In Vitro Antimalarial Activity of Different Inhibitors of the Plasmodial Isoprenoid Synthesis Pathway

Abstract

Previous studies have shown that fosmidomycin, risedronate, and nerolidol exert antimalarial activity in vitro. We included squalestatin, an inhibitor of the isoprenoid metabolism in Erwinia uredovora, and found that combinations of compounds which act on different targets of the plasmodial isoprenoid pathway possess important supra-additivity effects.M alaria expansion in some areas has been attributed to the failure of vector-control policies and, mainly, to the increase of parasite resistance to drugs commonly used for its therapy (1). This alarming scenario has fuelled research in new antimalarial drugs, and, with the sequencing of parasite genomes, new potential drug targets have emerged. Burrows et al. published a review discussing the strategies for designing the next generation of medicines for malaria control (2). A metabolic pathway that may be a likely target for evaluating potential antimalarials is the isoprenoid pathway. The first intermediates are biosynthesized in the apicoplast, which is an organelle present in protozoan parasites of the subphylum Apicomplexa, including Plasmodia (3).We have previously demonstrated that the isoprenoid biosynthesis pathway is functionally active in the intraerythrocytic stages of Plasmodium falciparum and that final products of this pathway such as dolichol of 11 to 12 isoprenic units (4), ubiquinones (5), isoprenylated/dolichylated proteins, carotenoids, menaquinone, and tocopherol are biosynthesized by this parasite (6). Several inhibitors of enzymes of the methyl erythritol diphosphate (MEP) and isoprenoid pathways were described as potential antimalarial drugs (6).Our aim in this work was to identify and validate different targets in the isoprenoid pathway in P. falciparum intraerythrocytic stages. We evaluated the combination of four drugs, three of them (fosmidomycin, nerolidol and risedronate) inhibiting parasite growth at some point in the MEP/isoprenoid pathway in intraerythrocytic stages of P. falciparum (6,7,8). We also evaluated the effect of squalestatin on P. falciparum growth. This drug is an inhibitor of the phytoene synthase in Erwinia uredovora (9, 10). Importantly, an enzyme with phytoene synthase activity was described in intraerythrocytic stages of P. falciparum (11).Parasites of P. falciparum clone 3D7 were cultured according to the protocol described by Trager and Jensen (12) where human serum was replaced by Albumax I (0.5%) (4). Stocks (1 mM) of squalestatin, risedronate, and fosmidomycin were prepared in RPMI medium, whereas nerolidol was diluted in ethanol, and all drugs were stored at Ϫ70°C.Assays were performed in 96-well microtiter plates containing 100 l of 3D7 culture at 1% parasitemia and 3% hematocrit and 100 l of the appropriate drug or drug combination in RPMI medium. Parasite growth was determined using Giemsa-stained smears immediately before the start of the assay and at intervals of 24 to 48 h. Vehicle controls were included on each plate. All tests were performed in triplicate for three independent experiments.Values for 5...

Terpenes as Potential Antimalarial Drugs

“…The activity of the risedronate was confirmed because only farnesyl-PP and geranylgeranyl-PP restored the growth intraerythrocytic stages of P. falciparum, after treatment with risedronate [55]. This drug also showed a significant inhibitory effect against murine blood stage malaria, without showing toxicity effects to the animals [55] and showed great results in synergism experiments with other drugs in P. falciparum [56]. Also, crystallography assays of the P. vivax GGPP enzyme inferred that GGPP could be a major target for the lipophilic bisphosphonates [57].…”

“…Different authors asked if using terpenes individually or in combination with other drugs could be a good strategy to treat malaria. It is well established that fosmidomycin and clindamycin, when combined, produce a synergistic activity in vitro and in vivo [88], and several drugs which act at different points of the MEP/isoprenoid showed supra-additive in vitro effects when combined [56]. These kinds of drug combination studies are useful for a better understanding of the interactions between the different intermediates of the MEP and isoprenoid pathways and to evaluate its antimalarial potential.…”

“…These kinds of drug combination studies are useful for a better understanding of the interactions between the different intermediates of the MEP and isoprenoid pathways and to evaluate its antimalarial potential. Fosmidomycin, risedronate, nerolidol and squalestatin (supposed to be a phytoene synthase inhibitor) are drugs which are believed to act in different cellular compartments, and all of them seem to inhibit at least one point related to isoprenoid metabolism [56]. Except nerolidol-risedronate, most binary combinations between fosmidomycin, risedronate, nerolidol and squalestatin showed a supra-additive effect [56].…”

“…Fosmidomycin, risedronate, nerolidol and squalestatin (supposed to be a phytoene synthase inhibitor) are drugs which are believed to act in different cellular compartments, and all of them seem to inhibit at least one point related to isoprenoid metabolism [56]. Except nerolidol-risedronate, most binary combinations between fosmidomycin, risedronate, nerolidol and squalestatin showed a supra-additive effect [56]. Probably it is because they target different enzymes in the same biosynthetic pathway.…”

“…On the other hand, nerolidol and risedronate when combined showed a sub-additive effect. It was suggested that this fact could be explained because both compounds affect the same target of isoprenoid pathways [56]. Nerolidol affects the synthesis of several isoprenoid compounds, including protein isoprenylation as well as risedronate [55,67].…”