A series of Spiropyrimidinetriones that enhances DNA cleavage mediated by Mycobacterium tuberculosis gyrase

The rise in drug-resistant tuberculosis has necessitated the search for alternative antibacterial treatments. Spiropyrimidinetriones (SPTs) represent an important new class of compounds that work through gyrase, the cytotoxic target of fluoroquinolone antibacterials. The present study analyzed the effects of a novel series of SPTs on the DNA cleavage activity of Mycobacterium tuberculosis gyrase. H3D-005722 and related SPTs displayed high activity against gyrase and increased levels of enzyme-mediated double-stranded DNA breaks. The activities of these compounds were similar to those of the fluoroquinolones, moxifloxacin, and ciprofloxacin and greater than that of zoliflodacin, the most clinically advanced SPT. All the SPTs overcame the most common mutations in gyrase associated with fluoroquinolone resistance and, in most cases, were more active against the mutant enzymes than wild-type gyrase. Finally, the compounds displayed low activity against human topoisomerase IIα. These findings support the potential of novel SPT analogues as antitubercular drugs

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

Voreloxin Is an Anticancer Quinolone Derivative that Intercalates DNA and Poisons Topoisomerase II

Topoisomerase II is critical for DNA replication, transcription and chromosome segregation and is a well validated target of anti-neoplastic drugs including the anthracyclines and epipodophyllotoxins. However, these drugs are limited by common tumor resistance mechanisms and side-effect profiles. Novel topoisomerase II-targeting agents may benefit patients who prove resistant to currently available topoisomerase II-targeting drugs or encounter unacceptable toxicities. Voreloxin is an anticancer quinolone derivative, a chemical scaffold not used previously for cancer treatment. Voreloxin is completing Phase 2 clinical trials in acute myeloid leukemia and platinum-resistant ovarian cancer. This study defined voreloxin's anticancer mechanism of action as a critical component of rational clinical development informed by translational research.Biochemical and cell-based studies established that voreloxin intercalates DNA and poisons topoisomerase II, causing DNA double-strand breaks, G2 arrest, and apoptosis. Voreloxin is differentiated both structurally and mechanistically from other topoisomerase II poisons currently in use as chemotherapeutics. In cell-based studies, voreloxin poisoned topoisomerase II and caused dose-dependent, site-selective DNA fragmentation analogous to that of quinolone antibacterials in prokaryotes; in contrast etoposide, the nonintercalating epipodophyllotoxin topoisomerase II poison, caused extensive DNA fragmentation. Etoposide's activity was highly dependent on topoisomerase II while voreloxin and the intercalating anthracycline topoisomerase II poison, doxorubicin, had comparable dependence on this enzyme for inducing G2 arrest. Mechanistic interrogation with voreloxin analogs revealed that intercalation is required for voreloxin's activity; a nonintercalating analog did not inhibit proliferation or induce G2 arrest, while an analog with enhanced intercalation was 9.5-fold more potent.As a first-in-class anticancer quinolone derivative, voreloxin is a toposiomerase II-targeting agent with a unique mechanistic signature. A detailed understanding of voreloxin's molecular mechanism, in combination with its evolving clinical profile, may advance our understanding of structure-activity relationships to develop safer and more effective topoisomerase II-targeted therapies for the treatment of cancer

Novel xanthone-polyamine conjugates as catalytic inhibitors of human topoisomerase II\uce\ub1

It has been proposed that xanthone derivatives with anticancer potential act as topoisomerase II inhibitors because they interfere with the ability of the enzyme to bind its ATP cofactor. In order to further characterize xanthone mechanism and generate compounds with potential as anticancer drugs, we synthesized a series of derivatives in which position 3 was substituted with different polyamine chains. As determined by DNA relaxation and decatenation assays, the resulting compounds are potent topoisomerase II alpha inhibitors. Although xanthone derivatives inhibit topoisomerase II alpha-catalyzed ATP hydrolysis, mechanistic studies indicate that they do not act at the ATPase site. Rather, they appear to function by blocking the ability of DNA to stimulate ATP hydrolysis. On the basis of activity, competition, and modeling studies, we propose that xanthones interact with the DNA cleavage/ligation active site of topoisomerase II alpha and inhibit the catalytic activity of the enzyme by interfering with the DNA strand passage step. (C) 2017 Elsevier Ltd. All rights reserved

Direct Monitoring of the Strand Passage Reaction of DNA Topoisomerase II Triggers Checkpoint Activation

<div><p>By necessity, the ancient activity of type II topoisomerases co-evolved with the double-helical structure of DNA, at least in organisms with circular genomes. In humans, the strand passage reaction of DNA topoisomerase II (Topo II) is the target of several major classes of cancer drugs which both poison Topo II and activate cell cycle checkpoint controls. It is important to know the cellular effects of molecules that target Topo II, but the mechanisms of checkpoint activation that respond to Topo II dysfunction are not well understood. Here, we provide evidence that a checkpoint mechanism monitors the strand passage reaction of Topo II. In contrast, cells do not become checkpoint arrested in the presence of the aberrant DNA topologies, such as hyper-catenation, that arise in the absence of Topo II activity. An overall reduction in Topo II activity (i.e. slow strand passage cycles) does not activate the checkpoint, but specific defects in the T-segment transit step of the strand passage reaction do induce a cell cycle delay. Furthermore, the cell cycle delay depends on the divergent and catalytically inert C-terminal region of Topo II, indicating that transmission of a checkpoint signal may occur via the C-terminus. Other, well characterized, mitotic checkpoints detect DNA lesions or monitor unattached kinetochores; these defects arise <i>via</i> failures in a variety of cell processes. In contrast, we have described the first example of a distinct category of checkpoint mechanism that monitors the catalytic cycle of a single specific enzyme in order to determine when chromosome segregation can proceed faithfully.</p></div

Analysis of G2/M Cell Cycle Checkpoint Activation in <i>top2</i> Mutants.

<p><b>a–c</b>, Population Assays: kinetics of cell cycle progression based on budding (DIC microscopy) and spindle morphology (Tub1-GFP). Cell populations were returned to growth after depletion of Top2^deg and synchronization in G1 (alpha-factor). The interval between spindle assembly and anaphase (<i>a</i>, arrow) is not significantly different with wild-type endogenously expressed <i>TOP2</i> or after Top2^deg degradation in G1. Panel <i>b</i> shows overlaid repetitions of each experiment to assess variation (green/blue/purple, indicate three experimental repeats). Panel <i>c</i>, histogram plot of average G2/M duration; see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003832#s3" target="_blank">Material and Methods</a> for statistical analysis. <b>d–f</b>, Single-cell Assays: kinetics of spindle assembly and elongation in single cells based on digital time-lapse microscopy of strains expressing Tub1-GFP. Images in panel <i>d</i> show representative images of each morphological state (also see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003832#pgen.1003832.s011" target="_blank">Movie S1</a>). Panel <i>e</i>, plot of average spindle length (or SPB diameter; first five time points) versus time for single cells aligned on the x-axis at the time of SPB separation (<i>i.e.</i> at time point 12 min). Standard deviation of lengths shows that SPB diameter and G2 spindle length are relatively constant. Standard deviation of spindle length increases markedly as some cells enter anaphase. Panel <i>f</i>, plots average time interval between SPB separation and the initiation of spindle elongation in anaphase B (+/− s.e.).</p

Defective Top2 ATP Binding and Hydrolysis Activate the Mad2-Depedent Checkpoint.

<p><b>a</b>, Cartoon describing catalytic defects in Top2^G144I which cannot bind ATP (green annotations) and Top2^E66Q which cannot hydrolyze ATP (blue annotations). (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003832#pgen-1003832-g001" target="_blank">Figure 1</a> for complete Strand Passage Reaction and Key). <b>b–j</b>, Cell cycle analyses showing that Top2^G144I and Top2^E66Q activate Mad2-dependent but Rad53-independent checkpoint signaling. Analysis of the kinetics of cell cycle progression (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003832#pgen-1003832-g003" target="_blank">Figure 3</a>) following depletion of Top2^deg and release from G1 synchrony in cells expressing endogenous levels of the indicated mutant Top2 proteins. (b–h) Population Assays: Histogram plots show average G2/M duration; see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003832#s3" target="_blank">Material and Methods</a> for statistical analysis. Western blots show each Top2 mutant relative to Tub1 loading control at G1 and G2. <i>a</i> values are significantly different to <i>b</i> values in the histogram plots. Strains with the same letter are not significantly different. (i,j) Single-cell assays: <i>i</i>, plots of average spindle length versus time for single cells aligned on the x-axis at the time of SPB separation (<i>i.e.</i> at time point 12 min). Error bars show standard deviation of lengths. <i>j</i>, Histogram plots of average time interval between SPB separation and the initiation of spindle elongation in anaphase B (+/− s.e.).</p