The novel hydroxynaphthoquinone 566C80 inhibits the development of liver stages of<i>Plasmodium berghei</i>cultured<i>in vitro</i>

Davies, Charlotte Aull; Pudney, Mary; Nicholas, J.; Sinden, Robert E.

doi:10.1017/s0031182000074746

Cited by 32 publications

(19 citation statements)

References 25 publications

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“…The doseresponse curve of atovaquone with a 50% inhibitory concentration (IC 50 ) of 3.95 nM (Fig. 3A) was consistent with previous results obtained by Davies et al for P. berghei in HepG2 cells (2). As expected, chloroquine was inactive on P. yoelii development even at high concentrations up to 5 M (Fig.…”

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confidence: 91%

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New Approach for High-Throughput Screening of Drug Activity onPlasmodiumLiver Stages

Gego

Silvie

Franetich

et al. 2006

Antimicrob Agents Chemother

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Plasmodium liver stages represent potential targets for antimalarial prophylactic drugs. Nevertheless, there is a lack of molecules active on these stages. We have now developed a new approach for the high-throughput screening of drug activity on Plasmodium liver stages in vitro, based on an infrared fluorescence scanning system. This method allowed us to count automatically and rapidly Plasmodium-infected hepatocytes, using different hepatic cells and different Plasmodium species, including Plasmodium falciparum. This new technique is well adapted for high-throughput drug screening and should facilitate the identification of new antimalarial compounds active on Plasmodium liver stages.Malaria remains one of the major human infectious diseases and is responsible for millions of deaths each year. As resistance to available drugs increases, there is an urgent need to identify new molecules. Targeting Plasmodium liver stage development is a valuable strategy for preventing malaria. Indeed, liver stages precede the appearance of blood stages, which are responsible for clinical symptoms and complications. The development of pharmaceutical products inhibiting the growth of Plasmodium hepatic forms is relevant for two main reasons: first, such compounds could be used as causal prophylactic agents by people exposed for a limited duration in an area where malaria is endemic (e.g., refugees and travelers) and, second, the emergence of drug-resistant strains is theoretically limited during the liver phase because of the lower parasitic load compared to the blood phase. Although hepatic stages provide attractive targets for antimalarial chemotherapy, the list of effective and widely applied drugs is still limited. The only currently available prophylactic drugs are atovaquone and two related drugs, primaquine and tafenoquine. Atovaquone has been demonstrated to be efficient on the first step of the parasite development (2); however, its use is limited by its unaffordable cost. Because of its hematological toxicity, the use of primaquine is restricted, particularly in Africa because of the frequency of G6PD deficiency (12). The identification of new drugs is slowed down by the lack of a reliable and sensitive method allowing a high-throughput screening. In vitro drug sensitivity assays are largely based on evaluating the number of liver schizonts in sporozoite-infected cultures (7, 10, 15). Even if alternative methods have been proposed (6), the number of infected cells is usually determined by microscopy analysis. Microscope-based quantification of infected cells is a very time-consuming method. Therefore, we have developed a new approach based on infrared fluorescence detection to automatically and rapidly quantify Plasmodium liver schizonts in vitro.Parasites and cells. Plasmodium berghei (ANKA strain), P. yoelii (265BY strain), and P. falciparum (NF54 strain) sporozoites were obtained from dissection of infected Anopheles stephensi mosquito salivary glands. Human hepatocarcinoma HepG2-A16 cells were cultured in Dulbecco ...

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confidence: 91%

“…Cultures of HepG2/CD81 cells in 96-well microplates were infected with P. yoelii sporozoites and treated with atovaquone or chloroquine. Atovaquone, but not chloroquine, is active on pre-erythrocytic parasites (2,9). Infection was then monitored by using the Odyssey method.…”

mentioning

confidence: 99%

New Approach for High-Throughput Screening of Drug Activity onPlasmodiumLiver Stages

Gego

Silvie

Franetich

et al. 2006

Antimicrob Agents Chemother

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“…Nevertheless, maintenance of ⌬⌿ m is likely to be critical for all stages of the malaria parasite's life cycle, and drugs that affect ⌬⌿ m are likely to act on all such stages. In accordance with this proposal atovaquone has been shown also to act against liver (54,55) and insect stages of malarial parasite (38). Further investigations on parasite mitochondrial physiology will be illuminating in our attempts to understand mechanisms of drug action and the development of newer strategies for chemotherapy of malaria.…”

Section: Discussionmentioning

confidence: 58%

Atovaquone, a Broad Spectrum Antiparasitic Drug, Collapses Mitochondrial Membrane Potential in a Malarial Parasite

Srivastava¹,

Rottenberg

Vaidya³

1997

Journal of Biological Chemistry

377

267

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At present, approaches to studying mitochondrial functions in malarial parasites are quite limited because of the technical difficulties in isolating functional mitochondria in sufficient quantity and purity. We have developed a flow cytometric assay as an alternate means to study mitochondrial functions in intact erythrocytes infected with Plasmodium yoelii, a rodent malaria parasite. By using a very low concentration (2 nM) of a lipophilic cationic fluorescent probe, 3,3dihexyloxa-carbocyanine iodide, we were able to measure mitochondrial membrane potential(⌬⌿ m ) in live intact parasitized erythrocytes through flow cytometry. The accumulation of the probe into parasite mitochondria was dependent on the presence of a membrane potential since inclusion of carbonyl cyanide m-chlorophenylhydrazone, a protonophore, dissipated the membrane potential and abolished the probe accumulation. We tested the effect of standard mitochondrial inhibitors such as myxothiazole, antimycin, cyanide and rotenone. All of them except rotenone collapsed the ⌬⌿ m and inhibited respiration. The assay was validated by comparing the EC 50 of these compounds for inhibiting ⌬⌿ m and respiration. This assay was used to investigate the effect of various antimalarial drugs such as chloroquine, tetracycline and a broad spectrum antiparasitic drug atovaquone. We observed that only atovaquone collapsed ⌬⌿ m and inhibited parasite respiration within minutes after drug treatment. Furthermore, atovaquone had no effect on mammalian ⌬⌿ m . This suggests that atovaquone, shown to inhibit mitochondrial electron transport, also depolarizes malarial mitochondria with consequent cellular damage and death.Plasmodium spp. are obligate intracellular parasites, spending a major portion of their life cycle within erythrocytes and converting these relatively inactive cells into metabolically thriving active hosts. At present, our knowledge of mechanisms by which the parasite accomplishes these changes is limited, as is our understanding of metabolic processes associated with parasitism. It is generally believed that glycolysis is the main source of ATP in erythrocytic stages of malarial parasites with little or no contribution by mitochondria to the cellular ATP pool (1, 2). A lack of tricarboxylic acid cycle enzymes (3-6) and an acristate mitochondrial morphology has led to the suggestion that mitochondria in malaria parasite act mainly to serve as an electron disposal sink for dihydroorotate dehydrogenase, a critical enzyme in pyrimidine biosynthesis (7-9). It is well established through studies in other systems that, in addition to oxidative phosphorylation, mitochondria are also central to many other physiological activities such as the metabolism of molecules such as amino acids, lipids, and heme, as well as intracellular Ca 2ϩ homeostasis (10). These functions are achieved by the action of gene products encoded by both mitochondrial and nuclear genomes. Because most of the mitochondrial proteins are encoded by the nuclear genome and imported into mitochon...

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“…35 The results thus support the conclusion that atovaquone destroys exoerythrocytic P. falciparum in humans, consistent with its reported activity against rodent malaria in vitro and in animals. 18,19 Plasma levels during the first 6.5 days of infection were rather modest, suggesting that the cycling of atovaquone through the liver by biliary excretion and reabsorption 36,37 may contribute to its causal efficacy. In addition, it is possible that differences in mitochondrial function 38 might render the hepatic stages more susceptible to this drug.…”

Section: Study Populationmentioning

confidence: 99%

“…11,12 Ample clinical evidence indicates that atovaquone and proguanil, individually or in combination, act against erythrocytic falciparum parasites, 11,[13][14][15][16] and that proguanil alone has activity against exoerythrocytic stages. 17 Although atovaquone affects P. berghei in cultured hepatocytes 18 and in rats, 19 no study has addressed the question of whether atovaquone itself has causal activity against P. falciparum. This issue is of considerable interest for several reasons.…”

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confidence: 99%

Prophylactic activity of atovaquone against Plasmodium falciparum in humans.

Shapiro

Ranasinha²,

Kumar

et al. 1999

The American Journal of Tropical Medicine and Hygiene

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Abstract. The prophylactic antimalarial activity of atovaquone was determined in a randomized, double-blind, placebocontrolled study of healthy volunteers who were challenged by the bite of Plasmodium falciparum-infected Anopheles stephensi. Subjects were randomly assigned to one of three groups: six received seven daily doses of 750 mg of atovaquone, starting the day before challenge; six received a single dose of 250 mg of atovaquone the day before challenge; and four received placebo. Polymerase chain reaction-and culture-confirmed parasitemia developed in all four placebo recipients, but in none of the drug recipients, indicating that either of the atovaquone regimens provides effective prophylaxis (P ϭ 0.005). However, in low-dose recipients, the drug levels by day 6.5 were profoundly subtherapeutic, indicating that parasites were eliminated prior to the establishment of erythrocytic infection. Atovaquone thus protects non-immune subjects against mosquito-transmitted falciparum malaria, and has causal prophylactic activity.

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The novel hydroxynaphthoquinone 566C80 inhibits the development of liver stages ofPlasmodium bergheiculturedin vitro

Cited by 32 publications

References 25 publications

New Approach for High-Throughput Screening of Drug Activity onPlasmodiumLiver Stages

New Approach for High-Throughput Screening of Drug Activity onPlasmodiumLiver Stages

Atovaquone, a Broad Spectrum Antiparasitic Drug, Collapses Mitochondrial Membrane Potential in a Malarial Parasite

Prophylactic activity of atovaquone against Plasmodium falciparum in humans.

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