Biochimica et Biophysica Acta 1759 (2006) 117 – 131 http://www.elsevier.com/locate/bba

Review

DNA topoisomerase I from parasitic protozoa: A potential target for chemotherapy R.M. Reguera, C.M. Redondo, R. Gutierrez de Prado, Y. Pérez-Pertejo, R. Balaña-Fouce ⁎ Dpto. Farmacología y Toxicología (INTOXCAL), Universidad de León, Campus de Vegazana s/n, 24071 León, Spain Received 14 February 2006; received in revised form 22 March 2006; accepted 30 March 2006 Available online 26 April 2006

Abstract The growing occurrence of drug resistant strains of unicellular prokaryotic parasites, along with insecticide-resistant vectors, are the factors contributing to the increased prevalence of tropical diseases in underdeveloped and developing countries, where they are endemic. Malaria, cryptosporidiosis, African and American trypanosomiasis and leishmaniasis threaten human beings, both for the high mortality rates involved and the economic loss resulting from morbidity. Due to the fact that effective immunoprophylaxis is not available at present; preventive sanitary measures and pharmacological approaches are the only sources to control the undesirable effects of such diseases. Current anti-parasitic chemotherapy is expensive, has undesirable side effects or, in many patients, is only marginally effective. Under this point of view molecular biology techniques and drug discovery must walk together in order to find new targets for chemotherapy intervention. The identification of DNA topoisomerases as a promising drug target is based on the clinical success of camptothecin derivatives as anticancer agents. The recent detection of substantial differences between trypanosome and leishmania DNA topoisomerase IB with respect to their homologues in mammals has provided a new lead in the study of the structural determinants that can be effectively targeted. The present report is an up to date review of the new findings on type IB DNA topoisomerase in unicellular parasites and the role of these enzymes as targets for therapeutic agents. © 2006 Elsevier B.V. All rights reserved. Keywords: DNA topoisomerase IB; Camptothecin; Indolocarbazole; Parasitic protozoa; Plasmodium; Cryptosporidium; Trypanosomatid

1. Introduction Tropical diseases caused by parasitic protozoa are among humanity's costliest banes both for the high mortality rates involved and the economic loss resulting from morbidity. Malaria, cryptosporidiosis, African and American trypanosomiasis and leishmaniasis, among others, are diseases that have an enormous impact on the health of populations living mostly in the world's poorest countries. Abbreviations: LdTOPIA, gene encoding the large subunit of L donovani topoisomerase I; LdTOPIB, gene encoding the small subunit of L donovani topoisomerase; PfTOPI, gene encoding P. falciparum topoisomerase I; NLS, nuclear localization signal; kDNA, kinetoplast DNA; Tdp-1, tyrosyl-phosphodiesterase-1; CPT, camptothecin; SUMO, Small Ubiquitin-like Modifiers; QSAR, Quantitative Structure Activity Relationship; DHBA, betulinic acid derivatives; SSB, single-strand break; DSB, double-strand break; RNAi, small interference RNA ⁎ Corresponding author. Tel.: +34987291257; fax: +34 987291252. E-mail address: [email protected] (R. Balaña-Fouce). 0167-4781/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbaexp.2006.03.006

Malaria constitutes a sanitary problem of enormous importance since more of 40 % of the world-wide population is exposed to suffer it. At present, it causes the death of more people than any other disease of obligatory declaration with the exception of tuberculosis. It is caused by unicellular parasites of the Plasmodium spp. (P. falciparum and P. vivax among others) and it is transmitted by the bite of an infected Anopheles mosquito. Inside the human host, the parasite undergoes a series of changes as part of its complex life-cycle. Nowadays, Malaria is endemic in more than 100 countries, where 2400 million people live. It mainly affects the most underdeveloped tropical regions of Africa, Asia and South America, where the control measures and economic conditions are clearly inadequate [1]. Trypanosomatid protozoa including African and American trypanosomes and leishmanias are characterized by the presence of one flagellum and a single mitochondrion housing a specialized organelle known as a kinetoplast that contains maxicircles and minicircles of DNA (kDNA) [2]. African trypanosomiasis – sleeping sickness – is a parasitic disease

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transmitted to humans by tsetse flies. Trypanosoma brucei gambiense, found in western and central Africa, causes chronic infections that may be asymptomatic for months or even years. T. brucei rhodesiense, by contrast, is more dangerous, causing acute and virulent outbreaks mostly in southern and eastern Africa [3]. American trypanosomiasis – Chagas' disease – is caused by T. cruzi, a parasite widespread in Central and South America. Humans contract Chagas' disease when bitten by infected triatomine (order Hemiptera) insects. After penetration, the parasite invades the bloodstream and multiplies inside host cells, particularly in the heart and smooth muscle, provoking severe cardiomyopathies and intestinal mega syndrome [4]. The term human leishmaniasis covers a complex of zoonotic diseases transmitted by the bite of female phlebotominae mosquitoes, originating cutaneous, mucocutaneous and visceral leishmaniasis – kala azar – in humans. Kala azar is, clinically speaking, the most harmful form of human leishmaniasis. Characterized by fever, swelling of the spleen and liver and anemia, it is usually fatal if not diagnosed and treated in time. Over 90% of Leishmania donovani-mediated visceral leishmaniasis is found in India, Bangladesh, Indonesia and Sudan [5]. Science still has no magic bullet for these diseases and many doubt that such a single solution will ever exist. The role played by the host immune system in resistance and healing of these diseases it is well established, however, no effective vaccine has been developed yet. Chloroquine, mefloquine, primaquine and quinine are still the first-line treatment for uncomplicated malaria in some countries, but resistance appears almost everywhere [6]. Sulfadoxine-pyrimethamine resistance occurred in most countries where the drug was introduced to replace chloroquine [7]. Effective up to date medicines, such as artemisinin-based combination therapies, are now clearly needed for the first-line treatment of falciparum malaria in Africa and Asia [8]. Antitrypanosomatid chemotherapy is often toxic and years of this treatment have led to the development of resistant strains. The aromatic diamidine pentamidine and the sulphonated naphtylamine suramin are the first-line drugs against the first stage of sleeping sickness [9]. Three drugs are used against the second stage of the disease; the organo-arsenical compound melarsoprol, the irreversible inhibitor of ornithine decarboxylase eflornithine and nifurtimox [10]. High toxicity, treatment failures, and the difficult administration have recommended the use of combination therapies [11]. No healing drugs are currently available against American trypanosomiasis [12]. The nitromidazole derivative benznidazole and the nitrofuran nifurtimox act via the generation of free oxygen radicals which selectively destroy the parasite cells due to their less effective detoxification system [13]. Both nifurtimox and benznidazole have significant activity in the acute phase, but fail in the treatment of the chronic disease. The main drug treatment against leishmaniasis includes pentavalent antimonials (Pentostam and Glucantime), the polyene antibiotic amphotericin B (including the lipid preparation AmBisome®) and pentamidine [14,15]. The most significant advance in the treatment of visceral leishmaniasis is the incorporation of an alkylphosphocholine derivative (miltefosine) which has been

recently registered by the Indian government. A phase IV trial is currently in progress [16]. Most of these drugs are not easy to manage, generally speaking, for the lengthy and costly treatment involved, nor are they free from undesirable side effects. On the other hand, the effects of tropical diseases threaten now developed countries as opportunistic infections in immuno-compromised patients [17]. For example, Leishmania/HIV co-infection causes cumulative immunodeficiency because Leishmania parasites and HIV infect and destroy the same cells [18]. Severe acute leishmaniasis is a serious problem in south-western European countries, where needle sharing by drug users is the most widespread form of transmission of the disease [19]. 2. DNA topoisomerases DNA topoisomerases are ubiquitous enzymes catalyzing changes in the topological state of duplex DNA during replication, transcription, recombination and DNA repair processes (for a general description of DNA topoisomerases, see reviews [20–22]). There are three categories of such enzymes: DNA topoisomerases types IA, IB (EC 5.99.1.2), and II (EC 5.99.1.3). Type II DNA topoisomerases are homodimeric ATP-dependent enzymes that introduce transient doublestranded breaks in the double helix, followed by passage and rejoining. These enzymes can relax, catenate/decatenate, knot/ unknot or introduce supercoils in the DNA molecule [23,24]. Type II topoisomerases have been cloned and functionally expressed from several parasitic sources, including P. falciparum [25], African [26] and American [27] trypanosomes, Cryptosporidium parvum [28] and Leishmania spp. [29]. The role played by these enzymes as antiparasitic targets is still under research but is out of the scope of this review. Type I topoisomerases are monomeric ATP-independent enzymes with relaxation activity for both positively and negatively supercoiled DNA. They introduce single-stranded breaks in DNA followed by passage and rejoining, thereby allowing single step changes in the linking number of circular DNA. They are subdivided into two distinct classes: type IA enzymes that bind covalently to the 5′ end, and type IB enzymes that form covalent bonds with the 3′ end of the broken DNA strand. Type IB topoisomerases include eukaryotic topoisomerases, archaebacterial topoisomerase V and vaccinia topoisomerase I. The activity of this enzyme can be resumed in a five-step catalytic cycle: (i) binding to DNA; (ii) cleavage of one of the DNA strands at the 3′ terminus, establishing a transient, covalently bonded enzyme–DNA intermediate complex; (iii) relaxation of superhelical tension; (iv) resealing of the DNA break; and (v) release of the DNA, restoring the integrity of the double-stranded duplex [30]. The covalent phosphodiester bond between the DNA and the enzyme is established by a tyrosine residue found on the active site of all topoisomerase I molecules described to date. DNA topoisomerase I from mammalian hosts is a monomeric protein consisting of 765 amino acids with a predicted molecular mass of 91 kDa. Crystallographic studies have confirmed the existence of four structural and functional

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domains required for the protein to be operational [31–33]: the highly (positive) charged N-terminus domain, which is not phylogenetically conserved, contains four putative nuclear localization signal (NLS) motifs. This portion of the enzyme is not required for DNA-relaxation activity, as truncated proteins lacking this region are fully active [32]. The core domain is essential for relaxation of supercoiled DNA and shows a high degree of phylogenetic conservation, particularly with respect to residues that interact closely with the double helix. In this region of human topoisomerase, an amino acid “tetrad” consisting of Arg-488, Lys-532, Arg-590 and His-632 constitutes the active site of the enzyme. The amino acid that establishes a transient covalent phosphodiester bond with DNA is found in the C-terminal domain. All type IB topoisomerases contain a conserved “SKXXY” signature in this region in which a tyrosine residue (Tyr-723 in the human topoisomerase I) is the DNA-cleaving amino acid. Finally, the core and C-terminal domains are connected to one another by a poorly conserved region, the linker, not functionally involved in DNA relaxation activity [34]. 3. Apicomplexan type IB DNA topoisomerase Type I DNA topoisomerase from the malaria parasite P. falciparum was firstly described by Riou et al. [35] who purified and characterized the enzyme from infected erythrocytes. The Plasmodium enzyme is a monomeric protein of 104 kDa corresponding to a peptide of 839 amino acids. The PfTopI gene appears as a single copy on chromosome 5 of the plasmodial genome [36]. Structurally, the enzyme well resembles other topoisomerases, keeping a 42% homology with the human enzyme. Three structural domains were described by the authors: (i) the N-terminus domain of 134 amino acids which is poorly conserved; (ii) a 500-amino acid length block of conserved amino acids with high homology to the core domain of the human protein; (iii) the C-terminal domain is smaller than the hosts' but conserves the active tyrosine at position 798 which makes the enzyme fully active. Sequence analysis show two extensive tracts of additional amino acids within the core domain, one constituted by 29–34 amino acids and the other by 79, whose functions were not described by the authors. Tosh et al. [37] have shown that type I DNA topoisomerase is developmentally regulated during the different stages of Plasmodium life cycle. Northern analyses demonstrate that the PfTOPI gene promoter is inactive in the ring forms, only activating during the asexual intraerythrocytic cycle. High levels of PfTOPI mRNA are found at the throphozoite stage but not in schizonts. 4. The unusual type IB DNA topoisomerases from trypanosomatids Trypanosoma and leishmania type I DNA topoisomerases differ significantly from their homologues described in all other organisms studied to date. Trypanosomatid type I DNA topoisomerases are heterodimers in which the genes encoding each protein subunit are located on different chromosomes [38].

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Genetic analyses identify a gene for a large subunit, namely LdTOPIA, on L. donovani chromosome 34, encoding for a 636amino acid polypeptide with an estimated molecular mass of 73 kDa. This subunit is closely homologous to the core domain of human topoisomerase I. In turn, LdTOPIB, the gene encoding for the small subunit, is found on L. donovani chromosome 4 and encodes for a 262-amino acid polypeptide with a predicted molecular mass of 28 kDa. The small subunit contains the phylogenetically conserved “SKXXY” motif placed at the Cterminal domain of all type I DNA topoisomerases, which conserves the tyrosine residue that plays a role in DNA cleavage (Fig. 1). The relaxation activity of negative supercoiled DNA can be reconstituted only when the two subunits are coexpressed in a Saccharomyces cerevisiae defective strain using a biscistronic yeast expression vector. This heterodimeric structure definitively rules out a previous hypothesis identifying an unusual monomeric enzyme bearing a Ser residue as the DNA-cleaving amino acid instead of the C-terminal domain Tyr observed in all type I DNA topoisomerases described to date [39]. The existence of dimeric type I DNA topoisomerases was reported later by Bodley et al. [40] in African trypanosomes. Also, a genetic search into the T. cruzi genome database shows the presence of two separate genes encoding for the core and catalytic domains, respectively, indicating that this singular feature is common to all trypanosomatids [41]. Previous studies carried out with human TopI have shown that the proteolytic cleavage of core and catalytic domains of human topoisomerase IB within the unconserved linker domain does not markedly affect catalysis. Stewart et al. [42] reconstituted the relaxation activity of human topoisomerase by adding to the core domain a series of peptides containing the C-terminal domain, in a proportion of 1:1 in the presence of DNA. This finding was reinforced by Park and Sternglanz [43]; by using a two-hybrid expression system, the authors identified proteins containing part of the linker and the C-terminal domain that supplemented the catalytic core of S. cerevisiae topoisomerase I. Two hypotheses were put forward to explain the reconstitution of dimeric type I DNA topoisomerases. The first stated that the two fragments are not properly folded and their association goes hand-in-hand with the conformational changes required to form the active enzyme, this hypothesis can been ruled out as shown by Davies et al. in their recent work [44]. According to the second, the core and C-terminal domains are independent folding units that form an active enzyme when united by non-covalent bonds. This last hypothesis is supported by the overall charge difference between monomers observed by Bodley et al. [40] in T. brucei. Since the pI of the large subunit is 9.47 and that of the small subunit is 5.21, the authors proposed that the protein regions bearing these differences may contribute to establishing ionic interactions that hold the subunits together. In any event, the presence of multiple putative NLSs at the C-terminal extension of the LdTOPIA subunit only, and not in the LdTOPIB sequence, provides evidence that enzyme assembly takes place in the cytosol before translocation to the nuclear compartment [40,41]. Other interesting and unexpected finding observed by these authors

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Fig. 1. Schematic lineal representations of the human DNA topoisomerase I compared with their counterparts in unicellular parasites. GeneBank accession numbers are as follows: Homo sapiens nuclear gene (hTOPI), K03077; P. falciparum (PfTOPI), Q26024; C. parvum (CpTOPI), Q5CY81; L. donovani large subunit encoding gene (LdTOPIA) AF303577; L. donovani small subunit encoding gene (LdTOPIB) AY062908; T. brucei large subunit encoding gene (TbTOPIA) Q581U8 and T. brucei small subunit encoding gene (TbTOPIB) AAP78905. From structural analysis of the human enzyme, type IB topoisomerases contain four domains [33]: (i) a nonconserved hydrophilic N-terminal domain; (ii) a highly conserved DNA-binding core domain; (iii) a small positively charged linker domain, and (iv) the highly conserved C-terminal domain, which contains the DNA-cleaving tyrosine. Unlike the Apicomplexan enzymes, leishmanial and trypanosome topoisomerases I contain all the catalytic residues divided between two proteins.

correspond to the multimeric status of T. brucei TopI. Molecular mass determination using gel filtration chromatography has suggested that the native enzyme might exist as a tetrameric active A2B2 form. Although the authors recognize that this finding may be artifactual, they have speculated that two heterodimers would facilitate the binding at the helical crossover points of DNA [40]. Despite the close resemblance in the amino acidic sequence between the trypanosomatid's heterodimer and other eukaryotic type IB enzymes, significant differences should be mentioned. The large subunit of L. donovani topoisomerase I contains a short non-conserved N-terminal domain (start-Met-Glu-43) followed by the conserved core domain (Arg-44–Lys-456) ending in a long C-terminal extension (Val-457–Val-635) which does not show any homology with its counterparts in other TopI proteins. The core region conserves all the amino acids that characterize the active site of TopIB topoisomerases, such as Arg-314, Lys-352, Arg-410 and His-453 in the leishmanial enzyme (corresponding to Arg-377, Lys-415, Arg-471 and His514 in T. brucei) which are homologous to the catalytic “tetrad” described before in the human enzyme (Fig. 2C). On the other hand, the small LdTOPIB subunit contains a large nonconserved N-terminal extension (start-Met-Asn-210), enriched

in serine residues which could be phosphorylated. TopI phosphorylation has been proposed as a triggering mechanism for possible post-translational down-regulation mediated by the ubiquitin/26S proteasome pathway in mammalian systems [45]. The C-terminal domain starts at Lys-211 and closely resembles the catalytic domain of eukaryotic topoisomerases, containing the Tyr-222 in the leishmanial enzyme (Tyr-233 in T. brucei), required for DNA cleavage. As noted above, although the N-terminal and core domains are found on the LdTOPIA subunit and the C-terminus on the small LdTOPIB monomer, the location of the non-conserved linker domain in dimeric topoisomerases is merely speculative. According to the scheme in Fig. 1, all or part of the C-terminal and N-terminal ends on the large LdTOPIA, and small LdTOPIB subunits may interact to generate a functional linker and to establish a cleavable complex involving the core, active site and DNA. One possibility is that this linker-like structure is shared by the two subunits that interact in order to stabilize the enzyme. Another is that part of the long carboxyl or amino extensions on the subunits are removed by post-translational modifications prior to assembly. A systematic partial deletion study of the two subunits is being carried out to assess the regions needed for activity in the

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Fig. 2. Simulated 3D structure of the assembled dimer of type I DNA topoisomerase from a trypanosomatid (L. donovani). (A) Viewed perpendicular to the pore. (B) Same structure viewed down the pore. Core subdomains I, II and III are shown in yellow, blue and red, respectively, the linker domain is in white and the C-terminal domain is shown in green. (C) Comparison between simulated leishmania topoisomerase active site, shown in red, and human topoisomerase active site, shown in green. Leishmanial topoisomerase structure was simulated, using Swiss pdb program, by analogy with crystal human topoisomerase; the domains were determined by comparison with those found by Redinbo and coworkers [33] in the human one.

leishmanial enzyme. It has been found that more than 70 amino acid residues sited at the C-terminal end of the large subunit are not required for either relaxation or cleaving activities, however, it must be noted that this region contains multiple putative NLSs required to direct the assembled protein to the nucleus. Similarly almost the entire N-terminal end of the small subunit is unnecessary for either relaxation or cleaving activities. Neither subunit contains a detectable mitochondrial targeting sequence, although in trypanosomatids these may be short and hardly recognizable. On the other hand, Das et al. [46] using a 39-amino acid truncated at the N-terminal end of leishmanial type I DNA topoisomerase, have suggested that the loss of residues 1–39 from the large subunit leads to slow cleavage and relaxation rates, pointing to their possible role in coordinating DNA contacts by other parts of the enzyme. Furthermore, a more extensive deletion of the 99 amino acids placed at the N-terminal end of the same subunit prevents relaxation activity, suggesting that amino acids 40–99 may be involved in the interaction between the large and small subunits of the heterodimer, in which case their role would be to correctly position the active site tyrosine for its nucleophilic attack on the DNA.

There is another important difference between trypanosomatid type I topoisomerase and its eukaryotic homologues. Immunocytochemical localization experiments in all three representative species of trypanosomatids show dual localization of the enzyme, associated both with genomic DNA in the nucleus and kDNA in the kinetoplast [40,41]. Although the trypanosomatid kDNA relaxation, knotting/unknotting and catenating/decatenating activities attributed to type II topoisomerase are well defined, the role played by type I topoisomerase in the organelle is not sufficiently clear. As in most eukaryotic cells, topoisomerases I and II are essential to cell life. Enzyme inhibition or gene disruption of trypanosomatid type II topoisomerase produces a singular phenotype lacking kDNA, called dyskinetoplastidy that leads to cell death [47]. Moreover, RNAi-mediated disruption of gene expression of either subunit of topoisomerase IB results in a drastic reduction of both DNA and RNA synthesis in African trypanosomes, mimicking the inhibition of nucleic acid biosynthesis observed when bloodstream-form trypanosomes are treated with the specific inhibitor camptothecin (CPT) [48].

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5. The phylogeny of protozoan type I DNA topoisomerase Unicellular eukaryotic type I DNA topoisomerases from parasitic protozoa are among the most evolutionarily remote DNA-cleaving proteins [49]. Unlike Apicomplexan topoisomerases which conserve the catalytic “pentad” in a unique protein, Trypanosomatid's type I DNA topoisomerases divide the active amino acid residues into two peptides which should be post-translationally assembled. Previous phylogenetic analysis carried out with the LdTOPIA gene and eukaryotic topoisomerase I sequences arose to branch very early evolutionarily [50]. This early divergence is caused by the absence of the C-terminal domain containing the tyrosine cleaving residue, which was annotated later on, in a separate chromosome of the leishmanial genome. When the comparison is carried with the LdTOPIA/LdTOPIB structure simulation (Fig. 3), the evolutionarily divergences with other topoisomerases still remain. The differential genetic location of the DNA-binding core and catalytic domains leads to speculation regarding the molecular evolution of topoisomerases and the possible advantages of this feature for kinetoplastids. Bodley et al. [40], based on the secondary and tertiary structural analogies with tyrosine recombinases [51], have proposed the hypothesis, supported by some phylogenetic evidence, of a common DNA-cleaving C-terminal motif which fused, over time, with different N-terminal domains providing distinct enzymatic activities. Despite scant amino acid sequence similarities, both catalytic domains of type IB topoisomerases and recombinases might come from a

common ancestral enzyme capable of transesterification to the 3′ phosphate at the site of DNA strand scission. Genes encoding separate domains may permit the subunits to function independently, or perhaps in conjunction with other partners. Under this perspective, the evolutionary pathway of DNA topoisomerases might derive from independent ancient catalytic and core domains remaining in kinetoplastids to the contemporary fused constructs. 6. Regulation of DNA topoisomerase I The critical role played by type I DNA topoisomerase in eukaryotic organisms means that this enzyme must be kept under strict cellular control. Different expression patterns have been described in the distinct life cycle stages of P. falciparum. Extracts derived from populations of parasites synchronized in the three major blood stages show an increase in relaxation activity during schizont stages compared with trophozoite ones, even though levels of the protein were comparable for each stage. These results may be an indirect evidence of potential post-translational mechanisms which could operate on Plasmodium topoisomerases at different stages of its life cycle [37]. It is well documented that Trypanosomatidae control gene expression exclusively at a post-transcriptional level synthesizing long polycistronic nascent RNA from a single transcription origin. These long RNA sequences are processed to single RNAs by a trans-splicing procedure which supplies a spliced-leader sequence at the 5′-end and a poly-A tail at the 3′-end of the mRNA molecule ready to be translated. 5′-UTR regions of each

Fig. 3. Phylogenetic tree carried out using the amino acid sequences of type I DNA topoisomerases from unicellular parasites and other organisms annotated into the GeneBank database. The phylogram is displayed on TreeView using the tree produced by CLUSTAL W. The evolutionary scale bar is shown on the left, it indicates the relative distance on the tree in arbitrary units.

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gene copy of type I DNA topoisomerase contain the putative signatures for the trans-splicing acceptor site required for the transcriptional control of this enzyme [52]. Due to the dimeric nature of the enzyme it is possible to postulate that the two encoding units of mRNAs would be translated into the cytosol and after folding properly they would carry on to associate in a full active dimeric enzyme which could translocate to the nucleus [40]. The presence of multiple putative NLSs only at the C-terminal extension of the LdTOPIA subunit, and not in the LdTOPIB sequence, provides evidence that enzyme assembly takes place in the cytosol before translocation to the nuclear compartment (Fig. 4). Indirect evidence of this process is supplied by heterologous transfections of dimeric LdTOPI in EKY-3 TopI defective S. cerevisiae strain using a biscistronic yeast expression vector. PSK-Ura-vector independently expresses the genes encoding both subunits of LdTOPI under different promoters stimulated by galactose [38]. The two folded subunits may then assemble with each other to originate an active relaxation complex. Type I DNA topoisomerases are crucial in the normal maintenance of the genome stability but they can also induce DNA damage apoptotic processes and cell death especially when their complexes with DNA are stabilized by CPT [53]. Therefore the cellular amount of these enzymes has to be rapidly and strictly regulated by the cell. Post-translational

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regulation of type I DNA topoisomerase has not been studied in any parasite yet, but the presence of most of the genes encoding the enzymatic machinery described in other eukaryotes points to many similarities that may occur between parasites and hosts. Type I DNA topoisomerase I is rapidly degraded in mammalian cells when exposed to CPT [54]. Enzyme degradation is prevented by 26S proteasome inhibitors, and ubiquitinconjugates are readily detected after treatment with MG-132 or other proteasome inhibitors [55]. This post-translational control mechanism of topoisomerase degradation occurs with the hyperphosphorylated enzyme and is replication independent. Pommier has proposed that topoisomerase degradation by 26S proteasome may be useful for (i) increasing tolerance to DNA cleaving poisons; (ii) facilitating the phosphodiesterase activity of tyrosyl-phosphodiesterase-1 (Tdp1) to excise Top1 from cleaved DNA [56]. Several authors have demonstrated that proteasome inhibitors and CPT act synergistically on mammalian cells increasing DNA damage and inducing apoptosis [57]. The ubiquitin/26S proteasome pathway has been described in several unicellular parasites including Giardia lamblia [58], African [59] and American [60] trypanosomes, Leishmania spp. [61], Entamoeba spp. [62] and the Apicomplexan P. falciparum [63], and Toxoplasma gondii [64]. The 28-subunits cylindrical structure, composed of four stacked seven-subunit rings, builds a 20S proteolytic complex called proteasome activator. This

Fig. 4. Hypothetical expression procedure of type I DNA topoisomerase from a trypanosomatid. (A) long polycistronic nascent RNAs are transcribed from both chromosomes and edited (B) by a trans-splicing mechanism which supplies a spliced-leader sequence at the 5′-end and a poly-A tail at the 3′-end of the mRNA. (C) mRNAs are translated to nascent polypeptides which are post-translationally assembled to a fully functional enzyme. The presence of NLSs motifs only in the large subunit supports the hypothesis that this process is carried out in the cytoplasmatic compartment before being translocated to the nucleus.

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structure is associated to a 11S regulatory subunit building up the active 26S proteasome which does not differ much from the host organelle [65]. On the other hand, several ubiquitinconjugating genes are described into different parasite Genome Projects [66–69], pointing to a universal pathway of protein degradation along the phylogeny of eukaryotic cells (unpublished results). Further to CPT-mediated TopI degradation an early and transient response to this drug involves the conjugation of DNA topoisomerase I to the so-called Small Ubiquitin-like Modifier (SUMO) proteins [70]. SUMO conjugation (sumoylation) is a DNA-replication independent process which, unlike ubiquitination, is carried out over the dephosphorylated protein and is not linked to TopI degradation [71]. Like ubiquitination however, a SUMO and SUMO-conjugating genes are annotated in distinct genome Projects [66–69] and may play a role in post-translational mechanisms of unicellular protozoa. Sumoylation is not a Top I down-regulation mechanism, on the contrary it seems to be a stimulatory system via relocation of the protein from nucleoplasm to the nucleolus thus preventing ubiquitin degradation of TopI [72]. Multiple putative ubiquitination/sumoylation acceptor sites consisting of a (I/V/L)KX(E/D) peptide [73] are distributed along the primary structure of the LdTOPIA subunit only, but not within the LdTOPIB sequence, providing evidence that enzyme modification takes place only in the large subunit. Next, the subunit will be recognized by a Tdp1 protein which will then remove the dimeric enzyme from the DNA cleaving complex for relocation to the nucleolus or degradation by the 26S proteasome [56]. 7. Inhibition of DNA topoisomerase I The structural differences between human and parasite type I DNA topoisomerases make this enzyme an attractive target for chemotherapeutic intervention [49,74,75]. Topoisomerase inhibitors fall into two general categories: compounds that stimulate the formation of covalent enzyme–DNA complexes, also called topoisomerase poisons (class I inhibitors), and products that interfere with the enzymatic functions of the enzyme (class II inhibitors) [76,77]. CPT is a good example of a class I topoisomerase poison. CPT is a pentacyclic natural alkaloid (Fig. 5A) produced by the plant Camptotheca accuminata. An enormous amount of antitumor agents derive from this compound and their clinical use is underway [78–80] but they fall however, beyond the scope of this review. CPT is a non-competitive micromolar and submicromolar range inhibitor which establishes ternary complexes with topoisomerase and DNA, preventing DNA religation. CPT generates covalent DNA–topoisomerase complexes with both nuclear and kinetoplastic preparations of DNA from trypanosomes, leishmanias [81] and other protozoan parasites of medical importance [82]. Therefore, CPT is cytotoxic for the erythrocytic forms of P. falciparum. Bodley et al. [82] showed that CPT enters into the intraerythrocytic stages of P. falciparum; promotes cleavable complex formation; inhibits nucleic acid biosynthesis; and ultimately kills the parasite within the micromolar range. Quantitative Structure Activity

Relationship (QSAR) studies conducted by Bodley et al. [83] to test a battery of CPT analogues for their in vitro effectiveness against African trypanosomes showed that their cytotoxicity was closely correlated to the ability to promote the formation of covalent protein–DNA complexes. This would indicate that the sole cellular target of these agents is topoisomerase I. Moreover, antitrypanosomal activity is increased by 9-substituted-10,11methylenedioxy analogues, which are selectively less cytotoxic to mammalian cells. Recent results revealed that the watersoluble derivatives of CPT, irinotecan and topotecan hydrochlorides, are hardly effective against bloodstream forms of T. brucei [84]. The authors concluded that the reduced efficacy of these compounds is due more to the low permeability of the parasite plasma membrane to the drugs, than to any inability to establish the cleavage complex with DNA. Furthermore, 7ethyl-10-hydroxy-CPT, a metabolite formed during the hydrolysis of irinotecan by a carboxylesterase non-active in the parasite, exhibits substantial trypanocidal activity [85]. Based on the co-purification of human topoisomerase I recombined with CPT and DNA, Staker et al. [86,87] proposed a series of interactions among the different components of the cleavage complex that, in the absence of the crystalline structure of the parasitic heterodimer, may be assumed in trypanosomatids. Derived from this model, CPT intercalates at the site of DNA cleavage mimicking a DNA base pair. Within the intercalation pocket site the side chain of Asp-533 in the human enzyme (corresponding to Asp-353 on the large subunit of leishmanial topoisomerase I) can establish a hydrogen bond with the 20(S)-hydroxyl moiety of the lactone form of CPT, whereas Arg-364 (corresponding to Arg-190 in Leishmania) establishes a second hydrogen bond with the nitrogen at CPT Bring. Asn-722, the amino acid adjacent to the DNA-cleaving Tyr, is also needed for CPT inhibition, although it does not establish hydrogen bonds with the drug. Other important residues involved in CPT interaction – Phe-361, Gly-363 and Arg-364 [88] (corresponding to a conserved FXGR motif in the core domain of Leishmania) – are conserved in the trypanosomatid enzyme. Furthermore, partially truncated human type I topoisomerases lacking the linker domain [34] conserve enzymatic activity but lose sensitivity to CPT in standard relaxation assays, which confirms the crucial role played by this domain in the interaction with the drug. Marquis et al. [89] recently described two amino acid substitutions in the large subunit of type I DNA topoisomerase from CPT-resistant strains of L. donovani. The LdTOPIA large unit became highly CPT-resistant when two substitutions, G185R and D325E, were made in the core domain of the protein. L. donovani topoisomerase I residues Gly-185 and Asp325 are both conserved among all known members of the eukaryotic topoisomerase family. These amino acid substitutions prevent the formation of the CPT–topoisomerase I–DNA complexes normally observed in wild-type CPT-treated kinetoplastids. Very few studies have been conducted on the efficacy of CPT and analogues in in vivo trypanosomatid infection. Proulx et al. [90] tested the efficacy of free and liposome-encapsulated CPT in a murine model of L. donovani visceral leishmaniasis. The

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Fig. 5. Chemical structure of DNA–topoisomerase I poisons: (A) CPT and derivatives; (B) non-camptothecin polycyclic chemicals and (C) minor groove binding compounds. 125

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parasite burden was significantly reduced when infected mice were treated with 2.5 mg/kg body weight CPT via intraperitoneal injections of free and liposomal CPT. Indolocarbazole derivatives (Fig. 5B) constitute a promising group of class I topoisomerase poisons with a polyheterocyclic aromatic structure. Many rebeccamycin analogues have been tested in in vitro QSAR studies for efficacy against tumor cells and some of them have been selected for clinical trials and development [91]. The planar structure of these molecules enables them to intercalate the pile of DNA base-pairs, where they eventually establish DNA–topoisomerase I cleavage complexes. The reaction between crystalline human topoisomerase I and rebeccamycin analogues shows that one of the two carbonyl groups from the maleimide ring can interact with Arg364 (conserved as Arg-190 in Leishmania) at the minor groove side of the intercalation complex. Indolocarbazole molecules are symmetrical molecules except for the sugar substituent bond to the nitrogen atom of one of the indolyl ring. This asymmetry situates the ternary intercalation pocket in such a way that the glycosylated indole stacks with bases on the undamaged DNA strand, whereas the nonglycosylated indole interacts with the cleaved strand of duplex DNA [87]. By contrast, the Asn-722, which is crucial to CPT inhibition, is irrelevant to the establishment of the indolocarbazole-induced DNA–topoisomerase I cleavage complex [92]. Deterding et al. [84] found rebeccamycin to have an in vitro cytotoxic effect on African trypanosomes at sub-micromolar concentrations. Our team (Diaz-González, unpublished results) has tested several rebeccamycin analogues lacking chlorine atoms (RM762) or sugar moieties (arcyriaflavin A; RM62) on L. donovani promastigotes. The results showed that the intercalation of indolocarbazole drugs in the DNA molecule is reinforced by the existence of the sugar moiety, which helps to stabilize the insertion complex by binding with the major groove of the ternary duplex DNA. The presence of chlorine atoms, on the contrary, diminishes topoisomerase I inhibition. Indenoisoquinolines are class I topoisomerase poisons with a polyheterocyclic structure (Fig. 5B). A number of these compounds, presently in pre-clinical development for use as anti-tumor agents, have been tested against L. major in vitro and found to exhibit a good cytotoxic profile in the sub-micromolar range (Balaña-Fouce et al., unpublished results). X-ray diffraction data show that once again the Arg-364 sited in the core domain establishes the crucial hydrogen bond with the O18 on the indenoisoquinoline analogue MJ-III-65 in the generation of the cleavage complex [93]. L. donovani has been observed to be sensitive to other antiprotozoan drugs such as the naphthoquinone derivative diospyrin that acts like a topoisomerase poison [94]. Diospyrin inhibition is relatively specific of leishmanial TopI, requiring 10-fold higher concentrations to inhibit calf thymus DNA topoisomerase I and failing to inhibit L. donovani DNA topoisomerase II [95]. Betulinic acid derivatives (DHBA) are pentacyclic triterpenoids that inhibit both parasite DNA types I and II topoisomerases by preventing enzyme–DNA binary complex formation. DHBA does not interact with substrate DNA and therefore is unable to stabilize the ternary cleavable

complex [96]. This compound has a drastic inhibitory effect on the growth of L. donovani promastigotes in the micromolar range. DHBA derivatives induce chromatin margination and dechromatinization of the leishmanial genome but not dyskinetoplastidy, providing support for the direct correlation between topoisomerase inhibition and apoptosis in L. donovani [97] (Fig. 5B). Other type I topoisomerase inhibitors include the first-line leishmanicide drugs derived from pentavalent antimony [98]. Sodium stibogluconate (Pentostam) but not meglumine antimoniate (Glucantime) can stabilize DNA–topoisomerase I cleavable complexes in both the purified leishmanial enzyme and intact promastigotes [99]. The drug's cytotoxicity is negligible at this stage of the parasitic life cycle, however, suggesting that the foregoing is a side-effect rather than the actual mode of action of the compound [100]. Berberine is a polyheterocyclic class I topoisomerase inhibitor with a structure resembling the intercalant drug benzo[a]acridine [101]. Several berberine analogues, tested against L. donovani [102] and bloodstream forms of T. brucei and T. congolense [103], exhibited micromolar range efficacy. In vivo trials with protoberberine analogues have also shown them to effectively treat golden hamster visceral leishmaniasis. Coralyne but not nitidine, both derivatives of berberine, has likewise been shown to effectively combat T. cruzi infection [104]. DNA minor groove binders (Fig. 5C) such as certain bisbenzymidazole dyes (Ho-33342 and its parent compound Ho33258) and a series of aromatic diamidines including the antiparasitic drugs pentamidine and berenil, can inhibit type I DNA topoisomerase I in vitro [105]. These compounds bind to AT-rich regions of DNA interfering with enzyme catalysis, but with the exception of Ho-33342 and Ho-33258, they do not stabilize the cleavable complexes between the enzyme and DNA. A recent report reveals that the leishmanicidal effect of these compounds is poorly related to type I DNA topoisomerase inhibition, pointing to a more complex pleiotropic effect [106]. 8. Biological consequences of DNA topoisomerase I inhibition The formation of covalent DNA–topoisomerase I complexes is readily reversible after short exposure to topoisomerase poison, resulting in low cytotoxicity. When the presence of such drugs is persistent, however, the cleavable complex is stabilized, trapping the enzyme in the phosphotyrosine bond (s) and preventing its dissociation from DNA. The outcome is the generation of single- (SSB) or double-stranded breaks (DSB) formed as the result of the collisions produced between the replication or transcription machinery and the cleavage complexes, which are believed to cause point mutations, fragmentation of the genome and cell death [107]. Topoisomerase poison toxicity depends heavily, therefore, on the ability of DNA repair systems to restore the basic cellular functions [108] (Fig. 6). In mammalian cells there are at least four routes involved in restoring topoisomerase I–DNA complexes to duplex DNA.

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Fig. 6. Genomics of DNA-repair in trypanosomatids (modified from Pommier et al. [108]). Both genes which function have been undoubtedly demonstrated and genes still in the in the Genome project of each species (L. major, T. cruzi and T. brucei) have been included in the table. No differences among the three species have been found [116].

Tdp-1 catalyzes the enzymatic cleavage of the tyrosine DNA– phosphodiester bond, yielding a 3′-phosphate which is further processed by polynucleotide kinase 3′-phosphatase (PNKP). This reaction is followed in turn by proteolysis of topoisomerase I adducts, with previous proteasome ubiquitination. 3′processing may be performed by three different endonuclease complexes—Rad1/Rad10, Mre11/Rad50 and Mus81/Mms4. Processing the 5′-ends involves the X-ray repair complementation group 1 (XRCC1) system, as well as the RAD52 (RAD50, RAD51, RAD54 and others) and RecQ helicase/ topoisomerase III homologous recombination proteins [56,108]. There is a paucity of information about DNA repair systems in kinetoplastid species, however. Most of the genes involved in the different DNA repair pathways are annotated as putative in the L. major, T. brucei and T. cruzi Genome Projects, but have not been functionally characterized. McKean et al.

[109] identified and characterized the DNA damage-inducible gene rad51 on L. major chromosome 35, which codifies for a 377-amino acid protein that contributes to the homologous recombination of DNA strains and may be involved in 5′-end processing after topoisomerase I-induced damage. In addition, an apurinic/apyrimidinic endonuclease that participates in the base-excision repair system (BER) has been found to protect against drug-induced oxidative stress in T. cruzi and L. major [110]. The fact that kDNA is not required to cell survival in some kinetoplastids [47,111] would appear to support the belief that class I topoisomerase poisons affect cell nuclei rather than their mitochondria. However, the results obtained by the Majumder group seem to rule out this hypothesis. Like other DNA damaging agents, class I topoisomerase poisons are efficient inducers of programmed cell death [112], which would explain

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the cytotoxic effect of these compounds in kinetoplastids. Sen et al. [113] found that CPT-mediated leishmanicidal effect appears after the mitochondrial function is inhibited, which is in turn followed by an increase in mitochondrial membrane potential. These present authors showed that CPT raises the intracellular concentration of reactive oxygen species, with the concomitant rise in lipid peroxidation and decline in the concentration of the free radical scavenger glutathione [114]. As in mammalian systems, caspase 3-like protease activation, poly(ADP-ribose) polymerase (PARP) cleavage and an increase in cytosolic Ca2+ released from cellular stores and followed by the rapid formation of the cytochrome c-mediated complex are common developments in CPT-induced apoptosis in L. donovani promastigotes [115]. 9. Conclusion and outlook Type I DNA topoisomerase is a well-recognized target for cancer therapy whose effectiveness against the diseases caused by parasitic protozoa has not been sufficiently exploited. The phylogenetically unique, anomalous dimeric structure of the trypanosomatid enzyme and its dual location – associated with both genomic DNA and kDNA – makes it an auspicious target for new antiparasitic drug development. Despite the multiple topoisomerase inhibitors tested against tumor cells, only scant QSAR information on the effect of these compounds on trypanosomatids is available. Moreover, there is a pressing need to perform in vivo trials to evaluate the actual efficiency of drug treatments targeting topoisomerase I. Recent findings relating to topoisomerase poison-induced programmed cell death make such compounds important tools in cell biology research focusing on these primitive eukaryotes. The crystalline structure of this protein will provide information of cardinal importance on the structural determinants involved in processes such as the assembly of the active heterodimer, enzyme kinetic machinery and interaction with inhibitors, that will play a key role in future drug discovery. Acknowledgements This research was supported in part by Comisión Interministerial de Ciencia y Tecnología (grants BMC 2002 04107-C0202 and AGL2003 06976/GAN). References [1] B.M. Greenwood, K. Bojang, C.J.M. Whitty, G.A.T. Targett, Malaria, Lancet 365 (2005) 1487–1498. [2] J. Shlomai, The structure and replication of kinetoplast DNA, Curr. Mol. Med. 4 (2004) 623–647. [3] D. Kioy, J. Jannin, N. Mattock, Human African trypanosomiasis, Nat. Rev., Microbiol. 2 (2004) 186–187. [4] M.A. Miles, M.D. Feliciangeli, A.R. de Arias, American trypanosomiasis (Chagas' disease) and the role of molecular epidemiology in guiding control strategies, BMJ 326 (2003) 1444–1448. [5] P. Desjeux, Leishmaniasis: current situation and new perspectives, Comp. Immunol. Microbiol. Infect. Dis. 27 (2004) 305–318. [6] J. May, C.G. Meyer, Chemoresistance in falciparum malaria, Trends Parasitol. 19 (2003) 432–435.

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