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Yves Pommier - One of the best experts on this subject based on the ideXlab platform.
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synthesis of methoxy methylenedioxy hydroxy and halo substituted benzophenanthridinone derivatives as dna topoisomerase ib top1 and tyrosyl dna phosphodiesterase 1 TDP1 inhibitors and their biological activity for drug resistant cancer
Journal of Medicinal Chemistry, 2021Co-Authors: Wenlin Tang, Wenjie Wang, Keli Agama, Yu Zhang, Hao Yang, Yves PommierAbstract:As a recently discovered DNA repair enzyme, tyrosyl-DNA phosphodiesterase 1 (TDP1) removes topoisomerase IB (TOP1)-mediated DNA protein cross-links. Inhibiting TDP1 can potentiate the cytotoxicity of TOP1 inhibitors and overcome cancer cell resistance to TOP1 inhibitors. On the basis of our previous study, herein we report the synthesis of benzophenanthridinone derivatives as TOP1 and TDP1 inhibitors. Seven compounds (C2, C4, C5, C7, C8, C12, and C14) showed a robust TOP1 inhibitory activity (+++ or ++++), and four compounds (A13, C12, C13, and C26) showed a TDP1 inhibition (half-maximal inhibitory concentration values of 15 or 19 μM). We also show that the dual TOP1 and TDP1 inhibitor C12 induces both cellular TOP1cc, TDP1cc formation and DNA damage, resulting in cancer cell apoptosis at a sub-micromolar concentration. In addition, C12 showed an enhanced activity in drug-resistant MCF-7/TDP1 cancer cells and was synergistic with topotecan in both MCF-7 and MCF-7/TDP1 cells.
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Inhibition of human tyrosyl-DNA phosphodiesterase by aminoglycoside antibiotics and ribosome
2020Co-Authors: Zhiyong Liao, Laurent Thibaut, Andrew Jobson, Yves PommierAbstract:ABSTRACT DNA topoisomerase I (Top1) is the target of camptothecin, and novel Top1 inhibitors are in development as anticancer agents. Top1 inhibitors damage DNA by trapping covalent complexes between the Top1 catalytic tyrosine and the 3Ј-end of the broken DNA. Tyrosyl-DNA phosphodiesterase (TDP1) can repair Top1-DNA covalent complexes by hydrolyzing the tyrosyl-DNA bond. Inhibiting TDP1 has the potential to enhance the anticancer activity of Top1 inhibitors (http://discover.nci.nih. gov/pommier/pommier.htm) and to act as antiproliferative agents. In the present study, we report that neomycin inhibits TDP1 more effectively than the related aminoglycosides paromomycin and lividomycin A. Inhibition of TDP1 by neomycin is observed both with single-and double-stranded substrates but is slightly stronger with duplex DNA, which is different from aclarubicin, which only inhibits TDP1 with the double-stranded substrate. Inhibition by neomycin can be overcome with excess TDP1 and is greatest at low pH. To our knowledge, aminoglycoside antibiotics and the ribosome inhibitors thiostrepton, clindamycin-2-phosphate, and puromycin are the first reported pharmacological TDP1 inhibitors
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identification of a ligand binding hot spot and structural motifs replicating aspects of tyrosyl dna phosphodiesterase i TDP1 phosphoryl recognition by crystallographic fragment cocktail screening
Nucleic Acids Research, 2019Co-Authors: George T Lountos, Evgeny Kiselev, Yves Pommier, Xue Zhi Zhao, Joseph E Tropea, D Needle, Terrence R Burke, David S WaughAbstract:Tyrosyl DNA-phosphodiesterase I (TDP1) repairs type IB topoisomerase (TOP1) cleavage complexes generated by TOP1 inhibitors commonly used as anticancer agents. TDP1 also removes DNA 3' end blocking lesions generated by chain-terminating nucleosides and alkylating agents, and base oxidation both in the nuclear and mitochondrial genomes. Combination therapy with TDP1 inhibitors is proposed to synergize with topoisomerase targeting drugs to enhance selectivity against cancer cells exhibiting deficiencies in parallel DNA repair pathways. A crystallographic fragment screening campaign against the catalytic domain of TDP1 was conducted to identify new lead compounds. Crystal structures revealed two fragments that bind to the TDP1 active site and exhibit inhibitory activity against TDP1. These fragments occupy a similar position in the TDP1 active site as seen in prior crystal structures of TDP1 with bound vanadate, a transition state mimic. Using structural insights into fragment binding, several fragment derivatives have been prepared and evaluated in biochemical assays. These results demonstrate that fragment-based methods can be a highly feasible approach toward the discovery of small-molecule chemical scaffolds to target TDP1, and for the first time, we provide co-crystal structures of small molecule inhibitors bound to TDP1, which could serve for the rational development of medicinal TDP1 inhibitors.
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TDP1 suppresses mis joining of radiomimetic dna double strand breaks and cooperates with artemis to promote optimal nonhomologous end joining
Nucleic Acids Research, 2018Co-Authors: Ajinkya S Kawale, Shar-yin N. Huang, Yves Pommier, Konstantin Akopiants, Kristoffer Valerie, Brian L Ruis, Eric A Hendrickson, Lawrence F PovirkAbstract:The Artemis nuclease and tyrosyl-DNA phosphodiesterase (TDP1) are each capable of resolving protruding 3'-phosphoglycolate (PG) termini of DNA double-strand breaks (DSBs). Consequently, both a knockout of Artemis and a knockout/knockdown of TDP1 rendered cells sensitive to the radiomimetic agent neocarzinostatin (NCS), which induces 3'-PG-terminated DSBs. Unexpectedly, however, a knockdown or knockout of TDP1 in Artemis-null cells did not confer any greater sensitivity than either deficiency alone, indicating a strict epistasis between TDP1 and Artemis. Moreover, a deficiency in Artemis, but not TDP1, resulted in a fraction of unrepaired DSBs, which were assessed as 53BP1 foci. Conversely, a deficiency in TDP1, but not Artemis, resulted in a dramatic increase in dicentric chromosomes following NCS treatment. An inhibitor of DNA-dependent protein kinase, a key regulator of the classical nonhomologous end joining (C-NHEJ) pathway sensitized cells to NCS, but eliminated the sensitizing effects of both TDP1 and Artemis deficiencies. These results suggest that TDP1 and Artemis perform different functions in the repair of terminally blocked DSBs by the C-NHEJ pathway, and that whereas an Artemis deficiency prevents end joining of some DSBs, a TDP1 deficiency tends to promote DSB mis-joining.
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prmt5 mediated arginine methylation of TDP1 for the repair of topoisomerase i covalent complexes
Nucleic Acids Research, 2018Co-Authors: Ishita Rehman, Benu Brata Das, Subhendu K. Das, Yves Pommier, Sangheeta Bhattacharjee, Suparna Mercy Basu, Arijit GhoshAbstract:Human tyrosyl-DNA phosphodiesterases (TDP) hydrolyze the phosphodiester bond between DNA and the catalytic tyrosine of Top1 to excise topoisomerase I cleavage complexes (Top1cc) that are trapped by camptothecin (CPT) and by genotoxic DNA alterations. Here we show that the protein arginine methyltransferase PRMT5 enhances the repair of Top1cc by direct binding to TDP1 and arginine dimethylation of TDP1 at residues R361 and R586. Top1-induced replication-mediated DNA damage induces TDP1 arginine methylation, enhancing its 3'- phosphodiesterase activity. TDP1 arginine methylation also increases XRCC1 association with TDP1 in response to CPT, and the recruitment of XRCC1 to Top1cc DNA damage foci. PRMT5 knockdown cells exhibit defective TDP1 activity with marked elevation in replication-coupled CPT-induced DNA damage and lethality. Finally, methylation of R361 and R586 stimulate TDP1 repair function and promote cell survival in response to CPT. Together, our findings provide evidence for the importance of PRMT5 for the post-translational regulation of TDP1 and repair of Top1cc.
Sherif F Elkhamisy - One of the best experts on this subject based on the ideXlab platform.
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studying TDP1 function in dna repair
Methods of Molecular Biology, 2018Co-Authors: Shihchieh Chiang, Kirsty Liversidge, Sherif F ElkhamisyAbstract:Topoisomerase poisons act by inducing abortive topoisomerase reactions, which generate stable protein-DNA breaks (PDBs) that interfere with transcription elongation and progression of replication forks. In vertebrates, Tyrosyl-DNA phosphodiesterase 1 (TDP1) plays a major role in removal of topoisomerase 1-associated PDBs in the nucleus and mitochondria by hydrolyzing the 3'-phosphotyrosine bond. Depletion of TDP1 sensitizes tumor cells with defective DNA repair capacity to the genotoxic effect of TOP1 poisons, while homozygous mutation of the catalytic residue of TDP1 is associated with cerebellar degeneration and ataxia. We describe here two fluorescence based biochemical assays for measuring TDP1 phosphodiesterase activity in cellular lysates. The Gyrasol assay is sensitive, high-throughput, and useful for screening potential TDP1 inhibitors or cell lines that are likely to develop resistance to TOP1 poisons. The gel-shift assay is low cost and simple to set up, and is also suitable for screening cell lines that are likely to develop resistance to TOP1 poisons, as well as for diagnostic screening for individuals with hereditary ataxias.
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TDP1 top1 ratio as a promising indicator for the response of small cell lung cancer to topotecan
Journal of Cancer Science & Therapy, 2014Co-Authors: Cornelia Meisenberg, Simon E. Ward, Peter Schmid, Sherif F ElkhamisyAbstract:BACKGROUND AND OBJECTIVE Small cell lung cancer (SCLC) is one of the most challenging tumors to treat due to high proliferation rate, early metastatic dissemination and rapid development of chemotherapy resistance. The current treatment protocols involve the use of topoisomerase 1 (TOP1) poisons such as irinotecan and topotecan in combination with platinum-based compounds. TOP1 poisons kill cancer cells by trapping TOP1 on DNA, generating lethal DNA double-strand breaks. A potential mechanism employed by cancer cells to resist killing by TOP1 poisons is to overexpress enzymes involved in the repair of TOP1-DNA breaks. Tyrosyl DNA phosphodiesterase 1 (TDP1) is a key player in this process and despite its importance, no data is currently available to correlate TDP1 protein and mRNA levels with catalytic activity in SCLC. In addition, it is not known if TDP1 and TOP1 protein levels correlate with the cellular response of SCLC to TOP1 based therapies. METHODS AND RESULTS We report a remarkable variation in TDP1 and TOP1 protein levels in a panel of SCLC cell lines. TDP1 protein level correlates well with TDP1 mRNA and TDP1 catalytic activity, as measured by two newly developed independent activity assays, suggesting the potential utility of immunohistochemistry in assessing TDP1 levels in SCLC tissues. We further demonstrate that whilst TDP1 protein level alone does not correlate with topotecan sensitivity, TDP1/TOP1 ratio correlates well with sensitivity in 8 out of 10 cell lines examined. CONCLUSION This study provides the first cellular analyses of TDP1 and TOP1 in SCLC and suggests the potential utility of TDP1/TOP1 ratio to assess the response of SCLC to topotecan. The establishment and validation of an easy-to-use TDP1 enzymatic assay in cell extracts could be exploited as a diagnostic tool in the clinic. These findings may help in stratifying patients that are likely to benefit from TOP1 poisons and TDP1 inhibitors currently under development.
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development of an oligonucleotide based fluorescence assay for the identification of tyrosyl dna phosphodiesterase 1 TDP1 inhibitors
Analytical Biochemistry, 2014Co-Authors: Sarah R Walker, Sherif F Elkhamisy, Cornelia Meisenberg, Rachel A Bibby, Trevor Askwith, Gareth Williams, Frauke H Rininsland, Laurence H Pearl, Antony W OliverAbstract:Topoisomerase 1 (TOP1) generates transient nicks in the DNA to relieve torsional stress encountered during the cellular processes of transcription, replication, and recombination. At the site of the nick there is a covalent linkage of TOP1 with DNA via a tyrosine residue. This reversible TOP1-cleavage complex intermediate can become trapped on DNA by TOP1 poisons such as camptothecin, or by collision with replication or transcription machinery, thereby causing protein-linked DNA single- or double-strand breaks and resulting in cell death. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a key enzyme involved in the repair of TOP1-associated DNA breaks via hydrolysis of 3′-phosphotyrosine bonds. Inhibition of TDP1 is therefore an attractive strategy for targeting cancer cells in conjunction with TOP1 poisons. Existing methods for monitoring the phosphodiesterase activity of TDP1 are generally gel based or of high cost. Here we report a novel, oligonucleotide-based fluorescence assay that is robust, sensitive, and suitable for high-throughput screening of both fragment and small compound libraries for the detection of TDP1 inhibitors. We further validated the assay using whole cell extracts, extending its potential application to determine of TDP1 activity in clinical samples from patients undergoing chemotherapy.
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TDP1 deficiency sensitizes human cells to base damage via distinct topoisomerase i and parp mechanisms with potential applications for cancer therapy
Nucleic Acids Research, 2014Co-Authors: Meryem Alagoz, Owen S Wells, Sherif F ElkhamisyAbstract:Base damage and topoisomerase I (Top1)-linked DNA breaks are abundant forms of endogenous DNA breakage, contributing to hereditary ataxia and underlying the cytotoxicity of a wide range of anti-cancer agents. Despite their frequency, the overlapping mechanisms that repair these forms of DNA breakage are largely unknown. Here, we report that depletion of Tyrosyl DNA phosphodiesterase 1 (TDP1) sensitizes human cells to alkylation damage and the additional depletion of apurinic/apyrimidinic endonuclease I (APE1) confers hypersensitivity above that observed for TDP1 or APE1 depletion alone. Quantification of DNA breaks and clonogenic survival assays confirm a role for TDP1 in response to base damage, independently of APE1. The hypersensitivity to alkylation damage is partly restored by depletion of Top1, illustrating that alkylating agents can trigger cytotoxic Top1-breaks. Although inhibition of PARP activity does not sensitize TDP1-deficient cells to Top1 poisons, it confers increased sensitivity to alkylation damage, highlighting partially overlapping roles for PARP and TDP1 in response to genotoxic challenge. Finally, we demonstrate that cancer cells in which TDP1 is inherently deficient are hypersensitive to alkylation damage and that TDP1 depletion sensitizes glioblastoma-resistant cancer cells to the alkylating agent temozolomide.
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tdp2 promotes repair of topoisomerase i mediated dna damage in the absence of TDP1
Nucleic Acids Research, 2012Co-Authors: Zhihong Zeng, Keith W Caldecott, Junko Murai, Yves Pommier, Shunichi Takeda, Abhishek Sharma, Lieve Umans, Liesbeth Vermeire, Danny Huylebroeck, Sherif F ElkhamisyAbstract:The abortive activity of topoisomerases can result in clastogenic and/or lethal DNA damage in which the topoisomerase is covalently linked to the 3'- or 5'-terminus of a DNA strand break. This type of DNA damage is implicated in chromosome translocations and neurological disease and underlies the clinical efficacy of an important class of anticancer topoisomerase 'poisons'. Tyrosyl DNA phosphodiesterase-1 protects cells from abortive topoisomerase I (Top1) activity by hydrolyzing the 3'-phosphotyrosyl bond that links Top1 to a DNA strand break and is currently the only known human enzyme that displays this activity in cells. Recently, we identified a second tyrosyl DNA phosphodiesterase (TDP2; aka TTRAP/EAPII) that possesses weak 3'-tyrosyl DNA phosphodiesterase (3'-TDP) activity, in vitro. Herein, we have examined whether TDP2 contributes to the repair of Top1-mediated DNA breaks by deleting TDP1 and Tdp2 separately and together in murine and avian cells. We show that while deletion of TDP1 in wild-type DT40 cells and mouse embryonic fibroblasts decreases DNA strand break repair rates and cellular survival in response to Top1-induced DNA damage, deletion of Tdp2 does not. However, deletion of both TDP1 and Tdp2 reduces rates of DNA strand break repair and cell survival below that observed in TDP1(-)(/)(-) cells, suggesting that Tdp2 contributes to cellular 3'-TDP activity in the absence of TDP1. Consistent with this idea, over-expression of human TDP2 in TDP1(-)(/)(-)/Tdp2(-)(/)(-)(/)(-) DT40 cells increases DNA strand break repair rates and cell survival above that observed in TDP1(-)(/)(-) DT40 cells, suggesting that Tdp2 over-expression can partially complement the defect imposed by loss of TDP1. Finally, mice lacking both TDP1 and Tdp2 exhibit greater sensitivity to Top1 poisons than do mice lacking TDP1 alone, further suggesting that Tdp2 contributes to the repair of Top1-mediated DNA damage in the absence of TDP1. In contrast, we failed to detect a contribution for TDP1 to repair Top2-mediated damage. Together, our data suggest that TDP1 and Tdp2 fulfil overlapping roles following Top1-induced DNA damage, but not following Top2-induced DNA damage, in vivo.
Christophe Marchand - One of the best experts on this subject based on the ideXlab platform.
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recifin a initial example of the tyr lock peptide structural family is a selective allosteric inhibitor of tyrosyl dna phosphodiesterase i
Journal of the American Chemical Society, 2020Co-Authors: Lauren R H Krumpe, Christophe Marchand, Wenjie Wang, Brice A P Wilson, Suthananda N Sunassee, Alun Bermingham, Edmund Price, Tad Guszczynski, James A Kelley, Kirk R GustafsonAbstract:Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a molecular target for the sensitization of cancer cells to the FDA-approved topoisomerase inhibitors topotecan and irinotecan. High-throughput screening of natural product extract and fraction libraries for inhibitors of TDP1 activity resulted in the discovery of a new class of knotted cyclic peptides from the marine sponge Axinella sp. Bioassay-guided fractionation of the source extract resulted in the isolation of the active component which was determined to be an unprecedented 42-residue cysteine-rich peptide named recifin A. The native NMR structure revealed a novel fold comprising a four strand antiparallel β-sheet and two helical turns stabilized by a complex disulfide bond network that creates an embedded ring around one of the strands. The resulting structure, which we have termed the Tyr-lock peptide family, is stabilized by a tyrosine residue locked into three-dimensional space. Recifin A inhibited the cleavage of phosphodiester bonds by TDP1 in a FRET assay with an IC50 of 190 nM. Enzyme kinetics studies revealed that recifin A can specifically modulate the enzymatic activity of full-length TDP1 while not affecting the activity of a truncated catalytic domain of TDP1 lacking the N-terminal regulatory domain (Δ1-147), suggesting an allosteric binding site for recifin A on the regulatory domain of TDP1. Recifin A represents both the first of a unique structural class of knotted disulfide-rich peptides and defines a previously unseen mechanism of TDP1 inhibition that could be productively exploited for potential anticancer applications.
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discovery synthesis and evaluation of oxynitidine derivatives as dual inhibitors of dna topoisomerase ib top1 and tyrosyl dna phosphodiesterase 1 TDP1 and potential antitumor agents
Journal of Medicinal Chemistry, 2018Co-Authors: Xiaoru Zhang, Christophe Marchand, Haowen Wang, Wenlin Tang, Yu Zhang, Hui Yang, Azhar Ravji, Evgeny Kiselev, Kwabena Oforiatta, Keli AgamaAbstract:Tyrosyl–DNA phosphodiesterase 1 (TDP1) is a recently discovered enzyme repairing DNA lesions resulting from stalled topoisomerase IB (TOP1)–DNA covalent complex. Inhibiting TDP1 in conjunction with TOP1 inhibitors can boost the action of the latter. Herein, we report the discovery of the natural product oxynitidine scaffold as a novel chemotype for the development of TOP1 and TDP1 inhibitors. Three kinds of analogues, benzophenanthridinone, dihydrobenzophenanthridine, and benzophenanthridine derivatives, were synthesized and evaluated for both TOP1 and TDP1 inhibition and cytotoxicity. Analogue 19a showed high TOP1 inhibition (+++) and induced the formation of cellular TOP1cc and DNA damage, resulting in cancer cells apoptosis at nanomolar concentration range. In vivo studies indicated that 19a exhibits antitumor efficiency in HCT116 xenograft model. 41a exhibited additional TDP1 inhibition with IC50 value of 7 μM and synergistic effect with camptothecin in MCF-7 cells. This work will facilitate future ef...
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probing the evolutionary conserved residues y204 f259 s400 and w590 that shape the catalytic groove of human TDP1 for 3 and 5 phosphodiester dna bond cleavage
DNA Repair, 2018Co-Authors: Evgeny Kiselev, Christophe Marchand, Thomas S Dexheimer, Sharyin Naomi Huang, Yves PommierAbstract:Tyrosyl-DNA phosphodiesterase 1 (TDP1) is an ubiquitous DNA repair enzyme present in yeast, plants and animals. It removes a broad range of blocking lesions at the ends of DNA breaks. The catalytic core of TDP1 consists in a pair of conserved histidine-lysine-asparagine (HKN) motifs. Analysis of the human TDP1 (hTDP1) crystal structure reveals potential involvement of additional residues that shape the substrate binding site. In this biochemical study, we analyzed four such conserved residues, tyrosine 204 (Y204), phenylalanine 259 (F259), serine 400 (S400) and tryptophan 590 (W590). We show that the F259 residue of hTDP1 is critical for both 3'- and 5'-phosphodiesterase catalysis. We propose that the double π-π interactions of the F259 residue with the -2 and -3 nucleobases serve to position the nucleopeptide substrate in phase with the active site histidines of hTDP1. Mutating Y204 of hTDP1 to phenylalanine (Y204F), as in fly and yeast TDP1 enzymes, had minor impact on TDP1 activity. In constrast, we find that S400 enhances 3'-processing activity while it suppresses 5'-processing activity, thereby promoting specificity for 3'-substrates. W590 is selectively important for 5'-processing. These results reveal the impact of conserved amino acid residues that participate in defining the DNA binding groove around the dual HKN catalytic core motif of TDP1, and their differential roles in facilitating the 3'- vs 5'-end processing activities of hTDP1.
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identification of natural products that inhibit the catalytic function of human tyrosyl dna phosphodiesterase TDP1
SLAS DISCOVERY: Advancing the Science of Drug Discovery, 2017Co-Authors: Alun Bermingham, Christophe Marchand, Adel Chergui, Alena Naumova, Lauren R H Krumpe, Edmund Price, Emily L Whitson, Ekaterina I Goncharova, Jason R Evans, Tawnya C MckeeAbstract:Tyrosyl-DNA phosphodiesterase 1 (TDP1) is an enzyme crucial for cleavage of the covalent topoisomerase 1-DNA complex, an intermediate in DNA repair. TDP1 plays a role in reversing inhibition of topoisomerase I by camptothecins, a series of potent and effective inhibitors used in the treatment of colorectal, ovarian, and small-cell lung cancers. It is hypothesized that inhibition of TDP1 activity may enhance camptothecin sensitivity in tumors. Here, we describe the design, development, and execution of a novel assay to identify inhibitors of TDP1 present in natural product extracts. The assay was designed to address issues with fluorescent "nuisance" molecules and to minimize the detection of false-positives caused by polyphenolic molecules known to nonspecifically inhibit enzyme activity. A total of 227,905 purified molecules, prefractionated extracts, and crude natural product extracts were screened. This yielded 534 initial positives (0.23%). Secondary prioritization reduced this number to 117 (0.05% final hit rate). Several novel inhibitors have been identified showing micromolar affinity for human TDP1, including halenaquinol sulfate, a pentacyclic hydroquinone from the sponge Xestospongia sp.
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synthesis and biological evaluation of the first triple inhibitors of human topoisomerase 1 tyrosyl dna phosphodiesterase 1 TDP1 and tyrosyl dna phosphodiesterase 2 tdp2
Journal of Medicinal Chemistry, 2017Co-Authors: Ping Wang, Christophe Marchand, Azhar Ravji, Evgeny Kiselev, Christophe E Redon, Mohamed S A Elsayed, Caroline B Plescia, Olga Zeleznik, Keli AgamaAbstract:TDP1 and Tdp2 are two tyrosyl–DNA phosphodiesterases that can repair damaged DNA resulting from topoisomerase inhibitors and a variety of other DNA-damaging agents. Both TDP1 and Tdp2 inhibition could hypothetically potentiate the cytotoxicities of topoisomerase inhibitors. This study reports the successful structure-based design and synthesis of new 7-azaindenoisoquinolines that act as triple inhibitors of Top1, TDP1, and Tdp2. Enzyme inhibitory data and cytotoxicity data from human cancer cell cultures establish that modification of the lactam side chain of the 7-azaindenoisoquinolines can modulate their inhibitory potencies and selectivities vs Top1, TDP1, and Tdp2. Molecular modeling of selected target compounds bound to Top1, TDP1, and Tdp2 was used to design the inhibitors and facilitate the structure–activity relationship analysis. The monitoring of DNA damage by γ-H2AX foci formation in human PBMCs (lymphocytes) and acute lymphoblastic leukemia CCRF-CEM cells documented significantly more DNA dama...
Thomas S Dexheimer - One of the best experts on this subject based on the ideXlab platform.
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synthesis anti cancer screening and tyrosyl dna phosphodiesterase 1 TDP1 inhibition activity of novel piperidinyl sulfamides
European Journal of Pharmaceutical Sciences, 2018Co-Authors: Jung Ho Jun, Thomas S Dexheimer, Vineet Kumar, Iwona Wedlich, Marc C Nicklaus, Yves Pommier, Sanjay V MalhotraAbstract:Abstract Novel piperidinyl-based sulfamide derivatives were designed and synthesized through various synthetic routes. Anticancer activities of these sulfamides were evaluated by phenotypic screening on National Cancer Institute's 60 human tumor cell lines (NCI-60). Preliminary screening at 10 μM concentration showed that piperidinyl sulfamide aminoester 26 (NSC 749204) was sensitive to most of the cell lines in the panel. Further dose-response studies showed that 26 was highly selective for inhibition of colon cancer cell lines with minimum GI50 = 1.88 μM for COLO-205 and maximum GI50 = 11.1 μM for SW-620 cells. These newly synthesized sulfamides were also screening for their TDP1 inhibition activity. Compound 18 (NSC 750706) showed significant inhibition of TDP1 with IC50 = 23.7 μM. Molecular-docking studies showed that 18 bind to TDP1 in its binding pocket similar to a known TDP1 inhibitor.
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probing the evolutionary conserved residues y204 f259 s400 and w590 that shape the catalytic groove of human TDP1 for 3 and 5 phosphodiester dna bond cleavage
DNA Repair, 2018Co-Authors: Evgeny Kiselev, Christophe Marchand, Thomas S Dexheimer, Sharyin Naomi Huang, Yves PommierAbstract:Tyrosyl-DNA phosphodiesterase 1 (TDP1) is an ubiquitous DNA repair enzyme present in yeast, plants and animals. It removes a broad range of blocking lesions at the ends of DNA breaks. The catalytic core of TDP1 consists in a pair of conserved histidine-lysine-asparagine (HKN) motifs. Analysis of the human TDP1 (hTDP1) crystal structure reveals potential involvement of additional residues that shape the substrate binding site. In this biochemical study, we analyzed four such conserved residues, tyrosine 204 (Y204), phenylalanine 259 (F259), serine 400 (S400) and tryptophan 590 (W590). We show that the F259 residue of hTDP1 is critical for both 3'- and 5'-phosphodiesterase catalysis. We propose that the double π-π interactions of the F259 residue with the -2 and -3 nucleobases serve to position the nucleopeptide substrate in phase with the active site histidines of hTDP1. Mutating Y204 of hTDP1 to phenylalanine (Y204F), as in fly and yeast TDP1 enzymes, had minor impact on TDP1 activity. In constrast, we find that S400 enhances 3'-processing activity while it suppresses 5'-processing activity, thereby promoting specificity for 3'-substrates. W590 is selectively important for 5'-processing. These results reveal the impact of conserved amino acid residues that participate in defining the DNA binding groove around the dual HKN catalytic core motif of TDP1, and their differential roles in facilitating the 3'- vs 5'-end processing activities of hTDP1.
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neuroprotection and repair of 3 blocking dna ends by glaikit gkt encoding drosophila tyrosyl dna phosphodiesterase 1 TDP1
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Dongyu Guo, Thomas S Dexheimer, Yves Pommier, Howard A NashAbstract:Tyrosyl-DNA phosphodiesterase (TDP1) is a phylogenetically conserved enzyme critical for the removal of blocking lesions at the 3′ ends of DNA or RNA. This study analyzes the Drosophila TDP1 gene ortholog glaikit (gkt) and its possible role(s) in the repair of endogenous DNA lesions and neuroprotection. To do so, we studied a homozygous PiggyBac insertion (c03958) that disrupts the 5′ UTR of gkt. Protein extracts of c03958 flies were defective in hydrolyzing 3′-DNA–tyrosyl residues, demonstrating that gkt is the Drosophila TDP1. Although the mutant is generally healthy and fertile, females exhibit reduced lifespan and diminished climbing ability. This phenotype was rescued by neuronal expression of TDP1. In addition, when c03958 larvae were exposed to bleomycin, an agent that produces oxidative DNA damage, or topoisomerase I-targeted drugs (camptothecin and a noncamptothecin indenoisoquinoline derivative, LMP-776), survivors displayed rough eye patches, which were rescued by neuronal expression of TDP1. Our study establishes that gkt is the Drosophila TDP1 gene, and that it is critical for neuroprotection, normal longevity, and repair of damaged DNA.
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biochemical assays for the discovery of TDP1 inhibitors
Molecular Cancer Therapeutics, 2014Co-Authors: Christophe Marchand, Shar-yin N. Huang, Adel Chergui, Andrew G. Stephen, Thomas S Dexheimer, Alena Naumova, Wendy A Lea, Bryan T Mott, Andrew S Rosenthal, Ganesha RaiAbstract:Drug screening against novel targets is warranted to generate biochemical probes and new therapeutic drug leads. TDP1 and TDP2 are two DNA repair enzymes that have yet to be successfully targeted. TDP1 repairs topoisomerase I-, alkylation-, and chain terminator-induced DNA damage, whereas TDP2 repairs topoisomerase II-induced DNA damage. Here, we report the quantitative high-throughput screening (qHTS) of the NIH Molecular Libraries Small Molecule Repository using recombinant human TDP1. We also developed a secondary screening method using a multiple loading gel-based assay where recombinant TDP1 is replaced by whole cell extract (WCE) from genetically engineered DT40 cells. While developing this assay, we determined the importance of buffer conditions for testing TDP1, and most notably the possible interference of phosphate-based buffers. The high specificity of endogenous TDP1 in WCE allowed the evaluation of a large number of hits with up to 600 samples analyzed per gel via multiple loadings. The increased stringency of the WCE assay eliminated a large fraction of the initial hits collected from the qHTS. Finally, inclusion of a TDP2 counter-screening assay allowed the identification of two novel series of selective TDP1 inhibitors.
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abstract c79 TDP1 repairs nuclear and mitochondrial dna damage induced by chain terminating anticancer and antiviral nucleoside analogs
Molecular Cancer Therapeutics, 2013Co-Authors: Shar-yin N. Huang, Junko Murai, Thomas S Dexheimer, Ilaria Dalla Rosa, William H Gmeiner, Yves PommierAbstract:Nucleoside analogs (NAs) that cause stalling or premature termination of DNA replication forks are widely used as anticancer and antiviral drugs. However, it is not well understood how cells repair the DNA damage induced by these chain-terminating NAs. Here we reveal the importance of tyrosyl-DNA phosphodiesterase 1 (TDP1) in the repair of nuclear and mitochondrial DNA damage induced by chain-terminating NAs. Upon investigating the effects of four NAs: acyclovir (ACV), cytarabine (Ara-C), zidovudine (AZT) and zalcitabine (ddC), we show that TDP1 is capable of removing the covalently-linked corresponding NAs from DNA 3’-ends. We also show that TDP1–/– cells are hypersensitive and accumulate more DNA damage when treated with ACV and Ara-C, implicating TDP1 in repairing NA-induced DNA damage. Since AZT and ddC are known to cause mitochondrial dysfunction, we examined whether TDP1 repairs the mitochondrial DNA damage they induced. We found that AZT and ddC treatment leads to greater depletion of mitochondrial DNA in TDP1–/– cells. Thus, TDP1 appears critical for repairing nuclear and mitochondrial DNA damage caused by chain-terminating NAs. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C79. Citation Format: Shar-Yin N. Huang, Junko Murai, Ilaria Dalla Rosa, Thomas S. Dexheimer, William H. Gmeiner, Yves Pommier. TDP1 repairs nuclear and mitochondrial DNA damage induced by chain-terminating anticancer and antiviral nucleoside analogs. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C79.
Keli Agama - One of the best experts on this subject based on the ideXlab platform.
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synthesis of methoxy methylenedioxy hydroxy and halo substituted benzophenanthridinone derivatives as dna topoisomerase ib top1 and tyrosyl dna phosphodiesterase 1 TDP1 inhibitors and their biological activity for drug resistant cancer
Journal of Medicinal Chemistry, 2021Co-Authors: Wenlin Tang, Wenjie Wang, Keli Agama, Yu Zhang, Hao Yang, Yves PommierAbstract:As a recently discovered DNA repair enzyme, tyrosyl-DNA phosphodiesterase 1 (TDP1) removes topoisomerase IB (TOP1)-mediated DNA protein cross-links. Inhibiting TDP1 can potentiate the cytotoxicity of TOP1 inhibitors and overcome cancer cell resistance to TOP1 inhibitors. On the basis of our previous study, herein we report the synthesis of benzophenanthridinone derivatives as TOP1 and TDP1 inhibitors. Seven compounds (C2, C4, C5, C7, C8, C12, and C14) showed a robust TOP1 inhibitory activity (+++ or ++++), and four compounds (A13, C12, C13, and C26) showed a TDP1 inhibition (half-maximal inhibitory concentration values of 15 or 19 μM). We also show that the dual TOP1 and TDP1 inhibitor C12 induces both cellular TOP1cc, TDP1cc formation and DNA damage, resulting in cancer cell apoptosis at a sub-micromolar concentration. In addition, C12 showed an enhanced activity in drug-resistant MCF-7/TDP1 cancer cells and was synergistic with topotecan in both MCF-7 and MCF-7/TDP1 cells.
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discovery synthesis and evaluation of oxynitidine derivatives as dual inhibitors of dna topoisomerase ib top1 and tyrosyl dna phosphodiesterase 1 TDP1 and potential antitumor agents
Journal of Medicinal Chemistry, 2018Co-Authors: Xiaoru Zhang, Christophe Marchand, Haowen Wang, Wenlin Tang, Yu Zhang, Hui Yang, Azhar Ravji, Evgeny Kiselev, Kwabena Oforiatta, Keli AgamaAbstract:Tyrosyl–DNA phosphodiesterase 1 (TDP1) is a recently discovered enzyme repairing DNA lesions resulting from stalled topoisomerase IB (TOP1)–DNA covalent complex. Inhibiting TDP1 in conjunction with TOP1 inhibitors can boost the action of the latter. Herein, we report the discovery of the natural product oxynitidine scaffold as a novel chemotype for the development of TOP1 and TDP1 inhibitors. Three kinds of analogues, benzophenanthridinone, dihydrobenzophenanthridine, and benzophenanthridine derivatives, were synthesized and evaluated for both TOP1 and TDP1 inhibition and cytotoxicity. Analogue 19a showed high TOP1 inhibition (+++) and induced the formation of cellular TOP1cc and DNA damage, resulting in cancer cells apoptosis at nanomolar concentration range. In vivo studies indicated that 19a exhibits antitumor efficiency in HCT116 xenograft model. 41a exhibited additional TDP1 inhibition with IC50 value of 7 μM and synergistic effect with camptothecin in MCF-7 cells. This work will facilitate future ef...
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synthesis and biological evaluation of the first triple inhibitors of human topoisomerase 1 tyrosyl dna phosphodiesterase 1 TDP1 and tyrosyl dna phosphodiesterase 2 tdp2
Journal of Medicinal Chemistry, 2017Co-Authors: Ping Wang, Christophe Marchand, Azhar Ravji, Evgeny Kiselev, Christophe E Redon, Mohamed S A Elsayed, Caroline B Plescia, Olga Zeleznik, Keli AgamaAbstract:TDP1 and Tdp2 are two tyrosyl–DNA phosphodiesterases that can repair damaged DNA resulting from topoisomerase inhibitors and a variety of other DNA-damaging agents. Both TDP1 and Tdp2 inhibition could hypothetically potentiate the cytotoxicities of topoisomerase inhibitors. This study reports the successful structure-based design and synthesis of new 7-azaindenoisoquinolines that act as triple inhibitors of Top1, TDP1, and Tdp2. Enzyme inhibitory data and cytotoxicity data from human cancer cell cultures establish that modification of the lactam side chain of the 7-azaindenoisoquinolines can modulate their inhibitory potencies and selectivities vs Top1, TDP1, and Tdp2. Molecular modeling of selected target compounds bound to Top1, TDP1, and Tdp2 was used to design the inhibitors and facilitate the structure–activity relationship analysis. The monitoring of DNA damage by γ-H2AX foci formation in human PBMCs (lymphocytes) and acute lymphoblastic leukemia CCRF-CEM cells documented significantly more DNA dama...
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design synthesis and biological evaluation of o 2 modified indenoisoquinolines as dual topoisomerase i tyrosyl dna phosphodiesterase i inhibitors
Journal of Medicinal Chemistry, 2014Co-Authors: Keli Agama, Christophe Marchand, Mark CushmanAbstract:Tyrosyl-DNA phosphodiesterase I (TDP1) repairs stalled topoisomerase I (Top1)–DNA covalent complexes and has been proposed to be a promising and attractive target for cancer treatment. Inhibitors of TDP1 could conceivably act synergistically with Top1 inhibitors and thereby potentiate the effects of Top1 poisons. This study describes the successful design and synthesis of 2-position-modified indenoisoquinolines as dual Top1–TDP1 inhibitors using a structure-based drug design approach. Enzyme inhibition studies indicate that indenoisoquinolines modified at the 2-position with three-carbon side chains ending with amino substituents show both promising Top1 and TDP1 inhibitory activity. Molecular modeling of selected target compounds bound to Top1 and TDP1 was used to rationalize the enzyme inhibition results and structure–activity relationship analysis.
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epigenetic and genetic inactivation of tyrosyl dna phosphodiesterase 1 TDP1 in human lung cancer cells from the nci 60 panel
DNA Repair, 2014Co-Authors: Rui Gao, Keli Agama, Benu Brata Das, Raghunath Chatterjee, Ogan D Abaan, Renata Matuo, Charles Vinson, Paul S Meltzer, Yves PommierAbstract:Abstract Tyrosyl-DNA-phosphodiesterase 1 (TDP1) repairs 3′-blocking DNA lesions by catalytically hydrolyzing the tyrosyl-DNA-phosphodiester bond of trapped topoisomerase I (Top1) cleavage complexes (Top1cc). It also removes 3′-blocking residues derived from oxidative damage or incorporation of chain terminating anticancer and antiviral nucleosides. Thus, TDP1 is regarded as a determinant of resistance to Top1 inhibitors and chain terminating nucleosides, and possibly of genomic stability. In the 60 cell lines of the NCI Developmental Therapeutic Anticancer Screen (the NCI-60), whose whole genome transcriptome and mutations have recently been characterized, we discovered two human lung cancer cell lines deficient for TDP1 (NCI_H522 and HOP_62). HOP_62 shows undetectable TDP1 mRNA and NCI_H522 bears a homozygous deleterious mutation of TDP1 at a highly conserved amino acid residue (K292E). Absence of TDP1 protein and lack of TDP1 catalytic activity were demonstrated in cell lysates from both cell lines. Lack of TDP1 expression in HOP_62 was shown to be due to TDP1 promoter hypermethylation. Our study provides insights into the possible inactivation of TDP1 in cancers and its relationship to cellular response to Top1-targeted drugs. It also reveals two TDP1 knockout lung cancer cell lines for further TDP1 functional analyses.
