES-20 suggested that the acylated REL is positioned in an unstable conformation as a result of a constrained Ω-loop.
Subject(s)
Pseudomonas Infections , Pseudomonas aeruginosa , Amino Acids , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Azabicyclo Compounds/pharmacology , Azabicyclo Compounds/therapeutic use , Drug Combinations , Humans , Imipenem/pharmacology , Imipenem/therapeutic use , Microbial Sensitivity Tests , Porins/genetics , Pseudomonas Infections/drug therapy , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/metabolism , United States , beta-Lactamases/metabolismABSTRACT
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.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Outer Membrane Proteins/metabolism , Escherichia coli Proteins/antagonists & inhibitors , Escherichia coli/drug effects , Protein Multimerization/drug effects , Triazines/pharmacology , Bacterial Outer Membrane Proteins/antagonists & inhibitors , Bacterial Outer Membrane Proteins/genetics , Biological Transport/physiology , Cell Membrane/drug effects , Cell Membrane Permeability/physiology , Drug Evaluation, Preclinical , Drug Resistance, Bacterial/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Microbial Sensitivity TestsABSTRACT
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.
Subject(s)
Azabicyclo Compounds/pharmacology , Imipenem/pharmacology , Pseudomonas aeruginosa/drug effects , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/drug effects , Drug Combinations , Drug Resistance, Multiple, Bacterial/drug effects , Humans , Kinetics , Microbial Sensitivity Tests , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/enzymology , beta-Lactamases/drug effectsABSTRACT
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.
Subject(s)
Bacterial Outer Membrane Proteins , Endopeptidases , Escherichia coli Proteins , Escherichia coli , Membrane Proteins , Protein Unfolding , Transcription Factors , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Endopeptidases/genetics , Endopeptidases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Transcription Factors/metabolismABSTRACT
To combat the threat of antibiotic-resistant Gram-negative bacteria, novel agents that circumvent established resistance mechanisms are urgently needed. Our approach was to focus first on identifying bioactive small molecules followed by chemical lead prioritization and target identification. Within this annotated library of bioactives, we identified a small molecule with activity against efflux-deficient Escherichia coli and other sensitized Gram-negatives. Further studies suggested that this compound inhibited DNA replication and selection for resistance identified mutations in a subunit of E. coli DNA gyrase, a type II topoisomerase. Our initial compound demonstrated weak inhibition of DNA gyrase activity while optimized compounds demonstrated significantly improved inhibition of E. coli and Pseudomonas aeruginosa DNA gyrase and caused cleaved complex stabilization, a hallmark of certain bactericidal DNA gyrase inhibitors. Amino acid substitutions conferring resistance to this new class of DNA gyrase inhibitors reside exclusively in the TOPRIM domain of GyrB and are not associated with resistance to the fluoroquinolones, suggesting a novel binding site for a gyrase inhibitor.
Subject(s)
Anti-Bacterial Agents/pharmacology , DNA Gyrase/metabolism , Topoisomerase II Inhibitors/pharmacology , Drug Resistance, Bacterial/genetics , Escherichia coli/drug effects , Escherichia coli/enzymology , Fluoroquinolones/pharmacology , Microbial Sensitivity Tests , Protein Domains , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/enzymologyABSTRACT
5'-Adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of energy homeostasis in eukaryotes. Despite three decades of investigation, the biological roles of AMPK and its potential as a drug target remain incompletely understood, largely because of a lack of optimized pharmacological tools. We developed MK-8722, a potent, direct, allosteric activator of all 12 mammalian AMPK complexes. In rodents and rhesus monkeys, MK-8722-mediated AMPK activation in skeletal muscle induced robust, durable, insulin-independent glucose uptake and glycogen synthesis, with resultant improvements in glycemia and no evidence of hypoglycemia. These effects translated across species, including diabetic rhesus monkeys, but manifested with concomitant cardiac hypertrophy and increased cardiac glycogen without apparent functional sequelae.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Cardiomegaly/chemically induced , Glucose/metabolism , Homeostasis/drug effects , Imidazoles/pharmacology , Pyridines/pharmacology , Animals , Benzimidazoles , Blood Glucose/drug effects , Fasting , Glycogen/metabolism , Hypoglycemia/chemically induced , Imidazoles/adverse effects , Imidazoles/chemistry , Insulin/pharmacology , Macaca mulatta , Male , Mice , Mice, Inbred C57BL , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Pyridines/adverse effects , Pyridines/chemistryABSTRACT
The growing prevalence of drug resistant bacteria is a significant global threat to human health. The antibacterial drug rifampin, which functions by inhibiting bacterial RNA polymerase (RNAP), is an important part of the antibacterial armamentarium. Here, in order to identify novel inhibitors of bacterial RNAP, we used affinity-selection mass spectrometry to screen a chemical library for compounds that bind to Escherichia coli RNAP. We identified a novel small molecule, MRL-436, that binds to RNAP, inhibits RNAP, and exhibits antibacterial activity. MRL-436 binds to RNAP through a binding site that differs from the rifampin binding site, inhibits rifampin-resistant RNAP derivatives, and exhibits antibacterial activity against rifampin-resistant strains. Isolation of mutants resistant to the antibacterial activity of MRL-436 yields a missense mutation in codon 622 of the rpoC gene encoding the RNAP ß' subunit or a null mutation in the rpoZ gene encoding the RNAP ω subunit, confirming that RNAP is the functional cellular target for the antibacterial activity of MRL-436, and indicating that RNAP ß' subunit residue 622 and the RNAP ω subunit are required for the antibacterial activity of MRL-436. Similarity between the resistance determinant for MRL-436 and the resistance determinant for the cellular alarmone ppGpp suggests a possible similarity in binding site and/or induced conformational state for MRL-436 and ppGpp.
Subject(s)
Anti-Bacterial Agents/pharmacology , DNA-Directed RNA Polymerases/antagonists & inhibitors , Drug Resistance, Bacterial/drug effects , Binding Sites , Drug Resistance, Bacterial/genetics , Enzyme Inhibitors/pharmacology , Escherichia coli/enzymology , Mass Spectrometry , Protein Binding , Rifampin/pharmacology , Small Molecule LibrariesABSTRACT
Resistance to existing classes of antibiotics drives the need for discovery of novel compounds with unique mechanisms of action. Nargenicin A1, a natural product with limited antibacterial spectrum, was rediscovered in a whole-cell antisense assay. Macromolecular labeling in both Staphylococcus aureus and an Escherichia coli tolC efflux mutant revealed selective inhibition of DNA replication not due to gyrase or topoisomerase IV inhibition. S. aureus nargenicin-resistant mutants were selected at a frequency of â¼1 × 10(-9), and whole-genome resequencing found a single base-pair change in the dnaE gene, a homolog of the E. coli holoenzyme α subunit. A DnaE single-enzyme assay was exquisitely sensitive to inhibition by nargenicin, and other in vitro characterization studies corroborated DnaE as the target. Medicinal chemistry efforts may expand the spectrum of this novel mechanism antibiotic.
Subject(s)
DNA Polymerase III/genetics , Drug Discovery , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , DNA Replication/drug effects , DNA-Directed DNA Polymerase/metabolism , Drug Resistance, Bacterial/genetics , Escherichia coli/drug effects , Inhibitory Concentration 50 , Lactones/chemistry , Lactones/metabolism , Lactones/pharmacology , Mutation , Nucleic Acid Synthesis Inhibitors/chemistry , Nucleic Acid Synthesis Inhibitors/pharmacology , Staphylococcus aureus/drug effectsABSTRACT
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.
Subject(s)
Pyrimidines/chemistry , Pyrimidines/pharmacology , RNA, Bacterial/chemistry , RNA, Bacterial/drug effects , Riboswitch/drug effects , Animals , Aptamers, Nucleotide/chemistry , Bacteria/cytology , Bacteria/drug effects , Bacteria/growth & development , Base Sequence , Crystallography, X-Ray , Escherichia coli Infections/drug therapy , Escherichia coli Infections/microbiology , Escherichia coli Proteins/genetics , Female , Flavin Mononucleotide/metabolism , Gene Expression Regulation, Bacterial/drug effects , Heat-Shock Proteins/genetics , Intramolecular Transferases/genetics , Ligands , Mice , Mice, Inbred DBA , Models, Molecular , Molecular Sequence Data , Pyrimidines/isolation & purification , Pyrimidines/therapeutic use , RNA, Bacterial/genetics , Reproducibility of Results , Riboflavin/biosynthesis , Riboswitch/genetics , Substrate SpecificityABSTRACT
ß-Lactamase inhibitors with a bicyclic urea core and a variety of heterocyclic side chains were prepared and evaluated as potential partners for combination with imipenem to overcome class A and C ß-lactamase mediated antibiotic resistance. The piperidine analog 3 (MK-7655) inhibited both class A and C ß-lactamases in vitro. It effectively restored imipenem's activity against imipenem-resistant Pseudomonas and Klebsiella strains at clinically achievable concentrations. A combination of MK-7655 and Primaxin® is currently in phase II clinical trials for the treatment of Gram-negative bacterial infections.
Subject(s)
Azabicyclo Compounds/chemistry , Azabicyclo Compounds/pharmacology , Cilastatin/chemistry , Drug Discovery , Enzyme Inhibitors/chemistry , Imipenem/chemistry , beta-Lactamase Inhibitors , Cilastatin/pharmacology , Cilastatin, Imipenem Drug Combination , Crystallography, X-Ray , Drug Combinations , Drug Resistance, Bacterial/drug effects , Imipenem/pharmacology , Inhibitory Concentration 50 , Klebsiella/drug effects , Microbial Sensitivity Tests , Models, Biological , Pseudomonas/drug effects , Structure-Activity RelationshipABSTRACT
The resistance of methicillin-resistant Staphylococcus aureus (MRSA) to all ß-lactam classes limits treatment options for serious infections involving this organism. Our goal is to discover new agents that restore the activity of ß-lactams against MRSA, an approach that has led to the discovery of two classes of natural product antibiotics, a cyclic depsipeptide (krisynomycin) and a lipoglycopeptide (actinocarbasin), which potentiate the activity of imipenem against MRSA strain COL. We report here that these imipenem synergists are inhibitors of the bacterial type I signal peptidase SpsB, a serine protease that is required for the secretion of proteins that are exported through the Sec and Tat systems. A synthetic derivative of actinocarbasin, M131, synergized with imipenem both in vitro and in vivo with potent efficacy. The in vitro activity of M131 extends to clinical isolates of MRSA but not to a methicillin-sensitive strain. Synergy is restricted to ß-lactam antibiotics and is not observed with other antibiotic classes. We propose that the SpsB inhibitors synergize with ß-lactams by preventing the signal peptidase-mediated secretion of proteins required for ß-lactam resistance. Combinations of SpsB inhibitors and ß-lactams may expand the utility of these widely prescribed antibiotics to treat MRSA infections, analogous to ß-lactamase inhibitors which restored the utility of this antibiotic class for the treatment of resistant Gram-negative infections.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Biphenyl Compounds/pharmacology , Depsipeptides/pharmacology , Glycopeptides/pharmacology , Glycosides/pharmacology , Lipopeptides/pharmacology , Membrane Proteins/antagonists & inhibitors , Methicillin-Resistant Staphylococcus aureus/drug effects , Oligopeptides/pharmacology , Staphylococcal Infections/drug therapy , beta-Lactams/pharmacology , Animals , Anti-Bacterial Agents/isolation & purification , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biological Transport , Biphenyl Compounds/chemical synthesis , Depsipeptides/isolation & purification , Drug Synergism , Drug Therapy, Combination , Female , Glycopeptides/chemical synthesis , Glycopeptides/isolation & purification , Glycosides/isolation & purification , Humans , Lipopeptides/isolation & purification , Membrane Proteins/genetics , Membrane Proteins/metabolism , Methicillin-Resistant Staphylococcus aureus/genetics , Methicillin-Resistant Staphylococcus aureus/growth & development , Mice , Mice, Inbred BALB C , Microbial Sensitivity Tests , Multigene Family , Oligopeptides/chemical synthesis , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Staphylococcal Infections/microbiology , beta-Lactam Resistance/drug effects , beta-Lactam Resistance/genetics , beta-Lactamases/genetics , beta-Lactamases/metabolismABSTRACT
Bacterial resistance to known therapeutics has led to an urgent need for new chemical classes of antibacterial agents. To address this we have applied a Staphylococcus aureus fitness test strategy to natural products screening. Here we report the discovery of kibdelomycin, a novel class of antibiotics produced by a new member of the genus Kibdelosporangium. Kibdelomycin exhibits broad-spectrum, gram-positive antibacterial activity and is a potent inhibitor of DNA synthesis. We demonstrate through chemical genetic fitness test profiling and biochemical enzyme assays that kibdelomycin is a structurally new class of bacterial type II topoisomerase inhibitor preferentially inhibiting the ATPase activity of DNA gyrase and topoisomerase IV. Kibdelomycin is thus the first truly novel bacterial type II topoisomerase inhibitor with potent antibacterial activity discovered from natural product sources in more than six decades.
Subject(s)
Actinomycetales/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Pyrroles/chemistry , Pyrroles/pharmacology , Pyrrolidinones/chemistry , Pyrrolidinones/pharmacology , Staphylococcus aureus/drug effects , Staphylococcus aureus/enzymology , Topoisomerase II Inhibitors/chemistry , Topoisomerase II Inhibitors/pharmacology , Anti-Bacterial Agents/isolation & purification , DNA Gyrase/metabolism , DNA Topoisomerase IV/antagonists & inhibitors , DNA Topoisomerase IV/metabolism , Drug Resistance, Bacterial , Humans , Microbial Sensitivity Tests , Models, Molecular , Pyrroles/isolation & purification , Pyrrolidinones/isolation & purification , Staphylococcal Infections/drug therapy , Staphylococcus aureus/genetics , Topoisomerase II Inhibitors/isolation & purificationABSTRACT
The bridged monobactam ß-lactamase inhibitor MK-8712 (1) effectively inhibits class C ß-lactamases. Side chain N-alkylated and ring-opened analogs of 1 were prepared and evaluated for combination with imipenem to overcome class C ß-lactamase mediated resistance. Although some analogs were more potent inhibitors of AmpC, none exhibited better synergy with imipenem than 1.
Subject(s)
Anti-Bacterial Agents/chemistry , Enzyme Inhibitors/chemistry , Monobactams/chemical synthesis , beta-Lactamase Inhibitors , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacology , Binding Sites , Computer Simulation , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Imipenem/pharmacology , Microbial Sensitivity Tests , Monobactams/pharmacology , Protein Structure, Tertiary , Structure-Activity Relationship , beta-Lactamases/metabolismABSTRACT
4,7-Dichloro-1-benzothien-2-yl sulfonylaminomethyl boronic acid (DSABA, Compound I) was discovered as the first boronic acid-based class D beta-lactamase inhibitor. It exhibited an IC(50) of 5.6 microM against OXA-40. The compound also inhibited class A and C beta-lactamases with sub to low microM IC(50), and synergized with imipenem against Acinetobacter baumannii.
Subject(s)
Anti-Bacterial Agents/pharmacology , Boronic Acids/pharmacology , Enzyme Inhibitors/pharmacology , beta-Lactamase Inhibitors , Acinetobacter baumannii/drug effects , Anti-Bacterial Agents/chemistry , Boronic Acids/chemistry , Enzyme Inhibitors/chemistry , Inhibitory Concentration 50 , Microbial Sensitivity TestsABSTRACT
Bridged monobactam beta-lactamase inhibitors were prepared and evaluated as potential partners for combination with imipenem to overcome class C beta-lactamase mediated resistance. The (S)-azepine analog 2 was found to be effective in both in vitro and in vivo assays and was selected for preclinical development.
Subject(s)
Carbapenems/chemistry , Drug Discovery/methods , Imipenem/chemistry , beta-Lactamase Inhibitors , Animals , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Carbapenems/pharmacology , Drug Combinations , Imipenem/pharmacology , Mice , Pseudomonas Infections/drug therapy , Pseudomonas Infections/enzymology , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/physiology , Structure-Activity Relationship , beta-Lactam Resistance/drug effects , beta-Lactam Resistance/physiology , beta-Lactamases/metabolismABSTRACT
From April 2000 to April 2001, 24 patients in intensive care units at Tisch Hospital, New York, N.Y., were infected or colonized by carbapenem-resistant Klebsiella pneumoniae. Pulsed-field gel electrophoresis identified a predominant outbreak strain, but other resistant strains were also recovered. Three representatives of the outbreak strain from separate patients were studied in detail. All were resistant or had reduced susceptibility to imipenem, meropenem, ceftazidime, piperacillin-tazobactam, and gentamicin but remained fully susceptible to tetracycline. PCR amplified a blaKPC allele encoding a novel variant, KPC-3, with a His(272)-->Tyr substitution not found in KPC-2; other carbapenemase genes were absent. In the outbreak strain, KPC-3 was encoded by a 75-kb plasmid, which was transferred in vitro by electroporation and conjugation. The isolates lacked the OmpK35 porin but expressed OmpK36, implying reduced permeability as a cofactor in resistance. This is the third KPC carbapenem-hydrolyzing beta-lactamase variant to have been reported in members of the Enterobacteriaceae, with others reported from the East Coast of the United States. Although producers of these enzymes remain rare, the progress of this enzyme group merits monitoring.