Single Amino Acid Substitutions at Specific Positions of the Heptad Repeat Sequence of Piscidin-1 Yielded Novel Analogs That Show Low Cytotoxicity and In Vitro and In Vivo Antiendotoxin Activity

Abstract

dPiscidin-1 possesses significant antimicrobial and cytotoxic activities. To recognize the primary amino acid sequence(s) in piscidin-1 that could be important for its biological activity, a long heptad repeat sequence located in the region from amino acids 2 to 19 was identified. To comprehend the possible role of this motif, six analogs of piscidin-1 were designed by selectively replacing a single isoleucine residue at a d (5th) position or at an a (9th or 16th) position with either an alanine or a valine residue. Two more analogs, namely, I5F,F6A-piscidin-1 and V12I-piscidin-1, were designed for investigating the effect of interchanging an alanine residue at a d position with an adjacent phenylalanine residue and replacing a valine residue with an isoleucine residue at another d position of the heptad repeat of piscidin-1, respectively. Single alanine-substituted analogs exhibited significantly reduced cytotoxicity against mammalian cells compared with that of piscidin-1 but appreciably retained the antibacterial and antiendotoxin activities of piscidin-1. All the single valine-substituted piscidin-1 analogs and I5F,F6A-piscidin-1 showed cytotoxicity greater than that of the corresponding alanine-substituted analogs, antibacterial activity marginally greater than or similar to that of the corresponding alanine-substituted analogs, and also antiendotoxin activity superior to that of the corresponding alanine-substituted analogs. Interestingly, among these peptides, V12I-piscidin-1 showed the highest cytotoxicity and antibacterial and antiendotoxin activities. Lipopolysaccharide (12 mg/kg of body weight)-treated mice, further treated with I16A-piscidin-1, the piscidin-1 analog with the highest therapeutic index, at a single dose of 1 or 2 mg/kg of body weight, showed 80 and 100% survival, respectively. Structural and functional characterization of these peptides revealed the basis of their biological activity and demonstrated that nontoxic piscidin-1 analogs with significant antimicrobial and antiendotoxin activities can be designed by incorporating single alanine substitutions in the piscidin-1 heptad repeat. Fish antimicrobial peptide (AMP) piscidin-1, which was discovered in 2001, possesses versatile biological activities. Piscidin-1 shows significant activity against bacteria, fungi, parasites, and cancer cells (1-7). It can also neutralize lipopolysaccharide (LPS)-induced proinflammatory responses in macrophage cells (5). Along with these desired biological activities, piscidin-1 also exhibits very significant lytic activity against normal mammalian cells, which is an obstacle for employing it as a lead molecule for the development of a new antimicrobial agent. Therefore, deciphering of the basis of cytotoxicity in piscidin-1 and the design of nontoxic analogs of piscidin-1 with desired biological activity were the objectives of the present investigation. Toward this end, we intended to identify the important primary sequence in piscidin-1 that could have a strong impact on its structural, functional, and ...

Piscidin-1-analogs with double L- and D-lysine residues exhibited different conformations in lipopolysaccharide but comparable anti-endotoxin activities

“…The localization of NBD-labeled piscidin-1, T15,21K-piscidin-1 and T15,21dK-piscidin-1 was also studied onto hRBCs by confocal microscopy14. Only NBD-labeled piscidin-1 localized effectively onto the hRBCs, as seen by the prominent green fluorescence on these cell membranes (Fig.…”

“…To comprehend the basis of cytotoxic and antibacterial activities of piscidin-1 and its analogs, peptide-induced permeabilization of mammalian membrane mimetic2728 PC/Chol (8:1 w/w) and bacterial membrane mimetic111429, PC/PG (3:1 w/w) lipid vesicles was studied. Matching with its haemolytic/cytotoxic properties, piscidin-1 induced the maximum permeabilization (expressed as the percentage of fluorescence recovery) (Fig.…”

“…The cellular localization of piscidin-1 and its analogs onto E.coli ATCC 25922 was probed by confocal scanning laser microscopy by employing NBD-labelled versions of piscidin-114 and its two least cytotoxic analogs, T15,21K-piscidin-1 and T15,21dK-piscidin-1. Confocal microscopic images of bacteria following the treatment of NBD-labeled piscidin-1 and its analogs showed appreciable green fluorescence (Fig.…”

“…Polymyxin B was taken as the positive control and the experiments were performed as reported earlier143031. We observed that experimental groups of LPS-treated (12 mg/kg) mice, further administered with single dose of T15,21K-piscidin-1 (0.5 mg/kg) or T15,21dK-piscidin-1 (1.0 mg/kg) or polymyxin B (1.0 mg/kg) resisted the lethal effects of LPS-toxicity with respect to mice groups treated with LPS (12 mg/kg) only (Fig.…”

“…NMR studies suggest that piscidins adopt an amphipathic α–helical conformation upon interaction with model membranes11 or detergent micelles512. Piscidin-1 possesses LPS-neutralizing property1314, and has potent activity against a variety of microbes, comprising of filamentous fungi, yeast, and Gram-positive and negative bacteria and their resistant versions. However, this peptide causes haemolysis of RBCs and is cytotoxic, which may limit its therapeutic applications1516171819.…”