ABSTRACT
Ramoplanin is a glycolipodepsipeptide antibiotic obtained from fermentation of Actinoplanes sp. ATCC 33076 that exhibits activity against clinically important multi-drug-resistant, Gram-positive pathogens including vancomycin-resistant Enterococcus (VRE), methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-intermediate resistant Clostridium difficile. It disrupts bacterial cell wall through a unique mechanism of action by sequestering the peptidoglycan intermediate Lipid II and therefore does not show cross-resistance with other antibiotics. However, while demonstrating excellent antimicrobial activity in systemic use in animal models of infection, ramoplanin presents low local tolerability when injected intravenously. As a consequence of this limitation, new derivatives are desirable to overcome this issue. During a natural product screening program developed to discover compounds that disrupt bacterial cell wall synthesis by inhibiting peptidoglycan transglycosylation through binding to the intermediate Lipid II, 49 actinomycete strains were identified by HR-LCMS as producers of ramoplanin-related compounds. The producing strains were isolated from environmental samples collected worldwide comprising both tropical and temperate areas. To assess the diversity of this microbial population, the 49 isolates were initially identified to the genus level on the basis of their micromorphology, and 16S sequencing confirmed the initial identification of the strains. These analyses resulted in the identification of members of genus Streptomyces, as well as representatives of the families Micromonosporaceae, Nocardiaceae, Thermomonosporaceae, and Pseudonocardiaceae, suggesting that the production of ramoplanins is relatively widespread among Actinomycetes. In addition, all of these isolates were tested against a panel of Gram-positive and Gram-negative bacteria, filamentous fungi, and yeast in order to further characterize their antimicrobial properties. This work describes the diversity of actinomycete strains that produced ramoplanin-related compounds, and the analysis of the antimicrobial activity exhibited by these isolates. Our results strongly suggest the presence of new ramoplanin-analogs among these actinomycete producers.
ABSTRACT
Emergence of bacterial resistance has eroded the effectiveness of many life saving antibiotics leading to an urgent need for new chemical classes of antibacterial agents. We have applied a Staphylococcus aureus fitness test strategy to natural products screening to meet this challenge. In this paper we report the discovery of kibdelomycin A, a demethylated congener of kibdelomycin, the representative of a novel class of antibiotics produced by a new strain of Kibdelosporangium. Kibdelomycin A is a potent inhibitor of DNA gyrase and topoisomerase IV, inhibits DNA synthesis and shows whole cell antibiotic activity, albeit, less potently than kibdelomycin. Kibdelomycin C-33 acetate and tetrahydro-bisdechloro derivatives of kibdelomycin were prepared which helped define a basic SAR of the family.
Subject(s)
Aminoglycosides/isolation & purification , Aminoglycosides/pharmacology , Anti-Bacterial Agents/chemistry , Naphthalenes/isolation & purification , Naphthalenes/pharmacology , Actinomycetales/chemistry , Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/pharmacology , DNA Gyrase/metabolism , DNA Topoisomerase IV/antagonists & inhibitors , DNA Topoisomerase IV/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/isolation & purification , Enzyme Inhibitors/pharmacology , Escherichia coli/drug effects , Escherichia coli/enzymology , Magnetic Resonance Spectroscopy , Microbial Sensitivity Tests , Molecular Conformation , Staphylococcus aureus/drug effects , Staphylococcus aureus/enzymology , Structure-Activity Relationship , Topoisomerase II InhibitorsABSTRACT
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
Bacterial resistance to antibiotics, particularly to multiple antibiotics, is becoming a cause for significant concern. The only really viable course of action to counter this is to discover new antibiotics with novel modes of action. We have recently implemented a new antisense-based chemical genetic screening technology to accomplish this goal. The discovery and antibacterial activity of coelomycin, a fully substituted 2,6-dioxo pyrazine, illustrates the application of the Staphylococcus aureus fitness test strategy to natural products discovery.
Subject(s)
Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/pharmacology , Ascomycota/metabolism , Pyrazines/isolation & purification , Pyrazines/pharmacology , Staphylococcus aureus/drug effects , Staphylococcus aureus/physiology , Anti-Bacterial Agents/chemistry , Ascomycota/isolation & purification , Drug Evaluation, Preclinical/methods , Humans , Juniperus/microbiology , Magnetic Resonance Spectroscopy , Molecular Structure , Pyrazines/chemistryABSTRACT
Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial and community-acquired pathogen for which few existing antibiotics are efficacious. Here we describe two structurally related synthetic compounds that potentiate beta-lactam activity against MRSA. Genetic studies indicate that these agents target SAV1754 based on the following observations: (i) it has a unique chemical hypersensitivity profile, (ii) overexpression or point mutations are sufficient to confer resistance, and (iii) genetic inactivation phenocopies the potentiating effect of these agents in combination with beta-lactams. Further, we demonstrate these agents inhibit peptidoglycan synthesis. Because SAV1754 is essential for growth and structurally related to the recently reported peptidoglycan flippase of Escherichia coli, we speculate it performs an analogous function in S. aureus. These results suggest that SAV1754 inhibitors might possess therapeutic potential alone, or in combination with beta-lactams to restore MRSA efficacy.
Subject(s)
Anti-Bacterial Agents/pharmacology , Carbapenems/pharmacology , Indoles/pharmacology , Methicillin-Resistant Staphylococcus aureus/drug effects , Peptidoglycan/chemistry , Piperidines/pharmacology , Anti-Bacterial Agents/chemistry , Carbapenems/chemistry , Drug Synergism , Indoles/chemistry , Methicillin-Resistant Staphylococcus aureus/genetics , Peptidoglycan/metabolism , Piperidines/chemistry , RNA Interference , RNA, Antisense/metabolismABSTRACT
Thiazolyl peptides are a class of thiazole-rich macrocyclic potent antibacterial agents. Recently, we described thiazomycin, a new member of thiazolyl peptides, discovered by a thiazolyl peptide specific chemical screening. This method also allowed for the discovery of a new thiazolyl peptide, thiazomycin A, which carries modification in the oxazolidine ring of the amino sugar moiety. Thiazomycin A is a specific inhibitor of protein synthesis (IC(50) 0.7 microg/mL) and a potent Gram-positive antibacterial agent with minimum inhibitory concentration (MIC) ranging 0.002-0.25 microg/mL. The isolation and structure elucidation and biological activities of thiazomycin A are described.
Subject(s)
Actinomycetales/chemistry , Anti-Bacterial Agents/pharmacology , Peptides, Cyclic/pharmacology , Staphylococcus aureus/drug effects , Thiazoles/pharmacology , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/isolation & purification , Inhibitory Concentration 50 , Microbial Sensitivity Tests , Oxazoles/chemistry , Oxazoles/pharmacology , Peptides, Cyclic/chemistry , Peptides, Cyclic/isolation & purification , Staphylococcus aureus/growth & development , Thiazoles/chemistry , Thiazoles/isolation & purificationABSTRACT
Bacterial resistance to antibiotics, particularly to multiple drug resistant antibiotics, is becoming cause for significant concern. The only really viable course of action is to discover new antibiotics with novel mode of actions. Thiazolyl peptides are a class of natural products that are architecturally complex potent antibiotics but generally suffer from poor solubility and pharmaceutical properties. To discover new thiazolyl peptides potentially with better desired properties, we designed a highly specific assay with a pair of thiazomycin sensitive and resistant strains of Staphylococcus aureus, which led to the discovery of philipimycin, a new thiazolyl peptide glycoside. It was isolated along with an acid-catalyzed degradation product by bioassay-guided fractionation. Structure of both compounds was elucidated by extensive application of 2D NMR, 1D TOCSY, and HRESIFT-MS/MS. Both compounds showed strong antibacterial activities against gram-positive bacteria including MRSA and exhibited MIC values ranging from 0.015 to 1 microg/mL. Philipimycin was significantly more potent than the degradation product. Both compounds showed selective inhibition of protein synthesis, indicating that they targeted the ribosome. Philipimycin was effective in vivo in a mouse model of S. aureus infection exhibiting an ED50 value of 8.4 mg/kg. The docking studies of philipimycin suggested that a part of the molecule interacts with the ribosome and another part with Pro23, Pro22, and Pro26 of L11 protein, which helped in explaining the differential of activities between the sensitive and resistant strains. The design and execution of the bioassay, the isolation, structure, in vitro and in vivo antibacterial activity, and docking studies of philipimycin and its degradation product are described.