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ABSTRACTMalaria remains a significant infectious disease that causes millions of clinical cases and >800,000 deaths per year. The Malaria Box is a collection of 400 commercially available chemical entities that have antimalarial activity. The collection contains 200 drug-like compounds, based on their oral absorption and the presence of known toxicophores, and 200 probe-like compounds, which are intended to represent a broad structural diversity. These compounds have confirmed activities against the asexual intraerythrocytic stages of Plasmodium falciparumand low cytotoxicities, but their mechanisms of action and their activities in other stages of the parasite's life cycle remain to be determined. The apicoplast is considered to be a promising source of malaria-specific targets, and its main function during intraerythrocytic stages is to provide the isoprenoid precursor isopentenyl diphosphate, which can be used for phenotype-based screens to identify compounds targeting this organelle. We screened 400 compounds from the Malaria Box using apicoplast-targeting phenotypic assays to identify their potential mechanisms of action. We identified one compound that specifically targeted the apicoplast. Further analyses indicated that the molecular target of this compound may differ from those of the current antiapicoplast drugs, such as fosmidomycin. Moreover, in our efforts to elucidate the mechanisms of action of compounds from the Malaria Box, we evaluated their activities against other stages of the life cycle of the parasite. Gametocytes are the transmission stage of the malaria parasite and are recognized as a priority target in efforts to eradicate malaria. We identified 12 compounds that were active against gametocytes with 50% inhibitory concentration values of <1 μM.

An N-acetylgalactosamine-specific lectin was isolated from Euphorbia heterophylla seeds by affinity chromatography on cross-linked arabinogalactan. It is a dimeric protein of two identical subunits of Mr 32 000, and differs structurally from all previously known Euphorbiaceae lectins. Its distribution over the seed is typical in that it is merely confined to the primary axes.

The thermodynamic parameters for the heat activation of the sporangiospores of Phycomyces blakesleeanuswere determined. For the apparent activation enthalpy (ΔH#) a value of 1,151 kJ/mol was found, whereas a value of 3,644 J./°K·mol was calculated for the apparent activation entropy (ΔS#). n-Alcohols (from methanol to octanol), phenethyl alcohol, and furfural lowered the activation temperature of P. blakesleeanusspores. The heat resistance of the spores was lowered concomitantly. The effect of the alcohols was a linear function of the concentration in the range that could be applied. When the log of the concentration needed to produce an equal shift of the activation temperature was plotted for each alochol against the log of the octanol/water partition coefficient, a straight line was obtained. The free energy of adsorption of the n-alcohols to their active sites was calculated to be −2,487 J/mol of CH2groups. Although still inconclusive, this points toward an involvement of protein in the activation process. The effect of phenethyl alcohol was similar to the effect of n-alcohols, but furfural produced a greater shift than would be expected from the value of its partition coefficient. When the heat activation of the spores was performed under high pressure, the activation temperature was raised by 2 to 4°K/1,000 atm. However, with pressures higher than 1,000 atm (1.013 × 105kPa) the activation temperature was lowered until the pressure became lethal (more than 2,500 atm). It is known that membrane phase transition temperatures are shifted upward by about 20°K/1,000 atm and that protein conformational changes are shifted upward by 2 to 6°K/1,000 atm. Consequently, heat activation of fungal spores seems to be triggered by a protein conformational change and not by a membrane phase transition. Activation volumes of −54.1 cm3/mol at 38°C and −79.3 cm2/mol at 40°C were found for the lowering effect of high pressure on the heat activation temperature.

A lectin has been isolated from embryos of a false brome grass species (Brachypodium sylvaticum) by affinity chromatography on immobilized N-acetylglucosamine. It is a dimeric protein of two identical subunits of mol.wt. 18 000. Although it resembles cereal lectins with respect to its biochemical and physicochemical properties, it differs structurally in several aspects from wheat-germ-agglutinin-like lectins.

Evidence is obtained for the existence of two different localizations of trehalase (a,a-trehalose glucohydrolase, EC 3.2.1.28) in Phycomyces spores: one inside the cell, and one in the periplasmic region. The latter enzyme is sensitive to 0.1 mol l-1 HCl treatment and its activity can be regulated by external pH changes. The periplasmic form of the enzyme is involved in the metabolism of added labelled trehalose. This sugar is hydrolyzed externally to glucose which is found mainly in the incubation medium and which is partly absorbed by the spores. During incubation trehalose leaks out from both dormant and activated spores and is subsequently hydrolyzed to glucose. The intracellular trehalase is probably involved in the breakdown of endogenous trehalose in spores. After heat activation the hydrolysis of endogenous trehalose is stimulated even without an important increase in activity of intracellular trehalase. Additional treatments which break dormancy of spores without a significant activation of trehalase are the following: heating of HCl-treated spores and treatment of spores with reducing substances (e.g. Na2S2O4 and NaHSO3).

The metabolism of [14C]glucose has been studied in Phycomyces spores during dormancy, activation, and the initial stages of germination. Dormant spores are able to take up and metabolize exogenous glucose into different products; the major part of it goes to trehalose synthesis (up to 60% when the external glucose concentration exceeds 10-3 M). During activation itself (i.e. a prolonged treatment at 50°) there is a general increase of glucose uptake and metabolism, without major changes in the relative rates of 14C-label distribution in the different fractions (as compared to the metabolism of dormant spores), except for a drop in material insoluble in 80% ethanol and a still higher percentage (73%) going to trehalose synthesis. In the early hours of germination we find an enhancement of the uptake and metabolism of glucose. Trehalose synthesis is practically switched off within 2 h whereas the major part of glucose (65%) is metabolized to CO2 and ethanol-insoluble proteinaceous material.

Lectins have been isolated from embryos of Secale cereale (rye) and Hordeum vulgare (barley) by affinity chromatography on immobilized N-acetylglucosamine. Both lectins are dimeric proteins of two identical subunits of mol.wt. 18000. They resemble strongly wheat-germ agglutinin with respect to their chemical, physical, biological and immunological properties.

Heat treatment of Phycomyces sporangiospores, which breaks dormancy, causes a very rapid 10- to 15fold increase in trehalase activity; soon after the heat shock the enzyme activity decays. This phenomenon can be repeated several times by repeating the heat shocks. Prolonging the heat treatment over the minimum required time delays the decay of enzyme activity. Cycloheximide does not prevent the rise in enzyme activity. It is suggested that heat treatment converts temporarily an inactive form of trehalase into an active one. Optimal enzyme activity is obtained at pH 7.5 and the enzyme requires metal ions for maximal activity. The possible role of trehalase in the spore-activation process is discussed.