Effects of the antimitotic natural product dolastatin 10, and related peptides, on the human malarial parasite Plasmodium falciparum

B. J. Fennell1, S. Carolan1, G. R. Pettit2 and A. Bell1,*

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Microtubule inhibitors from several chemical classes can block the growth and development of malarial parasites, reflecting the importance of microtubules in various essential parasite functions. With the spread of antimalarial drug resistance, there is an urgent need for new approaches to the chemotherapy of this devastating disease. We investigated the effects of two naturally occurring marine peptides, dolastatin 10 and dolastatin 15, and 10 synthetic dolastatin 10-based compounds (auristatins), on cultured malarial parasites of the species most lethal to humans, Plasmodium falciparum. Dolastatin 10 was a more potent inhibitor of P. falciparum than any other previously described microtubule inhibitor, with a median inhibitory concentration (IC50) of 10–10 M. Dolastatin 15 was less active, and compounds of the auristatin series had various potencies. Comparison of the concentrations required to inhibit P. falciparum and mammalian cell proliferation showed that the orders of potency were not the same. Dolastatin 10 and auristatin PE caused arrested nuclear division and apparent disassembly of mitotic microtubular structures in the parasite. The effects of these agents were, superficially at least, similar to those of vinblastine but different from those of paclitaxel. These studies indicate that compounds binding in the ‘Vinca domain’ of tubulin can be highly potent antimalarial agents.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The current portfolio of drugs for prophylaxis and treatment of malaria is limited, and the spread of resistance to chloroquine and some other agents limits the usefulness of these drugs in certain areas.1 With the exception of the artemisinins, there are few compounds in development for malaria that are not chemically related to drugs already well-established in the market.2 Given the massive morbidity and mortality associated with malaria, and the continuing absence of any viable vaccine, much effort has been devoted to identifying new drug targets and evaluating new chemical types for antimalarial activity.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Parasites and culture conditions

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 Sigma–Aldrich (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 18–24 h post-invasion, at 2.5% haematocrit, were grown in 24-well plates in the presence of 4–8 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 6–12, 21–27 and 36–42 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 15–30 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 {alpha}-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 Tris–HCl, 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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antimalarial activity of dolastatins

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 concentration–effect relationship. Instead, a plateau of ~30–50% of control growth (i.e. ~50–70% 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 ~10–20% 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 nM–8 µM) and the 72 h IC50 was 200 nM (Figure 1b).



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Figure 1. Susceptibilities of asynchronous cultures to dolastatin 10 (a), dolastatin 15 (b) and auristatin PE (c), as measured by the pLDH method. Mean values of four to eight determinations after 48 h (white circles) and 72 h (white squares) are shown; the initial parasitaemias were 0.8%. In (a) and (c), mean values after 72 h (black squares) and 96 h (black triangles) are shown for initial parasitaemias of 0.2%.

 
Antimalarial activity of auristatins

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 concentration–effect 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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Table 1.  Inhibitory effects on growth of P. falciparum and mammalian cells (various lines) by dolastatin 10 and derivatives
 
Data obtained with mammalian cell lines have shown closer potencies of dolastatin 10 and auristatin PE, and less difference in potency between the two dolastatins,10 than is evident here using malarial parasites. We therefore measured the antimalarial activity of several derivatives of the auristatin family in order to determine whether the structure–activity relationships found for mammalian cell proliferation were conserved when inhibition of parasite growth was tested. From the data presented in Table 1, the following structure–activity observations were apparent for parasite but not mammalian cells: (i) there is an advantage of the heterocyclic side chain extension in dolastatin 10 over auristatin PE; (ii) the chlorophenyl group in auristatin C confers lower activity than a phenyl (auristatin PE) or pyridyl (auristatin PYE); and (iii) the additional CH2 in auristatin MQ confers a disadvantage relative to GRP 18158. However, no agent had consistently, markedly stronger antimalarial than antimammalian cell activity.

Concentration–effect relationships using synchronized parasite cultures

The marked plateaux in concentration–effect 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 (~0–20 h post-invasion), trophozoites (~20–36 h) and schizonts (plus segmenters) (~36–48 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 ~0–6, 6–12 or 42–48 h post-invasion at the start of exposure were largely resistant to the inhibitors at the concentrations tested. In contrast, cultures starting at ~18–24, 24–30 or 30–36 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 ~12–18 and 36–42 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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Table 2.  Susceptibilities of synchronous cultures of different initial ages upon 48 h exposure to inhibitors
 
Effects of inhibitors on parasite morphology

Early trophozoite-stage (18–24 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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Figure 2. Development of parasites in the presence of inhibitors. (a) Cultures of 18–24 h post-invasion were exposed to no inhibitor, or 0.8 nM, 2.0 µM or 480 nM, respectively, of dolastatin 10, vinblastine or paclitaxel as indicated, and smears were counted at 6 or 12 h intervals, as described in Materials and methods. The ages of parasites shown on the x-axis are averages over an ~6 h range. The data are those of a representative experiment. (b) No inhibitor. (c) Morphology of parasites treated with dolastatin 10. Cultures were treated as described above, and the microscopic appearance of parasitized erythrocytes in Giemsa-stained smears was photographed. The numbers indicate the median ages of the parasites in hours post-invasion. The parasites shown are representative of those examined.

 
Effects of inhibitors on different developmental stages

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 (6–12 h post-invasion), trophozoite (21–27 h post-invasion) and schizont (36–42 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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Figure 3. Effects of microtubule inhibitors on different developmental stages. Cultures synchronized to the ring stage (6–12 h post-invasion), trophozoite stage (21–27 h) and schizont stage (36–42 h) were each incubated for 6 h in the presence of no inhibitor or 0.8 nM, 2.0 µM, 480 nM or 16 nM of dolastatin 10, vinblastine, paclitaxel or auristatin PE, respectively, as indicated. The cells were then washed, recultured for 48 h in inhibitor-free medium, and finally examined on Giemsa-stained smears; the parasitaemias obtained at this time are indicated on the y-axis. The ages of parasites shown on the x-axis are averages over the ~6 h range at the beginning of the experiment. The data shown are from a single, representative experiment.

 
Effects of inhibitors on mitotic microtubular structures

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 {alpha}-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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Figure 4. Mitotic microtubular structures of cultured parasites viewed by immunofluorescence using antibodies to P. falciparum ß-tubulin (left) and DAPI nuclear stain (right). (a) Untreated parasites: note microtubule-organizing centres (small arrow) and hemispindles (large arrow); (b) treated with 1 nM dolastatin 10: note diffuse staining (arrowheads) and loss of normal structures; (c) treated with 10 nM auristatin PE; (d) treated with 20 µM vinblastine; (e) treated with 1 µM paclitaxel: note thick rod of tubulin (block arrow) and loss of normal structures. In all cases exposure to inhibitor was for 6 h.

 
Asynchronous cultures exposed to dolastatin 10 or auristatin PE lost the normal microtubular structures and were labelled non-specifically, in a diffuse or micropunctate fashion, over the entire area of the parasite cell with the exception of the haemozoin granules (Figure 4). These effects were seen with concentrations as low as 1 nM dolastatin 10 and 10 nM auristatin PE, within 6 h of exposure. The effects were, superficially at least, similar to those of vinblastine (Figure 4). The effects were dissimilar to those of paclitaxel, which at concentrations down to 100 nM caused accumulation of thick rods of tubulin in place of the normal structures (Figure 4).

To assess the action of these agents more quantitatively, cultures were synchronized to the trophozoite stage (27–33 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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Table 3.  Quantitative analysis of effects of inhibitors on mitotic microtubular structuresa
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Previous studies with microtubule inhibitors and malarial parasites have indicated that potent growth-inhibitory activity can be observed with members of the ‘Vinca’ alkaloid and taxoid groups of compounds, and that less potent but more selective activity is seen with certain dinitroanilines.3 In this paper, the antimalarial activities of the marine organism-derived peptides dolastatin 10 and dolastatin 15, and a number of synthetic derivatives based on dolastatin 10, were examined. This constitutes to our knowledge the most extensive structure–antimalarial activity study ever carried out on microtubule inhibitors. The rationale was to investigate (i) whether the orders of activity of the series against parasite and mammalian cells might be distinct, and if so, (ii) whether a compound with marked selectivity for parasite over mammalian cells could be identified. The orders of activity were indeed quite different. However, no markedly selective compound was identified that might be progressed further as an antimalarial drug, and it seems that dolastatin 10 is an unpromising basis for further antimalarial evaluation. Perhaps the testing of such a (with one exception) potent series of anti-tumour compounds made (ii) a difficult target on this occasion. Nevertheless, among the series were found the most potent anti-malarial microtubule inhibitors so far described, superior even to Vinca alkaloids and taxoids, with activities in the 10–9–10–11 M range. From studies of these compounds on human cancer cell lines and antineoplastic evaluation in vivo, dolastatin 10 has proven to be one of the most potent anti-cancer drug presently known,14,15 and is currently in Phase II human cancer clinical trials under the auspices of the US National Cancer Institute.

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 dose–response 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 36–42 and 42–48 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 0–18 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.


    Acknowledgements
 
We thank G. J. Atkins for access to microscopic equipment. A.B. was supported by grant GA244 from the British Society for Antimicrobial Chemotherapy, and G.R.P. by Outstanding Investigator grants CA-44344 05-12 and R01-CA-90441-01 from the Division of Cancer Treatment and Diagnosis, National Cancer Institute, Department of Health and Human Services, the Arizona Disease Research Commission, and the Robert Dalton Endowment Fund.


    Footnotes
 
* Corresponding author. Tel: +353-1-608-1414; Fax: +353-1-679-9294; E-mail: abell{at}tcd.ie Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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