ABSTRACT
Nucleotide excision repair (NER) removes helix-destabilizing adducts including ultraviolet (UV) lesions, cyclobutane pyrimidine dimers (CPDs), and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). In comparison with CPDs, 6-4PPs have greater cytotoxicity and more strongly destabilizing properties of the DNA helix. It is generally believed that NER is the only DNA repair pathway that removes the UV lesions as evidenced by the previous data since no repair of UV lesions was detected in NER-deficient skin fibroblasts. Topoisomerase I (TOP1) constantly creates transient single-strand breaks (SSBs) releasing the torsional stress in genomic duplex DNA. Stalled TOP1-SSB complexes can form near DNA lesions including abasic sites and ribonucleotides embedded in chromosomal DNA. Here we show that base excision repair (BER) increases cellular tolerance to UV independently of NER in cancer cells. UV lesions irreversibly trap stable TOP1-SSB complexes near the UV damage in NER-deficient cells, and the resulting SSBs activate BER. Biochemical experiments show that 6-4PPs efficiently induce stable TOP1-SSB complexes, and the long-patch repair synthesis of BER removes 6-4PPs downstream of the SSB. Furthermore, NER-deficient cancer cell lines remove 6-4PPs within 24 h, but not CPDs, and the removal correlates with TOP1 expression. NER-deficient skin fibroblasts weakly express TOP1 and show no detectable repair of 6-4PPs. Remarkably, the ectopic expression of TOP1 in these fibroblasts led them to completely repair 6-4PPs within 24 h. In conclusion, we reveal a DNA repair pathway initiated by TOP1, which significantly contributes to cellular tolerance to UV-induced lesions particularly in malignant cancer cells overexpressing TOP1.
Subject(s)
DNA Breaks, Single-Stranded/radiation effects , DNA Repair , DNA Topoisomerases, Type I/metabolism , Ultraviolet Rays/adverse effects , CRISPR-Cas Systems/genetics , DNA Polymerase beta/genetics , DNA Polymerase beta/metabolism , Fibroblasts , Gene Knockout Techniques , Humans , MCF-7 Cells , Primary Cell Culture , Skin/cytology , Skin/pathology , Skin/radiation effects , X-ray Repair Cross Complementing Protein 1/genetics , X-ray Repair Cross Complementing Protein 1/metabolism , Xeroderma Pigmentosum/etiology , Xeroderma Pigmentosum/pathology , Xeroderma Pigmentosum Group A Protein/genetics , Xeroderma Pigmentosum Group A Protein/metabolismABSTRACT
Polynucleotides, DNA and RNA (mRNA and non-coding RNAs) are critically involved in the molecular pathways of disease. Small molecule binding interactions with polynucleotides can modify functional polynucleotide topologies and/or their interactions with proteins. Current approaches to library design (lead-like or fragment-like libraries) are based on protein-ligand interactions and often include careful consideration of the 3-dimensional orientation of binding motifs and exclude π-rich compounds (polyfused aromatics) to avoid off-target R/DNA interactions. In contrast to proteins, where π,π-interactions are weak, polynucleotides can form strong π,π-interactions with suitable π-rich ligands. To assist in designing a polynucleotide-biased library, a scaffold-divergent synthesis approach to polyfused aromatic scaffolds has been undertaken. Initial screening hits that form moderately stable polynucleotide-ligand-protein ternary complexes can be further optimized through judicious incorporation of substituents on the scaffold to increase protein-ligand interactions. An example of this approach is given for topoisomerase-1 (TOP1), generating a novel TOP1 inhibitory chemotype.
Subject(s)
Polynucleotides , RNA , Polynucleotides/chemistry , Ligands , DNA/chemistry , ProteinsABSTRACT
Tyrosyl-DNA phosphodiesterase 1 (TDP1) is an enzyme that repairs DNA lesions caused by the trapping of DNA topoisomerase IB (TOP1)-DNA break-associated crosslinks. TDP1 inhibitors have synergistic effect with TOP1 inhibitors in cancer cells and can overcome cancer cell resistance to TOP1 inhibitors. Here, we report the synthesis of 11-aminoalkoxy substituted benzophenanthridine derivatives as selective TDP1 inhibitors and show that six compounds 14, 16, 18, 20, 25 and 27 exhibit high TDP1 inhibition potency. The most potent TDP1 inhibitor 14 (IC50â¯=â¯1.7⯱â¯0.24⯵M) induces cellular TDP1cc formation and shows synergistic effect with topotecan in four human cancer cell lines MCF-7, A549, H460 and HepG2.
Subject(s)
Phosphoric Diester Hydrolases , Topoisomerase I Inhibitors , Benzophenanthridines , DNA Topoisomerases, Type I/metabolism , Humans , Phosphoric Diester Hydrolases/metabolism , Structure-Activity Relationship , Topoisomerase I Inhibitors/pharmacologyABSTRACT
Nature has been always a great source of possible lead compounds to develop new drugs against several diseases. Here we report the identification of a natural compound, membranoid G, derived from the Antarctic sponge Dendrilla antarctica displaying an in vitro inhibitory activity against human DNA topoisomerase 1B. The experiments indicate that membranoid G, when pre-incubated with the enzyme, strongly and irreversibly inhibits the relaxation of supercoiled DNA. This compound completely inhibits the cleavage step of the enzyme catalytic mechanism by preventing protein binding to the DNA. Membranoid G displays also a cytotoxic effect on tumour cell lines, suggesting its use as a possible lead compound to develop new anticancer drugs.
Subject(s)
Antineoplastic Agents , Neoplasms , Antarctic Regions , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , DNA/chemistry , DNA Topoisomerases, Type I/metabolism , DNA Topoisomerases, Type II/metabolism , Humans , Topoisomerase InhibitorsABSTRACT
In this study, different assortments of 2-arylquinolines and 2,6-diarylquinolines have been developed. Recently, we have developed a new series of 6,7-dimethoxy-4-alkoxy-2-arylquinolines as Topoisomerase I (TOP1) inhibitors with potent anticancer activity. Utilising the SAR outputs from this study, we tried to enhance anticancer and TOP1 inhibitory activities. Though target quinolines demonstrated potent antiproliferative effect, specifically against colorectal cancer DLD-1 and HCT-116, they showed weak TOP1 inhibition which may be attributable to their non-coplanarity. Thereafter, screening against kinase panel revealed their dual inhibitory activity against EGFR and FAK. Quinolines 6f, 6h, 6i, and 20f were the most potent EGFR inhibitors (IC50s = 25.39, 20.15, 22.36, and 24.81 nM, respectively). Meanwhile, quinolines 6f, 6h, 6i, 16d, and 20f exerted the best FAK inhibition (IC50s = 22.68, 14.25, 18.36, 17.36, and 15.36 nM, respectively). Finally, molecular modelling was employed to justify the promising EGFR/FAK inhibition. The study outcomes afforded the first reported quinolines with potent EGFR/FAK dual inhibition.
Subject(s)
Antineoplastic Agents/pharmacology , Focal Adhesion Kinase 1/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Quinolines/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/metabolism , Focal Adhesion Kinase 1/metabolism , Humans , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Quinolines/chemical synthesis , Quinolines/chemistry , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
A facile one-pot synthesis of C-ring substituted angular luotonins has been realized via a methanesulfonic acid mediated aza-Nazarov-Friedlander condensation sequence on quinazolinonyl enones. Topoisomerase I (topo-I) inhibition studies revealed that the angular luotonin library (7a-7l) and their regioisomeric analogs (linear luotonins, 8a-8l) are weak negative modulators, compared to camptothecin. These results would fare well for the design of topo-I-inert luotonins for non-oncological applications such as anti-fungal and insecticide lead developments. Surprisingly, the tricyclic vasicinones (9h, 9i, and 9j) showed better topo-I inhibition compared to pentacyclic C-aryl luotonins providing a novel pharmacophore for further explorations.
Subject(s)
Alkaloids/pharmacology , DNA Topoisomerases, Type I/metabolism , Drug Design , Pyrroles/pharmacology , Quinones/pharmacology , Topoisomerase I Inhibitors/pharmacology , Alkaloids/chemical synthesis , Alkaloids/chemistry , Dose-Response Relationship, Drug , Humans , Molecular Structure , Pyrroles/chemical synthesis , Pyrroles/chemistry , Quinones/chemical synthesis , Quinones/chemistry , Structure-Activity Relationship , Topoisomerase I Inhibitors/chemical synthesis , Topoisomerase I Inhibitors/chemistryABSTRACT
Based on our previous study on the development of the furoquinolinedione and isoxazoloquinolinedione TDP2 inhibitors, the further structure-activity relationship (SAR) was studied in this work. A series of furoquinolinedione and isoxazoloquinolinedione derivatives were synthesized and tested for enzyme inhibitions. Enzyme-based assays indicated that isoxazoloquinolinedione derivatives selectively showed high TDP2 inhibitory activity at sub-micromolar range, as well as furoquinolinedione derivatives at low micromolar range. The most potent 3-(3,4-dimethoxyphenyl)isoxazolo[4,5-g]quinoline-4,9-dione (70) showed TDP2 inhibitory activity with IC50 of 0.46 ± 0.15 µM. This work will facilitate future efforts for the discovery of isoxazoloquinolinedione TDP2 selective inhibitors.
Subject(s)
DNA-Binding Proteins/antagonists & inhibitors , Phosphodiesterase Inhibitors/pharmacology , Quinolones/pharmacology , Animals , DNA-Binding Proteins/metabolism , Dose-Response Relationship, Drug , Humans , Mice , Models, Molecular , Molecular Structure , Phosphodiesterase Inhibitors/chemical synthesis , Phosphodiesterase Inhibitors/chemistry , Phosphoric Diester Hydrolases/metabolism , Quinolones/chemical synthesis , Quinolones/chemistry , Structure-Activity RelationshipABSTRACT
Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs abortive topoisomerase II cleavage complexes. Here, we identify a novel short isoform of TDP2 (TDP2S) expressed from an alternative transcription start site. TDP2S contains a mitochondrial targeting sequence, contributing to its enrichment in the mitochondria and cytosol, while full-length TDP2 contains a nuclear localization signal and the ubiquitin-associated domain in the N-terminus. Our study reveals that both TDP2 isoforms are present and active in the mitochondria. Comparison of isogenic wild-type (WT) and TDP2 knockout (TDP2-/-/-) DT40 cells shows that TDP2-/-/- cells are hypersensitive to mitochondrial-targeted doxorubicin (mtDox), and that complementing TDP2-/-/- cells with human TDP2 restores resistance to mtDox. Furthermore, mtDox selectively depletes mitochondrial DNA in TDP2-/-/- cells. Using CRISPR-engineered human cells expressing only the TDP2S isoform, we show that TDP2S also protects human cells against mtDox. Finally, lack of TDP2 in the mitochondria reduces the mitochondria transcription levels in two different human cell lines. In addition to identifying a novel TDP2S isoform, our report demonstrates the presence and importance of both TDP2 isoforms in the mitochondria.
Subject(s)
Doxorubicin/pharmacology , Drug Resistance, Neoplasm/genetics , Neoplasms/drug therapy , Nuclear Proteins/genetics , Transcription Factors/genetics , Alternative Splicing/genetics , Cell Line, Tumor , DNA-Binding Proteins , Gene Expression Regulation, Enzymologic/drug effects , Gene Knockout Techniques , Humans , Mitochondria/drug effects , Mitochondria/genetics , Neoplasms/genetics , Neoplasms/pathology , Nuclear Proteins/antagonists & inhibitors , Phosphoric Diester Hydrolases , Protein Isoforms/genetics , Transcription Factors/antagonists & inhibitorsABSTRACT
DNA topoisomerase IB (TOP1) is a validated target for discovery and development of antitumor agents. Four TOP1 poisons are clinically used for tumor treatment now. In spite of their effectiveness in solid tumors, these camptothecin (CPT) poisons suffer from many shortcomings. Therefore, many investigations have focused on the discoveries of non-CPT poisons and catalytic inhibitors. Herein, we systematically study the antitumor activity of CYB-L10, a novel indolizinoquinolinedione TOP1 catalytic inhibitor discovered in our laboratory. The results indicated that CYB-L10 mainly acts on TOP1 in cancer cells and is not a substrate of the P-glycoprotein. In addition, CYB-L10 can induce apoptosis of HCT116 cells, shows high cytotoxicity against 60 human clinical cancer cell lines (NCI60) with the mean-graph midpoint for growth inhibition of all cancer cell lines of 0.050 µM concentration and obvious antitumor efficiency in vivo in the HCT116 xenograft model.
Subject(s)
Antineoplastic Agents/pharmacology , DNA Topoisomerases, Type I/metabolism , Indolizines/pharmacology , Quinolines/pharmacology , Topoisomerase I Inhibitors/pharmacology , Animals , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , Humans , Indolizines/chemistry , Mice , Mice, Nude , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/pathology , Quinolines/chemistry , Topoisomerase I Inhibitors/chemistry , Tumor Cells, CulturedABSTRACT
Fluorine and chlorine are metabolically stable, but generally less active replacements for a nitro group at the 3-position of indenoisoquinoline topoisomerase IB (Top1) poisons. A number of strategies were employed in the present investigation to enhance the Top1 inhibitory potencies and cancer cell growth inhibitory activities of halogenated indenoisoquinolines. In several cases, the new compounds' activities were found to rival or surpass those of similarly substituted 3-nitroindenoisoquinolines, and several unusually potent analogs were discovered through testing in human cancer cell cultures. A hydroxyethylaminopropyl side chain on the lactam nitrogen of two halogenated indenoisoquinoline Top1 inhibitors was found to also impart inhibitory activity against tyrosyl DNA phosphodiesterases 1 and 2 (TDP1 and TDP2), which are enzymes that participate in the repair of DNA damage induced by Top1 poisons.
Subject(s)
DNA Topoisomerases, Type I/metabolism , Indenes/pharmacology , Isoquinolines/pharmacology , Topoisomerase I Inhibitors/chemical synthesis , Topoisomerase I Inhibitors/pharmacology , Dose-Response Relationship, Drug , Humans , Indenes/chemical synthesis , Indenes/chemistry , Isoquinolines/chemical synthesis , Isoquinolines/chemistry , Models, Molecular , Molecular Structure , Structure-Activity Relationship , Topoisomerase I Inhibitors/chemistryABSTRACT
Mitochondrial topoisomerase I (Top1mt) is a type IB topoisomerase present in vertebrates and exclusively targeted to mitochondria. Top1mt relaxes mitochondrial DNA (mtDNA) supercoiling by introducing transient cleavage complexes wherein the broken DNA strand swivels around the intact strand. Top1mt cleavage complexes (Top1mtcc) can be stabilized in vitro by camptothecin (CPT). However, CPT does not trap Top1mtcc efficiently in cells and is highly cytotoxic due to nuclear Top1 targeting. To map Top1mtcc on mtDNA in vivo and to overcome the limitations of CPT, we designed two substitutions (T546A and N550H) in Top1mt to stabilize Top1mtcc. We refer to the double-mutant enzyme as Top1mt*. Using retroviral transduction and ChIP-on-chip assays with Top1mt* in Top1mt knock-out murine embryonic fibroblasts, we demonstrate that Top1mt* forms high levels of cleavage complexes preferentially in the noncoding regulatory region of mtDNA, accumulating especially at the heavy strand replication origin OH, in the ribosomal genes (12S and 16S) and at the light strand replication origin OL. Expression of Top1mt* also caused rapid mtDNA depletion without affecting mitochondria mass, suggesting the existence of specific mitochondrial pathways for the removal of damaged mtDNA.
Subject(s)
DNA Topoisomerases, Type I/metabolism , DNA, Mitochondrial/genetics , Mitochondria/enzymology , Animals , DNA Damage , DNA Topoisomerases, Type I/genetics , DNA, Mitochondrial/chemistry , DNA, Mitochondrial/metabolism , Mice , Mice, Knockout , Mitochondria/genetics , Regulatory Sequences, Nucleic AcidABSTRACT
Lamellarin D (Lam-D) is a hexacyclic pyrole alkaloid isolated from marine invertebrates, whose biologic properties have been attributed to mitochondrial targeting. Mitochondria contain their own DNA (mtDNA), and the only specific mitochondrial topoisomerase in vertebrates is mitochondrial topoisomerase I (Top1mt). Here, we show that Top1mt is a direct mitochondrial target of Lam-D. In vitro Lam-D traps Top1mt and induces Top1mt cleavage complexes (Top1mtcc). Using single-molecule analyses, we also show that Lam-D slows down supercoil relaxation of Top1mt and strongly inhibits Top1mt religation in contrast to the inefficacy of camptothecin on Top1mt. In living cells, we show that Lam-D accumulates rapidly inside mitochondria, induces cellular Top1mtcc, and leads to mtDNA damage. This study provides evidence that Top1mt is a direct mitochondrial target of Lam-D and suggests that developing Top1mt inhibitors represents a novel strategy for targeting mitochondrial DNA.
Subject(s)
Coumarins/pharmacology , DNA Topoisomerases, Type I/metabolism , Heterocyclic Compounds, 4 or More Rings/pharmacology , Isoquinolines/pharmacology , Mitochondria/drug effects , Mitochondria/metabolism , Cell Line, Tumor , DNA Topoisomerases, Type I/genetics , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , Humans , Mitochondria/geneticsABSTRACT
BLM, the helicase defective in Bloom syndrome, is part of a multiprotein complex that protects genome stability. Here, we show that Rif1 is a novel component of the BLM complex and works with BLM to promote recovery of stalled replication forks. First, Rif1 physically interacts with the BLM complex through a conserved C-terminal domain, and the stability of Rif1 depends on the presence of the BLM complex. Second, Rif1 and BLM are recruited with similar kinetics to stalled replication forks, and the Rif1 recruitment is delayed in BLM-deficient cells. Third, genetic analyses in vertebrate DT40 cells suggest that BLM and Rif1 work in a common pathway to resist replication stress and promote recovery of stalled forks. Importantly, vertebrate Rif1 contains a DNA-binding domain that resembles the αCTD domain of bacterial RNA polymerase α; and this domain preferentially binds fork and Holliday junction (HJ) DNA in vitro and is required for Rif1 to resist replication stress in vivo. Our data suggest that Rif1 provides a new DNA-binding interface for the BLM complex to restart stalled replication forks.
Subject(s)
Carrier Proteins/metabolism , DNA Replication , DNA-Binding Proteins/metabolism , DNA/metabolism , Nuclear Proteins/metabolism , RecQ Helicases/metabolism , Telomere-Binding Proteins/metabolism , Amino Acid Sequence , Animals , Blotting, Western , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/genetics , Cell Line , Chickens , DNA/genetics , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , HeLa Cells , Humans , Immunoprecipitation , Kidney/cytology , Kidney/metabolism , Molecular Sequence Data , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , RNA, Small Interfering/pharmacology , RecQ Helicases/antagonists & inhibitors , RecQ Helicases/genetics , Sequence Homology, Amino Acid , Telomere-Binding Proteins/antagonists & inhibitors , Telomere-Binding Proteins/geneticsABSTRACT
Purely structure-based pharmacophores (SBPs) are an alternative method to ligand-based approaches and have the advantage of describing the entire interaction capability of a binding pocket. Here, we present the development of SBPs for topoisomerase I, an anticancer target with an unusual ligand binding pocket consisting of protein and DNA atoms. Different approaches to cluster and select pharmacophore features are investigated, including hierarchical clustering and energy calculations. In addition, the performance of SBPs is evaluated retrospectively and compared to the performance of ligand- and complex-based pharmacophores. SBPs emerge as a valid method in virtual screening and a complementary approach to ligand-focussed methods. The study further reveals that the choice of pharmacophore feature clustering and selection methods has a large impact on the virtual screening hit lists. A prospective application of the SBPs in virtual screening reveals that they can be used successfully to identify novel topoisomerase inhibitors.
Subject(s)
DNA Topoisomerases, Type I/chemistry , DNA Topoisomerases, Type I/metabolism , DNA/metabolism , Drug Design , Drug Discovery , Enzyme Inhibitors/chemistry , High-Throughput Screening Assays , DNA/chemistry , Drug Evaluation, Preclinical , Enzyme Inhibitors/pharmacology , Humans , Ligands , Models, Molecular , Protein Binding , Protein ConformationABSTRACT
Poly(ADP-Ribose) (PAR) polymerase (PARP) inhibitors represent a promising class of novel anticancer agents. The present study explores the molecular rationale for combining veliparib (ABT-888) with camptothecin (CPT) and its clinical derivatives, topotecan and irinotecan. ABT-888 inhibited PAR induction by CPT and increased CPT-induced cell killing and histone γH2AX. Increased DNA breaks by ABT-888 were not associated with a corresponding increase of topoisomerase I cleavage complexes and were further increased by inactivation of tyrosyl-DNA phosphodiesterase 1. SiRNA knockdown for the endonuclease XPF-ERCC1 reduced the ABT-888-induced γH2AX response in non-replicating and replicating cells but enhanced the antiproliferative effect of ABT-888 in CPT-treated cells. Our findings indicate the involvement of XPF-ERCC1 in inducing γH2AX response and repairing topoisomerase I-induced DNA damage as an alternative pathway from PARP and tyrosyl-DNA phosphodiesterase 1.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Benzimidazoles/pharmacology , Camptothecin/toxicity , DNA-Binding Proteins/physiology , Endonucleases/physiology , Poly(ADP-ribose) Polymerase Inhibitors , Topoisomerase I Inhibitors/toxicity , Benzimidazoles/administration & dosage , Camptothecin/administration & dosage , Cell Line, Tumor , DNA Damage , DNA Repair , DNA Replication , DNA Topoisomerases, Type I/metabolism , Enzyme Inhibitors/pharmacology , Histones/analysis , Humans , Poly(ADP-ribose) Polymerases/physiology , Topoisomerase I Inhibitors/administration & dosage , Transcription, GeneticABSTRACT
Topoisomerase inhibitors such as camptothecin and etoposide are used as anti-cancer drugs and induce double-strand breaks (DSBs) in genomic DNA in cycling cells. These DSBs are often covalently bound with polypeptides at the 3' and 5' ends. Such modifications must be eliminated before DSB repair can take place, but it remains elusive which nucleases are involved in this process. Previous studies show that CtIP plays a critical role in the generation of 3' single-strand overhang at "clean" DSBs, thus initiating homologous recombination (HR)-dependent DSB repair. To analyze the function of CtIP in detail, we conditionally disrupted the CtIP gene in the chicken DT40 cell line. We found that CtIP is essential for cellular proliferation as well as for the formation of 3' single-strand overhang, similar to what is observed in DT40 cells deficient in the Mre11/Rad50/Nbs1 complex. We also generated DT40 cell line harboring CtIP with an alanine substitution at residue Ser332, which is required for interaction with BRCA1. Although the resulting CtIP(S332A/-/-) cells exhibited accumulation of RPA and Rad51 upon DNA damage, and were proficient in HR, they showed a marked hypersensitivity to camptothecin and etoposide in comparison with CtIP(+/-/-) cells. Finally, CtIP(S332A/-/-)BRCA1(-/-) and CtIP(+/-/-)BRCA1(-/-) showed similar sensitivities to these reagents. Taken together, our data indicate that, in addition to its function in HR, CtIP plays a role in cellular tolerance to topoisomerase inhibitors. We propose that the BRCA1-CtIP complex plays a role in the nuclease-mediated elimination of oligonucleotides covalently bound to polypeptides from DSBs, thereby facilitating subsequent DSB repair.
Subject(s)
BRCA1 Protein/metabolism , DNA Breaks, Double-Stranded , DNA Repair , Nuclear Proteins/metabolism , Animals , BRCA1 Protein/genetics , Cell Cycle , Cell Line, Tumor , Chickens , DNA/genetics , Nuclear Proteins/genetics , Protein Binding , Recombination, GeneticABSTRACT
Herein, a series of 11- or 12-substituted benzophenanthridinone derivatives was designed and synthesized for the discovery of dual topoisomerase IB (TOP1) and tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors. Enzyme-based assays indicated that two compounds 12 and 38 showed high TOP1 inhibitory potency (+++), and four compounds 35, 37, 39 and 43 showed good TDP1 inhibition with IC50 values ranging from 10 to 18â µM. 38 could induce cellular TOP1cc formation, resulting in the highest cytotoxicity against HCT-116â cells (0.25â µM). The most potent TDP1 inhibitor 43 (10â µM) could induce cellular TDP1cc formation and enhance topotecan-induced DNA damage and showed strong synergistic cytotoxicity with topotecan in both MCF-7 and MCF-7/TDP1â cells.
Subject(s)
DNA Topoisomerases, Type I , Phosphodiesterase Inhibitors , Humans , Phosphodiesterase Inhibitors/pharmacology , DNA Topoisomerases, Type I/metabolism , Topotecan , Phosphoric Diester Hydrolases/metabolism , Topoisomerase I Inhibitors/pharmacologyABSTRACT
Dibenzo[c,h][1,5]naphthyridinediones were prepared via a novel synthetic pathway. The compounds were designed as topoisomerase I (Top1) inhibitors based on the indenoisoquinoline series of drugs. The results of biological evaluation demonstrate that, unlike very closely related dibenzo[c,h][1,6]naphthyridinediones, dibenzo[c,h][1,5]naphthyridinediones retain the Top1 inhibitory activity of similarly substituted indenoisoquinolines.
Subject(s)
DNA Topoisomerases, Type I/metabolism , Isoquinolines/chemistry , Naphthyridines/chemistry , Naphthyridines/chemical synthesis , Topoisomerase I Inhibitors/chemistry , Topoisomerase I Inhibitors/chemical synthesis , Drug Screening Assays, Antitumor , Models, Molecular , Naphthyridines/pharmacology , Structure-Activity Relationship , Topoisomerase I Inhibitors/pharmacologyABSTRACT
Eukaryotic topoisomerases I (TOP1) are ubiquitous enzymes removing DNA torsional stress. However, there is little data concerning the three-dimensional structure of TOP1 in the absence of DNA, nor how the DNA molecule can enter/exit its closed conformation. Here, we solved the structure of thermostable archaeal Caldiarchaeum subterraneum CsTOP1 in an apo-form. The enzyme displays an open conformation resulting from one substantial rotation between the capping (CAP) and the catalytic (CAT) modules. The junction between these two modules is a five-residue loop, the hinge, whose flexibility permits the opening/closing of the enzyme and the entry of DNA. We identified a highly conserved tyrosine near the hinge as mediating the transition from the open to closed conformation upon DNA binding. Directed mutagenesis confirmed the importance of the hinge flexibility, and linked the enzyme dynamics with sensitivity to camptothecin, a TOP1 inhibitor targeting the TOP1 enzyme catalytic site in the closed conformation.
Subject(s)
DNA Topoisomerases, Type I/chemistry , DNA Topoisomerases, Type I/metabolism , Camptothecin/pharmacology , Catalytic Domain , Crystallography, X-Ray , DNA/chemistry , DNA/metabolism , DNA Damage , DNA Repair , DNA Topoisomerases, Type I/genetics , DNA-Binding Proteins , Humans , Models, Molecular , Protein Conformation , Sequence AlignmentABSTRACT
The present study demonstrates how TOP3B is involved in resolving R-loops. We observed elevated R-loops in TOP3B knockout cells (TOP3BKO), which are suppressed by TOP3B transfection. R-loop-inducing agents, the topoisomerase I inhibitor camptothecin, and the splicing inhibitor pladienolide-B also induce higher R-loops in TOP3BKO cells. Camptothecin- and pladienolide-B-induced R-loops are concurrent with the induction of TOP3B cleavage complexes (TOP3Bccs). RNA/DNA hybrid IP-western blotting show that TOP3B is physically associated with R-loops. Biochemical assays using recombinant TOP3B and oligonucleotides mimicking R-loops show that TOP3B cleaves the single-stranded DNA displaced by the R-loop RNA-DNA duplex. IP-mass spectrometry and IP-western experiments reveal that TOP3B interacts with the R-loop helicase DDX5 independently of TDRD3. Finally, we demonstrate that DDX5 and TOP3B are epistatic in resolving R-loops in a pathway parallel with senataxin. We propose a decatenation model for R-loop resolution by TOP3B-DDX5 protecting cells from R-loop-induced damage.