ABSTRACT
Multidrug-resistant Gram-negative pathogens are an emerging threat to human health, and addressing this challenge will require development of new antibacterial agents. This can be achieved through an improved molecular understanding of drug-target interactions combined with enhanced delivery of these agents to the site of action. Herein we describe the first application of siderophore receptor-mediated drug uptake of lactivicin analogues as a strategy that enables the development of novel antibacterial agents against clinically relevant Gram-negative bacteria. We report the first crystal structures of several sideromimic conjugated compounds bound to penicillin binding proteins PBP3 and PBP1a from Pseudomonas aeruginosa and characterize the reactivity of lactivicin and ß-lactam core structures. Results from drug sensitivity studies with ß-lactamase enzymes are presented, as well as a structure-based hypothesis to reduce susceptibility to this enzyme class. Finally, mechanistic studies demonstrating that sideromimic modification alters the drug uptake process are discussed.
Subject(s)
Gram-Negative Bacteria/metabolism , Peptides/metabolism , Receptors, Cell Surface/metabolism , Siderophores/metabolism , Gram-Negative Bacteria/drug effects , Gram-Negative Bacteria/enzymology , Microbial Sensitivity Tests , Peptides/drug effects , Peptides, Cyclic , beta-Lactamases/metabolismABSTRACT
Herein we describe the structure-aided design and synthesis of a series of pyridone-conjugated monobactam analogues with in vitro antibacterial activity against clinically relevant Gram-negative species including Pseudomonas aeruginosa , Klebsiella pneumoniae , and Escherichia coli . Rat pharmacokinetic studies with compound 17 demonstrate low clearance and low plasma protein binding. In addition, evidence is provided for a number of analogues suggesting that the siderophore receptors PiuA and PirA play a role in drug uptake in P. aeruginosa strain PAO1.
Subject(s)
Anti-Bacterial Agents/pharmacology , Gram-Negative Bacteria/drug effects , Monobactams/pharmacology , Pyridones/pharmacology , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacokinetics , Escherichia coli/drug effects , Inhibitory Concentration 50 , Klebsiella pneumoniae/drug effects , Male , Microbial Sensitivity Tests , Molecular Structure , Monobactams/chemistry , Monobactams/pharmacokinetics , Pseudomonas aeruginosa/drug effects , Pyridones/chemistry , Pyridones/pharmacokinetics , Rats , Rats, WistarABSTRACT
A structurally novel set of inhibitors of bacterial type II topoisomerases with potent in vitro and in vivo antibacterial activity was developed. Dual-targeting ability, hERG inhibition, and pharmacokinetic properties were also assessed.
Subject(s)
Anti-Bacterial Agents/pharmacology , DNA Topoisomerase IV/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Quinolines/pharmacology , Staphylococcus aureus/drug effects , Streptococcus pneumoniae/drug effects , Topoisomerase II Inhibitors , Animals , Anti-Bacterial Agents/administration & dosage , Anti-Bacterial Agents/chemistry , DNA Gyrase/metabolism , DNA Topoisomerase IV/metabolism , DNA Topoisomerases, Type II/metabolism , Dose-Response Relationship, Drug , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/chemistry , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Humans , Microbial Sensitivity Tests , Molecular Structure , Quinolines/administration & dosage , Quinolines/chemistry , Rats , Staphylococcus aureus/enzymology , Streptococcus pneumoniae/enzymology , Structure-Activity RelationshipABSTRACT
Acinetobacter baumannii is an increasingly problematic pathogen in United States hospitals. Antibiotics that can treat A. baumannii are becoming more limited. Little is known about the contributions of penicillin binding proteins (PBPs), the target of ß-lactam antibiotics, to ß-lactam-sulbactam susceptibility and ß-lactam resistance in A. baumannii. Decreased expression of PBPs as well as loss of binding of ß-lactams to PBPs was previously shown to promote ß-lactam resistance in A. baumannii. Using an in vitro assay with a reporter ß-lactam, Bocillin, we determined that the 50% inhibitory concentrations (IC(50)s) for PBP1a from A. baumannii and PBP3 from Acinetobacter sp. ranged from 1 to 5 µM for a series of ß-lactams. In contrast, PBP3 demonstrated a narrower range of IC(50)s against ß-lactamase inhibitors than PBP1a (ranges, 4 to 5 versus 8 to 144 µM, respectively). A molecular model with ampicillin and sulbactam positioned in the active site of PBP3 reveals that both compounds interact similarly with residues Thr526, Thr528, and Ser390. Accepting that many interactions with cell wall targets are possible with the ampicillin-sulbactam combination, the low IC(50)s of ampicillin and sulbactam for PBP3 may contribute to understanding why this combination is effective against A. baumannii. Unraveling the contribution of PBPs to ß-lactam susceptibility and resistance brings us one step closer to identifying which PBPs are the best targets for novel ß-lactams.
Subject(s)
Acinetobacter baumannii/chemistry , Acinetobacter/chemistry , Anti-Bacterial Agents/chemistry , Enzyme Inhibitors/chemistry , Penicillin-Binding Proteins/antagonists & inhibitors , beta-Lactam Resistance , Acinetobacter/enzymology , Acinetobacter baumannii/enzymology , Ampicillin/chemistry , Biological Assay , Boron Compounds/chemistry , Humans , Kinetics , Molecular Docking Simulation , Penicillin-Binding Proteins/chemistry , Penicillin-Binding Proteins/metabolism , Penicillins/chemistry , Solubility , Substrate Specificity , Sulbactam/chemistry , beta-Lactamase Inhibitors , beta-Lactamases/chemistry , beta-Lactamases/metabolism , beta-Lactams/chemistryABSTRACT
Multi-drug-resistant forms of the Gram-negative pathogen Acinetobacter baumannii are an emerging threat to human health and further complicate the general problem of treating serious bacterial infections. Meeting this challenge requires an improved understanding of the relationships between the structures of major therapeutic targets in this organism and the activity levels exhibited against it by different antibiotics. Here we report the first crystal structures of A. baumannii penicillin-binding proteins (PBPs) covalently inactivated by four ß-lactam antibiotics. We also relate the results to kinetic, biophysical, and computational data. The structure of the class A protein PBP1a was solved in apo form and for its covalent conjugates with benzyl penicillin, imipenem, aztreonam, and the siderophore-conjugated monocarbam MC-1. It included a novel domain genetically spliced into a surface loop of the transpeptidase domain that contains three conserved loops. Also reported here is the first high-resolution structure of the A. baumannii class B enzyme PBP3 in apo form. Comparison of this structure with that of MC-1-derivatized PBP3 of Pseudomonas aeruginosa identified differences between these orthologous proteins in A. baumannii and P. aeruginosa. Thermodynamic analyses indicated that desolvation effects in the PBP3 ligand-binding sites contributed significantly to the thermal stability of the enzyme-antibiotic covalent complexes. Across a significant range of values, they correlated well with results from studies of inactivation kinetics and the protein structures. The structural, biophysical, and computational data help rationalize differences in the functional performance of antibiotics against different protein targets and can be used to guide the design of future agents.
Subject(s)
Acinetobacter baumannii/drug effects , Anti-Bacterial Agents/pharmacology , beta-Lactams/pharmacology , Acinetobacter baumannii/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/drug effects , Bacterial Proteins/metabolism , Chromatography, Liquid , Crystallization , Mass Spectrometry , Microbial Sensitivity Tests , Models, Molecular , Molecular Sequence DataABSTRACT
SAR studies and optimization of various modified Hygromycin A fluoroalkyl ethers, which led to the discovery of the highly potent 4'-(2-cyclopropyl-2-fluoroethyl ether) antibacterial CE-156811 (1) derived from truncation of the ribose ring and difluorination of the phenyl found in Hygromycin A, are discussed.
Subject(s)
Anti-Bacterial Agents/chemistry , Cinnamates/chemistry , Dioxoles/chemistry , Hygromycin B/analogs & derivatives , Administration, Oral , Animals , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacokinetics , Dogs , Drug Evaluation, Preclinical , Haplorhini , Hygromycin B/chemistry , Mice , Microbial Sensitivity Tests , Rats , Structure-Activity RelationshipABSTRACT
Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes nosocomial infections for which there are limited treatment options. Penicillin-binding protein PBP3, a key therapeutic target, is an essential enzyme responsible for the final steps of peptidoglycan synthesis and is covalently inactivated by ß-lactam antibiotics. Here we disclose the first high resolution cocrystal structures of the P. aeruginosa PBP3 with both novel and marketed ß-lactams. These structures reveal a conformational rearrangement of Tyr532 and Phe533 and a ligand-induced conformational change of Tyr409 and Arg489. The well-known affinity of the monobactam aztreonam for P. aeruginosa PBP3 is due to a distinct hydrophobic aromatic wall composed of Tyr503, Tyr532, and Phe533 interacting with the gem-dimethyl group. The structure of MC-1, a new siderophore-conjugated monocarbam complexed with PBP3 provides molecular insights for lead optimization. Importantly, we have identified a novel conformation that is distinct to the high-molecular-weight class B PBP subfamily, which is identifiable by common features such as a hydrophobic aromatic wall formed by Tyr503, Tyr532, and Phe533 and the structural flexibility of Tyr409 flanked by two glycine residues. This is also the first example of a siderophore-conjugated triazolone-linked monocarbam complexed with any PBP. Energetic analysis of tightly and loosely held computed hydration sites indicates protein desolvation effects contribute significantly to PBP3 binding, and analysis of hydration site energies allows rank ordering of the second-order acylation rate constants. Taken together, these structural, biochemical, and computational studies provide a molecular basis for recognition of P. aeruginosa PBP3 and open avenues for future design of inhibitors of this class of PBPs.
Subject(s)
Anti-Bacterial Agents/chemistry , Models, Molecular , Penicillin-Binding Proteins/chemistry , Pseudomonas aeruginosa/chemistry , Siderophores/chemistry , beta-Lactams/chemistry , Amino Acids, Aromatic , Anti-Bacterial Agents/therapeutic use , Cross Infection/drug therapy , Cross Infection/microbiology , Crystallography, X-Ray , Humans , Protein Structure, Tertiary , Pseudomonas Infections/drug therapy , Pseudomonas Infections/microbiology , beta-Lactams/therapeutic useABSTRACT
We have developed an affinity purification of the large ribosomal subunit from Deinococcus radiodurans that exploits its association with FLAG-tagged 30S subunits. Thus, capture is indirect so that no modification of the 50S is required and elution is achieved under mild conditions (low magnesium) that disrupt the association, avoiding the addition of competitor ligands or coelution of common contaminants. Efficient purification of highly pure 50S is achieved, and the chromatography simultaneously sorts the 50S into three classes according to their association status (unassociated, loosely associated, or tightly associated), improving homogeneity.
Subject(s)
Deinococcus/ultrastructure , Ribosome Subunits, Large, Bacterial/chemistry , Bacterial Proteins/analysis , Centrifugation, Density Gradient , Chromatography, Affinity , Chromatography, High Pressure Liquid , Cloning, Molecular , Databases, Protein , Gene Expression , Magnesium Chloride , Oligopeptides , Peptide Fragments/analysis , Peptides/genetics , RNA, Bacterial/analysis , RNA, Ribosomal/analysis , Recombinant Fusion Proteins , Ribosomal Proteins/analysis , Ribosomal Proteins/genetics , Ribosome Subunits, Large, Bacterial/metabolism , Ribosome Subunits, Small, Bacterial/genetics , Ribosome Subunits, Small, Bacterial/metabolism , Spectrometry, Mass, Electrospray Ionization , Tandem Mass SpectrometryABSTRACT
We evaluated a novel truncated hygromycin A analog in which the furanose ring was replaced with a 2-fluoro-2-cyclopropylethyl substituent for its activity against multidrug resistant gram-positive bacteria and compared its activity to the activities of linezolid, quinupristin-dalfopristin, and vancomycin. CE-156811 demonstrated robust in vitro activity against gram-positive bacteria that was comparable to that of linezolid.