Chemical modulation of the biological activity of reutericyclin: a membrane-active antibiotic from Lactobacillus reuteri

Abstract

Whilst the development of membrane-active antibiotics is now an attractive therapeutic concept, progress in this area is disadvantaged by poor knowledge of the structure-activity relationship (SAR) required for optimizing molecules to selectively target bacteria. This prompted us to explore the SAR of the Lactobacillus reuteri membrane-active antibiotic reutericyclin, modifying three key positions about its tetramic acid core. The SAR revealed that lipophilic analogs were generally more active against Gram-positive pathogens, but introduction of polar and charged substituents diminished their activity. This was confirmed by cytometric assays showing that inactive compounds failed to dissipate the membrane potential. Radiolabeled substrate assays indicated that dissipation of the membrane potential by active reutericyclins correlated with inhibition of macromolecular synthesis in cells. However, compounds with good antibacterial activities also showed cytotoxicity against Vero cells and hemolytic activity. Although this study highlights the challenge of optimizing membrane-active antibiotics, it shows that by increasing antibacterial potency the selectivity index could be widened, allowing use of lower non-cytotoxic doses.

Figure 1. Studies on the membrane active agent reutericyclin.

A] Objectives of the study - 1] Perform structure-activity relationship (SAR) study by modifying the 1, 3 and 5-positions of the tetramic core 2] Determine selectivity index by measuring cytotoxicity against Vero cells and comparing to MIC against S. aureus Newman strain and 3] Determine the mechanism of action against S. aureus. B] Synthetic scheme for the preparation of the reutericyclin analogs.

Figure 2. Effects on membrane potential of S. aureus.

A] Reutericyclin (REU) acts in a concentration-dependent with full collapse of the membrane potential at 10 μM, similar to the control CCCP. B] Comparison of the ability of reutericyclin analogs at 10 μM to dissipate the membrane potential; complete dissipation of the membrane potential by CCCP (10 μM) is shown.

Figure 3. Effect of reutericyclins on the macromolecular synthesis of S. aureus.

The concentrations of compounds corresponding to 1 and 4 × their MICs are: 1 i.e. reutericyclin, (0.78 and 3.12 μg/ml), 5 (0.78 and 3.13 μg/ml), 13 (0.2 and 0.8 μg/ml). Controls were: novobiocin (8 μg/ml), rifampicin (0.08 μg/ml) and tetracycline (1.6 μg/ml).