Synthesis and biological characterization of retro-inverse analog of antimicrobial (RLFR)₃ peptide

The emerging bacterial resistance to conventional antibiotics has led to the search for new therapeutic alternatives. Antimicrobial cationic peptides are promising candidates, since they act on bacterial membranes causing their rapid destruction, with a low tendency to generate resistance. However, these compounds present low stability against proteases. This study aimed to design a retro inverse analog containing D amino acids of TA4R (RLFR)3 to evaluate the effect of substitution by D amino acids and inverted sequence on their biological properties and enzymatic stability.

For this, RI-dTA4R was synthesized by Fmoc chemistry. The Minimal inhibitory concentration (MIC) was determined against the following bacterial strains: Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25929, Staphylococcus aureus Methicillin Resistant SAMR1. Hemolytic activity and enzymatic stability were determined against human erythrocytes and digestive and serum proteases, respectively. DC analyses were performed in aqueous media and membrane mimetic vesicles.

RI-dTA4R showed antimicrobial activity against all bacteria strains tested (MIC=4.6 to 2.3 µM), increasing the inhibitory activity of TA4R, and improving their enzymatic stability against digestive and serum proteases. Nevertheless, this analog was hemolytic (~50% of hemolysis at 25 µM), but at MIC concentration presented less than 20% of hemolysis. CD analysis showed that the peptide is strongly structured in DPPG vesicles, adopting a levorotatory helical structure.

Based on these results, RI-dTA4R may be considered a potential therapeutic compound for treating infections produced by gram (+) and (-) bacteria with improved enzymatic stability.