Influence of metallocene substitution on the antibacterial activity of multivalent peptide conjugates.

Peptide dendrimers and derivatisation of peptides with metallocenes showed promising results in the search for new antibacterial agents. The two concepts are combined in this work leading to multivalent, metallocene-containing peptide derivates. These new peptides were synthesised utilising microwave assisted, copper(I) catalyzed alkyne-azide cycloaddition (CuAAC, "click" chemistry). Twelve new peptide conjugates, containing either a ferrocenoyl group or a ruthenocenoyl group on so-called ultrashort (i.e. < 5 amino acids) peptides, and ranging from monovalent to trivalent conjugates, were synthesised and their antibacterial activity was investigated by minimal inhibitory concentration (MIC) assays on five different bacterial strains. The antibacterial activity was compared to the same peptide conjugates without metallocenes. The resulting MIC values showed a significant enhancement of the antibacterial activity of these peptide conjugates against Gram-positive bacteria by the metallocenoyl groups. Additionally, the compounds with two metallocenoyl groups presented the best antibacterial activities overall.

Influence of the Multivalency of Ultrashort Arg-Trp-Based Antimicrobial Peptides (AMP) on Their Antibacterial Activity.

Peptide dendrimers are a class of molecules of high interest in the search for new antibiotics. We used microwave-assisted, copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC; "click" chemistry) for the simple and versatile synthesis of a new class of multivalent antimicrobial peptides (AMPs) containing solely arginine and tryptophan residues. To investigate the influence of multivalency on antibacterial activity, short solid-phase- synthesized azide-modified Arg-Trp-containing peptides were "clicked" to three different alkyne-modified benzene scaffolds to access scaffolds with one, two, or three peptides. The antibacterial activity of 15 new AMPs was investigated by minimal inhibitory concentration (MIC) assays on five different bacterial strains, including a multidrug-resistant Staphylococcus aureus (MRSA) strain. With ultrashort (2-3 residues) peptides, a clear synergistic effect of the trivalent display was observed, whereas this effect was not apparent with longer peptides. The best candidates showed activities in the low-micromolar range against Gram-positive MRSA. Surprisingly, the best activity against Gram-negative Acinetobacter baumannii was observed with an ultrashort dipeptide on the trivalent scaffold (MIC: 7.5 µM). The hemolytic activity was explored for the three most active peptides. At concentrations ten times the MIC values, <1 % hemolysis of red blood cells was observed.

Membrane-binding mechanism of a bacterial phospholipid N-methyltransferase.

The membrane lipid phosphatidylcholine (PC) is crucial for stress adaptation and virulence of the plant pathogen Agrobacterium tumefaciens. The phospholipid N-methyltransferase PmtA catalyzes three successive methylations of phosphatidylethanolamine to yield PC. Here, we asked how PmtA is recruited to its site of action, the inner leaflet of the membrane. We found that the enzyme attaches to the membrane via electrostatic interactions with anionic lipids, which do not serve as substrate for PmtA. Increasing PC concentrations trigger membrane dissociation suggesting that membrane binding of PmtA is negatively regulated by its end product PC. Two predicted alpha-helical regions (αA and αF) contribute to membrane binding of PmtA. The N-terminal helix αA binds anionic lipids in vitro with higher affinity than the central helix αF. The latter undergoes a structural transition from disordered to α-helical conformation in the presence of anionic lipids. The basic amino acids R8 and K12 and the hydrophobic amino acid F19 are critical for membrane binding by αA as well as for activity of full-length PmtA. We conclude that a combination of electrostatic and hydrophobic forces is responsible for membrane association of the phospholipid-modifying enzyme.