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 high-throughput 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 micro 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 phenyl-thiazolylurea-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.

FIG. 1.

Chemical structures of the phenyl-thiazolylurea-sulfonamides discussed in this study.

FIG. 2.

Inhibition of E. coli Phe-RS by compound 2. The rate of Phe-tRNA formation was determined at room temperature over 20 min. (A) Competitive binding with respect to Phe. Compound 2 concentrations were as follows: 0 μM (▴), 0.04 μM (▪), and 0.156 μM (⧫). The inset shows results in the form of a Lineweaver-Burk plot (K_m for Phe = 1.9 μM; K_i for compound 2 = 21 nM). (B) Noncompetitive binding with respect to ATP. Compound 2 concentrations were as follows: 0 μM (▴), 0.01 μM (▪), and 0.04 μM (⧫).

FIG. 3.

Effect of increasing Phe concentrations in the culture broth on the antibacterial activity of the phenyl-thiazolylurea-sulfonamides in vitro.

FIG. 4.

Time-kill study. Effect of increasing concentrations of compound 5 on the number of CFU of S. pneumoniae G9A.

FIG. 5.

In vivo activity of the phenyl-thiazolylurea-sulfonamides. (A) S. aureus 133 sepsis in the mouse. Bacterial loads in the organs after i.v. treatment with 100 mg of compound 2/kg of body weight at 0.5 h after infection are indicated. Antibacterial activity was compared in mice on normal or phenylalanine-free diets, resulting in blood phenylalanine concentrations of 70 or 15 μM, respectively. (B) S. pneumoniae 1707/4 sepsis in regularly fed rats without Phe limitation and with normal Phe blood levels (63 μM). Bacterial titers in the organs after i.p. treatment with 100 mg of compound 4/kg at 0.5 and 3 h after infection are indicated. Five animals were employed per group. The results of statistical analyses performed using the Mann-Whitney test were calculated using GraphPad Prism software (version 3.02). *, statistically significant (P < 0.05).