Potent Inhibitors of Plasmodium Phospholipid Metabolism with a Broad Spectrum of In Vitro Antimalarial Activities

Abstract

We characterized the potent in vitro antimalarial activity and biologic assessment of 13 phospholipid polar head analogs on a comparative basis. There was a positive relationship between the abilities of the drugs to inhibit parasite growth in culture and their abilities to specifically inhibit phosphatidylcholine biosynthesis of Plasmodium falciparum-infected erythrocytes. Maximal activity of G25 was observed for the trophozoite stage of the 48-h erythrocytic cycle (50% inhibitory concentration, 0.75 nM), whereas the schizont and ring stages were 12-and 213-fold less susceptible. The compounds exerted a rapid nonreversible cytotoxic effect, with complete clearance of parasitemia after 5 h of contact with the mature stages. The compounds were highly specific against P. falciparum, with much lower toxicity against three other mammalian cell lines, and the in vitro therapeutic indices ranged from 300 to 2,500,000. Finally, the monoquaternary ammonium E10 and two bis-ammonium salts, G5 and G25, were similarly active against multiresistant strains and fresh isolates of P. falciparum. This impressive selective in vitro toxicity against P. falciparum strongly highlights the clinical potential of these quaternary ammonium salts for malarial chemotherapy.The increasing polypharmacoresistance of Plasmodium falciparum to conventional antimalarials, combined with the resistance of mosquitoes to pesticides, partially accounts for the current dramatic resurgence of malaria (7, 16). Malaria has an enormous impact on world health, thus justifying the intensive research under way to design new therapeutic approaches to this disease and to develop a new range of drugs with original structures and modes of action (22,37).In the human host, the parasite undergoes cyclical development within red blood cells (RBC), which accounts for the clinical manifestations of this disease. The rate of multiplication within RBC, with 10 to 20 merozoites produced every 48 h, requires high metabolic activity, including a high membrane formation level. Phospholipids (PL) are essential lipids of Plasmodium membranes, and the erythrocyte PL content increases by as much as 500% after malarial infection (17, 27). Plasmodial PL biosynthesis, which is absent from normal mature human erythrocytes (29), was previously characterized as an ideal potential target for antimalarial chemotherapy due to its vital importance to the parasite (30, 32). Phosphatidylcholine (PC) is the major PL of infected erythrocytes, representing about 45% of total PL, much of which is provided by parasitedriven de novo biosynthesis from choline (4, 31). In the pathway, parasitic cholinephosphate cytidylyltransferase (38) and choline transport, which regulates the precursor supply, are a limiting and likely regulatory step (4, 33).More than 100 analogs of PL polar heads have been synthesized and tested in vitro for their activity against the intraerythrocytic stage of P. falciparum (2,8,9). More particularly, 36 compounds which possess one (or two) quaternary ammonium ions, such as...

Reliability of Antimalarial Sensitivity Tests Depends on Drug Mechanisms of Action

“…These assays were based on a modified Desjardins test (3,12). Metabolic activities of the parasites were assessed by measuring the incorporation of [ H]ethanolamine (1 Ci/well) in 30 l of complete medium was added in each well at the indicated time.…”

“…The compounds mimic the choline structure and prevent biosynthesis of the parasite phosphatidylcholine, which accounts for 40% of its lipid molecule (40)(41)(42). The compounds inhibit P. falciparum asexual blood stages at single-digit nanomolar concentrations and cure P. vinckei malaria infection in mice at doses under 1 mg/kg (1)(2)(3)45). The potency and specificity of these antiphospholipid effectors are likely associated with their unique property of accumulating in a nonreversible way inside intraerythrocytic parasites (43).…”

“…This mechanism likely induces erroneous results, according to the in vitro antimalarial activity test, and may be a problem for clinicians who do not detect rapid clearance of the parasites after treatment. To avoid problems linked with delayed parasite collapse, we routinely use a modified Desjardins test which measures [ 3 H]hypoxanthine incorporation into nucleic acids after a full infected-erythrocyte incubation cycle (3). With the aim of evaluating choline analogs under the most suitable conditions, we determined which of these tests were actually effective for determining the antimalarial potential of novel drug series, including antiphospholipid effectors.…”

Prodrugs of bisthiazolium salts are orally potent antimalarials

“…Chloroquine (CQ)-sensitive (3D7 and Nigerian) or -resistant (FCB1 and FCM29) strains of P. falciparum (12) were asexually cultured in human blood (14) and synchronized by using two successive 5% sorbitol treatments 26 h apart (15). Antimalarial activity of the compounds was routinely determined at 1.5% final hematocrit and 0.6% parasitemia (16).…”

In Vivo Antimalarial Activities of Mono- and Bis Quaternary Ammonium Salts Interfering with Plasmodium Phospholipid Metabolism

“…More particularly, 36 compounds which possess one (or two) quaternary ammoniums such as choline were found to be very efficient against P. falciparum, with a 50% inhibitory concentration (IC 50 ) ranging from the micromolar to picomolar range (6,7), and also against multidrug-resistant P. falciparum malaria. Biochemical characterizations indicate a probable link between PL biosynthesis inhibition and antimalarial activity (1,2,4,25).…”