New Class of Bacterial Phenylalanyl-tRNA Synthetase Inhibitors with High Potency and Broad-Spectrum Activity

Abstract

Phenylalanyl (Phe)-tRNA synthetase (Phe-RS) is an essential enzyme which catalyzes the transfer of phenylalanine to the Phe-specific transfer RNA (tRNA Phe ), a key step in protein biosynthesis. Phenyl-thiazolylurea-sulfonamides were identified as a novel class of potent inhibitors of bacterial Phe-RS by highthroughput screening and chemical variation of the screening hit. The compounds inhibit Phe-RS of Escherichia coli, Haemophilus influenzae, Streptococcus pneumoniae, and Staphylococcus aureus, with 50% inhibitory concentrations in the nanomolar range. Enzyme kinetic measurements demonstrated that the compounds bind competitively with respect to the natural substrate Phe. All derivatives are highly selective for the bacterial Phe-RS versus the corresponding mammalian cytoplasmic and human mitochondrial enzymes. Phenyl-thiazolylurea-sulfonamides displayed good in vitro activity against Staphylococcus, Streptococcus, Haemophilus, and Moraxella strains, reaching MICs below 1 g/ml. The antibacterial activity was partly antagonized by increasing concentrations of Phe in the culture broth in accordance with the competitive binding mode. Further evidence that inhibition of tRNA Phe charging is the antibacterial principle of this compound class was obtained by proteome analysis of Bacillus subtilis. Here, the phenyl-thiazolylurea-sulfonamides induced a protein pattern indicative of the stringent response. In addition, an E. coli strain carrying a relA mutation and defective in stringent response was more susceptible than its isogenic relA ؉ parent strain. In vivo efficacy was investigated in a murine S. aureus sepsis model and a S. pneumoniae sepsis model in rats. Treatment with the phenylthiazolylurea-sulfonamides reduced the bacterial titer in various organs by up to 3 log units, supporting the potential value of Phe-RS as a target in antibacterial therapy.

Role of the (p)ppGpp Synthase RSH, a RelA/SpoT Homolog, in Stringent Response and Virulence ofStaphylococcus aureus

“…However, the RSH synthase mutants certainly showed a higher sensitivity toward mupirocin than the wild type, similar to relA mutants of E. coli (5) or Streptococcus pneumoniae (26). One could speculate that the upregulation of genes involved in isoleucine biosynthesis observed in the wild type but not in the rsh syn mutant results in an increased intracellular isoleucine level.…”

Challenges of Antibacterial Discovery

“…With targeted screening, there is already a starting hypothesis for the mechanism of action and molecular target. Thus, initial work may be directed toward demonstration of phenotypes that should be associated with inhibition of that target, such as morphological changes (e.g., filamentation for inhibitors of FtsZ [81,277]), stress responses (stringent response for inhibitors of tRNA synthetases [38]), and specific promoter induction (gyrase inhibition leads to homeostatic upregulation of gyrase promoters as well as SOS promoters [7]). With these studies, it is necessary to use a wide variety of negative controls to show that the tested phenotypes are not caused by other classes of inhibitors.…”

Cell-free Determination of Binary Complexes That Comprise Extended Protein-Protein Interaction Networks of Yersinia pestis

“…6B-6F), whereas several previously reported interactions were recapitulated in the Y. pestis PPi network (Fig. 6B, 6D, 6E) (35)(36)(37). Of the annotated proteins within the network (n ϭ 218), one-quarter could be mapped to a protein class (Fig.…”