ABSTRACT
DNA gyrase is an essential enzyme in bacteria, and its inhibition results in the disruption of DNA synthesis and, subsequently, cell death. The pyrrolamides are a novel class of antibacterial agents targeting DNA gyrase. These compounds were identified by a fragment-based lead generation (FBLG) approach using nuclear magnetic resonance (NMR) screening to identify low-molecular-weight compounds that bind to the ATP pocket of DNA gyrase. A pyrrole hit with a binding constant of 1 mM formed the basis of the design and synthesis of a focused library of compounds that resulted in the rapid identification of a lead compound that inhibited DNA gyrase with a 50% inhibitory concentration (IC(50)) of 3 µM. The potency of the lead compound was further optimized by utilizing iterative X-ray crystallography to yield DNA gyrase inhibitors that also displayed antibacterial activity. Spontaneous mutants were isolated in Staphylococcus aureus by plating on agar plates containing pyrrolamide 4 at the MIC. The resistant variants displayed 4- to 8-fold-increased MIC values relative to the parent strain. DNA sequencing revealed two independent point mutations in the pyrrolamide binding region of the gyrB genes from these variants, supporting the hypothesis that the mode of action of these compounds was inhibition of DNA gyrase. Efficacy of a representative pyrrolamide was demonstrated against Streptococcus pneumoniae in a mouse lung infection model. These data demonstrate that the pyrrolamides are a novel class of DNA gyrase inhibitors with the potential to deliver future antibacterial agents targeting multiple clinical indications.
Subject(s)
Amides/pharmacology , Anti-Bacterial Agents/pharmacology , Pyrroles/pharmacology , Staphylococcus aureus/drug effects , Streptococcus pneumoniae/drug effects , Topoisomerase II Inhibitors , Amides/chemistry , Animals , Anti-Bacterial Agents/chemistry , Binding Sites , Crystallography, X-Ray , DNA Gyrase/chemistry , DNA Gyrase/metabolism , Drug Resistance, Bacterial , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Inhibitory Concentration 50 , Magnetic Resonance Spectroscopy , Mice , Microbial Sensitivity Tests , Models, Molecular , Mutation , Protein Binding , Pyrroles/chemistry , Staphylococcal Infections/drug therapy , Staphylococcal Infections/microbiology , Staphylococcus aureus/genetics , Staphylococcus aureus/growth & development , Streptococcus pneumoniae/growth & developmentABSTRACT
The pyrrolamides are a new class of antibacterial agents targeting DNA gyrase, an essential enzyme across bacterial species and inhibition results in the disruption of DNA synthesis and subsequently, cell death. The optimization of biochemical activity and other drug-like properties through substitutions to the pyrrole, piperidine, and heterocycle portions of the molecule resulted in pyrrolamides with improved cellular activity and in vivo efficacy.
Subject(s)
Amides/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Pyrroles/chemistry , Topoisomerase II Inhibitors , Amides/chemical synthesis , Amides/pharmacology , Anti-Bacterial Agents/chemical synthesis , Bacteria/drug effects , Binding Sites , Crystallography, X-Ray , DNA Gyrase/metabolism , Enzyme Inhibitors/chemical synthesis , Microbial Sensitivity Tests , Protein Structure, Tertiary , Structure-Activity RelationshipABSTRACT
Homologues of Escherichia coli bacA, encoding extremely hydrophobic proteins, were identified in the genomes of Staphylococcus aureus and Streptococcus pneumoniae. Allelic replacement mutagenesis demonstrated that the gene is not essential for in vitro growth in either organism, and the mutants showed no significant changes in growth rate or morphology. The Staph. aureus bacA mutant showed slightly reduced virulence in a mouse model of infection and an eightfold increase in bacitracin susceptibility. However, a Strep. pneumoniae bacA mutant was highly attenuated in a mouse model of infection, and demonstrated an increase in susceptibility to bacitracin of up to 160000-fold. These observations are consistent with the previously proposed role of BacA protein as undecaprenol kinase.
