uridine-diphosphate-n-acetylmuramic-acid and 6-carboxyfluorescein

The increasing resistance of human pathogens to conventional antibiotics presents a growing threat to the chemotherapeutic management of infectious diseases. The lanthionine antibiotics, still unused as therapeutic agents, have recently attracted significant scientific interest as models for targeting and management of bacterial infections. We investigated the action of one member of this class, subtilin, which permeabilizes lipid membranes in a lipid II-dependent manner and binds bactoprenyl pyrophosphate, akin to nisin. The role the C and N termini play in target recognition was investigated in vivo and in vitro by using the natural N-terminally succinylated subtilin as well as enzymatically truncated subtilin variants. Fluorescence dequenching experiments show that subtilin induces leakage in membranes in a lipid II-dependent manner and that N-succinylated subtilin is roughly 75-fold less active. Solid-state nuclear magnetic resonance was used to show that subtilin forms complexes with membrane isoprenyl pyrophosphates. Activity assays in vivo show that the N terminus of subtilin plays a critical role in its activity. Succinylation of the N terminus resulted in a 20-fold decrease in its activity, whereas deletion of N-terminal Trp abolished activity altogether.

Topics: Alanine; Anti-Bacterial Agents; Bacteriocins; Cell Membrane; Coated Vesicles; Diphosphates; Fluoresceins; Lactococcus lactis; Magnetic Resonance Spectroscopy; Microbial Sensitivity Tests; Peptides; Phosphatidylcholines; Phosphatidylglycerols; Succinic Acid; Sulfides; Tryptophan; Uridine Diphosphate N-Acetylmuramic Acid

This report describes the design, synthesis, and biochemical evaluation of alkene- and alkane-bridged AB(C)-ring mimics of the lantibiotic nisin. Nisin belongs to a class of natural antimicrobial peptides, and has a unique mode of action: its AB(C)-ring system binds to the pyrophosphate moiety of lipid II. This mode of action was the rationale for the design of smaller nisin-derived peptides to obtain novel potential antibiotics. As a conformational constraint the thioether bridge was mimicked by an alkene- or alkane isostere. The peptides of the linear individual ring precursors were synthesized on solid support or in solution, and cyclized by ring-closing metathesis in solution with overall yields of between 36 and 89 %. The individual alkene-bridged macrocycles were assembled in solution by using carbodiimide-based synthesis protocols for the corresponding AB(C)-ring mimics. These compounds were tested for their binding affinity toward lipid II by evaluation of their potency to inhibit nisin-induced carboxyfluorescein release from large unilamellar vesicles. It was found that these AB(C)-ring mimics were not able to induce membrane leakage; however, they acted by inhibiting nisin-induced carboxyfluorescein release; this indicates their affinity toward lipid II. These results imply that an alkene or alkane moiety is a suitable thioether bridge mimic.

Topics: Alkanes; Alkenes; Anti-Bacterial Agents; Catalysis; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Computers, Molecular; Cyclization; Drug Design; Fluoresceins; Models, Molecular; Molecular Mimicry; Nisin; Peptide Fragments; Phosphatidylcholines; Polyisoprenyl Phosphate Oligosaccharides; Stereoisomerism; Unilamellar Liposomes; Uridine Diphosphate N-Acetylmuramic Acid