Novel Synthetic, Salt-Resistant Analogs of Human Beta-Defensins 1 and 3 Endowed with Enhanced Antimicrobial Activity

Abstract

Human beta-defensins (hBDs) are antimicrobial peptides of human innate immunity. The antibacterial activities of hBDs 1, 2, and 4 but not the activity of hBD3 are impaired by high salt levels. We have designed and synthesized seven novel hBD analogs, constituted by different domains of hBD1 (which is constitutively expressed in humans) and of hBD3 (which is induced by microorganisms and inflammatory factors in humans), that would maintain and potentially increase the wild-type antimicrobial activities and be salt resistant. We have compared the antibacterial, antiviral, and chemotactic activities of the analogs with those of hBD1 and hBD3. We show that the hBD1 internal region and the hBD3 C-terminal region are critical for antibacterial activity also at high salt concentrations, whereas deletion of the N-terminal region of hBD3 results in an increase in antibacterial activity. All analogs inhibited herpes simplex virus; antiviral activity was enhanced by the hBD1 internal region and the hBD3 C-terminal region. Wild-type and analog peptides were chemotactic for granulocytes and monocytes, irrespective of the salt concentrations. These new peptides may have therapeutic potential.

Cytokeratins mediate epithelial innate defense through their antimicrobial properties

“…The bactericidal activity of synthetic K6A fragments was tested using a physiological salt (NaCl) concentration (150 mM) and a "lawn"/biofilmlike culture of bacteria. These conditions are known to reduce the activity of known cationic antimicrobial peptides, such as human β-defensins-1, -2, and -4 and cathelicidin LL-37 (23,24). The 19-mer KDAMP was almost equally effective in killing the cytotoxic P. aeruginosa clinical isolate 6206 in 150 mM NaCl solution or in water, in both cases reducing bacterial viable counts by approximately 3 to 4 logs after 3 hours at 200 μg/ml (P < 0.005 in each instance) ( Figure 3A).…”

Design and activity of a cyclic&nbsp;mini-&beta;-defensin analog: a novel antimicrobial tool

“…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.…”

“…These results confirm our previous results obtained with hBD1 and hBD3. 7 At all peptide concentrations used, cell viability was only slightly reduced after 24 hours (5%-20%), whereas it was reduced by approximately 25% after 48 and 72 hours ( Figure 3A). Moreover, we performed toxicity assays at a peptide concentration of 125 µM for 8, 24, and 72 hours of incubation ( Figure 3B).…”

“…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 )…”