In Vitro Antiplasmodium Effects of Dermaseptin S4 Derivatives

The 13-residue dermaseptin S4 derivative K₄S4(1-13)a (P) waspreviously shown to kill intraerythrocytic malaria parasitesthrough the lysis of the host cells. In this study, we havesought peptides that will kill the parasite without lysing theerythrocyte. To produce such peptides, 26 compounds of variablestructure and size were attached to the N terminus of P andscreened for antiplasmodium and hemolytic activities in culturesof Plasmodium falciparum. Results from this screen indicatedthat increased hydrophobicity results in amplified antiplasmodiumeffect, irrespective of the linearity or bulkiness of the additive.However, increased hydrophobicity also was generally associatedwith increased hemolysis, with the exception of two derivatives:propionyl-P (C3-P) and isobutyryl-P (iC4-P). Both acyl-peptideswere more effective than P, with 50% growth inhibition at 3.8,4.3, and 7.7 µM, respectively. The antiparasitic effectwas time dependent and totally irreversible, implying a cytotoxiceffect. The peptides were also investigated in parallel fortheir ability to inhibit parasite growth and to induce hemolysisin infected and uninfected erythrocytes. Whereas the dose dependenceof growth inhibition and hemolysis of infected cells overlappedwhen 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 dissipatethe parasite plasma membrane potential and cause depletion ofintraparasite potassium under nonhemolytic conditions. Theseresults clearly demonstrate that the acyl-peptides can affectparasite viability in a manner that is dissociated from lysisof the host cell. Overall, the data indicate the potential usefulnessof this strategy for development of selective peptides as investigativetools and eventually as antimalarial agents.

INTRODUCTION

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Introduction

Malaria still constitutes the most widespread infectious disease,affecting hundreds of millions of people, mostly in Third Worldcountries, and causing the death of one million children everyyear in Africa alone (40). This devastating situation stemsfrom the ever-increasing resistance of parasites to availabledrugs, and new chemotherapeutic means have to be developed.Recently, various animal-derived peptide-based antimicrobialshave emerged as interesting tools for exploring new potentialantiplasmodiams agents (8, 12, 13).

Antimicrobial peptides are generally made up of less than 50amino acids, with an excess of basic amino acids (lysine and/orarginine) over acidic residues and a substantial portion ofhydrophobic amino acids (3, 7, 10, 17, 27). They vary considerablyin structure, size, amino acid sequence, and spectrum of action.Structure-activity relationship studies have shown that themost potent peptides are highly basic, with a pronounced amphipathiccharacter (2, 4, 18, 24, 26, 35). These peptides exert antimicrobialaction through their effect on the membrane of target cellsby a mechanism the details of which remain to be understood.From available information, it can be suggested that they don'tinteract with stereospecific targets, such as protein receptors,enzymes, and so on (5, 38). Their charge and hydrophobicityare the main factors affecting antimicrobial activity. The strongsigmoid nature of their dose-response effect (25, 33) suggeststhat the active form of the peptide is an oligomer. Some ofthese peptides were stipulated to form ion channels or pores,as suggested by their ability to dissipate electric potentialacross energy-transducing membranes and ion conductance acrosslipid bilayers (32, 41). Various basic models for a membranolyticmechanism were proposed, ranging from pore formation to inductionof structural defects (11, 21, 28, 30, 36, 39). The perturbationsin the membrane structure lead to membrane permeabilization.Hence, essential ions and metabolites are free to leak in andout and dissipate electric potential across the membrane. Thisstops ATP production and arrests cellular metabolism, eventuallyleading to cell death.

Many antimicrobial peptides display a broad spectrum of activitythat can kill or neutralize gram-negative and -positive bacteria,yeast and filamentous fungi, and some enveloped viruses, aswell as many types of cancer cells (19, 23, 31). Yet, many ofthese peptides are quite inactive on normal eukaryotic cells.Although the basis for this discrimination is also unclear,it appears to be related to the lipid composition of the targetmembrane (i.e., fluidity, negative charge density, and absenceor presence of cholesterol) and the presence in the peptide-susceptibleorganisms of a large negative transmembrane electrical potential(1). Thus, while the precise mechanism of action of antimicrobialpeptides is yet to be better defined, microbicidal effect iswidely believed to result from their capacity to permeabilizethe membrane of target cells. Such a mechanism of action endowsthe peptide-based antimicrobial system with attractive advantagesover classical antibiotics, because it makes it extremely difficultfor microbes to develop resistance (4, 15, 22). However, a majordownside of such a mechanism is reflected in its unselectiveactivity over a wide range of cell types, which could be problematic—forinstance, in systemic routes of administration (9). In an ironicway, therefore, a major challenge of this field of researchconsists of figuring out how to endow specificity to a systemthat, by definition, is nonspecific.

The hemolytic antimicrobial peptide dermaseptin S4 has beenshown before to exert antiplasmodium activity (8). Subsequentstudies (5, 6, 13) with derivatives of S4 against Plasmodiumfalciparum, the most lethal human parasite, identified K₄K₂₀-S4as the most potent peptide (50% inhibitory concentration [IC₅₀]= 0.2 µM), with its shorter version, K₄-S4(1-13)a, displayingconsiderable effectiveness (IC₅₀ = 6 µM). Both peptidesacted more efficiently at the trophozoite stage. The antiplasmodiumaction was rapid and mediated by the lysis of the infected erythrocyte.Since these peptides were also lytic to normal erythrocytes,albeit at a 30-fold-higher concentration, it was deemed necessaryto develop additional derivatives that could affect the parasitewith minimal threat to erythrocytes. In this study, we investigatedthe antiplasmodium activities of various new dermaseptin S4derivatives. We have synthesized a peptide library based onthe 13-residue dermaseptin S4 derivative K₄-S4(1-13), and afterscreening for the most effective compounds, we investigatedtheir detailed mechanism of antiplasmodium action.

MATERIALS AND METHODS

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Materials and Methods

Preparation of dermaseptin S4 derivatives. The reference peptide K₄-S4(1-13) was synthesized by the solid-phasemethod as described previously (5), applying the Fmoc (9-fluorenylmethyloxycarbonyl)active ester chemistry on a fully automated, programmable AppliedBiosystems model 433A peptide synthesizer (Applied Biosystems).MBHA resin (Novabiochem, Darmstadt, Germany) was used to obtainan amidated peptide. Unless otherwise stated, all reagents werepurchased from Sigma (St. Louis, Mo.). The various analogs wereprepared by linking the N terminus of K₄-S4(1-13) to one ofthe compounds detailed in Table 1. Selective reaction with theamino-terminal group was ensured by selective deprotection (removalof Fmoc from the N terminus) of the Fmoc-protected resin-boundpeptide by mixing the resin with a solution of 20% piperidine-N-methylpyrrolidone(NMP), whereas all other potentially reactive groups remainedmasked by orthogonal protecting groups.

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TABLE 1. Structure of the parent peptide K₄-S4(1-13)a (P) and its derivatives

For amide-linked derivatives, the deprotected resin-bound peptide(20 mg) was washed with NMP to remove the piperidine and suspendedin 0.7 ml of dimethylformamide (DMF), to which a twofold molarexcess of the relevant acid compound was added followed by threefoldmolar excess of 1-ethyl-3-(dimethylaminopropyl)carbodiimide(EDAC). Using the same chemistry, part of the succinyl derivativewas reactivated to allow additional linkages to various amines,as detailed in Table 1. For sulfonamide derivatives, the deprotectedresin-bound peptide was suspended in 0.7 ml of anhydrous DMF,to which was added a 1.5-fold molar excess of the relevant alkylsulfonylchloride derivative, followed by 2-fold molar excess of diisopropylethylamine(DIPEA). For trimethyl-P synthesis the deprotected resin-boundpeptide was suspended in 1 ml of DMF-methanol (1:4), to whichwas added a 10-fold molar excess of both methyliodide and potassiumcarbonate.

The reaction mixtures were sonicated (5 min) and then agitatedfor 24 h at room temperature. Each mixture was then centrifugedfor 3 min (supernatant discarded), and the resin was washedfour times with DMF and then three times with ether-dichloromethane(1:1). The residue was dried for 1 h at room temperature andthen 4 h at 50°C.

Peptide cleavage from the resin was performed in a mixture of2.5% water, 2.5% triethylsilane, and 95% trifluoroacetic acid(TFA), and stirred in an ice bath for 15 min and then at roomtemperature for 2 h. After filtration of the resin, the peptide-TFAfiltrate was precipitated (added drop by drop) in ice-cold diethylether and placed in a fume hood to dry at 60°C for 2 h andthen dissolved in 10% acetic acid and lyophilized. The crudepeptide was purified to chromatographic homogeneity in the rangeof 95 to >99% by reverse-phase high-performance liquid chromatography(HPLC) (Alliance-Waters). HPLC runs were performed on a semipreparativeC₄ column (Vydac) with a linear gradient of acetonitrile inwater (1%/min); both solvents contained 0.1% TFA. Based on peakintegration of the HPLC profiles, the yield for each amide orsulfonamide linkage was generally not lower than 85%, with theexception of C16-P, where the yield was $~$ 65%. The yield for tM $~$ Pwas 63%.

The purified peptides were subjected to amino acid analysisand mass spectrometry in order to confirm their composition.Peptides were stocked as lyophilized powder at -20°C. Priorto testing, fresh solutions were prepared in water, brieflyvortexed, sonicated, centrifuged, and then diluted in the appropriatemedium. Buffers were prepared with distilled water (mQ; Millipore).All other reagents were analytical grade.

Parasite cultivation. The W2 strain of P. falciparum was used throughout this investigationwith human erythrocytes (RBCs). Parasites were cultivated bythe Jensen and Trager technique as modified in our laboratory(14). Culture was synchronized by the sorbitol method (16) withthe less toxic alanine, and infected cells were enriched fromculture by Percoll-alanine gradient centrifugation (14).

Drug screening test. Synchronized cultures at the ring stage were cultured at 1%hematocrit and 2% parasitemia in the presence of 10 µMdermaseptin derivatives. After 18 h of incubation, the cultureswere divided into two sets. [³H]hypoxanthine (final concentration,2 µCi/ml) was added to one set, and cells were harvested6 h later. The cell-associated radioactivity was determined,and inhibition of growth was calculated by comparison with thatof controls (without peptide). The second set was further incubatedfor 6 h, and the concentration of hemoglobin in the supernatantwas determined by absorption spectroscopy at 405 nm. Full lysiswas obtained by lysis of the same number of cells in an equalvolume of distilled water.

Determination of IC_50. Synchronized cultures at the ring stage were cultured at 1%hematocrit and 2% parasitemia in the presence of increasingconcentrations of lipodermaseptin derivatives. After 18 h ofincubation, parasite viability was determined by the hypoxanthineincorporation test. The IC₅₀ was determined by nonlinear regressionfitting of the data with the Sigmaplot program. By using thesame technique for the measurement of parasite viability andhemolysis, the stage and the time dependence of drug effectat each of the different stages was also determined. The preciseconditions are shown in the legends to the relevant figuresin the Results section.

Dissipation of parasite membrane potential by the acylated derivatives. Whole culture at the trophozoite stage in modified growth medium(wash medium containing 10 mM bicarbonate and 7% plasma) at0.5% hematocrit was incubated in the presence of 1 µMrhodamine 123 (R123) for 30 min at 37°C. R123 accumulatesinside cells in proportion to the membrane potential ( ${Delta}$ ${Psi}$ ) andhas been shown to respond to the dissipation of the plasma membrane ${Delta}$ ${Psi}$ in P. falciparum (34). Aliquots of this culture were then exposedto different peptides or to a mixture of 10 µM nigericin(K⁺:H⁺ exchanger) and 10 µM monensin (Na⁺:H⁺ exchanger)to dissipate the ion gradient across membranes (positive control).At different time intervals, samples were taken, and cells werepelleted by centrifugation, washed in phosphate-buffered saline(PBS), and resuspended in the original sample volume of PBS.Aliquots of 120 µl were placed in a 96-well plate andread in a fluorescence reader (excitation wavelength [ ${lambda}$ _ex] =530 nm, emission wavelength [ ${lambda}$ _em] = 585 nm). Relative fluorescence(as a percentage of that of the untreated control at the sametime) was plotted against the time of incubation.

Depletion of the parasite's intracellular potassium in the presence of dermaseptin derivatives. Infected cells at the young trophozoite stage were separatedfrom uninfected RBCs with the Percoll-alanine gradient and incubatedfor different time intervals in culture medium at 37°C.Infected cells ( $~$ 97% parasitemia, determined on Giemsa-stainedthin blood smears) were suspended at 0.5% hematocrit in culturemedium containing 15 µM dermaseptin derivatives. At timezero and at 4 h, aliquots were taken, and cells were pelletedby centrifugation, washed in PBS, and centrifuged again. Toobtain free parasite, cells were resuspended in PBS containing0.003% (wt/vol) saponin and after several minutes at room temperaturewere pelleted by centrifugation, washed in PBS several times,and finally washed with 110 mM MgCl₂ buffered with 10 mM HEPES.After disruption of the free parasites by freezing and thawingand centrifugation at 10,000 x g for 20 min, the supernatantliquid was analyzed for potassium content by inductive-coupledplasma-atomic emission spectroscopy on an Optima 3300 ICP-AESsystem (Perkin-Elmer, Norwalk, Conn.). Relative intracellularpotassium concentrations were calculated as a percentage ofthat of the untreated control at the same time.

RESULTS

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Results

We have shown previously that dermaseptins and their derivativesare able to kill intraerythrocytic malaria parasites (13). Thiseffect was demonstrably due to the lysis of the infected cell.Uninfected cells were similarly lysed, albeit at higher peptideconcentrations, but such differential lysis was insufficientto ensure safe therapy with no threat to the host. We have thereforesought peptides that will kill the parasite without lysing theerythrocyte. To produce such peptides, 25 compounds of variablestructure and size were attached to the N terminus of K₄-S4(1-13)a.The resulting peptide derivatives are shown in Table 1.

Screen of antiplasmodium and hemolytic activities. Results shown in Fig. 1 for group I indicate that various derivativesof K₄-S4(1-13)a have pronounced antiplasmodium activity. Comparedto the parent peptide, this series of compounds includes derivativeswith increased positive charge at the N terminus (tM $~$ P), increasednegative charge (Mal-P and Suc-P), and increased hydrophobicity,both linear (tME-Suc-P, C4-Suc-P, C6-Suc-P C12-Suc-P, and C8=P)and cyclic (cHA-P, Bz-P, Bz-Suc-P, and Fmoc-P).

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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.

From the antiplasmodium effect (Fig. 1A), we can conclude thatincreased hydrophobicity results in an amplified effect, irrespectiveof the linearity or bulkiness of the additive. Derivatives thatincreased the peptide's negative charge reduced the antiplasmodiumeffect, whereas the positive charge in tM $~$ P did not substantiallyimprove growth inhibition. Increased hydrophobicity, however,also was generally associated with increased hemolysis (Fig.1B), with the exception of two derivatives: Bz-Suc-P and C8=P.We have chosen to compare the ratio of relative inhibition torelative hemolysis as a selection criterion for the most suitablederivative. This analysis (Fig. 1C) revealed that Bz-Suc-P andC8=P were the most selective derivatives: compared to the parentpeptide, the ratio was up to twofold higher.

Since hydrophobicity emerged from this screen as a suitableproperty, we have screened a second series of compounds in whichthe length of the acyl chain was systematically increased fromacetyl (C2-P) to palmitoyl (C16-P). Results shown in Fig. 1for group II confirmed this notion, because the antiplasmodiumactivity increased with chain length (Fig. 1A). This was notthe case for the hemolytic activity (Fig. 1B). Increasing thelength from C2 up to C4 resulted in decreased activity, whereasa further increase in length led to a gradual increase in hemolysis.With the ratio criterion for selection of optimal compoundsin this series, it can be seen that C3-P and C4-P were at thetop of the list.

Following the results obtained with the first two series ofcompounds, we have screened a third series that consisted ofvarious C2-C4 derivatives (group III). In this series, whichincluded two sulfonamide and three amide-linked acyl derivatives,we have attempted to further modulate N-terminal hydrophobicity,assuming that this will reduce the hemolytic activity. Two derivatives(iC4-P and C4=P) exhibited increased antiplasmodium activitycompared to the parent peptide (Fig. 1A), but only iC4-P displayedboth increased antiplasmodium effect and decreased hemolysis(Fig. 1B). The calculated ratio (Fig. 1C) was the highest obtainedin this series of compounds and was therefore selected alongwith C3-P from the second series for further and more detailedinvestigations.

Detailed determination of antiplasmodium activity and stage dependence of selected compounds. The dose response of the two acyl-peptides C3-P and iC4-P wasinvestigated and compared to that of the parent peptide, P,by using synchronized cultures that were exposed to the peptideseither at the ring stage or at the trophozoite stage. Both acyl-peptideswere more effective than P at the ring stage, but not at thetrophozoite stage (Table 2). The stage dependence results indicatedthat ring-stage parasites were somewhat less sensitive thanthe more mature trophozoite stage. This was essentially foundwith P, as previously observed (13), but much less with iC4-Pand not at all with C3-P. The slopes of the dose-response curvesvaried with peptide and stage, indicating some fine differencesin the stoichiometry of drug and target relations.

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TABLE 2. Stage dependence of the growth inhibitory activity of the parent peptide K₄S4(1-13)a (P) and two derivatives

Time dependence and reversibility of antiplasmodial action. The time dependence of the antiplasmodium effect of the twoacyl-peptides was investigated by using synchronized cultureat the ring or at the trophozoite stage. Parasites were exposedto either C3-P or iC4-P at 10 µM for various lengths oftime. Thereafter, the peptides were washed, and the cultureswere tested for parasite viability either immediately or aftera period of recovery under the culture conditions. The preciseprotocols are shown in the upper part of Fig. 2. Less inhibitionafter a longer recovery time in the absence of peptides indicatesreversibility of drug action (a cytostatic effect), whereaswhen no change in parasite viability is observed due to removalof the peptide, an irreversible or cytotoxic effect is the cause.

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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.

As can be seen from Fig. 2, drug action is time dependent. Inhibitionof parasite growth increased with the time of exposure to thepeptide, reaching above 50% inhibition in ${approx}$ 24 h. This time courseof peptide action does not depend on the parasite stage thatwas exposed to the drug. Allowing the parasites to recover underculture conditions did not restore their viability, except forslightly reduced inhibition after recovery in the ring-stageparasites after 5 h of treatment with acyl-peptides. Thus, inhibitionof parasite growth was essentially irreversible, implying acytotoxic effect.

Hemolytic activity versus antiplasmodium activity. To elucidate the mechanism of antiplasmodium activity, C3-Pand iC4-P were investigated simultaneously for their abilityto induce hemolysis and to inhibit parasite growth. Infectedcells at the young trophozoite stage were separated from normalRBCs 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, [³H]hypoxanthine was added to one setof culture, and cells were harvested 4 h later. Hemolysis wasdetermined in two additional sets of cultured infected and normalRBCs by measuring the specific absorbance of hemoglobin in thesupernatants after 6 h of exposure to acylated peptides. C3-P(Fig. 3A) and iC4-P (Fig. 3B) were slightly more inhibitoryto parasite growth than P was (Fig. 3C). Under these conditionsof brief exposure of trophozoite-enriched cultures, the IC₅₀were essentially equal: 19 ± 4.4 and 21 ± 7.5µM, respectively, for C3-P and iC4-P and 19.6 ±1.6 µM for P.

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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; ${blacksquare}$ , hemolysis of infected RBCs; ${blacktriangleup}$ , 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.

Dissipation of the parasite plasma membrane potential. To assess whether the peptide can gain access to the parasitemembrane, the dissipation of R123 fluorescence was monitored.This fluorescent dye accumulates in parasites in correlationwith the parasite membrane ${Delta}$ ${Psi}$ . It was first ascertained that fluorescencemeasurement does indeed dissipate ${Delta}$ ${Psi}$ as measured by reduced accumulationof the dye with the ionophores nigericin and monensin (Fig.4). It was subsequently found that both C3-P and iC4-P depolarizedthe parasite membrane in a time-dependent manner, whereas Pdid not. These results were obtained in the presence of 10 µMacyl-peptides: i.e., at $~$ 2.5-fold the IC₅₀ of the acyl-peptides,where hemolysis was minimal. At this concentration, P neitherkilled the parasite nor lysed the infected cells. These resultsclearly demonstrate that the peptide can cross the host cellmembrane and interact with the parasite plasma membrane to permeabilizeit. As we saw previously, there was no significant differencebetween the potency of the two acyl-peptides to elicit thiseffect.

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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 ( ${lambda}$ _ex = 530 nm, ${lambda}$ _em = 585 nm). Relative fluorescence (as percentage of untreated control at the same time) was plotted against the time of exposure. ${circ}$ , PBS; •, P; ${square}$ , C3-P; ${blacksquare}$ , 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.

Measurement of intracellular potassium concentration of the parasite by atomic emission. Changes in the parasite's intracellular potassium concentrationwere investigated in the presence of P and C3-P. (Only one acyl-peptidewas examined, since so far, its action had been found to bealmost superimposed with that of iC4-P.) Infected cells at theyoung trophozoite stage were separated from normal RBCs by usingPercoll-alanine gradient. Infected cells ( $~$ 90% parasitemia) wereresuspended in culture medium containing 10 µM P or C3-P.After 4 h of incubation, the cell parasites were freed fromtheir host cell by saponin lysis and washed extensively to removehost cell material and finally in MgCl₂. Free parasites weredisrupted by freezing and thawing, and the potassium contentin the supernatant was determined. The results depicted in Fig.5 clearly indicate that treatment with C3-P, but not with P,resulted in marked depletion of parasite potassium. This addsto the previous demonstration that the acyl-peptide is ableto cross the host cell membrane and interact with that of theparasite to permeabilize it.

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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.

DISCUSSION

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Discussion

We have previously demonstrated that dermaseptin S4 derivativesdisplay potent antiparasitic activity that is mediated by thelysis of the host cell. Because the antiplasmodium activityparalleled the lytic activity, albeit 30-fold-higher concentrationswere needed to lyse uninfected cells, we have undertaken thedevelopment of additional derivatives with greater selectivity.We have been guided by the following rationale: selective antimicrobialactivity of peptides demonstrably depends on the membrane lipidcomposition (19, 23, 31). The lipid compositions of the hostand parasite membrane are different (37). Hence, if we can introducethe peptide into the host cell, it will be exposed to both thehost cell and the parasite membranes and exert its effect onthe more susceptible membrane. Increasing the lipophilicityof the peptide will render it more permeable through the hostcell membrane and therefore more accessible to the parasitemembrane.

This presumption has been tested experimentally by synthesizing26 peptides with various N termini. In general, the antiplasmodiumactivity increased with acyl chain length up to 6 carbons andthen leveled off. The impact of chain length on hemolysis displayeda minimum at C2-C4 for reasons that are not yet fully understood.We selected the most discriminating compounds based on the ratioof percent inhibition of parasite growth to percent hemolysis.For iC4-P, the ratio was 160, while for C3-P, it was 600, comparedto 18 for P. It is understood that the use of this ratio foridentifying the disadvantages of the most promising candidates,such as C4-Suc-P, which caused full growth inhibition at theconcentration tested, and which may have also done so (whilecausing less hemolysis) at much lower concentrations. In otherwords, peptides such as C4-Suc-P might have been better candidatesfor further characterization than those actually chosen. Nevertheless,while such peptides will be further characterized in the future,our choice is substantiated in view of the very large differencein the ratios. These results clearly demonstrate that the acyl-peptidescan affect parasite viability in a manner that is dissociatedfrom lysis of the host cell. This suggested that the acylatedpeptides can (somehow) cross the host cell membrane and interactwith the parasite plasma membrane to permeabilize it.

Further investigations therefore concentrated on these two acylpeptides. A detailed dose-response analysis revealed that theIC₅₀ of the acyl peptides was ${approx}$ 2.5-fold lower than that of P.In order to gain further insight into the selectivity effect,parallel determination of antiplasmodium activity and lysisof normal and infected erythrocytes were conducted for shortincubation times. The IC₅₀ values obtained in these experimentswere understandably higher than those obtained in the standarddose-response test that lasts 24 h (Table 2) because of thetime dependence effect. Most importantly, whereas with P, thedose dependence of growth inhibition and lysis of infected cellsoverlapped, for the two acyl-peptides, ${approx}$ 50% growth inhibitionoccurred at concentrations that did not cause lysis. This discrepancyis possibly even larger, since both growth inhibition and lysisare time-dependent processes, and exposure to peptides was only2 h in the first case and 6 h in the second. Noticeably, whereasP was more lytic to infected cells, both acylated peptides weresimilarly lytic to infected and uninfected erythrocytes. Thisis a further demonstration of lipid-dependent specificity ofpeptides, since the lipid composition of the membrane of infectederythrocytes is different from that of normal erythrocytes (37).

Unlike P (13), neither iC4-P nor C3-P was stage selective, beingequally inhibitory for both the young ring-stage and more maturetrophozoites. We interpret these results as follows: P actsessentially by lysing the host cell membrane. It is more lyticto host cells harboring mature parasite stages, indicating dependenceon changes induced by the parasite in the host cell membrane.Since the latter evolves with parasite maturation, trophozoitesare expected to be more sensitive than rings. In contrast, theselectivity of the acyl-peptides seems to depend on the differentialcomposition of the host and parasite membrane. Since this isestablished from the onset of parasite development, the permeableacyl-peptide is always sucked into the parasite membrane andaffects it. For this reason, stage dependence with acyl-peptidesis neither expected nor observed.

The antiplasmodium effect was clearly time dependent. It developedgradually with time and needed the continuous presence of peptidein the parasite culture for up to 24 h in order to elicit themaximal effect. However, even the partial effect was essentiallyirreversible, indicating that the damage was cytotoxic. Themutual progressive aspect of the inhibition and the need forprotracted exposure to the peptide may be the consequence ofthe gradual loss of the parasite's viability due to the membranepermeabilization. The lipophilic and membrane-trophic characterof the lipopetide insinuates that its interaction with the parasitemembrane would lead to its nonselective permeabilization. Theobserved dissipation of ${Delta}$ ${Psi}$ could result from increased permeabilityto protons, since the major generator of ${Delta}$ ${Psi}$ is presumably theV-type H⁺ pump (20, 29). Permeabilization to protons undercutsthe pump's function as the major regulator of cellular pH. Short-circuitingof the pump electrogenic function presumably underlies the observedloss of cellular potassium, the maintenance of which seems todepend on ${Delta}$ ${Psi}$ . While these presumptions could explain the gradualand irreversible loss of vital cellular functions, at the presenttime, we cannot exclude the possibility that the acyl-peptidesact on a different cellular target that mediates its cytotoxicaction.

In conclusion, we have demonstrated in this investigation thatmembrane active peptides can be engineered to act specificallyon the membrane of the intracellular parasite to perturb itsfunctions. This selective activity reduces the potential harmfrom inadvertent lysis of the host's erythrocytes. This is amajor achievement in the fine-tuning of peptide compositiontowards its further development as a potential antimalarialdrug. It remains to be shown that the acyl-petides are not toxicto mammalian cells or to whole animals and that their antiparasiticeffect is maintained in vivo. Investigations of these aspectsare under way in our laboratory.

ACKNOWLEDGMENTS

This research was supported by the Israel Science Foundation(grant no. 523/98).

The expert assistance of Josephina Silfen (Hebrew University)in peptide synthesis is gratefully acknowledged.

FOOTNOTES

* Corresponding author. Mailing address: Laboratory for Antimicrobial Peptides Investigation (L.A.P.I.), Wolfson Centre for Applied Structural Biology, The Hebrew University of Jerusalem, Givat Ram 91904, Jerusalem, Israel. Phone: (972 2) 65 85 295. Fax: (972 2) 65 85 573. E-mail: amor{at}macbeth.ls.huji.ac.il.

REFERENCES

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