Enhancement of the antimicrobial properties of KLR12 through charge addition and amino acid rearrangement

Abstract

 Background: Understanding structure-function relationships is critical to the rational design of antimicrobial peptides with enhanced activity and specificity for therapeutic applications. In particular, α-helical peptides, known for their broad-spectrum antibacterial activity, can be modifying physicochemical properties such as charge, hydrophobicity, and amphipathicity.

Objective: This study aims to enhance the antimicrobial potency of KLR12by modifying its charge and amino acid rearrangement.

Methods: Native KLR 12 and its derivatives, KLR12-4R and KLR12-4R,7L, were designed and analysed for their physicochemical properties using prediction tools, APD3, BACHEM, HeliQuest, MACREL, QSPpred, and AMPfun. Circular dichroism (CD) spectroscopy was used to assess secondary structure. Antibacterial activity against Staphylococcus aureus (S. aureus) ATCC 25923 and Escherichia coli (E. coli) ATCC 25922 was determined by broth microdilution, and cytotoxicity in L929 cells was evaluated in vitro.

Results: Prediction scores indicated that all peptides have broad-spectrum potential with low cytotoxicity. Helical structures were observed in the presence of sodium dodecyl sulphate (SDS) solution. In vitro studies illustrated that the KLR 12-4R, exhibited twice the activity against S. aureus but was not effective against E. coli compared to native KLR 12. Additionally, KLR12-4R,7L demonstrated 16- and 4-fold higher activity against S. aureus and E. coli, respectively, compared to the native KLR12. Furthermore, cytotoxicity in L929 cells was observed in a dosedependent manner.

Conclusion: Rational design of amphipathic KLR peptides led to enhanced antimicrobial potency. Among the three peptides, KLR12-4R,7L exhibited the highest potency. These peptides represent promising candidates for future drug development against infectious diseases.