Curr. Issues Mol. Biol. (2019) 31: 45-62. caister.com/cimb DNA Topoisomerases of Kinetoplastid Parasites: Brief Overview and Recent Perspectives

Sourav Saha†, Somenath Roy Chowdhury† and Hemanta K. Majumder*

Infectious Diseases and Immunology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India. *Correspondence: [email protected], [email protected] †Both authors contributed equally.

t DOI: https://dx.doi.org/10.21775/cimb.031.045

Abstract nucleic acid metabolic processes, permanent or Topoisomerases are a group of enzymes that resolve temporary separations of DNA strands cause dif- DNA topological problems and aid in diferent ferent topological constraints. Tese topological DNA transaction processes viz. replication, tran- constraints, if not removed, become disastrous and scription, recombination, etc. inside cells. Tese greatly afect cellular health (Chen et al., 2013). proteins accomplish their feats by steps of DNA DNA topoisomerases are the molecular machines strand(s) scission, strand passage or rotation and inside all kinds of living cells that resolve these subsequent rejoining activities. Topoisomerases of kinds of topological tensions. Not only that, DNA kinetoplastid parasites have been extensively stud- topoisomerases sometimes introduce measured ied because of their unusual features. Te unique topological tensions in the double helical DNA presence of heterodimeric Type IB topoisomerase molecules to aid DNA transaction processes and prokaryotic ‘TopA homologue’ Type IA topoi- (Champoux, 2001). somerase in kinetoplastids still generates immense Te mechanism of action of DNA topoisomer- interest among scientists. Moreover, because of ases involves following major steps: (1) binding of their structural dissimilarity with the host enzymes, topoisomerase to the substrate DNA, (2) cleavage topoisomerases of kinetoplastid parasites are atrac- by trans-esterifcation reaction accompanied by tive targets for chemotherapeutic interventions to the formation of a transient phosphodiester bond kill these deadly parasites. In this review, we sum- between a tyrosine residue in the protein and one marize historical perspectives and recent advances of the ends of the broken strand, (3) strand pas- in kinetoplastid topoisomerase research and how sage/rotation through/around the break leading these proteins are exploited for drug targeting. to change in the linking number and (4) strand religation and release of the enzyme as the DNA is religated (Stewart et al., 1998; Koster et al., 2005; Introduction Champoux, 2001). DNA topoisomerases are ubiquitous enzymes Based on structure and mechanism of action, that help to maintain proper topological states of topoisomerases are classifed as type I and type the double helical DNA molecules and prevent II. Tose enzymes that cleave a single strand of genomic instability inside cell. During replica- DNA and pass or rotate another strand through tion, transcription, recombination, and other vital or about the resultant gap are defned as type I

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topoisomerases and these enzymes change the Type IB topoisomerase of linking number of the substrate DNA in discrete kinetoplastid parasites steps of one or more than one (Champoux, 2001). Type IB topoisomerases are generally monomeric Tey are further classifed either as type IA or as enzymes. But in kinetoplastid parasites, type IB type IB enzyme. Tese two subfamilies have no topoisomerase is heterodimeric in nature (Das et sequence similarities between them and exhibit al., 2004a). Te discovery of bi-subunit type IB dissimilar structures and distinct region character- topoisomerase in kinetoplastid parasites opened istics. Type IA topoisomerase makes a transient up a new avenue in topoisomerase research related covalent complex with the 5′-end of the broken to evolution of type IB topoisomerase family and DNA strand. On the other hand, type IB topoi- its relevance as a drug target for trypanosomiasis somerase forms a covalent intermediate with the therapeutic interventions (Das et al., 2006). 3′-end of the DNA (Champoux, 2001). Te type Type IB DNA topoisomerase activities were frst II topoisomerases act in a manner fundamentally purifed from Leishmania donovani (Chakraborty diferent from either of the type I enzymes. Tey and Majumder, 1988; Chakraborty et al., 1993), require divalent metal ions for their activity and Trypanosoma cruzi (Riou et al., 1983) and Crithidia use ATP to actively transport DNA duplex through fasciculata (Melendy and Ray, 1987). Te purifed a transient enzyme-mediated break in the second active enzymes (65–79 kDa) were ATP independ- duplex of the same DNA strand thus changing ent and found to be sensitive to the topoisomerase linking numbers in steps of two. Prokaryotes I-specifc inhibitor camptothecin (Melendy and and lower eukaryotes encode only a single type Ray, 1987). II topoisomerase whereas vertebrates generally Topoisomerase IB-like gene from the kineto- express two discrete forms of the enzyme, topoi- plastid L. donovani was frst cloned and reported by somerase IIA and IIB (Champoux, 2001). Both Broccoli et al. (1999). Te amino acid sequence of Type IIA and IIB enzymes break the DNA by this gene showed homology with the N-terminal atacking and bonding to the 5′-phosphate, and and central core of other eukaryotic type IB have sequence similarities. Tey are encoded by topoisomerases, but had a variable C-terminus. separate genes, have diferent molecular masses, Interestingly, this ORF was lacking the SKINYL show distinct paterns of expression and perform motif that supplies the catalytic tyrosine (Broccoli diferent cellular functions. Topoisomerase IIA et al., 1999). Villa and co-workers frst reported helps mainly in the DNA replication and chro- the presence of a second small subunit harbouring mosome segregation in proliferating cells and the SKINYL motif in Leishmania parasites (Villa et topoisomerase IIβ is mainly required for proper al., 2003). Similar results were obtained in case of neural development (Deweese and Osherof, African trypanosomes in the same year (Bodley et 2009; Yang et al., 2000; Wang, 2002; Champoux, al., 2003). 2001; Velez-Cruz et al., 2004; McClendona and In general, type IB DNA topoisomerases of Osherof, 2007). kinetoplastid parasites are expressed from two diferent genes and form a heterodimeric active enzyme (Table 10.1). In L. donovani, gene for large Diferent topoisomerases of subunit (LdTOP1L) is present on chromosome kinetoplastid parasites 34 encoding a 636 amino acid polypeptide with Topoisomerases of kinetoplastid parasites are of an estimated molecular weight of 73 kDa and the following kinds: gene for the small subunit (LdTOP1S) is situated on chromosome 4 encoding a 262 amino acid poly- • type IB topoisomerase; peptide with a molecular weight of 29 kDa (Das et • type IA topoisomerase; al., 2004a). Bodley et al., 2003 have also identifed a • type II topoisomerase. heterodimer topoisomerase I in Trypanosoma brucei which is comprised of the 90 kDa large subunit and 36 kDa small subunit (Bodley et al., 2003). Large subunit (LdTOP1L) is closely homologous to the core domain of human topoisomerase I and small

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Table 10.1 Diferent topoisomerase genes of kinetoplastid parasites as annotated in GeneDB database (www. genedb.org/Homepage) (Logan-Klumpler et al., 2012) Protein coding gene Gene DB systematic IDs Length DNA topoisomerase IB LmjF.34.3440 (L. major Friedlin) 1905 base pairs large subunit (L) LdBPK_343220.1 (L. donovani BPK282A1) 1908 base pairs Tb927.4.133 (T. brucei 927) 2091 base pairs TcCLB.508693.20 (T. cruzi) 1995 base pairs DNA topoisomerase IB LmjF.04.0060 (L. major Friedlin) 789 base pairs small subunit (S) LdBPK_040070.1 (L. donovani BPK282A1) 789 base pairs Tb927.9.6940 (T. brucei 927) 822 base pairs TcCLB.506625.110 (T. cruzi) 819 base pairs DNA topoisomerase IA LmjF.21.0125 (L. major Friedlin) 2439 base pairs LdBPK_210180.1 (L. donovani BPK282A1) 2430 base pairs Tb927.10.1900 (T. brucei 927) 2421 base pairs TcCLB.510121.160 (T. cruzi) 2493 base pairs TcCLB.506493.80 (T. cruzi) 2490 base pairs DNA topoisomerase III LmjF.28.1780 (L. major Friedlin) 2601 base pairs LmjF.36.3200 (L. major Friedlin) 2844 base pairs LdBPK_281900.1 (L. donovani BPK282A1) 2604 base pairs LdBPK_363350.1 (L. donovani BPK282A1) 2844 base pairs Tb927.11.9520 (T. brucei 927) 2757 base pairs Tb927.11.9170 (T. brucei 927) 2559 base pairs TcCLB.510901.100 (T. cruzi) 2766 base pairs TcCLB.508851.170 (T. cruzi) 2526 base pairs TcCLB.511589.120 (T. cruzi) 2766 base pairs DNA topoisomerase II LmjF.28.2280 (L. major Friedlin) 4494 base pairs LdBPK_282450.1 (L. donovani BPK282A1) 4497 base pairs BSAL_28010 (B. saltans) 3741 base pairs Tb927.11.11550 (T. brucei 927) 4368 base pairs Tb927.11.11560 (T. brucei 927) 4275 base pairs TcCLB.508699.10 (T. cruzi) 4431 base pairs TcCLB.509203.70 (T. cruzi) 4455 base pairs Mitochondrial DNA LmjF.15.1290 (L. major Friedlin) 3711 base pairs topoisomerase II LdBPK_151310.1 (L. donovani BPK282A1) 3711 base pairs Tb927.9.5590 (T. brucei 927) 3666 base pairs TcCLB.508277.370 (T. cruzi) 3696 base pairs TcCLB.506445.60 (T. cruzi) 3693 base pairs

subunit LdTOP1S contains the phylogenetically the fact that LdTOPIB is a heterodimer whereas the conserved ‘SKXXY’ motif placed at the C-terminal human TOPIB is a monomer. domain of all topoisomerases IB (TOPIB) which Te in vitro reconstitution of the two recombinant harbours a conserved tyrosine residue playing a cru- proteins LdTOP1L and LdTOP1S correspond- cial role in DNA cleavage. Overall, the structure and ing to the large and small subunit forms active catalytic machinery of the two enzymes (LdTOPIB bi-subunit LdTOP1LS/LdTOPIB. LdTOP1L and and human TOPIB) are highly conserved, despite LdTOP1S form a direct 1 : 1 heterodimer complex

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through protein–protein interaction (Das et al., et al., 2004a; Das et al., 2008) and LdTOP1L 2004a). Interaction between the two subunits lead- navigates LdTOP1S by acting as a ‘molecular steer’ ing to the formation of an active complex could (Bose Dasgupta et al., 2008b). Another recent be exploited for development of new therapeutic study reported three nuclear localization signals agents with specifc selectivity (Das et al., 2004a). (NLSs) in LdTOP1B, one in LdTOP1L and two Tis observation leads to the concept that non– in LdTOP1S and both subunits are transported covalent interaction of both subunits is necessary to the nucleus separately. Tey also found that for the activity. Interestingly, deletion of 99 amino LdTOPIB–GFP constructs do not localize to mito- acids from the N-terminus of LdTOP1L results in chondria (Prada et al., 2013a). a protein which failed to interact with the smaller From bioinformatic analysis (multiple sequence subunit. Tis could be atributed to the pres- alignment) and crystal structure of LdTOP1LS ence of many polar residues in this region. Polar (L. donovani topoisomerase IB) captured as a interactions are common between the subunits vanadate complex linking the enzyme to a nicked of dimeric proteins. (Das et al., 2005). It was also DNA, Te crystal structure of the reconstituted L. found that the deletion of the frst 39 amino acids donovani topoisomerase IB (PDB ID 2B9S) reveals from N-terminus of LdTOP1L results in a protein that the three-dimensional confguration of the with decreased cleavage activity and sensitivity to active site is almost similar to that of human topoi- CPT (Das et al., 2005). Tese data argue in favour somerase I. Five amino acids have been identifed of the interpretation that N-terminal amino acids of as active site residues (Davies et al., 2006). Tese the large subunit regulates DNA dynamics during are Arg314, Lys352, Arg410, His453 and Tyr222. relaxation by controlling noncovalent DNA-bind- Functions of these amino acids in the L. donovani ing or by coordinating DNA contacts by the other topoisomerase IB have been confrmed by single parts of the enzyme. Further, it was shown that the site directed mutation studies (Díaz González et al., amino acids 39–456 of LdTOP1L and 210–262 2007b; Ganguly et al., 2009). Among these, Tyr222 of LdTOP1S constitute the minimal functionally resides in the SKXXY motif in the small subunit interacting fragments of LdTOP1LS (Bosedas- and is responsible for the nucleophilic atack on the gupta et al., 2008a) and the LdTOP1L navigates the scissile phosphate group in the DNA backbone and heterodimeric LdTOP1LS to its cellular DNA tar- subsequent formation of phosphotyrosine link- gets (Bose Dasgupta et al., 2008b). A recent report age. Arg410 activates Tyr222 for this nucleophilic also identifed the regions of both subunits that atack with water acting as a specifc base. Mutation interact to each other to form a functional linker in of the histidine at position 453 has been shown to L. donovani topoisomerase IB. Amino acids 175 and alter DNA binding of the enzyme. Of the remaining 180 (RPPVV) of the small subunit interact with a three, Arg314 and Lys352 stand as potential candi- region (amino acids 525–581) of the large subunit dates for acting as the general acid and thereby play (LdTopIL) and have role in sensitivity to campto- a vital role in transesterifcation reaction (Davies et thecin in Leishmania (Prada et al., 2012). Recently al., 2006; Ganguly et al., 2009). it was also shown that LdTOP1LS activity is stimu- Type IB topoisomerases including human lated by ATP (generally Type IB topoisomerases topoisomerase I and vaccinia virus topoisomerase I are ATP independent) in the absence of Mg2+ and share a common catalytic domain with the tyrosine this stimulation does not need ATP hydrolysis. Te recombinases that includes bacteriophage HP1 and ATP binds to a Arg190 residue on LdTOP1L and certain phage integrases and the XerC/D, Cre and stimulates the rate of strand rotation by the enzyme Flp recombinases (Corbet and Berger, 2004). In (Sengupta et al., 2011). prokaryotic tyrosine recombinases XerD, Cre, HP1 LdTOP1B localizes in both nucleus and kineto- integrase and eukaryotic topoisomerases the active plast of L. donovani. Multiple nuclear localization site residues RKHR (H/W)Y and RKRHY resides signals (NLSs) have been mapped in the large on a single monomer. In eukaryotic Flp recombi- subunit of Leishmania TOP1B but no NLS has nase and Leishmania heterodimeric topoisomerase been found in smaller subunits of the enzyme. So IB, the active site tyrosine is provided by one subunit it is likely that the subunits interact in the cytosol and the other residues implicated in catalysis comes before nuclear and kinetoplast importation (Das from another subunit (Chen and Rice, 2003). It may

48 DNA Topoisomerases of Kinetoplastid Parasites Kinetoplastid DNA TopoisomerasesSaha et al.| 155 be possible that the active site in case of kinetoplas- IA mainly prefers catalysing relaxation reaction tid topoisomerase I is created by the association of whereas topoisomerase III favours catenating/ subunits similar to Flp recombinase. Te structural decatenating DNA molecules (Li et al., 2000). diversity of the amino-terminal domain and the linker domain in various eukaryotic topoisomerase Topoisomerase IA IB argues that they have probably evolved by inde- Te presence of topoisomerase IA homologue in pendent gene fusion. Krogh and Shuman described the kinetoplastid parasites is very intriguing (Table that nuclear topoisomerase I enzymes evolved 10.1). Topoisomerase IA is of prokaryotic origin from the bacterial/poxvirus precursor (Krogh and and is not present in eukaryotes. However, kine- Shuman, 2002). However, in contrast to vaccinia toplastid parasites are exceptions in the eukaryotic topoisomerase I, the catalytic tyrosine of human world in this issue since they contain topoisomer- topoisomerase I resides on a small 6 kDa domain ase IA. Tis makes topoisomerase IA of these and is connected to the central core domain by a parasites a fantastic drug target due to its absence 7 kDa coil–coil linker element (Champoux, 2001). in the human host and warrants a thorough func- It now seems simpler to posit that during evolution tional and mechanistic investigation of the type of eukaryotic IB topoisomerase, the kinetoplastid IA topoisomerases of kinetoplastid parasites. Our parasites (Leishmania, Trypanosoma) which arose laboratory is currently involved in characterizing earlier than multicellular eukaryotes gained some topoisomerase IA of L. donovani. regulatory sequences (nuclear localization signals Another group reported topoisomerase IA from and sequences for CPT sensitivity) as split domain T. brucei. T. brucei topoisomerase IA gene is present architecture. Marcote et al. (1999) demonstrated on chromosome 10 (NCBI Reference Sequence: that the fusion of the subunits and insertion of XP_822446/Systematic Name: Tb927.10.1900), additional domains allow for the rapid evolution of 2418 bp in size and encodes an 88.9 kDa predicted new signalling pathways by the incorporation of a protein. Tis enzyme is mitochondrial, essential for novel interaction domain into a pre-existing pep- late theta structure resolution during kinetoplast tide. Horizontal gene transfer has been shown to DNA (kDNA) replication process and is important have an important role in the evolution of compos- for parasite survival (Scocca and Shapiro, 2008). ite proteins (Simonson et al., 2005). It is possible that horizontal gene transfer did play a key role in Topoisomerase IIIα and topoisomerase IIIβ propelling the topoisomerase IB protein among Like other higher eukaryotes, kinetoplastid para- the eukaryotes (Krogh and Shuman, 2002). Tus sites also have two putative DNA topoisomerase we can suggest that eukaryotic monomeric type IB III (paralogous isoforms of type IA topoisomerase) topoisomerase evolved from the common ancestral genes. Very recently, topoisomerase IIIα from T. bi-subunit enzyme by fusion of the two subunits at brucei has been shown to play a critical role in genetic level. antigenic variation by monitoring expression-site- associated VSG switching (Kim and Cross, 2010). Type IA topoisomerases of DNA topoisomerase IIIβ from kinetoplastid kinetoplastid parasites parasite L. donovani (LdTopIIIβ) has been well Type IA topoisomerases difer from type IB studied (Banerjee et al., 2011). LdTopIIIβ has high counterpart in their structures, requirement of sequence homology with human and drosophila Mg2+ for activity, substrate specifcity (type IA topoisomerase IIIβ. It has features typical for topoi- topoisomerases only relax negatively supercoiled somerase IIIβ proteins including the CXXC type DNA) and unlike type IB topoisomerases, type IA of motifs and a long stretch of G and R residues topoisomerases form 5′-phosphotyrosine catalytic at its C-terminus. Te recombinant protein was intermediate (Stewart et al., 1997; Champoux, purifed from in vitro transcription-translation 2001; Wang, 2002). Apart from type IB topoi- reaction and relaxation activity of the enzyme was somerase, three type IA topoisomerases are there characterized. GFP-fused LdTopIIIβ localizes both in the parasite genome (Table 10.1), termed as in nucleus and kinetoplast of L. donovani parasites topoisomerase IA, and two topoisomerase III indicating the involvement of LdTopIIIβ in DNA (topoisomerase IIIα and IIIβ). Topoisomerase processing inside both the parasite organelle.

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Wild-type LdTopIIIβ could complement the are identical in one way that they change the linking topoisomerase III mutant yeast with slow-growth number of DNA in an ATP-dependent manner, the phenotype whereas the C-terminal deletion con- eukaryotic type II enzymes are homodimers, while struct of LdTopIIIβ lacking its Zn-binding domain their bacterial counterparts such as gyrase and topo

was unable to rescue the mutant yeast revealing that IV are A2B2 tetramers; the B and A subunits being the C-terminal 258 amino acids were indispensable the N and C-terminal halves of their eukaryotic for functional complementation of LdTopIIIβ in counterparts (Lynn et al., 1996). vivo. Further investigations are required to deter- Topoisomerase II proteins of L. donovani and B. mine the role of C-terminal end of the LdTopIIIβ saltans were found to be localized both in the nucleus protein in DNA binding (Banerjee et al., 2011). and kinetoplast whereas topoisomerase II activity Te presence of functionally active topoisomer- isolated from C. fasciculata was shown to be immu- ase III proteins in parasites indicates towards its nolocalized in kinetoplast (Das et al., 2001; Gaziová role in DNA metabolism in the parasites, which and Lukes, 2003). Two nuclear topoisomerase II requires further studies and might emerge as a new genes from T. brucei brucei namely TbTOP2α and therapeutic target that can be exploited against the TbTOP2β which are separated by 1.7 kb intergenic deadly parasites (Kim and Cross, 2010; Banerjee et regions have been reported. TbTOP2α is ATP- al., 2011). dependent and is localized in the nucleus. Silencing of this gene by RNAi leads to growth arrest and Type II topoisomerases of causes severe defects in nuclear DNA although kinetoplastid parasites mitochondrial DNA remains unaltered. However, Eukaryotic type II topoisomerases are mainly silencing of TbTOP2β has no efect on the apparent involved in chromosome segregation and conden- phenotype of the cell and precise role of TbTOP2β sation via their decatenation activity. Eukaryotic remains unclear (Kulikowicz and Shapiro, 2006). topoisomerase II is available in two forms, topo TbTop2α is also essential in bloodstream form T. IIα and topo IIβ throughout the phylogeny which brucei as it is responsible for centromere-specifc exhibits strikingly diferent distributions among topoisomerase cleavage activity and has roles in the three domains of life (Gadelle et al., 2003). chromosome segregation. TbTop2β gene does not Te distribution of the enzyme among the kineto- have an obvious role in chromosome segregation plastid parasites is rather trivial. Te kinetoplastid (Obado et al., 2010). Mitochondrial topoisomerase parasites harbours two type II topoisomerases viz. II of Trypanosoma is involved in replication of the topoisomerase II and mitochondrial DNA topoi- massive kinetoplast DNA network. RNAi of mito- somerase II (Kulikowicz and Shapiro, 2006) (Table chondrial topoisomerase II leads to the progressive 10.1). degradation of mitochondria (kinetoplast) in Genes of topoisomerase II have been isolated, Trypanosoma (Wang and Englund, 2001). During cloned and sequenced from C. fasciculata, T. brucei, kDNA replication mini-circle is individually T. cruzi, L. donovani, L. infantum, L. chagasi, and B. released from the network and this phenomenon saltans and also its activity has been purifed from creates holes in the kDNA network. Afer deple- many kinetoplastid parasites (Das et al., 2001, tion of TbTOP2mt by RNAi these holes continue 2004b; Pasion et al., 1992; Strauss and Wang, 1990; to exist, indicating that this enzyme is responsible Fragoso and Goldenberg, 1992; Gaziová and Lukes, for remodelling the kDNA network during replica- 2003). Compared to other higher eukaryotes topoi- tion to maintain a proper mini-circle density and somerase II proteins of the parasites were found network structure (Lindsay et al., 2008). to be smaller, still they share the same functional Kinetoplastid parasites diverged early in the domains and are more homologous to eukary- eukaryotic evolution at the base of the evolution- otic than prokaryotic type II enzymes. Unlike E. ary tree well before the emergence of the metazoan coli DNA gyrase (Wang et al., 1996) none of the kingdom. In spite of having a similarity and identity parasitic type II enzymes are endowed with super- of 31% and 23% with yeast topoisomerase II, L. coiling activity and the enzymatic activities are donovani topoisomerase II (LdTOP2) was found to nearly same as other eukaryotic counterparts (Das complement a temperature-sensitive mutant yeast et al., 2004b). Tough all type IIA topoisomerases strain (Sengupta et al., 2003).

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Just like other eukaryotic topoisomerase II, al., 2005b) while the full-length protein generates the L. donovani enzyme can also be divided into more cleavable complex in the presence of etopo- an N-terminal ATPase, a central DNA-binding side. Like the human enzyme, the core domain of domain and an unconserved C-terminal domain LdTOP2 contains all the elements essential for (Sengupta et al., 2003, 2005a,b). Tough uncon- sequence preference in protein–DNA interaction served, the nuclear localization signal and the but unlike the human enzyme, the C-terminus dimerization domain of this homodimeric enzyme of the parasite enzyme plays an important role in have been mapped in the C-terminus. Te C-ter- the protein–drug interaction for the in vitro topoi- minus also contains a stretch of 60 amino acids not somerase II cleavage reaction. present in the human host. Terefore this region can be exploited to develop antileishmanial targets (Sengupta et al., 2003). Te parasite enzyme has a Exploiting the topoisomerases greater afnity for DNA and was also stable at a very of kinetoplastid parasites for high salt concentration compared with its human therapeutic interventions host. Tese fndings were quite consistent with the During its catalytic cycle, topoisomerase gener- greater susceptibility of the parasite protein to the ates breaks in the DNA and ataches itself to the anti-topoisomerase II agents. Tis is because of newly generated DNA 3′ (type IB topoisomerases) the fact that an enzyme with more afnity towards or 5′ termini (all other topoisomerases) via phos- DNA would perform more DNA cleavage and photyrosyl bonds. Under normal physiological thus a greater chance of being trapped in that state conditions, these covalent enzyme–DNA cleavage by an anti-topoisomerase II drug (Sengupta et al., complexes are short-lived catalytic intermediates 2005b). Te N-terminal 385 amino acid residues of and are tolerated by the cell. However, conditions LdTOP2 were found to possess the ATPase activity. that signifcantly increase the physiological con- Although the ATPase activity resides in the frst 385 centrations or lifetime of these breaks unleash a amino acid residues, only a larger protein was found multitude of deleterious side-efects. Tus, all to mimic the full-length enzyme kinetics in in vitro topoisomerases are fundamentally dualistic in assay (Sengupta et al., 2005a). Tis characteristic of nature. Although they catalyse essential reactions the parasite protein was found to be in contrast to in the cell, they possess an inherent dark side capa- the human enzyme where a smaller protein also has ble of inficting great harm to the genome of an ATPase kinetics similar to the full length enzyme organism. Topoisomerase-targeting therapeutics (Campbell and Maxwell, 2002) except for the fact currently in use act by stabilizing these covalent that a smaller fragment (1–420 amino acids) fails topoisomerase–DNA complexes. to be hyper stimulated by DNA. Most interestingly, Te known topoisomerase targeting compounds the N-terminal 385 amino acids of the parasite pro- can be divided into two classes, class I and class II tein were also found to be inhibited by etoposide (Liu, 1989; Wang, 1994). Te class I inhibitors have which showed that etoposide in addition to being a been referred to as ‘topoisomerase poisons’ where poison for the parasite enzyme was also a catalytic as the class II compounds are referred to as ‘cata- inhibitor. Te study identifes specifc amino acids lytic inhibitors’. Te class I drugs act by stabilizing such as Asn65, Asn69, Asn96 and Asp130 of the para- the covalent topoisomerase-DNA covalent com- site protein that are involved in the interaction with plexes. Te class II drugs interfere with catalytic ATP and etoposide (Sengupta et al., 2005a). Te function of DNA topoisomerase without trapping active site tyrosine implicated in DNA breakage and the covalent complexes. A major determinant of rejoining for L. donovani topoisomerase II has been cytotoxicity for the class I drug is the conversion of mapped to be Tyr775 (Sengupta et al., 2005b).Tis a latent single- or double-stranded break in a drug– tyrosine is the only residue in the parasite protein, topoisomerase-DNA complex into an irreversible which is involved in the trans-esterifcation reac- double-stranded break. Replication and transcrip- tion and is also homologous to the Tyr804 of human tion are the key cellular processes that drive this (Tsai-Pfugfelder et al., 1988). Surprisingly, the conversion. However, for class II topoisomerase II C-terminal truncation mutants of the parasite pro- drugs, processes other than replication might also tein failed to be inhibited by etoposide (Sengupta et be involved. Cell killing by class II topoisomerase II

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drugs may involve arresting cell cycle progression. Trypanosoma topoisomerase IB remain scarce. Traversing of eukaryotic cells through cell division 2–6 dimethyl-9-hydroxyellipticinium was shown in the absence of functional DNA topoisomerase to inhibit the relaxation activity of Trypanosoma II can lead to aneuploidy and chromosomal break- cruzi topoisomerase IB (Douc-Rasy et al., 1983). age. For class I drug, cytotoxicity increases with Nonetheless, few studies have been made to increasing cellular level of target enzyme where as observe the efect of diferent eukaryotic and bac- for class II drugs opposite is true. Tus increased terial topoisomerase I inhibitors upon the growth levels of topoisomerases render cells hypersensi- of Trypanosoma species (Zuma et al., 2011; Jobe tive to enzyme poisons but resistant to inhibitors. et al., 2012). Mostly, contribution has been made Conversely, decreased enzyme levels render cells to develop topoisomerase IB inhibitors targeted resistant to poison but hypersensitive to inhibitors. to Leishmania species. Such inhibitors come from a vast spectrum of chemical identity (Table 10.2). Topoisomerase IB inhibitors Te mode of inhibition primarily follows two Topoisomerase IB of kinetoplastids, being a het- distinct mechanisms as conferred in the preceding erodimeric enzyme, has added a new paradigm in section. Tey either hamper the catalytic activity the quest of antitrypanosomatid agents as we have of the enzyme (termed as ‘catalytic inhibitors’) or already discussed in the earlier section of this chap- stabilize DNA–topoisomerase cleavage complex ter. Continuous efort is being made to identify (termed as ‘poisons’). Tough such kind of classif- inhibitors, both from natural and synthetic origin, cation is not available for all the inhibitors reported which selectively target bi-subunit topoisomerase to date, few sporadic theories pertaining to the IB of trypanosomatids with negligible or no impact extent of inhibition have been established. Faty upon host enzyme. Te extent of enzyme inhibition acids are one of the major groups of compounds varies with structure and nature of the compound. studied thoroughly in this regard (Carballeira et Unfortunately, reports on specifc inhibitors of al., 2009). Two parameters viz. (1) carbon chain

Table 10.2 Compounds (natural/synthetic) targeting kinetoplastid topoisomerase IB Compound Chemical identity Kinetoplastid Reference (2R,5Z,9Z)-2-METHOXY-25-methyl- α-Methoxylated Δ5,9 Fatty L. infantum Carballeira et al. (2016) 5,9-hexacosadienoic acid and Acids (2R,5Z,9Z)-2-methoxy-24-methyl-5,9- hexacosadienoic acid 16α-Hydroxycleroda-3,13(14)Z-dien- Clerodane diterpenoids L. donovani Misra et al. (2010) 15,16-olide 2-methoxy-5,9-eicosadienoic acid, α-methoxylated fatty acid L. donovani Carballeira et al. (2013) 2-methoxy-5,9-eicosadiynoic acid and acetylinic analogue 2-Octadecynoic acid, 2-hexadecynoic 2-alkylnoic fatty acid L. donovani Carballeira et al. (2012a,b) acid, 2-tetradecynoic acid 3,4-Dihydroxyphenyl, derivative of Furopyridinediones L. donovani Mamidala et al. (2016) arylidenefuropyridinediones 3–3′-Di-indolylmethane Acid condensation product L. donovani Roy et al. (2008) of indole-3-carbinol 3β,6β,16β-trihydroxylup-20(29)-ene Lupane triterpene L. amazonensis Teles et al. (2015) 5-Nitrofuran and 5-nitroimidazole 1,3,4-Thiadiazole Leishmania Poorrajab et al. (2009) analogues of N-substituted-piperazinyl- derivatives spp. 1,3,4-thiadiazoles Anthra[1,2-d]imidazole-6,11-dione Imidazole fused L. donovani Chaudhuri et al. (2007) derivatives anthraquinone derivatives Baicalein, luteolin, quercetin Flavonoids L. donovani Das et al. (2006) Berberin Poliheterocyclics L. braziliensis Vennerstrom et al. (1990), L. donovani Marquis et al. (2003)

52 DNA Topoisomerases of Kinetoplastid Parasites Kinetoplastid DNA TopoisomerasesSaha et al.| 159

Table 10.2 Continued Compound Chemical identity Kinetoplastid Reference Bisbenzylisoquinoline alkaloid bisbenzylisoquinoline L. donovani Kumar et al. (2016) alkaloid Camptothecin Quinoline alkaloid T. brucei, Bodley and Shapiro (1995) L. donovani Copper salisylaldoxime (CuSAL) Transition metal complex L. donovani Singh et al. (2017) Diamidine compounds including Diamidines L. donovani Yang et al. (2016) pentamidine and its analogue DB75 Dichloroacetamido bromobenzyl Indolyl quinolines L. donovani Ray et al. (1997) bromoindolyl bromoquinoline Dihydrobetulinic acid Pentacyclic triterpenoid L. donovani Chowdhury et al. (2003) Diospyrin, naphthalene derivatives Naphthoquinones L. donovani Ray et al. (1998); T. brucei Ganapaty et al. (2006) T. cruzi Disuccinyl betulin, Diglutaryl Derivatives of betulin, a L. donovani Chowdhury et al. (2011) dihydrobetulin, Disuccinyl dihydrobetulin natural triterpene Hoechst-33258, Hoechst-33342 Bis-benzimidazoles L. donovani Walker and Saravia (2004) Indotecan (LMP400), AM13–55 Indenoisoquinolines L. infantum Balaña-Fouce et al. (2012) N-6 3-aminopropyl indenoisoquinolines, Indenoisoquinolines T. brucei Bakshi et al. (2009) N-6 3-imidazolylprolyl Indenoisoquinolines, alkylamine repeats N-6 N-benzyl 2, 2′α3,3′,5′, 6’,7′,7α,α′- Spirooxindole derivative L. donovani Saha et al. (2016) octahydro-2methoxycarbonyl-spiro [indole-3, 3′-pyrrolizidine]-2 one Niranthin, Lyoniside, Saracoside Lignan glycosides L. donovani Chowdhury et al. (2012), Saha et al. (2013) Peganine hydrochloride dihydrate Peganine hydrochloride L. donovani Misra et al. (2008) dihydrate Pentostam, glucantime Pentavalent antimony L. donovani Chakraborty and Majumder (1988) Putranoside-D, putranoside-A, Saponin L. donovani Kumar et al. (2014) putranoside-A methyl ester Rebeccamycin Indocarbazoles T. brucei Deterding et al. (2005) Tetrahydro indeno-1,5-naphthyridines Indeno-1,5-naphthyridines L. infantum Tejería et al. (2016) and indeno[1,5]naphthyridines Topotecan, gimatecan, pro-drug Quinoline alkaloid L. infantum Prada et al. (2013b) irinotecan and active metabolite SN38 Voacamine Indole alkaloid L. donovani Chowdhury et al. (2017) L. amazonensis T. cruzi length and, (2) amount of unsaturation are crucial al., 2011). Similarly, icosenoic acid is more potent that govern the DNA relaxation activity by faty than heptadecenoic acid (Carballeira et al., 2009). acids. If the nature of unsaturation is kept constant, On the other hand, if the chain length is kept con- inhibition capability becomes proportional with stant, unsaturated faty acids show more inhibition the carbon chain length. Tis correlation is exem- than their saturated counterparts. For this reason, plifed by 2-alkanoic acids. 2-Octadecynoic acid 2-methoxy-heptadecynoic acid shows higher inhi- shows highest inhibition followed by 2-hexacecy- bition than 2-methoxy-heptadecenoic acid. Teir noic acid and 2-tetradecynoic acid (Carballeira et saturated counterpart 2-methoxy-heptadecanoic

53 160 | DNASaha Topoisomeraseset al. of Kinetoplastid Parasites Saha et al.

acid shows no inhibition at all. Probably the weak required to establish these fndings in a form of a intermolecular interactions between enzyme active theory. In the succeeding subsections, we will now site and unsaturated bonds are responsible for deal with two fundamental models of topoisomer- this. In contrast, there are factors that negatively ase IB inhibition. regulate the enzyme inhibition capability of faty acids. Presence of ciscyclopropane group is one Catalytic inhibitors of topoisomerase IB such factor. Another theory suggests that rational Catalytic inhibitors show a general trend of inter- modifcation of core structure of known inhibi- acting with the free enzyme. Teir inhibitory efect tors improves inhibition efcacy. Chaudhuri et al. markedly increases when they are preincubated (2007) reported that if imidazole group is fused with the free enzyme prior to DNA substrate with anthraquinone derivatives, it improves the addition under in vitro conditions. Tese kinds of

efciency. Tis improved efcacy is probably due to inhibitors increase the KM and follow competitive the variation in pKa value and electronegativity of model of inhibition. However, they cannot bind the side-chain nitrogen. But it is to be remembered with DNA–topoisomerase cleavage complex and

all such inhibitors do not necessarily impart cyto- hence do not alter Vmax value. Several compounds toxicity upon parasites. Tough good inhibitors come under the aegis of catalytic inhibitors (Table generally refect the same trend with promastigotes 10.2). Derivatives of betulin such as disuccinyl and amastigotes, probability still exists that they betulin, diglutaryl dihydrobetulin, and disuccinyl may not be fruitful in all cases. Normally, faty acids, dihydrobetulin (Chowdhury et al., 2011), putra- being amphipathic, get incorporated into lipid noside-D, putranoside-A, putranoside-A methyl membranes. Tey perturb the structural integrity ester (Kumar et al., 2014), spirooxiindole derivative of lipid bilayer resulting in cytotoxicity (Carbal- (Saha et al., 2016), few bisbenzylisoquinoline alka- leira et al., 2016). Tis argument is corroborated by loids (Kumar et al., 2016) are some of the examples. the fnding that presence of triple bond along with All these compounds interact with topoisomerase α-methoxylation enhances enzyme inhibition. But IB of Leishmania. However their binding stoichiom- surprisingly, α-methoxylation decreases the cyto- etry varies. As an example, three betulin derivatives toxicity towards promastigotes. It is reported that developed by Chowdhury et al. (2011) show 1:1 despite being potential inhibitors of topoisomerase binding stoichiometry with the enzyme whereas IB, methoxylated faty acids are not signifcant another catalytic inhibitor, N-benzyl-octahydro-2- enough as far as parasite viability is concerned. methoxycarbonyl-spiro[indole-3,3ʹ-pyrrolizidine]- Tere is no general theory to establish the correla- 2-one, shows a 1:2 stoichiometry (Saha et al., tion between faty acids and their antiproriferative 2016). Tese catalytic inhibitors reversibly bind efect upon parasite. Few studies suggest that this with the enzyme and abrogate the DNA-enzyme efect is probably due to inhibited uptake of glu- covalent complex formation. cose or leucine by the protozoa (Chaudhuri et al., 1986). Finally, the most important concern is the Topoisomerase IB poisons host cytotoxicity. Situations ofen prevail where a Inhibitors falling under this group tend to bind particular inhibitor shows signifcant inhibition of either solely with DNA–enzyme covalent complex parasite enzyme and induces cell death. But at the or with both free enzyme and DNA–enzyme com-

same time, the compound exhibits similar or more plex. Tey may decrease both Km and Vmax values toxicity to host topoisomerase IB. Carballeira et al. acting as uncompetitive inhibitor or may decrease

(2016) recently reported that few methoxylated only Vmax value keeping Km unaltered acting as faty acids show more inhibition of human topoi- non-competitive inhibitor. Many such compounds, somerase IB than the parasite enzyme. Tey showed including camptothecin, have been established to that preference of an inhibitor is shifed from para- date as Leishmania topoisomerase IB poisons (Table site enzyme towards host enzyme due to increase 10.2). Unlike catalytic inhibitors, topoisomerase in alkyl chain length. Short chain faty acids are poisons do not abrogate the DNA-enzyme covalent beter inhibitors of Leishmania enzyme than human complex formation, rather stabilize the complex. topoisomerase and the case gets reversed for long Such kind of DNA–enzyme adducts ultimately chain faty acids. However, more investigation is cause DNA single-strand breaks. Tese breaks are

54 DNA Topoisomerases of Kinetoplastid Parasites Kinetoplastid DNA TopoisomerasesSaha et al.| 161 subsequently converted to double-strand breaks have been established to possess DNA intercalation upon collision with transcription or replication property at 300 μM (Das et al., 2006). machineries. Double-strand breaks are detrimental to the parasite. Te cell cycle progression is arrested Topoisomerase II inhibitors triggering programmed cell death of the parasite. All Topoisomerase II of the kinetoplastid parasites are these poisons can be further classifed into two sub- homodimeric enzymes similar to higher eukaryotes classes depending upon their capability to inhibit including human. But it is a crucial enzyme for the the religation reaction. Camptothecin (Das et al., parasites because of its involvement in replication 2006), lyoniside, saracoside (Saha et al., 2013), of kinetoplast DNA network (kDNA) inside mito- niranthin (Chowdhury et al., 2012), DIM (Roy et chondria (Shapiro et al., 1995; Das et al., 2008). al., 2008) are some of the poisons which inhibit Hence, topoisomerase II has also been explored the religation action. In contrast, baicalein, luteolin as a promising target for antiparasitic therapeutics cannot inhibit this topoisomerase IB-mediated reli- (Table 10.3). Interestingly, pentamidine was later gation step (Das et al., 2006). Moreover, there are found to also be an inhibitor of topoisomerase few examples of topoisomerase poisons which not II (Singh et al., 2007). A distinct aspect of this only binds with DNA-enzyme covalent complex enzyme in trypanosomatids is the discrete exist- but also intercalate into the DNA itself at a very ence of nuclear and kinetoplast topoisomerase II high concentration. Quercetin, bicalein, luteolin (Kulikowicz and Shapiro, 2006). Some compounds

Table 10.3 Compounds (natural/synthetic) targeting kinetoplastid topoisomerase II Compound Chemical identity Kinetoplastid Reference 3,5-Bis(4-chlorophenyl)–7- Isobenzofuranone L. donovani Mishra et al. (2014) hydroxyisobenzofuran-1(3H)-one derivatives and (4-bromo)-30-hydroxy-50-(4- bromophenyl)-benzophenone 5-Nitrofuran and 5-nitroimidazole 1,3,4-Thiadiazole Leishmania spp. Poorrajab et al. (2009) analogues of N-substituted-piperazinyl- derivatives 1,3,4-thiadiazoles Anilinoacridine compounds Anilinoacridine L. chagasi Werbovetz et al. (1994) Anilinoacridine compounds Anilinoacridine T. brucei Gamage et al. (1997) Dichloroacetamido bromo benzyl Indolyl quinolines L. donovani Ray et al. (1997) bromoindolyl bromo quinoline Dihydrobetulinic acid Pentacyclic triterpenoid L. donovani Chowdhury et al. (2003) Doxorrubicin Anthracyclins L. donovani Singh and Dey (2007) Enoxacin, Ciprofoxacin Fluoroquinolones L. panamensis Romero et al. (2005), Cortázar et al. (2007) Etoposide, teniposide Podophylotoxins L. donovani Sangeeta et al. (2005a,b) Lute Olin Flavonoids L. donovani Muttra et al. (2000) Monoacid and monoacid analogues 4-nitro-benzo- L. chagasi Slant et al. (1996) isoquinolinedione and derivatives Ofoading, ciprofoxacin, ofoading Fluoroquinolones T. brucei Neonates et al. (1999, 2003) Norfoxacin, ofoxacin Fluoroquinolones T. cruzi Gonzales-Perdomo et al. (1990) Novobiocin Aminocoumarin antibiotic L. donovani Singh et al. (2005) Novobiocin Aminocoumarin antibiotic T. cruzi Gonzales-Perdomo et al. (1990) Pentamidine Diamidines L. donovani Singh and Dey (2007)

55 162 | DNASaha Topoisomeraseset al. of Kinetoplastid Parasites Saha et al.

diferentially target nuclear and kinetoplast topoi- perform its catalytic cycle. Catalytic inhibitors of somerase II whereas some have similar preferences topoisomerase II have two diferent mechanisms. for both. As for example, one subset of mitonafde Tey either bind with the free enzyme preventing analogues promotes kDNA linearization in Leish- it to sit on DNA substrate or inhibit the ATPase mania and another subset afects activity of nuclear activity of the enzyme by interacting with ATPase topoisomerase II (Slunt et al., 1996). Moving onto domain. Since they abrogate topoisomerase–DNA anilinoacridines, they have similar efect for both interaction, naturally cleavage complex formation the enzymes. Tis diferential activity is primar- is blocked. Mishra et al. (2014) reported that two ily controlled by the presence and position of the derivatives of isobenzofuranone inhibit L. dono- nitro groups in the compounds. Now moving vani topoisomerase II by interfering at its catalytic onto host toxicity, similar scenario is encountered centre but neither have any efect in ATPase activity with topoisomerase II just like we saw with topoi- nor do they intercalate within substrate DNA. somerase IB. Host toxicity is a critical issue as far as topoisomerase II targeted therapeutic develop- Topoisomerase II poisons ment is concerned. Few researches have shown Topoisomerase II poisons stabilize enzyme- that parasitic topoisomerase II is more closely kinetoplast DNA cleavable complex and disrupt related to mammalian counterpart rather than cleavage-religation equilibrium. Tis irreversible prokaryotic topoisomerase II which has made the covalent association between topoisomerase II and situation more complex. Tis similarity increases DNA ultimately cause double-strand breaks which the possibility for a protozoan enzyme inhibitor to are extremely cytotoxic for the parasites. Luteolin, inhibit host enzyme (Werbovetz et al., 1992). Few quercetin (Mitra et al., 2000), some mitonafde compounds tend to inhibit both parasite and host analogues fall under this category. Tey are further enzyme at comparable range. Compounds such classifed as intercalating and non-intercalating as anilinoacridine, protoberberine coralyne, bis- poisons. Some poisons can intercalate into DNA by benzimidazoles, diamidine diminazene, etc. show virtue of their planar aromatic region (Slunt et al., equal efect on both parasite and human enzymes 1996). During interaction, a local distortion occurs whereas ellagic acid has preference for macrophage which resembles an intermediate of the topoi- topoisomerase II. Nevertheless second generation somerase II catalytic cycle. If an inhibitor favours fuoroquinolones such as enoxacin and ciprofoxa- that intermediate formation, cleavage complex is cin, favonoid quercetin target L. panamensis DNA stabilized. Amonafde and one mitonafde ana- topoisomerase II with substantially less toxicity logue have been reported to possess this property. towards macrophage enzyme (Cortazar et al., 2007). Naturally, all poisons do not have this capacity to Moving onto Trypanosoma, just like topoisomerase get intercalated. A classical example of non-inter- IB, reports on Trypanosoma-specifc topoisomer- calating Leishmania topoisomerase II poison is ase II inhibitors are substantially meagre. Efects etoposide (Sengupta et al., 2005b), which is a well- of few bacterial (ofoxacin, novibiocin, etc.) and known anticancer drug. Surprisingly, etoposide can eukaryotic (aclarubicin, idarubicin, mitoxantrone, also inhibit the ATPase activity of the recombinant merbarone, etc.) topoisomerase II inhibitors on N-terminal domain of L. donovani topoisomerase growth of Trypanosoma have been studied. But II (Sengupta et al., 2005a). But etoposide has no whether these compounds inhibit Trypanosoma impact upon parasite viability. topoisomerase II is not known so far. Topoisomer- ase II inhibitors are also fundamentally subdivided Dual inhibitors of topoisomerase IB into two subclasses; namely, catalytic inhibitors and and topoisomerase II poisons. In the succeeding section, we will take a Tere are few compounds which inhibit both closer view of these two kinds of inhibitors mostly type IB and II topoisomerases of Leishmania. in a Leishmania purview. Nonetheless, there is no theory available to date to functionally connect the structure of an inhibi- Catalytic inhibitors of topoisomerase II tor with dual inhibition potential. Few indolyl Topoisomerase II is fundamentally diferent from quinoline analogues namely 2-(2′-dichloro- topoisomerase IB in its requirement of ATP to acetamidobenzyl)-3-(3′-indolyl)-quinoline,

56 DNA Topoisomerases of Kinetoplastid Parasites Kinetoplastid DNA TopoisomerasesSaha et al.| 163

2-(2′-dichloroacetamido-5′-bromobenzyl)-3′- Te search also continues for novel RNA topoi- [3′-(5′-bromoindolyl]-6-bromo quinoline, and somerase activities in species of Leishmania and 2-(2′-acetamido benzyl)–3-(3′-indolyl)-quinoline Trypanosoma. Tis kind of non-canonical function (Ray et al., 1997), a pentacyclic triterpenoid viz. of type IA topoisomerases was frst reported for dihydrobetulinic acid (DHBA) (Chowdhury et al., Escherichia coli topoisomerase III (Wang et al., 2003) inhibit both the enzyme. DHBA is a catalytic 1996) and later in human topoisomerase 3β (Stoll inhibitor of both the enzymes. It is also cytotoxic et al., 2013; Xu et al., 2013). An extensive genetic for the parasites. Tough there are no reports on recombination among the 10,000 mini-circles, cre- Trypanosoma-specifc dual inhibitors, antiprolif- ating novel guide RNAs followed by RNA editing, erative efects of several eukaryotic dual inhibitors might require distinct RNA topoisomerase. have been investigated upon T. cruzi (Lacombe et Te major obstacle of kinetoplastid topoisomer- al., 2014). ase research is over-expressing purifed recombinant topoisomerases and elucidating their crystal struc- ture. In this way, structural information gained from Conclusion and future crystallographic studies can contribute to a beter perspectives understanding of the molecular mechanisms of Tere has been immense progress in the under- enzyme action in vivo, including their interaction standing of the biology of kinetoplastid parasites with inhibitors and poisons screened from natural especially of parasitic topoisomerases. Te avail- or synthetic sources. Tis will cause a paradigm shif ability of the Tritryp genome database has provided in the quest to target the topoisomerases selectively signifcant impetus towards the study of these as a means of therapeutic control of the parasites. enzymes and also provided necessary momen- tum to the much needed identifcation of new Acknowledgements targets for anti-leishmanial therapeutics. Scien- We thank Dr Samit Chatopadhyay, the Director of tists throughout the world including the author’s our Institute (CSIR-Indian Institute of Chemical laboratory have contributed signifcantly towards Biology), for his interest in this work. Tis work was the understanding of structures and activities of supported by Department of Science and Technol- these parasitic topoisomerases and established ogy (DST) Indo-Brazil project (DST/INT/Brazil/ Leishmania topoisomerases as potential drug tar- RPO-01/2009/2), Raja Ramanna Fellowship gets for leishmaniasis (kala-azar). Topoisomerase (DAE, Government of India) and NASI-Senior genes and proteins characterized from these lower Scientist Platinum Jubilee Fellowship to HKM. eukaryotes appear to share many characteristics associated with their human homologues. But References certain striking diferences, including diferent Bakshi, R.P., Sang, D., Morrell, A., Cushman, M., and enzyme activity requirements, variable catalytic Shapiro, T.A. (2009). Activity of indenoisoquinolines against African trypanosomes. Antimicrob. Agents sites and diferent sensitivities to topoisomerase Chemother. 53, 123–128. htps://doi.org/10.1128/ poisons, provide insight for the development of AAC.00650-07. topoisomerase-directed antiparasitic therapeutics. Balaña-Fouce, R., Prada, C.F., Requena, J.M., Cushman, M., It has been established by several studies that the Pommier, Y., Álvarez-Velilla, R., Escudero-Martínez, J.M., Calvo-Álvarez, E., Pérez-Pertejo, Y., and Reguera, inhibitors of topoisomerases convert these essential R.M. (2012). Indotecan (LMP400) and AM13–55: two enzymes into intracellular proliferating cell toxins novel indenoisoquinolines show potential for treating and thereby provide a good tool for preferentially visceral leishmaniasis. Antimicrob. Agents Chemother. killing of the highly replicative parasite cells within 56, 5264–5270. htps://doi.org/10.1128/AAC.00499- 12. the host. However, future challenges are to over- Banerjee, B., Sen, N., and Majumder, H.K. (2011). come the problem related to drug resistance for Identifcation of a functional type IA topoisomerase, development of novel anti-leishmanial therapeu- LdTopIIIβ, from Kinetoplastid parasite Leishmania tics. In this context, the approach of combinatorial donovani. Enz. Res. 2011 (Article ID 230542). htps:// doi.org/10.4061/2011/230542. therapy becomes very efective and identifcation of Berger, J.M., Gamblin, S.J., Harrison, S.C., and Wang, new drugs and new targets is absolutely warranted J.C. (1996). Structure and mechanism of DNA to combat parasite menace in the foreseeable future.

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