Novel bacterial acetyl coenzyme A carboxylase inhibitors with antibiotic efficacy in vivo

Abstract

The pseudopeptide pyrrolidinedione antibiotics, such as moiramide B, have recently been discovered to target the multisubunit acetyl coenzyme A (acetyl-CoA) carboxylases of bacteria. In this paper, we describe synthetic variations of each moiety of the modularly composed pyrrolidinediones, providing insight into structure-activity relationships of biochemical target activity, in vitro potency, and in vivo efficacy. The novel derivatives showed highly improved activities against gram-positive bacteria compared to those of previously reported variants. The compounds exhibited a MIC(90) value of 0.1 microg/ml against a broad spectrum of Staphylococcus aureus clinical isolates. No cross-resistance to antibiotics currently used in clinical practice was observed. Resistance mutations induced by pyrrolidinediones are exclusively located in the carboxyltransferase subunits of the bacterial acetyl-CoA carboxylase, indicating the identical mechanisms of action of all derivatives tested. Improvement of the physicochemical profile was achieved by salt formation, leading to aqueous solubilities of up to 5 g/liter. For the first time, the in vitro activity of this compound class was compared with its in vivo efficacy, demonstrating a path from compounds weakly active in vivo to agents with significant efficacy. In a murine model of S. aureus sepsis, the 100% effective dose of the best compound reported was 25 mg/kg of body weight, only fourfold higher than that of the comparator molecule linezolid. The obvious improvements achieved by chemical derivatization reflect the potential of this novel antibiotic compound class for future therapy.

FIG. 1.

Chemical structures of the natural products moiramide B and andrimid and of selected synthetic derivatives.

FIG. 2.

Part of the multiple amino acid sequence alignment of carboxyltransferase β-subunits (AccD) of bacterial acetyl-CoA carboxylases. The amino acid exchanges caused by mutations obtained by serial passaging of S. aureus 133 in the presence of the pyrrolidinedione variants 1, 3, and 7 are indicated below the alignment. The accD mutations are G400-A (compound 1), C332-A (compound 3), and T509-C (compound 7). The mutation isolated with compound 1 (the natural product moiramide B) was found in a strain with a fourfold increased MIC compared to that of the corresponding wild type. The mutation isolated with compound 3 was associated with a 16-fold elevated MIC, while the mutation isolated with compound 7 was identified in a mutant with a 256-fold increased MIC (see Results). Sequences of the following bacteria are included: Ecoli, E. coli; Hinf, Haemophilus influenzae; Paer, Pseudomonas aeruginosa; Bsub, B. subtilis (YttI is a synonym for AccD); Saur, S. aureus; Llact, Lactobacillus lactis; Spneu, S. pneumoniae; Efae, Enterococcus faecalis; and Aaeo, Aquifex aeolicus.

FIG. 3.

In vitro-in vivo efficacy correlation for variants of pyrrolidinedione antibiotics in a murine sepsis model with a reduced S. aureus inoculum. Compounds 2, 4, and 5 were dosed at 10 mg/kg i.v. at 30 and 90 min postinfection, and linezolid was given only at 30 min postinfection. The reductions of CFU in the blood, lungs, liver, and peritoneum determined 5 h after infection were plotted for the different derivatives (see Materials and Methods). Compound 5 (given twice) approximately reached the efficacy of linezolid (administered once). The in vivo efficacy ranking of pyrrolidinediones reflects the improvements of in vitro MIC data, as shown below the diagram.

FIG. 4.

Therapeutic effects of pyrrolidinedione derivatives 6 and 7 (see Fig. 1) and of the reference antibiotic linezolid in a murine model of S. aureus sepsis. The percentage of surviving mice among five animals per group was monitored over 5 days (see Materials and Methods).