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S. Liu, L. Zhou, J. Li, C. Tang, C. Verma, D. Tan, R. Beuerman; Design of C-Terminus Analogs and Dimers of Human Defensins to Enhance Specific Pathogen Killing and Reduce Cytotoxicity to Human Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):509.
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© ARVO (1962-2015); The Authors (2016-present)
Defensins have shown great versatility in their activity against a diverse range of microorganisms. As cytotoxicity to ocular tissue is a concern, bioengineering peptide design will be necessary, as well as strategies to enhance specific pathogen killing. Using a 10-residue fragment of hBD3 as a template, we have synthesized a series of 10-residue peptides and a dimer with decreased cytotoxicity.
Eight 10-residue analogs [coded W2, F2, Y2, V2, L2, I2 and H2, C2] and V2-dimer 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 products was done by LC-MS. Antibacterial activities of these analogs were determined for P. aeruginosa, E. coli, B. cereus, S. aureus and compared with gentamicin. Cytotoxicity of the analogs was analyzed on primary cultured human normal conjunctiva epithelial cells by measuring cell viability using CellTiter-blue. HBD3 was used as a control in all analyses.
Mass spectra confirmed the molecular weight of the nine analogs. Molecular hydrophobicity values of peptides based on HPLC retention times generally matched with the theoretical values calculated based on Hoop-Woods hydrophilicity scale. The order for hydrophobicity was as follows: W2 > F2 > L2 > I2 > Y2 > V2 > C2 > H2. The cytotoxicity of four peptides was much lower than that of hBD3 in the concentration of 6-200 ug/ml. V2, W2 and Y2 and V2-dimer show specificity against P. aeruginosa, and had potent antimicrobial activity against P. aeruginosa. Antimicrobial activity of V2-dimer against P. aeruginosa ATCC 9027 (Log Reduction) was >6.54, >6.54 and 2.82 at concentration of 25, 12.5 and 6.25 ug/ml, respectively, gentamicin had a log reduction of >5.88, >5.88, and 5.88 at concentration of 50, 25, 12.5 ug/ml, respectively.
This preliminary study shows that three of eight analogs, W2, Y2 and V2 exhibited potent antimicrobial activity against P. aeruginosa with remarkably reduced cytotoxicity, V2-dimer has very potent antimicrobial activity against P. aeruginosa, which was better than or comparable to that of gentamicin. The strategy of C-terminus analogs and dimers design may pave the way for a more efficient design of clinically useful antimicrobial peptides.
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