Type I DNA Topoisomerases - American Chemical Society

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Type I DNA Topoisomerases Giovanni Capranico,*,† Jessica Marinello,† and Giovanni Chillemi‡ †

Department of Pharmacy and Biotechnology, University of Bologna, Via Belmeloro 8/2, 40126 Bologna, Italy SCAI SuperComputing Applications and Innovation Department, Cineca, Via dei Tizii 6, 00185 Rome, Italy

‡

ABSTRACT: DNA topoisomerases constitute a large family of enzymes that are essential for all domains of life. Although they share general reaction chemistry and the capacity to govern DNA topology and resolve strand entanglements during fundamental molecular processes, they are characterized by differences in their structural organization, modes of enzymatic catalysis, and biological functions. Moreover, hundreds of compounds interfere with bacterial and/or eukaryotic enzymes, some of which are effective drugs for the treatment of infectious diseases and cancers. Research over the past decade has focused on the biological functions of DNA topoisomerases, and several findings have revealed unexpected roles of type I DNA topoisomerases, a subclass of these enzymes, in regulating gene expression and DNA and chromatin conformations. These new findings highlight that type I topoisomerases are still interesting targets for drug discovery for the treatment of several human diseases, including multidrugresistant infections and genetic disorders.

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DNA TOPOISOMERASES DNA topoisomerases constitute a unique family of nuclear enzymes that regulate basic functions of the genome by changing the topology of nucleic acid strands, particularly DNA. They are essential enzymes that are present in all living systems (eukaryotic, prokaryotic, and archaea cells) and certain viruses. The necessity of topoisomerases is due to the architectural organization of the genome. The long DNA duplex must be compacted in a much smaller nucleus of a eukaryotic cell or the nucleoid space of bacteria, increasing the likelihood of disordered DNA entanglements and knots that can constitute a serious obstacle for fundamental DNA functions. Thus, DNA topoisomerases are essential because they prevent the formation of such entanglements by governing supercoiling and other topological conformations of DNA. Additionally, the conformational dynamics of the DNA template can significantly affect transcription, replication, and other chromatin functions; therefore, these enzymes play basic roles in the regulation of many DNA transactions. Several types of DNA topoisomerases have been identified in different species since the discovery of the first enzyme, the Escherichia coli DNA topoisomerase I (the ω protein), in 1971.1 DNA topoisomerases constitute a family of five groups of enzymes that are based on structures and catalytic activity, which has been enlarged by the recent discovery of other members.2,3 DNA cutting is an obligatory step for the enzyme to change DNA topology, and all DNA topoisomerases cut DNA strands by nucleophilic attack of a tyrosine residue of the enzyme active site that remains covalently linked to a strand terminus (Figure 1). All topoisomerases are primarily classified based on the number of strands they cut: type I and type II enzymes cut one and two DNA strands, respectively. Enzymes with an odd Roman numeral belong to the type I class, whereas those with an even Roman numeral belong to the type II class. The enzymes are then subdivided into A, B, and C subfamilies based on their mechanism of catalysis and structural domain © 2017 American Chemical Society

organization. Type I-A enzymes have a tyrosine residue that remains linked to the 5′ end of the cut strand, whereas in types I-B and I-C enzymes, the tyrosine residue remains linked to the 3′ end (Table 1 and Figure 1). The transient covalent link of the protein to the broken DNA prevents the inadvertent release of broken DNA strands that could otherwise damage the genome. After DNA cleavage, the enzyme changes DNA topology by either one of two known mechanisms: by passing another strand through the cleaved DNA (type I-A) or by allowing the rotation of the cut strand around the uncut strand (type I-B) (Figure 2 and Table 1). All type I topoisomerases are monomeric enzymes and do not require ATP for catalysis, with the exception of reverse gyrases, which use ATP to introduce positive supercoils in a DNA duplex (Table 2). Currently, clinically relevant topoisomerase inhibitors are extremely selective for one type of enzyme and can be undoubtedly used as “targeted therapy.” Quinolones are good antibacterial drugs and specifically act on bacterial type II topoisomerases, whereas anthracyclines, epipodophyllotoxins, and amsacrines inhibit mammalian type II enzymes. In the case of type I enzymes, a large body of evidence accumulated over the past few decades has established that the anticancer drug camptothecin (compound 1 in Figure 11) and its derivatives specifically interfere with mammalian type I-B topoisomerases.4−6 The main property of these effective therapeutic agents is their capacity to inhibit the religation reaction of DNA topoisomerases, thus acting as highly cytotoxic agents that increase cellular DNA damage and cell death. The catalytic cycle of certain topoisomerases is indeed highly delicate and sensitive to several structural perturbations and has been extensively exploited by natural selection, as many topoisomerase-interfering compounds are natural products. Structural perturbations include not only several chemicals and drugs but Received: June 30, 2016 Published: January 10, 2017 2169

DOI: 10.1021/acs.jmedchem.6b00966 J. Med. Chem. 2017, 60, 2169−2192

Journal of Medicinal Chemistry

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Figure 1. DNA cleavage reaction by DNA topoisomerases. Type I-A and type I-B enzymes form a phosphotyrosine linkage with 5′ and 3′ ends, respectively, of the cut strand.

Table 1. Structural and Functional Features of Type I DNA Topoisomerases type I-A enzyme−DNA link enzymatic activity ATP requirement subunit cellular function

known poisons known catalytic inhibitors

5′-p-Y strand passage noa monomeric • relaxation of hypernegative supercoiled DNA • catenation/decatenation of DNA with singlestranded regions • dissolution of Holliday junctions • introduction of positive supercoils no yes

type I-B

type I-C

3′-p-Y strand rotation no monomericb • relaxation of positive and negative supercoils

3′-p-Y strand rotation no monomeric • relaxation of positive and negative supercoils

yes, anticancer drugs yes

no no

a ATP is required by particular type I-A enzymes, such as reverse gyrases (see Table 2). bThe enzyme is constituted by two subunits in some Trypanosoma species.

homologous to tyrosine recombinases and integrases.22 The capacity of these enzymes to relax both positive and negative supercoils without any energy cofactors, using only the free energy stored in DNA supercoils, denotes its capacity to perform complex coordinated structural rearrangements during the catalytic cycle, i.e., the enzyme goes from an open structure, which binds to DNA, to a closed conformation during the relaxation step and vice versa. In this respect, the linker domain, which is absent in type I-B topoisomerases from viruses and bacteria, plays a key role. In fact, this domain confers a highly processive character to the enzyme, i.e., the topoisomerase, after binding to a plasmid substrate, completely relaxes the substrate without dissociation.23,24 Single-molecule measurements of the relaxation velocities of human and vaccinia virus topoisomerase I-B show that the human enzyme is slower in the rotation of the scissile DNA strand than is the vaccinia enzyme,25 likely due to a greater number of protein−DNA contacts and the electrostatic interaction between the positively charged linker domain and the DNA. In a counterintuitive way, the linker domain, while slowing the substrate rotation, increases its processivity and therefore enzyme efficiency. Type I-B topoisomerases vary in size from 314 amino acids of the vaccinia enzyme to 972 amino acids of Drosophila melanogaster topoisomerase I (Table 2). The human enzyme is often used as a reference; the gene is approximately 96000 bp long, located on chromosome 20, and consists of 21 exons encoding a 765-amino-acid protein. The domain organization

also alterations of the template structures, such as base mismatches, DNA nicks and alkylations, and ribonucleotides incorporated into DNA strands, which are commonly found in the genomes of replicating cells. Interestingly, such perturbations can alter the enzyme activity, leading to not only increased DNA damage and cell death but also alterations of normal physiological processes at the cellular level. Several excellent reviews have been published on DNA topoisomerases7−12 and their inhibitors13−20 in the recent past. Here, we mostly provide an update focusing only on type I enzymes and discuss the recent findings that may be of interest for drug discovery and development, highlighting the structural features and unconventional functions of enzymes that may increase the potential to find novel targets and their uses in the therapy of diverse human diseases. Given these limitations and the high number of published papers in the field, we apologize to colleagues whose work could not be discussed owing to space restrictions.

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STRUCTURES OF TYPE I DNA TOPOISOMERASES Type I-B Topoisomerases. We first discuss type I-B enzymes, as they are targets of inhibitors that act with specific mechanisms of action and are clinically effective in anticancer therapeutic regimens (see below). Type I-B topoisomerases are ubiquitous in eukaryotes and are also present in certain viruses and bacteria21 (Table 2). The different forms of these enzymes share a similar architecture of the active site, with a fold 2170



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Figure 2. Catalytic mechanisms of type I DNA topoisomerases. To remove DNA entanglements, types I-A and I-B enzymes cleave one strand remaining linked to a cut strand end (see Figure 1). Then, type I-A enzymes catalyze the passage of the intact strand through the strand break (left), whereas type I-B enzymes remove DNA supercoils by controlling a rotation of the broken 5′ end around the intact strand (right). Finally, the enzyme reseals the strand cut with a reverse cleavage reaction.

Table 2. Genes Encoding Type I DNA Topoisomerases in Selected Organisms type I-A

type I-B

organisms

gene name

accession codea

protein lengthb

Escherichia coli

topA (ω protein) topB (Top III) Topo I-A

EcoGene:EG11013

865

EcoGene:EG11014

653

DR_1374 (NC_001263.1) PFU66557d

1021

Deinococcus radiodurans Thermophilic archea and bacteria Saccharomyces cerevisiae Vaccinia virus Leishmania donovani Drosophila melanogaster Homo sapiens

gene name

accession codea

Topo I-Bc

DR_0690 (NC_001263.1)

type I-C protein lengthb

gene name

Topo Ve

FungiDB: YLR234W

656

TOP1

TopIIIα TopIIIβ TOP3α

HQ694566 GQ499197 NM_078878.3

947 866 1250

TOP3β TOP3A TOP3B

NM_001298009.1 NM_004618 NM_001282112

875 1001 862

protein lengthb

UniProt: MK1436

984

346

1214

reverse gyrase TOP3

accession codea

TOP1 TOP1Lf TOP1Sf TOP1

FungiDB: YOL006C UniProt: P68697 XM_003864433 XM_003858022 NM_078606.4

769 314 636 262 972

TOP1 mtTOP1

NM_003286 NM_052963

765 601

a

Accession codes are from the NCBI database unless otherwise specified. bNumber of amino acids. cGenes encoding type I-B topoisomerase have been found in some genera of bacteria, including Deinococcus, Pseudomonas, Mycobacterium, Bordetella, Agrobacterium, Sinorhizobium, Sphingomonas, and Rhodobacter. dPyrococcus furiosus reverse gyrase. eTopo V is currently known to be present only in the archaeal Methanopyrus genus. fTOP1L and TOP1S are two subunits of a single enzyme (ref 57 in the text).

noncovalent complexes with a 22 base pair duplex DNA,22 together with the topo70 form, which also includes the linker domain.27 These structures show a cap region formed by core subdomains I and II (yellow and blue in Figures 3B and 4, respectively) that forms half of a clamp around the DNA. The other half is formed by core subdomain III (red in Figures 3B and 4), which contains four of the five catalytic residues, and the C-terminal domain (cyan in Figures 3B and 4), which

of human topoisomerase I-B has been established by several crystallographic investigations.26 The enzyme is composed of four major domains: N-terminus, core, linker, and C-terminus (Figure 3A,B). In eukaryotes, the N-terminal domain contains nuclear localization signals and is dispensable for its in vitro activity, as is the linker domain.24 A reconstituted enzyme consisting of the core and the C-terminal domain (named topo58/12) was crystallized in 1998 in both covalent and 2171



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Figure 3. Structural organization of topoisomerase I-B. (A) The human topoisomerase I-B gene (RefSeq database). (B) Organization of the 765 residues of the human topoisomerase I-B protein into four major regions. The core domain can be further subdivided in three subdomains. The conserved catalytic pentad is formed by four residues located in the core subdomain III and the catalytic tyrosine residue 723 in the C-terminal domain. (C) Comparison of topoisomerase I-B from D. radiodurans and Vaccinia virus. The domain organization is well conserved between them and the structural organization of the catalytic pentad is shared also with the human form, except for an asparagine residue in D. radiodurans, instead of a histidine. (D) Domain organization of the dimeric topoisomerase I-B from L. donovani. In the large subunit, an inactive serine is in the position corresponding to the catalytic tyrosine. The heterodimer complete the catalytic pentad with Tyr-222 from the small subunit.

negatively charged DNA (Figure 5B,C). For comparison purposes, Figure 5D represents the negative isosurface that, in previous panels, is completely embedded in the positive isosurface. Thus, the dominant positive surface of the whole enzyme explains the strong protein−DNA bonding force, while electrostatic interactions between the linker domain and the DNA scissile strand act as a brake that slows the strand rotation step, leading to the so-called “controlled rotation” mechanism.27 The linker regulation of the winding process, together with its additional strengthening of the DNA binding, likely determine the high processivity of the human enzyme. Indeed, topoisomerases I-B lacking a linker domain, such as the vaccinia enzyme, have a more distributive mode of catalytic activity. The domain rearrangements of human topoisomerase I-B enzymes during different steps of the catalytic cycle have been extensively studied by in silico modeling of the native protein31−35 and in several anticancer drug-resistant mutants. 36−42 Complete knowledge of the dynamics and conformational modifications of topoisomerase I-B enzymes may better define a rational design of compounds that efficiently interfere with the enzyme and its biological functions. The crystallization of topoisomerase I-B in a ternary complex with DNA and topotecan (compound 2 in Figure 11), a close derivative of compound 1, paved the way to study the structural mechanism of inhibition by anticancer drugs.43 Several combined in silico and biochemical studies clarified different

contains the catalytic tyrosine residue 723. The clamp around the DNA is closed by two lips located in subdomains I and III that interact via a salt bridge and a hydrogen bond22,28 (see Figure 4). Helices 8 and 9 in core subdomain III act as a hinge to open the enzyme, with a key role played by Glu445.29 The positively charged linker domain (green in Figures 3B and 4), inserted between core subdomain III and the C-terminal domain, is located parallel to the DNA, downstream of the scissile base, in the perfect position to control the rotation of the scissile strand during the relaxation step.23,27 In fact, during the relaxation step, the scissile DNA strand, located downstream of the cleavage site, relaxes the DNA supercoils by rotating around the intact DNA strand. An engineered linkerdeleted form of the human enzyme shows only a slight reduction in activity compared with that of the full-length form, accompanied by reduced DNA-binding affinity and a distributive relaxation mode, where the enzyme releases the DNA substrate after removing only a small number of supercoils at a time.24 Computer modeling of human topoisomerase I-B in interaction with a supercoiled DNA minicircle30 shows that electrostatic interactions between the positively charged enzyme and the DNA determine the DNA binding strength (Figure 5). Interestingly, visualization of the positive (blue) and negative (red) electrostatic surfaces at a contour level of ±0.5 KT/e shows how the global positively charged enzyme always presents a positive surface to the 2172



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Figure 5. 3D structure of the human topoisomerase I-B in interaction with a DNA minicircle. (A) The human enzyme is shown in cartoon representation. (B) Visualization of the positive (blue) and negative (red) electrostatic surfaces of human topoisomerase I-B at a contour level of ±0.5 KT/e. (D) Visualization of the negative electrostatic surface only. Comparison with the previous panel shows how the global positive charge of topoisomerase I-B produces a positive isosurface that completely embeds the negative one.

Figure 4. 3D structure of the human topoisomerase I-B−DNA complex. Color domains as in Figure 3B.The N-terminal domain is not present in the crystallographic structures of human topoisomerase I-B (here PDB 1a36). The DNA scissile strand, upstream of the cleavage site, is in purple color. The free scissile strand, downstream of the cleavage site, is in pink color. The intact DNA strand is in brown color. (A) The core domain and the C-terminal domain form a clamp around the DNA, with the positively charged linker domain parallel to the DNA. (B) The enzyme clamp is closed by lip1 (residues 361−369 in core subdomain I) and lip2 (residues 491−501 in core subdomain III). The hinge that allows the opening/closing around DNA is helix 8 (residues 434−453) in core subdomain III. A key role is played by Glu445. (C) The catalytic tyrosine 723 residue forms a covalent bond with the scissile DNA strand. The scissile strand downstream of the cleavage site releases the DNA supercoils by rotating around the intact DNA strand.

apoenzyme of Deinococcus radiodurans demonstrated that conformations with an assembled active site can exist even before DNA binding in an open conformation, able to bind the substrate56 (see Figure 6A). Moreover, common features between this apoenzyme and the human enzyme have been observed; in particular, the opening/closing mechanism around the DNA.56,57 Another interesting piece of information obtained by crystallography of D. radiodurans topoisomerase I-B was the identification of a secondary DNA binding site that likely plays a role in DNA condensation and synapsis by the enzyme without affecting DNA cleavage or supercoil relaxation56 (see Figure 6B). Notably, a secondary DNA binding site was also identified in the human topoisomerase30 by means of molecular dynamics simulation of topoisomerase in interaction with a supercoiled DNA minicircle involving four lysine residues (466, 468, 545, and 549) located in subdomain III (Figure 5). Altogether, these observations strongly indicate that a secondary DNA binding site is also present in mammalian topoisomerase I-B and likely explain an early preference of topoisomerase I-B for binding to supercoiled DNA molecules.58 Topoisomerase I-B of Leishmania donovani has a peculiar structural assembly, as it is the only known dimeric form of the topoisomerase I-B class.59 A topoisomerase I-B was identified in kinetoplastid parasites in 1999,60 but it was several years later that the peculiar dimeric nature of Leishmania enzymes was recognized.61 Topoisomerase I-B in protozoan parasites is encoded by two different genes located on chromosomes 4 and 34, and the domain organization of topoisomerase I of L. donovani is shown in Figure 3D. The enzyme is constituted of a

aspects of drug interactions with the protein and the role of different protein regions in drug resistance.44−53 These studies indicate three major drug resistance classes: mutations that alter the drug binding site, either by removing direct or watermediated interactions with the drug; mutations that alter the linker dynamics with a double effect on the DNA relaxation or long-range perturbations of the active site region; and mutations in the lips, particularly lip1 (Figure 4B), that produce drug resistance by altering the clamp around the DNA and therefore catalytic activity. Topoisomerases of type I-B from vaccinia and other cytoplasmic poxviruses have been extensively used as models for the eukaryotic topoisomerases, as they constitute the minimal functional unit of this class of enzymes.54 The domain organizations of poxviruses and bacteria are similar, with a reduced N-terminal domain, a lack of the nuclear localization signals, and the absence of a linker domain compared with the human enzyme (see Figure 3C). The first crystallographic structures of the vaccinia topoisomerases suggested that the active sites of topoisomerases I-B could be assembled only upon DNA binding.54,55 More recently, a structure of the 2173



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ing to Lys532 in humans.62 A general evolutionary conservation of the active site is consistent with the dimeric enzyme being sensitive to compound 1; however, substantial or subtle structural differences between parasitic and human enzymes can be defined and exploited to develop chemicals specifically targeted at Leishmania topoisomerase I-B. Type I-A Topoisomerases. Topoisomerases of type I-A bind to the 5′-phosphate group of the cleavable DNA during relaxation (Table 1 and Figure 1). E. coli possesses two type I-A topoisomerases, topoisomerase I and topoisomerase III, which have similar domain organizations (Figure 8A). The three-

Figure 6. 3D structure of the topoisomerase I-B from Deinococcus radiodurans. Domain colors as in Figure 3C. (A) The overall structure organization is different from the human enzyme, but the crystallographic structure (PDB 2f4q) shows the ability of the enzyme to form a preassembled active site, even in the absence of DNA, with the pentad residues in the same conformation than the human enzyme. (B) Another crystallographic structure of this enzyme (PDB 3m4a) revealed a secondary DNA binding site.

large subunit (73 kDa, 636 aa, Table 2), which forms the core domain, and a small subunit (28 kDa, 262 aa, Table 2), containing the active Tyr222. Interestingly, the enzyme active site is present only in the dimeric complex, as it is formed by four residues of the large subunit and Tyr222 of the small subunit (see Figures 3D and 7). Crystallographic structures of the L. donovani topoisomerase I-B heterodimer highlighted a conserved role for the catalytic residues, e.g., Arg410, corresponding to Arg590 in humans, and Lys352, correspond-

Figure 8. Topoisomerase I-A from Escherichia coli. (A) Domain organization of topoisomerase I and III. The catalytic residues in domains I and III are conserved. (B,C) 3D structure in two different views for topoisomerase III (PDB 1i7d). Domain colors as in (A). (D) Enlargement of the active site region. (E) Ten conformations projected along Essential Dynamics eigenvector 1 for a MD simulation of topoisomerase III.

dimensional (3D) structure of topoisomerase III is shown in Figure 8. Domain I (red) contains the catalytic TOPRIM fold, which is common among type I-A and II topoisomerases, DnaG-type primases, OLD family nucleases, and RecR proteins.63 Domain II (green in Figure 8) is an all-β fold that forms a central hole and is intercalated in sequence by domain III (cyan in Figure 8), which contains the catalytic tyrosine residue. Part B of Figure 8 shows the DNA single-strand binding site and the catalytic tyrosine.64 After covalent bond

Figure 7. 3D structure of the topoisomerase I-B from Leishmania donovani (PDB 2b9s). Domain colors as in Figure 3D. In this heterodimer, the catalydic pentad is formed by the core domain of the large subunit (red color) and Tyr-222 of the small subunit (cyan color). Note that these are the only protein domains present in the crystallographic structure. The DNA scissile and intact strands colored as in Figure 4. 2174



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formation between the tyrosine residue and the 5′-phosphate group, domain III opens and then closes during the catalytic cycle.65,66 This domain contains the so-called “decatenation loop” (Figure 8A−C), a region that plays a key role in the decatenation activity of this enzyme67 that is not present in topoisomerase I. Another difference between these two bacterial type I-A enzymes is in the three “zinc fingers” located in the C-terminal domain of topoisomerase I, which are required for relaxation activity.68 The catalytic cycle of topoisomerase III has been investigated by in silico calculations;66 however, to the best of our knowledge, no structural study on the interactions with their inhibitors has been performed for this topoisomerase or any other type I-A. Additional knowledge of the flexibility and longrange interactions in this class of topoisomerases may allow for the rational design of more specific inhibitors, thus complementing high-throughput screening studies aimed at identifying new active compounds against this class of enzyme.69 Figure 8E shows the projections along Essential Dynamics eigenvector 1 for topoisomerase III of E. coli. Essential Dynamics70 is a method equivalent to a principal component analysis on the coordinate fluctuations71,72 and is related to quasi-harmonic analysis.73,74 It allows for the separation of the configurational space visited by a protein into subspaces, i.e., an essential subspace, in which most of the positional fluctuations can be described by a few coordinates, and a remaining subspace that includes all of the coordinates with an approximate constrained harmonic character. Thus, the determination of the essential subspace by describing the great majority of protein fluctuations within few 3D directions allows for the characterization of the most biologically relevant motions. For example, Essential Dynamics has been applied for the characterization of the Thr718Ala mutant in human topoisomerase I, which enhances the stability of the cleavable complex with a mechanism similar to that of compound 1,34 demonstrating that the regions more altered in their dynamics compared to the wild-type (wt) enzyme are those that contact the DNA scissile strand and likely play a role in the DNA relaxation process. In the case of topoisomerase III, we observed great correlated movements of domain II along eigenvector 1 that likely function in the long-range protein communications necessary to continue the catalytic cycle. The domain organization and 3D structure of human topoisomerase IIIα has been solved, showing high conservation with respect to bacterial enzymes (Figure 9). Similarly to topoisomerase I of E. coli, the human enzyme includes a zinc finger motif whose crystal structure has not yet resolved75 (PDB 4cgy). One peculiar difference, however, is the absence of a loop equivalent to the decatenation loop of E. coli topoisomerase III in the human enzyme. This difference likely reflects different enzymatic functions, i.e., the E. coli topoisomerase III has the capacity to decatenate DNA molecules in vitro,76 while mammalian topoisomerase IIIα participates in the resolution of DNA Holliday junctions (intertwined single DNA strands generated during recombination) along with human RecQ helicases.77 The catalytic tyrosine (residue 362) is located in domain II. Figure 9 shows the positions of the acidic residues Asp148, Asp150, and Glu152 that bind to the metal ion cofactor; this binding group is typical of the Toprim domain.63 Type I-C Topoisomerases. In 1993, a topoisomerase from Methanopyrus kandleri was characterized78 that was later demonstrated to also exhibit DNA repair activity.79 The

Figure 9. Topoisomerase IIIα from Homo sapiens (PDB 4cgy). (A) Domain organization. This topoisomerase contains a zinc finger, as topoisomerase I from E. coli. The catalytic Tyr362 is in domain II, while residues Asp148, Asp150, and Glu152 bind a metal-ion cofactor (Mg(II) or Ca(II)) required for a DNA single-strand cleavage. (B) 3D structure (PDB 4cgy). Domain colors as in (A). Note that the Cterminal domain with the zinc finger region is not present in the crystal. The tertiary structure of this enzyme is very similar to E. coli topoisomerase III with the exception of the absence of the decatenation loop in the human enzyme (see Figure 8).

topoisomerases from the archaeal Methanopyrus genus were recently assigned to the new topoisomerase subtype I-C.80,81 Figure 10A shows the peculiar domain organization of type I-C topoisomerases in M. kandleri, with the catalytic residues located in the N-terminal region,82 followed by 12 tandem (HhH)2 domains with AP lyase activity (see Figure 10). The first catalytic residue in the AP lyase was identified as Lys571, located in (HhH)2-682 (see Figure 10). The presence of three distinct AP lyase active sites was recently demonstrated, identifying Lys809 as the catalytic residue of the second AP site, located in (HhH)2-10, while the third catalytic residue is located in (HhH)2-12 but has yet to be identified.83 Topoisomerase I-C is restricted to the Methanopyrus genus, and its pharmacological relevance is currently limited; however, it may have applications in other biotechnology fields.80 We do not discuss this subclass of enzymes further in the next part of this review. We have here highlighted structural peculiarities of type I DNA topoisomerases, in particular significative differences exist among specific members of the same subfamily, such as for type I-B enzymes. Investigations are therefore needed to characterize further structural specificities that may be exploited to develop novel inhibitors. Next, we discuss the cellular functions of type I 2175



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Figure 10. Topoisomerase I-C from Methanopyrus kandleri (PDB 5hm5). (A) Domain organization: the topoisomerase activity is in the N-terminal domain (the catalytic pentad residues are indicated); the DNA repair AP-lyase activity is in the (HhH)2 repeats at the C-terminal domain. Two of the three catalytic Lysines involved in the AP lyase activity are indicated in (HhH)2-6 and -10. The third catalytic lysine has not been still identified but is located in (HhH)2-12. (B,C) 3D structure (PDB 5hm5) in two different views. Domain colors as in (A). Note that (HhH)2-7, -11, and -12 are not resolved in the crystallographic structure. (D) Enlargement of the active site region.

DNA topoisomerases focusing on recent findings and unconventional molecular roles.

and elongation. A supercoiled template is more efficiently transcribed, and the genes are more efficiently activated than those located in a relaxed template.12,89 Topoisomerase I-B is enriched at transcribed genomic regions and is likely a main player in modulating the torsional stress generated during transcription elongation in mammalian cells. Genome mapping of single-stranded or torsionally stressed DNA regions has shown that the degree of DNA supercoiling is not homogeneous along the genome90−92 and that both RNA polymerase and topoisomerase activities create supercoiling domains that affect the folding of chromatin structures.92 In particular, active promoters are more negatively supercoiled than adjacent regions, and inhibition by compound 1 of human topoisomerase I-B increases negative supercoils specifically at the promoters of intermediately active genes.91 It has been suggested that topoisomerase I-B is the main relaxase for genes expressed at low and intermediate levels, whereas both topoisomerase I-B and topoisomerase II relax supercoils generated by RNA polymerases at highly active genes in human

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CELLULAR FUNCTIONS OF TYPE I DNA TOPOISOMERASES Type I-B Topoisomerases. Type I-B topoisomerases have been implicated in basic DNA-dependent processes, including replication, transcription, repair, chromatin modifications, and nucleosome assembly.84 Recent findings have provided insights into the functions of type I-B topoisomerases during transcription that may have an impact on research for new therapeutic approaches. Topoisomerases can dynamically regulate conformational transitions of the duplex during transcription by modulating the DNA supercoiling rate of the chromatin domain or locally at specific sites.85−88 In particular, DNA strands must separate and then reanneal behind RNA polymerases, and these strand dynamics modify DNA topology. If transcription elongation affects DNA topology, as in a feedback loop, DNA supercoiling affects transcription initiation 2176



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cells.91 It was recently proposed that topoisomerase I-B directly interacts with RNA polymerase II, particularly at the carboxyterminal domain (CTD) of its largest subunit.93 This interaction affects the activity of topoisomerase I-B, as the phosphorylation of the CTD by BRD4 kinase highly stimulates Top1 relaxation activity. Interestingly, BRD4 phosphorylation of the CTD also promotes RNA polymerase elongation; therefore, BRD4 coordinates topoisomerase I-B activity with transcription elongation to relieve the torsional stress generated by RNA polymerase II translocation along the template.93 Human topoisomerase I-B can facilitate transcription in different ways. First, topoisomerase I-B relaxes supercoiling generated by the transcription machinery that would otherwise accumulate and block transcription elongation. In the ‘‘twin domain model,’’94 the polymerase, while translocating along the template, generates positive and negative supercoils ahead of and behind the polymerase, respectively. Thus, accumulated supercoils, particularly positive supercoils, impede the progression of transcription; however, it is well-known that negative supercoils support DNA melting and favor initiation at promoters. Thus, as topoisomerase I-B can also relax negative supercoils, its activity can be regulated in a yet-to-bedetermined way to maintain negative supercoils at promoters and regulatory regions. Interestingly, negative supercoils can drive the DNA template into several types of non-B DNA structures that may affect gene expression. Among these structures, R loops are three-stranded nucleic acid structures with a DNA:RNA hybrid duplex and an unpaired single DNA strand that have been implicated in genomic instability and cell death caused by inhibition of topoisomerase I-B by compound 1 and in physiological cellular processes.95,96 As topoisomerase I-B can relieve free negative and positive supercoils, it is unknown whether its action can be regulated to distinguish torsional stress of different signs. Interestingly, some experimental data support the idea that type I topoisomerases influence and regulate non-B DNA structures in living mammalian and bacterial cells. Type I-A and type I-B topoisomerases can remove transcription-dependent negative supercoils while suppressing R loop formation. In parallel with the recent results for eukaryotic RNA polymerase and topoisomerase I-B, the C terminus of E. coli topoisomerase I (a type I-A enzyme) interacts directly with RNA polymerase, which may help recruit topoisomerase I to negative supercoils.88,97 Topoisomerases have also been linked to specific events during transcription, such as the activation or repression of particular promoters98 and nucleosome remodeling. Gene deletion of topoisomerase I-B in S. cerevisiae causes histone acetylation, specifically at subtelomeric regions, leading to the increased expression of telomere-proximal genes.86 Similarly, in Schizosaccharomyces pombe, topoisomerase I-B activity affects nucleosome disassembly/assembly at selected promoter regions.85 In human cells, topoisomerase I-B regulates gene expression by modulating histone modifications and nucleosome-free regions adjacent to the transcription start sites (TSSs).99 These findings indicate that, although it can be difficult to definitively determine whether the enzyme either directly or indirectly affects promoter function and structure, it is likely that topoisomerases affect transcriptional events in a manner that does not depend solely on the supercoiling relaxation activity but rather involves the modification of chromatin structures. Interestingly, the activity of topoisomerase I-B is not necessary for transcriptional activation in vitro,87 and more recent evidence has consistently shown that human

topoisomerase I-B is recruited as active promoters without being catalytically active.93 Therefore, one can speculate that topoisomerase I-B may affect chromatin architecture and histone modifications at promoter regions through specific protein−protein interactions. Nevertheless, the mechanism needs to be fully established with more investigations. Compound 1 has been very useful in defining topoisomerase I-B functions during transcription. In these types of studies, it is critical to use very short treatment times,84,100 as the half-life of the enzyme−DNA covalent complex (Top1ccs) in the presence of compound 2 is on the order of a few minutes, as established by single-molecule enzymology.85,86,101 Additionally, cellular uptake of compound 1 is very rapid.15 Therefore, the immediate molecular effects of compound 1 are more related to enzyme blocking on the DNA template than are the later drug effects, which can be due to molecular consequences and the response to transient enzyme blocking locally at the studied genomic regions. Early studies provided evidence that compound 1 immediately affects RNA polymerase II, favoring elongation from promoter-proximal pausing sites.84,100,102 This observation is in agreement with other early effects of compound 1: (i) hyperphosphorylation of RNA polymerase II, (ii) increased splicing of first introns of transcribed genes,102 and (iii) the release of active pTEFb.103 However, the pharmacological and biological impacts of these immediate effects are not yet clear. It would be interesting to assess whether the forced escape of RNA polymerase II from pausing sites has any consequence on chromatin and/or template structures. In more recent years, topoisomerase I-B has been proposed to have noncanonical functions such as in the regulation of non-B DNA structures. A previous study104 investigated the effects of stable topoisomerase I-B silencing in cancer mammalian cells in which genome instability is increased, likely due to a background level of DNA cleavage that is higher than that in parental wt cells. RNaseH1 overexpression partially rescued the observed impairment of DNA replication and higher levels of DNA cleavage. The authors suggested that topoisomerase I-B can suppress the interference of replication and transcription machineries by reducing transcriptionassociated R loop formation.104 Investigating the role of topoisomerase I-B in yeast S. cerevisiae clearly showed that topoisomerase I-B deletion causes impaired R loop formation at the 5′ end of the rDNA (rDNA) locus and impaired transcription, both of which are exacerbated by deletions of topoisomerase II and/or RNase H genes.105 These findings indicate that topoisomerase I-B deletion can enhance transcription-coupled R loop levels, inducing a longer pause of RNA polymerase at the rDNA gene locus. Further evidence has demonstrated the molecular and cellular consequences of topoisomerase I-B inhibition by compound 1 in human cells. R loops are increased by compound 1 after very short treatment times in cells expressing normal levels of topoisomerase I-B, whereas the drug is ineffective in cells with reduced enzyme levels.106 Interestingly, at longer treatment times, R loop levels decrease and are even reduced after 1 h of treatment compared to the levels in untreated cells. In parallel, Top1ccs also shows a biphasic curve over time, suggesting that R loop levels can be directly affected by topoisomerase I-B trapped on the DNA template. Moreover, other studies provided convincing evidence that compound 1 causes irreversible DNA double-strand breaks (DSBs) in resting (nonreplicating) cells in a manner that is dependent on R loop 2177



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formation.107,108 Thus, it is of high interests to establish more directly how topoisomerase I-B may affect R loop stability in human cells. Moreover, the inhibition of topoisomerase I-B activity by compound 1 rapidly induces an imbalance of sense/antisense transcripts at active promoters.102,106,109 In particular, the formation of Top1ccs by compound 1 enhances antisense RNA (aRNA) levels immediately upstream of the TSSs of promoters of intermediate activity. Interestingly, the affected gene promoters are composed of CpG islands and have symmetrical signatures typical of divergent transcription activity.106 As compound 1 has been proposed to trigger unscheduled R loop structures, we investigated the relationship between aRNAs and R loops.110 The aRNAs examined formed R loop structures normally at the CpG island promoters of untreated cells and, interestingly, R loops appeared to be extended at some of the studied promoters upon short treatment with compound 1, suggesting that topoisomerase I-B inhibition affects R loop formation or stability.110 However, upon longer treatment, R loop levels were undetectable, indicating that persistent Top1ccs markedly reduces the majority of them. The mechanisms of these effects remain to be determined; however, it is likely that the immediate increase of R loops may be due to the inhibition of topoisomerase I-B activity, whereas the later reduction of R loops is likely due to a marked decrease in the transcription rate by compound 1. The therapeutic role of these promoter-associated aRNAs has not yet been defined. However, additional evidence supports the idea that the topoisomerase I-B-mediated interference of compound 1 with antisense expression and R loop structures could be a new field of investigation to discover novel therapeutic solutions. An interesting screen of compounds in a murine cell model demonstrated that topoisomerase I-B poisons may revert the phenotype of Angelman syndrome patients, unsilencing the repressed paternal Ube3a allele.111 In this severe neurodevelopmental disorder, the maternal allele of the ubiquitin protein ligase E3A (Ube3A) is mutated or deleted, while the paternal wt allele is epigenetically repressed in cis by a large antisense transcript (Ube3a-ATS). The antisense Ube3A-ATS transcript is originated by extension of a proximal SNRPN/SNORD transcript from the SNORD116 locus up to the UBE3A gene.112 Compound screening revealed that compound 2 and Irinotecan (compound 3 in Figure 11), two FDA-approved derivatives of compound 1, restore persistent Ube3A expression in the neurons of a murine animal model of Angelman disease.111 Inhibition of topoisomerase I-B by compound 2 resulted in the upregulation of Ube3a expression and a concomitant downregulation of the Ube3a-ATS without altering the methylation status of the genomic region. Interestingly, the downregulation of the Ube3a-ATS by compound 1 derivatives has been proposed to be mediated by the stabilization of R loops and chromatin decondensation at the active paternal SNORD116 locus.112 Taken together, these findings point to a novel, fascinating pharmacological effect of topoisomerase I-B inhibition that connects Top1ccs formation to modifications of chromatin architecture, template structures, and transcription regulation. Further investigations are needed to fully establish the molecular mechanisms underlying these molecular actions. Type I-A Topoisomerases. Type I-A topoisomerases have been less studied than type I-B enzymes; therefore, the list of their cellular functions is far from complete. These enzymes are

Figure 11. Interfacial poisons of type I-B topoisomerases: camptothecin-like structures.

being actively investigated to identify inhibitors or poisons that may lead to the discovery of novel antibacterial drugs that are effective against drug-resistant human pathogens. Thus, it is relevant to fully understand their biological functions and the molecular pathways in which they play major roles. In E. coli, there are two type I-A enzymes: topoisomerase I and topoisomerase III (Table 2). They have overlapping but also distinct functions and catalytic activities and, interestingly, the eukaryotic type I-A enzymes are homologous to E. coli topoisomerase III. Several biochemical investigations have established that both bacterial topoisomerase I and topoisomerase III can relax negative but not positive supercoils even though they are not as efficient as other bacterial topoisomerases.94 In particular, topoisomerase I cannot fully relax a DNA molecule, and topoisomerase III is somewhat inefficient under physiological conditions. This insufficiency is due to the enzyme specificity for a substrate that is an unpaired DNA strand, a structure that can be present at significant levels only in highly negatively supercoiled DNA molecules. This limitation explains why topoisomerase I exhibits good activity only in cases of hyper-negatively supercoiled DNA, which has a high tendency to separate the two annealed strands. This biochemical feature of bacterial type I-A enzymes is shared with eukaryotic topoisomerases III. 2178



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topoisomerase III complex can efficiently complete DNA repair by homologous recombination without causing chromosomal rearrangements in vivo. The proposed model suggests that RecQ activity remodels the DNA into an accessible singlestranded conformation that is suitable for processing by topoisomerase IIIα. RMI proteins are an essential component of the complex that dissolve DHJs in vivo, as RMI1/2 dramatically increases the efficiency of the reaction by physically interacting with BLM/ topoisomerase IIIα.147−149 How these subunits affect the DHJ dissolution reaction is not completely known; however, a main role of RMI1/2 in the process is the direct modulation of topoisomerase IIIα enzymatic properties. RMI1 proteins significantly enhance topoisomerase IIIα binding to different substrates such as single-stranded DNA with the help of the RPA factor.130,149 Interestingly, RMI1 functions mainly in the decatenation phase of dissolution without affecting the convergent branch migration phase,147 possibly by modulating the dynamics of topoisomerase IIIα conformational changes.75,147 In each step of the reaction, the activities of RMI1/2 and topoisomerase IIIα are synergistically enhanced by BLM or other RecQ-type helicases,147,149,150 suggesting complex and highly coordinated subunit activities. Recently, several type I-A topoisomerases from all domains of life have been identified as RNA topoisomerases. Although human topoisomerase IIIβ is active with a DNA substrate, it also acts on RNA substrates. Interestingly, the enzyme forms a stoichiometric complex with fragile X syndrome factor and TDRD3 (Tudor domain-containing 3), interacting with several mRNAs.151 In contrast, topoisomerase IIIα has no activity with an RNA substrate, although the structural reasons for this inactivity have not fully established. However, an RGG RNAbinding motif is present in the CTD of topoisomerase IIIβ but not topoisomerase IIIα. Interestingly, the TDRD3−topoisomerase IIIβ complex associates with RNA-binding proteins and polyribosomes to regulate the translation of multiple mRNAs that encode proteins with neuronal functions related to schizophrenia and autism.140 Recent studies of several type IA enzymes with RNA topoisomerase activity have provided in vitro evidence that activity with a DNA or RNA substrate requires the conserved type I-A protein structures and the same catalytic residues, including the active site Tyr residue. Nevertheless, the variable CTD is not absolutely needed for RNA topoisomerase activity in bacterial enzymes but is required for human topoisomerase IIIβ.152 Several important questions remain: (i) Do these enzymes exhibit RNA topoisomerase activity in living cells? (ii) Which RNA species is the main substrate in vivo? (iii) What are the functions and molecular pathways of RNA topoisomerases, particularly human topoisomerase IIIβ? One may speculate that RNA topoisomerase affects the 3D structure of RNAs, thus modulating their activity. Therefore, these enzymes may regulate not only mRNA translation but several other RNA species, such as noncoding RNAs involved in gene expression and chromatin structure regulation. Type I-A DNA topoisomerases may thus function in specific molecular pathways such as homologous recombination repair (topoisomerase IIIα) or mRNA translation regulation (topoisomerase IIIβ) that likely require the formation or removal of interlinked nucleic acid strands. These enzymes likely work in complexes with other proteins factors in a manner that needs to be better defined and can be exploited to discover new inhibitors of cellular enzyme activities.

The cellular functions of bacterial topoisomerase I are consistent with its biochemical activities. The enzyme and DNA gyrase maintain the homeostasis of DNA supercoiling in the bacterial genome by relaxing excess negative and positive supercoils, respectively. As a consequence, plasmid DNA from bacteria expressing an inactive mutated topoisomerase I is hyper-negatively supercoiled; this excessive supercoiling is transcription-dependent and can lead to an accumulation of R loops and impaired cell growth.113−115 Consequently, mutations in the DNA gyrase and RNaseH genes can suppress the effects of inactive topoisomerase I, supporting a role for this enzyme in regulating DNA supercoiling and R loop structures in the bacterial genome. Beyond DNA relaxation activity, topoisomerase III can efficiently decatenate interlocked replication intermediates with single-strand nicks and gaps in vitro.76,116 Interestingly, in parallel with eukaryotic topoisomerases III (discussed below), bacterial RecQ helicases and single-strand DNA binding proteins favor E. coli topoisomerase III in unlinking the catenated replication intermediates at the end of replication,117 a catalytic reaction that most likely resembles the dissolution of recombination intermediates such as double Holliday junctions (DHJs).118 In higher eukaryotic and mammalian genomes, two different genes are present that encode type I-A topoisomerases, designated topoisomerase IIIα and topoisomerase IIIβ, which have similar enzymatic activities119,120 but likely different cellular functions. Topoisomerase IIIα is essential for viability and development in Drosophila and mice,119−122 and although topoisomerase IIIβ is dispensable for life, its deletion markedly affects genome stability and reduces fertility.123,124 Topoisomerase IIIα functions in the repair of doublestranded DNA cleavage, including homologous recombination. During the pathway, DHJs form with interlinked DNA strands that must be resolved to separate the two chromosomes. It is now well-established that topoisomerase IIIα contributes to the dissolution of DHJs. Topoisomerase IIIα breaks up DHJs in concert with a RecQ-type helicase (BLM) in mammals125,126 and the RMI1/2 factor.127−129 The dissolution of DHJs by the topoisomerase IIIα−BLM−RMI1/2 complex has an important feature: it avoids the generation of crossover products that would create chromosome rearrangements leading to genome instability in mitotic cells.130,131 Thus, topoisomerase IIIα plays an important function in completing the homologous recombination repair pathway by maintaining genome integrity. Although the distinct activities of topoisomerase IIIα, helicases, and RMI1/2 collaborate to disentangle interlinked strands in a manner that remains to be fully understood, the physical interaction of these three proteins is critical for the dissolution of DHJs. It has been clearly shown that topoisomerase III physically interacts with specific RecQ-type helicases in S. cerevisiae,132,133 S. pombe,134,135 and human cells.136,137 In particular, the N-terminal domain of human BLM mediates the physical association with topoisomerase IIIα; however, functional assays are somewhat contradictory. On one hand, DHJ dissolution can proceed in vitro when the helicase is truncated at the N terminus and cannot interact with topoisomerase IIIα, even though this reaction is much less efficient,138 suggesting that the formation of the complex is not required for DHJ dissolution. On the other hand, the formation of the helicase/topoisomerase III complex is required to rescue the phenotype of helicase-deleted yeast strains132,133,139−145 and the elevated sister chromatid exchange (SCE) phenotype in human cells.137,146 Thus, it is likely that only the helicase/ 2179



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CHEMICALS THAT INTERFERE WITH TYPE I DNA TOPOISOMERASES Research on topoisomerase inhibitors has been extremely active over the past few decades after the discovery that doxorubicin and other antitumor intercalators induce DNA cleavage mediated by human DNA topoisomerase II.153 Moreover, a wealth of knowledge has been accumulated on their modes of action and activities. To date, a rich amount of literature reports a plethora of small molecules as topoisomerase inhibitors, even though the data often do not clarify the mode of inhibition. It is well-known that topoisomerase inhibitors interfere with the enzyme through different mechanisms.154 In particular, many anticancer or antibacterial drugs, including doxorubicin and quinolones, act as poisons because they stabilize Top1ccs or Top2ccs and block the reclosure of the cut strand, a specific step of the catalytic cycle.155 Topoisomerase poisons increase DNA cleavage in cancer cells or microbial pathogens, leading to the activation of DNA damage response pathways and ultimately cell death.156 Other compounds do not poison topoisomerases but rather inhibit the catalytic activity of the enzyme in one of two modes. Some compounds bind to the DNA duplex, whereas others bind to the enzyme; both actions prevent topoisomerase binding to the DNA substrate. Basically, while the poison binds to a receptor site localized at the interface between the cleaved DNA and the enzyme active site,18 the catalytic inhibitor binds to either the enzyme or the DNA alone.154 The special mode of action of poisons has been further explored in more structural detail to improve our understanding of the molecular interactions and pharmacophores of interfacial poisons.18,20 Catalytic inhibitors are structurally heterogeneous compounds that include highly common drugs, such as sodium salicylate for topoisomerase II,13 and a few approved anticancer drugs, such as the cardioprotective bisdioxopiperazine derivatives, which antagonize the doxorubicin poisoning activity of topoisomerase IIβ, an established target of doxorubicin-induced cardiotoxicity.157 In general, enzyme binders may be more specific than DNA binders, as the latter likely inhibit several other DNA-dependent enzymes. For example, bisdioxopiperazine derivatives are enzyme binders that block the turnover of topoisomerase II by freezing a closed conformation of the protein, thus inhibiting the catalytic cycle and the action of doxorubicin and other interfacial topoisomerase II poisons.158 In general, catalytic inhibitors are less potent than poisons as cytotoxic agents. Unlike poisons that need to trap a relatively small subset of cellular enzyme molecules to trigger cell death, catalytic inhibitors must instead fully inhibit the cellular enzyme activity to be effective. Nevertheless, different modes of action are interesting and may be exploited for the development of new therapeutic strategies for diverse human diseases. The list of compounds that interfere with types II and/or I-B topoisomerases with a demonstrated mechanism of action is long.13,19 One may wonder why these enzymes are the targets of many compounds. One reason may be the intense research activities by many laboratories throughout the world over the past several years that have led to the identification of hundreds of enzyme inhibitors. Similarly, many kinase inhibitors have also been identified in another very active field of anticancer drug discovery. As many topoisomerase inhibitors are natural products or their derivatives, another reason is that the modulation of topoisomerase activity is a widespread physiological phenomenon in nature. It is tempting to speculate

whether such enzyme modulators may physiologically exist in the cell (or could be designed) to influence topoisomerase activity at selective genomic regions. For example, a physiological activator or inhibitor of a topoisomerase could increase or reduce the rate of relaxation activity of the enzyme at selective genomic regions, therefore affecting the kinetics and cross-talk of interconnected molecular processes that are highly coupled with DNA superhelical tension. This process may have important consequences on the regulation of DNA topology and basic cellular functions in a time- and space-dependent manner. Thus, it is important to precisely establish the mechanisms of action of current and novel topoisomerase inhibitors and modulators. To demonstrate that a small molecule is a topoisomerase inhibitor, it is not sufficient to perform an in vitro DNA relaxation assay, but it is essential to provide information on enzyme cleavage activity or biophysical/ biochemical data of molecular interactions. In the next sections, we discuss type I-B and type I-A topoisomerase interfacial poisons and inhibitors, focusing on the most recent and promising advances in the field. For previous compounds not included herein, we refer the reader to previously published reviews.12,14−17 Interfacial Poisons of Type I-B Topoisomerases. Chemicals that interfere with type I-B enzymes and are clinically important anticancer drugs belong to the interfacial poison category, as they stabilize Top1ccs in cancer cells. There is no doubt that compound 1 (Figure 11), a natural product derived from the plant Camptotheca acuminata, is the reference drug for type I-B topoisomerase poisons. Early after the discovery of its anticancer property, compound 1 was abandoned due to its high toxicity in patients and was then rediscovered in 1986 after it was demonstrated that it is a topoisomerase I-B poison.159 Two derivatives, 2 and 3, were developed and are now FDA-approved drugs for chemotherapy of human ovarian, lung, and gastrointestinal cancers. Compound 1 specifically target topoisomerase I-B, as cells lacking the gene or carrying specific single point mutations are completely resistant to the drug.4,6 The cellular effects of compound 1 are fast and reversible upon drug removal, and their high specificity and reversibility have allowed for the use of compound 1 as a tool to dissect the physiological roles of topoisomerase I-B and DNA damage response pathways in both normal and cancer cells. Much effort has been made in past years to improve pharmacokinetics of compound 1, as the drug’s lactone E-ring is not stable (Figure 11) and quickly opens to a carboxylate form that results in a completely inactive molecule. Compounds that limit E-ring opening, such as homocamptothecin diflomotecan (4) and the α-keto derivative S39625 (5), are highly potent in vitro, but clinical trials on most of these drugs have been discontinued. Derivatives on A and B rings, such as Gimatecan160 (6 in Figure 11), were also synthesized to improve solubility for oral administration and to overcome drug efflux-associated resistance. The clinical trials to date have not revealed improved patient tolerability or efficacy compared to that of FDA-approved parent drugs; however, one analogue, Belotecan (7 in Figure 11), has been approved for clinical use in South Korea. In an attempt to overcome the pharmacological limits of compound 1, several laboratories have developed synthetic interfacial poisons directed against topoisomerase I-B with noncamptothecin-like structures. Of them, only two classes are 2180



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activity in xenograft models than that of standard drugs.163 As these two classes of compounds have entered clinical development, they have been the focus of several investigations, and countless derivatives have been synthesized. In this effort, derivative structures have sometimes been extensively modified with respect to parent agents demonstrating moderate to strong topoisomerase I-B poisoning activity.164,165 In addition to these compounds, a number of different structures have been proposed to act as interfacial poisons of topoisomerase I-B. Vieira et al.166 recently published a study on berberine derivatives (13 in Figure 12) showing that the new tested compounds, inducing Top1ccs, inhibit topoisomerase IB relaxation, but at higher concentrations than compound 1. Interestingly, berberine agents are natural products used in traditional Chinese and ayurvedic medicines with a wide range of pharmacological properties and biochemical effects. Berberine derivatives may bind to DNA; thus, their activity against topoisomerases needs to be understood in terms of specificity and target binding. Thaspine (14 in Figure 12), an alkaloid from the South American tree Croton lechleri, is a dual catalytic inhibitor of both topoisomerase I-B and topoisomerase II-A.167 Recently, Castelli et al.168 demonstrated that the agent inhibits both the cleavage and religation steps of topoisomerase I-B. The authors further provided molecular docking results showing that thaspine can bind in the proximity of the enzyme active site and potentially insert into an enzyme active site pocket, suggesting that it may act as a poison of topoisomerase I-B similarly to compound 1. Zubovych et al.169 screened for chemicals that are selectively toxic to lung cancer cells but not normal cells; compound 15 (SW044248) was determined to be the most interesting agent (Figure 12). Experimental data demonstrate that compound 15 inhibits Top1 relaxation activity without trapping the enzyme on the DNA. Nevertheless, it blocks DNA replication, transcription, and protein translation and activates DNA damage response proteins, suggesting that it may induce DNA damage in living cells. Additional experiments in silenced cells confirmed that topoisomerase I-B is a target of 15, as topoisomerase I-B knockdown prevents the cellular response to DNA damage induced by the molecule. The discrepancies between in vitro and in vivo results and whether the agent is a poison of topoisomerase I-B or induces DNA damage by targeting a different enzyme remain to be understood. The importance of finding new drug targets against protozoan parasites, such as Trypanosoma brucei and L. donovani, has brought attention to topoisomerase I-B. Derivatives of compound 1 target topoisomerase I-B of Leishmania species, suggesting that the enzyme can be a target for the related disease. However, to find effective antitrypanosome drugs, one would need to discover a selective interfacial poison of Leishmania type I-B topoisomerase, which is a peculiar, dimeric enzyme (Figure 7). Majumdar et al.170 developed a series of thiohydantoin derivatives, one of which (compound 16 in Figure 12) exhibited DNA relaxation inhibition and cleavage activity of both human and Leishmania donovani topoisomerase I-B. The 50% inhibitory concentration (IC50) value of the compound is approximately 10 μM. As hydantoin derivatives are reported to have anticancer, antibacterial, antiviral, and antiparasitic activities with a mechanism of action not fully understood, they are often considered pan assay interference compounds (PAINS) and frequent hitters in screening campaigns.171 In the present study, compound 16 has been assessed for topoisomerase I-B

in clinical development and appear to be similarly active and tolerated in early phase clinical trials. The indenoisoquinolines were developed from screening the NCI compound database; two derivatives, indimitecan and indotecan (8 and 9 in Figure 12, respectively), are currently in clinical trials.161,162 These

Figure 12. Interfacial poisons of type I-B topoisomerases: noncamptothecin-like structures.

agents are particularly interesting, as they exhibit high chemical stability and the capacity to form more stable Top1ccs and to overcome drug efflux mechanisms of cells resistant to compound 1. The second class is constituted by derivatives developed from structure−activity relationship studies of the dibenzonaphthyridinone compound family (compound 10 in Figure 12). These agents are synthetic derivatives of the natural products nitidine and fagaronine (11 and 12 in Figure 12, respectively), which demonstrate greater efficacy than compound 1 in resistant cells expressing efflux pumps and greater 2181



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Figure 13. Inhibitors of type I-B topoisomerases.

inhibition is enhanced when the compound is preincubated with the enzyme, suggesting that the drug binding site is less accessible in the binary topoisomerase I-B−DNA complex. Compound 20 is effective at a relatively high dose (80 μM); however, molecular docking studies indicate that it may bind to an enzyme site that is close to the catalytic site. Thus, for this new series of compounds, it would be interesting to further explore their modes of action and binding sites in human topoisomerase I-B to better understand the structural determinants of the compounds’ activities. The use of metal compounds may be a novel strategy to target topoisomerase I-B and, possibly, topoisomerases of other subclasses. Akerman et al.173 synthesized several structurally unique gold complexes, showing that gold macrocycles are specific inhibitors of topoisomerase I-B. Using different assays, they put much effort into understanding whether the studied compounds are interfacial poisons or catalytic inhibitors. Gold complexes intercalate into DNA base pairs at the 5′-TA-3′ dinucleotide that is present at preferred DNA cleavage sequences of mammalian topoisomerase I-B.174 Akerman et al.173 discovered that all derivatives are catalytic inhibitors, with the exception of one gold-macrocycle complex (compound 21 in Figure 12) that has been indicated as an interfacial poison at low concentrations (up to 1 μM) and a catalytic inhibitor at higher concentrations. The authors showed that 21 has the lowest K a of DNA binding among the studied gold complexes,173 thus it is likely that a stronger DNA binding affinity of the compound much reduces (or abolishes) the other compound activity, i.e., the stabilization of Top1ccs. It should be noted that 21 can be a striking example of dual-mode topoisomerase I-B interference in a way that is similar to doxorubicin and other strong DNA-intercalating interfacial poisons of type II topoisomerases.153 It remains to be established whether these metal-derived agents affect the enzyme activity in a DNA sequence-selective manner and whether they exhibit significant biological activity. Janockova et al.175 developed a series of acridine derivatives (compound 22 in Figure 13) functioning as dual catalytic inhibitors against topoisomerase I-B and topoisomerase II-A. Their inhibitory activities are indirect, as the small molecules

inhibition both in vitro and ex vivo and the results are consistent, confirming the drug activity against the enzyme. Molecular modeling showed that the compound may fit into the topoisomerase I-B catalytic pocket, suggesting that its receptor site may be similar to that of compound 1. Thus, for this series of compounds, it would be interesting to explore whether the differential structure−activity relationships can lead to the discovery of more Leishmania-specific topoisomerase I-B inhibitors or interfacial poisons. Inhibitors of Type I-B Topoisomerases. In very recent years, a number of compounds have been reported to inhibit topoisomerase I-B at significant levels without forming Top1ccs. Some of the new structures may be of particular interest and could be developed to obtain promising leads of new drugs for oncological or neurological patients. As discussed above, an enzymatic inhibition test (such as DNA relaxation) cannot distinguish among the different modes of action of topoisomerase inhibitors. Thus, it is crucial to use appropriate assays to adequately characterize new potential inhibitors. We now discuss recent findings underlining the experimental evidence of the proposed mode of action of the tested inhibitor and its activity at the cellular level. β-Lapachone (compound 17 in Figure 13) has been investigated in the past as a topoisomerase I-B inhibitor,172 and one of its prodrugs (ARQ-761, structure not disclosed) is currently in clinical trials as it exhibits high antitumor activity. The exact mode of action of β-lapachone has not yet been defined; however, this compound is not selectively directed against topoisomerase I-B as it targets different proteins and cellular pathways. Topoisomerase I-B can be considered a catalytic inhibitor that binds to the free enzyme and, without impeding the binding to DNA, promotes the formation of a catalytically inactive ternary complex.172 Other compounds, including conjugated eicosapentaenoic acid and Erybraedin C (compounds 18 and 19 in Figure 13, respectively), have been proposed to act similarly to β-lapachone; interestingly, the latter can bind irreversibly to the enzyme.50 Another study168 provided evidence for a different structure (a kakuol analogue, compound 20 in Figure 13) that inhibits topoisomerase I-B activity without binding to the DNA. The 2182



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intercalate into the DNA and consequently prevent the binding of the protein to the DNA. Dual topoisomerase I-B and topoisomerase II inhibitors carrying the pharmacophore of trisubstituted pyridine (compound 23 in Figure 13) have also been described by Karki et al.,176 but no evidence of their mode of action has been shown. Fluorescein hydrazones have been synthesized and have also been reported to act as dual inhibitors177 (compound 24 in Figure 13). Some analogues are effective against only one enzyme, while others are similarly effective against both human enzymes. At the cellular level, the compounds induce both cell accumulation at the G1 phase of the cell cycle and apoptotic protein expression. The authors also provide evidence that the active fluorescein hydrazones do not intercalate into the DNA; however, they did not exclude the possibility that the agents may bind to the DNA in a different manner. Thus, it remains to be defined whether these dual topoisomerase inhibitors are enzyme or DNA binders. Inhibitors of Type I-A Topoisomerases. The search for type I-A topoisomerase inhibitors has not yet produced an effective drug that has entered clinical trials for cancer, infectious, or other diseases. Nevertheless, these enzymes can be valuable pharmacological targets, as they are essential in higher eukaryotes and heavily affect the vitality of bacteria. Moreover, a TA system (toxin−antitoxin operons) discovered in E. coli is constituted by the YjhX protein factor (TopAI), a toxin that inhibits topoisomerase I, and the YjhQ protein factor, which acts as the antitoxin.178 Interestingly, the protein factor genes are conserved in other species, including Salmonella, Caulobacter, Pseudomonas, and Myxococcus, but not in eukaryotes. The YjhX factor binds specifically to the N-terminal domain of a type I-A topoisomerase in complex with DNA, leading to bacterial cell death.178 In addition, another study179 demonstrated that the ectopic expression of the phage T4 ORFan gene 55.2 inhibits E. coli growth with irreversible activity. A screening approach used to discover the genes involved in this effect showed that the target of 55.2 was topoisomerase I, as its overexpression suppressed 55.2mediated bactericidal activity. For the first time, these studies provide evidence of proteins that specifically inhibit bacterial topoisomerase I (TopA), leading to death of the microbe. Therefore, in addition to genetic evidence showing that type IA topoisomerases have essential functions in pathogenic bacteria,180 these findings validate bacterial type I-A enzymes as valuable therapeutic targets for infectious diseases. Cheng et al.181 performed a fluorescence-based screening analysis of small compounds to identify potential topoisomerase inhibitors and found that two organo-mercury compounds (25 and 26 in Figure 14) were active (with IC50 values of 20 and 50 μM, respectively) at inhibiting Yersinia pestis and E. coli topoisomerase I-A. They also found that the compounds target the CTD of the enzymes, in particular the multiple tetra-cys zinc ribbon motifs. Supporting this mode of action, the two compounds do not inhibit topoisomerase I-A of Mycobacteria tuberculosis, which lacks these motifs. In this study, the compounds induced a redistribution of DNA cleavage sites along the oligonucleotide, likely interfering with the C-terminal DNA-binding domain that has been proposed to select for the genomic sites of enzyme activity in living microbes (Figure 4). Thus, the compounds may interfere in a complex manner with the enzyme, which is dependent on the sequences of the DNA sites. Moreover, these compounds are expected to interfere with other Zn finger-containing proteins; therefore, more selective agents remain to be identified.

Figure 14. Inhibitors of type I-A topoisomerases.

Using a different methodological approach, Leelaram et al.182 generated a panel of monoclonal antibodies (mAbs) against Mycobacterium smegmatis and Mycobacterium tuberculosis topoisomerase I and found two IgG1s (1E4F5 and 17B1IB4) with high affinity (4 and 22 nM, respectively) to the enzyme’s N-terminal region. The two mAbs inhibit the target enzyme, inducing a particular protein conformation. The mAbs inhibit the binding of the enzyme to the DNA substrate when added to the target before DNA addition. However, in the presence of preformed enzyme−DNA complexes, the two mAbs inhibit DNA cleavage and enhance strand religation without releasing the protein from the DNA substrate. These findings indicate that the two mAbs restrict enzyme movement, stabilizing Top1DNA heterocatenates by closure of the enzyme clamp around the DNA. These findings also indicate a different mode of action for a small molecule to inhibit topoisomerase I-A activity. Bisbenzimidazoles have been widely studied as human topoisomerase I-B inhibitors. Recently, Nimesh et al.183 aimed to identify bisbenzimidazole derivatives that are able to specifically inhibit bacterial topoisomerase I-A and not human topoisomerase I-B in an effort to bypass the extreme cytotoxicity of these compounds and to find safer molecules for human use. Interestingly, some compounds proved to be specific inhibitors of E. coli topoisomerase I with good antibacterial activity toward different strains (compound 27 in Figure 14). Molecular docking analyses suggested that 27 acts as a metal ion chelator and binds close to the enzyme active site, likely competing with the acidic triad Asp111, Asp113, and Glu115, and participates in metal coordination during the catalytic reaction. Experiments in animal models have indicated that the compound results in extended survival of infected mice. In a fluorescent-based high-throughput screening assay of small molecules and natural products, anziaic acid (compound 28 in Figure 14), extracted from the lichen Hypothachyna sp., was identified as an agent that is able to inhibit Yersinia pestis 2183



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and E. coli topoisomerase I among 10000 tested compounds.184 The compound has the capacity to chelate Mg2+, and experimental data demonstrated that topoisomerase I inhibition was not due to the chelation of free ions but rather to the compound binding at the active site and interference with ion interactions directly at this step. However, the compound was also able to inhibit E. coli DNA gyrase and human topoisomerase IIα relaxation and cleavage activities. Anziaic acid inhibits human topoisomerase I-B but only at concentrations 10-fold higher than that of bacterial topoisomerase I-A. The same group recently published a work in which they screened mixture-based combinatorial libraries of more than 30 million small molecules69 and identified four interesting polyamine-scaffold agents with bactericidal effects against M. smegmatis and M. tuberculosis through the inhibition of topoisomerase I-A. Streptococcus pneumoniae topoisomerase I is the unique type I topoisomerase present in the genome of the pathogen and is able to relax negatively but not positively supercoiled DNA. Thus, this enzyme may be a good candidate for a new therapeutic strategy for resistant infections. The sequence similarity between E. coli and S. pneumoniae topoisomerase I is approximately 47%. Garcia et al.185 analyzed 18 alkaloids derived from the natural alkaloid boldine and found that a phenanthrene analogue (compound 29 in Figure 14) inhibits S. pneumoniae topoisomerase I activity with an IC50 value of 17 μM. This compound was also active at inhibiting the bacterial growth of clinical isolates that are resistant to other antibiotics. The authors provide experimental evidence that the agents likely interact with the closed enzyme conformation at the DNA binding site. Human topoisomerase I-B was also partially inhibited but at concentrations at which pneumococcal topoisomerase I-A was inhibited fully,and no induction of human cell apoptosis was detected.185 These results suggest that the tested compounds are somewhat specific for bacterial enzymes. In the last part of the Perspective, we discuss interesting results obtained in attempts to improve the therapeutic index of established drugs. In particular, researchers have investigated DNA repair enzymes that affect the cellular outcome of Top1ccs, and protein factors that form functional complexes with topoisomerase IIIα, in order to discover novel pathway inhibitors as promising therapeutics. Moreover, targeteddelivery strategies are in clinical trials for established drugs such as derivatives of compound 1. Inhibitors of Topoisomerase Protein Partners or DNA Repair Pathways. Topoisomerase I-B. Through the covalent linkage of the catalytic tyrosine to the DNA (Figure 1), type I-B topoisomerases form Top1ccs, which lead to high-efficiency cell death. Thus, organisms have evolved specific repair pathways to tackle Top1ccs. Tyrosyl DNA phosphodiesterase I (Tdp1) efficiently catalyzes the removal of short peptides from DNA 3′ ends following the proteasome-dependent degradation of trapped enzymes. When topoisomerase I-B is blocked in a catalytically inactive cleavage complex, the enzyme undergoes post-translational modifications, i.e., sumoylation and/or ubiquitination, facilitating their partial proteolysis and recognition by Tdp1.186 Tdp1 contributes to a major repair pathway of Top1ccs that involves other protein factors, such as PARP1, DNA ligase 3, PNKP, and XRCC1. Human Tdp1 hydrolyzes the phosphodiester bond between a DNA 3′ end and a tyrosyl residue and can hydrolyze other 3′ end DNA alterations,

including 3′-phosphoglycolates and 3′-abasic sites, indicating that it functions as a general DNA 3′ end repair enzyme. The Tdp1 gene has been associated with spinocerebellar ataxia with axonal neuropathy (SCAN1), an autosomal recessive disorder clinically characterized by peripheral axonal motor and sensory neuropathy, distal muscular atrophy, and steppage gait. SCAN1 is likely caused by the H493R Tdp1 mutation that shows reduced enzymatic activity and accumulates Tdp1−DNA covalent intermediates.187 The capacity of wt Tdp1 to remove a stalled mutant protein from DNA can likely explain the recessive nature of SCAN1 neuropathy. All studies on Tdp1 and SCAN1 disease agree that SCAN1 cells are more sensitive to compound 1 and are defective at the repair of SSBs caused by topoisomerase I-B and oxidative damage.187,188 The importance of drugging Tdp1 has been supported by the synergistic effects of Tdp1 inhibition with topoisomerase I-B interfacial poisons that have been demonstrated extensively.189 To date, Tdp1 inhibitors have been identified using both highthroughput assays and virtual screenings. The most active compound NSC88915 (number 30 in Figure 15) has an IC50 of approximately 7 μM and belongs to the category of phosphotyrosine mimetics. However, compound 30 has limitations such as low cellular uptake and poor cytotoxicity. In addition, phosphotyrosine mimetics often demonstrate poor specificity, being simultaneous inhibitors of Tdp2, a structurally

Figure 15. Inhibitors of topoisomerase protein partners or DNA repair pathways. 2184



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Figure 16. Targeted-delivery topoisomerase I-B poisons.

BLM.194 Experimental data suggest that 34 competes with DNA for binding to BLM without interfering with ATPase activity. This molecule is a potent inhibitor of BLM strand separation activity rather than DNA junction branch migration or G-quadruplex DNA disruption activities. It inhibits BLM activity with an IC50 of approximately 3 μM but does not inhibit other human helicases such as RECQ1 and RECQ5. Instead, it inhibits WRN at a similar concentration (5 μM). However, WRN− and WRN+ fibroblasts are equally sensitive to 34 and both cell lines are sensitized to aphidicolin in the presence of the compounds, suggesting that, in human cells, 34 specifically inhibits the function of BLM and not WRN protein. A similar study was pursued to discover specific WRN helicase activity.195 Initially, the NCI Diversity Set library was screened using a radiometric assay to pinpoint possible helicase inhibitors. Seven compounds of particular interest were selected and screened for their specificity on WRN rather than on human RECQ1, BLM, and FANCJ or E. coli RecQ, UvrD, and DnaB, highlighting the potency and specificity of compound NSC19630 (compound 35 in Figure 15). 35 was identified as the best candidate, as it has an IC50 of approximately 3 μM and a cytotoxic effect that is strictly dependent on the cellular presence of WRN protein. This compound does not bind forked duplex DNA and is not active at inhibiting the WRN ATPase compared with its effect on WRN helicase activity. After drug treatment, cells remain in S-phase, activate ATM, accumulate DSBs and PCNA foci, and become apoptotic. Interestingly, the compound synergizes with compound 2, a Top1 poison, and with an inhibitor of PARP1 and Gquadruplex binders. Targeted-Delivery Topoisomerase I-B Poisons. Targeted-delivery topoisomerase I-B poisons constitute a new emerging therapeutic category in the topoisomerase field. The idea is not completely new, but its application to topoisomerase I-B poisons appears to be very promising. The tissue-specific delivery of antitumor drugs has the goal to increase the effective drug concentration at specific anatomical districts, therefore reducing drug side effects. Different site-selective drug delivery strategies have been developed, including the coupling of derivatives of compound 1 to metabolites, peptides, or synthetic polymers.

distinct tyrosyl DNA phosphodiesterase that removes peptides from DNA 5′ ends. Interestingly, synthetic dual Tdp1-topoisomerase I-B inhibitors may constitute a unique class of anticancer agents that could potentially exhibit synergistic activity due to the targeting of both targets. Nguyen et al.190 developed a series of derivatives based on the indenoisoquinoline pharmacophore. The examined sulfonates and sulfonamide indenoisoquinolines were all determined to be inactive at inhibiting Tdp1 and topoisomerase I-B, whereas the polyamino bisindenoisoquinoline in Figure 15 (compound 31 in Figure 15) was highly active against both enzymes. The same group recently published a novel series of dual inhibitors derived from the same pharmacophore; among the most active compounds, 8hydroxyindenoisoquinoline amine (compound 32 in Figure 15) showed comparable activity to previously published agents.191 Topoisomerase I-A. We have discussed above the cellular functions of topoisomerase IIIα in DHJ dissolution during homologous recombination repair pathways. In this process, it is essential that topoisomerase IIIα physically interacts with RecQ helicases (BLM in humans and Sgs1 in yeast). Interestingly, helicase mutants result in increased illegitimate recombination and gross chromosomal rearrangements in S. cerevisiae.140,192,193 In humans, Bloom syndrome, a recessive autosomal disorder due to BLM gene mutations, leads to a greatly increased risk of cancer that is associated with an elevated frequency of chromosomal rearrangements. Taken together, these results point to the essential contribution of helicase activity in the repair pathway and DHJ dissolution. Thus, the tight collaboration between helicases and topoisomerase IIIα suggests that targeting helicases may open a new strategy to potentiate the activity of topoisomerase poisons and inhibitors. Recently, fluorescent high-throughput screening of a library with more than 350000 compounds led to the discovery of MLS000559245 (compound 33 in Figure 15), a good pharmacophore for helicase inhibition. Further chemistry optimization with the aim to improve solubility and cell permeability led to the discovery of the analogue ML216 (compound 34 in Figure 15), a potent and selective inhibitor of 2185



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A well-designed example is the work of Cao and Yang196 that have taken advantage of folate receptors, overexpressed in many types of cancers. They synthesized an indenoisoquinoline covalently attached to a folate group via a NEBI linker (compound 36 in Figure 16), which undergoes accelerated hydrolysis in mild aqueous acidic solutions, therefore allowing the release of indenoisoquinoline molecules already internalized in cells through endocytosis. They demonstrated that the new synthesized compound has a higher level of cellular uptake and cytotoxicity (11-fold enhancement) in folate receptor-overexpressing KB cells as compared with those of folate receptorknockdown cells. Differently, Proia et at197 tested the efficacy of STA-12−8666 (compound 37 in Figure 16), carrying a resorcinol-based HSP90 inhibitor fused to the pharmacologically active metabolite of compound 3. HSP90 is a molecular chaperone overexpressed in tumor tissues with a role in regulating the maturation and stability of several cellular proteins. Interestingly, HSP90 is present in an activated configuration that has a high affinity for resorcinol inhibitors in cancer cells only. Thus, as the Top1 poison is attached to the HSP90 inhibitor via a carbamate linker, it is transported in an inactive state until an intracellular carboxylesterase activity regenerates the active molecule within tumor cells. Compound 37 was tested in breast cancer xenograft-bearing mice, not only confirming the efficacy of the Top1 poison but also showing a broader therapeutic window and higher durability of the response. In a different study, compound 2 was loaded in hyaluronic acid-grafted polyamidoamine dendrimers, demonstrating that drug accumulation in tumors of S-180 sarcoma-bearing mice was much improved.198 The functionalization of these dendrimers with HA is used to specifically recognized and interact with CD44 receptors, overexpressed in many tumor cells with a role in metastasis, invasion, adhesion, and angiogenesis. In HCT116 cells, a CD44-positive tumor line, the dendrimers-loaded Top1 poison was more cytotoxic than the free drug. Taken together, reported evidence demonstrates that it is possible to couple topoisomerase I-B interfacial poisons to chemical scaffolds to deliver the anticancer drug to cancer tissues limiting the systemic toxicities due to adverse effects on normal tissues and improving the therapeutic index. In addition, it is noteworthy that this strategy has also the advantage to overcome the instability of some topoisomerase IB poisons, which are generally administered in high doses to get the desired therapeutic effect.

molecular processes, such as genome stability and gene expression, in a number of organisms including microbial pathogens and parasites. The definition of such unexpected enzyme roles may indicate novel ways to treat cancer and other human pathological conditions including infectious and neurodegenerative diseases.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +39-0512091209. E-mail: giovanni.capranico@unibo. it. ORCID

Giovanni Capranico: 0000-0002-8708-6454 Notes

The authors declare no competing financial interest. Biographies Giovanni Capranico is currently a Professor of Molecular Biology in the Department of Pharmacy and Biotechnology, University of Bologna, Italy. He received his “Laurea” degree in Biological Sciences at the University of Perugia in 1982. He was a Fellow at the National Cancer Institute of Milan from 1983 to 1987 and a Guest Researcher for two years at the Laboratory of Molecular Pharmacology, NCI, NIH, Bethesda, MD, USA. In 1990, he returned to Italy and was appointed as a Senior Investigator at the National Cancer Institute, Milan. In 1998, he moved to the University of Bologna as an Associate Professor, where he was appointed as a Full Professor in 2006. His research has been supported by several grants from the “Associazione Italiana per la Ricerca sul Cancro” (AIRC), Milan. Jessica Marinello received her Master’s degree in Chemistry and Pharmaceutical Technology in 2005 from the University of Padova, Italy. She obtained a Ph.D. in Molecular Sciences in 2009 from the University of Padova, working on HIV integrase. In 2007 and 2008, she worked in the Laboratory of Molecular Pharmacology, National Cancer Institute, NIH, Bethesda, MD, USA, with Y. Pommier as part of her Ph.D. program. She was a postdoctoral fellow from 2009 to 2014 at the Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy, where she is currently a Research Assistant Professor. Giovanni Chillemi is currently Team Leader of the Specialist User Support group at the Cineca-SCAI (SuperComputing Applications and Innovation) Department. He received a Master’s Degree in Chemical Engineering at the University of Rome “La Sapienza” in 1992 and a Ph.D. in Biochemistry and Molecular Biology at the University of Rome “Tor Vergata” in 2004. From 1997 to 2013, he worked at CASPUR, the supercomputing center based in Rome. Since 2014, he has worked at Cineca-SCAI. From 2003 to 2012, he worked as an External Professor at the University of Rome “Tor Vergata” and, in 2012, at the University of Bologna. He received the 2012 Italian National Scientific Qualification as Associate Professor in “Molecular Biology” and “General and Clinical Biochemistry.”

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CONCLUSION Type I DNA topoisomerases are a family of essential enzymes present in all organisms. These topoisomerases share general reaction chemistry, nevertheless, they present degrees of variation in their structures, domain organizations, catalytic reactions, substrates, and molecular functions. Moreover, type I enzymes are involved in diverse cellular pathways that affect basic cellular functions and genome stability. Several compounds interfere mainly with the eukaryotic topoisomerase I-B enzymes, and some are highly effective anticancer drugs. Thus, the discovery of novel agents or new approaches to inhibit or poison type I-A topoisomerases may open significant opportunities for the treatment of human diseases such as cancers and multidrug-resistant infectious diseases. Novel mechanistic knowledge is also required to establish the notyet-understood and unusual roles of type I topoisomerases in

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ACKNOWLEDGMENTS This work has been supported by the grant IG 15886 to G.C. from the “Associazione Italiana per la Ricerca sul Cancro”, Milan, Italy, and the University of Bologna (FARB project “Modulation of Protein−DNA Interaction with Small Molecules: Novel Opportunities for Drug Design”).

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ABBREVIATIONS USED Top1ccs, topoisomerase I-DNA cleavage complex; DHJ, double Holliday junction; Tdp1, tyrosyl DNA phosphodiesterase I; 2186



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(22) Redinbo, M. R.; Stewart, L.; Kuhn, P.; Champoux, J. J.; Hol, W. G. Crystal structures of human topoisomerase I in covalent and noncovalent complexes with DNA. Science 1998, 279, 1504−1513. (23) Chillemi, G.; Redinbo, M.; Bruselles, A.; Desideri, A. Role of the linker domain and the 203-214 N-terminal residues in the human topoisomerase I DNA complex dynamics. Biophys. J. 2004, 87, 4087− 4097. (24) Stewart, L.; Ireton, G. C.; Champoux, J. J. Reconstitution of human topoisomerase I by fragment complementation. J. Mol. Biol. 1997, 269, 355−372. (25) Koster, D. A.; Croquette, V.; Dekker, C.; Shuman, S.; Dekker, N. H. Friction and torque govern the relaxation of DNA supercoils by eukaryotic topoisomerase IB. Nature 2005, 434, 671−674. (26) Champoux, J. J. DNA topoisomerases: structure, function, and mechanism. Annu. Rev. Biochem. 2001, 70, 369−413. (27) Stewart, L.; Redinbo, M. R.; Qiu, X.; Hol, W. G.; Champoux, J. J. A model for the mechanism of human topoisomerase I. Science 1998, 279, 1534−1541. (28) Chillemi, G.; Castrignano, T.; Desideri, A. Structure and hydration of the DNA-human topoisomerase I covalent complex. Biophys. J. 2001, 81, 490−500. (29) Chillemi, G.; Bruselles, A.; Fiorani, P.; Bueno, S.; Desideri, A. The open state of human topoisomerase I as probed by molecular dynamics simulation. Nucleic Acids Res. 2007, 35, 3032−3038. (30) D’Annessa, I.; Coletta, A.; Sutthibutpong, T.; Mitchell, J.; Chillemi, G.; Harris, S.; Desideri, A. Simulations of DNA topoisomerase 1B bound to supercoiled DNA reveal changes in the flexibility pattern of the enzyme and a secondary protein-DNA binding site. Nucleic Acids Res. 2014, 42, 9304−9312. (31) Chillemi, G.; Fiorani, P.; Benedetti, P.; Desideri, A. Protein concerted motions in the DNA-human topoisomerase I complex. Nucleic Acids Res. 2003, 31, 1525−1535. (32) Lillian, T. D.; Taranova, M.; Wereszczynski, J.; Andricioaei, I.; Perkins, N. C. A multiscale dynamic model of DNA supercoil relaxation by topoisomerase IB. Biophys. J. 2011, 100, 2016−2023. (33) Sari, L.; Andricioaei, I. Rotation of DNA around intact strand in human topoisomerase I implies distinct mechanisms for positive and negative supercoil relaxation. Nucleic Acids Res. 2005, 33, 6621−6634. (34) Szklarczyk, O.; Staron, K.; Cieplak, M. Native state dynamics and mechanical properties of human topoisomerase I within a structure-based coarse-grained model. Proteins: Struct., Funct., Genet. 2009, 77, 420−431. (35) Wereszczynski, J.; Andricioaei, I. Free energy calculations reveal rotating-ratchet mechanism for DNA supercoil relaxation by topoisomerase IB and its inhibition. Biophys. J. 2010, 99, 869−878. (36) Chillemi, G.; D’Annessa, I.; Fiorani, P.; Losasso, C.; Benedetti, P.; Desideri, A. Thr729 in human topoisomerase I modulates anticancer drug resistance by altering protein domain communications as suggested by molecular dynamics simulations. Nucleic Acids Res. 2008, 36, 5645−5651. (37) Chillemi, G.; Fiorani, P.; Castelli, S.; Bruselles, A.; Benedetti, P.; Desideri, A. Effect on DNA relaxation of the single Thr718Ala mutation in human topoisomerase I: a functional and molecular dynamics study. Nucleic Acids Res. 2005, 33, 3339−3350. (38) Fiorani, P.; Bruselles, A.; Falconi, M.; Chillemi, G.; Desideri, A.; Benedetti, P. Single mutation in the linker domain confers protein flexibility and camptothecin resistance to human topoisomerase I. J. Biol. Chem. 2003, 278, 43268−43275. (39) Fiorani, P.; Chillemi, G.; Losasso, C.; Castelli, S.; Desideri, A. The different cleavage DNA sequence specificity explains the camptothecin resistance of the human topoisomerase I Glu418Lys mutant. Nucleic Acids Res. 2006, 34, 5093−5100. (40) Fiorani, P.; Tesauro, C.; Mancini, G.; Chillemi, G.; D’Annessa, I.; Graziani, G.; Tentori, L.; Muzi, A.; Desideri, A. Evidence of the crucial role of the linker domain on the catalytic activity of human topoisomerase I by experimental and simulative characterization of the Lys681Ala mutant. Nucleic Acids Res. 2009, 37, 6849−6858.

TDRD3, Tudor domain-containing 3; SCAN1, spinocerebellar ataxia with axonal neuropathy; PARP1, poly[ADP-ribose]polymerase 1; PNKP, polynucleotide kinase 3′-phosphatase; XRCC1, X-ray repair cross-complementing protein 1; UBE3A, ubiquitin protein ligase E3A; SNRPN, small nuclear ribonucleoprotein polypeptide N; SNORD, small nucleolar RNAs

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