Functional Analysis of the Lipoglycodepsipeptide Antibiotic Ramoplanin

Abstract

The peptide antibiotic ramoplanin is highly effective against several drug-resistant gram-positive bacteria, including vancomycin-resistant Enterococcus faecium (VRE) and methicillin-resistant Staphylococcus aureus (MRSA), two important opportunistic human pathogens. Ramoplanin inhibits bacterial peptidoglycan (PG) biosynthesis by binding to Lipid intermediates I and II at a location different than the N-acyl-D-Ala-D-Ala dipeptide site targeted by vancomycin. Lipid I/II capture physically occludes these substrates from proper utilization by the late-stage PG biosynthesis enzymes MurG and the transglycosylases. Key structural features of ramoplanin responsible for antibiotic activity and PG molecular recognition have been discovered by antibiotic semisynthetic modification in conjunction with NMR analyses. These results help define a minimalist ramoplanin pharmacophore and introduce the possibility of generating ramoplanin-derived peptide or peptidomimetic antibiotics for use against VRE, MRSA, and related pathogens.

A crystal structure of a dimer of the antibiotic ramoplanin illustrates membrane positioning and a potential Lipid II docking interface

“…Positioning of the ligand's muramic acid group next to Hpg-7 implies that the charged side chain of Orn-4 can further stabilize the antibiotic:ligand complex by interacting with the carboxylate groups on Lipid II. Such a role would explain the 44-fold decrease in MIC associated with the ornithine-to-alanine substitution at this position (23) and is also consistent with chemical modification data that demonstrate a requirement for a positively charged side chain at position 4 (16).…”

“…For this reason the dimannosyl carbohydrate has not been included in the final refined model. The failure of the sugars to adopt a well-ordered conformation argues against any critical role for them, and in fact they do not contribute to antimicrobial activity or Lipid II binding (16,21,22,26). Indeed, the structurally related depsipeptide antibiotic enduracidin contains no carbohydrate, yet has comparable activity to that of ramoplanin (9,12).…”

“…Lipid II sequestration precludes formation of the mature, fully cross-linked peptidoglycan, leading to a mechanically weakened cell wall and bacterial death from osmotic lysis (11)(12)(13)(14). Thus, like the glycopeptide antibiotics, ramoplanin inhibits cell wall biosynthesis, but the molecular underpinnings are distinct, since the binding epitopes on Lipid II that are recognized by the two types of antibiotics are largely non-overlapping (3,4,10,15,16). This differential targeting likely explains ramoplanin's excellent activity against vancomycin-resistant microorganisms.…”

Construction of an Expression System for Site-DirectedMutagenesis of the LantibioticMersacidin

“…The mode of action of mersacidin and actagardine is not covered by any other antibiotics, since both substances interact with a novel target site that is present only on lipid II and do not bind to lipid I or intracellular peptidoglycan precursors (6). Although ramoplanin also binds to lipid II, this lipoglycodepsipeptide recognizes lipid I and UDP-N-acetylmuramylpentapeptide as well (10), and vancomycin and teicoplanin target the terminal D-Ala-D-Ala residues of the peptide side chain of lipid II. The promising activities of the lantibiotics against Staphylococcus and Streptococcus make them lead structures for the design of novel antibiotics against these problematic pathogens.…”

Cyclic Lipodepsipeptides in Novel Antimicrobial Drug Discovery

“…These common structural features indicate a possible similarity in mode of action and a common active pharmacophore. 75 The 3D NMR structure of ramoplanose revealed a double-stranded antiparallel -sheet with seven intramolecular hydrogen bonds and two reverse turns. 72 Almost identical structural characteristics were found in ramoplanin A2.…”