1 Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT; 2 Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK
Received 30 August 2001; returned 16 January 2002; revised 21 February 2002; accepted 18 April 2002
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Abstract |
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Introduction |
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In order to test the ability of compounds to interfere with haemin polymerization, two in vitro polymerization assays have been developed previously. One utilizes the formation of haemozoin from concentrated haemin solution in aqueous dimethylsulphoxide (DMSO)/Na acetate (pH 5), the polymer being quantified spectrophotometrically.13 The method is a good predictor of antimalarial activity, but takes 18 h and involves compoundhaemin interaction at millimolar concentration, which is not useful where there are limited quantities of test compound. A second method measures polymerized haemin radiochemically.14 This method allows compoundhaemin interaction with 0.1 mM [14C]haemin, and has been used with advanced robotic workstations to screen large numbers of compounds from a library.15 This method is suitable for a commercial operation, but is expensive and slow for academic studies and studies of natural products in short supply. Furthermore, the conditions of haemin concentration are still far above those able to differentiate a high-affinity interaction with haemin, which is likely to be in the sub-micromolar range, from many which occur in the 10100 µM range.
In view of the current major threat from drug-resistant malaria,16,17 both novel drug targets and structurally diverse drugs, active at validated targets like haemozoin formation, are urgently needed. In order to facilitate screening for novel haemin-binding antimalarials, two new assays are presented: one is based on the inhibition of the reaction(s) of GSH with haemin;9 the other on the reversal of the inhibition by haemin of the dopachrome tautomerase activity of human macrophage migration inhibitory factor (MIF).18 The assays were tested successfully using a series of 42 compounds isolated from antimalarial plant extracts.19 The methods offer improvements in sensitivity, speed and cost that should prove useful in antimalarial discovery.
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Materials and methods |
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Test compounds were extracted and purified from selected species of plant from six families used in traditional medicine for fever in South America: family Menispermaceae [Abuta grandifolia (Martius) Sandwith, Abuta rufescens Aublet, Cissampelos ovalifolia Candolle (DC)]; family Apocynamceae [Aspidosperma excelsum Bentham, Geissospermum sericeum (Sagot) Bentham & Hooker]; family Myrtaceae [Myrciaria dubia (Kunth) McVaugh]; family Simaroubaceae (Simarouba amara Aublet); family Lamiaceae (Plectranthus barbatus Andrews); and family Rutaceae [Zanthoxylum pentandrum (Aublet) R. A. Howard].19 The compounds were structurally characterized19 and tested for the ability to inhibit the proliferation, according to incorporation of [3H]hypoxanthine into nucleic acids, of a chloroquine-resistant (K1) and a sensitive (T9-96) strain of P. falciparum cultured in vitro.20
Scanning spectrophotometry
The effect of GSH upon haemin was analysed by repetitive scanning spectrophotometry (Perkin-Elmer 5) in the absence or presence of known haemin-binding antimalarial compounds. A solution (0.8 mL) of 10 µM haemin in 1 mM DETEPAC, 0.05 M Na phosphate pH 7.0 at 25°C in a cuvette of 1 cm light-path, was scanned from 480 to 340 nm. Neutralized GSH was added to 2 mM (using a 100 mM stock solution) and the sample scanned at 3 min intervals. The experiment was repeated in the presence of 18 µM chloroquine, mefloquine, quinine, bi-desethyl-amodiaquine, desbutyl-halofantrine or artemisinin. In each case the drug was added after the haemin scan, and an extra scan was made before the addition of GSH to show formation of a drughaemin complex.
Multiwell plate GSHhaemin interaction assay
Based upon the spectrophotometric data, a multiwell assay for the inhibition of GSHhaemin interaction was developed. The following stock solutions were prepared: 1 mM DETEPAC in 10 mM Na phosphate pH 7.0; 2 mM haemin in DMSO; and 100 mM GSH, 1 mM DETEPAC, 10 mM Na phosphate pH 6.8. Drug stock solutions (generally 2 mM) were prepared in water, methanol or DMSO as required. All stocks were stored frozen except the haemin which was prepared fresh daily. Working solutions were as follows: A, DETEPAC/phosphate stock 4 vol + 1 vol ethanol; B, 5 µL haemin stock solution per mL of solution A; and C, 0.15 mL GSH stock solution per mL solution A.
Assays were carried out in 96-well (400 µL) flat-bottomed polystyrene plates. Solution A (100 µL) was added, followed by drug or solvent control (e.g. 2 µL of 2 mM drug stock) in triplicate assays. Solution B (haemin) (200 µL) was then added to all wells using a multichannel pipette followed by 50 µL solution C (GSH). Final concentrations of drug and haemin were 11 µM and 5.7 µM, respectively. The absorbance at 360 nm (A360) was measured after c. 1 min using a plate reader (MCC Titertek, Thermo Life Sciences, Basingstoke, UK) set up to read at t = 0 and t = 30 min and to calculate the A360. The A360 of the controls (GSH + haemin + solvent ) decreased by about 0.08. The effect of the haemin-binding compounds was evaluated as the percentage decrease compared with control absorbance. Mean and S.D. of the triplicates were calculated and significance determined by Students t-test.
MIFhaemin interaction assay
The following stock solutions were prepared: D, 40% (v/v) ethane-1,2-diol, 1 mM EDTA, 20 mM Na phosphate pH 6.8; E, 1 mM EDTA, 10 mM Na phosphate pH 6.4; F, 10 mM L-dihydroxyphenylalanine-methyl ester in water; and G, 20 mM Na m-periodate in water. Recombinant human MIF was dissolved (0.5 mg/mL) in D. All solutions were stored at 4°C. A working solution of MIF (7.5 µg/mL) was prepared daily by diluting the stock with D, and kept on ice. Haemin (0.2 mM in DMSO) was also prepared daily. Tautomerase activity was assayed at 35°C in thermostatted 1 mL polystyrene cuvettes at 474 nm. To 740 µL solution E was added 32 µL solution F, stirred, then 24 µL solution G was added and stirred to form c. 0.4 mM L-dopachrome-methyl ester, all of the periodate (G) being consumed. A control volume of DMSO (4 µL) was added and the rate of spontaneous dopachrome decarboxylation was measured by the decrease in A474 over 0.4 min. MIF (2 µL i.e. 15 ng) was added with stirring and the dopachrome tautomerization followed similarly to obtain an uninhibited control rate. In order to check for direct inhibition of tautomerization by the test compound, which would preclude that compound from this type of assay, the assay was repeated using a maximal level of test compound (e.g. 2 µL of 2 mM in DMSO + 2 µL DMSO). If there was little or no inhibition, the original assay was repeated with c. 2 µL haemin, such that the activity due to MIF was inhibited by 7595%. The test compound (maximal amount) and haemin were then both included in the assay to see whether there was reversal of the haemin-dependent inhibition of the MIF tautomerase activity. In positive cases, successive two-fold reductions in the concentration of test compound gave a reversal of inhibition curve and the concentration causing 50% relief of the inhibition, RI50, was obtained graphically.
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Results |
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The percentage of GSH-dependent loss of A360 in the presence of each of the 42 compounds was compared with the IC50 of antiplasmodial activity in the T9-96 (Figure 4a) and K1 (Figure 4b) strains of P. falciparum. Compounds that inhibited growth by <50% at 40 µM were grouped as being not significantly growth inhibitory (IC50 > 40 µM). Twenty-one of the compounds showed 40% inhibition of the effect of GSH on haemin (13 bis-benzyl-isoquinoline, three ß-carboline, two protoberberine, two indole-alkaloid and one aporphine). Four compounds (two indole alkaloids and two ß-carbolines) were positive in the GSHhaemin interaction assay but failed to show significant inhibitory activity against either strain of P. falciparum (IC50 > 40 µM). In addition one compound (aporphine) was inhibitory only towards the K1 strain, and another (protoberberine) was inhibitory only towards the T9-96 strain. Inhibition of the GSHhaemin reactions correlated well with antiplasmodial activity (Table 1). Thus, only 3/19 antiplasmodial compounds failed to inhibit GSHhaemin reaction by >40%: an indole alkaloid of relatively low antiplasmodial activity, the benzyl-isoquinoline and the least potent of the antiplasmodial BBIQs.
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Eight of the 17 compounds showing both antiplasmodial activity and inhibition of GSHhaemin reaction were chosen to represent a range of potency for testing in the MIFhaemin interaction assay (i.e. the reversal of haemin inhibition of MIF dopachrome tautomerase activity). The log[RI50] of the test compounds, together with those of chloroquine and quinine were positively correlated with the log[antiplasmodial IC50] for theT9-96 strain (r = 0.824) and the K1 strain (r = 0.904) (Figure 5).
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Discussion |
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In order to test the practical performance of the assay, 42 natural compounds isolated from species of antimalarial plants were screened. The assay successfully detected 16 of the 19 showing antiplasmodial activity in vitro. Among the three false negatives, one was a derivative of the indole alkaloid geissoschizoline,23 which was only weakly antiplasmodial, and was similar structurally to ajmaline, another weak antimalarial. There was no obvious reason for failing to detect the other two, a benzyl-isoquinoline, (S)-6-methoxyjuziphine, and a partially characterized BBIQ from C. ovalifolia.19 Six compounds inhibited the GSHhaemin interaction without showing antiplasmodial activity towards either strain. However, two of these, including the only potent inhibitor, a protoberberine, fargarine IV, were antiplasmodial towards only one of the strains. The other four comprised two partially characterized indole alkaloids from A. excelsum and two ß-carbolines from S. amara, one of which was a glycoside.19 This test may clearly give positive results for various compounds with a hydrophilic moiety such as glycosides, which will be unlikely to have antiplasmodial activity in vitro. A group of 10 myricetin and quercetin glycosides were also isolated from the aqueous acetic acid-soluble fraction of methanol extracts (non-alkaloidal) of M. dubia.19 As expected, they showed no antiplasmodial activity in vitro; however, they were potent inhibitors of the GSHhaemin reaction, even at sub-stoichiometric levels of glycoside, which may be due to an antioxidant effect. Compounds of this type are expected to increase the numbers of false positives in the GSHhaemin interaction assay, but could perhaps be detected by their sub-stoichiometric potency.
The sensitivity of the GSHhaemin interaction assay is not high enough to cover the range of association constant of most useful haemin-binding antimalarials. In order to obtain information on the relative haemin affinity of compounds detected in the above assay, a more sensitive test was devised, namely the relief of haemin inhibition of the dopachrome tautomerase activity of human MIF, or MIFhaemin interaction assay. This assay successfully detected the predicted haemin binding of all the antimalarials tested (Figure 4) although the RI50 of lumefantrine could not be obtained due to its insolubility above 0.5 µM under these conditions. This assay is carried out in 99.5% aqueous solution generally in the range 50 nM to 5 µM of inhibitor with c. 200 nM haemin. The reversal of inhibition curve takes c. 1 h to obtain for each compound. The spectrophotometer is the only significant expense: only 150 ng MIF is needed per test compound at a cost of about £0.02.
Selecting eight of the compounds that gave a positive GSHhaemin interaction result for the MIFhaemin interaction assay, and adding chloroquine and quinine, RI50 values were obtained. The log10 of the RI50 for the compounds is reasonably proportional to the log10 of the antiplasmodial IC50 in the two strains (Figure 5) and is expected to be able to predict compounds capable of interacting with haemin at physiologically significant aqueous concentrations of haemin (sub-micromolar). The recombinant human MIF used in the assay is stable as a stock solution in 40% (v/v) ethan-1,2-diol, 10 mM Na phosphate for at least a year at 4°C.
Although the antimalarial activity of BBIQ compounds has been observed previously,24,25 their mode of action has not until now been related to their interaction with haemin.
It was considered that the GSHhaemin interaction assay could be used as a rapid screen for compounds binding in the micromolar range, and the inhibition of the MIFhaemin interaction assay could be used to test for sub-micromolar potency of interaction in order to select compounds that would render worthwhile the more complex assays that are better predictors of antimalarial activity.13,20
The MIFhaemin interaction assay, although not suitable for high-throughput screening, should be useful as an aid in the identification of the most potent haemin-binding natural or synthetic products, which could then be selected for structural characterization where needed, and further testing.13,20 The assay should also be most useful when a very small amount of test compound is available. The novel assays described are unlikely to predict antimalarial activity as well as those involving direct inhibition of haemin polymerization;13,14 however, they have important advantages in terms of sensitivity, speed, simplicity and cost.
Both assays depend on interactions thought to occur during plasmodial infection, and since MIF is present in human erythrocytes (c. 15 µg/mL, D. J. Meyer, unpublished) and may act as a proinflammatory cytokine, its release from parasitized or lysed erythrocytes could contribute to the symptoms of malaria. If this is the case, the interaction of MIF with haemin and its prevention by haemin-binding antimalarials might be of direct therapeutic significance.
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Acknowledgements |
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Footnotes |
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References |
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