Design of Analogs of Human Defensins to Reduce Cytotoxicity to Human Cells

Abstract

Purpose: : Defensins are small peptides able to kill a wide range of pathogens including fungi, bacteria and viruses. As cytotoxicity to ocular tissue is a concern, custom peptide design will be necessary, as well as strategies to enhance specific pathogen killing. Using human beta defensin 3 (hBD3) as a template, we have engineered a series of linear analogs with decreasing cytotoxicity.

Methods: : Six linear hBD3 analogs [coded A6, S6, C(Acm)6, Y6, F6 and W6] were synthesized by solid phase peptide synthesis using Fmoc chemistry. The purification of crude product was performed by semi–preparative HPLC. Molecular weight determination of final products was done by LC–MS. Antibacterial activities of the analogs were determined for E. coli, B. cereus, S. aureus and P. aeruginosa by measuring bacteria growth in liquid broth in the presence of the serially diluted peptides. Cytotoxicity of these analogs was analyzed on primary cultured human normal conjunctiva epithelial cells by measuring cell viability using CellTiter–blue. Wild type HBD3 was used as a control in all analyses.

Results: : Mass spectra confirmed the molecular weight of the six linear hBD3 analogs. The six peptides were classified into three groups based on their cytotoxicity and molecular hydrophobicity. Molecular hydrophobicity values of all peptides based on experimental HPLC retention times matched well with the theoretical hydrophobicity values calculated based on the hydrophobicity scale of Wimley and White. The cytotoxicity of the analogs correlated with the calculated hydrophobicity trend. A6 S6 and C(Acm)6 with low hydrophobicity had the lowest cytotoxicity at a high concentration of 25–100 ug/ml, Y6 with medium hydrophobicity had mid–level cytotoxicity while F6 and W6 with high hydrophobicity had relatively the highest cytotoxicity. The cytotoxicity of each of the six linear peptides was much lower than that of wild–type hBD3 in the concentration of 6–100 ug/ml.

Conclusions: : This preliminary study shows that these six linear hBD3 analogs exhibited reasonable antimicrobial activity with remarkably reduced cytotoxicity, which has positive implications for therapeutic development.

Keywords: antibiotics/antifungals/antiparasitics • wound healing • conjunctiva