To understand how peptide organization in aqueous solution might affect the activity of antimicrobial peptides, the potency of various dermaseptin S4 analogs was assessed against human red blood cells (RBC), protozoa, and several Gram-negative bacteria. Dermaseptin S4 had weak antibacterial activity but potent hemolytic or antiprotozoan effects. K 4 K 20 -S4 was 2-3-fold more potent against protozoa and RBC, yet K 4 K 20 -S4 was more potent by 2 orders of magnitude against bacteria. K 4 -S4 had similar behavior as K 4 K 20 -S4, but K 20 -S4 and analogous negative charge substitutions were as active as dermaseptin S4 or had reduced activity. Binding experiments suggested that potency enhancement was not the result of increased affinity to target cells. In contrast, potency correlated well with aggregation properties. Fluorescence studies indicated that K 20 -S4 and all negative charge substitutions were as aggregated as dermaseptin S4, whereas K 4 -S4 and K 4 K 20 -S4 were clearly less aggregated. Overall, the data indicated that N-terminal domain interaction between dermaseptin S4 monomers is responsible for the peptide's oligomerization in solution and, hence, for its limited spectrum of action. Moreover, bell-shaped dose-response profiles obtained with bacteria but not with protozoa or RBC implied that aggregation can have dramatic consequences on antibacterial activity. Based on these results, we tested the feasibility of selectivity reversal in the activity of dermaseptin S4. Tampering with the composition of the hydrophobic domains by reducing hydrophobicity or by increasing the net positive charge affected dramatically the peptide's activity and resulted in various analogs that displayed potent antibacterial activity but reduced hemolytic activity. Among these, maximal antibacterial activity was displayed by a 15-mer version that was more potent by 2 orders of magnitude compared with native dermaseptin S4. These results emphasize the notion that peptide-based antibiotics represent a highly modular synthetic antimicrobial system and provide indications of how the peptide's physico-chemical properties affect potency and selectivity.

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The hemolytic antimicrobial peptide dermaseptin S4 was recently shown to exert antimalarial activity. In this study, we attempted to understand the underlying mechanism(s) and identify derivatives with improved antimalarial activity. A number of dermaseptin S4 derivatives inhibited parasite growth with a 50% inhibitory concentration (IC 50 ) in the micromolar range. Among these, the substituted S4 analog K 4 K 20 -S4 was the most potent (IC 50 ‫؍‬ 0.2 M), while its shorter version, K 4 -S4(1-13)a, retained a considerable potency (IC 50 ‫؍‬ 6 M). Both K 4 K 20 -S4 and K 4 -S4(1-13)a inhibited growth of the parasites more at the trophozoite stage than at the ring stage. Significant growth inhibition was observed after as little as 1 min of exposure to peptides and proceeded with nearly linear kinetics. The peptides selectively lysed infected red blood cells (RBC) while having a weaker effect on noninfected RBC. Thus, K 4 K 20 -S4 lysed trophozoites at concentrations similar to those that inhibited their proliferation, but trophozoites were >30-fold more susceptible than normal RBC to the lytic effect of K 4 K 20 -S4, the most hemolytic dermaseptin. The same trend was observed with K 4 -S4(1-13)a. The D isomers of K 4 K 20 -S4 or K 4 -S4(1-13)a were as active as the L counterparts, indicating that antimalarial activity of these peptides, like their membrane-lytic activity, is not mediated by specific interactions with a chiral center. Moreover, dissipation of transmembrane potential experiments with infected cells indicated that the peptides induce damage in the parasite's plasma membrane. Fluorescence confocal microscopy analysis of treated infected cells also indicated that the peptide is able to find its way through the complex series of membranes and interact directly with the intracellular parasite. Overall, the data showed that dermaseptins exert antimalarial activity by lysis of infected cells. Dermaseptin derivatives are also able to disrupt the parasite plasma membrane without harming that of the host RBC.

An inherited deficiency of acid sphingomyelinase (ASM) activity results in the Type A and B forms of Niemann-Pick disease (NPD). Using the ASM-deficient mouse model (ASMKO) of NPD, we evaluated the efficacy of enzyme replacement therapy (ERT) for the treatment of this disorder. Recombinant human ASM (rhASM) was purified from the media of overexpressing Chinese Hamster ovary cells and i.v. injected into 16 five-month-old ASMKO mice at doses of 0.3, 1, 3, or 10 mg/kg every other day for 14 days (7 injections). On day 16, the animals were killed and the tissues were analyzed for their sphingomyelin (SPM) content. Notably, the SPM levels were markedly reduced in the hearts, livers, and spleens of these animals, and to a lesser degree in the lungs. Little or no substrate depletion was found in the kidneys or brains. Based on these results, three additional 5-month-old ASMKO animals were injected every other day with 5 mg/kg for 8 days (4 injections) and killed on day 10 for histological analysis. Consistent with the biochemical results, marked histological improvements were observed in the livers, spleens, and lungs, indicating a reversal of the disease pathology. A group of 10 ASMKO mice were then i.v. injected once a week with 1 mg/kg rhASM for 15 wk, starting at 3 wk of age. Although anti-rhASM antibodies were produced in these mice, the antibodies were not neutralizing and no adverse effects were observed from this treatment. Weight gain and rota-rod performance were slightly improved in the treated animals as compared with ASMKO control animals, but significant neurological deficits were still observed and their life span was not extended by ERT. In contrast with these CNS results, striking histological and biochemical improvements were found in the reticuloendothelial system organs (livers, spleens, and lungs). These studies indicate that ERT should be an effective therapeutic approach for Type B NPD, but is unlikely to prevent the severe neurodegeneration associated with Type A NPD.