Subject(s)
RNA, Bacterial/chemistry , Ribosomes/chemistry , 2-Aminopurine/chemistry , Aminoglycosides/chemical synthesis , Anti-Bacterial Agents/chemical synthesis , Crystallization , Crystallography, X-Ray , Gene Targeting , Models, Molecular , Nucleic Acid Conformation , Oligonucleotides/chemistry , RNA, Bacterial/biosynthesis , RNA, Bacterial/drug effects , Ribosomes/drug effects , Spectrometry, Fluorescence , Spectrophotometry, UltravioletABSTRACT
RNA recognition by natural aminoglycoside antibiotics depends on the 2-deoxystreptamine (2-DOS) scaffold which participates in specific hydrogen bonds with the ribosomal decoding-site target. Three-dimensional structure information has been used for the design of azepane-monoglycosides, building blocks for novel antibiotics in which 2-DOS is replaced by a heterocyclic scaffold. Azepane-glycosides showed target binding and translation inhibition in the low micromolar range and inhibited growth of Staphylococcus aureus, including aminoglycoside-resistant strains.
Subject(s)
Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Azepines/chemistry , Azepines/pharmacology , RNA, Ribosomal/antagonists & inhibitors , Staphylococcus aureus/drug effects , Staphylococcus aureus/growth & development , Aminoglycosides/adverse effects , Aminoglycosides/chemistry , Anti-Bacterial Agents/chemistry , Azepines/metabolism , Drug Design , Glycosides/chemistry , Glycosides/metabolism , Glycosides/pharmacology , Heterocyclic Compounds/chemistry , Heterocyclic Compounds/metabolism , Heterocyclic Compounds/pharmacology , Nucleic Acid Conformation , Paromomycin , Protein Biosynthesis/drug effects , RNA, Bacterial/antagonists & inhibitors , RNA, Ribosomal/chemistry , Structure-Activity RelationshipSubject(s)
Aminoglycosides/chemistry , Anti-Bacterial Agents/chemistry , Hexosamines/chemistry , RNA, Bacterial/metabolism , RNA, Ribosomal/metabolism , Aminoglycosides/metabolism , Aminoglycosides/pharmacology , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Binding Sites , Escherichia coli/drug effects , Escherichia coli/metabolism , Hexosamines/metabolism , Hexosamines/pharmacology , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , Staphylococcus aureus/drug effects , Staphylococcus aureus/metabolism , Structure-Activity RelationshipSubject(s)
Anti-Bacterial Agents/chemistry , Glycosides/chemistry , Piperidines/chemistry , RNA, Bacterial/metabolism , RNA, Ribosomal/metabolism , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Binding Sites , Escherichia coli/drug effects , Escherichia coli/metabolism , Glycosides/metabolism , Glycosides/pharmacology , Inhibitory Concentration 50 , Models, Molecular , Molecular Structure , Piperidines/metabolism , Piperidines/pharmacology , Staphylococcus aureus/drug effects , Staphylococcus aureus/metabolism , Structure-Activity RelationshipABSTRACT
Natural aminoglycoside antibiotics recognize an internal loop of bacterial ribosomal-decoding-site RNA by binding to the deep groove of the RNA structure. We have designed, synthesized, and tested RNA-targeted paromamine derivatives that exploit additional interactions on the shallow groove face of the decoding-site RNA. An in vitro transcription-translation assay of a series of 6'-derivatives showed the 6'-position to be very sensitive to substitution. This result suggests that the group at the 6'-position plays a pivotal role in RNA target recognition.
Subject(s)
Aminoglycosides/chemical synthesis , Aminoglycosides/pharmacology , RNA, Ribosomal/antagonists & inhibitors , Aminoglycosides/chemistry , Anti-Bacterial Agents/chemistry , Binding Sites , Cell-Free System , Drug Design , Protein Biosynthesis/drug effects , RNA, Bacterial/antagonists & inhibitors , RNA, Ribosomal/chemistry , RNA, Ribosomal/ultrastructure , Structure-Activity RelationshipABSTRACT
The ribosomal decoding site is the target of aminoglycoside antibiotics that specifically recognize an internal loop RNA structure. We synthesized RNA-targeted 2,5-dideoxystreptamine-4-amides in which a sugar moiety in natural aminoglycosides is replaced by heterocycles.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Hexosamines/chemistry , Hexosamines/pharmacology , RNA, Ribosomal/metabolism , Aminoglycosides , Binding Sites , Luciferases/genetics , Models, Molecular , Protein Biosynthesis/drug effects , RNA, Ribosomal/drug effects , Structure-Activity Relationship , Transcription, Genetic/drug effectsABSTRACT
Large-scale genotyping, mapping and expression profiling require affordable, fully automated high-throughput devices enabling rapid, high-performance analysis using minute quantities of reagents. In this paper, we describe a new combination of microwell polymerase chain reaction (PCR) based DNA amplification technique with automated ultrathin-layer gel electrophoresis analysis of the resulting products. This technique decreases the reagent consumption (total reaction volume 0.75-1 microL), the time requirement of the PCR (15-20 min) and subsequent ultrathin-layer gel electrophoresis based fragment analysis (5 min) by automating the current manual procedure and reducing the human intervention using sample loading robots and computerized real time data analysis. Small aliquots (0.2 microL) of the submicroliter size PCR reaction were transferred onto loading membranes and analyzed by ultrathin-layer gel electrophoresis which is a novel, high-performance and automated microseparation technique. This system employs integrated scanning laser-induced fluorescence-avalanche photodiode detection and combines the advantages of conventional slab and capillary gel electrophoresis. Visualization of the DNA fragments was accomplished by "in migratio" complexation with ethidium bromide during the electrophoresis process also enabling real time imaging and data analysis.
