The Topoisomerases of Protozoan Parasites

Reviews

The Topoisomerases of Protozoan Parasites S.J. Cheesman Protozoan parasites are responsible for a wide range of debilitating and fatal diseases that are proving notoriously difficult to treat. Many of the standard chemotherapies in use today are expensive, have toxic side effects and, in some cases have marginal efficacy because of the emergence of drug-resistant parasites. In the search for more effective treatments, protozoan topoisomerases are now being considered as potential drug targets, building on the clinical success of anticancer and antibacterial agents that target human and bacterial topoisomerases. In this review, Sandra Cheesman explores progress in this relatively new but potentially important Þeld of research. Topoisomerases are enzymes that resolve the complex topological situations arising from DNA metabolism. Type I enzymes function as monomeric proteins that catalyse the introduction of transient single-stranded breaks into a DNA duplex and alter the helical pitch of the DNA to resolve the torsional stresses associated with over- or underwinding of the template during processes such as DNA replication and transcription. Type I topoisomerases have been subdivided into two distinct families based on their reaction mechanisms and amino acid (aa) sequences. Type IA enzymes are grouped together on the basis that they bind covalently to the 59 end, whereas type IB enzymes bind covalently to the 39 end of the broken DNA strand. Members of the IA family include topoisomerases I and III of Escherichia coli, archaebacterial and eukaryotic topoisomerase III and reverse gyrase. Members of the IB family include eukaryotic and vaccinia topoisomerase I and archaebacterial topoisomerase V. Type II enzymes function as homodimeric (eukaryotic) or tetrameric (bacterial) proteins that introduce transient double-stranded breaks into the double helix, followed by transport of another intact duplex through the cleavage gap thus formed. These enzymes are able to relax, catenate/decatenate, knot/unknot or introduce supercoils (gyrase) into DNA substrates at the expense of ATP. Examples of type II enzymes include eukaryotic topoisomerase II (which in mammals encompasses two isoforms called a and b) and the bacterial enzymes DNA gyrase and topoisomerase IV. As a consequence of their differing mechanistic properties, topoisomerases exhibit functional divergence in the type of reaction they catalyse (Fig. 1), the biological roles they perform and their susceptibility to pharmacological agents. Topoisomerase function is required for nucleic acid synthesis, genome stability, chromosome condensation/decondensation and segregation of replicated chromosomes. Topoisomerase genes Human topoisomerase I has provisionally been divided into four domains based on its physical Sandra Cheesman is at the Institute of Cell and Molecular Biology, the University of Edinburgh, Darwin Building, King’s Buildings, Mayfield Road, Edinburgh, UK EH9 3JR. Tel: +44 131 650 5395, Fax: +44 131 668 3870, e-mail: [email protected] Parasitology Today, vol. 16, no. 7, 2000

properties, sensitivity to limited proteolysis, aa sequence alignments with other eukaryotic homologues and fragment complementation analysis. The N-terminal domain is species divergent, appears to be dispensable for activity and contains putative nuclear localization signals. The core domain spans aas 198Ð651 and is implicated in DNA binding and catalysis. Residues 697Ð765 form the C-terminal domain, which contains the activesite Tyr Ð the residue through which the protein becomes linked to the DNA when one strand of the duplex is cut by the enzyme. The core and the C-terminal domains are connected to a protease-sensitive region called the linker (residues ~652Ð696), which might function in DNA binding. Mutations that confer resistance to the eukaryotic topoisomerase I poison, camptothecin, have been mapped to regions of the core and linker domains1. Two genes encoding type IB topoisomerase have been isolated from protozoan sources. The sequence of Plasmodium falciparum TOP1 was published in 1995 (Ref. 2). The 2520 bp open reading frame (ORF) encodes a protein of 839 aas, which is slightly larger than the human enzyme. A comparison of the Plasmodium and human sequences show that they share 42% sequence identity and that the N-terminal domain probably encompasses residues 1Ð134 in the malarial homologue. Two stretches of non-repetitive aa insertions are predicted to fall within the core domain. The Þrst addition is 29Ð34 residues and the second is 79 residues long. However, the functional signiÞcance of these insertions remains an open question. More recently, a second gene that resembles those encoding topoisomerase I has been characterized from Leishmania donovani3. The gene has an ORF of 1905 bp, encoding a protein of only 635 residues. A large part of the protein shares considerable homology with the central core domain of other type IB topoisomerases, but intriguingly, there appears to be no active-site Tyr, the catalytic residue associated with strand breakage. The possibility that the sequence might be a pseudogene has been excluded. Eukaryotic topoisomerases II can be divided into three distinct domains based on homology to DNA gyrase (Fig. 2). The N-terminal domain is highly conserved between species. This conservation decreases markedly at the C-terminal domain, which in eukaryotes is believed to contain regulatory elements such as nuclear localization signals, phosphorylation sites and sequences that might direct dimerization or facilitate interactions with other proteins. Mutations that confer resistance to topoisomerase II poisons have been mapped to all three domains. Several type II topoisomerase genes have been isolated from protozoan sources. The Trypanosoma brucei and T. cruzi TOP2 genes encode the smallest of these enzymes, at 1221 aas, and 1232 aas per subunit, respectively4,5. Plasmodium falciparum topoisomerase II is a slightly larger enzyme composed of 1397 aas per subunit6. In addition, part of the TOP2 gene of Cryptosporidium parvum has also been isolated7. Residues that are reported to be important for speciÞc aspects of topoisomerase II function appear to be conserved in these proteins, and in line with higher eukaryotes, the

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Supercoiled

Relaxed

Catenated

Decatenated

Knotted

Unknotted Parasitology Today

Fig. 1. Schematic representation depicting some of the reactions catalysed by topoisomerases. Reactions involving circular covalently closed duplex DNA molecules are shown here. Relaxation of supercoiled substrates can be catalysed by type I enzymes, but they are unable to knot or unknot, catenate or decatenate these molecules unless they contain a single-stranded region or nick. Reactions that introduce negative supercoiling are catalysed by the bacterial type II topoisomerase, DNA gyrase. Catenation and decatenation, knotting and unknotting (which require breakage of both DNA strands) are catalysed by type II topoisomerases, such as bacterial topoisomerase IV or eukaryotic topoisomerase II.

greatest degree of sequence conservation occurs over the N-terminal region, whereas the C-terminus is most divergent. Furthermore, the Plasmodium homologue contains two unique polyasparagine-rich insertions that appear to be present in the processed message and therefore might be retained in the protein6. These Asnrich insertions are a puzzle: their existence in certain Plasmodium antigens present on the surface of the parasite has prompted the suggestion that they might play a role in immune evasion8, but it seems unlikely that they would serve this function in a topoisomerase. Amino acid sequence comparisons highlight speciÞc differences between human and protozoan topoisomerases. Human topoisomerase II a is signiÞcantly larger than the parasite homologues, at 1530 aas per subunit, and has a small unique N-terminal and larger C-terminal extension. Trypanosomatids share the lowest level of identity with human topoisomerase a, at around 30%; this value rises to around 45% for the P. falciparum homologue.

Patterns of gene expression In humans, levels of the nuclear topoisomerases I and II b do not appear to vary much throughout the cell cycle, whereas topoisomerase II a levels peak in proliferating cells. Post-translational modiÞcations that affect the stability of these proteins and inßuence enzyme activity have been reported, including polyADP ribosylation and phosphorylation9,10. Is anything known about the expression patterns of protozoan Amino acid topoisomerase genes throughout GyrB residues their life cycles? Although few studies have been undertaken, some inG 804 E. coli GyrA teresting Þndings are now beginCrystal structure Y 875 ning to emerge. SpeciÞc octomeric Crystal structure sequences have been identiÞed G Y S. cerevisiae 1429 within the 59 untranslated region of Crystal structure the TOP2 gene that regulate transcript cycling in Crithidia fascicuG 1530 Y Human a lata11, and nuclear extracts have been shown to contain a factor that G Human b Y 1621 binds to the 59 untranslated region periodically throughout the cell cycle, in parallel with mRNA and Y P. falciparum G 1397 protein accumulation12. Regulation of gene expression in Y G 1221 T. brucei the case of the P. falciparum TOP2 gene appears to rely on post-transcriptional control mechanisms. It N-terminal G Y has been established that the proextension ATPase DNA breakage/ C-terminal moter is active at a low level in the domain reunion domain domain stage that follows invasion of the Parasitology Today red blood cell (RBC) (ring), but increases in activity as the parasite Fig. 2. Schematic alignment of the domain structures of Escherichia coli DNA gyrase and enters the later stages of intrarepresentative eukaryotic type II topoisomerases from Saccharomyces cerevisiae, human, erythrocytic growth (trophozoites Plasmodium falciparum and Trypanosoma brucei. The eukaryotic enzymes comprise two identical subunits. DNA gyrase comprises two subunits of GyrA and two subunits of and schizonts). The accumulation of GyrB. Regions of the individual proteins for which crystal structures are available are mRNA peaks in the trophozoite but indicated45–47. Alignments are based on the positions of the known and postulated disappears completely at schizoactive-site tyrosines (Y) for each species. The relative positions of the ATPase active-site gony. Although very low levels of glycines (G) are indicated. Eukaryotic topoisomerases II have a three-domain structure: protein are present in rings, the the N-terminal region (shown here in white), which is associated with ATPase activity; highest levels are observed in trothe central region (light grey), which is associated with DNA breakage and reunion; and phozoite and schizont stages, and in the C-terminal domain (dark grey), which is species divergent. Short N- and C-terminal similar amounts13. These Þndings extensions or insertions are shown in black. contrast with similar studies with 278

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Reviews the P. falciparum TOP1 gene, whose promoter activity is upregulated in the trophozoite/schizont stages, in parallel with mRNA and protein accumulation14 (Fig. 3). In T. cruzi, although topoisomerase II is found predominantly in the replicative epimastigote stage, transcript levels are similar in both replicative- and non-replicative-stage parasites15. Although there does not appear to be any direct evidence to support post-translational modiÞcation of protozoal topoisomerases, indirect evidence suggests that this mechanism might operate in P. falciparum. Extracts derived from populations of parasites synchronized in the three major blood stages were used in standard decatenation (topoisomerase II) or relaxation (topoisomerase I) activity tests. In both cases, a marked increase in enzyme activity occurs in schizont stages compared with trophozoites, even though levels of the protein were comparable for each stage13,14. In addition, in the case of Plasmodium topoisomerase II, a triplet of bands was recognized by speciÞc antisera, which might be indicative of differential phosphorylation states of the protein, as has been reported in other species16.

Topoisomerases I and II

Topoisomerases I and II

Promoter activity - ++ mRNA + +/ Protein +/ - +/ Enzyme activity +/ - +/ -

Topoisomerase poison?

Ring

Schizont

Topoisomerases I and II mRNA Protein Enzyme activity

+++++ +++++

Merozoites Parasitology Today

Fig. 3. Schematic representation of malaria parasites multiplying within the red blood cells (RBCs) of the human host; only the asexual stages of parasite growth are shown. The cycle starts as infective merozoites released from the liver enter the RBCs. After a period of growth and differentiation, the ring form develops into a trophozoite, which matures to become a segmented schizont. The synthesis of topoisomerases I and II is highest during the trophozoite and schizont stages, where DNA replication occurs. In the schizont stage, where nuclear division takes place, the activity of both enzymes peak. Promoter activity for ring stages and a mixed population of trophozoites and schizont stages were measured by nuclear run-on. Levels of mRNA/protein or promoter/ enzyme activity are depicted by plus and minus signs, which range from zero (2) or a very low level (1/2) to high levels (11111). The intraerythrocytic stages that are likely to be most susceptible to topoisomerase poisons are indicated.

Enzyme activities and drug targets Type I and II activities have been isolated from several protozoan parasites. Type I enzymes have been puriÞed from L. donovani17,18, T. cruzi19 and Plasmodium bergei20; type II enzymes from P. bergei20, P. falciparum21, L. donovani22,23, Giardia lamblia24 and T. cruzi25. Generally speaking, for the few parameters that have been measured, these activities appear to resemble their more extensively characterized yeast and human homologues, at least in terms of their requirements for highenergy cofactors and divalent cations, but there are exceptions. The L. donovani type I enzyme resembles bacterial topoisomerase I in its requirement for divalent cations and inability to relax positively supercoiled DNA18, while remarkably, the T. cruzi and L. donovani type II enzymes are both reported to be ATP independent25,23. The reaction cycles of the type I and II enzymes are susceptible to various catalytic inhibitors and poisons26. Poisons can be classiÞed as agents that act by stabilizing an intermediate in the reaction cycle called the cleavable complex. This is normally a transient event, and is tolerated by the cell when present at normal physiological levels. However, in the presence of a topoisomerase poison, the cleavable complex is stabilized and the enzyme becomes trapped by the phosphotyrosine bond(s) and is unable to dissociate from the DNA duplex. This results in single- or doublestranded breaks, which are believed to cause fragmentation of the genome and cell death when DNA replication or transcription are attempted. The reaction cycles of these enzymes can be aborted by topoisomerase poisons such as camptothecin, which speciÞcally targets Parasitology Today, vol. 16, no. 7, 2000

Trophozoite

Topoisomerase poison?

Promoter activity* +++++ mRNA +++++ Protein +++++ Enzyme activity +++

eukaryotic topoisomerase I, by broad spectrum ATPase inhibitors like novobiocin, which is a catalytic inhibitor targeting bacterial topoisomerase II, or other molecules that can intercalate, distort or bind DNA. Topoisomerases as drug targets in protozoan parasites For protozoan topoisomerases to be targeted selectively by speciÞc agents, sufficient differences should exist between them and their human homologues to enable them to be pharmacologically distinguishable. Because inhibition of parasite growth is also related to the amount of enzyme available for the formation of DNAÐenzyme complexes, the pattern of expression, accumulation and activity are also important considerations. In species such as the trypanosomes and Leishmania, there might be another target not found in humans. This is a form of mitochondrial DNA called kinetoplast DNA, which comprises a huge network of topologically interlocking circles that can be divided into two distinct types. Maxicircles are analogous to the mitochondrial DNA of higher eukaryotes and number around 50 copies, whereas the more numerous minicircles are present at 5000Ð10 000 copies. Protozoan apicomplexan parasites such as Plasmodium and Toxoplasma also contain a unique type of DNA of 27Ð35 kb, called the plastid, which is believed to have arisen from endosymbiosis of a cyanobacterial-like prokaryote. Replication of these unique and unusual DNAs will require the presence of topoisomerase functions, providing further justiÞcation to pursue topoisomerases as drug targets. The ideal antiparasite topoisomerase agent should be speciÞc in its action against the parasite enzyme. 279

Reviews The traditional approach to testing this speciÞcity is to compare the sensitivities of the puriÞed enzymes of host and parasite to potential agents. However, obtaining sufficient amounts of the puriÞed enzymes from mammalian cells is expensive and time-consuming, and these problems are greatly increased for cells as small and expensive to culture as, for example, malaria parasites. The use of recombinant enzyme overexpressed in a more amenable system is one way out of this problem, but can also present its own difficulties. To our knowledge, only the gene encoding topoisomerase II of P. falciparum has been expressed in its fulllength form (in insect cells infected with baculovirus; S. Cheesman, unpublished), but the amounts of enzyme synthesized are small. Furthermore, separation of the recombinant and endogenous activities has proved very difficult. Another set of problems relates to the stability of these enzymes. Topoisomerase I undergoes rapid N-terminal degradation, yielding a fragment that is more catalytically active through loss of its regulatory domain. Topoisomerase II is also subject to rapid proteolysis of its regulatory domain, the C-terminus. For these reasons, alternative approaches to the screening of potential topoisomerase poisons that do not require protein isolation must also be developed. The most obvious of these rely on the production of host and parasite enzymes in parallel, in a drug permeable strain of yeast, which also carries conditional mutations that inactivate its own topoisomerase activities. Such a system would provide an inexpensive method to screen for compounds that are speciÞc for the parasite topoisomerase, but do not affect the human enzyme. Vast numbers of compounds have been synthesized as potential topoisomerase inhibitors, but have proved to be ineffective against human topoisomerases. These could be used as starting compounds in preliminary screens for antiprotozoal activity. However, classic eukaryotic topoisomerase poisons are often used as leads. For example, one of the Þrst groups of compounds reported to possess antitumour potential via topoisomerase II poisoning was the 9-anilinoacridines. High toxicity limited their immediate application, but they were used as lead compounds in the search for safer alternatives. The relative ease with which acridines can be chemically modiÞed led to the discovery of m-AMSA, a DNA intercalator and clinically relevant derivative with activity against leukaemia and malignant lymphoma27. In recent years, many anilinoacridine analogues have been evaluated as potential antiprotozoal agents. One of these, pyronaridine, an m-AMSA derivative synthesized in China in the 1970s as an alternative to chloroquine, has been used successfully there for around 20 years. This particular antimalarial has undergone advanced clinical trials in Africa28 and Thailand29. Acridine derivatives have been reported to promote cleavable complex formation in Leishmania chagas30, induce ultrastructural changes in both nuclear and kinetoplast DNA in L. donovani31 and inhibit partially puriÞed topoisomerase II in P. falciparum32,33. In P. falciparum, both pyronaridine and 3-6-diamino substitution of the acridine ring gave correspondingly high antimalarial activity and low mammalian cell cytotoxicity. Further work is needed to establish whether the intracellular target of these compounds is topoisomerase II; morphological studies of the effects of pyronaridine 280

show that the drug induces ultrastructural changes in the food vacuole, indicating that it might interfere with the digestive system of the parasite34. Epipodophyllotoxins, another group of eukaryotic topoisomerase II poison, have also been evaluated on protozoan parasites. These compounds are semisynthetic derivatives of the natural product podophyllotoxin. Their mechanism of action is related to the formation of cleavable complex but they are not DNA intercalators. Representatives VP16 (etoposide) and VM26 (teniposide) are clinically active against KaposiÕs sarcoma, leukaemia and small-cell lung cancer. VP-16 treatment of P. falciparum blood-stage parasites has been shown to promote cleavage of both nuclear and the 35 kb plastid organellar DNA. 59 covalent attachment of the protein to the DNA implicates type II topoisomerase targeting in this case35. In Trypanosoma equiperdum, VP-16 promotes cleavage of nuclear, mini- and maxicircle DNA36Ð38. Some drugs that poison bacterial type II enzymes by promoting cleavable complex formation have also been used against P. falciparum malaria. Ciproßoxacin (a ßuoroquinolone) is a derivative of nalidixic acid, which in turn was synthesized from 7-chloroquinoline, a byproduct of the antimalarial chloroquine. Fluoroquinolones are now widely used to target DNA gyrase and/or topoisomerase IV of pathogenic bacteria. Interestingly, ciproßoxacin treatment induces cleavage of the 35 kb plastid organelle of P. falciparum, but does not appear to target nuclear DNA, suggesting that there might be an additional uncharacterized type II enzyme present in the malaria parasite35. Classic antitrypanosomal drugs such as berenil [1,3-bis(49-amidinophenyl)triazene] have coincidentally turned out to be topoisomerase inhibitors. Berenil inhibits the mitochondrial, but not the nuclear, topoisomerase II of T. equiperdum39. An established minor-groove binder, the drug has also been shown to exhibit intercalative properties with both DNA and RNA substrates40. Fewer studies have been directed towards the effects of type I topoisomerase inhibitors on protozoans. Camptothecin, a pentacyclic plant alkaloid that promotes cleavable complex in mammalian cells has been shown to induce cleavage of both nuclear and mitochondrial DNA in trypanosomes and Leishmania41, and its antitrypanosomal activity is increased by 9-substituted-10,11-methylenedioxy analogues, which are less cytotoxic to mammalian cells42. More recent studies have demonstrated that camptothecin can also inhibit type I topoisomerase activity in extracts of P. falciparum blood-stage parasites14 and can also promote cleavable complex formation43. Other inhibitors of type I topoisomerase activity include the antileishmanial (L. donovani) pentavalent antimonials, sodium stibogluconate and ureastibamine17, and the bisnaphthoquinone Diospyrin, which has recently been reported to stabilize topoisomerase I cleavable complex formation in L. donovani44. Conclusions SigniÞcant advances have been made in the study of parasitic protozoan topoisomerases in recent years. Several genes encoding topoisomerases have been isolated and characterized, patterns of expression have been determined, and proteins have been puriÞed in small quantities directly from parasite sources. It will be of great interest in the future to see whether novel Parasitology Today, vol. 16, no. 7, 2000

Reviews topoisomerase genes or activities distinct from topoisomerase I and topoisomerase II will be discovered in these parasites. At Þrst sight, protozoan topoisomerases appear to share many characteristics associated with their human homologues, but closer inspection reveals that several differences do exist. Blocks of aa insertions are present in some species, enzyme activity requirements differ in others, and sensitivities to topoisomerase poisons are not always the same, encouraging the belief that topoisomerase-based therapies might be a realistic goal for the future. The challenge now is to devise new methods for rapidly screening existing poisons and their derivatives and purifying recombinant protozoan topoisomerases as a route to more detailed biochemical analysis and rational drug design. Acknowledgements Work in our laboratory has been supported by the Medical Research Council and the Wellcome Trust. I thank Brian Kilbey and Megan Porter for critical reading of the manuscript. Owing to space constraints, not all of the original work can appear in the reference list. References 1 Champoux, J.J. (1998) Domains of human topoisomerase I and associated functions. Prog. Nucleic Acids Res. Mol. Biol. 60, 111Ð132 2 Tosh, K. and Kilbey, B. (1995) The gene encoding topoisomerase I from the human malarial parasite Plasmodium falciparum. Gene 163, 151Ð154 3 Broccoli, S. et al. (1999) Characterisation of a Leishmania donovani gene encoding a protein that closely resembles a type IB topoisomerase. Nucleic Acids Res. 27, 2745Ð2752 4 Strauss, P.R. and Wang, J.C. (1990) The TOP2 gene of Trypanosoma brucei Ð a single-copy gene that shares extensive homology with other TOP2 genes encoding eukaryotic DNA topoisomerase II. Mol. Biochem. Parasitol. 38, 141Ð150 5 Fragoso, S.P. and Goldenberg, S. (1992) Cloning and characterisation of the gene encoding Trypanosoma-cruzi DNA topoisomerase II. Mol. Biochem. Parasitol. 55, 127Ð134 6 Cheesman, S. et al. (1994) The gene encoding topoisomerase II from Plasmodium falciparum. Nucleic Acids Res. 22, 2547Ð2551 7 Christopher, L.J. and Dykstra, C.C. (1994) IdentiÞcation of a type II topoisomerase gene from Cryptosporidium parvum. J. Euk. Microbiol. 41, 28S 8 Ridley, R.G. (1991) Proteins of unusual sequence composition from the malarial parasite Plasmodium falciparum. Biochem. Soc. Trans. 19, 525Ð528 9 Isaacs, R.J. et al. (1998) Physiological regulation of eukaryotic topoisomerase II. Biochim. Biophys. Acta 1400, 121Ð137 10 Pommier, Y. et al. (1998) Mechanisms of action of eukaryotic DNA topoisomerase I and drugs targeted to the enzyme. Biochim. Biophys. Acta 1400, 83Ð106 11 Pasion, S.G. et al. (1996) Sequences within the 59 untranslated region regulate the levels of a kinetoplast DNA topoisomerase mRNA during the cell cycle. Mol. Biochem. Parasitol. 16, 6724Ð6735 12 Mahmood, R. and Ray, D.S. (1998) Nuclear extracts of Crithidia fasciculata contain a factor(s) that binds to the 59 untranslated regions of TOP2 and RPAI mRNAÕs containing sequences required for their cell cycle regulation. J. Biol. Chem. 273, 23729Ð23734 13 Cheesman, S. et al. (1998) Intraerythrocytic expression of topoisomerase II from Plasmodium falciparum is developmentally regulated. Mol. Biochem. Parasitol. 92, 39Ð46 14 Tosh, K. et al. (1999) Plasmodium falciparum: stage-related expression of topoisomerase I. Exp. Parasitol. 91, 126Ð132 15 Fragoso, S.P. et al. (1998) Expression and cellular localisation of Trypanosoma cruzi type II DNA topoisomerase. Mol. Biochem. Parasitol. 94, 197Ð204 16 Heller, R.A. et al. (1986) Multiple forms and cellular localisation of Drosophila DNA topoisomerase II. J. Biol. Chem. 261, 8063Ð8069 17 Chakraborty, A.K. and Majumder, H.K. (1988) Mode of action of pentavalent antimonials: speciÞc inhibition of type I DNA topoisomerase of Leishmania donovani. Biochem. Biophys. Res. Commun. 152, 605Ð611 18 Chakraborty, A. et al. (1993) A type I DNA topoisomerase from the kinetoplast hemoßagellate Leishmania donovani. Indian J. Biochem. Biophys. 30, 257Ð263 19 Riou, G. et al. (1983) A type I topoisomerase from Trypanosoma cruzi. Eur. J. Biochem. 134, 479Ð484

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20 Riou, J-F. et al. (1986) PuriÞcation and characterisation of Plasmodium bergei DNA topoisomerases I and II: drug action, inhibition of decatenation and relaxation, and stimulation of DNA cleavage. Biochemistry 25, 1473Ð1479 21 Chavalitshewinkoon, P. et al. (1994) Partial puriÞcation and characterisation of DNA topoisomerase II from Plasmodium falciparum. Southeast Asian J. Trop. Med. Public Health 25, 32Ð36 22 Chakraborty, A.K. and Majumder, H.K. (1987) Decatenation of kinetoplast DNA by an ATP-dependent DNA topoisomerase from the kinetoplast hemoßagellate Leishmania donovani. Mol. Biochem. Parasitol. 26, 215Ð224 23 Chakraborty, A.K. and Mujumder, H.K. (1991) An ATP-independent catenating enzyme from the kinetoplast hemoßagellate Leishmania donovani. Biochem. Biophys. Res. Commun. 180, 279Ð285 24 Bell, C. et al. (1993) StructureÐactivity studies of dicationically substituted bis-benzimidazoles against Giardia lamblia: correlation of antigiardial activity with DNA binding affinity and giardial topoisomerase II inhibition. Antimicrob. Agents Chemother. 37, 2668Ð2673 25 Douc-Rasy, S. et al. (1986) ATP independent type II topoisomerase from trypanosomes. Proc. Natl. Acad. Sci. U. S. A. 83, 7152Ð7156 26 Sinha, B.K. (1996) Topoisomerase inhibitors. Drugs 49, 11Ð19 27 Cain, B.F. and Atwell, G.J. (1974) The experimental antitumor properties of three congeners of the acridylmethanesulphonanilide (AMSA) series. Eur. J. Cancer 10, 539Ð549 28 Ringwald, P. et al. (1996) Randomised trial of pyronaridine versus chloroquine for acute uncomplicated falciparum malaria in Africa. Lancet 347, 24Ð28 29 Looareesuwan, S. et al. (1996) Clinical study of pyronaridine for the treatment of acute uncomplicated falciparum malaria in Thailand. Am. J. Trop. Med. Hyg. 54, 205Ð209 30 Werbovetz, K.A. et al. (1994) Cleavable complex formation in Leishmania chagasi treated with anilinoacridines. Mol. Biochem. Parasitol. 65, 1Ð10 31 Mesa-Valle, C.M. et al. (1996) Activity and mode of action of acridine compounds against Leishmania donovani. Antimicrob. Agents Chemother. 40, 684Ð690 32 Chavalitshewinkoon, P. et al. (1993) StructureÐactivity relationships and modes of action of 9-anilinoacridines against chloroquine-resistant Plasmodium falciparum in vitro. Antimicrob. Agents Chemother. 37, 403Ð406 33 Gamage, S. et al. (1994) Synthesis and in vitro evaluation of 9-anilino-3,6,-diaminoacridines active against a multidrugresistant strain of the malaria parasite Plasmodium falciparum. J. Med. Chem. 37, 1486Ð1494 34 Kawai, S. et al. (1996) The effects of pyronaridine on the morphology of Plasmodium falciparum in Aotus trivirgatus. Am. J. Trop. Med. Hyg. 55, 223Ð229 35 Weissig, V. et al. (1997) Topoisomerase II inhibitors induce cleavage of nuclear and 35-kb plastid DNAs in the malarial parasite Plasmodium falciparum. DNA Cell. Biol. 16, 1483Ð1492 36 Shapiro, T.A. (1993) Inhibition of topoisomerases in African trypanosomes. Acta Trop. 54, 251Ð260 37 Shapiro, T. et al. (1989) Drug-promoted cleavage of kinetoplast DNA minicircles. J. Biol. Chem. 264, 4173Ð4178 38 Shapiro, T.A. and Showalter, A.F. (1994) In vivo inhibition of trypanosome mitochondrial topoisomerase II: effects on kinetoplast DNA maxicircles. Mol. Cell. Biol. 14, 5891Ð5897 39 Shapiro, T.A. and Englund, P.T. (1990) Selective cleavage of kinetoplast minicircles promoted by antitrypanosomal drugs. Proc. Natl. Acad. Sci. U. S. A. 87, 950Ð954 40 Pilch, D.S. et al. (1995) Berenil [1,3-bis (49-amidinophenyl)triazene] binding to DNA duplexes and to a RNA duplex: evidence for both intercalative and minor groove binding properties. Biochemistry 34, 9962Ð9976 41 Bodley, A.L. and Shapiro T.A. (1995) Molecular and cytotoxic effects of camptothecin, a topoisomerase I inhibitor, on trypanosomes and Leishmania. Proc. Natl. Acad. Sci. U. S. A. 92, 3726Ð3730 42 Bodley, A.L. et al. (1995) Antitrypanosomal sensitivity of camptothecin analogs. Biochem. Pharmacol. 50, 937Ð942 43 Bodley, A. et al. (1998) Effects of camptothecin, a topoisomerase I inhibitor, on Plasmodium falciparum. Biochem. Pharmacol. 55, 709Ð711 44 Ray, S. et al. (1998) Diospyrin, a bisnaphthoquinone: a novel inhibitor of type I DNA topoisomerase of Leishmania donovani. Mol. Pharmacol. 54, 994Ð999 45 Wigley, D.B. et al. (1991) Crystal structure of an N-terminal fragment of the DNA gyrase B protein. Nature 351, 624Ð629 46 Morais Cabral, J.H. et al. (1997) Crystal structure of the breakagereunion domain of DNA gyrase. Nature 388, 903Ð906 47 Berger, J.M. et al. (1996) Structure and mechanism of DNA topoisomerase II. Nature 379, 225Ð232

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