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Antimicrobial Agents and Chemotherapy, February 2009, p. 593-602, Vol. 53, No. 2
0066-4804/09/$08.00+0     doi:10.1128/AAC.00477-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Evaluation of Strategies for Improving Proteolytic Resistance of Antimicrobial Peptides by Using Variants of EFK17, an Internal Segment of LL-37{triangledown} ,{dagger}

Adam A. Strömstedt,1* Mukesh Pasupuleti,2 Artur Schmidtchen,2 and Martin Malmsten1

Department of Pharmacy, Uppsala University, Uppsala, Sweden,1 Department of Dermatology and Venereology, Lund University, Lund, Sweden2

Received 10 April 2008/ Returned for modification 7 October 2008/ Accepted 18 November 2008

Methods for increasing the proteolytic stability of EFK17 (EFKRIVQRIKDFLRNLV), a new peptide sequence with antimicrobial properties derived from LL-37, were evaluated. EFK17 was modified by four d-enantiomer or tryptophan (W) substitutions at known protease cleavage sites as well as by terminal amidation and acetylation. The peptide variants were studied in terms of proteolytic resistance, antibacterial potency, and cytotoxicity but also in terms their adsorption at model lipid membranes, liposomal leakage generation, and secondary-structure behavior. The W substitutions resulted in a marked reduction in the proteolytic degradation caused by human neutrophil elastase, Staphylococcus aureus aureolysin, and V8 protease but not in the degradation caused by Pseudomonas aeruginosa elastase. For the former two endoproteases, amidation and acetylation of the terminals also reduced proteolytic degradation but only when used in combination with W substitutions. The d-enantiomer substitutions rendered the peptides indigestible by all four proteases; however, those peptides displayed little antimicrobial potency. The W- and end-modified peptides, on the other hand, showed an increased bactericidal potency compared to that of the native peptide sequence, coupled with a moderate cytotoxicity that was largely absent in serum. The bactericidal, cytotoxic, and liposome lytic properties correlated with each other as well as with the amount of peptide adsorbed at the lipid membrane and the extent of helix formation associated with the adsorption. The lytic properties of the W-substituted peptides were less impaired by increased ionic strength, presumably by a combination of W-mediated stabilization of the largely amphiphilic helix conformation and a nonelectrostatic W affinity for the bilayer interface. Overall, W substitutions constitute an interesting means to reduce the proteolytic susceptibility of EFK17 while also improving antimicrobial performance.

* Corresponding author. Mailing address: Department of Pharmacy, Uppsala University, Husargatan 3, Box 580, Uppsala, Sweden. Phone: 46-18-4714370. Fax: 46-18-4714377. E-mail: adam.stromstedt{at}farmaci.uu.se

{triangledown} Published ahead of print on 24 November 2008.

{dagger} Supplemental material for this article may be found at http://aac.asm.org/.

Antimicrobial Agents and Chemotherapy, February 2009, p. 593-602, Vol. 53, No. 2
0066-4804/09/$08.00+0     doi:10.1128/AAC.00477-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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