RESUMEN
In some organisms, the replication of G-quadruplex (G4) structures is supported by the Rev1 DNA polymerase. We previously showed that residues in the insert-2 motif of human Rev1 (hRev1) increased the affinity of the enzyme for G4 DNA and mediated suppression of mutagenic replication near G4 motifs. We have now investigated the conservation of G4-selective properties in Rev1 from other species. We compared Rev1 from Danio rerio (zRev1), Saccharomyces cerevisiae (yRev1), and Leishmania donovani (lRev1) with hRev1, including an insert-2 mutant form of hRev1 (E466A/Y470A or EY). We found that zRev1 retained all of the G4-selective prowess of the human enzyme, but there was a marked attenuation of G4 binding affinity for the EY hRev1 mutant and the two Rev1 proteins lacking insert-2 (yRev1 and lRev1). Perhaps most strikingly, we found that insert-2 was important for disruption of the G4 structure and optimal stimulation of processive DNA synthesis across the guanine-rich motif by DNA polymerase kappa (pol κ). Our findings have implications for how Rev1 might contribute to G4 replication in different species spanning the evolutionary tree - signaling the importance of selection for enzymes with robust G4-selective properties in organisms where these non-B DNA structures may fulfill taxa-specific physiological functions.
RESUMEN
Glioblastoma (GBM) remains the most frequently diagnosed primary malignant brain cancer in adults. Despite recent progress in understanding the biology of GBM, the clinical outcome for patients remains poor, with a median survival of approximately one year after diagnosis. One factor contributing to failure in clinical trials is the fact that traditional models used in GBM drug discovery poorly recapitulate patient tumors. Previous studies have shown that monensin (MON) analogs, namely esters and amides on C-26 were potent towards various types of cancer cell lines. In the present study we have investigated the activity of these molecules in GBM organoids, as well as in a host:tumor organoid model. Using a mini-ring cell viability assay we have identified seven analogs (IC50 = 91.5 ± 54.4-291.7 ± 68.8 nM) more potent than parent MON (IC50 = 612.6 ± 184.4 nM). Five of these compounds induced substantial DNA fragmentation in GBM organoids, suggestive of apoptotic cell death. The most active analog, compound 1, significantly reduced GBM cell migration, induced PARP degradation, diminished phosphorylation of STAT3, Akt and GSK3ß, increased É£H2AX signaling and upregulated expression of the autophagy associated marker LC3-II. To investigate the activity of MON and compound 1 in a tumor microenvironment, we developed human cerebral organoids (COs) from human induced pluripotent stem cells (iPSCs). The COs showed features of early developing brain such as multiple neural rosettes with a proliferative zone of neural stem cells (Nestin+), neurons (TUJ1 +), primitive ventricular system (SOX2 +/Ki67 +), intermediate zone (TBR2 +) and cortical plate (MAP2 +). In order to generate host:tumor organoids, we co-cultured RFP-labeled U87MG cells with fully formed COs. Compound 1 and MON reduced U87MG tumor size in the COs after four days of treatment and induced a significant reduction of PARP expression. These findings highlight the therapeutic potential of MON analogs towards GBM and support the application of organoid models in anti-cancer drug discovery.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Células Madre Pluripotentes Inducidas , Adulto , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Glioblastoma/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Monensina/farmacología , Monensina/uso terapéutico , Organoides/metabolismo , Organoides/patología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Microambiente TumoralRESUMEN
The lipid peroxidation product malondialdehyde and the DNA peroxidation product base-propenal react with dG to generate the exocyclic adduct, M1dG. This mutagenic lesion has been found in human genomic and mitochondrial DNA. M1dG in genomic DNA is enzymatically oxidized to 6-oxo-M1dG, a lesion of currently unknown mutagenic potential. Here, we report the synthesis of an oligonucleotide containing 6-oxo-M1dG and the results of extension experiments aimed at determining the effect of the 6-oxo-M1dG lesion on the activity of human polymerase iota (hPol ι). For this purpose, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed to obtain reliable quantitative data on the utilization of poorly incorporated nucleotides. Results demonstrate that hPol ι primarily incorporates deoxycytidine triphosphate (dCTP) and thymidine triphosphate (dTTP) across from 6-oxo-M1dG with approximately equal efficiency, whereas deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) are poor substrates. Following the incorporation of a single nucleotide opposite the lesion, 6-oxo-M1dG blocks further replication by the enzyme.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Desoxiguanosina/metabolismo , Oligonucleótidos/metabolismo , Cromatografía Liquida , Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , Humanos , Estructura Molecular , Oligonucleótidos/síntesis química , Oligonucleótidos/química , Espectrometría de Masas en Tándem , ADN Polimerasa iotaRESUMEN
Li Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome caused by germline mutations in TP53. TP53 is the most common mutated gene in human cancer, occurring in 30-50% of glioblastomas (GBM). Here, we highlight a precision medicine platform to identify potential targets for a GBM patient with LFS. We used a comparative transcriptomics approach to identify genes that are uniquely overexpressed in the LFS GBM patient relative to a cancer compendium of 12,747 tumor RNA sequencing data sets, including 200 GBMs. STAT1 and STAT2 were identified as being significantly overexpressed in the LFS patient, indicating ruxolitinib, a Janus kinase 1 and 2 inhibitors, as a potential therapy. The LFS patient had the highest level of STAT1 and STAT2 expression in an institutional high-grade glioma cohort of 45 patients, further supporting the cancer compendium results. To empirically validate the comparative transcriptomics pipeline, we used a combination of adherent and organoid cell culture techniques, including ex vivo patient-derived organoids (PDOs) from four patient-derived cell lines, including the LFS patient. STAT1 and STAT2 expression levels in the four patient-derived cells correlated with levels identified in the respective parent tumors. In both adherent and organoid cultures, cells from the LFS patient were among the most sensitive to ruxolitinib compared to patient-derived cells with lower STAT1 and STAT2 expression levels. A spheroid-based drug screening assay (3D-PREDICT) was performed and used to identify further therapeutic targets. Two targeted therapies were selected for the patient of interest and resulted in radiographic disease stability. This manuscript supports the use of comparative transcriptomics to identify personalized therapeutic targets in a functional precision medicine platform for malignant brain tumors.
Asunto(s)
Glioblastoma/genética , Síndrome de Li-Fraumeni/genética , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT2/genética , Adolescente , Adulto , Niño , Femenino , Regulación Neoplásica de la Expresión Génica , Mutación de Línea Germinal/genética , Glioblastoma/complicaciones , Glioblastoma/patología , Humanos , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 1/genética , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/genética , Síndrome de Li-Fraumeni/complicaciones , Síndrome de Li-Fraumeni/patología , Masculino , Nitrilos/farmacología , Organoides/metabolismo , Medicina de Precisión , Pirazoles/farmacología , Pirimidinas/farmacología , RNA-Seq , Transcriptoma/genética , Adulto JovenRESUMEN
Glioblastoma (GBM) is highly resistant to treatment and invasion into the surrounding brain is a cancer hallmark that leads to recurrence despite surgical resection. With the emergence of precision medicine, patient-derived 3D systems are considered potentially robust GBM preclinical models. In this study, we screened a library of 22 anti-invasive compounds (i.e., NF-kB, GSK-3-B, COX-2, and tubulin inhibitors) using glioblastoma U-251 MG cell spheroids. We evaluated toxicity and invasion inhibition using a 3D Matrigel invasion assay. We next selected three compounds that inhibited invasion and screened them in patient-derived glioblastoma organoids (GBOs). We developed a platform using available macros for FIJI/ImageJ to quantify invasion from the outer margin of organoids. Our data demonstrated that a high-throughput invasion screening can be done using both an established cell line and patient-derived 3D model systems. Tubulin inhibitor compounds had the best efficacy with U-251 MG cells, however, in ex vivo patient organoids the results were highly variable. Our results indicate that the efficacy of compounds is highly related to patient intra and inter-tumor heterogeneity. These results indicate that such models can be used to evaluate personal oncology therapeutic strategies.
Asunto(s)
Bancos de Muestras Biológicas , Neoplasias Encefálicas/patología , Descubrimiento de Drogas , Glioblastoma/patología , Organoides , Medicina de Precisión , Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Línea Celular Tumoral , Descubrimiento de Drogas/métodos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Glioblastoma/tratamiento farmacológico , Humanos , Invasividad Neoplásica , Medicina de Precisión/métodos , Esferoides Celulares , Técnicas de Cultivo de TejidosRESUMEN
Breast cancer remains one of the leading cancers among women. Cancer stem cells (CSCs) are tumor-initiating cells which drive progression, metastasis, and reoccurrence of the disease. CSCs are resistant to conventional chemo- and radio-therapies and their ability to survive such treatment enables tumor reestablishment. Metastasis is the main cause of mortality in women with breast cancer, thus advances in treatment will depend on therapeutic strategies targeting CSCs. Salinomycin (SAL) is a naturally occurring polyether ionophore antibiotic known for its anticancer activity towards several types of tumor cells. In the present work, a library of 17 C1-single and C1/C20-double modified SAL analogs was screened to identify compounds with improved activity against breast CSCs. Six single- and two double-modified analogs were more potent (IC50 range of 1.1 ± 0.1-1.4 ± 0.2 µM) toward the breast cancer cell line MDA-MB-231 compared to SAL (IC50 of 4.9 ± 1.6 µM). Double-modified compound 17 was found to be more efficacious than SAL against the majority of cancer cell lines in the NCI-60 Human Tumor Cell Line Panel. Compound 17 was more potent than SAL in inhibiting cell migration and cell renewal properties of MDA-MB-231 cells, as well as inducing selective loss of the CD44+/CD24/low stem-cell-like subpopulation in both monolayer (2D) and organoid (3D) culture. The present findings highlight the therapeutic potential of SAL analogs towards breast CSCs and identify select compounds that merit further study and clinical development.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Piranos/farmacología , Antibióticos Antineoplásicos/síntesis química , Antígeno CD24 , División Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Receptores de Hialuranos/metabolismo , Células MCF-7 , Piranos/síntesis químicaRESUMEN
Expression of tryptophan 2,3-dioxygenase (TDO) is a determinant of malignancy in gliomas through kynurenine (KYN) signaling. We report that inhibition of TDO activity attenuated recovery from replication stress and increased the genotoxic effects of bis-chloroethylnitrosourea (BCNU). Activation of the Chk1 arm of the replication stress response (RSR) was reduced when TDO activity was blocked prior to BCNU treatment, whereas phosphorylation of serine 33 (pS33) on replication protein A (RPA) was enhanced-indicative of increased fork collapse. Analysis of quantitative proteomic results revealed that TDO inhibition reduced nuclear 53BP1 and sirtuin levels. We confirmed that cells lacking TDO activity exhibited elevated gamma-H2AX signal and defective recruitment of 53BP1 to chromatin following BCNU treatment, which corresponded with delayed repair of DNA breaks. Addition of exogenous KYN increased the rate of break repair. TDO inhibition diminished SIRT7 deacetylase recruitment to chromatin, which increased histone H3K18 acetylation-a key mark involved in preventing 53BP1 recruitment to sites of DNA damage. TDO inhibition also sensitized cells to ionizing radiation (IR)-induced damage, but this effect did not involve altered 53BP1 recruitment. These experiments support a model where TDO-mediated KYN signaling helps fuel a robust response to replication stress and DNA damage.
RESUMEN
We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.
Asunto(s)
Replicación del ADN , ADN/química , ADN/metabolismo , G-Cuádruplex , Nucleotidiltransferasas/química , Nucleotidiltransferasas/metabolismo , Línea Celular , ADN Polimerasa Dirigida por ADN/metabolismo , Genes myc , Humanos , Modelos Moleculares , Mutación , Motivos de Nucleótidos , Nucleotidiltransferasas/genética , Unión ProteicaRESUMEN
Garcinoic acid has been identified as an inhibitor of DNA polymerase ß (pol ß). However, no structure-activity relationship (SAR) studies of garcinoic acid as a pol ß inhibitor have been conducted, in part due to the lack of an efficient synthetic method for this natural product and its analogs. We developed an efficient semi-synthetic method for garcinoic acid and its analogs by starting from natural product δ-tocotrienol. Our preliminary SAR studies provided a valuable insight into future discovery of garcinoic acid-based pol ß inhibitors.
Asunto(s)
Benzopiranos/síntesis química , ADN Polimerasa beta/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Productos Biológicos/química , Técnicas de Química Sintética , Diseño de Fármacos , Humanos , Concentración 50 Inhibidora , Fenol/química , Relación Estructura-Actividad , Temperatura , Vitamina E/análogos & derivados , Vitamina E/químicaRESUMEN
Overexpression of human DNA polymerase kappa (hpol κ) in glioblastoma is associated with shorter survival time and resistance to the alkylating agent temozolomide (TMZ), making it an attractive target for the development of small-molecule inhibitors. We previously reported on the development and characterization of indole barbituric acid-derived (IBA) inhibitors of translesion DNA synthesis polymerases (TLS pols). We have now identified a potent and selective inhibitor of hpol κ based on the indole-aminoguanidine (IAG) chemical scaffold. The most promising IAG analogue, IAG-10, exhibited greater inhibitory action against hpol κ than any other human Y-family member, as well as pols from the A-, B-, and X-families. Inhibition of hpol κ by IAG analogues appears to proceed through a mechanism that is distinct from inhibition of hpol η based on changes in DNA binding affinity and nucleotide insertion kinetics. By way of comparison, both IAG and IBA analogues inhibited binary complex formation by hpol κ and ternary complex formation by hpol η. Decreasing the concentration of enzyme and DNA in the reaction mixture lowered the IC50 value of IAG-10 to submicromolar values, consistent with inhibition of binary complex formation for hpol κ. Chemical footprinting experiments revealed that IAG-10 binds to a cleft between the finger, little finger, and N-clasp domains on hpol κ and that this likely disrupts the interaction between the N-clasp and the TLS pol core. In cell culture, IAG-10 potentiated the antiproliferative activity and DNA damaging effects of TMZ in hpol κ-proficient cells but not in hpol κ-deficient cells, indicative of a target-dependent effect. Mutagenic replication across alkylation damage increased in hpol κ-proficient cells treated with IAG-10, while no change in mutation frequency was observed for hpol κ-deficient cells. In summary, we developed a potent and selective small-molecule inhibitor of hpol κ that takes advantage of structural features unique to this TLS enzyme to potentiate TMZ, a standard-of-care drug used in the treatment of malignant brain tumors. Furthermore, the IAG scaffold represents a new chemical space for the exploration of TLS pol inhibitors, which could prove useful as a strategy for improving patient response to genotoxic drugs.
Asunto(s)
ADN Polimerasa Dirigida por ADN/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Alquilación , Daño del ADN , Humanos , Concentración 50 InhibidoraRESUMEN
The tumor suppressor protein 53BP1 plays key roles in response to DNA double-strand breaks (DSBs) by serving as a master scaffold at the damaged chromatin. Current evidence indicates that 53BP1 assembles a cohort of DNA damage response (DDR) factors to distinctly execute its repertoire of DSB responses, including checkpoint activation and non-homologous end joining (NHEJ) repair. Here, we have uncovered LC8 (a.k.a. DYNLL1) as an important 53BP1 effector. We found that LC8 accumulates at laser-induced DNA damage tracks in a 53BP1-dependent manner and requires the canonical H2AX-MDC1-RNF8-RNF168 signal transduction cascade. Accordingly, genetic inactivation of LC8 or its interaction with 53BP1 resulted in checkpoint defects. Importantly, loss of LC8 alleviated the hypersensitivity of BRCA1-depleted cells to ionizing radiation and PARP inhibition, highlighting the 53BP1-LC8 module in counteracting BRCA1-dependent functions in the DDR. Together, these data establish LC8 as an important mediator of a subset of 53BP1-dependent DSB responses.
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Dineínas Citoplasmáticas/fisiología , Roturas del ADN de Doble Cadena , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteína BRCA1/genética , Línea Celular , Cromatina/metabolismo , Dineínas Citoplasmáticas/química , Dineínas Citoplasmáticas/metabolismo , Reparación del ADN , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Radiación IonizanteRESUMEN
Translesion DNA synthesis (TLS) performed by human DNA polymerase eta (hpol η) allows tolerance of damage from cis-diamminedichloroplatinum(II) (CDDP or cisplatin). We have developed hpol η inhibitors derived from N-aryl-substituted indole barbituric acid (IBA), indole thiobarbituric acid (ITBA), and indole quinuclidine scaffolds and identified 5-((5-chloro-1-(naphthalen-2-ylmethyl)-1H-indol-3-yl)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (PNR-7-02), an ITBA derivative that inhibited hpol η activity with an IC50 value of 8 µM and exhibited 5-10-fold specificity for hpol η over replicative pols. We conclude from kinetic analyses, chemical footprinting assays, and molecular docking that PNR-7-02 binds to a site on the little finger domain and interferes with the proper orientation of template DNA to inhibit hpol η. A synergistic increase in CDDP toxicity was observed in hpol η-proficient cells co-treated with PNR-7-02 (combination index values = 0.4-0.6). Increased γH2AX formation accompanied treatment of hpol η-proficient cells with CDDP and PNR-7-02. Importantly, PNR-7-02 did not impact the effect of CDDP on cell viability or γH2AX in hpol η-deficient cells. In summary, we observed hpol η-dependent effects on DNA damage/replication stress and sensitivity to CDDP in cells treated with PNR-7-02. The ability to employ a small-molecule inhibitor of hpol η to improve the cytotoxic effect of CDDP may aid in the development of more effective chemotherapeutic strategies.
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Antineoplásicos/farmacología , Cisplatino/farmacología , ADN Polimerasa Dirigida por ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Línea Celular Tumoral , Inhibidores Enzimáticos/química , Humanos , Indoles/química , Indoles/farmacología , Simulación del Acoplamiento Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Pirimidinas/química , Pirimidinas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Tiobarbitúricos/química , Tiobarbitúricos/farmacologíaRESUMEN
BACKGROUND: Naphthalene is a good structural replacement for the isovanillin moiety (i.e. the 3- hydroxy-4-methoxyphenyl unit) in the combretastatin A-4 molecule, a natural product structurally related to resveratrol, which consistently led to the generation of highly cytotoxic naphthalene analogues when combined with a 3,4,5-trimethoxyphenyl or related aromatic system. Also, the naphthalene ring system is present in many current drug molecules that are utilized for anti-tumor, anti-arrhythmia and antioxidant therapy. OBJECTIVE: In our continuing quest to improve the potencies of naturally occurring anti-cancer molecules through chemical modification, we have now synthesized a small library of 2-naphthaleno trans- stilbenes and cyanostilbenes that are structurally related to both resveratrol and DMU-212, and have evaluated these novel analogs against a panel of 54 human tumor cell lines. METHOD: A series of 2-naphthaleno-containing trans-stilbenes 3a-3h (Scheme 1) were synthesized by Wittig reaction of a variety of aromatic substituted benzyl-triphenylphosphonium bromide reactants with 2- naphthaldehyde using n-BuLi as a base in THF. A second series of 2-naphthaleno trans-cyanostilbenes analogs 5a-5h was synthesized by reaction of 2-naphthaldehyde (2; 1 mmol) with an appropriately substituted 2- phenylacrylonitrile 4a-4h; 1 mmol) in 5% sodium methoxide/methanol. The reaction mixture was stirred at room temperature for 2-3 hours and the reaction allowed to go to completion (TLC monitoring), during which time the desired product precipitated out of the solution as a solid. The resulting precipitate was filtered off, washed with water and dried to yield the desired compound in yields ranging from 70-95% (Scheme 2). RESULTS: The percentage growth inhibition of 54 human cancer cell lines in a primary NCI screen after exposure to compounds 3a, 3d, 5b and 5c was carried out. The results showed that only compounds 5b and 5c met the criteria for subsequent testing to determine growth inhibition values (GI50) in dose-response studies. At 10-5 M concentration, compounds 5b and 5c exhibited cytotoxic activity against leukemia cell lines HL-60(TB) and SR, lung cancer cell line NCI-H522, colon cancer cell lines COLO 205 and HCT-116, CNS-cancer cell line SF-539, melanoma cell line MDA-MB-435, and breast cancer cell line BT-549. The naphthalene trans-stilbene analogue 3a, exhibited significant growth inhibition against only one cell line, melanoma cell line MDA-MB-435 (96 % growth inhibition). Compound 3d was inactive in the 10-5 M single dose screen. CONCLUSION: We have synthesized a small set of novel 2-naphthaleno stilbenes and cyanostilbenes and evaluated several of these compounds for their anticancer properties against a panel of 54 human tumor cell lines. The most active analogs, 5b and 5c, showed significantly improved growth inhibition against the human cancer cells in the NCI panel when compared to DMU-212. Of these compounds, analog 5c was found to be the most potent anticancer agent and exhibited significant growth inhibitory effects against COLO 205, CNS SF 539 and melanoma SK-MEL 5 and MDA-MB-435 cell lines with GI50 values ≤ 25 nM. Analog 5b also exhibited GI50 values in the range 25-41 nM against CNS SF 295 and melanoma MDA-MB-435 and UACC-62 cell lines. Compounds 5b and 5c were also cytotoxic towards the MV4-11 leukemia cell line with LD50 value of 450 nM and 200 nM, respectively, and demonstrated >50% inhibition of tubulin polymerization at concentrations below their LD50 values in these cells. In silico docking studies suggest that compounds 5b and 5c bind favorably at the colchicine- binding pocket of the tubulin dimer, indicating that both 5b and 5c may inhibit tubulin polymerization through a mechanism similar to that exhibited by colchicine. Derivative 5c demonstrated more favorable binding based on the docking score and buried surface area, as compared to compound 5b, in agreement with the higher observed potency of 5c against a broader range of tumor cell lines. Based on these results, analog 5c is considered to be a lead compound for further optimization as a clinical candidate for treating a variety of cancers.
Asunto(s)
Antineoplásicos/farmacología , Naftalenos/farmacología , Estilbenos/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Naftalenos/química , Estilbenos/síntesis química , Estilbenos/química , Relación Estructura-ActividadRESUMEN
Uncovering the mechanisms by which single-stranded binding proteins both protect and expose single-stranded DNA has important implications for our understanding of DNA replication and repair. A new study serves up a master class in developing a full kinetic model for one such protein, mtSSB, showing how DNA can be reeled in and set free to control accessibility.
Asunto(s)
ADN Mitocondrial/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Mitocondriales/metabolismo , Modelos Moleculares , Sitios de Unión , Reparación del ADN , Replicación del ADN , ADN Mitocondrial/química , ADN de Cadena Simple/química , Proteínas de Unión al ADN/química , Proteínas de Escherichia coli/química , Humanos , Cinética , Proteínas Mitocondriales/química , Multimerización de ProteínaRESUMEN
The genomic landscape of cancer is one marred by instability, but the mechanisms that underlie these alterations are multifaceted and remain a topic of intense research. Cellular responses to DNA damage and/or replication stress can affect genome stability in tumors and influence the response of patients to therapy. In addition to direct repair, DNA damage tolerance (DDT) is an element of genomic maintenance programs that contributes to the etiology of several types of cancer. DDT mechanisms primarily act to resolve replication stress, and this can influence the effectiveness of genotoxic drugs. Translesion DNA synthesis (TLS) is an important component of DDT that facilitates direct bypass of DNA adducts and other barriers to replication. The central role of TLS in the bypass of drug-induced DNA lesions, the promotion of tumor heterogeneity, and the involvement of these enzymes in the maintenance of the cancer stem cell niche presents an opportunity to leverage inhibition of TLS as a way of improving existing therapies. In the review that follows, we summarize mechanisms of DDT, misregulation of TLS in cancer, and discuss the potential for targeting these pathways as a means of improving cancer therapies.
Asunto(s)
Replicación del ADN , ADN/genética , Neoplasias/genética , Animales , Antineoplásicos/farmacología , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Humanos , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Transducción de Señal/efectos de los fármacosRESUMEN
The structural and biophysical properties typically associated with G-quadruplex (G4) structures render them a significant block for DNA replication, which must be overcome for cell division to occur. The Werner syndrome protein (WRN) is a RecQ family helicase that has been implicated in the efficient processing of G4 DNA structures. The aim of this study was to identify the residues of WRN involved in the binding and ATPase-driven unwinding of G4 DNA. Using a c-Myc G4 DNA model sequence and recombinant WRN, we have determined that the RecQ-C-terminal (RQC) domain of WRN imparts a 2-fold preference for binding to G4 DNA relative to non-G4 DNA substrates. NMR studies identified residues involved specifically in interactions with G4 DNA. Three of the amino acids in the WRN RQC domain that exhibited the largest G4-specific changes in NMR signal were then mutated alone or in combination. Mutating individual residues implicated in G4 binding had a modest effect on WRN binding to DNA, decreasing the preference for G4 substrates by â¼25%. Mutating two G4-interacting residues (T1024G and T1086G) abrogated preferential binding of WRN to G4 DNA. Very modest decreases in G4 DNA-stimulated ATPase activity were observed for the mutant enzymes. Most strikingly, G4 unwinding by WRN was inhibited â¼50% for all three point mutants and >90% for the WRN double mutant (T1024G/T1086G) relative to normal B-form dsDNA substrates. Our work has helped to identify residues in the WRN RQC domain that are involved specifically in the interaction with G4 DNA.
Asunto(s)
ADN/metabolismo , G-Cuádruplex , Helicasa del Síndrome de Werner/metabolismo , Síndrome de Werner/enzimología , ADN/química , ADN/genética , Reparación del ADN , Replicación del ADN , Humanos , Modelos Moleculares , Mutación , Dominios Proteicos , Síndrome de Werner/genética , Síndrome de Werner/metabolismo , Helicasa del Síndrome de Werner/química , Helicasa del Síndrome de Werner/genéticaRESUMEN
This study determined the antinociceptive effects of morphine and morphine-6-O-sulfate (M6S) in both normal and diabetic rats, and evaluated the comparative role of mu-opioid receptors (mu-ORs) and delta-opioid receptors (delta-ORs) in the antinociceptive action of these opioids. In vitro characterization of mu-OR and delta-OR-mediated signaling by M6S and morphine in stably transfected Chinese hamster ovary (CHO-K1) cells showed that M6S exhibited a 6-fold higher affinity for delta-ORs and modulated G-protein and adenylyl cyclase activity via delta-ORs more potently than morphine. Interestingly, while morphine acted as a full agonist at delta-ORs in both functional assays examined, M6S exhibited either partial or full agonist activity for modulation of G-protein or adenylyl cyclase activity, respectively. Molecular docking studies indicated that M6S but not morphine binds equally well at the ligand binding site of both mu- and delta-ORs. In vivo analgesic effects of M6S and morphine in both normal and streptozotocin-induced diabetic Sprague-Dawley rats utilizing the hot water tail flick latency test showed that M6S produced more potent antinociception than morphine in both normal rats and diabetic rats. This difference in potency was abrogated following antagonism of delta- but not mu- or kappa (kappa-ORs) opioid receptors. During 9days of chronic treatment, tolerance developed to morphine-treated but not to M6S-treated rats. Rats that developed tolerance to morphine still remained responsive to M6S. Collectively, this study demonstrates that M6S is a potent and efficacious mu/delta opioid analgesic with a delayed tolerance profile when compared to morphine in both normal and diabetic rats. PERSPECTIVE: This study demonstrates that M6S acts at both mu- and delta-ORs, and adds to the growing evidence that the use of mixed mu/delta opioid agonists in pain treatment may have clinical benefit.
Asunto(s)
Analgésicos/farmacología , Derivados de la Morfina/farmacología , Morfina/farmacología , Receptores Opioides delta/metabolismo , Receptores Opioides mu/metabolismo , Analgésicos Opioides/metabolismo , Animales , Células CHO , Línea Celular , Cricetinae , Cricetulus , Diabetes Mellitus Experimental/complicaciones , Tolerancia a Medicamentos/fisiología , Masculino , Dolor/tratamiento farmacológico , Dolor/etiología , Ratas , Ratas Sprague-Dawley , Receptores Opioides kappa/metabolismoRESUMEN
We have investigated the in vitro properties of human Y-family polymerase κ (hpol κ) on G-quadruplex DNA (G4 DNA). Similar to hpol η, another Y-family member implicated in replication of G4 motifs, hpol κ bound G4 DNA with a 5.7-fold preference over control, non-G4 DNA. Results from pol extension assays are consistent with the notion that G-quadruplexes present a stronger barrier to DNA synthesis by hpol κ than they do to that by hpol η. However, kinetic analysis revealed that hpol κ activity increases considerably when the enzyme is 2-3 nucleotides from the G4 motif, a trend that was reported previously for hpol η, though the increase was less pronounced. The increase in hpol κ activity on G4 DNA was readily observed in the presence of either potassium or sodium but much less so when lithium was used in the buffer. The increased activity 2-3 nucleotides from the G4 motif was accompanied by a decrease in the fidelity of hpol κ when the counterion was either potassium or sodium but not in the presence of lithium. The activity of hpol κ decreased progressively as the primer was moved closer than 2 nucleotides from the G4 motif when either potassium or sodium was used to stabilize the G-quadruplex. Interestingly, the decrease in catalytic activity at the site of the quadruplex observed in potassium-containing buffer was accompanied by an increase in fidelity on G4 substrates versus control non-G4 substrates. This trend of increased fidelity in copying a tetrad-associated guanine was observed previously for hpol η, but not for the B-family member hpol ε, which exhibited a large decrease in both efficiency and fidelity in the attempt to copy the first guanine in the G4 motif. In summary, hpol κ activity was enhanced relative to those of other Y-family members when the enzyme is 2-3 nucleotides from the G4 motif, but hpol κ appears to be less competent than hpol η at copying tetrad-associated guanines.
Asunto(s)
Daño del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/metabolismo , G-Cuádruplex , Polarización de Fluorescencia , Humanos , CinéticaRESUMEN
Metabolism of the essential amino acid L-tryptophan (TRP) is implicated in a number of neurological conditions including depression, neurodegenerative diseases, and cancer. The TRP catabolite kynurenine (KYN) has recently emerged as an important neuroactive factor in brain tumor pathogenesis, with additional studies implicating KYN in other types of cancer. Often highlighted as a modulator of the immune response and a contributor to immune escape for malignant tumors, it is well-known that KYN has effects on the production of the coenzyme nicotinamide adenine dinucleotide (NAD(+)), which can have a direct impact on DNA repair, replication, cell division, redox signaling, and mitochondrial function. Additional effects of KYN signaling are imparted through its role as an endogenous agonist for the aryl hydrocarbon receptor (AhR), and it is largely through activation of the AhR that KYN appears to mediate malignant progression in gliomas. We have recently reported on the ability of KYN signaling to modulate expression of human DNA polymerase kappa (hpol κ), a translesion enzyme involved in bypass of bulky DNA lesions and activation of the replication stress response. Given the impact of KYN on NAD(+) production, AhR signaling, and translesion DNA synthesis, it follows that dysregulation of KYN signaling in cancer may promote malignancy through alterations in the level of endogenous DNA damage and replication stress. In this perspective, we discuss the connections between KYN signaling, DNA damage tolerance, and genomic instability, as they relate to cancer.
Asunto(s)
Inestabilidad Genómica/fisiología , Glioma/fisiopatología , Quinurenina/fisiología , Transducción de Señal/fisiología , Replicación del ADN/fisiología , Humanos , Neoplasias/fisiopatologíaRESUMEN
Cells engage numerous signaling pathways in response to oxidative stress that together repair macromolecular damage or direct the cell toward apoptosis. As a result of DNA damage, mitochondrial DNA or nuclear DNA has been shown to enter the cytoplasm where it binds to "DNA sensors," which in turn initiate signaling cascades. Here we report data that support a novel signaling pathway in response to oxidative stress mediated by specific guanine-rich sequences that can fold into G-quadruplex DNA (G4DNA). In response to oxidative stress, we demonstrate that sequences capable of forming G4DNA appear at increasing levels in the cytoplasm and participate in assembly of stress granules. Identified proteins that bind to endogenous G4DNA in the cytoplasm are known to modulate mRNA translation and participate in stress granule formation. Consistent with these findings, stress granule formation is known to regulate mRNA translation during oxidative stress. We propose a signaling pathway whereby cells can rapidly respond to DNA damage caused by oxidative stress. Guanine-rich sequences that are excised from damaged genomic DNA are proposed to enter the cytoplasm where they can regulate translation through stress granule formation. This newly proposed role for G4DNA provides an additional molecular explanation for why such sequences are prevalent in the human genome.