ABSTRACT
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
Thrombin is a serine protease that plays a key role in blood clotting. Pyrrolidine 1 is a potent thrombin inhibitor discovered at Merck several years ago. Seven analogs (2-8) of 1 in which the pyrrolidine core was replaced with various heterocycles were prepared and evaluated for activity against thrombin, clotting factors VIIa, IXa, Xa, and XIIa, and trypsin. The thiomorpholine analog 6 was the most active, essentially matching the thrombin inhibitory activity of 1 with slightly improved selectivity over trypsin.
Subject(s)
Enzyme Inhibitors/pharmacology , Heterocyclic Compounds/pharmacology , Thrombin/antagonists & inhibitors , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Heterocyclic Compounds/chemical synthesis , Heterocyclic Compounds/chemistry , Models, Molecular , Molecular Structure , Structure-Activity Relationship , Thrombin/metabolismABSTRACT
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.
