Novel Deazaflavin Analogues Potently Inhibited Tyrosyl DNA Phosphodiesterase 2 (TDP2) and Strongly Sensitized Cancer Cells toward Treatment with Topoisomerase II (TOP2) Poison Etoposide.
ABSTRACT: Topoisomerase II (TOP2) poisons as anticancer drugs work by trapping TOP2 cleavage complexes (TOP2cc) to generate DNA damage. Repair of such damage by tyrosyl DNA phosphodiesterase 2 (TDP2) could render cancer cells resistant to TOP2 poisons. Inhibiting TDP2, thus, represents an attractive mechanism-based chemosensitization approach. Currently known TDP2 inhibitors lack cellular potency and/or permeability. We report herein two novel subtypes of the deazaflavin TDP2 inhibitor core. By introducing an additional phenyl ring to the N-10 phenyl ring (subtype 11) or to the N-3 site of the deazaflavin scaffold (subtype 12), we have generated novel analogues with considerably improved biochemical potency and/or permeability. Importantly, many analogues of both subtypes, particularly compounds 11a, 11e, 12a, 12b, and 12h, exhibited much stronger cancer cell sensitizing effect than the best previous analogue 4a toward the treatment with etoposide, suggesting that these analogues could serve as effective cellular probes.
Project description:Tyrosyl-DNA phosphodiesterase 2 (TDP2) is a DNA repair enzyme that removes 5'-phosphotyrosyl blockages resulting from topoisomerase II (TOP2)-DNA cleavage complexes trapped by TOP2 inhibitors. TDP2 is a logical target for the development of therapeutics to complement existing treatments based on inhibition of TOP2. There is, however, no TDP2 inhibitor in clinical development at present. Of the reported TDP2 inhibitors, the deazaflavins are the most promising chemical class centered around the lead compound SV-5-153. Recently we reported new subtypes derived within the deazaflavin family with improved membrane permeability properties. In this work we characterize two representative analogues from two new deazaflavin subtypes based on their biochemical TDP2 inhibitory potency and drug-likeness. We demonstrate that the ZW-1288 derivative represents a promising direction for the development of deazaflavins as therapeutic agents. ZW-1288 exhibits potent inhibitory activity at low nanomolar concentrations against recombinant and cellular human TDP2 with profile similar to that of the parent analog SV-5-153 based on high resistance against murine TDP2 and human TDP2 mutated at residue L313H. While expressing weak cytotoxicity on its own, ZW-1288 potentiates the clinical TOP2 inhibitors etoposide (ETP) and mitoxantrone in human prostate DU145 and CCRF-CEM leukemia and chicken lymphoma DT40 cells while not impacting the activity of the topoisomerase I (TOP1) inhibitor camptothecin or the PARP inhibitor olaparib. ZW-1288 increases the uptake of ETP to a lesser extent than SV-5-153 and remained active in TDP2 knockout cells indicating that the deazaflavin TDP2 inhibitors have additional cellular effects that will have to be taken into account for their further development as TDP2 inhibitors.
Project description:Eukaryotic type II topoisomerases (Top2? and Top2?) are homodimeric enzymes; they are essential for altering DNA topology by the formation of normally transient double strand DNA cleavage. Anticancer drugs (etoposide, doxorubicin, and mitoxantrone) and also Top2 oxidation and DNA helical alterations cause potentially irreversible Top2·DNA cleavage complexes (Top2cc), leading to Top2-linked DNA breaks. Top2cc are the therapeutic mechanism for killing cancer cells. Yet Top2cc can also generate recombination, translocations, and apoptosis in normal cells. The Top2 protein-DNA covalent complexes are excised (in part) by tyrosyl-DNA-phosphodiesterase 2 (TDP2/TTRAP/EAP2/VPg unlinkase). In this study, we show that irreversible Top2cc induced in suicidal substrates are not processed by TDP2 unless they first undergo proteolytic processing or denaturation. We also demonstrate that TDP2 is most efficient when the DNA attached to the tyrosyl is in a single-stranded configuration and that TDP2 can efficiently remove a tyrosine linked to a single misincorporated ribonucleotide or to polyribonucleotides, which expands the TDP2 catalytic profile with RNA substrates. The 1.6-Å resolution crystal structure of TDP2 bound to a substrate bearing a 5'-ribonucleotide defines a mechanism through which RNA can be accommodated in the TDP2 active site, albeit in a strained conformation.
Project description:Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs topoisomerase II (TOP2) mediated DNA damages and causes cellular resistance to clinically used TOP2 poisons. Inhibiting TDP2 can potentially sensitize cancer cells toward TOP2 poisons. Commercial compound P10A10, to which the structure was assigned as 7-phenyl triazolopyrimidine analogue 6a, was previously identified as a TDP2 inhibitor hit in our virtual and fluorescence-based biochemical screening campaign. We report herein that the hit validation through resynthesis and structure elucidation revealed the correct structure of P10A10 (Chembridge ID 7236827) to be the 5-phenyl triazolopyrimidine regioisomer 7a. Subsequent structure-activity relationship (SAR) via the synthesis of a total of 47 analogues of both the 5-phenyl triazolopyrimidine scaffold (7) and its bioisosteric triazolopyridine scaffold (17) identified four derivatives (7a, 17a, 17e, and 17z) with significant TDP2 inhibition (IC50?<?50?µM), with 17z showing excellent cell permeability and no cytotoxicity.
Project description:Topoisomerase 2 (TOP2) DNA transactions proceed via formation of the TOP2 cleavage complex (TOP2cc), a covalent enzyme-DNA reaction intermediate that is vulnerable to trapping by potent anticancer TOP2 drugs. How genotoxic TOP2 DNA-protein cross-links are resolved is unclear. We found that the SUMO (small ubiquitin-related modifier) ligase ZATT (ZNF451) is a multifunctional DNA repair factor that controls cellular responses to TOP2 damage. ZATT binding to TOP2cc facilitates a proteasome-independent tyrosyl-DNA phosphodiesterase 2 (TDP2) hydrolase activity on stalled TOP2cc. The ZATT SUMO ligase activity further promotes TDP2 interactions with SUMOylated TOP2, regulating efficient TDP2 recruitment through a "split-SIM" SUMO2 engagement platform. These findings uncover a ZATT-TDP2-catalyzed and SUMO2-modulated pathway for direct resolution of TOP2cc.
Project description:Tyrosyl-DNA phosphodiesterase 2 (TDP2) is a 5'-tyrosyl DNA phosphodiesterase important for the repair of DNA adducts generated by non-productive (abortive) activity of topoisomerase II (TOP2). TDP2 facilitates therapeutic resistance to topoisomerase poisons, which are widely used in the treatment of a range of cancer types. Consequently, TDP2 is an interesting target for the development of small molecule inhibitors that could restore sensitivity to topoisomerase-directed therapies. Previous studies identified a class of deazaflavin-based molecules that showed inhibitory activity against TDP2 at therapeutically useful concentrations, but their mode of action was uncertain. We have confirmed that the deazaflavin series inhibits TDP2 enzyme activity in a fluorescence-based assay, suitable for high-throughput screen (HTS)-screening. We have gone on to determine crystal structures of these compounds bound to a 'humanized' form of murine TDP2. The structures reveal their novel mode of action as competitive ligands for the binding site of an incoming DNA substrate, and point the way to generating novel and potent inhibitors of TDP2.
Project description:The compaction of DNA and the continuous action of DNA transactions, including transcription and DNA replication, create complex DNA topologies that require Type IIA Topoisomerases, which resolve DNA topological strain and control genome dynamics. The human TOP2 enzymes catalyze their reactions via formation of a reversible covalent enzyme DNA-protein crosslink, the TOP2 cleavage complex (TOP2cc). Spurious interactions of TOP2 with DNA damage, environmental toxicants and chemotherapeutic "poisons" perturbs the TOP2 reaction cycle, leading to an accumulation of DNA-protein crosslinks, and ultimately, genomic instability and cell death. Emerging evidence shows that TOP2-DNA protein crosslink (DPC) repair entails multiple strand break repair activities, such as removal of the poisoned TOP2 protein and rejoining of the DNA ends through homologous recombination (HR) or non-homologous end joining (NHEJ). Herein, we discuss the molecular mechanisms of TOP2-DPC resolution, with specific emphasis on the recently uncovered ZATT<sup>Znf451</sup>-licensed TDP2-catalyzed TOP2-DPC reversal mechanism.
Project description:Topoisomerase II (TOP2) relieves topological stress in DNA by introducing double-strand breaks (DSBs) via a transient, covalently linked TOP2 DNA-protein intermediate, termed TOP2 cleavage complex (TOP2cc). TOP2ccs are normally rapidly reversible, but can be stabilized by TOP2 poisons, such as the chemotherapeutic agent etoposide (ETO). TOP2 poisons have shown significant variability in their therapeutic effectiveness across different cancers for reasons that remain to be determined. One potential explanation for the differential cellular response to these drugs is in the manner by which cells process TOP2ccs. Cells are thought to remove TOP2ccs primarily by proteolytic degradation followed by DNA DSB repair. Here, we show that proteasome-mediated repair of TOP2cc is highly error-prone. Pre-treating primary splenic mouse B-cells with proteasome inhibitors prevented the proteolytic processing of trapped TOP2ccs, suppressed the DNA damage response (DDR) and completely protected cells from ETO-induced genome instability, thereby preserving cellular viability. When degradation of TOP2cc was suppressed, the TOP2 enzyme uncoupled itself from the DNA following ETO washout, in an error-free manner. This suggests a potential mechanism of developing resistance to topoisomerase poisons by ensuring rapid TOP2cc reversal.
Project description:The camptothecin derivatives topoisomerase I (TOP1) inhibitors, irinotecan and topotecan, are FDA approved for the treatment of colorectal, ovarian, lung and breast cancers. Because of the chemical instability of camptothecins, short plasma half-life, drug efflux by the multidrug-resistance ABC transporters, and the severe diarrhea produced by irinotecan, indenoisoquinoline TOP1 inhibitors (LMP400, LMP776, and LMP744), which overcome these limitations, have been developed and are in clinical development. Further modifications of the indenoisoquinolines led to the fluoroindenoisoquinolines, one of which, LMP517, is the focus of this study. LMP517 showed better antitumor activity than its parent compound LMP744 against H82 (small cell lung cancer) xenografts. Genetic analyses in DT40 cells showed a dual TOP1 and TOP2 signature with selectivity of LMP517 for DNA repair-deficient tyrosyl DNA phosphodiesterase 2 (TDP2)- and Ku70-knockout cells. RADAR assays revealed that LMP517, and to a lesser extent LMP744, induce TOP2 cleavage complexes (TOP2cc) in addition to TOP1ccs. Histone ?H2AX detection showed that, unlike classical TOP1 inhibitors, LMP517 targets cells independently of their position in the cell cycle. Our study establishes LMP517 as a dual TOP1 and TOP2 inhibitor with therapeutic potential.
Project description:Anticancer topoisomerase "poisons" exploit the break-and-rejoining mechanism of topoisomerase II (TOP2) to generate TOP2-linked DNA double-strand breaks (DSBs). This characteristic underlies the clinical efficacy of TOP2 poisons, but is also implicated in chromosomal translocations and genome instability associated with secondary, treatment-related, haematological malignancy. Despite this relevance for cancer therapy, the mechanistic aspects governing repair of TOP2-induced DSBs and the physiological consequences that absent or aberrant repair can have are still poorly understood. To address these deficits, we employed cells and mice lacking tyrosyl DNA phosphodiesterase 2 (TDP2), an enzyme that hydrolyses 5'-phosphotyrosyl bonds at TOP2-associated DSBs, and studied their response to TOP2 poisons. Our results demonstrate that TDP2 functions in non-homologous end-joining (NHEJ) and liberates DSB termini that are competent for ligation. Moreover, we show that the absence of TDP2 in cells impairs not only the capacity to repair TOP2-induced DSBs but also the accuracy of the process, thus compromising genome integrity. Most importantly, we find this TDP2-dependent NHEJ mechanism to be physiologically relevant, as Tdp2-deleted mice are sensitive to TOP2-induced damage, displaying marked lymphoid toxicity, severe intestinal damage, and increased genome instability in the bone marrow. Collectively, our data reveal TDP2-mediated error-free NHEJ as an efficient and accurate mechanism to repair TOP2-induced DSBs. Given the widespread use of TOP2 poisons in cancer chemotherapy, this raises the possibility of TDP2 being an important etiological factor in the response of tumours to this type of agent and in the development of treatment-related malignancy.
Project description:Tyrosyl DNA phosphodiesterase II (TDP2) is a recently discovered enzyme that specifically repairs DNA damages induced by topoisomerase II (Top2) poisons and causes resistance to these drugs. Inhibiting TDP2 is expected to enhance the efficacy of clinically important Top2-targeting anticancer drugs. However, TDP2 as a therapeutic target remains poorly understood. We report herein the discovery of isoquinoline-1,3-dione as a viable chemotype for selectively inhibiting TDP2. The initial hit compound 43 was identified by screening our in-house collection of synthetic compounds. Further structure-activity relationship (SAR) studies identified numerous analogues inhibiting TDP2 in low micromolar range without appreciable inhibition against the homologous TDP1 at the highest testing concentration (111 ?M). The best compound 64 inhibited recombinant TDP2 with an IC50 of 1.9 ?M. The discovery of this chemotype may provide a platform toward understanding TDP2 as a drug target.