Chloroquine-resistant Plasmodium falciparum accumulate significantly less chloroquine than susceptible parasites, and this is thought to be the basis of their resistance. However, the reason for the lower accumulation of chloroquine was unknown. The resistant parasite has now been found to release chloroquine 40 to 50 times more rapidly than the susceptible parasite, although their initial rates of chloroquine accumulation are the same. Verapamil and two other calcium channel blockers, as well as vinblastine and daunomycin, each slowed the release and increased the accumulation of chloroquine by resistant (but not susceptible) Plasmodium falciparum. These results suggest that a higher rate of chloroquine release explains the lower chloroquine accumulation, and thus the resistance observed in resistant Plasmodium falciparum.

The parasite Plasmodium falciparum, like neoplastic cells, develops resistance to multiple structurally unrelated drugs. If the mechanisms by which P. falciparum and neoplastic cells become resistant are similar, then it may be possible to reverse the resistance in the two types of cells by the same pharmacological agents. Verapamil, a calcium channel blocker, completely reversed chloroquine resistance in two chloroquine-resistant P. falciparum clones from Southeast Asia and Brazil. Verapamil reversed chloroquine resistance at the same concentration (1 X 10(-6)M) as that at which it reversed resistance in multidrug-resistant cultured neoplastic cells. This same concentration of verapamil had no effect on chloroquine-sensitive parasites. Hence, chloroquine resistance in P. falciparum may fit the criteria for the multidrug-resistant phenotype.

Proguanil and pyrimethamine are antifolate drugs with distinct chemical structures that are used commonly in the prophylaxis and treatment of Plasmodium falciparum malaria. Clinical reports and field studies have suggested that some parasites refractory to proguanil can be treated with pyrimethamine, and vice versa. Analysis of the P. falciparum dihydrofolate reductase (DbFR) from different parasites reveals the-structural basis for differential susceptibility to these antifolate drugs. Parasites harboring a pair of point mutations from Ala-16 to Val-16 and from Ser-108 to Thr-108 are resistant to cycloguanil (the active metabolite of proguanil) but not to pyrimethamine. A single Asn-108 mutation, on the other hand, confers resistance to pyrimethamine with only a moderate decrease in susceptibility to cycloguanil. Significant crossresistance to both drugs occurs in parasites having mutations that include Ser-108lOS Asn-108 and Ile-164 -Leu-164. These results reflect the distinct structures of pyrimethamine and cycloguanil and suggest fine differences in binding within the active site cavity of DHFR. Alternative inhibitors, used alone or in combination, may be effective against some strains of cycloguanil-or pyrimethamine-resistant malaria.Proguanil and pyrimethamine, introduced nearly 40 years ago (1, 2), were powerful additions to the spectrum ofantimalarial agents. Both drugs are specific inhibitors of the enzyme dihydrofolate reductase (DHFR;5,6,7, DNA Extraction and Nucleotide Sequence Analysis. Parasites were obtained from infected erythrocytes by lysis in TSE buffer (100 mM NaCl/50 mM EDTA/20 mM Tris, pH 8.0) containing 0.15% saponin. After centrifugation at 5000 x g, the parasites were resuspended in TSE buffer and lysed by addition of sodium lauryl sulfate (SDS) to 1% and NaCI04 to 0.5 M. The lysate was gently mixed at room temperature for 1 hr, extracted twice with phenol equilibrated in TSE buffer (pH 8.0), and finally extracted twice with 1:1 (vol/vol) phenol/chloroform. The solution was brought 0.2 M in sodium acetate, 1.5 volumes of 95% EtOH were added, and the DNA was spooled out of solution.In P. falciparum the DHFR domain is part of the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR/TS). Oligonucleotide primers were designed from the gene sequence (23) and used in the polymerase chain reaction to amplify the DHFR domain as described (7). Fifty nanograms of P. falciparum DNA (representing -1.5 X 106 copies of the genome) was used as the starting material. Both strands of amplified DNA were sequenced by a modification of the method reported by Innis et al. (24). Two microliters of the polymerase chain reaction product were used in a Abbreviations: DHFR, dihydrofolate reductase; PABA, p-aminobenzoic acid. 3018The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Parasites of the phylum Apicomplexa cause substantial morbidity, mortality and economic losses, and new medicines to treat them are needed urgently. The shikimate pathway is an attractive target for herbicides and antimicrobial agents because it is essential in algae, higher plants, bacteria and fungi, but absent from mammals. Here we present biochemical, genetic and chemotherapeutic evidence for the presence of enzymes of the shikimate pathway in apicomplexan parasites. In vitro growth of Toxoplasma gondii, Plasmodium falciparum (malaria) and Cryptosporidium parvum was inhibited by the herbicide glyphosate, a well-characterized inhibitor of the shikimate pathway enzyme 5-enolpyruvyl shikimate 3-phosphate synthase. This effect on T. gondii and P. falciparum was reversed by treatment with p-aminobenzoate, which suggests that the shikimate pathway supplies folate precursors for their growth. Glyphosate in combination with pyrimethamine limited T. gondii infection in mice. Four shikimate pathway enzymes were detected in extracts of T. gondii and glyphosate inhibited 5-enolpyruvyl shikimate 3-phosphate synthase activity. Genes encoding chorismate synthase, the final shikimate pathway enzyme, were cloned from T. gondii and P. falciparum. This discovery of a functional shikimate pathway in apicomplexan parasites provides several targets for the development of new antiparasite agents.