Design of potent, non-toxic antimicrobial agents based upon the structure of the frog skin peptide, pseudin-2.
Pseudin-2, a naturally occurring 24 amino-acid-residue antimicrobial peptide first isolated from the skin of the South American paradoxical frog Pseudis paradoxa, has weak hemolytic and cytolytic activity but also relatively low potency against microorganisms. In a membrane-mimetic environment, the peptide exists in an amphipathic alpha-helical conformation. Analogs of the peptide with increased cationicity and alpha-helicity were chemically synthesized by progressively substituting neutral and acidic amino acid residues on the hydrophilic face of the alpha-helix by lysine. Analogs with up to three L-lysine substitutions showed increased potency against a range of gram-negative and gram-positive bacteria (up to 16-fold) whilst retaining low hemolytic activity. The analog [D-Lys3, D-Lys10, D-Lys14]pseudin-2 showed potent activity against gram-negative bacteria (minimum inhibitory concentration, MIC=5 microM against several antibiotic-resistant strains of Escherichia coli) but very low hemolytic activity (HC50>500 microM) and cytolytic activity against L929 fibroblasts (LC50=215 microM). Increasing the number of l-lysines to four and five did not enhance antimicrobial potency further but increased hemolytic activity towards human erythrocytes. Time-kill studies demonstrated that the analog [Lys3, Lys10, Lys14, Lys21]pseudin-2 at a concentration of 1 x MIC was bacteriocidal against E. coli (99.9% cell death after 96 min) but was bacteriostatic against S. aureus. Increasing the hydrophobicity of pseudin-2, while maintaining the amphipathic character of the molecule, by substitution of neutral amino acids on the hydrophobic face of the alpha-helix by L-phenylalanine, had only minor effects on antimicrobial and hemolytic activities.
Pál
Tibor
T
Department of Medical Microbiology, Faculty of Medicine and Health Sciences, United Arab Emirates University, PO Box 17666 Al-Ain, United Arab Emirates.
Sonnevend
Agnes
A
Galadari
Sehamuddin
S
Conlon
J Michael
JM
D029845:Amphibian Proteins / Q000737:chemistry* / Q000633:toxicity; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000633:toxicity; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry* / Q000633:toxicity; D002460:Cell Line; D006090:Gram-Negative Bacteria / Q000254:growth & development; D006094:Gram-Positive Bacteria / Q000254:growth & development; D051379:Mice; D008826:Microbial Sensitivity Tests; D017433:Protein Structure, Secondary; D011898:Ranidae; D013329:Structure-Activity Relationship
D029845:Amphibian Proteins; D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; C441477:pseudin-2 protein, Pseudis paradoxa