Chimeric Beta-Defensin Analogs, Including the Novel 3NI Analog, Display Salt-Resistant Antimicrobial Activity and Lack Toxicity in Human Epithelial Cell Lines

Abstract

i Human beta-defensins (hBDs) are crucial peptides for the innate immune response and are thus prime candidates as therapeutic agents directed against infective diseases. Based on the properties of wild-type hBD1 and hBD3 and of previously synthesized analogs (1C, 3I, and 3N), we have designed a new analog, 3NI, and investigated its potential as an antimicrobial drug. Specifically, we evaluated the antimicrobial activities of 3NI versus those of hBD1, hBD3, 1C, 3I, and 3N. Our results show that 3NI exerted greater antibacterial activity against Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecalis than did hBD1 and hBD3, even with elevated salt concentrations. Moreover, its antiviral activity against herpes simplex virus 1 was greater than that of hBD1 and similar to that of hBD3. Subsequently, we investigated the cytotoxic effects of all peptides in three human epithelial carcinoma cell lines: A549 from lung, CaCo-2 from colon, and Capan-1 from pancreas. None of the analogs significantly reduced cell viability versus wild-type hBD1 and hBD3. They did not induce genotoxicity or cause an increase in the number of apoptotic cells. Using confocal microscopy, we also investigated the localization of the peptides during their incubation with epithelial cells and found that they were distributed on the cell surface, from which they were internalized. Finally, we show that hBD1 and hBD3 are characterized by high resistance to serum degradation. In conclusion, the new analog 3NI seems to be a promising anti-infective agent, particularly given its high salt resistance-a feature that is relevant in diseases such as cystic fibrosis.

Design and activity of a cyclic mini-β-defensin analog: a novel antimicrobial tool

“…We used two concentrations of peptides, ie, 2.5 µM and 12.5 µM. For salt-dependence assay, 0, 50, 100, and 200 mM NaCl was included in the incubation PBS buffer for 2 hours at 37°C under non-proliferating conditions (see also Scudiero et al 7 and Scudiero et al 8 )…”

“…8 Supernatants were analyzed by LC/MS using a combined system constituted by a reversephase HPLC (Gilson, Middleton, USA) interfaced with a Flexar SQ 300 mass spectrometer with electrospray ionization (PerkinElmer Inc.). Components were separated using a reverse-phase Jupiter C 18 analytical column (5 µm particle size, 300 Å pore size, 4.6×250 mm) (Phenomenex) with a linear gradient of 5%-60% acetonitrile in acidified water (0.1% TFA) over 30 minutes.…”

“…5,6 In this scenario, we previously designed a number of β-defensin analogs that have increased potency and/or reduced sensitivity to high ionic strength, and investigated their antibacterial, antiviral, and chemotactic activities, as well as their salt resistance. 7,8 The results showed that the charged C-terminal domain (RRKK) of hBD3 and the internal domain of hBD1 (PIFTKIQGT) are crucial for defensin activity and that deletion of six residues at the N-terminus of hBD3 did not reduce the activity. 7,8 Consequently, the internal region of hBD1 and the C-terminus of hBD3 are attractive domains in terms of engineering functional truncated defensin analogs.…”

“…7,8 The results showed that the charged C-terminal domain (RRKK) of hBD3 and the internal domain of hBD1 (PIFTKIQGT) are crucial for defensin activity and that deletion of six residues at the N-terminus of hBD3 did not reduce the activity. 7,8 Consequently, the internal region of hBD1 and the C-terminus of hBD3 are attractive domains in terms of engineering functional truncated defensin analogs. Notwithstanding these promising features, natural defensins have several properties that detract from their suitability as drug candidates, such as the fact that they are rapidly cleaved in human fluids by proteases.…”

“…7,8 We tested two concentrations of each peptide (2.5 and 12.5 µM) and four concentrations of NaCl (0, 50, 100, and 200 mM) against P. aeruginosa, E. coli, and E. faecalis, which are the most frequent causes of many human infections. Firstly, we tested the antibacterial properties of AMC in the reduced and oxidized forms against E.coli.…”