Topoisomerase II poisons inhibit vertebrate DNA replication through distinct mechanisms.

Topoisomerase II (TOP2) unlinks chromosomes during vertebrate DNA replication. TOP2 "poisons" are widely used chemotherapeutics that stabilize TOP2 complexes on DNA, leading to cytotoxic DNA breaks. However, it is unclear how these drugs affect DNA replication, which is a major target of TOP2 poisons. Using Xenopus egg extracts, we show that the TOP2 poisons etoposide and doxorubicin both inhibit DNA replication through different mechanisms. Etoposide induces TOP2-dependent DNA breaks and TOP2-dependent fork stalling by trapping TOP2 behind replication forks. In contrast, doxorubicin does not lead to appreciable break formation and instead intercalates into parental DNA to stall replication forks independently of TOP2. In human cells, etoposide stalls forks in a TOP2-dependent manner, while doxorubicin stalls forks independently of TOP2. However, both drugs exhibit TOP2-dependent cytotoxicity. Thus, etoposide and doxorubicin inhibit DNA replication through distinct mechanisms despite shared genetic requirements for cytotoxicity.

Basis for the discrimination of supercoil handedness during DNA cleavage by human and bacterial type II topoisomerases.

To perform double-stranded DNA passage, type II topoisomerases generate a covalent enzyme-cleaved DNA complex (i.e. cleavage complex). Although this complex is a requisite enzyme intermediate, it is also intrinsically dangerous to genomic stability. Consequently, cleavage complexes are the targets for several clinically relevant anticancer and antibacterial drugs. Human topoisomerase IIα and IIß and bacterial gyrase maintain higher levels of cleavage complexes with negatively supercoiled over positively supercoiled DNA substrates. Conversely, bacterial topoisomerase IV is less able to distinguish DNA supercoil handedness. Despite the importance of supercoil geometry to the activities of type II topoisomerases, the basis for supercoil handedness recognition during DNA cleavage has not been characterized. Based on the results of benchtop and rapid-quench flow kinetics experiments, the forward rate of cleavage is the determining factor of how topoisomerase IIα/IIß, gyrase and topoisomerase IV distinguish supercoil handedness in the absence or presence of anticancer/antibacterial drugs. In the presence of drugs, this ability can be enhanced by the formation of more stable cleavage complexes with negatively supercoiled DNA. Finally, rates of enzyme-mediated DNA ligation do not contribute to the recognition of DNA supercoil geometry during cleavage. Our results provide greater insight into how type II topoisomerases recognize their DNA substrates.

Telling Your Right Hand from Your Left: The Effects of DNA Supercoil Handedness on the Actions of Type II Topoisomerases.

Type II topoisomerases are essential enzymes that modulate the topological state of DNA supercoiling in all living organisms. These enzymes alter DNA topology by performing double-stranded passage reactions on over- or underwound DNA substrates. This strand passage reaction generates a transient covalent enzyme-cleaved DNA structure known as the cleavage complex. Al-though the cleavage complex is a requisite catalytic intermediate, it is also intrinsically dangerous to genomic stability in biological systems. The potential threat of type II topoisomerase function can also vary based on the nature of the supercoiled DNA substrate. During essential processes such as DNA replication and transcription, cleavage complex formation can be inherently more dangerous on overwound versus underwound DNA substrates. As such, it is important to understand the profound effects that DNA topology can have on the cellular functions of type II topoisomerases. This review will provide a broad assessment of how human and bacterial type II topoisomerases recognize and act on their substrates of various topological states.

No Time to Relax and Unwind: Exploration of Topoisomerases and a Growing Field of Study.

With the topoisomerase field in its sixth decade [...].

Actions of a Novel Bacterial Topoisomerase Inhibitor against Neisseria gonorrhoeae Gyrase and Topoisomerase IV: Enhancement of Double-Stranded DNA Breaks.

Novel bacterial topoisomerase inhibitors (NBTIs) are an emerging class of antibacterials that target gyrase and topoisomerase IV. A hallmark of NBTIs is their ability to induce gyrase/topoisomerase IV-mediated single-stranded DNA breaks and suppress the generation of double-stranded breaks. However, a previous study reported that some dioxane-linked amide NBTIs induced double-stranded DNA breaks mediated by Staphylococcus aureus gyrase. To further explore the ability of this NBTI subclass to increase double-stranded DNA breaks, we examined the effects of OSUAB-185 on DNA cleavage mediated by Neisseria gonorrhoeae gyrase and topoisomerase IV. OSUAB-185 induced single-stranded and suppressed double-stranded DNA breaks mediated by N. gonorrhoeae gyrase. However, the compound stabilized both single- and double-stranded DNA breaks mediated by topoisomerase IV. The induction of double-stranded breaks does not appear to correlate with the binding of a second OSUAB-185 molecule and extends to fluoroquinolone-resistant N. gonorrhoeae topoisomerase IV, as well as type II enzymes from other bacteria and humans. The double-stranded DNA cleavage activity of OSUAB-185 and other dioxane-linked NBTIs represents a paradigm shift in a hallmark characteristic of NBTIs and suggests that some members of this subclass may have alternative binding motifs in the cleavage complex.

Recognition of DNA Supercoil Handedness during Catenation Catalyzed by Type II Topoisomerases.

Although the presence of catenanes (i.e., intermolecular tangles) in chromosomal DNA stabilizes interactions between daughter chromosomes, a lack of resolution can have serious consequences for genomic stability. In all species, from bacteria to humans, type II topoisomerases are the enzymes primarily responsible for catenating/decatenating DNA. DNA topology has a profound influence on the rate at which these enzymes alter the superhelical state of the double helix. Therefore, the effect of supercoil handedness on the ability of human topoisomerase IIα and topoisomerase IIß and bacterial topoisomerase IV to catenate DNA was examined. Topoisomerase IIα preferentially catenated negatively supercoiled over positively supercoiled substrates. This is opposite to its preference for relaxing (i.e., removing supercoils from) DNA and may prevent the enzyme from tangling the double helix ahead of replication forks and transcription complexes. The ability of topoisomerase IIα to recognize DNA supercoil handedness during catenation resides in its C-terminal domain. In contrast to topoisomerase IIα, topoisomerase IIß displayed little ability to distinguish DNA geometry during catenation. Topoisomerase IV from three bacterial species preferentially catenated positively supercoiled substrates. This may not be an issue, as these enzymes work primarily behind replication forks. Finally, topoisomerase IIα and topoisomerase IV maintain lower levels of covalent enzyme-cleaved DNA intermediates with catenated over monomeric DNA. This allows these enzymes to perform their cellular functions in a safer manner, as catenated daughter chromosomes may be subject to stress generated by the mitotic spindle that could lead to irreversible DNA cleavage.

Topoisomerase II Poisons: Converting Essential Enzymes into Molecular Scissors.

The extensive length, compaction, and interwound nature of DNA, together with its controlled and restricted movement in eukaryotic cells, create a number of topological issues that profoundly affect all of the functions of the genetic material. Topoisomerases are essential enzymes that modulate the topological structure of the double helix, including the regulation of DNA under- and overwinding and the removal of tangles and knots from the genome. Type II topoisomerases alter DNA topology by generating a transient double-stranded break in one DNA segment and allowing another segment to pass through the DNA gate. These enzymes are involved in a number of critical nuclear processes in eukaryotic cells, such as DNA replication, transcription, and recombination, and are required for proper chromosome structure and segregation. However, because type II topoisomerases generate double-stranded breaks in the genetic material, they also are intrinsically dangerous enzymes that have the capacity to fragment the genome. As a result of this dualistic nature, type II topoisomerases are the targets for a number of widely prescribed anticancer drugs. This article will describe the structure and catalytic mechanism of eukaryotic type II topoisomerases and will go on to discuss the actions of topoisomerase II poisons, which are compounds that stabilize DNA breaks generated by the type II enzyme and convert these essential enzymes into "molecular scissors." Topoisomerase II poisons represent a broad range of structural classes and include anticancer drugs, dietary components, and environmental chemicals.

1,2-Naphthoquinone as a Poison of Human Type II Topoisomerases.

1,2-Naphthoquinone, a secondary metabolite of naphthalene, is an environmental pollutant found in diesel exhaust particles that displays cytotoxic and genotoxic properties. Because many quinones have been shown to act as topoisomerase II poisons, the effects of this compound on DNA cleavage mediated by human topoisomerase IIα and IIß were examined. The compound increased the levels of double-stranded DNA breaks generated by both enzyme isoforms and did so better than a series of naphthoquinone derivatives. Furthermore, 1,2-naphthoquinone was a more efficacious poison against topoisomerase IIα than IIß. Topoisomerase II poisons can be classified as interfacial (which interact noncovalently at the enzyme-DNA interface and increase DNA cleavage by blocking ligation) or covalent (which adduct the protein and increase DNA cleavage by closing the N-terminal gate of the enzyme). Therefore, experiments were performed to determine the mechanistic basis for the actions of 1,2-naphthoquinone. In contrast to results with etoposide (an interfacial poison), the activity of 1,2-naphthoquinone against topoisomerase IIα was abrogated in the presence of sulfhydryl and reducing agents. Moreover, the compound inhibited cleavage activity when incubated with the enzyme prior to the addition of DNA and induced virtually no cleavage with the catalytic core of the enzyme. It also induced stable covalent topoisomerase IIα-DNA cleavage complexes and was a partial inhibitor of DNA ligation. Findings were also consistent with 1,2-naphthoquinone acting as a covalent poison of topoisomerase IIß; however, mechanistic studies with this isoform were less conclusive. Whereas the activity of 1,2-naphthoquinone was blocked in the presence of a sulfhydryl reagent, it was much less sensitive to the presence of a reducing agent. Furthermore, the reduced form of 1,2-naphthoquinone, 1,2-dihydroxynaphthalene, displayed high activity against the ß isoform. Taken together, results suggest that 1,2-naphthoquinone increases topoisomerase II-mediated double-stranded DNA scission (at least in part) by acting as a covalent poison of the human type II enzymes.

Genome-wide TOP2A DNA cleavage is biased toward translocated and highly transcribed loci.

Type II topoisomerases orchestrate proper DNA topology, and they are the targets of anti-cancer drugs that cause treatment-related leukemias with balanced translocations. Here, we develop a high-throughput sequencing technology to define TOP2 cleavage sites at single-base precision, and use the technology to characterize TOP2A cleavage genome-wide in the human K562 leukemia cell line. We find that TOP2A cleavage has functionally conserved local sequence preferences, occurs in cleavage cluster regions (CCRs), and is enriched in introns and lincRNA loci. TOP2A CCRs are biased toward the distal regions of gene bodies, and TOP2 poisons cause a proximal shift in their distribution. We find high TOP2A cleavage levels in genes involved in translocations in TOP2 poison-related leukemia. In addition, we find that a large proportion of genes involved in oncogenic translocations overall contain TOP2A CCRs. The TOP2A cleavage of coding and lincRNA genes is independently associated with both length and transcript abundance. Comparisons to ENCODE data reveal distinct TOP2A CCR clusters that overlap with marks of transcription, open chromatin, and enhancers. Our findings implicate TOP2A cleavage as a broad DNA damage mechanism in oncogenic translocations as well as a functional role of TOP2A cleavage in regulating transcription elongation and gene activation.

Coupling the core of the anticancer drug etoposide to an oligonucleotide induces topoisomerase II-mediated cleavage at specific DNA sequences.

Etoposide and other topoisomerase II-targeted drugs are important anticancer therapeutics. Unfortunately, the safe usage of these agents is limited by their indiscriminate induction of topoisomerase II-mediated DNA cleavage throughout the genome and by a lack of specificity toward cancer cells. Therefore, as a first step toward constraining the distribution of etoposide-induced DNA cleavage sites and developing sequence-specific topoisomerase II-targeted anticancer agents, we covalently coupled the core of etoposide to oligonucleotides centered on a topoisomerase II cleavage site in the PML gene. The initial sequence used for this 'oligonucleotide-linked topoisomerase inhibitor' (OTI) was identified as part of the translocation breakpoint of a patient with acute promyelocytic leukemia (APL). Subsequent OTI sequences were derived from the observed APL breakpoint between PML and RARA. Results indicate that OTIs can be used to direct the sites of etoposide-induced DNA cleavage mediated by topoisomerase IIα and topoisomerase IIß. OTIs increased levels of enzyme-mediated cleavage by inhibiting DNA ligation, and cleavage complexes induced by OTIs were as stable as those induced by free etoposide. Finally, OTIs directed against the PML-RARA breakpoint displayed cleavage specificity for oligonucleotides with the translocation sequence over those with sequences matching either parental gene. These studies demonstrate the feasibility of using oligonucleotides to direct topoisomerase II-mediated DNA cleavage to specific sites in the genome.

Bimodal Actions of a Naphthyridone/Aminopiperidine-Based Antibacterial That Targets Gyrase and Topoisomerase IV.

Gyrase and topoisomerase IV are the targets of fluoroquinolone antibacterials. However, the rise in antimicrobial resistance has undermined the clinical use of this important drug class. Therefore, it is critical to identify new agents that maintain activity against fluoroquinolone-resistant strains. One approach is to develop non-fluoroquinolone drugs that also target gyrase and topoisomerase IV but interact differently with the enzymes. This has led to the development of the "novel bacterial topoisomerase inhibitor" (NBTI) class of antibacterials. Despite the clinical potential of NBTIs, there is a relative paucity of data describing their mechanism of action against bacterial type II topoisomerases. Consequently, we characterized the activity of GSK126, a naphthyridone/aminopiperidine-based NBTI, against a variety of Gram-positive and Gram-negative bacterial type II topoisomerases, including gyrase from Mycobacterium tuberculosis and gyrase and topoisomerase IV from Bacillus anthracis and Escherichia coli. GSK126 enhanced single-stranded DNA cleavage and suppressed double-stranded cleavage mediated by these enzymes. It was also a potent inhibitor of gyrase-catalyzed DNA supercoiling and topoisomerase IV-catalyzed decatenation. Thus, GSK126 displays a similar bimodal mechanism of action across a variety of species. In contrast, GSK126 displayed a variable ability to overcome fluoroquinolone resistance mutations across these same species. Our results suggest that NBTIs elicit their antibacterial effects by two different mechanisms: inhibition of gyrase/topoisomerase IV catalytic activity or enhancement of enzyme-mediated DNA cleavage. Furthermore, the relative importance of these two mechanisms appears to differ from species to species. Therefore, we propose that the mechanistic basis for the antibacterial properties of NBTIs is bimodal in nature.

6,6'-Dihydroxythiobinupharidine as a poison of human type II topoisomerases.

A number of natural products with medicinal properties increase DNA cleavage mediated by type II topoisomerases. In an effort to identify additional natural compounds that affect the activity of human type II topoisomerases, a blind screen of a library of 341 Mediterranean plant extracts was conducted. Extracts from Nuphar lutea, the yellow water lily, were identified in this screen. N. lutea has been used in traditional medicine by a variety of indigenous populations. The active compound in N. lutea, 6,6'-dihydroxythiobinupharidine, was found to enhance DNA cleavage mediated by human topoisomerase IIα and IIß âˆ¼8-fold and ∼3-fold, respectively. Mechanistic studies with topoisomerase IIα indicate that 6,6'-dihydroxythiobinupharidine is a "covalent poison" that acts by adducting the enzyme outside of the DNA cleavage-ligation active site and requires the N-terminal domain of the protein for its activity. Results suggest that some of the medicinal properties of N. lutea may result from the interactions between 6,6'-dihydroxythiobinupharidine and the human type II enzymes.

Smoothed Potential MD Simulations for Dissociation Kinetics of Etoposide To Unravel Isoform Specificity in Targeting Human Topoisomerase II.

Human type II topoisomerases (TopoII) are essential for controlling DNA topology within the cell. For this reason, there are a number of TopoII-targeted anticancer drugs that act by inducing DNA cleavage mediated by both TopoII isoforms (TopoIIα and TopoIIß) in cells. However, recent studies suggest that specific poisoning of TopoIIα may be a safer strategy for treating cancer. This is because poisoning of TopoIIß appears to be linked to the generation of secondary leukemia in patients. We recently reported that enzyme-mediated DNA cleavage complexes (in which TopoII is covalently linked to the cleaved DNA during catalysis) formed in the presence of the anticancer drug etoposide persisted approximately 3-fold longer with TopoIIα than TopoIIß. Notably, enhanced drug-target residence time may reduce the adverse effects of specific TopoIIα poisons. However, it is still not clear how to design drugs that are specific for the α isoform. In this study, we report the results of classical molecular dynamics (MD) simulations to comparatively analyze the molecular interactions formed within the TopoII/DNA/etoposide complex with both isoforms. We also used smoothed potential MD to estimate etoposide dissociation kinetics from the two isoform complexes. These extensive classical and enhanced sampling simulations revealed stabilizing interactions of etoposide with two serine residues (Ser763 and Ser800) in TopoIIα. These interactions are missing in TopoIIß, where both amino acids are alanine residues. This may explain the greater persistence of etoposide-stabilized cleavage complexes formed with Topo TopoIIα. These findings could be useful for the rational design of specific TopoIIα poisons.

Activities of gyrase and topoisomerase IV on positively supercoiled DNA.

Although bacterial gyrase and topoisomerase IV have critical interactions with positively supercoiled DNA, little is known about the actions of these enzymes on overwound substrates. Therefore, the abilities of Bacillus anthracis and Escherichia coli gyrase and topoisomerase IV to relax and cleave positively supercoiled DNA were analyzed. Gyrase removed positive supercoils ∼10-fold more rapidly and more processively than it introduced negative supercoils into relaxed DNA. In time-resolved single-molecule measurements, gyrase relaxed overwound DNA with burst rates of ∼100 supercoils per second (average burst size was 6.2 supercoils). Efficient positive supercoil removal required the GyrA-box, which is necessary for DNA wrapping. Topoisomerase IV also was able to distinguish DNA geometry during strand passage and relaxed positively supercoiled substrates ∼3-fold faster than negatively supercoiled molecules. Gyrase maintained lower levels of cleavage complexes with positively supercoiled (compared with negatively supercoiled) DNA, whereas topoisomerase IV generated similar levels with both substrates. Results indicate that gyrase is better suited than topoisomerase IV to safely remove positive supercoils that accumulate ahead of replication forks. They also suggest that the wrapping mechanism of gyrase may have evolved to promote rapid removal of positive supercoils, rather than induction of negative supercoils.

Interlinked DNA nano-circles for measuring topoisomerase II activity at the level of single decatenation events.

DNA nano-structures present appealing new means for monitoring different molecules. Here, we demonstrate the assembly and utilization of a surface-attached double-stranded DNA catenane composed of two intact interlinked DNA nano-circles for specific and sensitive measurements of the life essential topoisomerase II (Topo II) enzyme activity. Topo II activity was detected via the numeric release of DNA nano-circles, which were visualized at the single-molecule level in a fluorescence microscope upon isothermal amplification and fluorescence labeling. The transition of each enzymatic reaction to a micrometer sized labeled product enabled quantitative detection of Topo II activity at the single decatenation event level rendering activity measurements in extracts from as few as five cells possible. Topo II activity is a suggested predictive marker in cancer therapy and, consequently, the described highly sensitive monitoring of Topo II activity may add considerably to the toolbox of individualized medicine where decisions are based on very sparse samples.

Fluoroquinolone interactions with Mycobacterium tuberculosis gyrase: Enhancing drug activity against wild-type and resistant gyrase.

Mycobacterium tuberculosis is a significant source of global morbidity and mortality. Moxifloxacin and other fluoroquinolones are important therapeutic agents for the treatment of tuberculosis, particularly multidrug-resistant infections. To guide the development of new quinolone-based agents, it is critical to understand the basis of drug action against M. tuberculosis gyrase and how mutations in the enzyme cause resistance. Therefore, we characterized interactions of fluoroquinolones and related drugs with WT gyrase and enzymes carrying mutations at GyrA(A90) and GyrA(D94). M. tuberculosis gyrase lacks a conserved serine that anchors a water-metal ion bridge that is critical for quinolone interactions with other bacterial type II topoisomerases. Despite the fact that the serine is replaced by an alanine (i.e., GyrA(A90)) in M. tuberculosis gyrase, the bridge still forms and plays a functional role in mediating quinolone-gyrase interactions. Clinically relevant mutations at GyrA(A90) and GyrA(D94) cause quinolone resistance by disrupting the bridge-enzyme interaction, thereby decreasing drug affinity. Fluoroquinolone activity against WT and resistant enzymes is enhanced by the introduction of specific groups at the C7 and C8 positions. By dissecting fluoroquinolone-enzyme interactions, we determined that an 8-methyl-moxifloxacin derivative induces high levels of stable cleavage complexes with WT gyrase and two common resistant enzymes, GyrA(A90V) and GyrA(D94G). 8-Methyl-moxifloxacin was more potent than moxifloxacin against WT M. tuberculosis gyrase and displayed higher activity against the mutant enzymes than moxifloxacin did against WT gyrase. This chemical biology approach to defining drug-enzyme interactions has the potential to identify novel drugs with improved activity against tuberculosis.

Polyamine-containing etoposide derivatives as poisons of human type II topoisomerases: Differential effects on topoisomerase IIα and IIß.

Etoposide is an anticancer drug that acts by inducing topoisomerase II-mediated DNA cleavage. Despite its wide use, etoposide is associated with some very serious side-effects including the development of treatment-related acute myelogenous leukemias. Etoposide targets both human topoisomerase IIα and IIß. However, the contributions of the two enzyme isoforms to the therapeutic vs. leukemogenic properties of the drug are unclear. In order to develop an etoposide-based drug with specificity for cancer cells that express an active polyamine transport system, the sugar moiety of the drug has been replaced with a polyamine tail. To analyze the effects of this substitution on the specificity of hybrid molecules toward the two enzyme isoforms, we analyzed the activity of a series of etoposide-polyamine hybrids toward human topoisomerase IIα and IIß. All of the compounds displayed an ability to induce enzyme-mediated DNA cleavage that was comparable to or higher than that of etoposide. Relative to the parent drug, the hybrid compounds displayed substantially higher activity toward topoisomerase IIß than IIα. Modeling studies suggest that the enhanced specificity may result from interactions with Gln778 in topoisomerase IIß. The corresponding residue in the α isoform is a methionine.

Recognition of DNA Supercoil Geometry by Mycobacterium tuberculosis Gyrase.

Mycobacterium tuberculosis encodes only a single type II topoisomerase, gyrase. As a result, this enzyme likely carries out the cellular functions normally performed by canonical gyrase and topoisomerase IV, both in front of and behind the replication fork. In addition, it is the sole target for quinolone antibacterials in this species. Because quinolone-induced DNA strand breaks generated on positively supercoiled DNA ahead of replication forks and transcription complexes are most likely to result in permanent genomic damage, the actions of M. tuberculosis gyrase on positively supercoiled DNA were investigated. Results indicate that the enzyme acts rapidly on overwound DNA and removes positive supercoils much faster than it introduces negative supercoils into relaxed DNA. Canonical gyrase and topoisomerase IV distinguish supercoil handedness differently during the DNA cleavage reaction: while gyrase maintains lower levels of cleavage complexes on overwound DNA, topoisomerase IV maintains similar levels of cleavage complexes on both over- and underwound substrates. M. tuberculosis gyrase maintained lower levels of cleavage complexes on positively supercoiled DNA in the absence and presence of quinolone-based drugs. By retaining this important feature of canonical gyrase, the dual function M. tuberculosis type II enzyme remains a safe enzyme to act in front of replication forks and transcription complexes. Finally, the N-terminal gate region of the enzyme appears to be necessary to distinguish supercoil handedness during DNA cleavage, suggesting that the capture of the transport segment may influence how gyrase maintains cleavage complexes on substrates with different topological states.

Interactions between Quinolones and Bacillus anthracis Gyrase and the Basis of Drug Resistance.

Gyrase appears to be the primary cellular target for quinolone antibacterials in multiple pathogenic bacteria, including Bacillus anthracis, the causative agent of anthrax. Given the significance of this type II topoisomerase as a drug target, it is critical to understand how quinolones interact with gyrase and how specific mutations lead to resistance. However, these important issues have yet to be addressed for a canonical gyrase. Therefore, we utilized a mechanistic approach to characterize interactions of quinolones with wild-type B. anthracis gyrase and enzymes containing the most common quinolone resistance mutations. Results indicate that clinically relevant quinolones interact with the enzyme through a water-metal ion bridge in which a noncatalytic divalent metal ion is chelated by the C3/C4 keto acid of the drug. In contrast to other bacterial type II topoisomerases that have been examined, the bridge is anchored to gyrase primarily through a single residue (Ser85). Substitution of groups at the quinolone C7 and C8 positions generated drugs that were less dependent on the water-metal ion bridge and overcame resistance. Thus, by analyzing the interactions of drugs with type II topoisomerases from individual bacteria, it may be possible to identify specific quinolone derivatives that can overcome target-mediated resistance in important pathogenic species.

Novel trifluoromethylated 9-amino-3,4-dihydroacridin-1(2H)-ones act as covalent poisons of human topoisomerase IIα.

A number of topoisomerase II-targeted anticancer drugs, including amsacrine, utilize an acridine or related aromatic core as a scaffold. Therefore, to further explore the potential of acridine-related compounds to act as topoisomerase II poisons, we synthesized a series of novel trifluoromethylated 9-amino-3,4-dihydroacridin-1(2H)-one derivatives and examined their ability to enhance DNA cleavage mediated by human topoisomerase IIα. Derivatives containing a H, Cl, F, and Br at C7 enhanced enzyme-mediated double-stranded DNA cleavage ∼5.5- to 8.5-fold over baseline, but were less potent than amsacrine. The inclusion of an amino group at C9 was critical for activity. The compounds lost their activity against topoisomerase IIα in the presence of a reducing agent, displayed no activity against the catalytic core of topoisomerase IIα, and inhibited DNA cleavage when incubated with the enzyme prior to the addition of DNA. These findings strongly suggest that the compounds act as covalent, rather than interfacial, topoisomerase II poisons.