Short-Chain Aliphatic Polysulfonates Inhibit the Entry of Plasmodium into Red Blood Cells

Several steps in the pathogenesis of a Plasmodium falciparuminfection depend on interactions of parasite surface proteinswith negatively charged sugars on the surface of host cellssuch as sialate residues or glycosaminoglycans. For these reasons,our previous studies examining agents that interfere with heparansulfate-protein binding during amyloidogenesis suggested thatshort-chain aliphatic polysulfonates may prove useful as antimalarialagents. A series of related polysulfonates were synthesizedand assessed both in tissue culture with the asexual stagesof P. falciparum in human red blood cells and in vivo by useof Plasmodium berghei infections in mice. Poly(vinylsulfonatesodium salt) (molecular weight range, 1,500 to 3,000) provedeffective in interfering with P. falciparum merozoite entryinto human red blood cells and significantly delaying the increasein the level of P. berghei parasitemia in mice. The conceptthat anionic molecules that mimic large polysaccharide structuresmay have antimalarial properties has been suggested and examinedpreviously. Our results suggest that related anionic agents[poly(vinylsulfonate sodium salt)-like molecules] orders ofmagnitude smaller than those previously considered may proveuseful in abrogating merozoite entry into erythrocytes and maypotentially block sporozoite entry into liver cells. Structure-activitystudies conducted to enhance these properties may provide compoundswith scope for significant further analysis and development.

INTRODUCTION

Top

Introduction

Chloroquine-resistant Plasmodium falciparum malaria was firstrecognized over 40 years ago and has since spread to almostall areas where malaria is endemic (30). When chloroquine-resistantmalaria extended into the areas of Africa with high rates ofmalaria parasite transmission, it raised fears of an impendingpublic health crisis since switching to alternative antimalarialssuch as mefloquine, artemisinin derivatives, halofantrine, orquinine is unaffordable for many countries in sub-Saharan Africa(41). Recent reports indicate that the rates of mortality dueto a widespread resurgence of malaria are now escalating (21).To meet this challenge, novel and inexpensive antimalarialsmust be found. The asexual schizogonic blood stage is the mostfrequently targeted point in the parasite's life cycle (10).Symptoms of the disease begin to occur during this stage; andthe consequences of the parasite's presence can include anemia,organ dysfunction, coma, and even death (3).

The majority of the antimalarials available at present (e.g.,quinine, mefloquine, quinidine, and chloroquine) are quinolinesthat function by inhibiting the polymerization and detoxificationof heme into an insoluble pigment (hemazoin) (11). This hasled to problems with cross-resistance between related antimalarialsand limits the time during which the parasite is vulnerableto treatment to the second half of its intraerythrocytic lifecycle. Other classes of antimalarials exist, such as antifolates,which target the parasite's bifunctional dihydrofolate reductase-thymidylatesynthase enzyme (17), and atovaquone, which inhibits the parasite'smitochondrial electron transport and mitochondrial membranepotential. However, inhibitors of other phases of the asexuallife cycle would be useful. Another potential target in thedevelopment of antimalarials is the merozoite invasion process(6).

The invasion process involves a release of merozoites from aparasitized erythrocyte (RBC), followed by an initial attachmentand reorientation event, in part dependent on an ionic interactionbetween components on the merozoite and those on the RBC (7).Irreversible attachment and junction formation ensue, followedby formation of the parasitophorous vacuole and insertion ofthe merozoite into the RBC. This process relies on specificligands and receptors; however, the identities of these combinationsare still being determined (6). To date, no commercially availableantimalarial agents that specifically inhibit this step in thelife cycle have been identified. Nevertheless, there have beenseveral reports that saccharide anions inhibit both the invasionof RBCs by merozoites (8) and the invasion of hepatic cellsby sporozoites (28, 29). Furthermore, heparin, dextran sulfate,fucoidin, and chondroitin sulfate all inhibit one or severalparasite-host interactions; however, their large polydispersenature and other in vivo activities have hampered their developmentas therapeutic agents (35).

We have noted that several stages of malaria parasite infectioninvolve binding interactions between proteins on the surfaceof the parasite and negatively charged sugars on the surfaceof host cells, such as sialate residues or sulfated glycosaminoglycans(GAGs). Examples include sporozoite binding to hepatocyte heparansulfate (42) and infected RBC binding to endothelial chondroitinsulfate (14, 16, 23, 34) and placental chondroitin sulfate (1,15, 22, 33). Several of us (R.K., W.A.S., and S.B.) have hadextensive experience exploring negatively charged sugar polymer(GAG)-protein interactions related to amyloidogenesis (4, 19,27). In the latter case, the GAG-protein binding rapidly changesthe conformation of the amyloidogenic protein, leading to protofilaments(19, 24) and amyloid fibrils which adversely affect adjacentparenchymal cells (e.g., in patients with Alzheimer's disease).Agents that prevent this GAG-protein interaction have provedof value in inhibiting both the deposition of amyloid and themobilization of preexisting deposits (19). One such class ofcompounds is short-chain aliphatic anionic sulfonates.

As part of a program extending our GAG-protein binding studiesto the anionic sugar polymer-cationic malarial protein bindingissue, we have been examining structural details of GAG bindingto sporozoite and merozoite surface proteins. We have determinedthat aliphatic polysulfonates have antimalarial properties bothin vitro and in vivo at concentrations that may lead to potentiallyuseful therapeutic agents. While the exact mechanism of theiraction is not yet known, they appear to be potent inhibitorsof the process whereby the merozoite invades the host RBC, andthey may therefore constitute a novel class of antimalarials.

MATERIALS AND METHODS

Top

Materials and Methods

Chemical synthesis. (i) Poly(vinylsulfonate sodium salt) I (compound 1). A supply of poly(vinylsulfonate sodium salt) was obtained fromAldrich Chemical Company (catalog no. 27,842-4) as a 25% (wt/wt)solution in water, and the solution was processed as followsto provide a sample of compound 1. The solution (2 liters) wasconcentrated under reduced pressure to half of its volume. Ethanol(200 ml) was added, followed by the addition of activated carbon(50 g). The mixture was warmed on a steam bath for 20 min andthen filtered through Celite, and the solvent was removed underreduced pressure to give a light-yellowish oil. The oil wasdried in a vacuum oven (50°C) for 3 days to give an amorphoussolid (486 g). The molecular weight distribution was determinedby gel permeation chromatography by the American Polymer StandardsCorporation, as follows: number-average molecular weight (M_n)= 1,800; weight-average molecular weight (M_w) = 3,050; z-averagemolecular weight (M_z) = 5,400; M_w/M_n (polydispersity index)= 1.69.

(ii) Poly(vinylsulfonate sodium salt) II (compound 2). Poly(vinylsulfonate sodium salt) II was prepared by a radicalpolymerization of sodium vinylsulfonate in the presence of sodiumpersulfate. Each of the solutions, sodium vinylsulfonate (catalogno. 27,841-6; Aldrich) as 160 ml of a 25% (wt/wt) solution inwater (160 ml) and sodium persulfate (1.6 g) in H₂O (30 ml),was purged with argon for 30 min, and then the solutions weremixed. The mixture was heated (under argon) at 80°C in anoil bath for 17 h. The reaction mixture was concentrated underreduced pressure to 100 ml; addition of methanol (1 liter) gavea white solid which was dried in a vacuum oven (75°C) overnightto give a sample of compound 2 (45 g). The molecular weightdistribution was determined by gel permeation chromatographyby the American Polymer Standards Corporation, as follows: M_n= 1,600; M_w = 2,000; M_z = 2,700; M_w/M_n = 1.25.

(iii) Sodium 1,2-ethanedisulfonate (compound 3), sodium 1,3-propanedisulfonate (compound 4), sodium 1,4-butanedisulfonate (compound 5), sodium 1,5-pentanedisulfonate (compound 6), sodium 1,6-hexanedisulfonate (compound 7), and sodium 1,9-nonanedisulfonate (compound 8). The disulfonates (compounds 3 to 8) were prepared as their sodiumsalts by way of the reaction of sodium sulfite with the correspondingdibromide (see the report by Stone [39]). In each case, the¹H and ¹³C nuclear magnetic resonance spectra and the electron-sprayionization-mass spectrometry spectrum were consistent with theassigned structure.

The structures of the candidate compounds (compounds 1 to 8)are shown in Fig. 1.

View larger version (9K):
[in this window]
[in a new window]
FIG. 1. Structures of candidate compounds 1 to 8. The boldface numbers represent the compound numbers

Parasite lines and culture. P. falciparum cultures were grown in human type A-positive bloodobtained by venipuncture of volunteers. Cultures of the laboratoryline ItG were maintained by the method of Trager and Jensen(40) with RPMI 1640 supplemented with 10% human serum and 50µM hypoxanthine (RPMI-A). In vitro P. falciparum susceptibilitytesting was performed by a lactate dehydrogenase (LDH) enzymeassay specific for the LDH found in Plasmodium (pLDH) (20, 31).Briefly, the compounds to be tested were dissolved in RPMI-A,and the solutions were filtered by passing them through a 0.22-µm-pore-sizepolycarbonate filter and then serially diluted in duplicatein a 96-well plate to produce a compound gradient with twofolddilutions. Equal amounts of parasite culture (5% hematocrit,5% parasitemia) were added to each well, and the plates werethen incubated at 37°C in 95% N₂-3% CO₂-2%O₂ for 72 h. Thecontents of the wells were then resuspended, and a 10-µlsample was removed and added to 100 µl of the pLDH enzymeassay mixture. After 1 h the absorbance of the wells at 650nm was determined with a microplate reader. The 50% inhibitoryconcentrations (IC₅₀s) of individual compounds were determinedby a nonlinear regression analysis of the dose-response curve.The initial concentrations used in vitro were determined byserially diluting 1-mg/ml solutions of the candidate compounds.

In some experiments asynchronous parasite cultures (1 ml) wereeither untreated (controls) or treated with 500 µg ofcompound 1, 4, or 7 per ml for 24 h. The RBCs were then washedonce in RPMI-A prior to the addition of 10-µl aliquotsto the plates for the LDH assay and were used to produce bloodfilms that were stained with Giemsa. The percentage of parasitespresent as ring or mature stages were then determined for eachsample.

Determination of compound activity in mice. Female BALB/c mice (age, 6 to 8 weeks; Charles Rivers, Montreal,Quebec, Canada) were used in the study, and for each experimentthe mice were divided into four groups of eight mice each. Eachgroup was infected with Plasmodium berghei as described below.Twenty-four hours after infection the compound to be testedwas administered by intraperitoneal (i.p.) injection every 12h (9 a.m. and 9 p.m.) for at least 10 days. The quantities administeredin vivo were based on the effective concentrations determinedin culture, the assumption that the compound was distributedevenly within the animal, and a mouse weight of 25 g (i.e.,an animal volume of 25 ml). In some experiments a previouslydemonstrated effective concentration either was administered24 h before parasite inoculation and treatment was continuedevery 12 h (9 a.m. and 9 p.m.) for at least 10 days or was administered24 h after parasite inoculation and treatment was discontinuedafter 7 days. Animals were killed at the termination of theexperiment by CO₂ narcosis.

The compounds were dissolved in distilled water and injectedi.p. in a volume of 100 µl. Sufficient compound was preparedin water for entire experiments, and 5-ml aliquots were storedfrozen at -20°C until they were used. Aliquots were thawedslowly at 4°C before use. Unfrozen compound was always keptat 4°C, but at no time did the compound remain unfrozenfor more than 48 h.

The P. berghei organisms used for the i.p injections were obtainedfrom the blood of previously infected untreated mice that hadbeen infected with the organism for 10 to 14 days. Their parasiteloads were of the order of 40 to 50 intracellular organisms/100RBCs. Such mice were exsanguinated by cardiac puncture afterCO₂ narcosis, and the blood was immediately mixed with Alsever'ssolution (9.5 ml of glycerol, 2.33 g of glucose, 0.52 g of NaCl,and 1.0 g of sodium citrate made to 100 ml with distilled water)at a volume ratio of 1:3 (blood:solution). It was then dividedinto 0.5-ml aliquots and frozen at -70°C until it was required.When they were required, aliquots of diluted blood were thawedand mixed, and 100 µl was injected i.p. into each mouse.In most experiments a single injection was sufficient to raiseinfection levels to over 40 intracellular parasites/100 RBCsin untreated animals in 10 to 11 days. When the aliquots werestored for longer periods, the postinoculation increase in parasiteload was slower than the loads obtained when fresher aliquotswere used.

To monitor the progression of the parasitemia in each mouse,10 to 15 µl of blood was collected from the tail tip onceevery 24 h and placed into a heparinized glass capillary tube;and a blood smear was prepared, air dried, fixed in a 100% methanolbath for 2 min, immediately stained with Giemsa for 30 min,and rinsed in water. The slides were then air dried and dippedin Hemo-D solvent (a clearing agent; Fisher), and a coverslipwas placed with Permount.

The number of intracellular parasites per 100 RBCs was determinedby image analysis. The imaging system consisted of a Leitz Diaplanmicroscope with a x40 objective and a x10 eyepiece. A Hitachimodel HV-C10U CCD television camera was mounted on the microscope,and the image was transferred to the screen of a computer witha Pentium-type processor and digitized. The individual RBC imageson the screen were sharp and considerably larger than thoseseen under a microscopic high-power field at a x1,000 magnification.The parasites in infected cells were easily recognized. Themorphological parameters describing an average RBC were determinedexperimentally and entered into the program to be used witheach field examined. When tested and validated against manualcounts, in regions where the cells were well dispersed, theprogram determined the number of RBCs per field to a level withan error of 1 to 2%. The intraerythrocytic parasites were easilyrecognized, and the numbers were counted manually from the screenof the computer monitor. The total numbers of RBCs per fieldwere determined by image analysis. A minimum of 500 cells werecounted for each slide. The result was expressed as the numbersof intracellular parasites/100 RBCs. The image analysis program(MC-5, version 4.0, beta 2.0) is from MCID Imaging ResearchInc., Brock University, St. Catharines, Ontario, Canada L2S3A1.

RESULTS

Top

Results

Effects of compounds on P. falciparum in culture. An initial evaluation of the compounds was undertaken to determineif the compounds had antimalarial properties in vitro. Whenthe compounds were used in a 48-h antimalarial activity assaythat measured compound concentration versus pLDH activity inasynchronous cultures, it was observed that compounds 4 and7 had no detectable effect on the outcome of the assay. Similarexperiments indicated that compound 1 had a detectable effect(Fig. 2). Microscopic examination of the parasites in the wellswith the highest concentration of compound 1 indicated thatthere were an unusual number of free merozoites. Merozoitesthat had successfully invaded RBCs appeared to have developednormally. When the assay period was extended to 72 h and theparasite cultures were synchronized to the ring stage of developmentby sorbitol lysis, a more dramatic effect of compound 1 on thecultures was observed (Fig. 3). From this assay it was determinedthat compounds 4 and 7 had IC₅₀s of >20 mM and that compound1 had an IC₅₀ of 0.2 µM on the basis of a molecular weightof 3,050 (Table 1). Examination of synchronized cultures withcompounds 4 and 1 that were allowed to mature to the point ofmerozoite release suggested that merozoites failed to adhereto human RBCs in the presence of compound 1 (Fig. 4). Amonga series of compounds related to compound 1, compound 2 hadan IC₅₀ of 6 ± 2 µM on the basis of a molecularweight of 2,000 (Table 1).

View larger version (20K):
[in this window]
[in a new window]
FIG. 2. Effects of chloroquine and compounds 1, 4, and 7 on the viabilities of nonsynchronized P. falciparum merozoites in tissue culture. P. falciparum cultures were exposed to various concentrations of either a candidate compound (lower scale) or chloroquine (upper scale) for 48 h before the degree of parasite viability was determined by an LDH assay (see Materials and Methods). The percentage of the control activity was determined by comparing the values obtained in treated wells to those obtained in untreated wells. The datum points indicate the results obtained with compounds 1 (•), 4 ( ${blacktriangleup}$ ), and 7 ( ${blacktriangledown}$ ) and chloroquine ( ${circ}$ ), with lines of best fit shown.

View larger version (17K):
[in this window]
[in a new window]
FIG. 3. Effects of candidate compounds on the viabilities of sorbitol-lysed synchronized P. falciparum merozoites in tissue culture. P. falciparum cultures were exposed to various concentrations of a candidate compound for 48 h before the degree of parasite viability was determined by an LDH assay (see Materials and Methods). The percentage of the control activity was determined by comparing the values obtained in treated wells to those obtained in untreated wells. The datum points indicate the results obtained with compound 4 (•), compound 7 ( ${blacktriangledown}$ ), no treatment ( ${triangledown}$ ), and compound 1 ( ${circ}$ ), with lines of best fit shown. Compounds 4 and 7 had IC₅₀s greater than 1 mg/ml. The IC₅₀ of compound 1 was 0.2 µM.

View this table:
[in this window]
[in a new window]
TABLE 1. IC₅₀s of a series of candidate compounds for P. falciparum viability in human RBC cultures

View larger version (80K):
[in this window]
[in a new window]
FIG. 4. Effects of candidate compounds on invasion of merozoites. P. falciparum cultures were grown in the presence of 6 µg of compound 1 per ml (left panel) or 6 µg of compound 4 per ml (right panel). After 24 h, samples of the cultures were removed and thin films were produced, fixed with methanol, and stained with 3% Giemsa stain in water for 30 min. The panels indicate representative fields of cultured RBCs-malarial parasites observed by microscopy. Note the extracellular locations of the parasites in the left panel. Magnifications, x1,000.

Assessment of the proportions of ring and mature stages of theparasites after 24 h of treatment with compound 1, 4, or 7 revealedthat the last two compounds had little effect on the differentstages compared to the effect of no treatment. In contrast,compound 1 had an effect on the viability of the parasites andcaused a significant reduction in the proportion of ring forms(Fig. 5).

View larger version (24K):
[in this window]
[in a new window]
FIG. 5. Asynchronous parasite cultures (1 ml) were either untreated or treated with 500 µg of compound 1, 4, or 7 per ml for 24 h. The RBCs were then washed once in RPMI-A prior to addition of 10-µl aliquots to plates for the LDH assay (the values are the averages ± standard deviations of four values) and were used to produce blood films that were stained with Giemsa. The percentages of parasites present as ring or mature stages were then determined for each sample (n = 200 cells in each case).

In vivo observations with P. berghei. Figure 6A illustrates the effects of different doses of compound1 on the progression of the parasitemia in vivo. Of significantinterest was the effect of compound 1 at doses of 4.8 and 38.4mg twice daily. There was considerable inhibition of the parasitemiaover the first 7 days in the treated animals compared to thatin the untreated animals and those receiving a dose of 0.6 mgtwice daily. After 7 days the parasites appeared to break freeof the inhibitory effects of this compound, with parasite levelsincreasing steadily. A two-way analysis of variance (ANOVA)examining between-group variations indicated a highly significantinhibition (P < 0.001) by the two higher doses of compound1.

View larger version (12K):
[in this window]
[in a new window]
FIG. 6. Effects of compounds 1 (A), 4 (B), and 7 (C) on the time course of P. berghei parasitemia expressed as numbers of intracellular parasites/100 RBCs. Parasites were inoculated i.p., and treatment with compound 1, 4, or 7 was started 24 h later. Each dose of agent was given i.p. at 12-h intervals for a period of 9 days. •, no treatment; ${blacktriangledown}$ , treatment with a dose of 0.6 mg; ${blacktriangleup}$ , treatment with a dose of 4.8 mg; ${blacksquare}$ , treatment with a dose of 38.4 mg. (A) Each point is the mean ± standard error of the mean for eight animals per time point. An ANOVA of between-group comparisons revealed significant differences (P < 0.001). (B) Each point is the mean ± standard error of the mean for eight animals per time point. An ANOVA of between-group comparisons revealed significant differences (P < 0.012). (C) Each point is the mean ± standard error of the mean for eight animals per time point. An ANOVA of between-group comparisons revealed no significant differences.

In contrast to the effects of compound 1, compound 4 (Fig. 6B),the monomeric unit of compound 1, at the two higher doses hada transient lesser inhibitory effect, but an inhibitory effectthat was nonetheless significant (P < 0.012), between days5 and 9. Compound 7, an additional compound related to compound1 in which the charged groups are spaced farther apart, hadrelatively little effect on progression of the infection (Fig.6C). Such a compound serves as a good negative control.

Figure 7A illustrates the effect of compound 1 administeredat 4.8 mg twice daily beginning either 24 h before or 24 h afterparasite inoculation. This compound also considerably inhibitedthe parasitemia over the first 7 days after inoculation comparedto the level of parasitemia in untreated animals (P < 0.001),but it was not more effective than treatment postinoculation.Figure 7B illustrates the effects of different doses of compound2.

View larger version (15K):
[in this window]
[in a new window]
FIG. 7. (A) Effects of poly(vinylsulfonate sodium salt) I (compound 1; 4.8 mg/dose) administered either before or after parasite inoculation on the time course of P. berghei parasitemia, expressed as the numbers of intracellular parasites/100 RBCs. Parasites were inoculated i.p., and treatment with compound 1 was started either 24 h before or 24 h after inoculation. Compound 1 was given i.p. at 12-h intervals for a period of 10 days. •, no treatment; ${blacksquare}$ , treatment with compound 1 starting 24 h after inoculation; ${blacktriangledown}$ , treatment with compound 1 starting 24 h after inoculation and ending after day 7; ${blacktriangleup}$ , treatment with compound 1 starting 24 h before inoculation. Each point is the mean ± standard error of the mean for eight animals per time point. An ANOVA of between-group comparisons revealed significant differences (P < 0.001). (B) Effects of different doses of poly(vinylsulfonate sodium salt) II (compound 2) on the time course of P. berghei parasitemia, expressed as the numbers of intracellular parasites/100 RBCs. Parasites were inoculated i.p., and treatment with compound 2 was started 24 h later. Each dose of compound 2 was given i.p. at 12-h intervals for a period of 9 days. •, no treatment; ${blacktriangledown}$ , treatment with a dose of 125 µg; ${blacktriangleup}$ , treatment with a dose of 500 µg; ${blacksquare}$ , treatment with a dose of 2 mg. The highest dose proved to have an adverse effect on animal well-being, and these animals were euthanized after 8 days. Each point is the mean ± standard error of the mean for eight animals per time point. An ANOVA of between-group comparisons revealed significant differences (P < 0.001).

The infection dynamics appear to be slightly different in eachof the in vivo experiments, with a more gradual increase inthe level of parasitemia in some experiments compared with therate of increase in others. This is likely due to the viabilitiesof the parasites in aliquots of infected mouse blood that arestored for different periods of times and that are used to inoculatethe mice.

As early as the second day of treatment compounds 1 and 2 hadadverse effects on the health of the mice. Although the parasitelevels were significantly lower in mice treated with compounds1 and 2 than in the untreated control mice, animals treatedwith compounds 1 and 2 were much less active than the animalsin the untreated group, their fur was ruffled, and at the timethey were killed their tissues appeared to be dehydrated. Grossinspection of the organs of such animals failed to reveal anyobvious pathological features such as differences in size, color,or shape. No such adverse effects could be demonstrated whencompounds 1 and 2 were given orally in antiamyloid studies (19)rather than by the i.p. route used in the present studies. Theroute of administration of compounds 1 and 2 plays a role inthe toxic manifestations. The antimalarial properties of compounds1 and 2 were likely not dependent on their in vivo toxic effects,as the antimalarial effect was also seen in vitro. Furthermore,compound 7 exhibited toxicity but was not effective in vivo.

DISCUSSION

Top

Discussion

The compounds studied were originally designed to explore theirinhibitory properties on heparan sulfate-amyloid protein binding.This heparan sulfate-protein interaction rapidly alters theconformation of amyloidogenic proteins such as serum amyloidA, the precursor to inflammation-associated amyloid (24), andAß, the peptide in Alzheimer's disease-associatedamyloid (12, 19, 25, 26), conferring upon them significant increasesin ß-sheet structure, the characteristic protein conformationof amyloids. Our initial attempts to find heparan sulfate-proteinbinding inhibitors focused on sulfonated and sulfated disaccharidesas analogues of the heparan sulfate structure, and they provedto have the required activities (unpublished data). Unfortunately,they also had significant anticoagulant properties. However,the acyclic compounds retained the antiamyloid properties butdid not display the unwanted anticoagulant effects (19).

In several ways many of the steps in the malarial parasite invasionprocess are analogous to the GAG-amyloid protein interactionseen in amyloidogenesis. There is robust evidence indicatingthat the P. falciparum sporozoite interacts with the host'shepatocyte through a heparan sulfate proteoglycan on the livercell surface and its own circumsporozoite protein during itsentry into the liver cell (37, 42). Native remnant lipoproteinparticles which use the same binding process with hepatocytescompete effectively with the sporozoites for entry into livercells (13, 38). P. falciparum RBC membrane protein 1 (PfEMP-1)is expressed on the surfaces of parasitized RBCs. It possessesheparan sulfate-like binding properties that may be importantin the formation of RBC rosettes (5). Furthermore, infectedRBCs expressing PfEMP-1 on their surfaces can adhere to thechondroitin sulfate present on endothelial thrombomodulin (32,34), can potentially adhere to endothelial cell surface heparansulfate (9), and can adhere to the chondroitin sulfate in theplacenta during pregnancy (1, 15, 22, 33). Moreover, merozoiteentry into RBCs may also depend, in part, on ionic interactions(7). The success that we had interfering with amyloidogenesisby using agents with low molecular weights that blocked GAG-proteininteractions suggested that the same strategy might be usefulin interfering with some of these steps during the malarialparasite invasion process. Such a concept has, in fact, beenconsidered in the past (8, 28, 29), but the previous approachesused high-molecular-weight compounds (compounds with molecularweights of the order of 500,000) very similar in structure tothe GAGs themselves (8). In this context our present findingsindicate that molecules orders of magnitude smaller than thosepreviously considered as antimalarials have many desirable propertiesand should be considered lead compounds for additional explorationand optimization. Such studies are ongoing with agents structurallyrelated to poly(vinylsulfonate), with promising results (unpublisheddata). The poly(vinylsulfonate) appears to exert its effectby inhibiting merozoite entry into RBCs, a step which may involvean ionic heparan sulfate-like binding interaction (5). Preciselywhich merozoite surface-RBC surface molecular interaction(s)is involved is not apparent at present. Given the nature ofpoly(vinylsulfonate), ionic binding likely plays a role, butit is not the only important factor. As shown previously (4,18), molecular conformation as well as charge spacing influenceprotein-GAG binding and the inhibitory properties of GAG mimeticcompounds. It may also make sense to use such compounds as inhibitorsof sporozoite entry into hepatocytes (13, 37, 38, 42).

Compound 1 (and compound 2) was found to be effective both ina human in vitro malarial system and in a rodent in vivo model.This finding suggests that compound 1 inhibits a step in theinvasion process that is common to multiple Plasmodium speciesand does not depend on the presence of a species-specific receptor-ligandcombination on either the host or the parasite cell surface.The effect of compound 1 on the parasite in vitro and in vivoappears to be limited to the prevention of merozoite invasion,since merozoites that successfully invaded RBCs appeared todevelop normally. It is noteworthy that compound 1 was unableto completely inhibit the invasion process in vitro, since compound1 was able to slow the increase in the level of parasitemiabut was not able to halt the infection. The incomplete inhibitionmay be due to the presence of a redundant, non-compound 1-relatedinhibitable invasion mechanism, may be due to a relatively lowefficiency of blockade of merozoite entry into RBCs, allowingthe eventual growth of an overwhelming merozoite population,or may reflect the fact that compound 1 has no intraerthrocyticantimalarial activity. The observation that compound 1 becameless effective after 8 days in vivo suggests that a normallyredundant mechanism may be present and may be being selectedduring the course of the infection. This possibility is beingexplored.

Our results also demonstrate a satisfying correlation betweenin vitro and in vivo activities. The compounds exerting themost significant inhibitory properties against P. falciparumin vitro proved to be the most effective against P. bergheiin vivo. Our in vitro and in vivo experiments were not designedto identify the receptor-ligand combination that is being inhibited;however, observation of merozoites in culture suggests thatthe compounds are inhibiting the initial step of the invasionprocess. Further work is required to determine if the compoundsinteract with a merozoite surface protein, such as EBA-175,or whether they interact with an RBC receptor such as glycophorinA or B (2, 36).

It should be recognized that the samples of poly(vinylsulfatesodium salt) (compounds 1 and 2) are mixtures of oligomericspecies with slightly different chain lengths and stereochemistries.Work is in progress to synthesize oligomers with defined lengthsand configurations at stereogenic centers.

Our results provide significant scope for further development.Among the questions that need to be addressed are the minimumsizes of poly(vinylsulfate sodium salt)-like molecules thatare effective in abrogating merozoite entry into RBCs, theirpossible efficacies in blocking sporozoite entry into livercells, and whether there are specific stereoisomers among themultiple forms which poly(vinylsulfate sodium salt) can takethat may be more effective than others.

ACKNOWLEDGMENTS

We gratefully acknowledge the generous support of the Bill andMelinda Gates Foundation (to R.K.) and the able technical assistanceof Michelle Ciach in the execution of this work.

FOOTNOTES

* Corresponding author. Mailing address: Department of Pathology, Queen's University, Kingston, Ontario K7L 3N6, Canada. Phone: (613) 533-6411. Fax: (613) 533-2907. E-mail: Kisilevsky{at}cliff.path.queensu.ca.

REFERENCES

Top