Inhibition of Plasmodium Falciparum Malaria Using Antisense Oligodeoxynucleotides (Phosphorothioate Deoxynucleotides/Drug Resistance) ROBERT H

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 514-518, January 1996 Medical Sciences

Inhibition of Plasmodium falciparum malaria using antisense oligodeoxynucleotides (phosphorothioate deoxynucleotides/drug resistance) ROBERT H. BARKER, JR.*t#, VALERI METELEVt, ELIEZER RAPAPORTt, AND PAUL ZAMECNIKt *Hybridon, Inc., One Innovation Drive, Worcester, MA 01605; and tWorcester Foundation for Biomedical Research, 222 Maple Avenue, Shrewsbury, MA 01545. Contributed by Paul Zamecnik, August 22, 1995

ABSTRACT We studied inhibition of growth of the ma- (8), and their use against cytomegalovirus and human immu- laria parasite Plasmodium falciparum in in vitro culture using nodeficiency virus (HIV) in humans is currently under clinical antisense (AS) oligodeoxynucleotides (ODNs) against differ- investigation. ent target genes. W2 and W2mef strains of drug-resistant Using chloroquine-resistant parasites cultured in vitro, pre- parasites were exposed to AS ODNs over 48 hr, and growth vious experiments in our laboratory showed that AS ODNs was determined by microscopic examination and [3H]hypox- were taken up by infected, not uninfected, erythrocytes and anthine incorporation. At ODN concentrations of 1 ,LM, that AS ODNs directed against the DHFR gene inhibited phosphorothioate (PS) ODNs inhibited growth in a target- parasites in culture (9). However, those studies and others (10) independent manner. However, between 0.5 and 0.005 ,LM, also revealed significant non-DNA sequence-dependent inhi- ODNs against dihydrofolate reductase, dihydropteroate syn- bition of parasites when the nuclease-resistant phosphorothio- thetase, ribonucleotide reductase, the schizont multigene fam- ate (PS) form of the ODN was used. The present studies were ily, and erythrocyte binding antigen EBA175 significantly undertaken to clarify the issue of sequence-dependent inhibi- inhibited growth compared with a PS AS ODN against human tion and to examine alternative target genes. immunodeficiency virus, two AS ODNs containing eight mismatches, or the sense strand controls (P < 0.0001). The IC50 was -0.05 IAM, whereas that for non-sequence-specific con- MATERIALS AND METHODS trols was 15-fold higher. PS AS ODNs against DNA poly- Parasite Culture. P. falciparum strains W2 and W2mef were merase ai showed less activity than that for other targets, maintained as described (11). All strains were kindly provided whereas a single AS ODN against triose-phosphate isomerase by Dyann F. Wirth, Harvard School of Public Health. did not differ significantly from controls. We conclude that at ODN Preparation. AS ODNs were synthesized on a Bio- concentrations below 0.5 ,LM, PS AS ODNs targeted against search model 8700 synthesizer (MilliGen). Assembly of oligo- several malarial genes significantly inhibit growth of drug- nucleotides was performed using standard phosphoroamidite resistant parasites in a nucleotide sequence-dependent man- chemistry. PS bonds were introduced by oxidation with the ner. This technology represents an alternative method for Beaucage thiolating reagent as described (12). Full-length identifying malarial genes as potential drug targets. oligonucleotides were purified by HPLC, and final purity was assayed using a WAX column (13). Before use in vitro, 10 ,uM Malaria afflicts "200 million people each year, making it a ODN stock solutions were prepared by dilution in unsupple- major cause of human morbidity and mortality worldwide. mented RPMI lacking human plasma and were filter sterilized Because of the increased prevalence ofPlasmodiumfalciparum through a 0.45-,um filter. Serial 10-fold dilutions were then strains resistant to current chemotherapy, treatment and con- prepared for charging microtiter plates. trol of this disease are becoming progressively more difficult. Gene Targets. The sequences of ODNs are shown in Table This trend is made more alarming because of the apparent 1. ODN HIV is directed against the gag protein of HIV-1 (14) cross-resistance between mefloquine, halofantrine, and arte- and was used as a sequence control. PS AS ODN 105 is against misinin, even in areas where the latter two drugs are unavail- a conserved sequence in the DHFR gene from 1153 to able (1). New antimalarial compounds are urgently needed. bp bp Most antimalarial drugs act posttranslationally by interfer- 1179 (2); PS AS ODNs RB36 and RB37 target the same region ing with protein function. Examples include antifolate com- but contain eight mismatches. PS ODN RB58 is the "sense" pounds, which inhibit dihydrofolate reductase (DHFR) (2), strand complementary to 105. PS AS ODNs 86a, 86c, and 104 and sulfonamide drugs, which inhibit dihydropteroate syn- target different portions of the DHFR gene. RB1 (bp 535-568) thetase (DHPS) (3). A disadvantage of this approach is that in targets the 5' cysteine-rich conserved region of the erythrocyte these cases, one or two point mutations can confer drug binding antigen EBA175 (15). RB10 is from a conserved resistance (4). region (bp 71-97) of the triose-phosphate isomerase (TPI) Use of antisense (AS) oligodeoxynucleotides (ODNs) to gene. RB29 (AGT start codon), RB30 (bp 1944-1970), RB31 inhibit specific mRNA synthesis and translation represents a (bp 1976-2003), RB60 (bp 2066-2091), and RB65 (bp 637- promising alternative chemotherapeutic strategy. Unlike the 661) target DHPS (17). RB46 (bp 161-187), RB50 (bp 2-24), cases described above, more sequence alterations are required and RB62 (bp 200-226) target the schizont multigene family to eliminate AS ODN function (5), making it in principle more (RHO) thought to include the rhoptry proteins (18). PS AS difficult to develop drug resistance mediated by point muta- ODNs RB47 (bp 112-136), RB48 (bp 987-1011), and RB61 tions. (bp 593-621) are sequences from the ribonucleotide reductase In vitro, AS ODNs have been shown to inhibit replication of both Trypanosoma brucei (6) and Leishmania amazonensis (7). Abbreviations: PS, phosphorothioate; AS, antisense; ODN, oligode- In vivo, AS ODNs have been used to cure ducks of hepatitis B oxynucleotide; DHFR, dihydrofolate reductase; DHPS, dihydropteroate synthetase; RR, ribonucleotide reductase; RHO, schizont multigene family; TPI, triose-phosphate isomerase; HIV, human The publication costs of this article were defrayed in part by page charge immunodeficiency virus. payment. This article must therefore be hereby marked "advertisement" in tTo whom reprint requests should be addressed at the Worcester accordance with 18 U.S.C. §1734 solely to indicate this fact. address. 514 Medical Sciences: Barker et al. Proc. Natl. Acad. Sci. USA 93 (1996) 515

Table 1. ODNs used against different targets ODN Sequence Target HIV TCT TCC TCT CTC TAC CCA CGC TCT C HIV 86a ATA AAT ATC GAA AAC GTC GCA DHFR 86c ATA AAT ATC GAA AAC GTC GCA C DHFR 104 TAA AAT ATG ACA AGG AGG TAA TGC CAT DHFR 105 TCT TAA AAA TAA TTT CTT CGT AGT TAA DHFR RB58* TTA ACT ACG AAG AAA TTA TTT TTA AGA DHFR RB36t TCA TAT AAT TAT TTA CTA CGT TGT AAA DHFR RB37t T-jT TAT ATA AAA ATT GTT CCT A-CT TTT DHFR RB1 GCA TAA TTG GAT TCT ACG ATC AGG AAT ACA TAC EBA175 RB10 CAA AAT CCA AAT TGT TAA AAC TGT TTG TPI RB29 GAA AGT ATT AGT TCT TGT ATA GTT TCC DHPS RB30 TAA CAT TTA ATA TTC CAA CAA TAT TTG DHPS RB31 CAA AAA TAC CTC CAT CTG AAA AAG AAT C DHPS RB60 GGA GCA GAG GAT TCT CCA CCT ATA TC DHPS RB65 CAT TTG TTC CTA AGT TTA ATA CGG C DHPS RB46 ATT TGG TAC ATC CAG TGG TTC ACT TTG RHO RB50 CCA CTA GGT TCT AGT ACC ACT TCA CC RHO RB62 CGG TTG TGT ATT GAA TGG AAT ATC TCC RHO RB47 GGG AAA ACA TCA GGA TAT AAT ATT GG RR RB48 AAG ACA TGA TCC TTT CGT TGA GCC ATG RR RB61 CCG TGT AAT TTA TTT TGT TTC TTA AAC C RR RB49 TTT TAA TAA ATA AAT TAA TAT ATA CAT DNA Pol RB51 CCT CAT TTT CTT TAA TTT GAA TTT CC DNA Pol RB52 GAA TTT CTT CTT TGC TTA ATT TAT TTC C DNA Pol RB53 TAA TTA TTA TTA TTA TTA TTA TTT TCG DNA Pol RB63 CCC TAG TTT TCC TTT TTC TTG TTC TCG DNA Pol RB64 CCC TTT TGA AAC TTT TCA TGT ATA GG DNA Pol The sequences were obtained from the following references: HIV (gag protein), 14; DHFR (DHFR/ thymidylate synthase), 2; EBA175 merozoite surface protein, 15; triose-phosphate isomerase (TPI), 16; DHPS, 17; schizont multigene family (RHO), 18; ribonucleatide reductase (RR), 19; DNA polymerase a (DNA pol), 20. *Sequence corresponds to the sense strand for 105. tUnderlining indicates mismatches with 105. (RR) gene (21). PS AS ODNs RB49 (bp 752-778; initiation RESULTS codon), RB51 (bp 1426-1451), RB52 (bp 893-920), RB53 (bp 1132-1158), RB63 (bp 984-1010), and RB64 (bp 2113-2140) Initial experiments examined four different PS AS ODNs all target DNA polymerase a (20). directed against the DHFR gene for their capacity to inhibit Parasite Inhibition Assays. Effects of ODNs on parasite parasite growth using the W2 (Fig. 1) or W2mef (data not growth were measured by microscopic examination of thin shown) strain. DHFR catalyzes the reaction of 7,8- smears and by [3H]hypoxanthine incorporation after drug dihydrofolate plus NADPH in formation of 5,6,7,8- treatment. For microscopy, 48-well microtiter plates were tetrahydrofolate, which the parasite requires since it does not charged with PS AS ODNs at different dilutions, unsynchronized parasites were added to a total of 1 ml (0.4% parasitemia, Target: Dihydrofolate Reductase 5% red blood cells), cultures were incubated 48 hr at 37°C, and *105 Microscopy then slides were prepared. The proportion of infected cells was measured by counting at least 500 red blood cells. Results were expressed as the percent reduction compared with controls receiving medium alone without PS AS ODNs. c For hypoxanthine incorporation assays, 96-well microtiter plates were charged with ODNs, using four wells for each Two dilution. hundred microliters of unsyncronized parasites 0. was added (0.4% parasitemia, 5% red blood cells), and cultures 0 were incubated at 37°C. After 20-24 hr, 0.5 ,uCi (1 Ci = 37 cL GBq) of [G-3H]hypoxanthine was added to each well, cultures were incubated overnight, and cells were harvested for scin- tillation counting using a PHD cell harvester (Cambridge Technology, Watertown, MA). After counting, values were 0.01 0.05 0.1 0.5 averaged for the four samples at each dilution, and the average ODN Concentration (uM) was multiplied by the proportion of parasites present at that dilution of ODNs relative to control (determined by micros- FIG. 1. Effect of PS AS ODNs against DHFR. W2 strain parasites copy) and then was expressed as percent reduction compared were cultured 48 hr in the presence of PS AS ODNs at the concentrations with control no (IxM) indicated and then were examined microscopically and counted. samples receiving PS AS ODNs. Results are averages from five experiments; error bars indicate the Statistical Analysis. All experiments were repeated a min- standard deviation. Percent reduction was calculated relative to controls imum of five times. Average percent reduction in parasite cultured in the absence ofPS AS ODNs. ODN HIV, corresponding to the growth was compared with controls using Student's t test. gag region of HIV, was used as a sequence control. 516 Medical Sciences: Barker et al. Proc. Natl. Acad. Sci. USA 93 (1996) appear to utilize pyrimidine salvage pathways (22) and there- The recently described RHO is thought to include the fore synthesizes pyrimidines de novo. Results showed that rhoptry proteins (18) and is therefore presumably involved in inhibition by PS AS ODNs 104 and 105 differed significantly merozoite invasion. Three different PS AS ODNs against (P < 0.001) from that of the HIV sequence control, whereas RHO were tested (Fig. 4A). Although the average percent the other two (86a and 86c) did not (Fig. 1). Because the reduction was generally lower than that using 105, this differ- average inhibition by ODN 105 was slightly higher, it was ence was not statistically significant, whereas at concentrations selected as a positive reference for all subsequent experiments. above 0.005 ,uM, all three anti-RHO PS AS ODNs specifically We then examined the target specificity of parasite inhibi- inhibited parasite growth compared with the HIV sequence tion using PS AS ODNs. PS AS ODN 105 was compared with control (P < 0.001). Similar results were obtained with RB1, three different types of negative controls. PS AS ODN HIV an AS ODN directed against EBA175 (Fig. 4B), a merozoite targets the gag region of HIV and shares no known homology protein related to the Plasmodium vivax Duffy blood group with P. falciparum. PS AS ODNs RB36 and RB37 were derived antigen involved in erythrocyte binding (15). In contrast, PS from the ODN 105 sequence but contain eight mismatches AS ODN RB10, against TPI, a glycolytic enzyme, was signif- (Table 1). RB58 corresponds to the sense sequence of 105. icantly less inhibitory (P < 0.01), differing from 105 especially Preliminary results showed that at concentrations of 1 ,uM or at higher concentrations (Fig. 4B). greater, PS ODNs inhibited parasites by about 90%, regardless of the PS ODN sequence used (Fig. 2A and data not shown). DISCUSSION at between 0.5 and 0.01 ,uM, there However, concentrations to target was a highly significant (P < 0.001) difference between PS AS Studies presented here were undertaken reexamine DHFR and controls. The value for specificity of PS AS ODN inhibition of malarial growth and to ODNs targeted against IC50 examine use of alternative gene targets. Previous work in our PS AS ODN 105 was between 0.05 and 0.01 ,uM, whereas the laboratory (9) and others (10) showed that at high concentra- IC50 values for negative controls were 10- to 15-fold higher tions, PS ODNs inhibit growth of P. falciparum in culture (Fig. 2B). independent of the sequence used. Because of this, Clark and Experiments compared the efficacy of PS AS ODNs against coworkers (10) concluded that virtually all PS ODN inhibition a variety of other gene targets. Five different PS AS ODNs of malaria was due to non-sequence-dependent effects. Results targeted against DHPS, an enzyme in the folate biosynthetic from the present study (Fig. 2A) confirmed that at concen- pathway, were examined for inhibition of malarial growth (Fig. trations of 1 ,uM or higher, all PS ODNs tested reduced 3A). Like PS AS ODNs targeted against DHFR, all DHPS- parasites about 90% relative to controls. Thus, studies that only targeted PS AS ODNs specifically inhibited parasites at 0.5 ,uM examine high ODN concentrations have found little sequence (P < 0.0001). However, at lower concentrations (0.1-0.01 ,uM) dependence for AS-mediated inhibition (10). three PS AS ODNs (RB31, RB60, and RB65) retained signif- However, at concentrations between 0.005 and 0.5 ,uM, icant antimalarial activity (P < 0.0001), whereas the remaining parasite inhibition by PS AS ODNs specifically directed against two PS AS ODNs (RB29 and RB30) did not differ statistically DHFR was 10- to 30-fold greater (P < 0.0001) than that of from PS ODN HIV. sense sequence controls (RB58), mismatch controls (RB36 Three ODNs (RB47, RB48, and RB61) were tested against and RB37), or a PS AS ODN against HIV (Fig. 2B). Similar RR (21), which catalyzes the rate-limiting step in de novo sequence-specific effects were observed for most of the other synthesis of deoxyribonucleotides. Although the average per- AS ODNs tested here against other target genes. Thus, the cent reduction for each of these was slightly lower than that of inhibitory effects of PS ODNs against malaria are not simply 105 (against DHFR), the difference was not significant, nonspecific, as has been concluded by others (10), but at low whereas all three did differ significantly (P < 0.001) from PS concentrations can be used to identify critical genes, which can AS ODN HIV, the HIV sequence control (Fig. 3B). be exploited for both drug and vaccine development. DNA polymerase a (20) is an enzyme essential for DNA Reasons for the non-target-specific inhibition by PS ODNs replication in most organisms. Six PS AS ODNs were exam- are not clear, although a number of hypotheses have been ined. The efficacy of these PS AS ODNs was less than that of advanced (23). PS ODNs are charged polyanions, which in 105 at 0.5 ,uM and fell more rapidly as the concentration other systems have been shown to bind and sequester a variety decreased (Fig. 3C). of growth factors (24). In P. falciparum, non-sequence-specific Anti-Malarial Specificity Anti-Malarial Specificity A Microscopy B 3H CPM loo1

c c 0 0 ts *0 0 a1)

CL 0La)

0.001 0.005 0.01 0.05 0.1 0.5 ODN Concentration (uM) ODN Concentration (uM) FIG. 2. Sequence specificity of PS ODNs against P. falciparum, determined by microscopic examination after 48 hr (A) or [3H]hypoxanthine incorporation (B). Results are averages from eight experiments comparing the HIV sequence control (ODN HIV) with ODN 105 against DHFR. Error bars indicate the standard deviation. The nucleotide sequences of ODNs RB36 and RB37 are derived from that of ODN 105 but contain mismatches, and the nucleotide sequence of ODN 58 corresponds to the sense strand of ODN 105 (Table 1). Medical Sciences: Barker et al. Proc. Natl. Acad. Sci. USA 93 (1996) 517

Target: Dihydropteroate Synthetase Target: Schizont Multi-Gene Family A 3H CPM A 3H CPM

C c .o 0 *0 e a C 8 a.

0.005 0.01 0.05 0.1 0.5

Target: Ribonucleotide Reductase Targets: TPI and EBAl 75 B 3H CPM B 3H CPM

C a 0 0. 15 0 :3

CD eL

0.005 0.01 0.05 0.1 0.5 ODN Concentration (uM) Target: DNA Polymerase FIG. 4. Effect of AS ODNs against RHO (18) (A), the erythrocyte C 105 3H CPM binding antigen (EBA175; B), and TPI (B). Results are averages of (A) or five experiments (B) assayed by [3H]hypox- - eight experiments 100 E HIV anthine incorporation. Error bars indicate standard deviation. eaRB83 80 controls (Fig. 2B). This value is significantly below those de-

0 scribed previously (9, 10). This difference may be specific for the 't El RB52 PS AS ODN 105 sequence, since the inhibitory efficacy of 660 different ODN sequences clearly varies (Figs. 2-4), and the IC50 40 values we obtained for three different AS ODNs directed against RR (Fig. 3B) were similar to those reported for another ODN against that target (21). Altematively, the lower IC50 of AS ODN 20 105 determined here may reflect differences in the way the assays were performed: studies by Rapaport and colleagues (9) were assayed after 24 hr, instead of the 48 hr used in the present case, 0 0.005 0.01 0.05 0.1 0.5 whereas the Clark et al. studies (10) were performed in the ODN Concentration (uM) absence of human plasma, which was included in our medium. Some combination of these three factors may explain the lower FIG. 3. Effect of AS ODNs against DHPS (A), RR (B), and DNA IC50 observed in the present studies. polymerase (C). Results are averages from eight experiments assayed Seven different genes were examined as possible targets for by [3H]hypoxanthine incorporation; error bars denote standard devi- PS AS ODNs. Functionally, these fell into three categories: (i) ation. genes involved with folate biosynthesis and DNA replication (DHFR, DHPS, RR, and DNA polymerase); (ii) genes po- effects have been shown to be proportional to length, concen- tentially involved in merozoite invasion (EBA175 and RHO); tration, and to the time of exposure to PS AS ODNs during the and (iii) an enzyme in the glycolytic pathway (TPI). Genes malaria life cycle (10). It has been suggested that the polyanion involved in folate biosynthesis and DNA replication were effect of PS ODNs may inhibit merozoite invasion (9, 10) in a logical choices since they are required for parasite growth and manner similar to that observed for dextran sulfate (25). because several of these enzymes are targets of presently used However, since this inhibition could also be explained by antimalarial compounds. The second group of targets was intracellular nonspecific polyanion effects, the actual mecha- selected because of the requirement for merozoite invasion, nism by which PS ODNs achieve a non-target-specific effect whereas the third seemed appropriate because of the parasite's remains unclear. metabolic requirements. In studies presented here, the IC50 for ODN 105 was -0.05 ,uM In all but one case (PS AS ODN 10; Fig. 4B) PS AS ODNs for both strains tested, which is more than 10-fold below those of targeted against a variety of genes showed some efficacy com- 518 Medical Sciences: Barker et al. Proc. Natl. Acad. Sci. USA 93 (1996) pared with the HIV control in inhibiting malarial growth. How- swering questions associated with clinical application, such as ever, there was considerable variation among different PS AS frequency and timing of dosage. It also seems possible that by ODNs against the same target gene and between PS AS ODNs combining AS ODNs against different targets, it may be targeted to different genes. Variation in efficacy between PS AS possible to achieve a synergistic effect, thereby increasing the ODNs targeting different regions of the same gene may reflect potency of AS ODNs against malaria. differences in hybridization efficiency or the extent of localized This methodology represents a powerful tool for identifying protein complexing, which may inhibit AS ODN annealing (26). essential malarial genes as targets for drug development. While Preliminary experiments (R.H.B., unpublished results) suggested it is obviously our goal at present to focus on development of that AS ODNs directed against the start site of transcription are AS ODNs for this purpose, such identification is necessary for less effective than those targeted downstream (data not shown); development of other drugs as well. Although transfection however, this requires further examination. studies may yield similar kinds of results, those experiments Generally speaking, results obtained using either micro- seem at present to be far more difficult. We therefore feel that scopic examination or 3H incorporation assays were similar. use of AS ODNs will provide an alternative means for exam- However, the two assays measure different endpoints: micros- ining questions of gene function central to the biology of the copy allows scoring of parasite numbers but provides limited parasite, as well being possible chemotherapeutic agents. information concerning metabolic condition, whereas hypoxanthine incorporation measures the parasites' ability to take P. Zamecnik is a member of the Board of Directors and Chairman up and utilize a metabolic precursor. In the present case, of the Scientific Advisory Committee of Hybridon. exposure of parasites to PS ODNs stimulated hypoxanthine uptake compared with untreated controls (data not shown), 1. Gay, F., Bustos, D. G., Diquet, B., Rivero, L. R., Litaudon, M., which is why cpm values were "corrected" prior to determining Pichet, C., Danis, M. & Gentilini, M. (1990) Lancet 336, 1262. the percent reduction by multiplying average cpm by the 2. Bzik, D. J., Li, W.-B., Horii, T. & Inselburg, J. (1987) Proc. Natl. proportion of parasites present at each PS ODN concentration Acad. Sci. USA 84, 8360-8364. 3. Brooks, D. R., Wang, P., Read, M., Watkins, W. M., Sims, determined microscopically. P. F. G. & Hyde, J. E. (1994) Eur. J. Biochen. 224, 397-405. The efficacy of PS AS ODNs directed against different gene 4. Basco, L. K., Eldin de Pecoulas, P., Wilson, C. M., Le Bras, J. & targets varied. Some targets, like DHFR, DHPS, and RR, show Mazabraud, A. (1995) Mol. Biochem. Parasitol. 69, 135-138. approximately equivalent sensitivity to PS AS ODNs, while 5. Agrawal, S. (1992) Trends Biotechnol. 10, 152-157. others (DNA polymerase and TPI) were less sensitive. The 6. Verspieren, P., Cornelissen, A. W. C. A., Thuong, N. T., Helene, sensitivity to PS AS ODNs may depend upon several different C. & Toulme, J. J. (1987) Gene 61, 307-315. factors: (i) the stability/half-life of the protein whose mRNA 7. Ramazeilles, C., Mishra, R. K., Morreau, S., Pascolo, E. & is targeted, (ii) the existence of alternative biochemical path- Toulme, J.-J. (1994) Proc. Natl. Acad. Sci. USA 91, 7859-7863. ways to circumvent ones blocked by PS AS ODNs, (iii) the 8. Offensperger, W.-B., Offensperger, S., Walter, E., Teubner, K., stage of the parasite life cycle during which specific mRNA Igloi, G., Blum, H. E. & Gerok, W. (1993) EMBO J. 12, 1257-1262. 9. Rapaport, E., Misiura, K., Agrawal, S. & Zamecnik, P. (1992) occurs, and differences in synthesis (iv) possible stage-specific Proc. Natl. Acad. Sci. USA 89, 8577-8580. ODN uptake by the parasite. 10. Clark, D. L., Chrisey, L. A., Campbell, J. R. & Davidson, E. A. In contrast with drugs such as chloroquine and mefloquine, (1994) Mol. Biochem. Parasitol. 63, 129-134. where parasite inhibition can approach 100%, this does not 11. Volkman, S. K., Wilson, C. M. & Wirth, D. F. (1993) Mol. seem to be the case for PS AS ODNs under the conditions Biochem. Parasitol. 57, 203-212. assayed here. PS AS ODNs presumably inhibit cells by inter- 12. Padmapriya, A. A., Tang, J. & Agrawal, S. (1994) Antiseise Res. fering with translation or by an RNase H-mediated mechanism Dev. 4, 185-199. in which mRNA is destroyed. Conventional drugs inhibit by 13. Metelev, V. & Agrawal, S. (1992) Anal. Biochem. 200, 342-346. binding to the target enzyme, thereby preventing its function. 14. Lisziewicz, J., Sun, D., Weichold, F. F., Thierry, A. R., Lusso, P., In the case of PS AS ODNs, any enzyme present at the time Tang, J., Gallo, R. C. & Agrawal, S. (1994) Proc. Natl. Acad. Sci. USA 7942-7946. of initial exposure will continue to be functional, and the 91, 15. Sim, B. K. L., Orlandi, P. A., Haynes, J. D., Klotz, F. W., Carter, inhibitory effects of the PS AS ODNs will therefore be J. M., Camus, D., Zegans, M. E. & Chulay, J. D. (1990) J. Cell dependent upon both the initial concentration and half-life of Biol. 111, 1877-1884. the target enzyme. Since these studies were performed using 16. Ranie, J., Kumar, V. P. & Balaram, H. (1993) Mol. Biochem. unsynchronized parasites exposed to AS ODNs over a period Parasitol. 61, 159-170. of only one erythrocytic stage replication cycle, parasites that 17. Triglia, T. & Cowman, A. F. (1994) Proc. Natl. Acad. Sci. USA 91, had already synthesized sufficient enzyme would not be inhib- 7149-7153. ited until the next replication cycle. In contrast, conventional 18. Carcy, B., Bonnefoy, S., Guillotte, M., Le Scanf, C., Grellier, P., antimalarial drugs directly inhibit enzyme function. This hy- Schrevel, J., Fandeur, T. & Mercereau-Puijalon, 0. (1994) Mol. pothesis is currently being tested by examining the effects of Biochem. Parasitol. 68, 221-233. 19. Rubin, H., Salem, J. S., Li, L.-S., Yang, F.-D., Mama, S., Wang, longer exposure times. Z.-M., Fisher, A., Hamann, C. S. & Cooperman, B. S. (1993) Previous work from our laboratory demonstrated that Proc. Natl. Acad. Sci. USA 90, 9280-9284. ODNs specifically enter infected, not uninfected, erythrocytes 20. White, J. H., Kilbey, B. J., de Vries, E., Goman, M., Alano, P., (9). While the mechanism by which this occurs is not under- Cheesman, S., McAleese, S. & Ridley, R. G. (1993) Nucleic Acids stood, it is known that the parasite induces changes in eryth- Res. 21, 3643-3646. rocyte permeability to a variety of solutes (27) and that uptake 21. Chakrabarti, D., Schuster, S. S. & Chakrabarti, R. (1993) Proc. increases as parasites mature (28). If, as seems likely, uptake Natl. Acad. Sci. USA 90, 12020-12024. of ODNs is also greater during later stages, target mRNAs that 22. Elford, B. C., Cowan, G. M. & Ferguson, J. P. (1995) Biochem. J. are transcribed early should be less susceptible to AS inhibition 308, 361-374. than mRNAs that are transcribed late. Similarly, constitutively 23. Stein, C. A. & Kreig, A. M. (1994) Antisense Res. Dev. 4, 67-69. 24. S. & enzymes with a relatively long half-life might be Wellstein, A., Zugmaier, G., Califano, J., Kern, F., Paik, expressed Lippman, M. (1991) J. Natl. Cancer Inst. 83, 716-720. comparatively resistant to AS ODNs over the time course 25. Beuria, M. K. & Das, M. K. (1991) Indian J. Exp. Biol. 29, 284-285. examined here (48 hr). 26. Leonetti, J. P., Mechti, N., Degols, G., Gagnor, C. & Lebleu, B. Variation notwithstanding, these experiments demonstrate (1991) Proc. NatI. Acad. Sci. USA 88, 2702-2706. that PS AS ODN inhibition at lower concentrations is highly 27. Cabantchik, Z. I. (1989) Blood 74, 1464-1471. sequence specific. Elucidating why gene targets differ in 28. Kutner, S., Breuer, W. V., Ginsburg, H., Aley, S. B. & Ca- susceptibility to ODNs will be especially important for an- bantchik, Z. I. (1985) J. Cell. Physiol. 125, 521-527.