Molecular characterization of type II topoisomerase in the endosymbiont-bearing Trypanosomatid Blastocrithidia culicis

DNA topoisomerases are involved in DNA metabolism. These enzymes are inhibited by antimicrobial and antitumoral agents and might be important targets in the chemotherapy of diseases caused by parasites. We have cloned and characterized the gene encoding topoisomerase II from the monoxenic trypanosomatid Blastocrithidia culicis (BcTOP2). The BcTOP2 gene has a 3693 nucleotide-long open reading frame that encodes a 138 kDa polypeptide. The B. culicis topoisomerase II (BctopoII) amino-acid sequence shares high similarity (>74%) with topoisomerases from other trypanosomatids, and shares a lower similarity (41%) with other eukaryotic topoisomerases II from yeast to humans. BcTOP2 is a single copy gene and encodes a 4.4 kb mRNA. Western blotting of B. culicis extracts using the antiserum raised against a C-terminal portion of BctopoII showed a 138 kDa polypeptide. Immunolocalization assays showed that the antiserum recognized the nuclear topoisomerase II.     

Topoisomerases of kinetoplastid parasites as potential chemotherapeutic targets

The protozoan parasites Trypanosoma, Leishmania and Crithidia, which belong to the order kinetoplastidae, emerge from the most ancient eukaryotic lineages. The diversity found in the life cycle of these organisms must be directed by genetic events, wherein topoisomerases play an important role in cellular processes affecting the topology and organization of intracellular DNA. Topoisomerases are valuable as potential drug targets because they have indispensable function in cell biology. This review summarizes what is known about topoisomerase genes and proteins of kinetoplastid parasites and the roles of these enzymes as targets for therapeutic agents.     

Biochemical composition and metabolic pathways of filarial worms Setaria cervi: search for new antifilarial agents

The main problem regarding the chemotherapy of filariasis is that no safe and effective drug is available yet to combat the adult human filarial worms. Setaria cervi, the causal organism of setariasis and lumbar paralysis in cattle, is routinely employed as a model organism for conducting biochemical and enzymatic studies on filarial parasites. In view of the practical difficulties in procuring human strains of Wuchereria bancrofti and Brugia malayi for drug screening, the bovine filarial parasite S. cervi, resembling the human species in having microfilarial periodicity and chemotherapeutic response to known antifilarial agents, is widely used as a model in such studies. For a rational approach to antifilarial chemotherapy, knowledge of the biochemical composition and metabolic pathways of this helminth parasite may be of paramount importance, so that more potent antifilarial agents based on specific drug targets can be identified in drug discovery programmes. The present review provides an update on the biochemistry of the important metabolic pathways functioning within this potentially important bovine parasite, that have so far been studied, and on those that need to be investigated further so as to identify novel drug targets that can be exploited for designing new antifilarial drugs.     

Purification and characterization of Plasmodium berghei DNA topoisomerases I and II: drug action, inhibition of decatenation and relaxation, and stimulation of DNA cleavage

It has recently been suggested that topoisomerases could be important targets for drugs used in several diseases. This prompted us to purify and characterize the topoisomerases I and II present in the erythrocytes of protozoan parasites of the genus Plasmodium, the causative agent of malaria, in order to later use these enzymatic systems in antimalarial drug assays. The topoisomerases were purified from Plasmodium berghei, a parasite of mouse red cells. The Plasmodium topoisomerase II consists of two subunits with a molecular weight of about 160K. The enzyme is ATP- and Mg2+-dependent. The conditions for the reactions of relaxation, unknotting, decatenation, and catenation were found to be similar to those observed with enzymes from other eukaryotic cells. The Plasmodium topoisomerase I is a monomeric enzyme with a Mr of 70K-100K. It is ATP-independent and K+- or Na-dependent. Mg2+ is not required for relaxation but stimulates the reaction. Topoisomerase II was more sensitive to drug action than topoisomerase I. The most active drugs were the ellipticine derivatives. The antimalarial drugs, currently used in human clinical therapy, were poor inhibitors. Some antitumoral drugs stimulated the double-stranded DNA cleavage activity of Plasmodium topoisomerase II, like that of mammalian topoisomerases II. Antimalarial drugs had no stimulating activity. It is therefore suggested that Plasmodium topoisomerases are not good targets for antimalarial drugs.     

Topoisomerases in kinetoplastids

Topoisomerases are enzymes that mediate topological changes in DNA that are essential for nucleic acid biosynthesis and for cell survival. The kinetoplastid protozoa, which include pathogenic trypanosomes and Leishmania, have yielded an interesting variety of purified topoisomerase activities as well as several topoisomerase genes. In these parasites, topoisomerases are involved in the metabolism of both nuclear and mitochondrial (kinetoplast) DNA. In this review, Christian Burri, Armette Bodley and Theresa Shapiro summarize what is known about topoisomerases in kinetoplastids, and consider the intriguing possibility that these enzymes may act as valuable antiparasite drug targets.     

A tyrosyl DNA phosphodiesterase 1 from kinetoplastid parasite Leishmania donovani (LdTdp1) capable of removing topo I–DNA covalent complexes

Tyrosyl DNA phosphodiesterase 1 (Tdp1) is a member of phospholipase D superfamily, which cleaves a broad range of 3′‐DNA adducts, the best characterized of which is the phosphodiester bond formed between DNA and topoisomerase IB. This study describes cloning and functional characterization of the enzyme, termed as LdTdp1 in the kinetoplastid parasite Leishmania donovani. Sequence analysis confirmed conservation of the active site motifs typical for all Tdp1 proteins. LdTdp1 activity was detected in the parasite nucleus as well as in the kinetoplast. The enzyme harbours a nuclear localization signal at its C‐terminus. Overexpression of the active enzyme protected the parasites against topoisomerase IB inhibitor camptothecin (CPT) and oxidative agent H₂O₂‐mediated cytotoxicity and its downregulation rendered the parasites hypersensitive to CPT. Trapping of mutant LdTdp1 on DNA takes place following CPT treatment in L. donovani cells. The expression level and associated activity of LdTdp1 were found to be higher in CPT‐resistant L. donovani parasites. Altogether, this is the first report of Tdp1 from the kinetoplastid parasite L. donovani, which actively participates in topoisomerase I‐mediated DNA damage repair process and thereby counteracts the cytotoxic effect of topoisomerase I inhibitors.     

Thirteen-exon-motif signature for vertebrate nuclear and mitochondrial type IB topoisomerases

DNA topoisomerases contribute to various cellular activities that involve DNA. We previously identified a human nuclear gene that encodes a mitochondrial DNA topoisomerase. Here we show that genes for mitochondrial DNA topoisomerases (type IB) exist only in vertebrates. A 13-exon topoisomerase motif was identified as a characteristic of genes for both nuclear and mitochondrial type IB topoisomerases. The presence of this signature motif is thus an indicator of the coexistence of nuclear and mitochondrial type IB DNA topoisomerases. We hypothesize that the prototype topoisomerase IB with the 13-exon structure formed first, and then duplicated. One topoisomerase specialized for nuclear DNA and the other for mitochondrial DNA.     

Novel DNA Topoisomerase IIα Inhibitors from Combined Ligand- and Structure-Based Virtual Screening

DNA topoisomerases are enzymes responsible for the relaxation of DNA torsional strain, as well as for the untangling of DNA duplexes after replication, and are important cancer drug targets. One class of topoisomerase inhibitors, “poisons”, binds to the transient enzyme-DNA complex which occurs during the mechanism of action, and inhibits the religation of DNA. This ultimately leads to the accumulation of DNA double strand breaks and cell death. Different types of topoisomerases occur in human cells and several poisons of topoisomerase I and II are widely used clinically. However, their use is compromised by a variety of side effects. Recent studies confirm that the inhibition of the α-isoform of topoisomerase II is responsible for the cytotoxic effect, whereas the inhibition of the β-isoform leads to development of adverse drug reactions. Thus, the discovery of agents selective for topoisomerase IIα is an important strategy for the development of topoisomerase II poisons with improved clinical profiles. Here, we present a computer-aided drug design study leading to the identification of structurally novel topoisomerase IIα poisons. The study combines ligand- and structure-based drug design methods including pharmacophore models, homology modelling, docking, and virtual screening of the National Cancer Institute compound database. From the 8 compounds identified from the computational work, 6 were tested for their capacity to poison topoisomerase II in vitro: 4 showed selective inhibitory activity for the α- over the β-isoform and 3 of these exhibited cytotoxic activity. Thus, our study confirms the applicability of computer-aided methods for the discovery of novel topoisomerase II poisons, and presents compounds which could be investigated further as selective topoisomerase IIα inhibitors.     

人类非洲锥虫病的治疗研究进展

人类非洲锥虫病是一种被忽视的热带寄生虫病,威胁着撒哈拉以南非洲人民的生命健康。目前临床使用的治疗药物有四种,这些药物副作用多,用药困难并产生了一定的耐药性,因此有必要开发安全有效且利于服用的药物。目前有三类候选药物相继进入临床实验阶段,在不久的将来可能会进入临床应用。另外,还有许多正在研究的治疗药物,包括天然产物、磷酸二酯酶抑制剂、蛋白酶抑制剂、拓扑异构酶抑制剂、亮氨酰t RNA合成酶抑制剂、微管蛋白抑制剂以及一些靶点尚不明确却有很好的锥虫抑制活性的化合物。其中,壳二孢呋喃酮选择性较好,喹啉-3-羧酸类衍生物毒性低,5-硝基-2-呋喃酰胺类化合物对硝呋莫司耐受的锥虫仍有活性。这些研究无疑都为新型抗锥虫药物的出现提供了可能,相信在不久的将来越来越多的药物会在临床使用。     

Sequence-specific base pair mimics are efficient topoisomerase IB inhibitors

Topoisomerase IB controls DNA topology by cleaving DNA transiently. This property is used by inhibitors, such as camptothecin, that stabilize, by inhibiting the religation step, the cleavage complex, in which the enzyme is covalently attached to the 3'-phosphate of the cleaved DNA strand. These drugs are used in clinics as antitumor agents. Because three-dimensional structural studies have shown that camptothecin derivatives act as base pair mimics and intercalate between two base pairs in the ternary DNA-topoisomerase-inhibitor complex, we hypothesized that base pairs mimics could act like campthotecin and inhibit the religation reaction after the formation of the topoisomerase I-DNA cleavage complex. We show here that three base pair mimics, nucleobases analogues of the aminophenyl-thiazole family, once targeted specifically to a DNA sequence were potent topoisomerase IB inhibitors. The targeting was achieved through covalent linkage to a sequence-specific DNA ligand, a triplex-forming oligonucleotide, and was necessary to position and keep the nucleobase analogue in the cleavage complex. In the absence of triplex formation, only a weak binding to the DNA and topoisomerase I-mediated DNA cleavage was observed. The three compounds were equally active once conjugated, implying that the intercalation of the nucleobase upon triplex formation is the essential feature for the inhibition activity.     

Stimulation of topoisomerase II-mediated DNA cleavage by three DNA-intercalating plant alkaloids: cryptolepine, matadine, and serpentine.

Cryptolepine, matadine, and serpentine are three indoloquinoline alkaloids isolated from the roots of African plants: Cryptolepis sanguinolenta, Strychnos gossweileri, and Rauwolfia serpentina, respectively. For a long time, these alkaloids have been used in African folk medicine in the form of plant extracts for the treatment of multiple diseases, in particular as antimalarial drugs. To date, the molecular basis for their diverse biological effects remains poorly understood. To elucidate their mechanism of action, we studied their interaction with DNA and their effects on topoisomerase II. The strength and mode of binding to DNA of the three alkaloids were investigated by spectroscopy. The alkaloids bind tightly to DNA and behave as typical intercalating agents. All three compounds stabilize the topoisomerase II-DNA covalent complex and stimulate the cutting of DNA by topoisomerase II. The poisoning effect is more pronounced with cryptolepine than with matadine and serpentine, but none of the drugs exhibit a preference for cutting at a specific base. Cryptolepine which binds 10-fold more tightly to DNA than the two related alkaloids proves to be much more cytotoxic toward B16 melanoma cells than matadine and serpentine. The cellular consequences of the inhibition of topoisomerase II by cryptolepine were investigated using the HL60 leukemia cell line. The flow cytometry analysis shows that the drug alters the cell cycle distribution, but no sign of drug-induced apoptosis was detected when evaluating the internucleosomal fragmentation of DNA in cells. Cryptolepine-treated cells probably die via necrosis rather than via apoptosis. The results provide evidence that DNA and topoisomerase II are the primary targets of cryptolepine, matadine, and serpentine.     

Binding of etoposide to topoisomerase II in the absence of DNA: decreased affinity as a mechanism of drug resistance

Despite the prevalence of topoisomerase II-targeted drugs in cancer chemotherapy and the impact of drug resistance on the efficacy of treatment, interactions between these agents and topoisomerase II are not well understood. Therefore, to further define interactions between anticancer drugs and the type II enzyme, a nitrocellulose filter assay was used to characterize the binding of etoposide to yeast topoisomerase II. Results indicate that etoposide binds to the enzyme in the absence of DNA. The apparent Kd value for the interaction was approximately 5 microM drug. Etoposide also bound to ytop2H1012Y, a mutant yeast type II enzyme that is approximately 3-4-fold resistant to etoposide. However, the apparent Kd value for the drug (approximately 16 microM) was approximately 3 times higher than that determined for wild-type topoisomerase II. Although it has been widely speculated that resistance to topoisomerase II-targeted anticancer agents results from a decreased drug-enzyme binding affinity, these data provide the first direct evidence in support of this hypothesis. Finally, the ability of yeast topoisomerase II to bind etoposide was dependent on the presence of the hydroxyl moiety of Tyr783, suggesting specific interactions between etoposide and the active site residue that is involved in DNA scission.     

Structure of the N-terminal fragment of topoisomerase V reveals a new family of topoisomerases

Topoisomerases are involved in controlling and maintaining the topology of DNA and are present in all kingdoms of life. Unlike all other types of topoisomerases, similar type IB enzymes have only been identified in bacteria and eukarya. The only putative type IB topoisomerase in archaea is represented by Methanopyrus kandleri topoisomerase V. Despite several common functional characteristics, topoisomerase V shows no sequence similarity to other members of the same type. The structure of the 61 kDa N-terminal fragment of topoisomerase V reveals no structural similarity to other topoisomerases. Furthermore, the structure of the active site region is different, suggesting no conservation in the cleavage and religation mechanism. Additionally, the active site is buried, indicating the need of a conformational change for activity. The presence of a topoisomerase in archaea with a unique structure suggests the evolution of a separate mechanism to alter DNA.     

Application of a Novel Microtitre Plate-Based Assay for the Discovery of New Inhibitors of DNA Gyrase and DNA Topoisomerase VI

DNA topoisomerases are highly exploited targets for antimicrobial drugs. The spread of antibiotic resistance represents a significant threat to public health and necessitates the discovery of inhibitors that target topoisomerases in novel ways. However, the traditional assays for topoisomerase activity are not suitable for the high-throughput approaches necessary for drug discovery. In this study we validate a novel assay for screening topoisomerase inhibitors. A library of 960 compounds was screened against Escherichia coli DNA gyrase and archaeal Methanosarcina mazei DNA topoisomerase VI. Several novel inhibitors were identified for both enzymes, and subsequently characterised in vitro and in vivo. Inhibitors from the M. mazei topoisomerase VI screen were tested for their ability to inhibit Arabidopsis topoisomerase VI in planta. The data from this work present new options for antibiotic drug discovery and provide insight into the mechanism of topoisomerase VI.     

An insight into the active site of a type I DNA topoisomerase from the kinetoplastid protozoan Leishmania donovani

DNA topoisomerases are ubiquitous enzymes that govern the topological interconversions of DNA thereby playing a key role in many aspects of nucleic acid metabolism. Recently determined crystal structures of topoisomerase fragments, representing nearly all the known subclasses, have been solved. The type IB enzymes are structurally distinct from other known topoisomerases but are similar to a class of enzymes referred to as tyrosine recombinases. A putative topoisomerase I open reading frame from the kinetoplastid Leishmania donovani was reported which shared a substantial degree of homology with type IB topoisomerases but having a variable C-terminus. Here we present a molecular model of the above parasite gene product, using the human topoisomerase I crystal structure in complex with a 22 bp oligonucleotide as a template. Our studies indicate that the overall structure of the parasite protein is similar to the human enzyme; however, major differences occur in the C-terminal loop, which harbors a serine in place of the usual catalytic tyrosine. Most other structural themes common to type IB topoisomerases, including secondary structural folds, hinged clamps that open and close to bind DNA, nucleophilic attack on the scissile DNA strand and formation of a ternary complex with the topoisomerase I inhibitor camptothecin could be visualized in our homology model. The validity of serine acting as the nucleophile in the case of the parasite protein model was corroborated with our biochemical mapping of the active site with topoisomerase I enzyme purified from L.donovani promastigotes.     

Analysis of bisdioxopiperazine dexrazoxane binding to human DNA topoisomerase II alpha: decreased binding as a mechanism of drug resistance

Topoisomerase II is an ATP-operated clamp that effects topological changes by capturing a double stranded DNA segment and transporting it through another DNA molecule. Despite the extensive use of topoisomerase II-targeted drugs in cancer chemotherapy and the impact of drug resistance on the efficacy of treatment, much remains unknown concerning the interactions between these agents and topoisomerase II. To identify the interaction of the bisdioxopiperazine dexrazoxane (ICRF-187) with topoisomerase II, we developed a rapid gel-filtration assay and characterized the binding of ((3)H)-dexrazoxane to human topoisomerase II alpha. Dexrazoxane binds to human topoisomerase II alpha in the presence of DNA and ATP with an apparent K(d) of 23 microM and a stoichiometry of 1 drug molecule per enzyme dimer. Various N-terminal single amino acid substitutions in human topoisomerase II alpha that were previously shown to confer specific bisdioxopiperazine resistance either totally abolished drug binding or resulted in less efficient binding. The effect of the various mutations on drug binding correlated well with their effect on drug resistance in vivo and in vitro. Interestingly, an altered active site tyrosine mutant of human topoisomerase II alpha, which is incapable of carrying out DNA strand passage, was unable to bind dexrazoxane, which agrees with the drug's proposed mechanism of action late in the topoisomerase II catalytic cycle. The direct correlation between the level of drug binding and dexrazoxane resistance is consistent with a decreased drug binding mechanism of action for these dexrazoxane resistance conferring mutations.     

Induction of mammalian DNA topoisomerase I mediated DNA cleavage by antitumor indolocarbazole derivatives.

DNA topoisomerases have been shown to be important therapeutic targets in cancer chemotherapy. We found that KT6006 and KT6528, synthetic antitumor derivatives of indolocarbazole antibiotic K252a, were potent inducers of a cleavable complex with topoisomerase I. In DNA cleavage assay using purified calf thymus DNA topoisomerase I and supercoiled pBR322 DNA, KT6006 induced topoisomerase I mediated DNA cleavage in a dose-dependent manner at drug concentrations up to 50 microM, while DNA cleavage induced by KT6528 was saturated at 5 microM. The maximal amount of nicked DNA produced by KT6006 was more than 50% of substrate DNA, which was comparable to that of camptothecin. Heat treatment (65 degrees C) of the reaction mixture containing these compounds and topoisomerase I resulted in a substantial reduction in DNA cleavage, suggesting that topoisomerase I mediated DNA cleavage induced by KT6006 and KT6528 is through the mechanism of stabilizing the reversible enzyme-DNA "cleavable complex". Both KT6006 and KT6528 did not induce topoisomerase II mediated DNA cleavage in vitro. KT6006 and KT6528 were found to induce nearly identical topoisomerase I mediated DNA cleavage patterns, which was distinctly different from that with camptothecin. In contrast to the similarity between KT6006 and KT6528 in their structures and the nature of their cleavable complex with topoisomerase I, these drugs have different properties with respect to their interaction with DNA: KT6006 is a very weak intercalator whereas KT6528 is a strong intercalator with potentials comparable to that of adriamycin. These results indicate that KT6006 and KT6528 represent a new distinct class of mammalian DNA topoisomerase I active antitumor drugs.     

Crystal structure of a bacterial type IB DNA topoisomerase reveals a preassembled active site in the absence of DNA

Type IB DNA topoisomerases are found in all eukarya, two families of eukaryotic viruses (poxviruses and mimivirus), and many genera of bacteria. They alter DNA topology by cleaving and resealing one strand of duplex DNA via a covalent DNA-(3-phosphotyrosyl)-enzyme intermediate. Bacterial type IB enzymes were discovered recently and are described as poxvirus-like with respect to their small size, primary structures, and bipartite domain organization. Here we report the 1.75-A crystal structure of Deinococcus radiodurans topoisomerase IB (DraTopIB), a prototype of the bacterial clade. DraTopIB consists of an amino-terminal (N) beta-sheet domain (amino acids 1-90) and a predominantly alpha-helical carboxyl-terminal (C) domain (amino acids 91-346) that closely resemble the corresponding domains of vaccinia virus topoisomerase IB. The five amino acids of DraTopIB that comprise the catalytic pentad (Arg-137, Lys-174, Arg-239, Asn-280, and Tyr-289) are preassembled into the active site in the absence of DNA in a manner nearly identical to the pentad configuration in human topoisomerase I bound to DNA. This contrasts with the apoenzyme of vaccinia topoisomerase, in which three of the active site constituents are either displaced or disordered. The N and C domains of DraTopIB are splayed apart in an "open" conformation, in which the surface of the catalytic domain containing the active site is exposed for DNA binding. A comparison with the human topoisomerase I-DNA cocrystal structure suggests how viral and bacterial topoisomerase IB enzymes might bind DNA circumferentially via movement of the N domain into the major groove and clamping of a disordered loop of the C domain around the helix.