Isoprenoid biosynthesis and drug targeting in the Apicomplexa.
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Animals↗
Biomedical subjects
H J Vial
Publications and source records attributed to H J Vial.
Transport of phospholipid synthesis precursors and lipid trafficking into malaria-infected erythrocytes.
Phospholipid biosynthesis in Plasmodium is of crucial importance considering the high degree of membrane biogenesis. In the de novo phosphatidylcholine pathway, the major plasmodial phospholipid, choline, first enters infected erythrocytes by a transport-mediated process, whose main kinetic characteristics are the same as in normal cells except for a considerable increase in Vm. The kinetic and functional characterizations of the choline carrier (affinity, specificity, stereoselectivity, asymmetric cyclic model, ionic dependence, limiting step in carrier translocation) have now been done, although there is no information concerning its nature and structure, despite the fact that it is likely an outstanding pharmacological target. Other unanswered questions concern the mechanisms for choline entry into the parasite. The intense lipid trafficking between the intracellular parasite and the host cell membrane also indicates that Plasmodium controls its own lipid composition as well as that of its host cell. Organelles that house the machinery for lipid synthesis, and mechanisms for trafficking and sorting, have not yet been described because of the lack of appropriate tools, but they could address fundamental questions in the contemporary cell biology of this parasite.
Animals↗
Renewed strategies for drug development against parasitic diseases.
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Antimalarials↗
Antimalarial activity of 77 phospholipid polar head analogs: close correlation between inhibition of phospholipid metabolism and in vitro Plasmodium falciparum growth.
Seventy-seven potential analogs of phospholipid polar heads, choline and ethanolamine, were evaluated in vitro as inhibitors of Plasmodium falciparum growth. Their IC50 ranged from 10(-3) to 10(-7) mol/L. Ten compounds showed similar antimalarial activity when tested against three different parasite strains (2 chloroquine-sensitive strains and 1 chloroquine-resistant strain). Compounds showing marked antimalarial activity were assayed for their effects on phospholipid metabolism. The most active compounds (IC50 of 1 to 0.03 micromol/L) were inhibitors of de novo phosphatidylcholine (PC) biosynthesis from choline. For a series of 50 compounds, there was a close correlation between impairment of phospholipid biosynthesis and inhibition of in vitro malaria parasite growth. High choline concentrations caused a marked specific shift in the curves for PC biosynthesis inhibition. Concentrations inhibiting 50% PC metabolism from choline were in close agreement with the Ki of these compounds for the choline transporter in Plasmodium knowlesi-infected erythrocytes. By contrast, measurement of the effects of 12 of these compounds on rapidly dividing lymphoblastoid cells showed a total absence of correlation between parasite growth inhibition and human lymphoblastoid cell growth inhibition. Specific antimalarial effects of choline or ethanolamine analogs are thus likely mediated by their alteration of phospholipid metabolism. This indicates that de novo PC biosynthesis from choline is a very realistic target for new malaria chemotherapy, even against pharmacoresistant strains.
Animals↗
Plasmodium falciparum CTP:phosphocholine cytidylyltransferase expressed in Escherichia coli: purification, characterization and lipid regulation.
The Plasmodium falciparum CTP:phosphocholine cytidylyltransferase (PfCCT) has been isolated from an overexpressing strain of Escherichia coli. The plasmid pETPfCCT mediated the overexpression of the full-length polypeptide directly. The recombinant protein corresponded to 6-9% of the total cellular proteins and was found essentially in the insoluble membrane fraction. Urea at 6 M was used to solubilize the recombinant protein from the insoluble fraction. The CCT activity was restored upon the removal of urea, and the protein was subsequently purified to homogeneity on a Q-Sepharose column. Approx. 1.4 mg of pure enzyme was obtained from a 250 ml culture of E. coli. Biochemical properties, including in vitro substrate specificity and enzymic characterization, were assessed. The lipid regulation of the recombinant plasmodial CCT activity was characterized for the first time. The Km values were 0.49+/-0.03 mM (mean+/-S.E.M.) for phosphocholine and 10.9+/-0.5 mM for CTP in the presence of lipid activators (oleic acid/egg phosphatidylcholine vesicles), whereas the Km values were 0.66+/-0.07 mM for phosphocholine and 28.9+/-0.8 mM for CTP in the absence of lipid activators. The PfCCT activity was stimulated to the same extent in response to egg phosphatidylcholine vesicles containing anionic lipids, such as oleic acid, cardiolipin and phosphatidylglycerol, and was insensitive or slightly sensitive to PC vesicles containing neutral lipids, such as diacylglycerol and monoacylglycerol. Furthermore, the stimulated enzyme activity by oleic acid was antagonized by the cationic aminolipid sphingosine. These lipid-dependence properties place the parasite enzyme intermediately between the mammalian enzymes and the yeast enzyme.
Animals↗
Phospholipid metabolism of serine in Plasmodium-infected erythrocytes involves phosphatidylserine and direct serine decarboxylation.
Erythrocytes infected with Plasmodium falciparum or Plasmodium knowlesi efficiently incorporated radioactive serine into phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho). Serine was also metabolized into ethanolamine (Etn) and phosphorylethanolamine (P-Etn) via direct serine decarboxylation; this is a major phenomenon since together these metabolites represent 60% of total radioactive water-soluble metabolites. They were identified by reverse-phase HPLC and two TLC-type analyses and confirmed by alkaline phosphatase treatment, which depleted the radioactive P-Etn peak completely with a concomitant increase in that of Etn. In the presence of 5 microM labelled serine, radioactivity appeared in Etn and P-Etn after a 25 min lag period, and isotopic equilibrium was reached at 40 and 95 min respectively. There was a similar lag period for PtdEtn formation, which accumulated steadily for at least 180 min. Incorporation of serine into phospholipids and water-soluble metabolites increased in the presence of up to 500 microM external serine. An apparent plateau was then reached for all metabolites except intracellular serine and Etn. Exogenous Etn (at 20 microM) induced a concomitant dramatic decrease in serine incorporation into P-Etn and all phospholipids, but not into Etn. Increasing exogenous serine to 100 microM decreased the incorporation of radioactive Etn into PtdEtn by only 30%, and the PtdCho level was not affected. 2-Hydroxyethylhydrazine significantly decreased serine incorporation into P-Etn and PtdEtn, whereas Etn was accumulated. No concomitant inhibition of PtdSer or PtdCho labelling from serine occurred, even when PtdEtn formation was decreased by 95%. This indicates that the PtdEtn pool derived from direct serine decarboxylation differed from that derived from PtdSer decarboxylation, and the latter appeared to be preferentially used for PtdCho biosynthesis. Hydroxylamine also inhibited phosphorylation of serine-derived Etn but not that of exogenous Etn. The rate of PtdSer synthesis from 10 microM L-serine was 3.1+/-0.5 and 2.95+/-1.3 nmol/5 h per 10(10) infected cells, whereas L-serine decarboxylation accounted for 7.1+/-1.5 and 9.9+/-3 nmol/5 h per 10(10) infected cells for P. falciparum and P. knowlesi respectively (means+/-S.E.M.). The serine decarboxylating reaction was not detected in other higher eukaryotic cells such as mouse fibroblasts and human lymphocytes. Finally, these results also indicate compartmentalization of phospholipid metabolism in Plasmodium-infected erythrocytes.
Animals↗
Characterization of the potent in vitro and in vivo antimalarial activities of ionophore compounds.
Large-scale in vitro screening of different types of ionophores previously pinpointed nine compounds that were very active and selective in vitro against Plasmodium falciparum; their in vitro and in vivo antimalarial effects were further studied. Addition of the ionophores to synchronized P. falciparum suspensions revealed that all P. falciparum stages were sensitive to the drugs. However, the schizont stages were three- to ninefold more sensitive, and 12 h was required for complete parasite clearance. Pretreatment of healthy erythrocytes with toxic doses of ionophores for 24 to 48 h showed that the activity was not due to an irreversible effect on the host erythrocyte. No preferential ionophore adsorption in infected or uninfected erythrocytes occurred. On the other hand, ionophore molecules strongly bound to serum proteins since increasing the serum concentration from 2 to 50% led to almost a 25-fold parallel increase in the ionophore 50% inhibitory concentration. Mice infected with the malaria parasites Plasmodium vinckei petteri or Plasmodium chabaudi were successfully treated with eight ionophores in a 4-day suppressive test. The 50% effective dose after intraperitoneal administration ranged from 0.4 to 4.1 mg/kg of body weight, and the therapeutic indices were about 5 for all ionophores except monensin A methyl ether, 5-bromo lasalocid A, and gramicidin D, whose therapeutic indices were 12, 18, and 344, respectively. These three compounds were found to be curative, with no recrudescence. Gramicidin D, which presented impressive antimalarial activity, requires parenteral administration, while 5-bromo lasalocid A has the major advantage of being active after oral administration. Overall, the acceptable levels of toxicity and the good in vivo therapeutic indices in the rodent model highlight the interesting potential of these ionophores for the treatment of malaria in higher animals.
Animals↗
Differential in vitro activities of ionophore compounds against Plasmodium falciparum and mammalian cells.
Twenty-two ionophore compounds were screened for their antimalarial activities. They consisted of true ionophores (mobile carriers) and channel-forming quasi-ionophores with different ionic specificities. Eleven of the compounds were found to be extremely efficient inhibitors of Plasmodium falciparum growth in vitro, with 50% inhibitory concentrations of less than 10 ng/ml. Gramicidin D was the most active compound tested, with 50% inhibitory concentration of 0.035 ng/ml. Compounds with identical ionic specificities generally had similar levels of antimalarial activity, and ionophores specific to monovalent cations were the most active. Compounds were further tested to determine their in vitro toxicities against mammalian lymphoblast and macrophage cell lines. Nine of the 22 compounds, i.e., alborixin, lonomycin, nigericin, narasin, monensin and its methylated derivative, lasalocid and its bromo derivative, and gramicidin D, most specific to monovalent cations, were at least 35-fold more active in vitro against P. falciparum than against the two other mammalian cell lines. The enhanced ability to penetrate the erythrocyte membrane after infection could be a factor that determines ionophore selectivity for infected erythrocytes.
Animals↗
Molecular cloning of CTP:phosphocholine cytidylyltransferase from Plasmodium falciparum.
CTP:phosphocholine cytidylyltransferase (CCT) is the rate-limiting and regulatory enzyme in the synthesis of phosphatidylcholine, the major membrane phospholipid, in Plasmodium. The structural gene encoding CCT was isolated from the human malaria parasite Plasmodium falciparum. This was achieved using the PCR to amplify genomic DNA with degenerate primers constructed on the basis of conserved regions identified within yeast and rat liver CCT molecules, and using the PCR product to screen a genomic library. The P. falciparum CCT gene encodes a protein of 370 amino acids (42. 6 kDa) and displays 41-43% similarity (28-29% identity) to CCT molecules of the other organisms cloned to date. The central domain of CCT, proposed as the catalytic domain of the CTP-transfer reaction, shows 68-72% similarity and 48-55% identity among P. falciparum, human, rat and yeast enzymes. This gene is present in a single copy, as determined by Southern-blotting of genomic DNA, and located on chromosome 13 of P. falciparum. Large transcripts were detected by Northern analysis and indicate that this gene is expressed in the asexual intraerythrocytic stages. The coding region of the P. falciparum CCT gene was inserted into an Escherichia coli expression vector to confirm the function of the CCT product. The recombinant CCT expressed in E. coli is catalytically active, as evidenced by the conversion of phosphocholine to CDP-choline.
Amino Acid Sequence↗
Characterization of phosphatidylinositol synthase and evidence of a polyphosphoinositide cycle in Plasmodium-infected erythrocytes.
Plasmodium knowlesi-infected erythrocytes possess a membranous cytidine 5'-diphospho-1,2-diacyl-sn-glycerol: myoinositol 3-phosphatidyl transferase (PI synthase) (EC 2.7.8.11) activity of 10 +/- 1.7 nmol min-1 per 10(10) infected cells. The activity was successfully solubilized with 40 mM n-octyl-beta-D-glucopyranoside in the presence of bivalent metal ions which were absolutely required for activity. The optimal pH was 8 and the apparent Ks for Mn2+ was 0.1 mM. Mg2+ allowed two-fold higher PI synthase activity, with an optimum above 100 mM. Calcium alone was ineffective while at 2 mM it inhibited solubilized PI synthase activity in the presence of 100 mM Mg2+. Enzymatic activity was fully dependent on CDP-diacylglycerol and inositol with apparent Km of 0.16 +/- 0.1 mM and 1 +/- 0.5 mM respectively. Affinity chromatography clearly showed CDP-diacylglycerol-dependent interactions of PI synthase with CDP-diacylglycerol Sepharose. However, elution of enzymatic activity in an active form was unsuccessful while SDS-PAGE of the eluate showed one apparent band. Incubations of Plasmodium falciparum-infected erythrocytes with 32P or [3H]inositol revealed de novo biosynthesis of phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate which appeared to predominate in the second half of the asexual cellular cycle. Ionomycin, a calcium ionophore, induced Li(+)-sensitive production of radioactive inositol phosphates, with neo-synthesized inositol 1,4,5-trisphosphate accumulation being the highest.
Animals↗
Present development concerning antimalarial activity of phospholipid metabolism inhibitors with special reference to in vivo activity.
The systematic screening of more than 250 molecules against Plasmodium falciparum in vitro has previously shown that interfering with phospholipid metabolism is lethal to the malaria parasite. These compounds act by impairing choline transport in infected erythrocytes, resulting in phosphatidylcholine de novo biosynthesis inhibition. A thorough study was carried out with the leader compound G25, whose in vitro IC50 is 0.6 nM. It was very specific to mature parasites (trophozoïtes) as determined in vitro with P. falciparum and in vivo with P. chabaudi -infected mice. This specificity corresponds to the most intense phase of phospholipid biosynthesis activity during the parasite cycle, thus corroborating the mechanism of action. The in vivo antimalarial activity (ED50) against P. chabaudi was 0.03 mg/kg, and a similar sensitivity was obtained with P. vinckei petteri, when the drug was intraperitoneally administered in a 4 day suppressive test. In contrast, P. berghei was revealed as less sensitive (3- to 20-fold, depending on the P. berghei-strain). This difference in activity could result either from the degree of synchronism of every strain, their invasion preference for mature or immature red blood cells or from an intrinsically lower sensitivity of the P. berghei strain to G25. Irrespective of the mode of administration, G25 had the same therapeutic index (lethal dose 50 (LD50)/ED50) but the dose to obtain antimalarial activity after oral treatment was 100-fold higher than after intraperitoneal (or subcutaneous) administration. This must be related to the low intestinal absorption of these kind of compounds.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Infected erythrocyte choline carrier inhibitors: exploring some potentialities inside Plasmodium phospholipid metabolism for eventual resistance acquisition.
We have developed a model for designing antimalarial drugs based on interference with an essential metabolism developed by Plasmodium during its intraerythrocytic cycle, phospholipid (PL) metabolism. The most promising drug interference is choline transporter blockage, which provides Plasmodium with a supply of precursor for synthesis of phosphatidylcholine (PC), the major PL of infected erythrocytes. Choline entry is a limiting step in this metabolic pathway and occurs by a facilitated-diffusion system involving an asymmetric carrier operating according to a cyclic model. Choline transport in the erythrocytes is not sodium dependent nor stereospecific as demonstrated using stereoisomers of alpha and beta methylcholine. These last two characteristics along with distinct effects of nitrogen substitution on transport rate demonstrate that choline transport in the infected erythrocyte possesses characteristics quite distinct from that of the nervous system. This indicates a possible discrimination between the antimalarial activity (inhibition of choline transport in the infected erythrocyte) and a possible toxic effect through inhibition of choline entry in synaptosomes. Apart from the de novo pathway of choline, PC can be synthesized by N-methylation from phosphatidylethanolamine (PE). There is a de novo pathway for PE biosynthesis from ethanolamine in infected cells but phosphatidylserine (PS) decarboxylation also occurs. In addition, PE can be directly and abundantly synthesized from serine decarboxylation into ethanolamine, a pathway which is absent from the host. The variety of the pathways that exist for the biosynthesis of one given PL led us to investigate whether an equilibrium can occur between all PL metabolic pathways.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
The design of original antimalarial drugs. An example of phospholipid metabolism.
The aim of our program was to find an original chemotherapeutical treatment (and eventually a preventive treatment) of malaria, an illness largely predominant in developing countries, by interfering on an essential metabolism developed by Plasmodium during its erythrocytic phase. Apart from what has been learnt about metabolism and the pharmacological target, a crucial step has been taken during this contract by passing from micromolar in vitro active concentrations (during 1986-1990) to nanomolar ones (during 1990). These compounds should naturally short-circuit resistance phenomena already established against drugs in current use, as has already been verified on polypharmacoresistant strains or isolates of P. falciparum. The administration of a therapeutic dose of our molecules would now appear to be possible in all cases.
Animals↗
Saturable and non-saturable components of choline transport in Plasmodium-infected mammalian erythrocytes: possible role of experimental conditions.
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Biological Transport↗
Use of radioactive ethanolamine incorporation into phospholipids to assess in vitro antimalarial activity by the semiautomated microdilution technique.
Phospholipid biosynthetic activity is intense in the erythrocytic stage of Plasmodium falciparum because of the parasite's own enzymatic machinery. The incorporation of various labeled phospholipid precursors in comparison with the incorporation of nucleic acid and protein precursors was tested to evaluate P. falciparum growth in vitro. These precursors, namely, [3H]ethanolamine, [3H]hypoxanthine, [3H]palmitate, [14C]serine, [3H]choline, [3H]inositol, and [3H]isoleucine, were all accurate indicators of parasite growth. However, because of its high level of incorporation, [3H]ethanolamine proved to be the best tool for assessing parasite viability. When culture parameters were carefully controlled, [3H]ethanolamine incorporation into phospholipids was proportional to pulse time, precursor concentration, and initial parasitemia and was sensitive to the number of uninfected erythrocytes (hematocrit). It can be used for a wide range of infected erythrocytes, from 2 x 10(4) to 5 x 10(5). The use of [3H]ethanolamine for in vitro antimalarial drug screening is a good alternative to the method of Desjardins et al. (R. E. Desjardins, C. J. Canfield, J. D. Haynes, and J. D. Chulay, Antimicrob. Agents. Chemother. 16:710-718, 1979). The major advantage is that the culture medium can be supplemented with hypoxanthine, which results in better parasite growth. [3H]ethanolamine is also an ideal tool when compounds that interfere with DNA and/or RNA metabolism are to be investigated for their effect on Plasmodium growth.
Animals↗
Basic biochemical investigations as rationale for the design of original antimalarial drugs. An example of phospholipid metabolism.
The future of antimalarial chemotherapy is particularly alarming in view of the spread of parasite cross-resistances to drugs that are not even structurally related. Only the availability of new pharmacological models will make it possible to select molecules with novel mechanisms of action, thus delaying resistance and allowing the development of new chemotherapeutic strategies. We reached this objective in mice. Our approach is hunged on fundamental and applied research begun in 1980 to investigate the phospholipid (PL) metabolism of intraerythrocytic Plasmodium. This metabolism is abundant, specific and indispensable for the production of Plasmodium membranes. Any drug able to interfere with this Plasmodium membranes. Any drug able to interfere with this metabolism blocks parasitic development. The most effective interference yet found involves blockage of the choline transporter, which supplies Plasmodium with choline for the synthesis of phosphatidylcholine, its major PL, this is a limiting step in the pathway. The drug sensitivity threshold is much lower for the parasite, which is more dependent on this metabolism than host cells. The compounds show in vitro activity against P. falciparum at 1 to 10 nM. They show a very low toxicity against a lymphoblastoid cell line, demonstrating a total absence of correlation between growth inhibition of parasites and lymphoblastoid cells. They show antimalarial activity in vivo, in the P. berghei or P. chabaudi/mouse system, at doses 20- to 100-fold lower than their acute toxicity limit. The bioavailability of a radiolabeled form of the product seemed to be advantageous (slow blood clearance and no significant concentration in tissues). Lastly, the compounds are inexpensive to produce. They are stable and water-soluble.
Animals↗
Increased permeability to choline in simian erythrocytes after Plasmodium knowlesi infection.
The permeability of simian erythrocytes to choline was found to be considerably increased after infection by the malaria parasite, Plasmodium knowlesi. Choline entry occurs by a facilitated-diffusion system involving a carrier, which displays temperature-dependence, saturability with choline (Km = 8.5 +/- 0.7 microM) and specificity. This carrier can also be inhibited by a thiol reagent, N-ethylmaleimide, at an inactivation rate which is, in the absence of choline, the same as in normal erythrocytes. Inactivation by N-ethylmaleimide can be accelerated by external choline and prevented by decamethonium, which acts as an inhibitor of choline entry in infected cells (as with dodecyltrimethylammonium). Both ethanolamine and imidazole act as inhibitors or activators of choline entry in infected erythrocytes, depending on the relative concentrations of choline and of the competing compound (i.e. ethanolamine or imidazole). After infection, the maximum velocity reached 2.84 +/- 0.5 nmol/min per 10(10) infected cells, which is more than 10 times the Vmax. of normal erythrocytes. Impairing the biosynthesis of phosphatidylcholine de novo in Plasmodium-infected erythrocytes by various methods (glucose or ATP depletion, high ethanolamine concentrations) did not result in any alteration of choline transport (Km or Vmax.), indicating that the constant triggering and transformation of choline into phosphatidylcholine by the parasite is not directly responsible for the increase in the choline transport rate after infection. This high increase in choline transport activity is more likely related to modifications in choline carriers and/or in their environment after Plasmodium infection.
Animals↗