Linear Analogues of Human β‐Defensin 3: Concepts for Design of Antimicrobial Peptides with Reduced Cytotoxicity to Mammalian Cells

Abstract

A series of engineered linear analogues [coded as F6, W6, Y6, A6, S6 and C(Acm)6] were modeled, designed, synthesized and structurally characterized by mass spectra, circular dichroism, hydrophobicity analysis and molecular modeling. We have screened antimicrobial activity, hemolysis to rabbit erythrocytes, and cytotoxicity to human conjunctival epithelial cells. No significant hemolytic effect was observed for hBD3 or from five of the six analogues [F6, Y6, A6, S6 and C(Acm)6] over the range of 3-100 microg mL(-1). The six linear analogues have reduced cytotoxicity to human conjunctival epithelial cells over the range of 6-100 microg mL(-1) compared to hBD3. By tuning the overall hydrophobicity of linear hBD3 analogues, reduced cytotoxicity and hemolysis were obtained while preserving the antimicrobial properties. The decreased cytotoxicity of the linear analogues is suggested to be structurally related to the removal of disulfide bridges, and the flexible structure of the linear forms, which seem to be associated with loss of secondary structure. These results suggest a new approach for guiding the design of new linear analogues of defensin peptides with strong antibiotic properties and reduced cytotoxicity to mammalian cells.

Native Thrombocidin-1 and Unfolded Thrombocidin-1 Exert Antimicrobial Activity via Distinct Structural Elements

“…Similar conformational changes have been reported for hBD-3 and GCP-2/CXCL6 in membranemimicking conditions (14,15,44). It has been suggested that these structural modifications are involved in the mode of action of antimicrobial proteins (14,15).…”

“…Several antimicrobial peptides and antimicrobial chemokines retain activity as linearized molecules despite the loss of their characteristic ␤-sheet structures (14,15). This questions the requirement of the three-dimensional patch of antimicro- bial chemokines and shows that other structural elements can also be sufficient for antimicrobial activity of these proteins.…”

“…Moreover, reduction and unfolding are even required to reveal the full antimicrobial activity of human ␤-defensin 1 (13). Upon linearization, these proteins can lose their characteristic ␤-sheet secondary structures (14,15) that will substantially affect their three-dimensional structure (16). These observations question the necessity of the three-dimensional positive patch for antimicrobial activity and suggest that other structural elements, at least in the linearized proteins, are involved in this antimicrobial activity.…”

Progressive Structuring of a Branched Antimicrobial Peptide on the Path to the Inner Membrane Target

“…B2088 and the monomer peptide were incubated with a human conjunctiva epithelial cell line (IOBA-NHC) as described previously (31,32). The luminescent signal was read by a microplate reader (Tecan GeniosPro, Tecan Asia, Singapore).…”

Pulmonary Surfactant Protein A Protects Lung Epithelium from Cytotoxicity of Human β-Defensin 3

“…Several reports have indicated a correlation between hydrophobicity of AMPs and cytotoxicity (31)(32)(33). Consistent with the proposed hydrophobicity-cytotoxicity relationship, more hydrophobic analogous peptides of hBD3 exhibit greater cytotoxic activity (10,34,35). With respect to the structural aspect, the solution structure of hBD3 is characterized by an amphipathic dimeric structure, which is different from those of hBD1 and hBD2 (36).…”