Nigro, Ersilia (2011) “Antimicrobial Activity and Cell Toxicity of Human Beta Defensins and Their Newly Synthesized Analogs”. [Tesi di dottorato] (Unpublished)
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|Item Type:||Tesi di dottorato|
|Uncontrolled Keywords:||Antimicrobial peptides, Defensins,Defensin Analogs|
|Date Deposited:||09 Dec 2011 12:25|
|Last Modified:||17 Jun 2014 06:03|
Human Beta Defensins (hBD-s) are a class of antimicrobial peptides (AMPs) that play an important role both in innate and adaptive immunity. In fact, they directly kill bacteria (Gram+, -), viruses and fungi and exert chemotaxis against dendritic cells, lymphocytes and monocytes. Thanks to their antimicrobial and chemotactic activity, hBD-s represent a good target to develop new therapeutic molecules (Scudiero et al. 2010). Previously, Scudiero et al. synthesized the wild type hBD-1, hBD-3 and designed eight analogs selecting the sequences of hBD-1 and -3 that are crucial tracts for their activity. They demonstrated that the C-terminal domain of hBD-3 and the internal portion of hBD-1 are necessary for the antibacterial, antiviral and chemotactic activity; they demonstrated that 1C and 3N are the only two analogs with higher activity respect to the wild type confirming that this is a potent strategy to develop new pharmacological molecules. In the present study, we tested the possibility to find a new defensin analog with enhanced antimicrobial properties. We designed a new peptide, 3NI, with the following features: (a) shorter total sequence in order to provide greater stability and ability to penetrate bacteria and viruses; (b) presence of the C-terminus portion of hBD-3 selected for the antimicrobial activity; (c) presence of inner portion of hBD-1 selected for the chemotactic activity. At this aim, we used 3NI to perform antibacterial tests on E.coli, P. aeruginosa, E. faecalis and demonstrated that it suppresses bacterial growth already at a concentration of 12.5 M. We also tested the antiviral activity on herpes simplex virus type 1 and found that 3NI suppresses the infectivity at a concentration of 50 M. Therefore, our results indicate that 3NI analog represents a new attractive target for therapeutic approaches. In addition, we investigated two others pharmacological properties of these peptides: the stability in human serum and the potential cytotoxic effects using human cell lines. We demonstrated that hBD-3, used as a reference, is a peptide with pronounced stability; in fact it resulted not degraded after 1 hour of serum incubation and with a degradation rate of 45% after 24 hours. Successively, to evaluate the cytotoxic effects of hBD-1, hBD-3 and 1C, 3N and 3NI analogs, we tested cell viability, apoptosis and DNA damage using three human epithelial cell lines: lung carcinoma (A549), colon carcinoma (CaCo-2) and pancreas adenocarcinoma (Capan-1). In all cell lines, we demonstrated that the peptides cause 30% reduction of cell viability and 20% of apoptosis only after 72 hours treatment. In addition, we demonstrated that the peptides do not induce DNA damage. Finally, in order to investigate whether defensins interact with plasma membranes and penetrate into human epithelial cell lines, we labeled our peptides with NBD fluorochrome and performed confocal microscopy experiments. We observed that peptides bind to plasma membranes and penetrate into A549 and CaCo-2 cells through active mechanisms involving early endosomes. We observed that they are partially digested through lysosomes. Altogether our results confirm that the beta defensins analogs are attractive factors to develop new antimicrobial molecules with therapeutic applicability for the treatment of infectious diseases. In fact, 1C, 3N and 3NI are analogs that show an increased antimicrobial activity compared to wild type defensins, do not exert relevant cytotoxic effects and have pronounced stability in human serum.
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