1 Department of Microbiology, Moyne Institute, Trinity College, Dublin 2, Ireland; 2 Cancer Research Institute, Arizona State University, Tempe, AZ 85287-2404, USA
Received 10 April 2002; returned 13 September 2002; revised 2 December 2002; accepted 13 January 2003
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Abstract |
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Introduction |
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Microtubules are essential components of almost all eukaryotic cells. In malarial parasites they have various functions depending on the stage of the life cycle.3 In the asexual, erythrocytic stage, which is the one associated with the symptoms of malaria, microtubules are apparently required for nuclear division, partitioning of organelles and cytosol into new merozoites and invasion of erythrocytes by merozoites.3,4 Much of the evidence for these roles has accumulated through experimentation with compounds such as colchicine, vinblastine and paclitaxel (Taxol), which are known to disrupt microtubular functions in other cells39. Whereas these compounds are clearly useful as research tools, their potential clinical utility for malaria is limited by their high potency against mammalian cells and the high degree of amino acid sequence conservation between human and P. falciparum tubulins, the target proteins for these agents.3 Nonetheless, data from the Vinca alkaloid and taxoid groups show that it is possible to have higher potency against cultured parasites than host cells. Also, certain dinitroaniline herbicides such as trifluralin have very low host cell toxicity but enjoy moderate activity against the parasite. Therefore it seems possible that one could find microtubule inhibitors with potent and selective antimalarial activity, perhaps as a result of subtle differences in tubulin structures or different uptake kinetics.
In this study, we have examined the antimalarial activity of two peptides from the sea hare Dolabella auricularia, dolastatin 10 and dolastatin 15.10 They are already under investigation as potential anticancer agents, and are believed to interact close to the binding site of vinblastine (the Vinca domain) on tubulin. In addition, several related synthetic compounds known as auristatins10 were tested. We demonstrate here the high antimalarial potency of dolastatin 10 and some of the auristatins, the differences in order of activity in this series against mammalian and P. falciparum cells, and the apparent vinblastine-like action of dolastatin 10 and auristatin PE on nuclear division and mitotic microtubules of the parasite.
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Materials and methods |
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P. falciparum FCH5.C2, a cloned subline of FCH5/Tanzania adapted to growth in horse serum,11 was cultured and synchronized in human A+ erythrocytes as described previously.11 Synchronization was carried out by two-step sorbitol treatment.12
Inhibitors
Dolastatins 10 and 15 and the 10 auristatins were obtained by total synthesis as previously described.10,1317 Other chemicals were purchased from SigmaAldrich (Dublin, Ireland) unless otherwise stated.
Inhibitor susceptibility
Susceptibility to inhibitors was determined in 96-well microplates using the parasite lactate dehydrogenase (pLDH) method.18 For the examination of parasite morphology, synchronized 0.5 mL cultures of 1824 h post-invasion, at 2.5% haematocrit, were grown in 24-well plates in the presence of 48 x IC50 concentrations of each inhibitor or the corresponding concentration of solvent alone. Smears were made every 6 h, stained with Giemsa, examined by bright-field microscopy and photographed. For the determination of susceptibility of different stages, parasites were first synchronized to 612, 2127 and 3642 h post-invasion. One millilitre cultures at 2.5% haematocrit and 2% parasitaemia were then exposed to inhibitors at 8 x IC50, or solvent alone, for 6 h. The cultures were then washed three times in 10 mL of warm wash medium (RPMI 1640 with L-glutamine, supplemented with 25 mM HEPES, 50 mg/L hypoxanthine and 0.18% w/v sodium bicarbonate) and recultured for 48 h in inhibitor-free medium, with a change of medium after 24 h, before counting Giemsa-stained smears as described above. Slides were prepared in duplicate and at least 1000 erythrocytes were counted per slide.
Inhibition of proliferation of cultured mammalian cell lines was measured as described previously.19
Immunofluorescence microscopy
Cultured parasites were exposed to inhibitors or solvent alone in 24-well dishes for the times indicated below. One hundred and fifty microlitre portions were then removed into 9 vols of warm wash medium and centrifuged at 800g for 5 min. Each erythrocyte pellet was then resuspended in 140 µL of wash medium. Eight millimetre diameter windows of printed slides (Hendley, Essex, UK) were coated for 10 min with 1 mg/mL poly-L-lysine and washed twice in wash medium before application of 20 µL of fixative [4% paraformaldehyde/0.2% v/v Triton X-100 in phosphate-buffered saline (PBS)]. Ten microlitres of parasitized erythrocyte suspension was then mixed into each drop of fixative and the slides were left for 30 min in a moist chamber at room temperature. The parasite-coated windows were then subjected to the following steps, using a capillary pipette under suction for changing the solutions: (1) washing 5 x 3 min in PBS; (2) blocking for 1530 min (or overnight at 4°C) in 5% normal swine serum in PBS; (3) probing for 1 h with primary antibody (affinity-purified rabbit antiserum to a synthetic P. falciparum ß-tubulin peptide,20 or DM1A mouse monoclonal antibody to -tubulin); (4) washing as in step 1; (5) probing for 1 h with secondary antibody (anti-rabbit or -mouse Ig conjugated to fluorescein isothiocyanate); (6) washing as in step 1; and (7) mounting under a cover-slip with 5.0 mg/mL n-propylgallate/90% glycerol/100 mM TrisHCl, pH 8.0. Fluorescent nuclear staining was carried out as required using 1 µg/mL diamidinophenylindole (DAPI) for 30 s after the secondary antibody step. Suitable dilutions of antibodies were determined empirically. After sealing, the slides were examined by phase contrast and epifluorescence with the 100x oil-immersion lens of a Nikon Eclipse E400 microscope. Photographs were taken with a Coolpix 950 digital camera using full auto-exposure mode and Best Shot Selection.
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Results |
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Both dolastatin 10 and dolastatin 15 had inhibitory activity on P. falciparum grown asynchronously in culture, as measured by the pLDH method (Figure 1). The former was more than three orders of magnitude more potent than the latter and is superior to any other microtubule inhibitor for which data have been published3 or which we have tested. The IC50 after 48 h exposure to dolastatin 10 was difficult to determine, as there was no clear sigmoidal concentrationeffect relationship. Instead, a plateau of 3050% of control growth (i.e.
5070% inhibition) over a very wide concentration range between
250 pM and
8 µM was observed (Figure 1a). After 72 h exposure, the plateau was reduced to
1020% of control growth over the same concentration range, and an IC50 of 100 pM was evident. In order to abolish completely the plateau of incomplete inhibition above the IC50 and obtain a sigmoidal curve, it was necessary to extend the time of exposure to 96 h, using a lower initial parasitaemia (Figure 1a). A similar phenomenon was seen with dolastatin 15, except that the plateau was much narrower (
500 nM8 µM) and the 72 h IC50 was 200 nM (Figure 1b).
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In view of the extremely potent antimalarial activity of dolastatin 10, we investigated a series of synthetic compounds, known as auristatins, which are closely related to this natural product. Auristatin PE (see Table 1 for structure) was also a potent antimalarial agent with a 72 h IC50 of 2 nM (Figure 1c). The plateau effect seen with the dolastatins was evident with this compound too, and was reduced after 72 h and abolished after 96 h. Interestingly, for all three drugs, the 48 h curve turns from partial to full inhibition at about the same concentration (between 10 and 100 µM), in spite of the different width of plateau. This suggested that all three drugs had roughly equal potency, in the micromolar range, against some low-affinity target, but that they differed greatly in their potency against a separate, high-affinity target. We observed similar concentrationeffect relationships with the structurally unrelated microtubule inhibitors vinblastine (72 h IC50, 250 nM; 48 and 72 h plateaux from 500 nM to 16 µM) and paclitaxel (72 h IC50, 60 nM; 48 h plateau from
100 nM to 8 µM), but not with other agents such as chloroquine (data not shown).
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Concentrationeffect relationships using synchronized parasite cultures
The marked plateaux in concentrationeffect curves for all the inhibitors tested at 48 h suggested that two distinct populations of parasites with differing susceptibilities were present. The disappearance of these plateaux upon longer exposure to inhibitors further indicated that the distinct populations may relate to the developmental stage of the parasite inside the erythrocyte, i.e. ring forms (020 h post-invasion), trophozoites (
2036 h) and schizonts (plus segmenters) (
3648 h). If the inhibitors acted on a specified period (window of susceptibility) of the developmental cycle, only parasites exposed to sufficient concentrations during this window would be fully susceptible. Although all parasites were exposed for 48 h, there may be a lag while the drug penetrates to its site of action and takes full effect, therefore not all parasites may have been exposed to sufficient concentrations during the window of susceptibility.
To test this idea, parasite cultures were synchronized to produce eight populations with age ranges of 6 h covering the whole cycle, and the inhibitor susceptibility determinations over 48 h were repeated with each. The data in Table 2 show that susceptibilities to dolastatin 10 and auristatin PE were highly dependent on the age of the parasites at the time of initial exposure. Cultures that were
06, 612 or 4248 h post-invasion at the start of exposure were largely resistant to the inhibitors at the concentrations tested. In contrast, cultures starting at
1824, 2430 or 3036 h post-invasion were highly susceptible, giving curves that were similar to those obtained after asynchronous cultures were exposed for 72 h (Figure 1). The results for
1218 and 3642 h post-invasion were intermediate between these extremes. This indicated that dolastatin 10 and auristatin PE were maximally active when initially applied to trophozoite or early schizont stage cultures. Similar results were obtained with vinblastine and paclitaxel (Table 2).
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Early trophozoite-stage (1824 h post-invasion) parasites exposed to dolastatin 10 and auristatin PE were examined for obvious morphological or developmental abnormalities on Giemsa-stained smears. At the time when new ring forms appeared in untreated cultures, there was a marked reduction in parasitaemias, a transient rise in number of schizonts and an almost complete lack of new rings in the dolastatin 10- or auristatin PE-treated cultures (Figure 2a and data not shown). Similar effects were observed with both vinblastine and paclitaxel (Figure 2a). This indicated that the inhibitors caused developmental delay or arrest at the schizont stage. In addition, some of the dolastatin 10-treated, developmentally delayed schizonts were distorted or irregular in morphology (Figure 2b, c and data not shown).
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To confirm that dolastatin 10 and auristatin PE acted primarily on the schizont stage and to investigate the reversibility of the inhibitors, cultures were synchronized to the ring (612 h post-invasion), trophozoite (2127 h post-invasion) and schizont (3642 h post-invasion) stages, and subjected to brief (6 h) exposure to inhibitors. They were then washed and recultured for 48 h in inhibitor-free medium. The ring stages were not affected by any of the inhibitors at concentrations equivalent to 8x (Figure 3) or 80x (not shown) IC50. The schizont stages were irreversibly damaged at 8 x IC50, as very few parasites of any stage were observed (Figure 3). The results for trophozoites were intermediate between those for rings and schizonts (Figure 3). A similar profile of stage-dependent killing was seen with vinblastine and paclitaxel (Figure 3).
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Mitotic and post-mitotic microtubular structures similar to those described by Read et al.21 were observed using antibodies raised to a synthetic peptide specific for P. falciparum ß-tubulin and the immunofluorescence protocol described above (Figure 4). Similar structures were observed using monoclonal antibody DM1A to mouse -tubulin, but these were less clear. No structures were seen when the protocol of Read et al.21 was used with these antibodies.
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To assess the action of these agents more quantitatively, cultures were synchronized to the trophozoite stage (2733 h post-invasion) and exposed to inhibitors for 6 h, after which most control parasites had entered schizogony, with its associated mitotic divisions. The numbers of parasites containing normal and various abnormal structures were counted (Table 3). The results indicated that under these conditions, 1 nM dolastatin or 10 nM auristatin PE caused complete loss of normal mitotic structures.
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Discussion |
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In addition, some interesting observations were made on the effects of these agents on P. falciparum cells in comparison with other classes of microtubule inhibitor.
The effects of the dolastatin 10/auristatin series were similar to those of vinblastine in three respects: (i) the presence of a plateau of partial inhibition in 48 h doseresponse curves using asynchronous parasites, which disappeared upon longer incubation (Figure 1); (ii) delayed or arrested development during schizogony, resulting in a decrease in new ring forms (Figure 2); and (iii) the loss of the normal mitotic microtubular structures seen by immunofluorescent microscopy and their replacement by diffuse or fragmented tubulin labelling (Figure 4). The last effect presumably reflects an accumulation of tubulin in the disassembled state. Dolastatin 10 is one of those microtubule inhibitors believed to bind to tubulin in the Vinca domain,22 i.e. close to the binding site of vinblastine and other Vinca alkaloids. It is known to inhibit Vinca alkaloid binding non-competitively, and like vinblastine it inhibits microtubule polymerization, associated GTP hydrolysis and nucleotide exchange,22 although there are fine differences in the effects of these two agents. Dolastatin 10 and vinblastine also have broadly similar effects on cultured mammalian cells, causing mitotic arrest and, at high concentrations, disappearance of microtubules. However, it is likely that at low concentrations dolastatin 10, like vinblastine, can inhibit mitosis in the absence of gross changes in polymerized microtubule mass, via subtle effects on dynamic behaviour. This may also be the case with P. falciparum microtubules, since growth-inhibitory effects were observed at concentrations below those that visibly affected microtubular structures.
Based on the presumption that members of the dolastatin 10/auristatin series act primarily on mitotic microtubules, one would expect both types of agent to be most active against the later (schizont) stage of intraerythrocytic development, in which mitosis occurs. Although the precise target stage is difficult to determine in the absence of knowledge of the kinetics of inhibitor uptake, the data are consistent with a primary effect of dolastatin 10 and auristatin PE on schizogony. The likely explanation for the plateau effect after 48 h exposure of asynchronous cultures is that at the beginning of exposure a subpopulation of the parasites was not in the age range most appropriate for complete inhibition. They may have already entered schizogony or have been sufficiently close to it as not to allow time for accumulation of inhibitory amounts of the agent during division (the 3642 and 4248 h post-invasion populations in Table 2). Upon longer exposure (72 or 96 h), all or almost all parasites would have had an opportunity to pass through this window of susceptibility. In the case of parasites initially aged 018 h post-invasion, the reduced effect (Table 2) may be caused by arrest in schizogony and the fact that schizonts contain higher pLDH activity per parasite than rings.23 Only after the control parasites had entered the more metabolically active trophozoite stage, with its higher pLDH levels, would the inhibitory effect be apparent. In agreement with this conclusion, schizonts were more susceptible to irreversible damage upon short-term exposure to dolastatin 10 or auristatin PE than trophozoites, which were in turn more susceptible than rings (Figure 3). These results may reflect the fact that the concentration of tubulins is lowest in rings, higher in trophozoites and highest in schizonts (B. J. Fennell & A. Bell, unpublished data), and that schizonts are deploying the tubulins in chromosome segregation. However, there is also a plausible alternative explanation, which suggests that ring-stage parasites are simply less permeable to the inhibitors.
At higher concentrations, all parasites were inhibited by dolastatin 10 or auristatin PE after 48 h, suggesting the presence of a lower-affinity target. The susceptibility of the putative lower-affinity target appears to be much less variable between different agents of the dolastatin/auristatin series than that of the high-affinity target, and may not involve tubulin binding. A similar phenomenon is seen with docetaxel (Taxotere), and in this case the low-affinity target is believed to be contained not in the parasite itself but in the host erythrocyte, which lacks tubulin.24 The plateau effect and greater susceptibility of parasites in the trophozoite stage was also observed with vinblastine and paclitaxel. Similar observations were made previously using docetaxel.24 These findings could explain the widely differing IC50 values that have been reported for certain microtubule inhibitors against P. falciparum cultures using different exposure times and degrees of synchrony.3 In terms of possible therapy, they indicate that microtubule inhibitors might be fast- or slow-acting in malaria patients, depending on the age range of the parasites present.
Can compounds binding in the Vinca domain be obtained that have the antimalarial potency of the compounds described here but with little effect on host cells? Comparative screening of large combinatorial libraries of compounds, and/or determination of the structures of the drug-binding sites on mammalian and parasite tubulins, could give the answer to this question.
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Acknowledgements |
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Footnotes |
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References |
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