Inhibitors and Poisons of Mammalian Type II Topoisomerases

CHAPTER FIVE

Inhibitors and Poisons of Mammalian Type II Topoisomerases Matthew B. Murphy*, Susan L. Mercer*,†, Joseph E. Deweese*,†,1 *Lipscomb University College of Pharmacy, Nashville, TN, United States † Vanderbilt University School of Medicine, Nashville, TN, United States 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction to Topoisomerases 2. Clinically Approved Poisons and Their Derivatives 2.1 Podophyllotoxin derivatives 2.2 Anthracyclines 2.3 Acridines 2.4 Ellipticines 3. Catalytic Inhibitors and Derivatives 3.1 Bisdioxopiperazines 3.2 Merbarone 3.3 Novobiocin 4. Recent Updates in Novel Analogs and Drug Metabolites 4.1 Etoposide metabolites and analogs 4.2 Acridine analogs 4.3 Anthracyclines 4.4 Salicylate 5. Recent Updates in Natural Products and Dietary Compounds 5.1 A berberine derivative 5.2 Curcumin 5.3 Eusynstyelamide B 5.4 HU-331 5.5 Resveratrol 5.6 Xanthones 6. Recent Updates in Synthetic Compounds 6.1 Benzo[a]phenazine derivatives 6.2 Naphthalimide derivatives and conjugates 6.3 Phenanthriplatin 6.4 Pyridine 6.5 1,3-Benzoazolyl-substituted pyrrolo[2,3-b]pyrazine 6.6 Quinoline aminopurine compound 1 6.7 Quinolone-based anticancer agent: Vosaroxin Advances in Molecular Toxicology, Volume 11 ISSN 1872-0854 http://dx.doi.org/10.1016/B978-0-12-812522-9.00005-1

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6.8 Quinoxaline analogs 6.9 Thiadiazoles 6.10 Thiochromanone 6.11 Thiosemicarbazones 6.12 Triazines 7. The Path Forward: Strategies for Targeting Topoisomerase II Acknowledgments References

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Abstract Topoisomerases are critical cellular enzymes involved in the regulation of DNA topology. These enzymes generate transient single- (type I) and double-strand (type II) DNA breaks in order to relieve topological strain due to replication, transcription, and chromosome segregation. Disruption of topoisomerase activity has been used as a target of antineoplastic therapy for several decades. While some agents have been used for over 30 years, many new compounds continue to be explored. This chapter will focus on reviewing the function, mechanism, and targeting of mammalian type II topoisomerases. In particular, we will highlight newer compounds that are under examination and explore new strategies for targeting topoisomerase II in humans that may provide alternatives to existing therapies.

1. INTRODUCTION TO TOPOISOMERASES Therapeutic treatment of cancer traditionally focuses on targeting critical cellular processes involved in DNA replication and cell division. This approach involves a diverse set of agents each targeting different pathways and enzymes. One class of agents, particularly effective at disrupting cancer cell growth, are drugs targeting DNA topoisomerases [1,2]. To understand the critical functions of these enzymes, it will be important to briefly review the structure of DNA and chromatin. In a eukaryotic cell, DNA exists as a double helix that winds around histone proteins forming nucleosomes. Nucleosomes bundle together in loops to form fibers. Various states of DNA compaction are found in the genome in a context-dependent manner. For example, during interphase active regions of chromosomes decondense to varying degrees to allow access for transcription of essential genes. Such dynamic opening and closing of the genome is known as chromatin remodeling. This process is still being elucidated but involves epigenetic, cell-state, and cell-type-specific regulation. Transcription, replication, and chromatin remodeling all impact the

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topology of DNA. DNA topology refers to the relationship between the two strands of the double helix and includes the concept of supercoiling [3]. DNA topoisomerases are a family of enzymes found in the nucleus and the mitochondria that are responsible for maintaining DNA topology during the critical processes mentioned earlier [4]. Supercoiling is an important feature of DNA topology [3,4]. As such, it must be properly maintained for cellular processes to take place. Positive supercoils build up in front of transcription and replication complexes. In addition, knots and tangles occur during replication, DNA repair, recombination, and mitosis [3,4]. For example, sister chromatids become intertwined or catenated during replication. Supercoiling and tangling must be alleviated in order for cells to survive, replicate, divide, and grow. DNA topoisomerases manage supercoiling and tangling by a mechanism that involves transient breaks in the DNA [3,4]. There are two broad classes of enzymes: type I and type II. Type I topoisomerases make a transient single-strand DNA break to alleviate DNA supercoiling, which occurs ahead of replication forks and transcription bubbles. There are three families of type I topoisomerases: IA, IB, and IC, which differ based upon structure, mechanism, and cofactor involvement [4,5]. Type II topoisomerases, known generically as topoisomerase II, form a transient double-strand DNA break in one segment and are able to pass another segment of DNA through the break prior to ligating the cleaved DNA ends. Currently, two families of type II topoisomerases, known as IIA and IIB, are found in living organisms [5]. They differ based upon structure, mechanism, and cofactor involvement. Type II enzymes operate by manipulating separate chromosomes (e.g., for chromosome segregation and unknotting) or sections of the same chromosome (e.g., during transcription and replication) [4]. While the role of topoisomerase I in cells is important, it appears that topoisomerase II is essential to cellular survival because of the need to unlink (decatenate) sister chromatids after replication and before chromosome segregation [4]. All known life forms encode at least one type I and one type II topoisomerase [5]. However, many organism express several topoisomerases. Humans, for example, have six topoisomerases including four type I and two type II enzymes [4]. The type IA enzymes in humans are topoisomerase IIIα and topoisomerase IIIβ, and the type IB enzymes are topoisomerase IB and mitochondrial topoisomerase IB [4]. Humans and other mammals encode two distinct type IIA topoisomerases (Top2) that have become known as topoisomerase IIα and IIβ [4]. The genes for the Top2 isoforms are found on separate

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chromosomes and share a large amount of sequence homology with the exception of a small portion at the N-terminus and most of the C-terminus [2]. While these enzymes exhibit similar catalytic properties, they fill distinct roles in the cell. Topoisomerase IIα (Top2A) expression coincides with replication and mitosis, while topoisomerase IIβ (Top2B) is expressed throughout the cell cycle and in nondividing cells [4,6]. Based upon expression patterns, analyses of localization and trafficking, and other observations, Top2A operates during replication and mitosis, while Top2B facilitates transcription [6]. This knowledge has led to the hypothesis that targeting Top2A rather than Top2B would be more effective in killing cancer cells [1,6]. The mechanism of Top2 is still being explored, but many features have been examined. To explore the mechanism, it is important to understand that Top2 has three “gates” (Fig. 5.1). The N-terminus forms the N-gate, which is also the location of the ATPase domain. Second, the central domain including the active site is known as the DNA gate, and it is the T-segment 1

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3 Catalytic inhibitor (Merbarone)

Catalytic inhibitor (inh. DNA binding) ATP

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Catalytic inhibitor (ATP Comp. Inh.) ADP

6

5

ATP

ADP DP

Catalytic inhibitor (ICRF-193)

Poison (Etoposide)

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Fig. 5.1 Eukaryotic Top2 is a homodimer (shown at left). The upper N-terminal gate (N-gate) is the location of the ATPase domain. The central portion includes the TOPRIM metal ion coordination domain, the active site tyrosine, and the gate segment DNAbinding domain (DNA-gate). The lower portion includes the C-terminal gate (C-gate). Note that this diagram does not include the far C-terminus. Top2 binds to a helix–helix crossovers. The G-segment is severely bent and cleaved in the presence of Mg(II). The transport segment (T-segment) is captured by the N-terminal domain in the presence of ATP. Once the DNA-gate opens, the T-segment is transported through the break which coincides with the hydrolysis of one ATP molecule. After strand passage, the DNA-gate closes, and ligation can take place. Upon the hydrolysis of the second ATP molecule, the C-gate opens to release the T-segment. The enzyme can then reset for another round of catalysis. Examples of points of inhibition are indicated throughout the cycle.

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location of DNA cleavage/ligation. Within this region, there is a TOPRIM (Topoisomerase–Primase) metal-ion-binding domain and the active site tyrosine [7]. Third, the lower portion of the enzyme forms the C-gate, which includes a portion of the protein near the C-terminus. Generally, the mechanism involves a series of steps as shown in Fig. 5.1 [8]. First, the enzyme binds to helix–helix crossovers (either between the same or adjacent chromosomes) [9,10]. One of the double helices, positioned in the DNA-gate, is called the gate segment (or G-segment). The active site bends the G-segment, which may facilitate cleavage [11,12]. The other double helix, captured by the N-terminus, is called the transport segment (or T-segment). Second, the enzyme forms a transient, doublestranded DNA break in the G-segment [11,13–16]. This process involves two metal ion mechanisms for DNA cleavage [11,17–20]. Third, in the presence of ATP, the enzyme undergoes a conformational change that opens the break in the G-segment [10,21–25]. The T-segment is then passed through the break [26]. This involves a conformational change in the N-terminus and evidence from yeast Top2 suggests the hydrolysis of one of the two ATP molecules [21,22,27–29]. While ATP is required for strand passage as described earlier, it does not provide energy for strand passage but functions in maintaining the contact at the N-gate [21,29,30]. Fourth, the G-segment is brought back together and ligated, closing the DNA gate [17,31,32]. Fifth, the C-gate opens and allows release of the T-segment, which is accompanied by the hydrolysis of the remaining ATP molecule [22,27,28]. Sixth, the enzyme is now able to release the G-segment and reset for another round of catalysis. This review intends to focus specifically on the α and β isoforms of Top2 found in humans along with the agents and environmental compounds that impact these enzymes. Disruption of Top2 activity may lead to cellular death via apoptosis or via mitotic failure. For example, agents that increase enzyme:DNA complexes by inhibiting DNA ligation or increasing DNA cleavage, known as poisons, lead to an increase in single- and double-strand DNA breaks [1,33]. The consequence of increased breaks is that cells die via activation of apoptosis [1]. Conversely, some cells do not die but rather repair the damage, which results in translocations [34,35]. Some of the translocations have been shown to cause the development of therapy-induced leukemias including acute myeloid leukemia (t-AML) [34,35]. It is of great interest to identify the molecular mechanisms behind new translocations in healthy hematopoietic stem cells that result in leukemogenic potential and to

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determine what pathways will reduce the incidence of these events [36–38]. As will be discussed in this chapter, most of the approved agents targeting topoisomerase II are poisons. Furthermore, some compounds display the ability to inhibit overall topoisomerase II activity. This class of compounds has been classified as catalytic inhibitors [1]. Catalytic inhibitors do not necessarily share a common mechanism of action (see Fig. 5.1) but instead may have differing effects on the enzyme—primarily on the ATPase domain at the N-terminus of the protein. While many potential compounds have catalytic inhibitory properties, relatively few have been approved for therapeutic use. While the full explanation for this may not be apparent, the answer may lie in the specificity of the mechanism. One possible explanation is that agents targeting ATPase activity or other functions may utilize mechanisms that are less specific to topoisomerase II, while poisons primarily impact topoisomerase II [1]. These mechanisms will be explored in the sections dealing with those agents. It is of note that some compounds do not display a binary mechanism of action as either a poison or a catalytic inhibitor. As will be noted, some compounds appear to act as covalent inhibitors or poisons [8]. Previously, some of these compounds were referred to as redox-dependent poisons and are now recognized as being able to covalently adduct to Top2 [8]. The first few sections will focus on clinically approved agents. Then, the focus will shift to describing various novel compounds, natural products, environmental and industrial chemicals, drug metabolites, and other molecules that impact the function of topoisomerase II. Many of these compounds were identified from a literature review of Top2 inhibitors and poisons from late 2014 through early 2017. These sections will be followed by a discussion of challenges and opportunities for targeting topoisomerase II going forward.

2. CLINICALLY APPROVED POISONS AND THEIR DERIVATIVES As discussed earlier, topoisomerase II poisons are characterized by their ability to stabilize the enzyme–DNA complex (termed the cleavage complex) in a state, where the DNA is cleaved on one or both strands by the enzyme [33]. Due to the covalent attachment that the enzyme forms with DNA, topoisomerase II becomes a roadblock on DNA to any DNA tracking systems (such as replication or transcription complexes). These are highly effective anticancer agents that have been used in a broad array

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of cancer types. Note that some poisons are intercalative (e.g., anthracyclines, mitoxantrone, mAMSA, and ellipticine), while others are nonintercalative (e.g., epipodophyllotoxins). Structures of some established Top2 poisons are shown in Fig. 5.2.

2.1 Podophyllotoxin derivatives The origin of etoposide began underground as the natural product podophyllotoxin. Podophyllotoxin can primarily be found within the roots and rhizomes of the Podophyllum species. Two prominent species deserve attention regarding historical therapeutic uses: P. emodi, located among India’s Himalayan region, and P. peltatum, scattered across North America [39]. Cultures have utilized such plants for generations as evidenced by the Leech Book of Bald (AD 900–950). Interestingly, Native Americans used podophyllotoxin for its supposed antihelminthic and cathartic properties in addition to rudimentary cancer therapies [40]. Such medicinal uses quickly garnered scientific scrutiny. Podophyllotoxin was determined to show H3C

O O HO O A O

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O OH B

O C

H3C

O O

O

HO O

OH

O

O O

O

HO O

D O

OH

O O

O O

O

O

O

O

E O

O

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Etoposide

O

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O

OH

Doxorubicin

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Etopophos

O O OH P OH

OH O OH

O

O

Teniposide

OH O

O

O OH

O

O

OH

O

OH

Epirubicin

OH

O

O OH

NH2

O O

OH

H N N

N m-AMSA

Fig. 5.2 Structures of Top2 poisons.

Ellipticine

OH

O

OH

Idarubicin

H O N S O

HN

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NH2

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antineoplastic properties through an antimitotic mechanism of action in 1946. However, gastrointestinal toxicity prevented podophyllotoxin from being considered a viable antineoplastic therapy. Wanting to avoid these toxicities yet maintain activity, chemists at Sandoz created podophyllotoxin derivatives. Sandoz produced several prominent compounds during the late 1960s, most notably, etoposide (VP-16) and teniposide (VM-26) [39]. Eventually, these compounds were licensed to Bristol-Myers Squibb who brought etoposide and teniposide to the US market in 1983 and 1993, respectively [41]. Etoposide is also available in a phosphorylated prodrug form known as Etopophos®, which increases solubility. However, the prodrug undergoes dephosphorylation before it works against Top2. As discussed later, continual examination and derivatization has yielded a number of newer metabolites and analogs of etoposide, which continue to be studied.

2.2 Anthracyclines Anthracyclines make up a second major category of topoisomerase II poisons. Falling into a larger grouping known as antitumor antibiotics, anthracyclines were discovered in Streptomyces and were found to have antitumor activity as early as the 1950s and 1960s [42]. The founding member of this class was identified by two separate European labs and became known as daunorubicin, which was a combination of the original names each lab used: daunomycin and rubidomycin [42]. Many analogs have been produced, but only a small number have developed into approved agents. Some of the most prominent approved agents are doxorubicin, epirubicin, and idarubicin. Another agent in this class that is approved internationally is aclarubicin. Doxorubicin is one of the most widely used anticancer agents with activity in a variety of cancer types and solid tumors. In addition, more agents of this family of compounds are being explored, an example will be discussed later in this chapter. While the anthracyclines inhibit religation, similar to etoposide, they also exert antitumor effects via other mechanisms including DNA intercalation, free-radical generation, and binding to membranes [43]. As noted earlier, these agents can inhibit ligation by topoisomerase II, but they also impact cellular function in other ways. DNA intercalation blocks replication and transcription and disrupts topoisomerase II function which results in strand breaks. Additionally, oxidation of anthracyclines leads to free radical formation, which can lead to cellular damage [43]. Interestingly, the main toxicity associated with this

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class of agents is cardiotoxicity [43]. Coadministration of the topoisomerase II catalytic inhibitor and iron chelator, dexrazoxane (ICRF-187), counteracts the cardiotoxicity [44]. However, it is debated whether this effect is due to the impact on topoisomerase II or the reduction of free-radical generation. Mitoxantrone (dihydroxyanthracenedione) is an anthracene, which has structural similarities to anthracyclines. It also acts in a very similar manner in terms of interacting both with topoisomerase II as a poison and with the DNA as an intercalator. Evidence supports the ability of mitoxantrone to form DNA adducts and possibly to cause condensation of nucleic acids [42].

2.3 Acridines Amsacrine (m-AMSA) belongs to the class of compounds known as acridines. Initially isolated over 100 years ago, the acridine ring structure exists in a number of compounds [45]. Acridine derivatives show various types of activities including anticancer, antimalarial, and antiseptic among others [45]. Amsacrine is used in acute myeloid leukemias and continues to be studied in combination with various agents for the treatment of several cancers. Amsacrine intercalates into DNA, but it also acts as a topoisomerase II poison by inhibition of ligation [46].

2.4 Ellipticines The planar alkaloid ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]cabazole) comes from the Apocyanaceae plants, which includes the leaves of the evergreen tree (Ochrosia elliptica) [47]. Ellipticine and derivatives exhibit activity in several tumor types and apparently lack hematological toxicity [47]. Ellipticines act using at least two mechanisms: DNA binding/intercalation and increasing DNA cleavage by topoisomerase II. Recent evidence suggests these compounds can be activated by CYPs (cytochrome P450s) and cellular oxidases (e.g., myeloperoxidase, cyclooxygenase, etc.) into hydroxylated forms that can generate DNA adducts [48]. While this family of compounds is not in clinical use, it continues to be examined for potential therapeutic application.

3. CATALYTIC INHIBITORS AND DERIVATIVES While the “traditional” mechanism of action against topoisomerase II is poisoning, there are other ways to interact with and disrupt the function of

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these enzymes. Compounds known as catalytic inhibitors act by inhibiting catalysis and generally do so without increasing levels of DNA cleavage. This includes mechanisms of action such as decreasing enzyme:DNA binding, inhibiting the ATPase function of topoisomerase II, blocking the N-terminal clamp, and other possible effects [1]. Clinically approved agents will be reviewed first followed by compounds that are useful in research (Fig. 5.3).

3.1 Bisdioxopiperazines Bisdioxopiperazines such as dexrazoxane (ICRF-187), razoxane (ICRF159), and ICRF-193 are catalytic inhibitors of topoisomerase II [49–52]. This family of compounds generally inhibits the enzyme without inducing or stabilizing strand breaks. The mechanism of action likely involves trapping the N-terminal protein clamp in a closed conformation [49]. Experimental evidence indicates that these compounds inhibit ATP hydrolysis and trap the enzyme in a closed-clamp conformation preventing strand passage and catalytic activity [53–55]. Dexrazoxane has been clinically approved as an antitumor agent with action similar to other bisdioxopiperazines [55–57]. Interestingly, coadministration of dexrazoxane with doxorubicin reduces the cardiotoxicity associated with doxorubicin [44]. This protective mechanism may involve the ability of dexrazoxane to undergo ring-opening hydrolysis into an EDTA analog form, which chelates iron atoms thereby decreasing free radical damage [53]. The FDA has limited the use of dexrazoxane to specific sets of patients who have a large total cumulative OH H N

O

H N

O

O

O

N

N

O O

HN N

N O

N H

O

ICRF-187 dexrazone

O

N H

NH

O

O

N H

O O

ICRF-193

HO NH

O

S

Merbarone

O

O

O

OH O

O

NH2

Fig. 5.3 Structures of catalytic inhibitors of Top2.

Novobiocin

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exposure to doxorubicin based upon observations of secondary leukemia development in patients receiving dexrazoxane [58]. As mentioned earlier, these agents have not been considered to cause DNA strand breaks but perhaps allow strand breaks to form from blocked DNA replication and transcription machineries [52]. However, a recent study provides evidence that dexrazoxane can induce double-stranded breaks, presumably via Top2 [59]. It will be important to see how this evidence fits with previous and future biochemical work on these compounds in order to determine the mechanism more clearly. Evidence from purified enzyme systems using the standard methods demonstrates catalytic inhibition [49]. A different set of experimental conditions found evidence with ICRF-193 and Top2B for stabilized strand breaks but required harsh denaturation conditions [60]. For now, bisdioxopiperazines clearly disrupt Top2 function and are generally considered as Top2 catalytic inhibitors [1].

3.2 Merbarone Merbarone (5-[N-phenyl carboxamido]-2-thiobarbituric acid) is a catalytic inhibitor of topoisomerase II. The compound underwent Phase I and Phase II Clinical Trials primarily in the late 1980s and 1990s [61–64]. Biochemical studies demonstrate that the compound is a catalytic inhibitor of Top2 [65]. Interestingly, the effect of merbarone on Top2A is not due to inhibition of ATP hydrolysis nor DNA binding [65]. Not only is relaxation inhibited, but DNA cleavage levels decrease as well in the presence of merbarone [65]. The exact mechanism for these effects is not known. While this drug is not approved for use, it remains a useful tool for studying Top2.

3.3 Novobiocin Novobiocin is an antibiotic designed to target DNA gyrase, a bacterial type IIA topoisomerase [66]. Novobiocin acts by competitive inhibition of DNA gyrase ATP hydrolysis [67]. While this compound is a catalytic inhibitor of DNA gyrase, it is also a weak catalytic inhibitor of mammalian Top2 [1]. It should be noted that in human cells, alternate mechanisms of toxicity exist including interactions with histones and mitochondria [68,69]. As previously suggested, this compound may serve as a potential pharmacophore for further medicinal chemistry and rational design of Top2 catalytic inhibitors [46].

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4. RECENT UPDATES IN NOVEL ANALOGS AND DRUG METABOLITES Clinically approved Top2 agents continue to be the focus of intense study. This focus allows for a detailed understanding of metabolism and for the design and testing of novel analogs. Recent FDA guidance suggests that human metabolites formed at greater than 10% of total drug-related exposure at steady state can raise a safety concern [70]. This guidance provides evidence for the importance of studying drug metabolites and their potential for further desirable pharmacological activity or toxic effects. The following discussion explores the activity and relevance of some metabolites and analogs of clinically used Top2 agents.

4.1 Etoposide metabolites and analogs Etoposide is known to be metabolized by demethylation and oxidation of the E-ring into several forms including active catechol and quinone forms by CYPs and cellular oxidases [71–76]. Recent evidence indicates that the quinone is very potent and causes a high level of double-strand breaks through a dual mechanism of traditional and covalent poisoning [77–80]. Since the quinone form displays higher activity than etoposide and is formed by cellular oxidases such as myeloperoxidase, it has been implicated in secondary leukemias such as therapy-related acute myeloid leukemia or t-AML [35,36,38]. There is ongoing interest in the structure–activity relationship of etoposide as well as its metabolites and analogs. From a drug targeting perspective, there is interest in narrowing the cells affected by etoposide. To that end, one strategy is to take advantage of the polyamine transport system, which is active in proliferating cells [81]. Barrett and colleagues designed a version of etoposide with a polycation spermine moiety in place of the carbohydrate group (Fig. 5.4), which has been designated F14512 [81]. This addition appears to have increased solubility, cellular uptake, and cell death [81]. Further analysis has also confirmed that the spermine group also results in a stronger poisoning effect on Top2A and Top2B, with three- to fivefold increases in double-stranded DNA cleavage [82]. Molecular modeling of F14512 suggests that the structure is fairly optimized for binding to the Top2:DNA cleavage complex rather than DNA alone as might be expected for a polycation [83]. Taken together, this modification may serve as a means for increasing the effectiveness of drug targeting.

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O O

H N

HN

H N

O

N H N H

NH2

HN

O O

O O

O

O

O O

O

F14512

O

O

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OH

NPRL-Z-1

O OH

N N N N

N N N N

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O

O O

O O

O O O

O OH

4␤-(4-N,N-dimethylaminomethyl-1,2,3triazolyl)-4¢-demethyl-epi podophyllotoxin Ref. #86 — compound 6

O O

O OH

4b-(1-N,N-dimethylaminopropyl-4-methyl1,2,3-triazolyl)-4¢-demethyl-epipodophyllotoxin Ref. #86 — compound 7

Fig. 5.4 Selected etoposide derivatives are shown.

NPRL-Z-1 was formed as a product in a series of 4β-[(400 -benzamido)amino]-40 -O-demethyl-epipodophyllotoxin derivatives, which were aiming to increase Top2 inhibition, evade drug resistance, and modulate water solubility [84]. Follow-up analyses provide evidence for the ability of this analog to inhibit relaxation and cause the buildup of Top2:DNA complexes in cancer cells [85]. In studies on renal cancer cells (ACHN, A498), NPRLZ-1 appears to require lower levels of drug to achieve cell kill and causes stabilization of Top2:DNA complexes when compared to etoposide [85]. It appears that this compound is under further development with the latest study noting that reactive oxygen species generation may also play a role in the mechanism [85]. With the crystal structure of Top2B in complex with etoposide found in 2011, structural details of the interaction between etoposide and Top2B were clarified. Taking advantage of this information and of the differences between Top2A and Top2B, researchers have aimed at identifying structural features that would allow for selective targeting of each isoform. To that end, a key difference in the active sites was identified between Top2A and Top2B [86]. The carbohydrate group of etoposide is near a glutamine (Q778) in Top2B. This position in Top2A is a methionine (M762), which is nonpolar

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compared to the polar glutamine. From this, two strategies were used to modify etoposide—both focused on replacing the carbohydrate moiety with a triazole group, allowing for various substitutions [86]. The authors argue based upon cellular data, that at least two of the compounds (Fig. 5.4, compounds 6 and 7) have higher IC50 values when Top2B is knocked down, which suggests that these compounds are selective for Top2B [86]. Unfortunately, no experiments in a purified system were provided to examine direct activity against both isoforms. This approach, however, has highlighted that there are subtle but important distinctions between the active sites of Top2A and Top2B.

4.2 Acridine analogs Building upon the success of acridine-based compounds like amsacrine, studies have also pursued modifications to the core structure in an effort to increase activity against Top2 and cancer cells (Fig. 5.5). A recent study utilized trifluoromethylated 9-amino-3,4-dihydroacridin-1(2H)-one derivatives of acridine with several substitutions including halogenations [87]. Results with Top2A indicated that some modifications (C7 H, Cl, F, and Br) were tolerated, but all were less potent than amsacrine [87]. Interestingly, these compounds were redox sensitive and appeared to act as covalent poisons and lost activity against the catalytic core of Top2A—which is not typically seen for traditional interfacial poisons such as amsacrine [87]. Further work to clarify this mechanism will be needed, but it provides an interesting example of the impact of structural modifications on drug targeting. An imidazoacridinone known as C-1311 (now Symadex) was identified in the 1990s as being active against Top2 in purified systems and against cancer cells possessing EC50 values similar to m-AMSA and doxorubicin [88]. More recently, this compound has been shown to also inhibit the receptor tyrosine kinase FLT3 and induce apoptosis [89]. Further evidence indicates

N NH O

NH2 O

OH

CI N

N

CF3

Trifluoromethylated acridine derivative

Fig. 5.5 Structures of selected acridine analogs.

N

Imidazoacridinone C-1311

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that cellular responses to this compound are affected by the presence or absence of p53, and it can sensitize cells to radiation therapy [90].

4.3 Anthracyclines Doxorubicin’s success as an anticancer agent has been somewhat tarnished by the threat of cardiotoxicity associated with higher cumulative doses. This has fueled interest in new anthracyclines with reduced side effects. Recently, a novel doxorubicin analog, 5-imino, 13-deoxydoxorubicin (GPX-150), has been identified as having less cardiotoxicity in a rabbit model [91]. GPX-150 (Fig. 5.6) also displayed no impact in a purified Top2B decatenation assay when compared with doxorubicin [91]. Interestingly, the lack of activity against Top2B combined with the decrease in cardiotoxicity may actually lend support to the role of Top2B in doxorubicin-induced cardiotoxicity. However, this study did not show any data for activity against Top2A. So, the cellular target of GPX-150 is not entirely clear. GPX-150 has proceeded through Phase 1 and recently (late 2016) completed a Phase 2 clinical trial for soft tissue sarcoma. The results of the Phase 2 trial are not available as of the time of this writing. While there are undoubtedly many other novel anthracycline analogs being examined in various contexts, the literature indicates that at least two other areas appear to be in focus: (1) antioxidant usage for cardioprotection during anthracycline therapy and (2) targeted packaging and delivery of anthracyclines (e.g., antibody–drug conjugates and nanoparticles). Antioxidant usage may be a short-term solution to the cardiotoxicity of these compounds. However, packaging and delivery systems may enable a higher specificity of targeting these compounds to cancer cells. The next decade will undoubtedly see a number of new strategies developed for both of these issues. OH O

OH O

OH

OH

HO O

NH

NH2

OH O O

GPX-150

OH

2-Hydroxybenzoate

Fig. 5.6 Structures of doxorubicin analog GPX-150 and salicylate (2-hydroxybenzoate).

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4.4 Salicylate Salicylates are among a family of compounds found in a number of products including aspirin (acetylsalicylic acid) and oil of wintergreen (methyl salicylate). Salicylate (2-hydroxybenzoate; Fig. 5.6) is formed enzymatically (cyclooxygenase) or nonenzymatically from aspirin [92]. Evidence from biochemical studies indicates that sodium salicylate decreases the accumulation of doxorubicin-induced DNA strand breaks [93]. The evidence suggests that sodium salicylate is a catalytic inhibitor of Top2 [93]. Further analysis demonstrates that salicylate does not prevent Top2:DNA binding but does inhibit Top2A ATP hydrolysis [94]. The authors propose that the noncompetitive inhibition of ATP hydrolysis is secondary to the inhibition of Top2 DNA cleavage [94]. Interestingly, the salicylate inhibition appears to be stronger against Top2A than against Top2B [94]. In an additional study, the researchers identify positions on salicylate (20 and 500 ) that appear to be important in the interaction with Top2 [95].

5. RECENT UPDATES IN NATURAL PRODUCTS AND DIETARY COMPOUNDS Over the last two decades a number of compounds found in plants and various food sources have been identified that impact the activity of Top2. For example, dietary polyphenols or bioflavonoids are naturally occurring compounds in plants that are often part of the human diet. Numerous bioflavonoids have been examined for activity against topoisomerase II including isoflavones such as genistein, flavonols such as myricetin and quercetin, and catechins such as EGCG, ECG, and GCG [96–99]. Other families of compounds have been explored as well. The following is a brief review of some recent explorations into the activity of dietary and natural product compounds and their derivatives (Fig. 5.7).

5.1 A berberine derivative Berberine is a plant alkaloid derived from roots, rhizomes, and stem bark of several plants with a historical use in various ancient medicines. It has a characteristic yellow color, which can be useful as a dye. A recent study of cyclizing berberine formed a compound referred to as A35, which was studied as a dual Top1 and Top2 inhibitor [100]. The authors provide evidence that A35 inhibits Top1 and Top2A relaxation [100]. Additionally, A35 appears to increase Top2A DNA cleavage and inhibits religation, which

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3-(3-Butylamino-2-hydroxypropoxy)-1-(4chlorophenethoxy)-9H-xanthen-9-one Ref. 113 — compound 37

Fig. 5.7 Structures of selected dietary and natural product derivatives are shown.

provides evidence that A35 is a Top2 poison. The authors do show a cleavage assay with Top2B that displays no cleavage activity in the presence of A35 at concentrations up to 180 μM [100]. Based upon the DNA cleavage and other cellular data, they conclude that A35 is selective for Top2A. While this may be accurate, it will be interesting to see a more complete biochemical analysis including Top2B relaxation assays. Nevertheless, this compound appears to be active in cells against both Top1 and Top2A.

5.2 Curcumin Curcumin (diferuloylmethane) has been used for medicinal, food, and domestic purposes in the Indian subcontinent. Current day medicinal use is under increasing interest and exploration with numerous Phase I and Phase II clinical trials either completed or ongoing for a wide range of indications from Alzheimer’s disease to cancer to kidney disease (for the latest, see: https://clinicaltrials.gov/). Curcumin is being examined as both a chemopreventative and a curative agent. Evidence indicates that curcumin can

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cause apoptosis and cancer cell death [101,102]. It was initially found that curcumin does increase DNA cleavage levels, but the mechanism was unclear [101]. Additionally, it was found that curcumin induces both Top1 and Top2 complexes in K562 cells [103]. Further biochemical analysis confirmed the proposal by Lopez-Lazaro and colleagues that redox cycling and oxidation is involved in the ability of curcumin to affect Top2 [104,105]. These experiments support the hypothesis that oxidation of curcumin is critical in the ability to increase DNA cleavage by Top2 and allows the compound to act as a redox-dependent poison and not as an interfacial poison of Top2A and Top2B [104,105]. Examination of autoxidation of curcumin and the other forms of curcumin found in turmeric extract, demethoxycurcumin and disdemethoxycurcumin, also demonstrates the important contribution oxidation plays in increasing Top2-mediated DNA cleavage [105].

5.3 Eusynstyelamide B Eusynstyelamide B (EB) is an alkaloid from a Marine ascidian, which was identified in a screen for anticancer natural products [106]. EB inhibited cell growth and induced a G2 cell cycle arrest in prostate (LNCaP) and breast (MDA-MB-231) cell lines [106]. Based upon DNA damage induced in prostate cells along with other evidence, the researchers examined Top2 activity in the presence of EB and found EB may be disrupting Top2 activity [106]. The data did not clarify the exact mechanism nor the isoform used in these studies, but there appeared to be an inhibition of relaxation and decatenation, which would be consistent with an G2 arrest prior to cell division [106].

5.4 HU-331 Cannabidiol is a major component of cannabis that apparently lacks psychotropic effects. However, derivatives of cannabidiol have been synthesized over the last 50 years for various purposes. More recently, cannabinoid quinone derivatives have been synthesized for analysis of medicinal properties [107]. Studies identified HU-331, an oxidation product of cannabidiol, as being active against cancer cells in culture [107]. Further examination identified Top2 as a possible target of HU-331s activity [108]. Initial evidence suggested catalytic inhibition, and this mechanism was supported by additional biochemical data demonstrating inhibition of Top2A ATP hydrolysis and a decrease in enzyme:DNA binding [109]. Additional studies of HU-331 and cannabidiol have been performed to clarify the mechanism,

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and those results support a mechanism that involves locking the N-terminal clamp similar to dexrazoxane (J.E. Deweese, unpublished results). Taken together, HU-331 interacts with the N-terminal domain of Top2, which inhibits ATP hydrolysis and disrupts the catalytic cycle.

5.5 Resveratrol As discussed earlier, polyphenols provide a rich source of compounds to explore for medicinal purposes. Resveratrol is found in grapes and demonstrates anticancer activities. Initial studies with Top2 show the ability of resveratrol to inhibit relaxation [110]. Additional studies support an inhibitory effect on Top2A-mediated decatenation and also an increase in cellular double-strand breaks in the presence of resveratrol [111]. More recently, studies of biologically relevant Phase II metabolites of resveratrol, including reserveratrol-3-sulfate, resvertraol-3-glucuronide, and resveratrol disulfate, show retention of activity against Top2 [112]. Importantly, these authors provide cellular evidence that resveratrol and the metabolites are not acting as poisons against Top2A and Top2B [112]. Based upon their use of the in vivo complex of enzyme bioassay, no increase in enzyme:DNA complexes occurs in the presence of resveratrol or the metabolites [112]. Further work is needed to clarify the impact of resveratrol on the activity of Top2.

5.6 Xanthones Xanthones or xanthonoids are heterotricyclic phytochemicals isolated from a number of plant and fungal sources. These compounds appear to have a diversity of cellular and biochemical effects. A recent study of a series of C1-O-substituted-3-(3-butylamino-2-hydroxy-propoxy)-xanthen-9-ones explores the activity of these derivatives against Top2 [113]. The study focuses on compound 37 from this series, which shows the best ability to inhibit relaxation but does not increase DNA cleavage levels. Further analysis indicates that this compound is a catalytic inhibitor likely by inhibition of ATP binding and/or hydrolysis [113]. Given the structure of the compound, the authors performed molecular docking to examine the fit of the compound to the ATP-binding site and provide some analysis of the docking [113]. While this compound provides an interesting possibility, further work will need to keep in mind the promiscuity of compounds from this series. The xanthone core structure may bind many ATPase domains, which may make the impact of this compound more general and less specific.

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6. RECENT UPDATES IN SYNTHETIC COMPOUNDS Numerous strategies have been used to target Top2. The relatively recent availability of eukaryotic and mammalian Top2 crystal structures that include partial to nearly complete forms has opened the door to newer structure-based drug design approaches. Taking advantage of the knowledge of active agents in combination with structural data, synthetic strategies have sought to target Top2 more selectively, in particular, target Top2A while avoiding Top2B. While truly selective inhibitors have yet to be identified and used clinically, some compounds are moving closer to that goal. NK314 is a synthetic benzo[c]phenathridine alkaloid, which shows selective activity against Top2A in vitro and in cells [114]. This may be the first among many synthetic compounds to aim at selectively inhibiting Top2A. The following is a brief review of an array of synthetic compounds targeting Top2 (Fig. 5.8).

6.1 Benzo[a]phenazine derivatives Benzo[a]phenazine derivatives have been explored in cancer therapy for over two decades. In the 1990s, the benzo[a]phenazine derivative 2+ N N H N

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Fig. 5.8 Representative structures of synthetic compounds.

7-(4-(Dimethylamino)butyl) benzo[a]phenazin-5(7H)-one Ref. 119 — compound 6c-1

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NC-190 was characterized and found to be a Top2 poison [115]. XR11576 is another derivative identified as a dual poison of Top1 and Top2 [116,117]. Further work on XR11576 demonstrated that cellular Top:DNA complexes were formed with both Top1 and Top2 [118]. Novel 7-alkylamino-substituted benzo[a]phenazines were recently synthesized and characterized [119]. Among the derivatives, several compounds displayed the ability to inhibit both Top1- and Top2-mediated DNA relaxation at the same concentrations as camptothecin and etoposide [119]. While the active derivative 6c-1 was a Top1 poison, it did not poison Top2 cleavage, but it was found to be a competitive inhibitor of Top2 ATP hydrolysis [119]. This could serve as an interesting model for a dual Top1 poison, Top2 catalytic inhibitor.

6.2 Naphthalimide derivatives and conjugates Amonafide (benzisoquinolinedione) is a naphthalimide that has been examined as an anticancer agent for approximately 30 years. The activity of amonafide against Top2 as a poison and DNA intercalator was identified by the late 1980s [120]. A positive feature of this agent is that it is not a substrate for the gp170 (MRD1) efflux transporter, which would allow it to avoid resistance mechanisms that confound other Top2 agents in some cancers [121,122]. A recent phase III trial found amonafide in combination with cytarabine to be as effective in inducing complete remission as daunorubicin and cytarabine in patients with secondary AML [123]. Numerous derivatives have been formed based upon amonafide, but a recent study examined a naphthalimide—cyclam conjugate that has the potential to inhibit both Top1 and Top2 [124]. The authors provide data from purified enzyme assays that some forms of the conjugate inhibited relaxation by Top1 and decatenation by Top2 [124]. Unfortunately, the biochemical evidence is limited but appears promising. Given the critical role that topoisomerases have in cells, inhibition of both classes is certainly disruptive to replication, gene expression, and cell division. However, it is unclear whether these derivatives would be safe in vivo. Naphthalimides are also being conjugated to metal complexes such as ruthenium (II) polypyridyl complexes. A recent study examined the Top1 and Top2 inhibitory activity of two forms of a Ru (II) polypyridyl complex fused with a naphthalimide group [125]. The authors provide biochemical data indicating the ability of these complexes to inhibit both Top1and Top2-mediated relaxation. Intriguingly, these complexes are also

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photoactivatable and can cause DNA cleavage in the absence of Top1 or Top2 [125]. It will be interesting to see the further development of novel conjugates such as these. It is also of note that nonnaphthalimide Ru (II) complexes are also being examined as Top2 poisons and DNA-damaging agents [126].

6.3 Phenanthriplatin Phenanthriplatin is a monofunctional cationic platinum (II) complex that is able to bind to DNA on a single base rather than cross-linking DNA strands/ bases [127]. A recent study examined the impact of phenanthriplatin on Top2A activity [128]. Biochemical evidence indicates that Top2Amediated DNA cleavage is increased in the presence of phenanthriplatin and that sites of cleavage are consistent with sites of alkylation of the DNA [128]. Cellular evidence is consistent with this result showing an increase in cellular Top2:DNA complexes formed in the presence of phenathriplatin [128]. Taken together, the authors conclude that phenanthriplatin is acting as a covalent Top2 poison, and this may serve as a prototype molecule combining the impact of platinum (II)-mediated alkylation and Top2 poisoning.

6.4 Pyridine Triaryl pyridine derivatives have been studied as dual Top1 and Top2 inhibitors [129,130]. The same group has hydroxylated this series of compounds to improve activity against Top1 and Top2 [131]. While there are a large number of compounds being examined, the compounds span the range from showing no activity to high inhibitory activity against Top1- and Top2Amediated DNA relaxation. Selected compounds were assayed for DNAdamaging capability in cells (comet assay) and for the ability to poison Top2A-mediated DNA cleavage. The data suggest that at least one compound has the ability to poison Top2A and to cause DNA strand breaks in cells [131]. Further work is being done on this series to refine the best compounds for increased activity.

6.5 1,3-Benzoazolyl-substituted pyrrolo[2,3-b]pyrazine With a unique strategy, Li and coauthors utilized 1,3-benzoazole derivatives (catalytic inhibitor of Top2) fused to aloisine derivatives (kinase inhibitor) to design a Top2 catalytic inhibitor [132]. The resulting compounds are a series of 1,3-benzoazolyl-substituted pyrrolo[2,3-b]pyrazines (BPPs). Functional

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assays demonstrate that members of this series show variable ability to inhibit Top2-mediated DNA relaxation [132]. Additionally, among the selected BPPs tested, there was no evidence of Top2 poisoning [132]. Further biochemical analysis suggests that these compounds inhibit Top2 ATP hydrolysis [132]. Unfortunately, the published study does not specify the isoform used in the biochemical assays. Cellular assays showed an increase in apoptosis in the presence of selected BPPs [132]. Additional work is likely ongoing with this series.

6.6 Quinoline aminopurine compound 1 Using a structure-based design approach, Chene and colleagues developed a pharmacophore based upon the ATP-binding site within the ATPase domain of Top2 [133]. The compound they generated with the most activity is a purine diamine analog called quinoline aminopurine compound 1 (QAP 1), which was found to be a catalytic inhibitor of Top2A and Top2B [133]. In order to determine whether QAP 1 is specific for Top2, the authors used a panel of 20 different serine/threonine/tyrosine protein kinases and determined that only c-Src and Ret had IC50 values lower than 10 μM and the IC50 for Top2 was an order of magnitude lower than for c-Src and Ret [133]. They also found that QAP 1 disrupted chromosome segregation in HL-60 cells [133]. In a separate study, 18F-labeled QAP 1 derivatives were generated as PET imaging probes for Top2 overexpression and tested in mice [134]. The utility of QAP 1 as a diagnostic may prove to be useful in the clinic.

6.7 Quinolone-based anticancer agent: Vosaroxin Quinolone-based drugs are typically antibacterial in nature and cause DNA damage by poisoning bacterial type II topoisomerases: DNA gyrase and topoisomerase IV. However, efforts have been underway to develop quinolone-based anticancer agents. One agent from this category that has shown promise in vitro and in clinical trials is vosaroxin (previously voreloxin or SNS-595). Biochemical and cellular evidence indicates that vosaroxin poisons Top2A and Top2B [135]. It also is an intercalative agent due to the planar nature of the thiazole group, and the evidence indicates that this key structural feature is critical to the cytotoxicity of the compound [135]. This compound appears to have activity in acute myeloid leukemia, and it is undergoing additional Phase II/III clinical trials to validate activity in patients.

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6.8 Quinoxaline analogs XK469 is a peptidomimetic quinoxaline anticancer agent with clinical efficacy [136,137]. A series of analogs based upon XK469 were synthesized and analyzed [138]. Based upon biochemical evidence, one of the analogs displayed the ability to inhibit decatenation by Top2A [138]. Additional data utilized molecular docking studies and did not necessarily validate the mechanism. More data will be needed to clarify the mechanism of action of these compounds on Top2.

6.9 Thiadiazoles 1,3,4-Thiadiazoles have been shown to have anticancer properties [139]. Additionally, some derivatives also impact DNA synthesis [140]. Plech and colleagues synthesized a series of 2,5-substituted 1,3,4-thiadiazoles and tested them for activity against cancer cells and Top2 [141]. While some of these compounds do impact cancer cell growth (MCF-7 and MDA-MB231), the authors do not show the data for the biochemical assays and do not appear to specify which isoform was examined. Therefore, it is difficult to comment on the reliability of this data and of the interpretation of it.

6.10 Thiochromanone Thiochromanones have a diverse array of biological activities including anticancer, antibacterial, and antiinflammatory. A recent published study examined the activity of the cis/trans isomers of the halogenated thiochromanone, 3-chloromethylene-6-fluorochroman-4-one (CMFT) against both Top1 and Top2 [142]. Assays explored both cellular and biochemical analyses. Specifically, the biochemical data support that the trans-isomer (E-CMFT) inhibits relaxation by Top1 and Top2 [142]. Further, the authors provide evidence that this compound serves as a Top1 poison [142]. They also argue that E-CMFT is a Top2 poison. However, the data provided are insufficient to support that claim since there was not a proper positive control, and the authors do not specify the isoform of enzyme used [142]. It will be interesting to explore this compound further and additional data will help clarify the mechanism against Top2.

6.11 Thiosemicarbazones Thiosemicarbazones (TSCs) are a class of inorganic metal chelators, and numerous forms have been synthesized for examination of anticancer

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properties. TSCs form complexes with various transition metals including Cu, Pd, and Ni. The impact of TSCs on Top2 has been noted several times, and the exact mechanism was initially unclear, though it appeared to involve increases in DNA cleavage [143]. The ability to impact Top2 function was also used to design Cu-radiolabeled TSC complexes in order to track Top2 expression in tumor tissue [144]. TSC24 was identified as a catalytic inhibitor of Top2A and showed the ability to inhibit ATP hydrolysis [145]. Further, evidence from subsequent analysis indicated that the presence of the metal ion (Cu(II) in this study) was essential for TSC activity against Top2 [146]. To this end, analysis of the TSC ligands Dp44mT and Triapine demonstrated that the ligands alone do not show activity against Top2A [147]. More recently, analysis of quinolone-2-carboxaldehyde TSCs in both Cu(II) and Ni(II) forms demonstrate that the Cu(II) forms are more active [148]. Studies of acetylpyridine-ethylthiosemicarbazone and acetylpyrazinemethylthiosemicabazone Cu(II) complexes confirm the ability of Cu(II) TSC complexes to inhibit ATP hydrolysis by Top2A [149]. Interestingly, not only do these compounds inhibit relaxation and ATP hydrolysis, but they also increase Top2A-mediated DNA cleavage without inhibiting ligation [149]. This combination of results is a bit puzzling and suggests the mechanism is not as simple as direct inhibition of ATP hydrolysis. Additional analysis is needed to more clearly understand how the compounds are resulting in higher levels of DNA cleavage without inhibiting ligation. Recent cellular studies continue to support the antiproliferative effect of Cu(II) TSCs, and additional work is underway on this interesting class of compounds [150,151]. The data in the previous papers have focused on Top2A. It will be interesting to see if these results are consistent for Top2B.

6.12 Triazines Initial studies by one group identified 1,3,5-triazine compounds as a viable pharmacophore for Top2 catalytic inhibition [152]. As a follow-up to that study, they generated 4,6-substituted 1,3,5-triazines and examined these compounds for activity [153]. Biochemical evidence indicates that these compounds do not increase DNA cleavage or poison Top2A. They do provide evidence with Top2A that these compounds inhibit relaxation, decatenation, and ATP hydrolysis [153]. Additional studies are likely ongoing based upon the potential for further refinement.

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7. THE PATH FORWARD: STRATEGIES FOR TARGETING TOPOISOMERASE II What is the future of Top2-targeting in anticancer therapy? Since Top2 remains an effective target for disrupting cell growth, the clinical relevance of this enzyme persists. However, a desire to reduce adverse events and off-target toxicities associated with Top2 agents fuels their refinement. Such refinements focus on greater selectivity for targeting Top2A, decreasing an agent’s impact on normal cells and tissues, and in some cases decreasing the direct damage to DNA. While some compounds display a level of selectivity, it continues to be a challenge to unambiguously demonstrate selectivity along with activity against cancer cells and in animal models. The two isoforms share a high amino acid identity (80+%) throughout the ATPase and catalytic core regions (including the active site and the metal-ion-binding TOPRIM domain). Nevertheless, structure-based design methods are pursuing this goal and will likely produce a number of new lead compounds in the coming years. Given the challenges of structure-based design in targeting the DNA cleavage/ligation active site where these proteins share such similarity, what other options are available? As noted earlier, cancer cell or cell proliferationspecific targeting by using chemical moieties added on to Top2 agents shows promise (e.g., F14512). However, this solution does not completely solve the problem of differentiating between the Top2A and Top2B. In addition, it does not settle the issue of secondary leukemias, which are associated with etoposide and several other Top2 drugs. The challenge of selectivity will not likely be solved by one strategy. Instead, multiple strategies from different angles will be needed to isolate and target Top2A while avoiding Top2B. One such strategy involves an alternative approach that identifies functional and regulatory differences between the two enzymes. As discussed earlier in this chapter, these two isoforms fulfill distinct, yet sometimes overlapping, cellular roles. Amino acid sequence analysis of Top2A and Top2B provides insight into key differences between these isoforms, which provide information on some of the functional distinctions. As mentioned earlier, the ATPase and catalytic core share very high sequence identities. In contrast, the C-terminal domain of these proteins shares little identity overall. While these do share isolated pockets of high similarity, there are large regions of sequence that do not

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align at all. Studies on the C-terminus have demonstrated that this region is the location of the nuclear localization sequence, a nuclear export sequence, multiple phosphorylation sites, and other modification sites. Additionally, while this region does not appear to play a direct role in catalysis, it does appear to regulate the substrate selection in vitro and the localization and functional role in cells [154–159]. Protein–protein interactions may also be mediated by this region of the protein [6]. As such, the C-terminus is apparently a critical regulator of function for Top2. However, it remains one of the least understood portions of the protein. Based upon the difficulty of generating a crystal structure of this domain, it has been considered to be relatively unstructured or at least highly movable and flexible. In recent years, at least one research group has tried to take advantage of these additional properties of Top2A in order to make the enzyme a more responsive target. Both isoforms have nuclear import and export signals found in their C-terminal domains, and there is some evidence that these proteins may have some ability to be shuttled in and out of the nucleus, though that evidence is not conclusive [154,157]. Top2 drug resistance in multiple myeloma appears to involve exporting Top2A from the nucleus [160,161]. Therefore, it was hypothesized that by adding a nuclear export inhibitor, Top2A would remain in the nucleus and persist as a drug target, effectively sensitizing cells to Top2 drugs [160,161]. This was tested using agents targeting CRM1 [162,163]. CRM1 (XPO1) is directly involved with Top2 export from the nucleus, and CRM1 inhibitors do prevent the export of Top2 from the nucleus [162,163]. This method is showing promise at overcoming drug resistance due to nuclear exporting of Top2. It will be interesting to see if this strategy has broader applications. For example, is it possible to prevent import of Top2A into the nucleus? This strategy may reduce the need for DNA-damaging Top2 poisons, but it remains unclear what the cellular and systemic consequences would be from this type of strategy. Additionally, it will require a much more detailed understanding of the import/export process to be able to effectively assess this strategy. On a separate note, Top2A and Top2B are the targets of a number of posttranslational modifications (PTMs). According to Phosphosite Plus (phosphosite.org), the most common PTM of Top2 is phosphorylation, but the possible modifications also include sumoylation, acetylation, ubiquitination, and methylation. However, given that much of the data on PTMs is high-throughput, the level of confidence in this data remains low until these modifications can be examined biochemically and functionally. This

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assumes, of course, that methodologies exist to appropriately test and examine these modifications, which is not necessarily the case. Will it be possible to selectively target Top2A via the posttranslational modifications? Perhaps there is evidence for this from a medication used for African sleeping sickness, Suramin, which is known to be a kinase inhibitor. Interestingly, studies from the 1990s provide evidence that Suramin decreases activity of Top2 via inhibition of protein kinase C [164,165]. Clearly, additional studies will be required to put a proper context on how specific PTMs modulate Top2 function in cells. In conclusion, the number of new compounds that impact Top2 activity continues to rise each year. Both catalytic inhibitors and poisons are under development including some that are isoform selective and some that target both Top1 and Top2. In the modern genomic area of precision medicine, classical targets like Top2 are not likely to be completely replaced or removed, at least not in the near future. Therefore, it is critical that the topoisomerase field continue to examine new compounds, refine existing compounds, and explore new strategies for targeting Top2 in cancer cells.

ACKNOWLEDGMENTS We would like to thank J. Myles Keck for reviewing the manuscript. We also would like to thank the Lipscomb University College of Pharmacy, the Pharmaceutical Sciences Summer Research Program, and the AFPE Gateway Scholars Program (M.B.M.) for support.

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