Proteasome inhibition for treatment of leishmaniasis, Chagas disease and sleeping sickness.

Chagas disease, leishmaniasis and sleeping sickness affect 20 million people worldwide and lead to more than 50,000 deaths annually. The diseases are caused by infection with the kinetoplastid parasites Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp., respectively. These parasites have similar biology and genomic sequence, suggesting that all three diseases could be cured with drugs that modulate the activity of a conserved parasite target. However, no such molecular targets or broad spectrum drugs have been identified to date. Here we describe a selective inhibitor of the kinetoplastid proteasome (GNF6702) with unprecedented in vivo efficacy, which cleared parasites from mice in all three models of infection. GNF6702 inhibits the kinetoplastid proteasome through a non-competitive mechanism, does not inhibit the mammalian proteasome or growth of mammalian cells, and is well-tolerated in mice. Our data provide genetic and chemical validation of the parasite proteasome as a promising therapeutic target for treatment of kinetoplastid infections, and underscore the possibility of developing a single class of drugs for these neglected diseases.

The enigmatic RNase MRP of kinetoplastids.

The ribonucleoprotein RNase MRP is responsible for the processing of ribosomal RNA precursors. It is found in virtually all eukaryotes that have been examined. In the Euglenozoa, including the genera Euglena, Diplonema and kinetoplastids, MRP RNA and protein subunits have so far escaped detection using bioinformatic methods. However, we now demonstrate that the RNA component is widespread among the Euglenozoa and that these RNAs have secondary structures that conform to the structure of all other phylogenetic groups. In Euglena, we identified the same set of P/MRP protein subunits as in many other protists. However, we failed to identify any of these proteins in the kinetoplastids. This finding poses interesting questions regarding the structure and function of RNase MRP in these species.

DNA Topoisomerases of Kinetoplastid Parasites: Brief Overview and Recent Perspectives.

Topoisomerases are a group of enzymes that resolve DNA topological problems and aid in different DNA transaction processes viz. replication, transcription, recombination, etc. inside cells. These proteins accomplish their feats by steps of DNA strand(s) scission, strand passage or rotation and subsequent rejoining activities. Topoisomerases of kinetoplastid parasites have been extensively studied because of their unusual features. The unique presence of heterodimeric Type IB topoisomerase and prokaryotic 'TopA homologue' Type IA topoisomerase in kinetoplastids still generates immense interest among scientists. Moreover, because of their structural dissimilarity with the host enzymes, topoisomerases of kinetoplastid parasites are attractive targets for chemotherapeutic interventions to kill these deadly parasites. In this review, we summarize historical perspectives and recent advances in kinetoplastid topoisomerase research and how these proteins are exploited for drug targeting.

Comparative Metabolism of Free-living Bodo saltans and Parasitic Trypanosomatids.

Comparison of the genomes of free-living Bodo saltans and those of parasitic trypanosomatids reveals that the transition from a free-living to a parasitic life style has resulted in the loss of approximately 50% of protein-coding genes. Despite this dramatic reduction in genome size, B. saltans and trypanosomatids still share a significant number of common metabolic traits: glycosomes; a unique set of the pyrimidine biosynthetic pathway genes; an ATP-PFK which is homologous to the bacterial PPi -PFKs rather than to the canonical eukaryotic ATP-PFKs; an alternative oxidase; three phosphoglycerate kinases and two GAPDH isoenzymes; a pyruvate kinase regulated by fructose-2,6-bisphosphate; trypanothione as a substitute for glutathione; synthesis of fatty acids via a unique set of elongase enzymes; and a mitochondrial acetate:succinate coenzyme A transferase. B. saltans has lost the capacity to synthesize ubiquinone. Among genes that are present in B. saltans and lost in all trypanosomatids are those involved in the degradation of mureine, tryptophan and lysine. Novel acquisitions of trypanosomatids are components of pentose sugar metabolism, pteridine reductase and bromodomain-factor proteins. In addition, only the subfamily Leishmaniinae has acquired a gene for catalase and the capacity to convert diaminopimelic acid to lysine.

Evolutionary comparison of prenylation pathway in kinetoplastid Leishmania and its sister Leptomonas.

BACKGROUND: Leptomonas is monogenetic kinetoplastid parasite of insects and is primitive in comparison to Leishmania. Comparative studies of these two kinetoplastid may share light on the evolutionary transition to dixenous parasitism in Leishmania. In order to adapt and survive within two hosts, Leishmania species must have acquired virulence factors in addition to mechanisms that mediate susceptibility/resistance to infection in the pathology associated with disease. Rab proteins are key mediators of vesicle transport and contribute greatly to the evolution of complexity of membrane transport system. In this study we used our whole genome sequence data of these two divergent kinetoplastids to analyze the orthologues/paralogues of Rab proteins. RESULTS: During change of lifestyle from monogenetic (Leptomonas) to digenetic (Leishmania), we found that the prenyl machinery remained unchanged. Geranylgeranyl transferase-I (GGTase-I) was absent in both Leishmania and its sister Leptomonas. Farnesyltransferase (FTase) and geranylgeranyl transferase-II (GGTase-II) were identified for protein prenylation. We predict that activity of the missing alpha-subunit (α-subunit) of GGTase-II in Leptomonas was probably contributed by the α-subunit of FTase, while beta-subunit (ß-subunit) of GGTase-II was conserved and indicated functional conservation in the evolution of these two kinetoplastids. Therefore the ß-subunit emerges as an excellent target for compounds inhibiting parasite activity in clinical cases of co-infections. We also confirmed that during the evolution to digenetic life style in Leishmania, the parasite acquired capabilities to evade drug action and maintain parasite virulence in the host with the incorporation of short-chain dehydrogenase/reductase (SDR/MDR) superfamily in Rab genes. CONCLUSION: Our study based on whole genome sequences is the first to build comparative evolutionary analysis and identification of prenylation proteins in Leishmania and its sister Leptomonas. The information presented in our present work has importance for drug design targeted to kill L. donovani in humans but not affect the human form of the prenylation enzymes.

Trypanosoma brucei harbours a divergent XPB helicase paralogue that is specialized in nucleotide excision repair and conserved among kinetoplastid organisms.

Conserved from yeast to humans, TFIIH is essential for RNA polymerase II transcription and nucleotide excision repair (NER). TFIIH consists of a core that includes the DNA helicase Xeroderma pigmentosum B (XPB) and a kinase subcomplex. Trypanosoma brucei TFIIH harbours all core complex components and is indispensable for RNA polymerase II transcription of spliced leader RNA genes (SLRNAs). Kinetoplastid organisms, however, possess two highly divergent XPB paralogues with only the larger being identified as a TFIIH subunit in T. brucei. Here we show that a knockout of the gene for the smaller paralogue, termed XPB-R (R for repair) resulted in viable cultured trypanosomes that grew slower than normal. XPB-R depletion did not affect transcription in vivo or in vitro and XPB-R was not found to occupy the SLRNA promoter which assembles a RNA polymerase II transcription pre-initiation complex including TFIIH. However, XPB-R(-/-) cells were much less tolerant than wild-type cells to UV light- and cisplatin-induced DNA damage, which require NER. Since XPB-R(-/-) cells were not impaired in DNA base excision repair, XPB-R appears to function specifically in NER. Interestingly, several other protists possess highly divergent XPB paralogues suggesting that XPBs specialized in transcription or NER exist beyond the Kinetoplastida.

Therapeutic potential of phosphodiesterase inhibitors in parasitic diseases.

Protozoan parasites of the order kinetoplastida are the causative agents of three of the world's most important neglected human diseases: African trypanosomiasis, American trypanosomiasis, and leishmaniasis. Current therapies are limited, with some treatments having serious and sometimes lethal side effects. The growing number of cases that are refractory to treatment is also of concern. With few new drugs in development, there is an unmet medical need for new, more effective, and safer medications. Recent studies employing genetic and pharmacological techniques have begun to shed light on the role of the cyclic nucleotide phosphodiesterases in the life cycle of these pathogens and suggest that these important regulators of cyclic nucleotide signaling may be promising new targets for the treatment of parasitic diseases.

Phage Origin of Mitochondrion-Localized Family A DNA Polymerases in Kinetoplastids and Diplonemids.

Mitochondria retain their own genomes as other bacterial endosymbiont-derived organelles. Nevertheless, no protein for DNA replication and repair is encoded in any mitochondrial genomes (mtDNAs) assessed to date, suggesting that the nucleus primarily governs the maintenance of mtDNA. As the proteins of diverse evolutionary origins occupy a large proportion of the current mitochondrial proteomes, we anticipate finding the same evolutionary trend in the nucleus-encoded machinery for mtDNA maintenance. Indeed, none of the DNA polymerases (DNAPs) in the mitochondrial endosymbiont, a putative α-proteobacterium, seemingly had been inherited by their descendants (mitochondria), as none of the known types of mitochondrion-localized DNAP showed a specific affinity to the α-proteobacterial DNAPs. Nevertheless, we currently have no concrete idea of how and when the known types of mitochondrion-localized DNAPs emerged. We here explored the origins of mitochondrion-localized DNAPs after the improvement of the samplings of DNAPs from bacteria and phages/viruses. Past studies have revealed that a set of mitochondrion-localized DNAPs in kinetoplastids and diplonemids, namely PolIB, PolIC, PolID, PolI-Perk1/2, and PolI-dipl (henceforth designated collectively as "PolIBCD+") have emerged from a single DNAP. In this study, we recovered an intimate connection between PolIBCD+ and the DNAPs found in a particular group of phages. Thus, the common ancestor of kinetoplastids and diplonemids most likely converted a laterally acquired phage DNAP into a mitochondrion-localized DNAP that was ancestral to PolIBCD+. The phage origin of PolIBCD+ hints at a potentially large contribution of proteins acquired via nonvertical processes to the machinery for mtDNA maintenance in kinetoplastids and diplonemids.

Phosphoglycerate kinase: structural aspects and functions, with special emphasis on the enzyme from Kinetoplastea.

Phosphoglycerate kinase (PGK) is a glycolytic enzyme that is well conserved among the three domains of life. PGK is usually a monomeric enzyme of about 45 kDa that catalyses one of the two ATP-producing reactions in the glycolytic pathway, through the conversion of 1,3-bisphosphoglycerate (1,3BPGA) to 3-phosphoglycerate (3PGA). It also participates in gluconeogenesis, catalysing the opposite reaction to produce 1,3BPGA and ADP. Like most other glycolytic enzymes, PGK has also been catalogued as a moonlighting protein, due to its involvement in different functions not associated with energy metabolism, which include pathogenesis, interaction with nucleic acids, tumorigenesis progression, cell death and viral replication. In this review, we have highlighted the overall aspects of this enzyme, such as its structure, reaction kinetics, activity regulation and possible moonlighting functions in different protistan organisms, especially both free-living and parasitic Kinetoplastea. Our analysis of the genomes of different kinetoplastids revealed the presence of open-reading frames (ORFs) for multiple PGK isoforms in several species. Some of these ORFs code for unusually large PGKs. The products appear to contain additional structural domains fused to the PGK domain. A striking aspect is that some of these PGK isoforms are predicted to be catalytically inactive enzymes or 'dead' enzymes. The roles of PGKs in kinetoplastid parasites are analysed, and the apparent significance of the PGK gene duplication that gave rise to the different isoforms and their expression in Trypanosoma cruzi is discussed.

Bodo sp., a free-living flagellate, expresses divergent proteolytic activities from the closely related parasitic trypanosomatids.

We report the characterization of cell-associated and extracellular peptidases of Bodo sp., a free-living flagellate of the Bodonidae family, order Kinetoplastida, which is considered ancestral to the trypanosomatids. This bodonid isolate is phylogenetically related to Bodo caudatus and Bodo curvifilus. The proteolytic activity profiles of Bodo sp. were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing co-polymerized gelatin, casein, hemoglobin, or bovine serum albumin as substrates. The enzymatic complex degraded gelatin better in acidic pH, and under these conditions four proteolytic bands (120, 100, 90, and 75 kDa) were detected in the cellular or extracellular extracts. Two peptidases (250 and 200 kDa) were exclusively detected with the substrate casein. All these enzymes belong to the serine peptidase class, based on inhibition by aprotinin and phenylmethylsulfonyl fluoride. This is the first biochemical characterization of peptidases in a free-living Bodo sp., potentially providing insight into the physiology of these protozoa and the evolutionary importance of peptidases to the order Kinetoplastida as some of these enzymes are important virulence factors in pathogenic trypanosomatids.

Unassigned MURF1 of kinetoplastids codes for NADH dehydrogenase subunit 2.

BACKGROUND: In a previous study, we conducted a large-scale similarity-free function prediction of mitochondrion-encoded hypothetical proteins, by which the hypothetical gene murf1 (maxicircle unidentified reading frame 1) was assigned as nad2, encoding subunit 2 of NADH dehydrogenase (Complex I of the respiratory chain). This hypothetical gene occurs in the mitochondrial genome of kinetoplastids, a group of unicellular eukaryotes including the causative agents of African sleeping sickness and leishmaniasis. In the present study, we test this assignment by using bioinformatics methods that are highly sensitive in identifying remote homologs and confront the prediction with available biological knowledge. RESULTS: Comparison of MURF1 profile Hidden Markov Model (HMM) against function-known profile HMMs in Pfam, Panther and TIGR shows that MURF1 is a Complex I protein, but without specifying the exact subunit. Therefore, we constructed profile HMMs for each individual subunit, using all available sequences clustered at various identity thresholds. HMM-HMM comparison of these individual NADH subunits against MURF1 clearly identifies this hypothetical protein as NAD2. Further, we collected the relevant experimental information about kinetoplastids, which provides additional evidence in support of this prediction. CONCLUSION: Our in silico analyses provide convincing evidence for MURF1 being a highly divergent member of NAD2.

Trypanoplasma borreli cysteine proteinase activities support a conservation of function with respect to digestion of host proteins in common carp.

Trypanoplasma borreli is an extracellular parasite that is transmitted by a leech vector and is naturally found in the blood of cyprinid fish. High parasitemia and associated severe anemia together with splenomegaly are typical of infection of common carp, Cyprinus carpio L. Papain-like cysteine proteinases expressed by trypanosome parasites contribute to the pathogenicity of trypanosomes, and are considered an important target for the development of new trypanocidal drugs. T. borreli is a member of the Parabodonida, sharing a common ancestor with the other Kinetoplastida. We demonstrate the presence of a cysteine proteinase expressed by T. borreli. Alignment of the sequence with other kinetoplastid cysteine proteinase sequences supports the phylogenetic hypotheses based on analyses of ribosomal RNA genes. We expressed the T. borreli cysteine proteinase in Escherichia coli, refolded the purified protein into a biologically active proteinase and showed it has cathepsin L-like activity. Addition of the (non)active proteinase to in vitro-derived carp head kidney-derived macrophages did not significantly modulate macrophage activity. Immunization of carp with the recombinant proteinase did induce a very high increase in proteinase-specific antibodies but only slightly lowered parasitemia. Digestion of host hemoglobin and immunoglobulin by the cysteine proteinase likely contribute to the pathogenicity of T. borreli. The possibility that digestion by the cysteine proteinase of host transferrin could contribute to an innate activation profile of macrophages in vivo is discussed. Our findings suggest a conservation of function with respect to cysteine proteinase activity in the Parabodonida in support of the hypotheses on the phylogeny of the Kinetoplastida.

Protozoan protein tyrosine phosphatases.

The aim of this review is to provide a synthesis of the published experimental data on protein tyrosine phosphatases from parasitic protozoa, in silico analysis based on the availability of completed genomes and to place available data for individual phosphatases from different unicellular parasites into the comparative and evolutionary context. We analysed the complement of protein tyrosine phosphatases (PTP) in several species of unicellular parasites that belong to Apicomplexa (Plasmodium; Cryptosporidium, Babesia, Theileria, and Toxoplasma), kinetoplastids (Leishmania and Trypanosoma spp.), as well as Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis and a microsporidium Encephalitozoon cuniculi. The analysis shows distinct distribution of the known families of tyrosine phosphatases in different species. Protozoan tyrosine phosphatases show considerable levels of divergence compared with their mammalian homologues, both in terms of sequence similarity between the catalytic domains and the structure of their flanking domains. This potentially makes them suitable targets for development of specific inhibitors with minimal effects on physiology of mammalian hosts.

Shedding light on lipid metabolism in Kinetoplastida: A phylogenetic analysis of phospholipase D protein homologs.

Unicellular flagellates that make up the class Kinetoplastida include multiple parasites responsible for public health concerns, including Trypanosoma brucei and T. cruzi (agents of African sleeping sickness and Chagas disease, respectively), and various Leishmania species, which cause leishmaniasis. These diseases are generally difficult to eradicate, with treatments often having lethal side effects and/or being effective only during the acute phase of the diseases, when most patients are still asymptomatic. Phospholipid signaling and metabolism are important in the different life stages of Trypanosoma, including playing a role in transitions between stages and in immune system evasion, thus, making the responsible enzymes into potential therapeutic targets. However, relatively little is understood about how the pathways function in these pathogens. Thus, in this study we examined evolutionary history of proteins from one such signaling pathway, namely phospholipase D (PLD) homologs. PLD is an enzyme responsible for synthesizing phosphatidic acid (PA) from membrane phospholipids. PA is not only utilized for phospholipid synthesis, but is also involved in many other signaling pathways, including biotic and abiotic stress response. 37 different representative Kinetoplastida genomes were used for an exhaustive search to identify putative PLD homologs. The genome of Bodo saltans was the only one of surveyed Kinetoplastida genomes that encoded a protein that clustered with plant PLDs. The representatives from other Kinetoplastida species clustered together in two different clades, thought to be homologous to the PLD superfamily, but with shared sequence similarity with cardiolipin synthases (CLS), and phosphatidylserine synthases (PSS). The protein structure predictions showed that most Kinetoplastida sequences resemble CLS and PSS, with the exception of 5 sequences from Bodo saltans that shared significant structural similarities with the PLD sequences, suggesting the loss of PLD-like sequences during the evolution of parasitism in kinetoplastids. On the other hand, diacylglycerol kinase (DGK) homologs were identified for all species examined in this study, indicating that DGK could be the only pathway for the synthesis of PA involved in lipid signaling in these organisms due to genome streamlining during transition to parasitic lifestyle. Our findings offer insights for development of potential therapeutic and/or intervention approaches, particularly those focused on using PA, PLD and/or DGK related pathways, against trypanosomiasis, leishmaniasis, and Chagas disease.

Novel 8-nitroquinolin-2(1H)-ones as NTR-bioactivated antikinetoplastid molecules: Synthesis, electrochemical and SAR study.

To study the antiparasitic 8-nitroquinolin-2(1H)-one pharmacophore, a series of 31 derivatives was synthesized in 1-5 steps and evaluated in vitro against both Leishmania infantum and Trypanosoma brucei brucei. In parallel, the reduction potential of all molecules was measured by cyclic voltammetry. Structure-activity relationships first indicated that antileishmanial activity depends on an intramolecular hydrogen bond (described by X-ray diffraction) between the lactam function and the nitro group, which is responsible for an important shift of the redox potential (+0.3 V in comparison with 8-nitroquinoline). With the assistance of computational chemistry, a set of derivatives presenting a large range of redox potentials (from -1.1 to -0.45 V) was designed and provided a list of suitable molecules to be synthesized and tested. This approach highlighted that, in this series, only substrates with a redox potential above -0.6 V display activity toward L. infantum. Nevertheless, such relation between redox potentials and in vitro antiparasitic activities was not observed in T. b. brucei. Compound 22 is a new hit compound in the series, displaying both antileishmanial and antitrypanosomal activity along with a low cytotoxicity on the human HepG2 cell line. Compound 22 is selectively bioactivated by the type 1 nitroreductases (NTR1) of L. donovani and T. brucei brucei. Moreover, despite being mutagenic in the Ames test, as most of nitroaromatic derivatives, compound 22 was not genotoxic in the comet assay. Preliminary in vitro pharmacokinetic parameters were finally determined and pointed out a good in vitro microsomal stability (half-life > 40 min) and a 92% binding to human albumin.

Novel scaffolds for inhibition of Cruzipain identified from high-throughput screening of anti-kinetoplastid chemical boxes.

American Trypanosomiasis or Chagas disease is a prevalent, neglected and serious debilitating illness caused by the kinetoplastid protozoan parasite Trypanosoma cruzi. The current chemotherapy is limited only to nifurtimox and benznidazole, two drugs that have poor efficacy in the chronic phase and are rather toxic. In this scenario, more efficacious and safer drugs, preferentially acting through a different mechanism of action and directed against novel targets, are particularly welcome. Cruzipain, the main papain-like cysteine peptidase of T. cruzi, is an important virulence factor and a chemotherapeutic target with excellent pre-clinical validation evidence. Here, we present the identification of new Cruzipain inhibitory scaffolds within the GlaxoSmithKline HAT (Human African Trypanosomiasis) and Chagas chemical boxes, two collections grouping 404 non-cytotoxic compounds with high antiparasitic potency, drug-likeness, structural diversity and scientific novelty. We have adapted a continuous enzymatic assay to a medium-throughput format and carried out a primary screening of both collections, followed by construction and analysis of dose-response curves of the most promising hits. Using the identified compounds as a starting point a substructure directed search against CHEMBL Database revealed plausible common scaffolds while docking experiments predicted binding poses and specific interactions between Cruzipain and the novel inhibitors.

Carbamoyl-phosphate synthetase II in kinetoplastids.

Genes for carbamoyl-phosphate synthetase II (CPS II), the first enzyme of de novo pyrimidine biosynthesis, were cloned from kinetoplastids, Trypanosoma cruzi and Leishmania mexicana. T. cruzi CPS II gene encodes a protein of 1524 amino acids that encompasses the glutaminase and CPS domains, but incorporates neither aspartate carbamoyltransferase nor dihydroorotase. The residue corresponding to lysine 993 of Escherichia coli CPS, a residue that characterizes the CPS inhibited by UMP and that is replaced by tryptophan in those inhibited by UTP, is in kinetoplastids a hydrophilic glutamine, in line with the preferential inhibition by UDP of kinetoplastid CPS II.

cAMP signalling in the kinetoplastid protozoa.

Several species of kinetoplastid protozoa cause major human infectious diseases. Trypanosoma cruzi is responsible for the fatal Chagas disease in large parts of South America, the various species of Leishmania cause a number of different human diseases with millions of patients world-wide, and the African trypanosome Trypanosoma brucei is the agent of human sleeping sickness, a disastrously re-emerging epidemic of fatal infections in Sub-Saharan Africa. Chemotherapy of all of these infections is in a very unsatisfactory state. cAMP signalling pathways in humans have provided interesting drug targets for a number of clinical conditions, from asthma to impotency. Similarly, cAMP signalling in kinetoplastids might offer useful targets for the development of novel antiparasitic drugs, which makes their exploration an urgent need. Current knowledge suggests that cAMP signalling proceeds along very similar pathways in all kinetoplastid pathogens (T. cruzi, the Leishmanias and T. brucei). Their adenylyl cyclases are structurally very different from the human enzymes and appear to function as enzyme-linked cell surface receptors. They might represent the major sensory apparatus of the kinetoplastids, guiding much of their environmental sensing and host/parasite interaction. The cAMP-specific phosphodiesterases of the kinetoplastids are rather similar to those of human cells and might function in similar ways. Essentially nothing is known on downstream effectors of cAMP in the kinetoplastids. Homologues of protein kinase A and its regulatory subunits have been identified, but their biochemical properties seem to be disctinct from that of mammalian protein kinase A.

The structure and replication of kinetoplast DNA.

Kinetoplast DNA (kDNA), the mitochondrial DNA of flagellated protozoa of the order Kinetoplastida, is unique in its structure, function and mode of replication. It consists of few dozen maxicircles, encoding typical mitochondrial proteins and ribosomal RNA, and several thousands minicircles, encoding guide RNA molecules that function in the editing of maxicircles mRNA transcripts. kDNA minicircles and maxicircles in the parasitic species of the family Trypanosomatidae are topologically linked, forming a two dimensional fishnet-type DNA catenane. Studies of early branching free-living and parasitic species of the Bodonidae family revealed various other forms of this remarkable DNA structure and suggested the evolution of kDNA from unlinked DNA circles and covalently-linked concatamers into a giant topological catenane. The replication of kDNA occurs during nuclear S phase and includes the duplication of free detached minicircles and catenated maxicircle and the generation of two progeny kDNA networks that segregate upon cell division. Recent reports of sequence elements and specific proteins that regulate the periodic expression of replication proteins advanced our understanding of the mechanisms that regulate the temporal link between mitochondrial and nuclear DNA synthesis in trypanosomatids. Studies on kDNA replication enzymes and binding proteins revealed their remarkable organization in clusters at defined sites flanking the kDNA disk, in correlation with the progress in the cell cycle and the process of kDNA replication. In this review I describe the recent advances in the study of kDNA and discuss some of the major challenges in deciphering the structure, replication and segregation of this remarkable DNA structure.

DNA topoisomerases in life and death: implications in kinetoplastid protozoa.

Current biomedical research has its focus on the search for newer intervention strategies to control public health impact of parasitic diseases. The dramatic advances of molecular and cellular biology in recent times have provided opportunities for discovering and evaluating molecular targets for drug designing, which now form a rational basis for the development of improved anti parasitic therapy. DNA topoisomerases, the "cellular magicians" involved in nearly all biological processes governing DNA, have emerged as one such biological target. Over the last two decades, interest in topoisomerases has expanded beyond the realm of the basic science laboratory into the clinical arena. This review aims at providing a comprehensive insight into the biology of DNA topoisomerases and also focus on its evolution as a drug target in the unicellular kinetoplastids.