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1.
Bioorg Med Chem ; 52: 116500, 2021 12 15.
Article in English | MEDLINE | ID: mdl-34801826

ABSTRACT

The accumulation of epigenetic alterations is one of the major causes of tumorigenesis. Aberrant DNA methylation patterns cause genome instability and silencing of tumor suppressor genes in various types of tumors. Therefore, drugs that target DNA methylation-regulating factors have great potential for cancer therapy. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) is an essential factor for DNA methylation maintenance. UHRF1 is overexpressed in various cancer cells and down-regulation of UHRF1 in these cells reactivates the expression of tumor suppressor genes, thus UHRF1 is a promising target for cancer therapy. We have previously shown that interaction between the tandem Tudor domain (TTD) of UHRF1 and DNA ligase 1 (LIG1) di/trimethylated on Lys126 plays a key role in the recruitment of UHRF1 to replication sites and replication-coupled DNA methylation maintenance. An arginine binding cavity (Arg-binding cavity) of the TTD is essential for LIG1 interaction, thus the development of inhibitors that target the Arg-binding cavity could potentially repress UHRF1 function in cancer cells. To develop such an inhibitor, we performed in silico screening using not only static but also dynamic metrics based on all-atom molecular dynamics simulations, resulting in efficient identification of 5-amino-2,4-dimethylpyridine (5A-DMP) as a novel TTD-binding compound. Crystal structure of the TTD in complex with 5A-DMP revealed that the compound stably bound to the Arg-binding cavity of the TTD. Furthermore, 5A-DMP inhibits the full-length UHRF1:LIG1 interaction in Xenopus egg extracts. Our study uncovers a UHRF1 inhibitor which can be the basis of future experiments for cancer therapy.


Subject(s)
CCAAT-Enhancer-Binding Proteins/antagonists & inhibitors , DNA Ligase ATP/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Molecular Dynamics Simulation , Pyridines/pharmacology , Ubiquitin-Protein Ligases/antagonists & inhibitors , Animals , CCAAT-Enhancer-Binding Proteins/genetics , CCAAT-Enhancer-Binding Proteins/metabolism , Cell Line, Tumor , DNA Ligase ATP/metabolism , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemistry , Humans , Molecular Structure , Pyridines/chemistry , Structure-Activity Relationship , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Xenopus
2.
Int J Radiat Oncol Biol Phys ; 111(2): 515-527, 2021 10 01.
Article in English | MEDLINE | ID: mdl-34044093

ABSTRACT

PURPOSE: Artemis and DNA Ligase IV are 2 critical elements in the nonhomologous end joining pathway of DNA repair, acting as the nuclease and DNA ligase, respectively. Enhanced cellular radiosensitivity by inhibition of either protein contributes to a promising approach to develop molecular targeted radiosensitizers. The interaction between Artemis and DNA Ligase IV is required for the activation of Artemis as nuclease at 3'overhang DNA; thus, we aim to generate an inhibitory peptide targeting the interaction between Artemis and DNA Ligase IV for novel radiosensitizer development. METHODS AND MATERIALS: We synthesized the peptide BAL, which consists of the interaction residues of Artemis to DNA Ligase IV. The radiosensitization effect of BAL was evaluated by colony formation assay. The effects of BAL on radiation-induced DNA repair were evaluated with Western blotting and immunofluorescence. The effects of BAL on cell proliferation, cell cycle arrest, and cell apoptosis were assessed via CCK-8 and flow cytometry assays. The potential synergistic effects of BAL and irradiation in vivo were investigated in a xenograft mouse model. RESULTS: The generated peptide BAL blocking the interaction between Artemis and DNA Ligase IV significantly enhanced the radiosensitivity of GBC-SD and HeLa cell lines. BAL prolonged DNA repair after irradiation; BAL and irradiation showed synergistic effects on cell proliferation, cell cycle, and cell apoptosis, and these functions are all DNA Ligase IV-related. Finally, we confirmed the endogenous radiosensitization effect of BAL in a xenograft mouse model. CONCLUSIONS: The inhibitory peptide BAL targeting the binding of Artemis and DNA Ligase IV successfully functions as a novel radiosensitizer that delays DNA repair and synergizes with irradiation to inhibit cell proliferation, induce cell cycle arrest, and promote cell apoptosis.


Subject(s)
DNA Ligase ATP/metabolism , DNA Repair/drug effects , DNA-Binding Proteins/metabolism , Endonucleases/metabolism , Neoplasms/radiotherapy , Peptides/pharmacology , Radiation-Sensitizing Agents/pharmacology , Animals , Apoptosis/drug effects , Apoptosis/radiation effects , Cell Cycle Checkpoints/drug effects , Cell Cycle Checkpoints/radiation effects , DNA Ligase ATP/antagonists & inhibitors , DNA-Binding Proteins/antagonists & inhibitors , Endonucleases/antagonists & inhibitors , HeLa Cells , Humans , Male , Mice , Neoplasms/pathology
3.
Cancer Rep (Hoboken) ; 4(3): e1341, 2021 06.
Article in English | MEDLINE | ID: mdl-33496064

ABSTRACT

BACKGROUND: DNA double-strand breaks (DSBs) are harmful to the cell as it could lead to genomic instability and cell death when left unrepaired. Homologous recombination and nonhomologous end-joining (NHEJ) are two major DSB repair pathways, responsible for ensuring genome integrity in mammals. There have been multiple efforts using small molecule inhibitors to target these DNA repair pathways in cancers. SCR7 is a very well-studied anticancer molecule that blocks NHEJ by targeting one of the critical enzymes, Ligase IV. RECENT FINDINGS: In this review, we have highlighted the anticancer effects of SCR7 as a single agent and in combination with other chemotherapeutic agents and radiation. SCR7 blocked NHEJ effectively both in vitro and ex vivo. SCR7 has been used for biochemical studies like chromosomal territory resetting and in understanding the role of repair proteins in cell cycle phases. Various forms of SCR7 and its derivatives are discussed. SCR7 is also used as a potent biochemical inhibitor of NHEJ, which has found its application in improving genome editing using a CRISPR-Cas system. CONCLUSION: SCR7 is a potent NHEJ inhibitor with unique properties and wide applications as an anticancer agent. Most importantly, SCR7 has become a handy aid for improving genome editing across different model systems.


Subject(s)
DNA Breaks, Double-Stranded , DNA End-Joining Repair/drug effects , Neoplasms/drug therapy , Pyrimidines/pharmacology , Schiff Bases/pharmacology , Animals , CRISPR-Cas Systems/genetics , Cell Line, Tumor , DNA Ligase ATP/antagonists & inhibitors , DNA Ligase ATP/metabolism , Disease Models, Animal , Gene Editing/methods , Humans , Mice , Neoplasms/genetics , Pyrimidines/therapeutic use , Schiff Bases/therapeutic use
4.
Bioorg Med Chem Lett ; 36: 127787, 2021 03 15.
Article in English | MEDLINE | ID: mdl-33460740

ABSTRACT

SmltD is an ATP-dependent ligase that catalyzes the condensation of UDP-MurNAc-l-Ala and l-Glu to form UDP-MurNAc-l-Ala-l-Glu, in the newly discovered peptidoglycan biosynthesis pathway of a Gram-negative multiple-drug-resistant pathogen, Stenotrophomonas maltophilia. Phytochemical investigation of the 70% ethanol extract from Woodfordia fruticosa flowers collected in Myanmar led to the identification of anti-SmltD active flavonoids, kaempferol 3-O-(6''-galloyl)-ß-d-glucopyranoside (1), astragalin (2), and juglalin (3). Among them, 1 showed the most potent SmltD inhibitory activity. An enzyme steady-state kinetic study revealed that 1 exerted competitive inhibition with respect to ATP. The results of this study provided an attractive foundation for the further development of novel inhibitors of SmltD.


Subject(s)
DNA Ligase ATP/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Peptidoglycan/biosynthesis , Woodfordia/chemistry , DNA Ligase ATP/metabolism , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/isolation & purification , Flavonoids/chemistry , Flavonoids/isolation & purification , Molecular Structure , Peptidoglycan/chemistry , Stenotrophomonas maltophilia/enzymology , Structure-Activity Relationship
5.
Int J Mol Sci ; 21(21)2020 Nov 05.
Article in English | MEDLINE | ID: mdl-33167404

ABSTRACT

Poly(ADP-ribosyl)polymerase (PARP) synthesizes poly(ADP-ribose) (PAR), which is anchored to proteins. PAR facilitates multiprotein complexes' assembly. Nuclear PAR affects chromatin's structure and functions, including transcriptional regulation. In response to stress, particularly genotoxic stress, PARP activation facilitates DNA damage repair. The PARP inhibitor Olaparib (OLA) displays synthetic lethality with mutated homologous recombination proteins (BRCA-1/2), base excision repair proteins (XRCC1, Polß), and canonical nonhomologous end joining (LigIV). However, the limits of synthetic lethality are not clear. On one hand, it is unknown whether any limiting factor of homologous recombination can be a synthetic PARP lethality partner. On the other hand, some BRCA-mutated patients are not responsive to OLA for still unknown reasons. In an effort to help delineate the boundaries of synthetic lethality, we have induced DNA damage in VERO cells with the radiomimetic chemotherapeutic agent bleomycin (BLEO). A VERO subpopulation was resistant to BLEO, BLEO + OLA, and BLEO + OLA + ATM inhibitor KU55933 + DNA-PK inhibitor KU-0060648 + LigIV inhibitor SCR7 pyrazine. Regarding the mechanism(s) behind the resistance and lack of synthetic lethality, some hypotheses have been discarded and alternative hypotheses are suggested.


Subject(s)
Bleomycin/pharmacology , Chromones/pharmacology , Morpholines/pharmacology , Phthalazines/pharmacology , Piperazines/pharmacology , Pyrimidines/pharmacology , Pyrones/pharmacology , Schiff Bases/pharmacology , Thiophenes/pharmacology , Animals , Antineoplastic Agents/pharmacology , Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors , Chlorocebus aethiops , DNA Ligase ATP/antagonists & inhibitors , DNA Repair/drug effects , DNA-Activated Protein Kinase/antagonists & inhibitors , Drug Combinations , Drug Resistance, Neoplasm/drug effects , Drug Synergism , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Vero Cells
6.
Biochem Biophys Res Commun ; 533(3): 449-457, 2020 12 10.
Article in English | MEDLINE | ID: mdl-32972746

ABSTRACT

CRISPR/Cas9-mediated gene knock-in in in vivo neurons using in utero electroporation is a powerful technique, but the knock-in efficiency is generally low. We previously demonstrated that co-transfection with RAD51, a key molecule of the initial step of homology-directed repair (HDR), expression vector increased EGFP knock-in efficiency in the ß-actin site up to 2.5-fold in the pyramidal neurons in layer 2/3 of the somatosensory cortex of mouse brain. To further improve the efficiency, we examined the effect of inhibition of DNA ligase IV (LIG4) that is an essential molecule for non-homologous end joining (NHEJ). Co-transfection with small hairpin RNA for LIG4 (shlig4) expression vector increased the EGFP knock-in efficiency in the ß-actin site up to 3.6-fold compared to the condition without shlig4. RAD51 and shlig4 expression vector co-transfection further increased the knock-in efficiency up to 4.7-fold of the control condition. These results suggest that the inhibition of LIG4 is more effective than RAD51 overexpression, and it enhances the effect of RAD51 overexpression on HDR-mediated gene knock-in in vivo neurons.


Subject(s)
Brain/metabolism , CRISPR-Cas Systems , DNA Ligase ATP/antagonists & inhibitors , Gene Knock-In Techniques/methods , Neurons/metabolism , Animals , Cells, Cultured , DNA Ligase ATP/genetics , Electroporation , Green Fluorescent Proteins/genetics , Mice , Mice, Inbred C57BL , Neurons/cytology , Neurons/physiology , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Recombinational DNA Repair , Transfection
7.
Gene ; 763: 144997, 2020 Dec 30.
Article in English | MEDLINE | ID: mdl-32783992

ABSTRACT

The CRISPR-Cas system currently stands as one of the best multifaceted tools for site-specific genome engineering in mammals. An important aspect of research in this field focusses on improving the specificity and efficacy of precise genome editing in multiple model systems. The cornerstone of this mini-review is one of the extensively investigated small molecule inhibitor, SCR7, which abrogates NHEJ, a Ligase IV-dependent DSB repair pathway, thus guiding integration of the foreign DNA fragment via the more precise homology directed repair during genome editing. One of our recent studies sheds light on properties of different forms of SCR7. Here, we give a succinct account on the use of SCR7 and its different forms in CRISPR-Cas system, highlighting their chemical properties and biological relevance as potent efficiency-enhancing CRISPR tools.


Subject(s)
CRISPR-Cas Systems , Enzyme Inhibitors/pharmacology , Gene Editing/methods , Pyrimidines/pharmacology , Recombinational DNA Repair/drug effects , Schiff Bases/pharmacology , Animals , DNA Ligase ATP/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Humans , Pyrimidines/chemistry , Schiff Bases/chemistry
8.
Mol Carcinog ; 59(6): 618-628, 2020 06.
Article in English | MEDLINE | ID: mdl-32189406

ABSTRACT

Targeting DNA repair with small-molecule inhibitors is an attractive strategy for cancer therapy. Majority of DNA double-strand breaks in mammalian cells are repaired through nonhomologous end-joining (NHEJ). It has been shown that small-molecule inhibitors of NHEJ can block efficient repair inside cancer cells, leading to cell death. Previously, we have reported that SCR7, an inhibitor of NHEJ can induce tumor regression in mice. Later studies have shown that different forms of SCR7 can inhibit DNA end-joining in Ligase IV-dependent manner. Recently, we have derivatized SCR7 by introducing spiro ring into core structure. Here, we report the identification of a novel inhibitor of NHEJ, named SCR130 with 20-fold higher efficacy in inducing cytotoxicity in cancer cell lines. SCR130 inhibited DNA end-joining catalyzed by rat tissue extract. Specificity analysis revealed that while SCR130 was specific to Ligase IV, it showed minimal or no effect on Ligase III and Ligase I mediated joining. Importantly, SCR130 exhibited the least cytotoxicity in Ligase IV-null cell line as compared with wild type, confirming Ligase IV-specificity. Furthermore, we demonstrate that SCR130 can potentiate the effect of radiation in cancer cells when used in combination with γ-radiation. Various cellular assays in conjunction with Western blot analysis revealed that treatment with SCR130 led to loss of mitochondrial membrane potential leading to cell death by activating both intrinsic and extrinsic pathways of apoptosis. Thus, we describe a novel inhibitor of NHEJ with higher efficacy and may have the potential to be developed as cancer therapeutic.


Subject(s)
Antineoplastic Agents/pharmacology , Cell Death , DNA Breaks, Double-Stranded/drug effects , DNA End-Joining Repair/drug effects , DNA Ligase ATP/antagonists & inhibitors , Pyrimidines/pharmacology , Schiff Bases/pharmacology , Small Molecule Libraries/pharmacology , Animals , HeLa Cells , Humans , Mice , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/pathology , Rats
9.
Mutagenesis ; 35(1): 51-60, 2020 02 13.
Article in English | MEDLINE | ID: mdl-31630206

ABSTRACT

The joining of interruptions in the phosphodiester backbone of DNA is critical to maintain genome stability. These breaks, which are generated as part of normal DNA transactions, such as DNA replication, V(D)J recombination and meiotic recombination as well as directly by DNA damage or due to DNA damage removal, are ultimately sealed by one of three human DNA ligases. DNA ligases I, III and IV each function in the nucleus whereas DNA ligase III is the sole enzyme in mitochondria. While the identification of specific protein partners and the phenotypes caused either by genetic or chemical inactivation have provided insights into the cellular functions of the DNA ligases and evidence for significant functional overlap in nuclear DNA replication and repair, different results have been obtained with mouse and human cells, indicating species-specific differences in the relative contributions of the DNA ligases. Inherited mutations in the human LIG1 and LIG4 genes that result in the generation of polypeptides with partial activity have been identified as the causative factors in rare DNA ligase deficiency syndromes that share a common clinical symptom, immunodeficiency. In the case of DNA ligase IV, the immunodeficiency is due to a defect in V(D)J recombination whereas the cause of the immunodeficiency due to DNA ligase I deficiency is not known. Overexpression of each of the DNA ligases has been observed in cancers. For DNA ligase I, this reflects increased proliferation. Elevated levels of DNA ligase III indicate an increased dependence on an alternative non-homologous end-joining pathway for the repair of DNA double-strand breaks whereas elevated level of DNA ligase IV confer radioresistance due to increased repair of DNA double-strand breaks by the major non-homologous end-joining pathway. Efforts to determine the potential of DNA ligase inhibitors as cancer therapeutics are on-going in preclinical cancer models.


Subject(s)
DNA Ligase ATP/genetics , DNA Ligase ATP/metabolism , Disease/genetics , Animals , DNA Damage , DNA Ligase ATP/antagonists & inhibitors , DNA Repair , Humans , Immunologic Deficiency Syndromes/etiology , Mice , Neoplasms/genetics , Poly-ADP-Ribose Binding Proteins/genetics , Poly-ADP-Ribose Binding Proteins/metabolism
10.
Biochimie ; 168: 241-250, 2020 Jan.
Article in English | MEDLINE | ID: mdl-31756402

ABSTRACT

Prostate cancer (PCa) progression is characterized by increased expression and transcriptional activity of the androgen receptor (AR). In the advanced stages of prostate cancer, AR significantly upregulates the expression of genes involved in DNA repair. Upregulation of expression for base excision repair (BER) related genes is associated with poor patient survival. Thus, inhibition of the BER pathway may prove to be an effective therapy for prostate cancer. Using a high throughput BER capacity screening assay, we sought to identify BER inhibitors that can synergize with castration therapy. An FDA-approved drug library was screened to identify inhibitors of BER using a fluorescence-based assay suitable for HTS. A gel-based secondary assay confirmed the reduction of BER capacity by compounds identified in the primary screen. Five compounds were then selected for further testing in the independently derived, androgen-dependent prostate cancer cell lines, LNCaP and LAPC4, and in the nonmalignant prostate derived cell lines PNT1A and RWPE1. Further analysis led to the identification of a lead compound, natamycin, as an effective inhibitor of key BER enzymes DNA polymerase ß (pol ß) and DNA Ligase I (LIG I). Natamycin significantly inhibited proliferation of PCa cells in an androgen depleted environment at 1 µM concentration, however, growth inhibition did not occur with nonmalignant prostate cell lines, suggesting that BER inhibition may improve efficacy of the castration therapies.


Subject(s)
Cell Proliferation/drug effects , DNA Ligase ATP/antagonists & inhibitors , DNA Polymerase beta/antagonists & inhibitors , DNA Repair/drug effects , Natamycin/pharmacology , Prostatic Neoplasms, Castration-Resistant/drug therapy , Cell Line, Tumor , Databases, Pharmaceutical , Humans , Male
11.
Eur J Med Chem ; 182: 111657, 2019 Nov 15.
Article in English | MEDLINE | ID: mdl-31499361

ABSTRACT

The emergence of drug resistance, coupled with the issue of low tumor selectivity and toxicity is a major pitfall in cancer chemotherapy. It has necessitated the urgent need for the discovery of less toxic and more potent new anti-cancer pharmaceuticals, which target the interactive mechanisms involved in division and metastasis of cancer cells. Human DNA ligase I (hligI) plays an important role in DNA replication by linking Okazaki fragments on the lagging strand of DNA, and also participates in DNA damage repair processes. Dysregulation of the functioning of such ligases can severely impact DNA replication and repair pathways events that are generally targeted in cancer treatment. Although, several human DNA ligase inhibitors have been reported in the literature but unfortunately not a single inhibitor is currently being used in cancer chemotherapy. Results of pre-clinical studies also support the fact that human DNA ligases are an attractive target for the development of new anticancer agents which work by the selective inhibition of rapidly proliferating cancer cells. In this manuscript, we discuss, in brief, the structure, synthesis, structure-activity-relationship (SAR) and anticancer activity of recently reported hLigI inhibitors.


Subject(s)
Antineoplastic Agents/pharmacology , DNA Ligase ATP/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Neoplasms/drug therapy , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , DNA Ligase ATP/metabolism , Enzyme Inhibitors/chemistry , Humans , Neoplasms/metabolism , Neoplasms/pathology , Structure-Activity Relationship
12.
J Pharm Biomed Anal ; 162: 205-214, 2019 Jan 05.
Article in English | MEDLINE | ID: mdl-30265980

ABSTRACT

S012-1332 is the first DNA ligase I inhibitor that demonstrated in vivo anti-breast cancer activity. The present study aimed to assess the in vivo pharmacokinetics of S012-1332 in rats and interpret them with in vitro findings. A sensitive and selective liquid chromatography-tandem mass spectrometry bioanalytical method was developed and validated to determine S012-1332. Following oral administration, the absolute bioavailability was 7.04%. The absorption was prolonged which can be explained by low solubility in simulated gastric fluid and several folds higher solubility in simulated intestinal fluid. The effective permeability across the intestinal membrane in in situ single pass perfusion study for S012-1332 was 5.58 ± 1.83 * 10-5 cm/sec compared to 5.99 ± 0.65 * 10-5 cm/sec for carbamazepine, with no significant difference, indicating S012-1332 has high permeability. It was rapidly partitioning into plasma in blood, where it was stable. Plasma protein binding was moderate which may have attributed to the rapid distribution out of the vascular compartment. The pharmacokinetics of S012-1332 was characterized by extensive clearance as seen with rat liver and intestinal microsomes. In vitro results elucidate the in vivo pharmacokinetic data. These findings provide crucial information for further development of S012-1332 as anti-breast cancer agent.


Subject(s)
Antineoplastic Agents/pharmacokinetics , DNA Ligase ATP/antagonists & inhibitors , Enzyme Inhibitors/pharmacokinetics , Administration, Oral , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/blood , Biological Availability , Chromatography, Liquid , DNA Ligase ATP/metabolism , Drug Stability , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/blood , Inactivation, Metabolic , Intestinal Absorption , Intestinal Mucosa/metabolism , Male , Microsomes, Liver/metabolism , Permeability , Protein Binding , Rats, Sprague-Dawley , Reproducibility of Results , Solubility , Spectrometry, Mass, Electrospray Ionization , Tandem Mass Spectrometry
13.
Cell Death Dis ; 8(12): 3208, 2017 12 13.
Article in English | MEDLINE | ID: mdl-29238067

ABSTRACT

Neuroblastoma is a cancer of neural crest stem cell (NCSC) lineage. Signaling pathways that regulate NCSC differentiation have been implicated in neuroblastoma tumorigenesis. This is exemplified by MYCN oncogene targets that balance proliferation, differentiation, and cell death similarly in normal NCSC and in high-risk neuroblastoma. Our previous work discovered a survival mechanism by which MYCN-amplified neuroblastoma circumvents cell death by upregulating components of the error-prone non-canonical alternative nonhomologous end-joining (alt-NHEJ) DNA repair pathway. Similar to proliferating stem cells, high-risk neuroblastoma cells have enhanced DNA repair capacity, overcoming DNA damage with higher repair efficiency than somatic cells. Adequate DNA maintenance is required for lineage protection as stem cells proliferate and during tumor progression to overcome oncogene-induced replication stress. On this basis, we hypothesized that alt-NHEJ overexpression in neuroblastoma is a cancer cell survival mechanism that originates from DNA repair systems of NCSC, the presumed progenitor cell of origin. A human NCSC model was generated in which inducible MYCN triggered an immortalized phenotype capable of forming metastatic neuroectodermal tumors in mice, resembling human neuroblastoma. Critical alt-NHEJ components (DNA Ligase III, DNA Ligase I, and Poly [ADP-ribose polymerase 1]) were highly expressed in normal early NCSC, and decreased as cells became terminally differentiated. Constitutive MYCN expression maintained high alt-NHEJ protein expression, preserving the expression pattern of the immature neural phenotype. siRNA knockdown of alt-NHEJ components reversed MYCN effects on NCSC proliferation, invasion, and migration. DNA Ligase III, Ligase I, and PARP1 silencing significantly decreased neuroblastoma markers expression (TH, Phox2b, and TRKB). These results utilized the first human NCSC model of neuroblastoma to uncover an important link between MYCN and alt-NHEJ expression in developmental tumor initiation, setting precedence to investigate alt-NHEJ repair mechanics in neuroblastoma DNA maintenance.


Subject(s)
Cell Transformation, Neoplastic/genetics , DNA End-Joining Repair , Gene Expression Regulation, Neoplastic , N-Myc Proto-Oncogene Protein/genetics , Neural Stem Cells/metabolism , Neuroblastoma/genetics , Animals , Cell Differentiation , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , DNA Ligase ATP/antagonists & inhibitors , DNA Ligase ATP/genetics , DNA Ligase ATP/metabolism , Disease Models, Animal , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/genetics , Isoenzymes/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mice , N-Myc Proto-Oncogene Protein/metabolism , Neural Crest/metabolism , Neural Crest/pathology , Neural Stem Cells/pathology , Neuroblastoma/metabolism , Neuroblastoma/pathology , Poly (ADP-Ribose) Polymerase-1/antagonists & inhibitors , Poly (ADP-Ribose) Polymerase-1/genetics , Poly (ADP-Ribose) Polymerase-1/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptor, trkB/genetics , Receptor, trkB/metabolism , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolism , Transgenes
14.
PLoS Pathog ; 13(12): e1006784, 2017 12.
Article in English | MEDLINE | ID: mdl-29287110

ABSTRACT

Hepadnavirus covalently closed circular (ccc) DNA is the bona fide viral transcription template, which plays a pivotal role in viral infection and persistence. Upon infection, the non-replicative cccDNA is converted from the incoming and de novo synthesized viral genomic relaxed circular (rc) DNA, presumably through employment of the host cell's DNA repair mechanisms in the nucleus. The conversion of rcDNA into cccDNA requires preparation of the extremities at the nick/gap regions of rcDNA for strand ligation. After screening 107 cellular DNA repair genes, we herein report that the cellular DNA ligase (LIG) 1 and 3 play a critical role in cccDNA formation. Ligase inhibitors or functional knock down/out of LIG1/3 significantly reduced cccDNA production in an in vitro cccDNA formation assay, and in cccDNA-producing cells without direct effect on viral core DNA replication. In addition, transcomplementation of LIG1/3 in the corresponding knock-out or knock-down cells was able to restore cccDNA formation. Furthermore, LIG4, a component in non-homologous end joining DNA repair apparatus, was found to be responsible for cccDNA formation from the viral double stranded linear (dsl) DNA, but not rcDNA. In conclusion, we demonstrate that hepadnaviruses utilize the whole spectrum of host DNA ligases for cccDNA formation, which sheds light on a coherent molecular pathway of cccDNA biosynthesis, as well as the development of novel antiviral strategies for treatment of hepatitis B.


Subject(s)
DNA Ligases/metabolism , DNA, Circular/biosynthesis , DNA, Viral/biosynthesis , Hepadnaviridae/metabolism , Cell Line , DNA Ligase ATP/antagonists & inhibitors , DNA Ligase ATP/genetics , DNA Ligase ATP/metabolism , DNA Ligases/antagonists & inhibitors , DNA Ligases/genetics , DNA Repair/genetics , Gene Knockdown Techniques , Gene Knockout Techniques , HEK293 Cells , Hep G2 Cells , Hepadnaviridae/genetics , Hepadnaviridae/pathogenicity , Hepatitis B virus/genetics , Hepatitis B virus/metabolism , Hepatitis B virus/pathogenicity , Hepatocytes/metabolism , Hepatocytes/virology , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/physiology , Humans , Metabolic Networks and Pathways , Poly-ADP-Ribose Binding Proteins/antagonists & inhibitors , Poly-ADP-Ribose Binding Proteins/genetics , Poly-ADP-Ribose Binding Proteins/metabolism
15.
DNA Repair (Amst) ; 60: 29-39, 2017 12.
Article in English | MEDLINE | ID: mdl-29078112

ABSTRACT

In human cells, there are three genes that encode DNA ligase polypeptides with distinct but overlapping functions. Previously small molecule inhibitors of human DNA ligases were identified using a structure-based approach. Three of these inhibitors, L82, a DNA ligase I (LigI)-selective inhibitor, and L67, an inhibitor of LigI and DNA ligases III (LigIII), and L189, an inhibitor of all three human DNA ligases, have related structures that are composed of two 6-member aromatic rings separated by different linkers. Here we have performed a structure-activity analysis to identify determinants of activity and selectivity. The majority of the LigI-selective inhibitors had a pyridazine ring whereas the LigI/III- and LigIII-selective inhibitors did not. In addition, the aromatic rings in LigI-selective inhibitors had either arylhydrazone or acylhydrazone, but not vinyl linkers. Among the LigI-selective inhibitors, L82-G17 exhibited increased activity against and selectivity for LigI compared with L82. Notably. L82-G17 is an uncompetitive inhibitor of the third step of the ligation reaction, phosphodiester bond formation. Cells expressing LigI were more sensitive to L82-G17 than isogenic LIG1 null cells. Furthermore, cells lacking nuclear LigIIIα, which can substitute for LigI in DNA replication, were also more sensitive to L82-G17 than isogenic parental cells. Together, our results demonstrate that L82-G17 is a LigI-selective inhibitor with utility as a probe of the catalytic activity and cellular functions of LigI and provide a framework for the future design of DNA ligase inhibitors.


Subject(s)
DNA Ligase ATP/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Pyridazines/pharmacology , DNA Ligase ATP/metabolism , DNA Replication , Humans , Kinetics , Pyridazines/chemical synthesis , Structure-Activity Relationship
16.
Sci Rep ; 7(1): 10715, 2017 09 06.
Article in English | MEDLINE | ID: mdl-28878282

ABSTRACT

Existing cancer therapies are often associated with drug resistance and toxicity, which results in poor prognosis and recurrence of cancer. This necessitates the identification and development of novel therapeutics against existing as well as novel cellular targets. In this study, a novel class of Benzocoumarin-Stilbene hybrid molecules were synthesized and evaluated for their antiproliferative activity against various cancer cell lines followed by in vivo antitumor activity in a mouse model of cancer. The most promising molecule among the series, i.e. compound (E)-4-(3,5-dimethoxystyryl)-2H-benzo[h]chromen-2-one (19) showed maximum antiproliferative activity in breast cancer cell lines (MDA-MB-231 and 4T1) and decreased the tumor size in the in-vivo 4T1 cell-induced orthotopic syngeneic mouse breast cancer model. The mechanistic studies of compound 19 by various biochemical, cell biology and biophysical approaches suggest that the compound binds to and inhibits the human DNA ligase I enzyme activity that might be the cause for significant reduction in tumor growth and may constitute a promising next-generation therapy against breast cancers.


Subject(s)
Anthracenes , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , DNA Ligase ATP/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Stilbenes , Animals , Anthracenes/chemistry , Apoptosis/drug effects , Breast Neoplasms , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , DNA Damage , Disease Models, Animal , Female , Humans , Mice , Molecular Structure , Signal Transduction/drug effects , Stilbenes/chemistry , Xenograft Model Antitumor Assays
17.
Mol Carcinog ; 56(2): 550-566, 2017 02.
Article in English | MEDLINE | ID: mdl-27312791

ABSTRACT

The terminal step of ligation of single and/or double-strand breaks during physiological processes such as DNA replication, repair and recombination requires participation of DNA ligases in all mammals. DNA Ligase I has been well characterised to play vital roles during these processes. Considering the indispensable role of DNA Ligase I, a therapeutic strategy to impede proliferation of cancer cells is by using specific small molecule inhibitors against it. In the present study, we have designed and chemically synthesised putative DNA Ligase I inhibitors. Based on various biochemical and biophysical screening approaches, we identify two prospective DNA Ligase I inhibitors, SCR17 and SCR21. Both the inhibitors blocked ligation of nicks on DNA in a concentration-dependent manner, when catalysed by cell-free extracts or purified Ligase I. Docking studies in conjunction with biolayer interferometry and gel shift assays revealed that both SCR17 and SCR21 can bind to Ligase I, particularly to the DNA Binding Domain of Ligase I with KD values in nanomolar range. The inhibitors did not show significant affinity towards DNA Ligase III and DNA Ligase IV. Further, addition of Ligase I could restore the joining, when the inhibitors were treated with testicular cell-free extracts. Ex vivo studies using multiple assays showed that even though cell death was limited in the presence of inhibitors in cancer cells, their proliferation was compromised. Hence, we identify two promising DNA Ligase I inhibitors, which can be used in biochemical and cellular assays, and could be further modified and optimised to target cancer cells. © 2016 Wiley Periodicals, Inc.


Subject(s)
DNA Ligase ATP/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Animals , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , DNA Ligase ATP/chemistry , DNA Ligase ATP/metabolism , DNA Replication/drug effects , Drug Design , HEK293 Cells , Humans , Male , Molecular Docking Simulation , Rats , Rats, Wistar
18.
DNA Repair (Amst) ; 43: 18-23, 2016 07.
Article in English | MEDLINE | ID: mdl-27235626

ABSTRACT

DNA ligases are attractive therapeutics because of their involvement in completing the repair of almost all types of DNA damage. A series of DNA ligase inhibitors with differing selectivity for the three human DNA ligases were identified using a structure-based approach with one of these inhibitors being used to inhibit abnormal DNA ligase IIIα-dependent repair of DNA double-strand breaks (DSB)s in breast cancer, neuroblastoma and leukemia cell lines. Raghavan and colleagues reported the characterization of a derivative of one of the previously identified DNA ligase inhibitors, which they called SCR7 (designated SCR7-R in our experiments using SCR7). SCR7 appeared to show increased selectivity for DNA ligase IV, inhibit the repair of DSBs by the DNA ligase IV-dependent non-homologous end-joining (NHEJ) pathway, reduce tumor growth, and increase the efficacy of DSB-inducing therapeutic modalities in mouse xenografts. In attempting to synthesize SCR7, we encountered problems with the synthesis procedures and discovered discrepancies in its reported structure. We determined the structure of a sample of SCR7 and a related compound, SCR7-G, that is the major product generated by the published synthesis procedure for SCR7. We also found that SCR7-G has the same structure as the compound (SCR7-X) available from a commercial vendor (XcessBio). The various SCR7 preparations had similar activity in DNA ligation assay assays, exhibiting greater activity against DNA ligases I and III than DNA ligase IV. Furthermore, SCR7-R failed to inhibit DNA ligase IV-dependent V(D)J recombination in a cell-based assay. Based on our results, we conclude that SCR7 and the SCR7 derivatives are neither selective nor potent inhibitors of DNA ligase IV.


Subject(s)
Antineoplastic Agents/pharmacology , DNA Ligase ATP/genetics , DNA/genetics , Enzyme Inhibitors/pharmacology , Pyrimidines/pharmacology , Schiff Bases/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Cell Line, Tumor , Cell Survival/drug effects , DNA/metabolism , DNA Breaks, Double-Stranded , DNA End-Joining Repair/drug effects , DNA Ligase ATP/antagonists & inhibitors , DNA Ligase ATP/metabolism , Enzyme Inhibitors/chemical synthesis , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Cells/pathology , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Leukocytes/drug effects , Leukocytes/metabolism , Leukocytes/pathology , Mice , Neurons/drug effects , Neurons/metabolism , Neurons/pathology , Pyrimidines/chemical synthesis , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Schiff Bases/chemical synthesis , Substrate Specificity , Tumor Burden/drug effects , V(D)J Recombination/drug effects , Xenograft Model Antitumor Assays
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