In vitro antiplasmodium effects of dermaseptin S4 derivatives

Abstract

The 13-residue dermaseptin S4 derivative K(4)S4(1-13)a (P) was previously shown to kill intraerythrocytic malaria parasites through the lysis of the host cells. In this study, we have sought peptides that will kill the parasite without lysing the erythrocyte. To produce such peptides, 26 compounds of variable structure and size were attached to the N terminus of P and screened for antiplasmodium and hemolytic activities in cultures of Plasmodium falciparum. Results from this screen indicated that increased hydrophobicity results in amplified antiplasmodium effect, irrespective of the linearity or bulkiness of the additive. However, increased hydrophobicity also was generally associated with increased hemolysis, with the exception of two derivatives: propionyl-P (C3-P) and isobutyryl-P (iC4-P). Both acyl-peptides were more effective than P, with 50% growth inhibition at 3.8, 4.3, and 7.7 microM, respectively. The antiparasitic effect was time dependent and totally irreversible, implying a cytotoxic effect. The peptides were also investigated in parallel for their ability to inhibit parasite growth and to induce hemolysis in infected and uninfected erythrocytes. Whereas the dose dependence of growth inhibition and hemolysis of infected cells overlapped when cells were treated with P, the acyl-peptides exerted 50% growth inhibition at concentrations that did not cause hemolysis. Noticeably, the acyl derivatives, but not P, were able to dissipate the parasite plasma membrane potential and cause depletion of intraparasite potassium under nonhemolytic conditions. These results clearly demonstrate that the acyl-peptides can affect parasite viability in a manner that is dissociated from lysis of the host cell. Overall, the data indicate the potential usefulness of this strategy for development of selective peptides as investigative tools and eventually as antimalarial agents.

FIG. 1.

Screen of antiplasmodium and hemolytic activities for three groups of peptides. Synchronized cultures of infected human RBCs at the ring stage were cultured at 1% hematocrit and 2% parasitemia in the presence of a 10 μM concentration of each of the designated peptides (Table 1). After 18 h of incubation, the cultures were divided into two sets. [³H]hypoxanthine was added to one set, and cells were harvested 6 h later. The cell-associated radioactivity was determined, and inhibition of growth was calculated by comparison with that of controls (A). The second set was further incubated for 6 h, and the concentration of hemoglobin in the supernatant was determined by absorption spectroscopy at 405 nm. Hemolysis was calculated by comparison with fully hemolyzed cultures (B). Error bars represent the standard deviation from the mean, calculated from at least two independent experiments performed in quadruplicates. Panel C shows the ratio (A/B) calculated from the mean values from panels A and B. Note the logarithmic scale of the ratio axis.

FIG. 2.

Stage dependence of antiplasmodium activity. Infected cells at the ring or at the trophozoite stage (1% hematocrit and 2% parasitemia) were exposed to either C3-P or iC4-P (10 μM), and parasite growth was determined by hypoxanthine (Hx) incorporation. The various protocols for time of exposure to the peptides, recovery without peptide, and hypoxanthine incorporation (A to D) are depicted in the upper part. Open bars, ring stage; solid bars, trophozoite stage. Each experiment was performed twice in triplicates. Error bars represent the standard deviation from the mean.

FIG. 3.

Hemolytic activity versus antiplasmodium activity. Infected cells at the young trophozoite stage were separated from normal RBCs by the Percoll-alanine gradient method. Infected (≥90% parasitemia) and uninfected cells were resuspended in culture medium (0.5% hematocrit) containing increasing concentrations of the peptides. After 2 h of incubation, hypoxanthine was added to one set of the culture, cells were harvested 4 h later, and the cell-associated radioactivity was determined. Hemolysis was determined in two additional sets of cultured infected and normal RBCs by measuring the specific absorbance of hemoglobin in the supernatants after 6 h of exposure to the peptides. The continuous lines depict the best fit of the data to the dose response. •, inhibition of hypoxanthine uptake; ▪, hemolysis of infected RBCs; ▴, hemolysis of normal RBCs. Each experiment was performed at least twice in quadruplicates. Error bars represent the standard deviation from the mean. If no error bar is shown, the standard deviation was smaller than the diameter of the symbol.

FIG. 4.

Dissipation of the parasite plasma membrane potential. Trophozoites that had been preincubated with R123 were exposed to one of the designated peptides and to a mixture of known ionophores (monensin and nigericin). Samples were taken at different time intervals, washed, and resuspended in the original sample volume of PBS. The fluorescence was read (λ_ex = 530 nm, λ_em = 585 nm). Relative fluorescence (as percentage of untreated control at the same time) was plotted against the time of exposure. ○, PBS; •, P; □, C3-P; ▪, iC4-P; ✳, nigericin plus monensin. Each experiment was performed twice in duplicates. Error bars represent the standard deviation from the mean. If no error bar is shown, the standard deviation was smaller than the diameter of the symbol.

FIG. 5.

Measurement of potassium intracellular concentration of the parasite. Infected cells at the young trophozoite stage (∼97% parasitemia) were cultured in the absence or presence of peptide (10 μM). After 4 h of incubation, parasites were freed from their host cell by saponin lysis followed by extensive washes in MgCl₂. Free parasites were disrupted by freezing and thawing, and the potassium content in the supernatant was determined by inductive-coupled plasma-atomic emission spectroscopy. Results are shown as percent potassium content relative to that of the control at time zero. Error bars represent the standard deviation from the mean calculated from two independent experiments performed in duplicates. If no error bar is shown, the standard deviation was smaller than the diameter of the symbol.