Host-defense cationic antimicrobial peptides (Ϸ12-50 aa long) play an essential protective role in the innate immune system of all organisms. Lipopeptides, however, are produced only in bacteria and fungi during cultivation, and they are composed of specific lipophilic moieties attached to anionic peptides (six to seven amino acids). Here we report the following. (i) The attachment of an aliphatic chain to otherwise inert, cationic D,L tetrapeptides endows them with potent activity against various microorganisms including antibiotic resistance strains. (ii) Cell specificity is determined by the sequence of the short peptidic chain and the length of the aliphatic moiety. (iii) Despite the fact that the peptidic chains are very short, their mode of action involves permeation and disintegration of membranes, similar to that of many long antimicrobial peptides. Besides adding important information on the parameters necessary for host-defense lipopeptides to kill microorganisms, the simple composition of these lipopeptides and their diverse specificities should make them economically available, innate immunity-mimicking antimicrobial and antifungal compounds for various applications.antimicrobial peptides ͉ innate immunity ͉ peptide-membrane interaction ͉ carpet model ͉ lytic peptides

Antimicrobial lipopeptides are produced nonribosomally in bacteria and fungi during cultivation. They are composed of a cationic or an anionic peptide covalently bound to a specifically modified aliphatic chain. Most of the peptidic moieties have complex cyclic structures. Here we report that conjugation of a palmitic acid to the N-terminus of very short cationic di- and tripeptides composed of all l- and d, l-amino acids endowed them with potent antimicrobial activities. Interestingly, cell specificity was determined by the sequence of the short peptidic chain. Palmitoyllysine served as a control and was inactive toward all microorganisms tested. Replacing an l-amino acid with its d-enantiomer did not affect the activity of the corresponding lipopeptides. Importantly, selected lipopeptides were also potent in vivo in a mouse model of Candida albicans infection. Bacterial leakage experiments and negative staining electron microscopy suggest that their mode of action involves permeation and disintegration of the microorganism's membrane, similar to many long antimicrobial peptides and lipopeptides. Interestingly, each lipopeptide assembled in solution into a nanostructure with a unique morphology which could partially explain differences in their biological activity. Besides adding important information on the parameters necessary for antimicrobial lipopeptides to kill microorganisms, the simple composition of these minilipopeptides and their diverse cell specificities make them attractive candidates for various applications.

We report on a short host defense-like peptide that targets and arrests the growth of aggressive and hormone-resistant primary human prostate and breast tumors and prevents their experimental and spontaneous metastases, respectively, when systemically inoculated to immuodeficient mice. These effects are correlated with increased necrosis of the tumor cells and a significant decrease in the overall tumor microvessel density, as well as newly formed capillary tubes and prostate-specific antigen secretion (in prostate tumors). Growth inhibition of orthotopic tumors derived from stably transfected highly fluorescent human breast cancer cells and prevention of their naturally occurring metastases were visualized in real time by using noninvasive whole-body optical imaging. The exclusive selectivity of the peptide towards cancer derives from its specific binding to surface phosphatidylserine and the killing of the cancer cells via cytoplasmic membrane depolarization. These data indicate that membrane disruption can provide a therapeutic means of inhibiting tumor growth and preventing metastases of various cancers. (Cancer Res 2006; 66(10): 5371-8)

Previously, we reported that intratumor or systemic inoculation of a cationic 15-mer, innate immunity-like lytic peptide composed of D-and L-amino acids ([D]-K 6 L 9 ) caused growth arrest of 22RV1 prostate carcinoma xenografts in a mouse model. However, despite its therapeutic potential, this peptide has significant systemic toxicity at concentrations slightly higher than the therapeutic one. Here, we used the acidic environment created by solid tumors as a trigger to activate anticancer lytic peptides by making them cationic only at low pH levels. We achieved this selectivity by substituting lysines (pKa, f10.5) for histidines (pKa, f6.1) in the parental peptide [D]-K 6 L 9 . Histidine is protonated below pH 7. For that purpose, we replaced either three or all six lysines in the parental peptide with histidines to obtain the peptides [D]-K 3 H 3 L 9 and [D]-H 6 L 9 . Interestingly, in vitro experiments showed pH-dependent activity only with [D]-H 6 L 9 mainly toward cancer cell lines. However, both peptides showed reduced systemic toxicity compared with the parental peptide. Intratumor and systemic inoculation of these peptides resulted in a significant decrease in the 22RV1 prostate cancer tumor volume and systemic secretion of prostate-specific antigen in a xenograft mice model. Moreover, histologic modifications revealed a significant reduction in new blood vessels selectively in tumor tissues after treatment with the peptides compared with the untreated tumors. The lytic mode of action of these new peptides, which makes it difficult for the cancer cells to develop resistance, and their selective and pH-dependent activity make them potential candidates for treatment of solid cancer tumors.

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Antimicrobial peptides and lipopeptides play an essential protective role in the innate immune system of all organisms. Despite many studies, the factors that dictate their cell-selectivity and pH-dependent activity are yet not clear. This is important because various organs of the human body have an acidic pH environment, for example, the vagina, gastric lumen, cryogenic dental foci, and lung-lining fluids in cystic fibrosis and asthma. In this study we synthesized a new group of lipopeptides by conjugating dodecanoic acid (DDA) to the N-termini of 12-mer peptides LXXLLXXLLXXL (L(6)X(6), X = Lys, His, Arg, and all the leucines are d-amino acid enantiomers) and investigated their pH-dependent biological activity and a plausible mode of action by using model phospholipids mimicking bacterial, mammalian, and fungal membranes. The data revealed that, depending on the basic amino acid incorporated, the lipopeptides are active against both bacteria and fungi or solely toward fungi. Furthermore, their activity is expressed at an acidic pH alone, neutral pH alone, or at both environments. Determination of secondary structure, membrane leakage experiments, surface plasmon resonance (SPR) binding experiments, and transmission electron microscopy suggest the involvement of a membranolytic effect. This mode of action, which should make it hard for the microorganism to develop resistance, their selective and pH-dependent activity, as well as pharmacological advantages due to the presence of d-amino acids, make them potential candidates for the treatment of mycoses in organs, under various pH environments, especially in cases where the bacterial flora should not be harmed.

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Cationic polysaccharides based on spermine-dextran conjugates were synthesized and tested as vectors for gene transfection. Dextrans of 10-380 kDa were oxidized under mild conditions by potassium periodate to obtain the respective polyaldehydes in 90% overall yield. The oxidized dextrans were reacted by reductive amination with increasing amounts of spermine, and the efficacy of conjugation between the oligoamine and polysaccharides was studied as a function of spermine/aldehyde mole ratio, pH, and temperature of medium. The optimal conjugation yields were obtained at 1.25 mole ratio (spermine/ aldehyde groups) and pH 11 at room temperature. Under these conditions, ∼2 µmol/mg (spermine/ polysaccharide) conjugation was achieved with 25-30% of the spermine moieties were conjugated in both sides to form branched polymers. The water-soluble polymers obtained were interacted with pCMV-GFP plasmid to form nanoparticles that were introduced to HEK293 and NIH3T3 cells in vitro for transfection efficacy assessment. Out of about 50 different polymer structures, only spermine-dextran of 6000-8000 Da, spermine content of ∼2 µmol/mg, and degree of branching of 25-30% was active in transfecting about 50% of the cells while all other polymers were significantly less active.