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We have designed a hybrid peptide by combining sequences of human ␤-defensin-1 (HBD-1) and -defensin, in an attempt to generate a molecule that combines the diversity in structure and biological activity of two different peptides to yield a promising therapeutic candidate. HBD-1 was chosen as it is a natural defensin of humans that is constitutively expressed, but its antibacterial activity is considerably impaired by elevated ionic strength. -Defensins are expressed in human bone marrow as a pseudogene and are homologous to rhesus monkey circular minidefensins. Retrocyclins are synthetic human -defensins. The cyclic nature of the -defensin peptides makes them salt resistant, nonhemolytic, and virtually noncytotoxic in vitro. However, a nonhuman circular molecule developed for clinical use would be less viable than a linear molecule. In this study, we have fused the C-terminal region of HBD-1 to the nonapeptide sequence of a synthetic retrocyclin. Cyclization was achieved by joining the terminal ends of the hybrid peptide by a disulfide bridge. The hybrid peptide with or without the disulfide bridge exhibited enhanced antimicrobial activity against both Gram-negative and Gram-positive bacteria as well as against fungi, including clinical bacterial isolates from eye infections. The peptide retained activity in the presence of NaCl and serum and was nonhemolytic in vitro. Thus, the hybrid peptide generated holds potential as a new class of antibiotics. Defensins are a group of peptides that are important components of host defense. They have the ability to recognize and neutralize invading microorganisms quickly and specifically (1-3). These peptides are diverse members of a large family of cationic host defense peptides (HDPs) that are widely distributed throughout the animal kingdom (2, 4, 5). These cysteine-rich peptides vary in their length, the spacing of their cysteine residues, and their disulfide connectivities (4, 5). Defensins show antimicrobial activities against Gram-negative and Gram-positive bacterial strains, fungi, as well as some parasites and enveloped viruses (1, 2, 4, 6). Mammalian defensins can be classified into ␣-, ␤-, and -defensins on the basis of their disulfide connectivities (2, 4, 6-8).Only ␣-and ␤-defensins are expressed in humans. -Defensin (found in rhesus monkey) displays the same connectivity associated with that of the ␣-defensins but is not functionally present in humans (7). -Defensins are much smaller (18 amino acid residues) than ␣-and ␤-defensins (29 to 45 residues), and their antiviral properties are considerably more pronounced than their antibacterial and antifungal effects (9, 10).␤-Defensins are found in epithelial cells that line mucosal surfaces and that provide the first line of defense between an organism and the environment (2, 6). To date, four human ␤-defensins (human ␤-defensin-1 [HBD-1] to HBD-4) have been characterized (5,(11)(12)(13)(14)(15)(16)(17)(18)(19). HBD-1 is expressed in epithelia that are directly exposed to the environment or microbial flora (e.g.,...

We have examined the antimicrobial activity of C-terminal analogs of human β-defensins HBD-1and-3 wherein lysines have been selectively replaced by L- and D-arginines and L-isoleucine substituted with its D-enantiomer. The analogs exhibited antibacterial and antifungal activities. Physiological concentration of NaCl did not attenuate the activity of the peptides against Gram-negative bacteria considerably, while some attenuation of activity was observed against S. aureus. Variable attenuation of activity was observed in the presence of Ca2+ and Mg2+. Introduction of D-amino acids abrogated the need for a disulfide bridge for exhibiting activity. Confocal images of carboxyfluorescein (CF) labeled peptides indicated initial localization on the membrane and subsequent translocation into the cell. Analogs corresponding to cationic rich segments of human defensins substituted with L- and D-arginine, could be attractive candidates for development as future therapeutic drugs.

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