Backbone Cyclization and Dimerization of LL-37-Derived Peptides Enhance Antimicrobial Activity and Proteolytic Stability.

Sunithi Gunasekera,Taj Muhammad,Adam A Strömstedt,Ulf Göransson,K Johan Rosengren

doi:10.3389/fmicb.2020.00168

Sunithi Gunasekera, Taj Muhammad + Show 3 more

Open Access

https://doi.org/10.3389/fmicb.2020.00168

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Abstract

Can antimicrobial activity and peptide stability of alpha-helical peptides be increased by making them into dimers and macrocycles? Here, we explore that concept by using KR-12 as the starting point for peptide engineering. KR-12 has previously been determined as the minimalized antimicrobial fragment of the human host defense peptide LL-37. Backbone-cyclized KR-12 dimers, tethered by linkers of two to four amino acid residues, were synthesized and their antimicrobial activity, proteolytic stability and structures characterized. A modified KR-12 sequence, with substitutions at previously identified key residues, were also included in the screening panel. The backbone cyclized KR-12 dimers showed improved antimicrobial activity and increased stability compared to monomeric KR-12. The most active cyclic dimer displayed 16-fold higher antibacterial activity compared to KR-12 against Pseudomonas aeruginosa and Staphylococcus aureus, and 8-fold increased fungicidal activity against Candida albicans. It also showed increased hemolytic and cytotoxic activity. Enhanced antimicrobial activity coincided with increased membrane permeabilization of liposomes with one distinct discrepancy: monomeric KR-12 was much less disruptive of liposomes with bacterial lipid composition compared to liposomes from fungal lipid extract. Circular dichroism showed that the four-residue linked most active cyclic dimer had 65% helical content when bound to lyso-phosphatidylglycerol micelles, indicating that the helical propensity of the parent peptide is maintained in the new macrocyclic form. In conclusion, the current work on KR-12 suggests that dimerization together with backbone cyclization is an effective strategy for improving both potency and stability of linear antimicrobial peptides.

Highlights

Antimicrobial peptides (AMPs) are gaining interest for antibiotic discovery and development, but despite natural functions as host defense peptides they come with inherent challenges in stability and potency
A series of KR-12 derivatives were synthesized to investigate whether the antimicrobial potency and biological stability can be enhanced by dimerization and head-to-tail cyclization
The design strategy included tandem repeats and reverse tandem repeats

Summary

Introduction

Antimicrobial peptides (AMPs) are gaining interest for antibiotic discovery and development, but despite natural functions as host defense peptides they come with inherent challenges in stability and potency. KR-12 is the shortest antimicrobial epitope of the human host defense peptide LL-37. LL-37 is reported to inhibit biofilms (Overhage et al, 2008; Mishra et al, 2016), induce chemotaxis of immune cells (Agerberth et al, 2000), and promote wound healing (Heilborn et al, 2003; Koczulla et al, 2003). These activities have led LL-37 into the clinic: topical treatment of venous leg ulcers with

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