Topological State Of DNA Research Articles

Molecular Dynamics Simulation Insights into Hydrogen Bonding‐Mediated Conformational Changes Topoisomerase‐IB

AbstractTopoisomerase‐IB changes the topological state of DNA during central dogma by the formation of intermediate bond in between active amino acid and DNA strand. How the hydrogen bonding provides hooks restriction during the topological change and gives the conformational change. This study employs a 200 ns Molecular Dynamics (MD) simulation to analyze the conformational changes of Topo‐IB resulting from artificially created intermediate bonds. Specifically, the study focuses on hydrogen bonding interactions between Tyrosine at position Y509 in the protein and Thymine at DT561 in the DNA. The investigation reveals that these artificial intermediate bonds act as temporary hooks, stabilizing the complex's structure, and driving significant conformational changes. The analysis of Root Mean Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF) values underscores the dynamic nature of the complex, with artificial intermediate bonds playing a pivotal role.

Simple and Fast Rolling Circle Amplification-Based Detection of Topoisomerase 1 Activity in Crude Biological Samples.

Isothermal amplification-based techniques such as the rolling circle amplification have been successfully employed for the detection of nucleic acids, protein amounts, or other relevant molecules. These methods have shown to be substantial alternatives to PCR or ELISA for clinical and research applications. Moreover, the detection of protein amount (by Western blot or immunohistochemistry) is often insufficient to provide information for cancer diagnosis, whereas the measurement of enzyme activity represents a valuable biomarker. Measurement of enzyme activity also allows for the diagnosis and potential treatment of pathogen-borne diseases. In all eukaryotes, topoisomerases are the key DNA-binding enzymes involved in the control of the DNA topological state during important cellular processes and are among the important biomarkers for cancer prognosis and treatment. Over the years, topoisomerases have been substantially investigated as a potential target of antiparasitic and anticancer drugs with libraries of natural and synthetic small-molecule compounds that are investigated every year. Here, the rolling circle amplification method, termed rolling circle enhanced enzyme activity detection (REEAD) assay that allows for the quantitative measurement of topoisomerase 1 (TOP1) activity in a simple, fast, and gel-free manner is presented.By cleaving and ligating a specially designed DNA substrate, TOP1 converts a DNA oligonucleotide into a closed circle, which becomes the template for rolling circle amplification, yielding ~103 tandem repeat rolling circle products. Depending on the nucleotide incorporation during the amplification, there is the possibility of different readout methods, from fluorescence to chemiluminescence to colorimetric. As each TOP1-mediated cleavage-ligation generates one closed DNA circle, the assay is highly sensitive and directly quantitative.

Topoisomerase activity is linked to altered nucleosome positioning and transcriptional regulation in the fission yeast fbp1 gene.

Chromatin structure, including nucleosome positioning, has a fundamental role in transcriptional regulation through influencing protein-DNA interactions. DNA topology is known to influence chromatin structure, and in doing so, can also alter transcription. However, detailed mechanism(s) linking transcriptional regulation events to chromatin structure that is regulated by changes in DNA topology remain to be well defined. Here we demonstrate that nucleosome positioning and transcriptional output from the fission yeast fbp1 and prp3 genes are altered by excess topoisomerase activity. Given that lncRNAs (long noncoding RNAs) are transcribed from the fbp1 upstream region and are important for fbp1 gene expression, we hypothesized that local changes in DNA topological state caused by topoisomerase activity could alter lncRNA and fbp1 transcription. In support of this, we found that topoisomerase overexpression caused destabilization of positioned nucleosomes within the fbp1 promoter region, which was accompanied by aberrant fbp1 transcription. Similarly, the direct recruitment of topoisomerase, but not a catalytically inactive form, to the promoter region of fbp1 caused local changes in nucleosome positioning that was also accompanied by altered fbp1 transcription. These data indicate that changes in DNA topological state induced by topoisomerase activity could lead to altered fbp1 transcription through modulating nucleosome positioning.

Insight into Mechanism of Action of Anticancer Benzazoles.

Targeting the DNA topoisomerase II enzyme (topo II) is a promising anticancer treatment approach. TopoII controls and modifies the topological states of DNA and plays key roles in DNA replication, transcription, and chromosome segregation. The DNA binding and cleavage domain is one of the active sites of this enzyme. It is known that topoisomerase inhibitors, also known as topoisomerase poisons, bind to the transient enzyme-DNA complex and inhibit the religation of DNA, generating single- and double-stranded breaks that harm the integrity of the genome. This ultimately leads to the accumulation of DNA strand breaks and cell death. Our previously synthesized benzazole derivatives were tested for their eukaryotic DNA topoisomerase II inhibitory activity in a cell-free system. Their interactions with the enzyme were studied by carrying out molecular docking studies using and comparing two different docking programs. The results of the docking studies clarified binding modes of these compounds to the topoisomerase II enzyme. This study also provides guidelines to design novel and more potent antitumor agents functioning as human topoisomerase II enzyme inhibitors.

Exploitation of Pare Topoisomerase IV as Drug Target for the Treatment of Multidrug-Resistant Bacteria: A Review

The antibacterial resistance (ABR) is a growing phenomenon and global threat to mankind. To circumvent the ABR, many approaches have been put forth, but none of them meet the pre-requisites associated with the resistance mechanisms. In this review, we focused on the importance of unexploited enzyme, ParE, a topoisomerase responsible for the bacterial survival. The bacterial topoisomerases maintain the topological state of DNA. The gyrases and topoisomerases IV are validated targets for the antibacterial activity. Both these enzymes are structurally similar and possess high degree conservation in the catalytic domain of the N-terminal region, which make them appealing targets for broad spectrum antibacterial activity. Despite being an attractive target for the development of new antibacterials, there are currently no antibiotics targeting gyrases and topoisomerase (topo) IV in the market. Availability of the high-resolution crystal structure data for ParE made it possible to design new classes of antibacterials. Here, we discuss the importance of targeting topo IV enzyme as it is less prone to bacterial resistance which has been disclosed in the literature.

The reverse gyrase TopR1 is responsible for the homeostatic control of DNA supercoiling in the hyperthermophilic archaeon Sulfolobus solfataricus.

Maintaining an appropriate DNA topology with DNA-based processes (DNA replication, transcription and recombination) is crucial for all three domains of life. In bacteria, the homeostatic regulation for controlling DNA supercoiling relies on antagonistic activities of two DNA topoisomerases, TopoI and gyrase. In hyperthermophilic crenarchaea, the presence of such a regulatory system is suggested as two DNA topoisomerases, TopoVI and reverse gyrase, catalyze antagonistic activities. To test this hypothesis, we estimated and compared the number of the TopoVI with that of the two reverse gyrases, TopR1 and TopR2, inSulfolobus solfataricuscells maintained either at 80 or at 88°C, or reciprocally shifted from one temperature to the other. From the three DNA topoisomerases, TopR1 is the only one exhibiting significant quantitative variations in response to the up- and down-shifts. In addition, the corresponding intrinsic activities of these three DNA topoisomerases were tested in vitro at both temperatures. Although temperature modulates the three DNA topoisomerases activities, TopR1 is the sole topoisomerase able to function at high temperature. Altogether, results presented in this study demonstrate, for the first time, that the DNA topological state of a crenarchaeon is regulated via a homeostatic control, which is mainly mediated by the fine-tuning of TopR1.

New Insights into Structural and Functional Roles of Indole-3-acetic acid (IAA): Changes in DNA Topology and Gene Expression in Bacteria.

Indole-3-acetic acid (IAA) is a major plant hormone that affects many cellular processes in plants, bacteria, yeast, and human cells through still unknown mechanisms. In this study, we demonstrated that the IAA-treatment of two unrelated bacteria, the Ensifer meliloti 1021 and Escherichia coli, harboring two different host range plasmids, influences the supercoiled state of the two plasmid DNAs in vivo. Results obtained from in vitro assays show that IAA interacts with DNA, leading to DNA conformational changes commonly induced by intercalating agents. We provide evidence that IAA inhibits the activity of the type IA topoisomerase, which regulates the DNA topological state in bacteria, through the relaxation of the negative supercoiled DNA. In addition, we demonstrate that the treatment of E. meliloti cells with IAA induces the expression of some genes, including the ones related to nitrogen fixation. In contrast, these genes were significantly repressed by the treatment with novobiocin, which reduces the DNA supercoiling in bacterial cells. Taking into account the overall results reported, we hypothesize that the IAA action and the DNA structure/function might be correlated and involved in the regulation of gene expression. This work points out that checking whether IAA influences the DNA topology under physiological conditions could be a useful strategy to clarify the mechanism of action of this hormone, not only in plants but also in other unrelated organisms.

Gyrase containing a single C-terminal domain catalyzes negative supercoiling of DNA by decreasing the linking number in steps of two.

The topological state of DNA in vivo is regulated by topoisomerases. Gyrase is a bacterial topoisomerase that introduces negative supercoils into DNA at the expense of ATP hydrolysis. According to the strand-passage mechanism, a double-strand of the DNA substrate is cleaved, and a second double-stranded segment is passed through the gap, converting a positive DNA node into a negative node. The correct orientation of these DNA segments for strand passage is achieved by wrapping of the DNA around gyrase, which involves the C-terminal domains (CTDs) of both GyrA subunits in the A2B2 heterotetramer. Gyrase lacking both CTDs cannot introduce negative supercoils into DNA. Here, we analyze the requirements for the two CTDs in individual steps in the supercoiling reaction. Gyrase that contains a single CTD binds, distorts, and cleaves DNA similarly to wildtype gyrase. It also shows wildtype-like DNA-dependent ATPase activity, and undergoes DNA-induced movement of the CTD as well as N-gate narrowing. Most importantly, the enzyme still introduces negative supercoils into DNA in an ATP-dependent reaction, with a velocity similar to wildtype gyrase, and decreases the linking number of the DNA in steps of two. One CTD is thus sufficient to support DNA supercoiling.

Covalent Complex Model of DNA Topoisomerase and DNA for Molecular Dynamics Simulation

DNA topoisomerase is responsible for regulation of DNA topological state. The relaxation of supercoiled DNA is catalyzed by DNA topoisomerase. Upon interaction, DNA topoisomerase form a transient covalent linkage with DNA. This transient covalent complex can be trapped using topoisomerase inhibitors and these inhibitors have a potential to be developed as a new class of drugs targeting topoisomerase. In Escherichia coli (E. coli), accumulation of topoisomerase I (topoI)-DNA complex leads to bacterial cell death. We have designed a model of the complex to be investigated using molecular dynamics (MD) simulations. This model allowed us to study topoisomerase-DNA covalent system using computer simulations incorporating structural flexibility as well as effect of small molecule in the topoisomerase-DNA interaction. As an example, we used E. coli topoisomerase I (EctopoI), which is the most extensively studied bacterial type IA topoI and a target for antibacterial drug discovery. Utilizing our model, we first simulated the EctopoI-DNA covalent complex and then performed screening of a library of compounds against the minimized EctopoI target, using virtual screening, to identify potential EctopoI inhibitors. Finally, taking one of the identified inhibitors, we investigated the dynamics of ternary complex of EctopoI, inhibitor, and DNA. Our simulation results show that both the EctopoI-DNA binary complex as well as EctopoI-inhibitor-DNA ternary complex were stable for 50 ns of MD simulations. Our results further imply that the identified inhibitor prevents relegation of the cleaved DNA, which leads to accumulation of the EctopoI-inhibitor-DNA ternary complex.

DNA supercoiling is a fundamental regulatory principle in the control of bacterial gene expression.

Although it has become routine to consider DNA in terms of its role as a carrier of genetic information, it is also an important contributor to the control of gene expression. This regulatory principle arises from its structural properties. DNA is maintained in an underwound state in most bacterial cells and this has important implications both for DNA storage in the nucleoid and for the expression of genetic information. Underwinding of the DNA through reduction in its linking number potentially imparts energy to the duplex that is available to drive DNA transactions, such as transcription, replication and recombination. The topological state of DNA also influences its affinity for some DNA binding proteins, especially in DNA sequences that have a high A + T base content. The underwinding of DNA by the ATP-dependent topoisomerase DNA gyrase creates a continuum between metabolic flux, DNA topology and gene expression that underpins the global response of the genome to changes in the intracellular and external environments. These connections describe a fundamental and generalised mechanism affecting global gene expression that underlies the specific control of transcription operating through conventional transcription factors. This mechanism also provides a basal level of control for genes acquired by horizontal DNA transfer, assisting microbial evolution, including the evolution of pathogenic bacteria.

A Checkpoint-Related Function of the MCM Replicative Helicase Is Required to Avert Accumulation of RNA:DNA Hybrids during S-phase and Ensuing DSBs during G2/M.

The Mcm2-7 complex is the catalytic core of the eukaryotic replicative helicase. Here, we identify a new role for this complex in maintaining genome integrity. Using both genetic and cytological approaches, we find that a specific mcm allele (mcm2DENQ) causes elevated genome instability that correlates with the appearance of numerous DNA-damage associated foci of γH2AX and Rad52. We further find that the triggering events for this genome instability are elevated levels of RNA:DNA hybrids and an altered DNA topological state, as over-expression of either RNaseH (an enzyme specific for degradation of RNA in RNA:DNA hybrids) or Topoisomerase 1 (an enzyme that relieves DNA supercoiling) can suppress the mcm2DENQ DNA-damage phenotype. Moreover, the observed DNA damage has several additional unusual properties, in that DNA damage foci appear only after S-phase, in G2/M, and are dependent upon progression into metaphase. In addition, we show that the resultant DNA damage is not due to spontaneous S-phase fork collapse. In total, these unusual mcm2DENQ phenotypes are markedly similar to those of a special previously-studied allele of the checkpoint sensor kinase ATR/MEC1, suggesting a possible regulatory interplay between Mcm2-7 and ATR during unchallenged growth. As RNA:DNA hybrids primarily result from transcription perturbations, we suggest that surveillance-mediated modulation of the Mcm2-7 activity plays an important role in preventing catastrophic conflicts between replication forks and transcription complexes. Possible relationships among these effects and the recently discovered role of Mcm2-7 in the DNA replication checkpoint induced by HU treatment are discussed.

DNA gyrase with a single catalytic tyrosine can catalyze DNA supercoiling by a nicking-closing mechanism.

The topological state of DNA is important for replication, recombination and transcription, and is regulated in vivo by DNA topoisomerases. Gyrase introduces negative supercoils into DNA at the expense of ATP hydrolysis. It is the accepted view that gyrase achieves supercoiling by a strand passage mechanism, in which double-stranded DNA is cleaved, and a second double-stranded segment is passed through the gap, converting a positive DNA node into a negative node. We show here that gyrase with only one catalytic tyrosine that cleaves a single strand of its DNA substrate can catalyze DNA supercoiling without strand passage. We propose an alternative mechanism for DNA supercoiling via nicking and closing of DNA that involves trapping, segregation and relaxation of two positive supercoils. In contrast to DNA supercoiling, ATP-dependent relaxation and decatenation of DNA by gyrase lacking the C-terminal domains require both tyrosines and strand passage. Our results point towards mechanistic plasticity of gyrase and might pave the way for finding novel and specific mechanism-based gyrase inhibitors.

DNA supercoiling is a fundamental regulatory principle in the control of bacterial gene expression.

Although it has become routine to consider DNA in terms of its role as a carrier of genetic information, it is also an important contributor to the control of gene expression. This regulatory principle arises from its structural properties. DNA is maintained in an underwound state in most bacterial cells and this has important implications both for DNA storage in the nucleoid and for the expression of genetic information. Underwinding of the DNA through reduction in its linking number potentially imparts energy to the duplex that is available to drive DNA transactions, such as transcription, replication and recombination. The topological state of DNA also influences its affinity for some DNA binding proteins, especially in DNA sequences that have a high A + T base content. The underwinding of DNA by the ATP-dependent topoisomerase DNA gyrase creates a continuum between metabolic flux, DNA topology and gene expression that underpins the global response of the genome to changes in the intracellular and external environments. These connections describe a fundamental and generalised mechanism affecting global gene expression that underlies the specific control of transcription operating through conventional transcription factors. This mechanism also provides a basal level of control for genes acquired by horizontal DNA transfer, assisting microbial evolution, including the evolution of pathogenic bacteria.

Abstract B10: Increased expression of EZH2 and TOP2A predicts for a poorer prognostic outcome in Genitourinary Cancers

Abstract The enhancer of Zeste homolog 2 (EZH2), a component of the Polycomb Repressive Complex 2 (PRC2), is an epigenetic silencer that is known to be highly expressed in both metastatic and primary prostate cancer (PCa). More recently, topoisomerase IIα (TOP2A), an enzyme that controls the topologic states of DNA during the cell cycle, has also been identified as being highly expressed in PCa. Both EZH2 and TOP2A have been associated with more aggressive disease and poorer prognostic outcome. During genome-wide analysis, using The Cancer Genome Atlas (TCGA), EZH2 and Top2A were found to be highly positively correlated, most noticeably in PCa and clear cell Renal Cell Carcinoma (ccRCC). As a result, we predicted increased expression of both EZH2 and TOP2A in an individual, would predict for a worse prognostic outcome than either gene being highly expressed alone. Further analysis of the TCGA and Memorial Sloan Kettering Cancer Center (MSKCC) datasets, demonstrated that increased expression of these two genes predicted for a poorer prognostic outcome in genitourinary cancers. In tumor samples with increased expression of EZH2 and TOP2A, as compared to the tumor samples that did not concurrently overexpress both EZH2 and TOP2A, patients displayed worse overall Regression Free Survival (RFS), which were associated with increase hazard ratio, and tumor staging and grades. . As these poor prognostic markers were consistent across multiple cancer subtypes, we are beginning to explore those genes that are differentially expressed in individuals with concurrent high TOP2A and EZH2 as a means of further characterizing this novel phenotype. Since these two genes are targetable by drugs such as etoposide (a TOP2 poison) and DZNep (an EZH2 inhibitor), it makes them attractive candidates for treatment in those individuals who not only have tumors that highly express these two genes, but also those individuals that have exacerbated existing treatment options. Citation Format: Spencer Rosario, Zafardjan Dalimov, Jason Kirk, Kayla Morrell, Kevin Eng, Leigh Ellis. Increased expression of EZH2 and TOP2A predicts for a poorer prognostic outcome in Genitourinary Cancers. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B10.

Knotting and Unknotting Dynamics of DNA Strands in Nanochannels.

The self-knotting dynamics of DNA strands confined in nanochannels is studied with Brownian simulations. The model DNA chains are several microns long and placed inside channels that are 50-300 nm wide. This width range covers the transition between different metric scaling regimes and the concomitant drop of DNA knotting probability for channel widths below ∼75 nm. We find that knots typically originate from deep looping and backfoldings of the chain ends. Upon lowering the channel width, backfoldings become shallower and rarer and the lifetime of knots decreases while that of unknots increases. This lifetimes interplay causes the dramatic reduction of knots incidence for increasing confinement. The results can aid the design of nanochannels capable of harnessing the self-knotting dynamics to quench or relax the DNA topological state as desired.

Abstract 2519: A class of biometallo-organic compounds that target human topoisomerase IIa

Abstract Topoisomerase IIa (topo II) is a nuclear enzyme that changes the topological state of DNA by breaking and rejoining the phosphodiester backbone of DNA. It is an important target for anticancer therapy and a number of agents are known to influence its action in vivo. These agents operate by either disrupting the cleavage-resealing cycle (interfacial poisons) or by simply blocking catalytic action of topo II (catalytic inhibitors). A new class of metallo-drugs is described that is unique in acting by both mechanisms. These are a series of fully characterized crystalline d8 coordination compounds with tetradentate ligands that we have evaluated for their targeting potential with topo II. These compounds are quite toxic to cells in culture with inhibitory values in low micromolar range. Mechanistic in vitro data with purified enzyme reveal these compounds to act as both catalytic inhibitors and as interfacial poisons over the same range of drug concentration. For example, CC50 values (drug concentrations yield 50% maximum DNA cleavage) were 3.47 µM for the prototype metallo-agent (compared to 5.47 µM for etoposide). Acting as catalytic inhibitors, the IC50 value was 3.17 µM. In sum, these compounds exhibit dual mechanisms in vitro, by stabilizing the covalent cleavage complex as well as blocking catalytic function. Analysis of endogenous topo II activity in HeLa cells confirms that the compounds target endogenous topo II by stabilizing the covalent cleavage complex and therefore acting as a poison in a cellular context. Endogenous topo I was not affected by the drug showing that these novel metallo-agents are type II specific. The dual mechanism of targeting of topo II suggests significant anti-tumor potential, and further studies are being conducted to evaluate clinical potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2519. doi:10.1158/1538-7445.AM2011-2519

Atomic Force Microscopy Studies on the Effect of DNA Supercoiling on Nucleosome Structure

The genome of eukaryotes is folded into chromatin. Nucleosomes are its basic unit. The compaction of DNA in nucleosomes impedes nuclear processes that require DNA as a template such as transcription, replication, and DNA damage repair. It has been proposed that changes in DNA topology influence the accessibility of nucleosomal DNA. Such changes in DNA topology occur e.g. during transcription when reverse torsional tensions are being built up within the DNA during elongation in front of and behind the polymerase complex. This leads to positive or negative supercoiling of the DNA strand possibly affecting chromatin structure. Here we investigated the influence of different topological states of DNA on nucleosomal structure by qualitative analysis of atomic force microscopy (AFM) images. Recombinant histone octamers were reconstituted via salt dialysis on PUC 18 plasmids of superhelical densities σ = −0.046, −0.02, 0, +0.03 and observed by AFM. The obtained images were edited and the nucleosomal DNA opening angle (α) analysed by a half automated technique. The results show a clear dependence between the size of α and the strength and type of superhelical density of the DNA. With decreasing negative torsional strain alpha is found to be increasing whereas an increase in positive torsional strain leads to further increase of the opening angle.

Kinetics of comet formation in single‐cell gel electrophoresis: Loops and fragments

We investigated the mechanisms of DNA exit during single-cell gel electrophoresis (the comet assay) by measuring the kinetics of the comet tail formation. In the neutral comet assay, the rate of DNA exit was found to be dependent on the topological state of DNA, which was influenced by either ethidium bromide or a low radiation dose. The results clearly show that the comet tail is formed by extended DNA loops: the loop extension, being reversible when the DNA torsional constraint remains in the loops, is favored when the constraint is relaxed. The kinetics of the comet formation in the case of a high radiation dose points out that accumulation of the single-strand breaks causes DNA fragmentation. In contrast to the neutral comet assay, the alkaline comet assay is not related to the chromatin loops. Our results imply that the alkaline treatment induces detachment of the loops from the nuclear matrix, and the comet tail is formed by ssDNA fragments, the ends of which are pulled out from the comet head by electric force. We suggest that the kinetic approach can be considered as an important improvement of the comet assay.

DNA Topoisomerases.

DNA topoisomerases are enzymes that control the topological state of DNA in all cells; they have central roles in DNA replication and transcription. They are classified into two types, I and II, depending on whether they catalyze reactions involving the breakage of one or both strands of DNA. Structural and mechanistic distinctions have led to further classifications: IA, IB, IC, IIA, and IIB. The essence of the topoisomerase reaction is the ability of the enzymes to stabilize transient breaks in DNA, via the formation of tyrosyl-phosphate covalent intermediates. The essential nature of topoisomerases and their ability to stabilize DNA breaks has led to them being key targets for antibacterial and anticancer agents. This chapter reviews the basic features of topoisomerases focussing mainly on the prokaryotic enzymes. We highlight recent structural advances that have given new insight into topoisomerase mechanisms and into the molecular basis of the action of topoisomerase-specific drugs.

Recent Advances and Developments in the Inhibitors of DNA Topoisomerases

Recent years have seen the development of the latest generation of inhibitors of the topoisomerase enzymes. New discoveries and improvements to methodologies underpinning drug design and discovery have caused a renewed interest in the ubiquitously expressed DNA topoisomerases. Virtually every aspect of nucleic acid physiology is influenced by the topological state of DNA, which is managed predominantly by these enzymes. For this reason, they have been the targets of clinically important anti-cancer drugs for a number of years. Although the emergence of targeted therapies may have diminished the interest in topoisomerases, clinically established drugs such as irinotecan and etoposide will remain the mainstay treatment of cancer both as single agents and more recently, in combinatorial programs. By todays standards, these first generation inhibitors are crude, and their applications limited by non-ideal drug characteristics. However, the new inhibitors, such as diflomotecan, gimatecan, ICRF-193 and XR5000 offer enhanced activity through increased metabolic stability and improvements in pharmacokinetic and pharmacodynamic profiles. These developments have resulted in improved drug delivery and in reductions in adverse effects. This review will revisit this class of anti-cancer agent, and will summarise and update the exciting developments in the field.