Engineering of KR-12: A minimalized domain derived from human host defense peptide LL-37 into a potent antimicrobial drug lead

The human cathelicidin LL-37 is a multifunctional host defence molecule that mediates various host responses including antimicrobial action, chemotaxis, epithelial cell activation, angiogenesis, and activation of chemokine secretion [1]. However, LL-37 encounters the generic problems common to most linear counter parts such as short biological half-life, primarily arising from proteolytic susceptibility. A minimalized bacteriolytic domain of LL-37, referred to as KR-12, has selective toxicity toward bacteria [2]. In our previous work, we have performed alanine/lysine scans on KR-12 to identify critical residues for antimicrobial activity. Using the insights obtained from the previous studies, we designed 2^nd generation cyclic analogs to improve peptide stability further and reduce cytotoxicity. The stable peptide leads were engineered by using a combination of peptide stabilization approach, namely backbone cyclization, disulfide bond formation and dimerization. The new peptide analogues showed more potent antimicrobial activity against all the tested strains (E. coli, P. aeruginosa, S. aureus and C. albicans) than the 1^st generation cyclic analogs. The activity was equivalent to the parent peptide LL-37. Moreover, the new series of peptides showed more potency on E. coli membrane permabilization as compared to the 1^st generation cyclic analogs. Notably, NMR analysis revealed that the 2^nd generation cyclic analogs were unstructured, presumably because backbone cyclization restricts conformational freedom. In addition to increasing our understanding of the structure activity relationship of KR-12, this study highlights several viable peptide stabilization strategies to improve both potency and stability of the enzyme susceptible KR-12, which could serve as a template for novel antibiotic development.

Acknowledgements: Swedish NMR Centre Gothenburg.

Keywords: LL-37, KR-12, dimerization, membrane permabilization, NMR.

References:

[1] Patricia MS. The human cathelicidin hCAP18/LL-37: A multifunctional peptide involved in mycobacterial infections. Peptides 2010; 31: 1791 – 1798

[2] Wang G. Structures of human host defense cathelicidin LL-37 and its smallest antimicrobial peptide KR-12 in lipid micelles. J Biol Chem 2008; 283: 32637 – 32643