ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 1990, p. 1529-1534
Vol. 34, No. 8
0066-4804/90/081529-06$02.00/0 Copyright © 1990, American Society for Microbiology
Mode of Action of Tubulozoles against Plasmodium falciparum In Vitro ANGELA DIECKMANN-SCHUPPERTt* AND RICHARD M. FRANKLIN Department of Structural Biology, Biocenter, University of Basel, CH4056 Basel, Switzerland Received 4 January 1990/Accepted 24 May 1990 The mode of action of the tubulozole isomers, recently recognized as a new class of potential antimalarial agents, was investigated. Whereas neither glycolysis, protease activity, or nucleic acid biosynthesis was primarily affected, protein biosynthesis decreased soon after addition of the drug. Inhibitors of protein biosynthesis, however, did not show synergistic activity with tubulozole. Colcemid, on the other hand, had an effect on protein synthesis similar to that seen with the tubulozoles. Furthermore, combinations of the tubulozole isomers with compounds known to interact with tubulin inhibited malaria in a synergistic or antagonistic fashion. Therefore, the inhibition might be elicited by interaction with tubulin or some other component of the microtubules. This is remarkable insofar as only one of the tubulozole isomers affects mammalian cells by binding to tubulin. New antimalarial agents are urgently required since a suitable vaccine has not been developed and drug resistance is an ever-increasing problem. Tubulozoles were recently identified as a new class of potential antimalarial agents (4). The aim of the present study was to investigate the mechanism of action of tubulozoles in inhibiting the malarial parasite. Tubulozoles represent a class of chemical compounds which are unique in that in mammalian cells only the C (cis) isomer inhibits tubulin polymerization, whereas the T (trans) isomer does not (8). Tubulozole C-treated mammalian cells experience a mitotic and transport block. This primary cytostatic effect becomes cytocidal after sufficiently long exposure. Since both isomers show the same dose dependency in the inhibition of intraerythrocytic growth of Plasmodiumfalciparum, their mode of action had to be carefully reconsidered, not excluding a priori possibilities other than an interaction with tubulin. We therefore investigated the effect of the tubulozoles on lactate production, activity of parasitic proteases, nucleic acid synthesis, and protein synthesis to define, in broad terms, an area of metabolism which might be a target of the drugs. MATERIALS AND METHODS Parasites. P. falciparum Ro58, highly resistant to chloroquine and pyrimethamine-sulfadoxine (Fansidar), was cultivated in RPMI 1640 with 10% horse serum, as reported previously (4). The multiplication of parasites was monitored by evaluation of Giemsa-stained thin smears. Growth of the parasites was synchronized to +4 h by repeated sorbitol treatment (14). Sources of inhibitors. Taxol was kindly provided by M. Suffness, National Cancer Institute, Bethesda, Md.; mebendazole and the tubulozole isomers were provided by H. Vanden Bossche, Janssen Research Foundation, Beerse, Belgium; and pyrimethamine was provided by H. Matile, F. Hoffmann-La Roche & Co., Basel, Switzerland. Chloramphenicol, tetracycline, and nalidixic acid were purchased * Corresponding author. t Present address: Zentrum fur Hygiene und Medizinische Mikrobiologie, Philipps-Universitat Marburg, Robert-Koch-Strasse 17, D-3550 Marburg, Federal Republic of Germany.
from Boehringer GmbH; vincristine was purchased from Eli Lilly; colchicine (CLC) and rifampin were purchased from Serva; colcemid and vinblastine (VBL) were purchased from Sigma; albendazole was purchased from SmithKline; and dactinomycin (actinomycin D) and cycloheximide were purchased from Calbiochem-Behring. Lumicolchicine was prepared from CLC by UV irradiation as previously described (19). Sterile stock solutions were prepared in dimethyl sulfoxide or saline as appropriate and kept at -20°C. Working dilutions were prepared in culture medium immediately before use. Lactate determination. Lactate was assayed in the supernatants of 3-ml cultures by the enzymatic procedure employing lactate dehydrogenase and glutamate-pyruvate transaminase (15). Briefly, culture supernatants were deproteinized by the addition of perchloric acid and neutralized with potassium hydroxide. Aliquots were added in a cuvette to glutamate buffer containing 24 mM NAD and 80 U of glutamate-pyruvate transaminase per ml. The reaction was started by the addition of 550 U of lactate dehydrogenase per ml, and the increase in A339 was monitored. Protease assay. Proteolytic activity in a crude P. falciparum lysate, prepared as described below, was estimated by spectrophotometric measurement of dye release from Hide Powder Azure (Calbiochem-Behring) (16) in the pH range between 4 and 9. Briefly, 1 ml of a 10-mg/ml substrate suspension was incubated with 0.1 ml of lysate at 37°C for 30 min. The activity was estimated after centrifugation by the amount of blue dye released into the supernatant, the resultant increase in A595 being measured. [3H]hypoxanthine and [35S]methionine incorporation. Cultures (2-ml) at 5% hematocrit and 8% parasitemia (32 + 4 h-old trophozoites) were exposed to the appropriate drug and labeled with 30-min pulses of 10 ,uCi of [3H]hypoxanthine (specific activity, 3.8 to 8.6 Ci/mmol; Amersham) or [35S]methionine (specific activity, >1,000 Ci/mmol; Amersham) per ml in hypoxanthine- or methionine-free RPMI 1640. After the cultures were washed three times with 20 ml of ice-cold phosphate-buffered saline, the pellet was lysed by the addition of 500 ,ul of distilled water, followed by ultrasonication. Precipitation of nucleic acids and proteins was done by the addition of 1 ml of 20o ice-cold trichloroacetic acid. The insoluble material was washed three times with 8% 1529
1530
ANTIMICROB. AGENTS CHEMOTHER.
DIECKMANN-SCHUPPERT AND FRANKLIN
trichloroacetic acid and collected on glass fiber filters, and the radioactivity was determined by liquid scintillation counting. Drug interaction studies. The dose-response relationship of each of the compounds under study was evaluated after exposing microtiter plate cultures to serial dilutions of the compounds. The final volume in each well was 200 ,u. Parasitemia was determined on Giemsa-stained smears after 48 h of exposure to the drug. Untreated cultures served as controls. Fifty percent inhibitory concentrations (IC50s) were determined graphically. Paired-drug interaction studies were performed by checkerboard titration, following the same protocol as for the study of single drugs. In this case, each drug was tested at eight concentrations, yielding eight times eight wells per experiment. IC50s for single rows and lines were obtained as described above. Isoboles (lines connecting points of the same extent of inhibition, i.e., IC50) were constructed graphically. If any two drugs, A and B, act additively, the isobole will be a straight line connecting the IC50 points on the axes. In the case of synergism a concave line is obtained, and in the case of antagonism a convex one is obtained (1). The extent of interaction is given by the sum of the fractional inhibition constants (IFIC), which is defined (1) as follows: IC50 of A in combination + IC50 of B in combination XFIC =
IC50 of A alone
IC50 of B alone
If A and B act additively, the IFIC will be 1 because their respective IC50s are not affected by mixing A and B. If A and B act synergistically, the :FIC will be less than 1; if they act antagonistically, it will be greater than 1 (1). RESULTS Several metabolic parameters of intraerythrocytic P. falciparum were investigated to trace the effects of tubulozoles. Each tubulozole isomer was tested separately in all assays. The results were always identical, irrespective of the isomer. We therefore use the term tubulozoles whenever both isomers are meant. Lactate accumulates in the culture supernatant and is an indicator of the glycolytic activity of the parasite and the erythrocyte. Over 4 h the rate of lactate production, amounting to 1.3 mM/h, was not affected significantly by the presence of 10 p.M tubulozole (Fig. 1). The amount of lactate produced agreed with values reported by others (21). The proteolytic activity of crude malarial lysates was not abolished by the addition of 10 p.M tubulozole. Typically, absorbance values ranging between 0.5 and 0.8 optical density units were obtained, irrespective of the presence of tubulozole. The incorporation of [3H]hypoxanthine, indicative of nucleic acid biosynthesis, reached a plateau after more than 3 h (Fig. 2). This, however, is longer than the minimal exposure time required for killing of the parasite by 10 p.M tubulozole (4) and was therefore regarded as an unspecific or secondary effect. Correspondingly, no inhibitor of nucleic acid biosynthesis (rifampin, nalidixic acid, dactinomycin, or pyrimethamine) displayed more than additivity when combined with tubulozoles (IFIC = 1) (Table 1). [35S]methionine incorporation, indicating protein biosynthesis, was inhibited by either tubulozole isomer at 10 p.M from the onset of drug treatment (Fig. 3A). Drug interaction studies, however, again revealed mere additivity between protein biosynthesis inhibitors (tetracycline, chlorampheni-
E2
7~
c
6
o
75
0/
._
3
32
4 h
FIG. 1. Effect of 10 I1M tubulozole T on lactate production in erythrocytic cultures of P. falciparum. The inhibitor was added at time zero. *, Control culture; O, treated culture; O, uninfected erythrocytic culture. All values are averages from three experiments.
col, cycloheximide) and the tubulozoles (Table 1). Furthermore, neither tubulozole isomer inhibited the staining of P. falciparum mitochondria with rhodamine 123 (6) during 6 h of drug exposure, whereas the above-mentioned 70S ribosomal inhibitors did. The antimicrotubular agent colcemid had an effect on 30-
20E 0
10-
1
2
3
4h
FIG. 2. Effect of 10 p.M tubulozole T on synthesis of nucleic acids in erythrocytic cultures of P. falciparum. The inhibitor was added at time zero. 0, Control culture; 0, treated culture. All values are averages from three experiments.
VOL. 34, 1990
ACTION OF TUBULOZOLES AGAINST P. FALCIPARUM
TABLE 1. Inhibitors of protein and nucleic acid biosynthesis tested for interaction with tubulozoles in P. falciparum in vitro Compounda IC50 0.3 FM Rifampin ........... Nalidixic acid ........... 30 mM 50 pM Dactinomycin ........... 26 ,uM Pyrimethamine ........... 40 ,uM Tetracycline ........... 40 ,uM Chloramphenicol ........... 4 nM Cycloheximide ........... a
1531
mebendazole, albendazole, and taxol (Table 2, Fig. 4). The antimalarial activities of taxol and CLC are reported here for the first time, the IC50s for strain Ro58 being 1 and 10 ,uM, respectively. Lumicolchicine, a derivative of CLC which does not bind to tubulin, was inactive at corresponding concentrations. Only additivity could be detected between tubulozoles and Vinca alkaloids (Table 2). Interaction between any other pair of the established tubulin-binding inhibitors tested was, however, markedly antagonistic in the malarial system, as summarized in Table 2.
All the compounds displayed additivity when combined with tubulozoles.
[35S]methionine incorporation (Fiig. 3B) similar to that of the tubulozoles (Fig. 3A) and, moireover, its effect on [3H] hypoxanthine incorporation was comparable to that of the tubulozoles (data not shown). The antimalarial activity of a range of antimicrotubular agents was reported by us ea rlier (4). Drug interaction studies using these agents and tubulozoles demonstrated pronounced synergism (IFIC = 0.25) between both tubulozole isomers and each of the f"ollowing: colcemid, CLC, 1501
A
* * E
*
100
a.
0
......-o
50 -
°0 10-
, I
, 3
2
,
, 4 h
* 50
B *
40E a.
30 -
7/
~
cfo
0
20
,0
7'
X-1/0
10
, , , 1 2 4 h 3 FIG. 3. Effects of 10 ,uM tubulozo)le T (A) and 100 ,uM colcemid (B) on incorporation of [35S]methioniine into protein in erythrocytic cultures of P. falciparum. The inhibiltor was added at time zero. , Control culture; 0, treated culture. All values are averages from ,
three experiments.
DISCUSSION
All mammalian cell lines studied so far are affected only by the cis isomer of tubulozole. In contrast, malarial parasites are inhibited to the same extent by both the cis and trans isomers. The wide phylogenetic distance between protozoa and higher eucaryotes prompted us to reevaluate the mode of action of the tubulozole isomers in malaria. Their effects on several biochemical parameters were measured, and drug interaction studies employing inhibitors of the aforementioned metabolic pathways, as well as inhibitors of microtubule formation, were done. The fact that cis- and transtubulozoles behaved identically in all the biochemical and drug interaction studies suggests that they, in contrast to the situation in mammalian cells, have a common molecular target in malaria. Glycolysis, the main pathway for energy production in intraerythrocytic P. falciparum, was not affected by tubulozoles, as indicated by unaltered lactate production. This indicates that glycolysis is not the molecular target of tubulozole in malaria. A spectrum of different proteases serves to degrade the erythrocytic stroma, a process which is essential for the survival of the malarial parasite. The antimalarial drug chloroquine is believed by many researchers to inhibit, although possibly in an indirect way, proteolytic breakdown in the plasmodial food vacuole (20), but this concept was recently questioned (9). In our assay system chloroquine inhibited the proteolytic activity only at concentrations in the millimolar range (data not shown). Both tubulozole isomers failed to inhibit malarial proteases in the cell-free assay at a concentration of 10 ,uM. These results are consistent with the lack of interaction between chloroquine and tubulozole in drug-interaction studies. We therefore conclude that the tubulozoles do not interfere with malarial protease activity. Tubulozoles do not interfere specifically, i.e., within the first 3 h (4), with nucleic acid biosynthesis, since we found neither a marked and early reduction in hypoxanthine uptake nor an interaction with rifampin, nalidixic acid, dactinomycin, or pyrimethamine, all being either direct or indirect inhibitors of nucleic acid biosynthesis (7). Furthermore, in vitro translation of malarial RNA by a rabbit reticulocyte lysate is not affected by either tubulozole isomer (C. Sartorius and R. M. Franklin, unpublished results), suggesting that direct interaction with malarial mRNA does not take
place. Malarial protein biosynthesis in either the mitochondrion or the cytoplasm is not primarily affected by tubulozoles, since synergism with tetracycline, chloramphenicol, or cycloheximide cannot be demonstrated. These compounds are well-established inhibitors of mitochondrial protein synthesis, in the case of tetracycline and chloramphenicol, or cytoplasmic protein synthesis, in the case of cycloheximide,
1532
ANTIMICROB. AGENTS CHEMOTHER.
DIECKMANN-SCHUPPERT AND FRANKLIN
TABLE 2. Interaction of tubulozoles and various established tubulin-binding substances as studied by their effect upon the growth of P. falciparum in vitro
Tubulozoles
3
CLC or colcemid
IFM
Colchicum alkaloids
Benzimidazoles
Synergistic (0.25)
Synergistic (0.25)
Additive (1)
Synergistic (0.25)
Antagonistic (>4)
Antagonistic (>4)
Antagonistic (2)
Antagonistic (>4)
Antagonistic (2)
10 P,M
Mebendazole Albendazole
200 ,uM 30 pLM
Vincristine VBL
6 nM 100 nM
a
Interactiona
IC50
Compound(s)
Vinca alkaloids
Taxol
Antagonistic (2)
Values given in parentheses are IFICs.
and are also known to inhibit malarial growth (5). The hypothesis that protein biosynthesis is not the primary target of tubulozoles is further supported by the lack of tubulozole inhibition of rhodamine dye uptake, which, as an indicator of mitochondrial function, is rapidly affected by tetracycline treatment (11). Since the effect of tubulozoles on 35S incorporation can be mimicked by colcemid, it may be a secondary phenomenon-keeping in mind that there are other as-yet-unexplained secondary effects of Colchicum alkaloids. Since we do not, as yet, have sufficient P. falciparum tubulin available for studies on interaction with tubulozoles, we have resorted to studies of pairwise interactions of compounds known to bind to tubulin with each other and with tubulozole (with respect to inhibition of malaria). Such
interactions have been extensively studied with purified mammalian tubulin (12). There are two binding sites on tubulin-a CLC site and a VBL site. Among the compounds which bind to the CLC site are CLC, colcemid, and the benzimidazoles. The benzimidazoles depress binding of CLC to tubulin (12). VBL, on the other hand, enhances the binding of CLC to tubulin by a mechanism not yet understood (13). Taxol induces tubulin to polymerize, and the binding site is believed to be different from the CLC and VBL sites (12). It inhibits both CLC and VBL binding, albeit the concentration dependency of this inhibition is rather complicated (13). There is no evidence as to the binding site of tubulozole, but this compound does have a carbamate moiety and some complex carbamates, including the benzimidazoles, do compete with CLC for binding (2, 12). This 3.2
3.2 -
c
Go N 0 3 .0
N
0 :3 .0 :3 1-
1.6
I-
2.5
5
Colcemid [PM]
10
3.2 r
r-
G
N
.0 D :3
I-
0 N 0
1.6-
0
0
0.8 -
.0 I-
W: _4M
50 do0 Mebendozole [,uM]
200
3 1.5 Vincristine [nM]
6
FIG. 4. Synergistic effect of tubulozole T with colcemid, mebendazole, and taxol and additivity with vincristine on inhibition of the in vitro growth of P. falciparum. All values represent average IC50s from three determinations.
ACTION OF TUBULOZOLES AGAINST P. FALCIPARUM
VOL. 34, 1990
TABLE 3. Parasitic protozoa surveyed for susceptibility to tubulozole and benzimidazoles (mebendazole and albendazole) in vitro and comparison with mamnmalian cells (Chinese hamster ovary cell line) Effecta of:
Organism
10 ,LM
10 pLM
trans-
cis-
tubulozole
tubulozole
Benzimidazoles
Giardia lamblia (axenic culture)
-
-+b
Trypanosoma brucei brucei (procyclic and bloodstream
+C
+C
+C
+C
-
-
Not done
-
-
Not done
-
-
Not done
Sarcocystis muris (invasion of CRFK cells, gametocytogenesis, and zygote formation)
-
-
Not done
CHO cells
-
+
+
forms) Leishmania infantum (promastigotes) Babesia bovis (erythrocytic stage) Babesia caballi (erythrocytic stage) Babesia divergens (erythrocytic stage)
a
b
_d
1533
genes and one species of transcript for P-tubulin (3, 18), one group reported to have found several transcripts (17). These findings suggest some heterogeneity of malarial tubulin species which may form different microtubule populations with different drug susceptibilities. This might also be an explanation of our results. Although our considerations suggest that the inhibition of malaria by both tubulozole isomers may be the result of an interaction of these compounds with tubulin, drug-binding studies using malarial tubulin are necessary to clarify this issue. We have extended our observations on the activity of tubulozoles to other parasitic protozoa, some of which are known to possess large amounts of tubulin. The results of a number of preliminary studies on drug inhibition suggest prominent differences both between the tubulins from these lower eucaryotes and between these tubulins as a whole and mammalian tubulins (Table 3). The effect of tubulozoles on parasitic protozoa living in nucleate cells is difficult, if not impossible, to assess, since in two cases (Theileria parva and Toxoplasma gondii, the former studied in our laboratory; the latter studied by A. Hassl and H. Aspock, University of Vienna, Vienna, Austria, unpublished results) the host cells were markedly affected by the drug. Invasion of CRFK cells in vitro by cyst merozoites of Sarcocystis muris and subsequent gametocytogenesis and zygote formation were, however, not affected by either tubulozole at 10 ,uM. Considering their diversity and divergence from mammalian tubulins, protozoal tubulins as a whole thus constitute a promising area of research toward new chemotherapeutic agents.
-, Not affected; +, affected. R. C. A. Thompson, Murdoch University, Murdoch, Australia, personal
communication. c Inhibited at a concentration higher than that needed for malaria. d T. Seebeck, University of Bern, Bern, Switzerland, personal communication.
suggests that tubulozole might bind at the CLC site, but this conclusion must await direct experimental evidence. From the considerations discussed above, we should suspect that there would be synergism between CLC and VBL site-specific binders and antagonism between taxol and both CLC and VBL binders, as well as between CLC-CLC and VBL-VBL binders. This is not, however, what our data indicate. Synergism of tubulozoles with Colchicum alkaloids, benzimidazoles, and taxol suggests interaction of tubulozoles with malarial microtubules. The antagonistic effects on the growth of P. falciparum seen between the other inhibitors alone (Table 2), however, seem to rule out a direct interaction of the two tubulozole isomers with malarial tubulin if that tubulin, in this respect, was comparable to the tubulins of higher eucaryotes. Alternatively, accessory proteins interacting with tubulin could be involved in the direct mechanism of action of the tubulozoles. A molecular or functional difference in the respective targets of colcemid and tubulozoles would help to explain the earlier onset of tubulozole inhibition during the intraerythrocytic developmental cycle of P. falciparum (4). The suggestion of some divergence between malarial tubulin and that of higher eucaryotes is supported further by the finding of a significant deviation from all other species studied of malarial a-tubulin in amino acids 41 to 46 (10) and a number of differences between the ,-tubulin amino acid sequence of P. falciparum and the amino acid sequence derived from the human M40 gene (3). Heterogeneity of P. falciparum a-tubulin, in both the genes and their transcripts, has been reported by several laboratories (10, 17). Whereas two groups reported single
ACKNOWLEDGMENTS We are indebted to H. Aspock, University of Vienna, Vienna, Austria; R. Brun, Swiss Tropical Institute, Basel, Switzerland; D. Dobbelaere, Nuclear Research Center, Karlsruhe, Federal Republic of Germany; K. T. Friedhoff, School of Veterinary Medicine, Hannover, Federal Republic of Germany; P. Kohler, University of Zurich, Zurich, Switzerland; H. Mehlhorn, University of Bochum, Bochum, Federal Republic of Germany; and their collaborators for their excellent cooperation and for kind assistance with culture material of parasites other than P. falciparum. A.D.-S. was the recipient of a postdoctoral fellowship in the special Molecular Parasitology program of the German Academic Exchange Service. This work was supported in part by funds from the Canton City of Basel and the Swiss National Fund (grant 3.049-0.87 to R.M.F.).
1.
2.
3. 4. 5.
6.
7.
LITERATURE CITED Berenbaum, M. C. 1978. A method for testing for synergy with any number of agents. J. Infect. Dis. 137:122-130. Bowdon, B. J., W. R. Waud, G. P. Wheeler, R. Hain, L. Dansby, and C. Temple, Jr. 1987. Comparison of 1,2-dihydropyrido[3,4b]pyrazines (1-deaza-7,8-dihydropteridines) with several other inhibitors of mitosis. Cancer Res. 47:1621-1626. Delves, C. J., R. G. Ridley, M. Goman, S. P. Holloway, J. E. Hyde, and J. G. Scaife. 1989. Cloning of a 1-tubulin gene from Plasmodium falciparum. Mol. Microbiol. 3:1511-1519. Dieckmann-Schuppert, A., and R. M. Franklin. 1989. Compounds binding to cytoskeletal proteins are active against Plasmodium falciparum in vitro. Cell Biol. Int. Rep. 13:411-418. Divo, A. A., T. G. Geary, and J. B. Jensen. 1985. Oxygen- and time-dependent effects of antibiotics and selected mitochondrial inhibitors on Plasmodium falciparum in culture. Antimicrob. Agents Chemother. 27:21-27. Divo, A. A., T. G. Geary, J. B. Jensen, and H. Ginsburg. 1985. The mitochondrion of Plasmodium falciparum visualized by rhodamine 123 fluorescence. J. Protozool. 32:442-446. Geary, T. G., and J. B. Jensen. 1983. Effects of antibiotics on
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Plasmodium falkiparum in vitro. Am. J. Trop. Med. Hyg. 32:221-225. Genens, G. M. A., R. M. Nuydens, R. E. Willebrords, R. M. L. Van de Veire, F. Goossens, C. H. Dragonetti, M. M. K. Mareel, and M. J. De Brabander. 1985. Effects of tubulozole on the microtubule system of cells in culture and in vivo. Cancer Res. 45:733-742. GInsbUrg, H., E. Nssani, and M. Knaglak. 1989. Alkaliniization of the food vacuole of malaria parasites by quinoline drugs and alkylamines is not correlated with their antimalarial activity. Biochem. Pharmacol. 38:2645-2654. Holloway, S. PI,, P. F. G. Sims, C. J. Delves, J. G. Scalfe, and J. E. Hyde. 1989. Isolation of a-tubulin genes from the human malaria parasite, Plasmodium falciparum: sequence analysis of a-tubulin. Mol. Microbiol. 3:1501-1510. Kiatuengfoo, K., T. Suthiphongchai, P. Prapunwattana, and Y. Yuthavong. 1989. Mitochondria as the site of action of tetracycline on Plasmodium falciparum. Mol. Biochem. Parasitol. 34:109-116. Lacey, E. i988. The role of the cytoskeletal protein, tubulin, in the mode of action and mechanism of drug resistance to benzimidazoles. Int. J. Parasitol. 18:885-936. Lacey, E., J. A. Edgar, and C. C. J. Culvenor. 1987. Interaction of phomopsin A and related compounds with purified sheep brain tubulin. Biochem. Pharmacol. 36:2133-2138. Lambros, C., and J. P. Vanderberg. 1979. Synchronization of
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Plasmodiumfalciparum erythrocytic stages in culture. J. Parasitol. 65:418-420. Noil, F. 1984. L (+)-Lactate, p. 582-588. In H. U. Bergmeyer, J. Bergmeyer, and M. Grassl (ed.), Methods of enzymatic analysis, vol. 6, 3rd ed. Verlag Chemie, Weinheim, Federal Republic of Germany. Rinderknecht, H., M. C. Geokas, P. Silverman, and B. J. Haverback. 1968. A new ultrasensitive method for the determination of proteolytic activity. Clin. Chim. Acta 21:197-203. Sen, K., and G. N. Godson. 1990. Isolation of a- and ,-tubulin genes of Plasmodium falciparum using a single oligonucleotide probe. Mol. Biochem. Parasitol. 39:173-182. Wesseling, J. G., R. Dirks, M. A. Smits, and J. G. G. Schoenmakers. 1989. Nucleotide sequence and expression of a 3tubulin gene from Plasmodium falciparum, a malarial parasite of man. Gene 83:301-309. Wilson, L., and M. Friedkin. 1966. The biochemical events of mitosis. I. Synthesis and properties of colchicine labeled with tritium in its acetyl moiety. Biochemistry 5:2463-2468. Yayon, A., and H. Ginsburg. 1983. Chloroquine inhibits the degradation of endocytic vesicles in human malaria parasites. Cell Biol. Int. Rep. 7:895. ZoIg, J. W., A. J. MacLeod, J. G. Scaife, and R. L. Beaudoin. 1984. The accumulation of lactic acid and its influence on the growth of Plasmodium falciparum in synchronized cultures. In Vitro (Rockville) 20:205-215.
Vol. 34, No. 8
0066-4804/90/081529-06$02.00/0 Copyright © 1990, American Society for Microbiology
Mode of Action of Tubulozoles against Plasmodium falciparum In Vitro ANGELA DIECKMANN-SCHUPPERTt* AND RICHARD M. FRANKLIN Department of Structural Biology, Biocenter, University of Basel, CH4056 Basel, Switzerland Received 4 January 1990/Accepted 24 May 1990 The mode of action of the tubulozole isomers, recently recognized as a new class of potential antimalarial agents, was investigated. Whereas neither glycolysis, protease activity, or nucleic acid biosynthesis was primarily affected, protein biosynthesis decreased soon after addition of the drug. Inhibitors of protein biosynthesis, however, did not show synergistic activity with tubulozole. Colcemid, on the other hand, had an effect on protein synthesis similar to that seen with the tubulozoles. Furthermore, combinations of the tubulozole isomers with compounds known to interact with tubulin inhibited malaria in a synergistic or antagonistic fashion. Therefore, the inhibition might be elicited by interaction with tubulin or some other component of the microtubules. This is remarkable insofar as only one of the tubulozole isomers affects mammalian cells by binding to tubulin. New antimalarial agents are urgently required since a suitable vaccine has not been developed and drug resistance is an ever-increasing problem. Tubulozoles were recently identified as a new class of potential antimalarial agents (4). The aim of the present study was to investigate the mechanism of action of tubulozoles in inhibiting the malarial parasite. Tubulozoles represent a class of chemical compounds which are unique in that in mammalian cells only the C (cis) isomer inhibits tubulin polymerization, whereas the T (trans) isomer does not (8). Tubulozole C-treated mammalian cells experience a mitotic and transport block. This primary cytostatic effect becomes cytocidal after sufficiently long exposure. Since both isomers show the same dose dependency in the inhibition of intraerythrocytic growth of Plasmodiumfalciparum, their mode of action had to be carefully reconsidered, not excluding a priori possibilities other than an interaction with tubulin. We therefore investigated the effect of the tubulozoles on lactate production, activity of parasitic proteases, nucleic acid synthesis, and protein synthesis to define, in broad terms, an area of metabolism which might be a target of the drugs. MATERIALS AND METHODS Parasites. P. falciparum Ro58, highly resistant to chloroquine and pyrimethamine-sulfadoxine (Fansidar), was cultivated in RPMI 1640 with 10% horse serum, as reported previously (4). The multiplication of parasites was monitored by evaluation of Giemsa-stained thin smears. Growth of the parasites was synchronized to +4 h by repeated sorbitol treatment (14). Sources of inhibitors. Taxol was kindly provided by M. Suffness, National Cancer Institute, Bethesda, Md.; mebendazole and the tubulozole isomers were provided by H. Vanden Bossche, Janssen Research Foundation, Beerse, Belgium; and pyrimethamine was provided by H. Matile, F. Hoffmann-La Roche & Co., Basel, Switzerland. Chloramphenicol, tetracycline, and nalidixic acid were purchased * Corresponding author. t Present address: Zentrum fur Hygiene und Medizinische Mikrobiologie, Philipps-Universitat Marburg, Robert-Koch-Strasse 17, D-3550 Marburg, Federal Republic of Germany.
from Boehringer GmbH; vincristine was purchased from Eli Lilly; colchicine (CLC) and rifampin were purchased from Serva; colcemid and vinblastine (VBL) were purchased from Sigma; albendazole was purchased from SmithKline; and dactinomycin (actinomycin D) and cycloheximide were purchased from Calbiochem-Behring. Lumicolchicine was prepared from CLC by UV irradiation as previously described (19). Sterile stock solutions were prepared in dimethyl sulfoxide or saline as appropriate and kept at -20°C. Working dilutions were prepared in culture medium immediately before use. Lactate determination. Lactate was assayed in the supernatants of 3-ml cultures by the enzymatic procedure employing lactate dehydrogenase and glutamate-pyruvate transaminase (15). Briefly, culture supernatants were deproteinized by the addition of perchloric acid and neutralized with potassium hydroxide. Aliquots were added in a cuvette to glutamate buffer containing 24 mM NAD and 80 U of glutamate-pyruvate transaminase per ml. The reaction was started by the addition of 550 U of lactate dehydrogenase per ml, and the increase in A339 was monitored. Protease assay. Proteolytic activity in a crude P. falciparum lysate, prepared as described below, was estimated by spectrophotometric measurement of dye release from Hide Powder Azure (Calbiochem-Behring) (16) in the pH range between 4 and 9. Briefly, 1 ml of a 10-mg/ml substrate suspension was incubated with 0.1 ml of lysate at 37°C for 30 min. The activity was estimated after centrifugation by the amount of blue dye released into the supernatant, the resultant increase in A595 being measured. [3H]hypoxanthine and [35S]methionine incorporation. Cultures (2-ml) at 5% hematocrit and 8% parasitemia (32 + 4 h-old trophozoites) were exposed to the appropriate drug and labeled with 30-min pulses of 10 ,uCi of [3H]hypoxanthine (specific activity, 3.8 to 8.6 Ci/mmol; Amersham) or [35S]methionine (specific activity, >1,000 Ci/mmol; Amersham) per ml in hypoxanthine- or methionine-free RPMI 1640. After the cultures were washed three times with 20 ml of ice-cold phosphate-buffered saline, the pellet was lysed by the addition of 500 ,ul of distilled water, followed by ultrasonication. Precipitation of nucleic acids and proteins was done by the addition of 1 ml of 20o ice-cold trichloroacetic acid. The insoluble material was washed three times with 8% 1529
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ANTIMICROB. AGENTS CHEMOTHER.
DIECKMANN-SCHUPPERT AND FRANKLIN
trichloroacetic acid and collected on glass fiber filters, and the radioactivity was determined by liquid scintillation counting. Drug interaction studies. The dose-response relationship of each of the compounds under study was evaluated after exposing microtiter plate cultures to serial dilutions of the compounds. The final volume in each well was 200 ,u. Parasitemia was determined on Giemsa-stained smears after 48 h of exposure to the drug. Untreated cultures served as controls. Fifty percent inhibitory concentrations (IC50s) were determined graphically. Paired-drug interaction studies were performed by checkerboard titration, following the same protocol as for the study of single drugs. In this case, each drug was tested at eight concentrations, yielding eight times eight wells per experiment. IC50s for single rows and lines were obtained as described above. Isoboles (lines connecting points of the same extent of inhibition, i.e., IC50) were constructed graphically. If any two drugs, A and B, act additively, the isobole will be a straight line connecting the IC50 points on the axes. In the case of synergism a concave line is obtained, and in the case of antagonism a convex one is obtained (1). The extent of interaction is given by the sum of the fractional inhibition constants (IFIC), which is defined (1) as follows: IC50 of A in combination + IC50 of B in combination XFIC =
IC50 of A alone
IC50 of B alone
If A and B act additively, the IFIC will be 1 because their respective IC50s are not affected by mixing A and B. If A and B act synergistically, the :FIC will be less than 1; if they act antagonistically, it will be greater than 1 (1). RESULTS Several metabolic parameters of intraerythrocytic P. falciparum were investigated to trace the effects of tubulozoles. Each tubulozole isomer was tested separately in all assays. The results were always identical, irrespective of the isomer. We therefore use the term tubulozoles whenever both isomers are meant. Lactate accumulates in the culture supernatant and is an indicator of the glycolytic activity of the parasite and the erythrocyte. Over 4 h the rate of lactate production, amounting to 1.3 mM/h, was not affected significantly by the presence of 10 p.M tubulozole (Fig. 1). The amount of lactate produced agreed with values reported by others (21). The proteolytic activity of crude malarial lysates was not abolished by the addition of 10 p.M tubulozole. Typically, absorbance values ranging between 0.5 and 0.8 optical density units were obtained, irrespective of the presence of tubulozole. The incorporation of [3H]hypoxanthine, indicative of nucleic acid biosynthesis, reached a plateau after more than 3 h (Fig. 2). This, however, is longer than the minimal exposure time required for killing of the parasite by 10 p.M tubulozole (4) and was therefore regarded as an unspecific or secondary effect. Correspondingly, no inhibitor of nucleic acid biosynthesis (rifampin, nalidixic acid, dactinomycin, or pyrimethamine) displayed more than additivity when combined with tubulozoles (IFIC = 1) (Table 1). [35S]methionine incorporation, indicating protein biosynthesis, was inhibited by either tubulozole isomer at 10 p.M from the onset of drug treatment (Fig. 3A). Drug interaction studies, however, again revealed mere additivity between protein biosynthesis inhibitors (tetracycline, chlorampheni-
E2
7~
c
6
o
75
0/
._
3
32
4 h
FIG. 1. Effect of 10 I1M tubulozole T on lactate production in erythrocytic cultures of P. falciparum. The inhibitor was added at time zero. *, Control culture; O, treated culture; O, uninfected erythrocytic culture. All values are averages from three experiments.
col, cycloheximide) and the tubulozoles (Table 1). Furthermore, neither tubulozole isomer inhibited the staining of P. falciparum mitochondria with rhodamine 123 (6) during 6 h of drug exposure, whereas the above-mentioned 70S ribosomal inhibitors did. The antimicrotubular agent colcemid had an effect on 30-
20E 0
10-
1
2
3
4h
FIG. 2. Effect of 10 p.M tubulozole T on synthesis of nucleic acids in erythrocytic cultures of P. falciparum. The inhibitor was added at time zero. 0, Control culture; 0, treated culture. All values are averages from three experiments.
VOL. 34, 1990
ACTION OF TUBULOZOLES AGAINST P. FALCIPARUM
TABLE 1. Inhibitors of protein and nucleic acid biosynthesis tested for interaction with tubulozoles in P. falciparum in vitro Compounda IC50 0.3 FM Rifampin ........... Nalidixic acid ........... 30 mM 50 pM Dactinomycin ........... 26 ,uM Pyrimethamine ........... 40 ,uM Tetracycline ........... 40 ,uM Chloramphenicol ........... 4 nM Cycloheximide ........... a
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mebendazole, albendazole, and taxol (Table 2, Fig. 4). The antimalarial activities of taxol and CLC are reported here for the first time, the IC50s for strain Ro58 being 1 and 10 ,uM, respectively. Lumicolchicine, a derivative of CLC which does not bind to tubulin, was inactive at corresponding concentrations. Only additivity could be detected between tubulozoles and Vinca alkaloids (Table 2). Interaction between any other pair of the established tubulin-binding inhibitors tested was, however, markedly antagonistic in the malarial system, as summarized in Table 2.
All the compounds displayed additivity when combined with tubulozoles.
[35S]methionine incorporation (Fiig. 3B) similar to that of the tubulozoles (Fig. 3A) and, moireover, its effect on [3H] hypoxanthine incorporation was comparable to that of the tubulozoles (data not shown). The antimalarial activity of a range of antimicrotubular agents was reported by us ea rlier (4). Drug interaction studies using these agents and tubulozoles demonstrated pronounced synergism (IFIC = 0.25) between both tubulozole isomers and each of the f"ollowing: colcemid, CLC, 1501
A
* * E
*
100
a.
0
......-o
50 -
°0 10-
, I
, 3
2
,
, 4 h
* 50
B *
40E a.
30 -
7/
~
cfo
0
20
,0
7'
X-1/0
10
, , , 1 2 4 h 3 FIG. 3. Effects of 10 ,uM tubulozo)le T (A) and 100 ,uM colcemid (B) on incorporation of [35S]methioniine into protein in erythrocytic cultures of P. falciparum. The inhibiltor was added at time zero. , Control culture; 0, treated culture. All values are averages from ,
three experiments.
DISCUSSION
All mammalian cell lines studied so far are affected only by the cis isomer of tubulozole. In contrast, malarial parasites are inhibited to the same extent by both the cis and trans isomers. The wide phylogenetic distance between protozoa and higher eucaryotes prompted us to reevaluate the mode of action of the tubulozole isomers in malaria. Their effects on several biochemical parameters were measured, and drug interaction studies employing inhibitors of the aforementioned metabolic pathways, as well as inhibitors of microtubule formation, were done. The fact that cis- and transtubulozoles behaved identically in all the biochemical and drug interaction studies suggests that they, in contrast to the situation in mammalian cells, have a common molecular target in malaria. Glycolysis, the main pathway for energy production in intraerythrocytic P. falciparum, was not affected by tubulozoles, as indicated by unaltered lactate production. This indicates that glycolysis is not the molecular target of tubulozole in malaria. A spectrum of different proteases serves to degrade the erythrocytic stroma, a process which is essential for the survival of the malarial parasite. The antimalarial drug chloroquine is believed by many researchers to inhibit, although possibly in an indirect way, proteolytic breakdown in the plasmodial food vacuole (20), but this concept was recently questioned (9). In our assay system chloroquine inhibited the proteolytic activity only at concentrations in the millimolar range (data not shown). Both tubulozole isomers failed to inhibit malarial proteases in the cell-free assay at a concentration of 10 ,uM. These results are consistent with the lack of interaction between chloroquine and tubulozole in drug-interaction studies. We therefore conclude that the tubulozoles do not interfere with malarial protease activity. Tubulozoles do not interfere specifically, i.e., within the first 3 h (4), with nucleic acid biosynthesis, since we found neither a marked and early reduction in hypoxanthine uptake nor an interaction with rifampin, nalidixic acid, dactinomycin, or pyrimethamine, all being either direct or indirect inhibitors of nucleic acid biosynthesis (7). Furthermore, in vitro translation of malarial RNA by a rabbit reticulocyte lysate is not affected by either tubulozole isomer (C. Sartorius and R. M. Franklin, unpublished results), suggesting that direct interaction with malarial mRNA does not take
place. Malarial protein biosynthesis in either the mitochondrion or the cytoplasm is not primarily affected by tubulozoles, since synergism with tetracycline, chloramphenicol, or cycloheximide cannot be demonstrated. These compounds are well-established inhibitors of mitochondrial protein synthesis, in the case of tetracycline and chloramphenicol, or cytoplasmic protein synthesis, in the case of cycloheximide,
1532
ANTIMICROB. AGENTS CHEMOTHER.
DIECKMANN-SCHUPPERT AND FRANKLIN
TABLE 2. Interaction of tubulozoles and various established tubulin-binding substances as studied by their effect upon the growth of P. falciparum in vitro
Tubulozoles
3
CLC or colcemid
IFM
Colchicum alkaloids
Benzimidazoles
Synergistic (0.25)
Synergistic (0.25)
Additive (1)
Synergistic (0.25)
Antagonistic (>4)
Antagonistic (>4)
Antagonistic (2)
Antagonistic (>4)
Antagonistic (2)
10 P,M
Mebendazole Albendazole
200 ,uM 30 pLM
Vincristine VBL
6 nM 100 nM
a
Interactiona
IC50
Compound(s)
Vinca alkaloids
Taxol
Antagonistic (2)
Values given in parentheses are IFICs.
and are also known to inhibit malarial growth (5). The hypothesis that protein biosynthesis is not the primary target of tubulozoles is further supported by the lack of tubulozole inhibition of rhodamine dye uptake, which, as an indicator of mitochondrial function, is rapidly affected by tetracycline treatment (11). Since the effect of tubulozoles on 35S incorporation can be mimicked by colcemid, it may be a secondary phenomenon-keeping in mind that there are other as-yet-unexplained secondary effects of Colchicum alkaloids. Since we do not, as yet, have sufficient P. falciparum tubulin available for studies on interaction with tubulozoles, we have resorted to studies of pairwise interactions of compounds known to bind to tubulin with each other and with tubulozole (with respect to inhibition of malaria). Such
interactions have been extensively studied with purified mammalian tubulin (12). There are two binding sites on tubulin-a CLC site and a VBL site. Among the compounds which bind to the CLC site are CLC, colcemid, and the benzimidazoles. The benzimidazoles depress binding of CLC to tubulin (12). VBL, on the other hand, enhances the binding of CLC to tubulin by a mechanism not yet understood (13). Taxol induces tubulin to polymerize, and the binding site is believed to be different from the CLC and VBL sites (12). It inhibits both CLC and VBL binding, albeit the concentration dependency of this inhibition is rather complicated (13). There is no evidence as to the binding site of tubulozole, but this compound does have a carbamate moiety and some complex carbamates, including the benzimidazoles, do compete with CLC for binding (2, 12). This 3.2
3.2 -
c
Go N 0 3 .0
N
0 :3 .0 :3 1-
1.6
I-
2.5
5
Colcemid [PM]
10
3.2 r
r-
G
N
.0 D :3
I-
0 N 0
1.6-
0
0
0.8 -
.0 I-
W: _4M
50 do0 Mebendozole [,uM]
200
3 1.5 Vincristine [nM]
6
FIG. 4. Synergistic effect of tubulozole T with colcemid, mebendazole, and taxol and additivity with vincristine on inhibition of the in vitro growth of P. falciparum. All values represent average IC50s from three determinations.
ACTION OF TUBULOZOLES AGAINST P. FALCIPARUM
VOL. 34, 1990
TABLE 3. Parasitic protozoa surveyed for susceptibility to tubulozole and benzimidazoles (mebendazole and albendazole) in vitro and comparison with mamnmalian cells (Chinese hamster ovary cell line) Effecta of:
Organism
10 ,LM
10 pLM
trans-
cis-
tubulozole
tubulozole
Benzimidazoles
Giardia lamblia (axenic culture)
-
-+b
Trypanosoma brucei brucei (procyclic and bloodstream
+C
+C
+C
+C
-
-
Not done
-
-
Not done
-
-
Not done
Sarcocystis muris (invasion of CRFK cells, gametocytogenesis, and zygote formation)
-
-
Not done
CHO cells
-
+
+
forms) Leishmania infantum (promastigotes) Babesia bovis (erythrocytic stage) Babesia caballi (erythrocytic stage) Babesia divergens (erythrocytic stage)
a
b
_d
1533
genes and one species of transcript for P-tubulin (3, 18), one group reported to have found several transcripts (17). These findings suggest some heterogeneity of malarial tubulin species which may form different microtubule populations with different drug susceptibilities. This might also be an explanation of our results. Although our considerations suggest that the inhibition of malaria by both tubulozole isomers may be the result of an interaction of these compounds with tubulin, drug-binding studies using malarial tubulin are necessary to clarify this issue. We have extended our observations on the activity of tubulozoles to other parasitic protozoa, some of which are known to possess large amounts of tubulin. The results of a number of preliminary studies on drug inhibition suggest prominent differences both between the tubulins from these lower eucaryotes and between these tubulins as a whole and mammalian tubulins (Table 3). The effect of tubulozoles on parasitic protozoa living in nucleate cells is difficult, if not impossible, to assess, since in two cases (Theileria parva and Toxoplasma gondii, the former studied in our laboratory; the latter studied by A. Hassl and H. Aspock, University of Vienna, Vienna, Austria, unpublished results) the host cells were markedly affected by the drug. Invasion of CRFK cells in vitro by cyst merozoites of Sarcocystis muris and subsequent gametocytogenesis and zygote formation were, however, not affected by either tubulozole at 10 ,uM. Considering their diversity and divergence from mammalian tubulins, protozoal tubulins as a whole thus constitute a promising area of research toward new chemotherapeutic agents.
-, Not affected; +, affected. R. C. A. Thompson, Murdoch University, Murdoch, Australia, personal
communication. c Inhibited at a concentration higher than that needed for malaria. d T. Seebeck, University of Bern, Bern, Switzerland, personal communication.
suggests that tubulozole might bind at the CLC site, but this conclusion must await direct experimental evidence. From the considerations discussed above, we should suspect that there would be synergism between CLC and VBL site-specific binders and antagonism between taxol and both CLC and VBL binders, as well as between CLC-CLC and VBL-VBL binders. This is not, however, what our data indicate. Synergism of tubulozoles with Colchicum alkaloids, benzimidazoles, and taxol suggests interaction of tubulozoles with malarial microtubules. The antagonistic effects on the growth of P. falciparum seen between the other inhibitors alone (Table 2), however, seem to rule out a direct interaction of the two tubulozole isomers with malarial tubulin if that tubulin, in this respect, was comparable to the tubulins of higher eucaryotes. Alternatively, accessory proteins interacting with tubulin could be involved in the direct mechanism of action of the tubulozoles. A molecular or functional difference in the respective targets of colcemid and tubulozoles would help to explain the earlier onset of tubulozole inhibition during the intraerythrocytic developmental cycle of P. falciparum (4). The suggestion of some divergence between malarial tubulin and that of higher eucaryotes is supported further by the finding of a significant deviation from all other species studied of malarial a-tubulin in amino acids 41 to 46 (10) and a number of differences between the ,-tubulin amino acid sequence of P. falciparum and the amino acid sequence derived from the human M40 gene (3). Heterogeneity of P. falciparum a-tubulin, in both the genes and their transcripts, has been reported by several laboratories (10, 17). Whereas two groups reported single
ACKNOWLEDGMENTS We are indebted to H. Aspock, University of Vienna, Vienna, Austria; R. Brun, Swiss Tropical Institute, Basel, Switzerland; D. Dobbelaere, Nuclear Research Center, Karlsruhe, Federal Republic of Germany; K. T. Friedhoff, School of Veterinary Medicine, Hannover, Federal Republic of Germany; P. Kohler, University of Zurich, Zurich, Switzerland; H. Mehlhorn, University of Bochum, Bochum, Federal Republic of Germany; and their collaborators for their excellent cooperation and for kind assistance with culture material of parasites other than P. falciparum. A.D.-S. was the recipient of a postdoctoral fellowship in the special Molecular Parasitology program of the German Academic Exchange Service. This work was supported in part by funds from the Canton City of Basel and the Swiss National Fund (grant 3.049-0.87 to R.M.F.).
1.
2.
3. 4. 5.
6.
7.
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