Mechanism of action and specificity of antimicrobial peptides designed based on buforin IIb

Abstract

Buforin IIb-a synthetic analog of buforin II that contains a proline hinge between the two alpha-helices and a model a-helical sequence at the C-terminus (3 x RLLR)-is a potent cell-penetrating antimicrobial peptide. To develop novel antimicrobial peptides with enhanced activities and specificity/therapeutic index, we designed several analogs (Buf III analogs) by substitutions of amino acids in the proline hinge region and two a-helices of buforin IIb, and examined their antimicrobial activity and mechanism of action. The substitution of hydrophobic residues ([F-6] and [V-8]) in the proline hinge region with other hydrophobic residues ([W-6] and [I-8]) did not affect antimicrobial activity, while the substitution of the first four amino acids RAGL with a model alpha-helical sequence increased the antimicrobial activity up to 2-fold. Like buforin IIb, Buf III analogs penetrated the bacterial cell membranes without significantly permeabilizing them and were accumulated inside Escherichia coli. Buf III analogs were shown to bind DNA in vitro and the DNA binding affinity of the peptides correlated linearly with their antimicrobial potency. Among the Buf III analogs, the therapeutic index of Buf IIIb and IIIc (RVVRQWPIG[RVVR](3) and KLLKQWPIG[KLLK](3), respectively) were improved 7-fold compared to that of buforin IIb. These results indicate that Buf III analogs appear to be promising candidates for future development as novel antimicrobial agents. (C) 2012 Elsevier Inc. All rights reserved.