anoplin and Hemolysis

Anoplin is a short natural cationic antimicrobial peptide which is derived from the venom sac of the solitary wasp, Anoplius samariensis. Due to its short sequence G

Topics: Amino Acid Substitution; Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Candida; Erythrocytes; Gram-Negative Bacteria; Gram-Positive Bacteria; Hemolysis; Humans; Hydrophobic and Hydrophilic Interactions; Insect Proteins; Microbial Sensitivity Tests; Protein Stability; Protein Structure, Secondary; Proteolysis; Solid-Phase Synthesis Techniques; Structure-Activity Relationship; Wasp Venoms; Wasps

Here we report that grafting of a short antimicrobial peptide, anoplin, to chitosan polymers is a strategy for abolishing the hemolytic propensity, and at the same time increasing the activity of the parent peptide. Anoplin-chitosan conjugates were synthesized by CuAAC reaction of multiple peptides through 2-azidoacetyl groups on chitosan.

Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Chitosan; Enterococcus faecalis; Erythrocytes; Escherichia coli; Hemolysis; Humans; Microbial Sensitivity Tests; Organosilicon Compounds; Staphylococcus aureus; Wasp Venoms

Microbial resistance is an increasing health concern and a true danger to human well-being. A worldwide search for new compounds is ongoing, and antimicrobial peptides are promising lead candidates for tomorrow's antibiotics. The decapeptide anoplin (GLLKRIKTLL-NH2) is an especially interesting candidate because of its small size as well as its antimicrobial and nonhemolytic properties. Optimization of the properties of an antimicrobial peptide such as anoplin requires multidimensional searching in a complex chemical space. Typically, such optimization is performed by labor-intensive and costly trial-and-error methods. In this study, we show the benefit of fractional factorial design for identification of the optimal antimicrobial peptide in a combination matrix. We synthesized and analyzed a training set of 12 anoplin analogs, representative of 64 analogs in total. Using MIC, hemolysis, and high-performance liquid chromatography retention time data, we constructed analysis-of-variance models that describe the relationship between these properties and the structural characteristics of the analogs. We show that the mathematical models derived from the training set data can be used to predict the properties of other analogs in the chemical space. Hence, this method provides an efficient means of identification of the optimal peptide in the searched chemical space.

Topics: Algorithms; Amino Acid Sequence; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Candida albicans; Chromatography, High Pressure Liquid; Enterococcus faecium; Erythrocytes; Escherichia coli; Factor Analysis, Statistical; Hemolysis; Humans; Hydrophobic and Hydrophilic Interactions; Microbial Sensitivity Tests; Molecular Sequence Data; Pseudomonas aeruginosa; Staphylococcus aureus; Wasp Venoms

Isolated from the venom sac of solitary spider wasp, Anoplius samariensis, anoplin is the smallest linear α-helical antimicrobial peptide found naturally with broad spectrum activity against both Gram-positive and Gram-negative bacteria, and little hemolytic activity toward human erythrocytes. Deamidation was found to decrease the peptide's antibacterial properties. In the present work, interactions of amidated (Ano-NH2) and deamidated (Ano-OH) forms of anoplin as well as Ano-NH2 composed of all D-amino acids (D-Ano-NH2) with model cell membranes were investigated by means of Langmuir Blodgett (LB) technique, atomic force microscopy (AFM), X-ray photoemission electron microscopy (X-PEEM) and carboxyfluorescein leakage assay in order to gain a better understanding of the effect of these peptide modifications on membrane binding and lytic properties. According to LB, all three peptides form stable monolayers at the air/water interface with Ano-NH2 occupying a slightly greater area per molecule than Ano-OH. All three forms of the peptide interact preferentially with anionic 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG), rather than zwitterionic 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid monolayer. Peptides form nanoscale clusters in zwitterionic but not in anionic monolayers. Finally, membrane lytic activity of all derivatives was found to depend strongly on membrane composition and lipid/peptide ratio. The results suggest that amidated forms of peptides are likely to possess higher membrane binding affinity due to the increased charge.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Amides; Amino Acid Substitution; Animals; Antimicrobial Cationic Peptides; Bacillus subtilis; Cell Membrane; Escherichia coli; Fluoresceins; Hemolysis; Humans; Hymenoptera; Lipid Bilayers; Microscopy, Atomic Force; Microscopy, Electron; Phosphatidylglycerols; Protein Binding; Wasp Venoms

We have synthesised nine analogues of the antibacterial peptide anoplin with a peptoid residue at position 5 (H-GLLKXIKTLL-NH(2)). The most active compounds showed MIC-values of 12.5 and 25 microM against E.coli and S.aureus. These MIC-values are comparable with anoplin which showed 23 microM and 11 microM against E. coli and S.aureus. However, the selectivity was reversed. Our results indicate that peptoid analogues of anoplin are promising lead structures for developing new antibacterial agents.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Circular Dichroism; Escherichia coli; Hemolysis; Humans; Microbial Sensitivity Tests; Peptoids; Staphylococcus aureus; Wasp Venoms

Anoplin is a decapeptide amide, GLLKRIKTLL-NH2 derived from the venom sac of the solitary spider wasp, Anoplius samariensis. It is active against Gram-positive and Gram-negative bacteria and is not hemolytic towards human erythrocytes. The present paper reports a structure-activity study of anoplin based on 37 analogues including an Ala-scan, C- and N-truncations, and single and multiple residue substitutions with various amino acids. The analogues were tested for antibacterial activity against both S. aureus ATCC 25923 and E. coli ATCC 25922, and several potent antibacterial analogues were identified. The cytotoxicity of the analogues against human erythrocytes was assessed in a hemolytic activity assay. The antibacterial activity and selectivity of the analogues against S. aureus and E. coli varied considerably, depending on the hydrophobicity and position of the various substituted amino acids. In certain cases the selectivity for Gram-positive and Gram-negative bacteria was either reversed or altogether eliminated. In addition, it was generally found that antibacterial activity coincided with hemolytic activity.

Topics: Amino Acids; Antimicrobial Cationic Peptides; Circular Dichroism; Drug Design; Escherichia coli; Hemolysis; Humans; Hydrophobic and Hydrophilic Interactions; Staphylococcus aureus; Structure-Activity Relationship; Wasp Venoms