RESUMO
Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment.
Assuntos
Neoplasias do Colo/tratamento farmacológico , DNA Glicosilases/genética , DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Poli(ADP-Ribose) Polimerase-1/imunologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/mortalidade , Dano ao DNA , DNA Glicosilases/antagonistas & inibidores , DNA Glicosilases/metabolismo , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Guanina/análogos & derivados , Guanina/metabolismo , Células HCT116 , Humanos , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Estresse Oxidativo , Poli(ADP-Ribose) Polimerase-1/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Análise de Sobrevida , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cornelia de Lange syndrome (CdLS) is a congenital developmental disorder characterized by craniofacial dysmorphia, growth retardation, limb malformations, and intellectual disability. Approximately 60% of patients with CdLS carry a recognizable pathological variant in the NIPBL gene, of which two isoforms, A and B, have been identified, and which only differ in the C-terminal segment. In this work, we describe the distribution pattern of the isoforms A and B mRNAs in tissues of adult and fetal origin, by qPCR (quantitative polymerase chain reaction). Our results show a higher gene expression of the isoform A, even though both seem to have the same tissue distribution. Interestingly, the expression in fetal tissues is higher than that of adults, especially in brain and skeletal muscle. Curiously, the study of fibroblasts of two siblings with a mild CdLS phenotype and a pathological variant specific of the isoform A of NIPBL (c.8387A > G; P.Tyr2796Cys), showed a similar reduction in both isoforms, and a normal sensitivity to DNA damage. Overall, these results suggest that the position of the pathological variant at the 3´ end of the NIPBL gene affecting only isoform A, is likely to be the cause of the atypical mild phenotype of the two brothers.
Assuntos
Síndrome de Cornélia de Lange/genética , Polimorfismo de Nucleotídeo Único , Proteínas/genética , Adolescente , Encéfalo/embriologia , Encéfalo/metabolismo , Proteínas de Ciclo Celular , Criança , Síndrome de Cornélia de Lange/diagnóstico , Humanos , Masculino , Músculo Esquelético/embriologia , Músculo Esquelético/metabolismo , Fenótipo , Isoformas de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
We describe a spontaneously derived mouse line that completely failed to induce Ig class switching in vitro and in vivo. The mice inherited abolished IgG serum titers in a recessive manner caused by a spontaneous G â A transition mutation in codon 112 of the aicda gene, leading to an arginine to histidine replacement (AID(R112H)). Ig class switching was completely reconstituted by expressing wild-type AID. Mice homozygous for AID(R112H) had peripheral B cell hyperplasia and large germinal centers in the absence of Ag challenge. Immunization with SRBCs elicited an Ag-specific IgG1 response in wild-type mice, whereas AID(R112H) mice failed to produce IgG1 and had reduced somatic hypermutation. The phenotype recapitulates the human hyper-IgM (HIGM) syndrome that is caused by point mutations in the orthologous gene in humans, and the AID(R112H) mutation is frequently found in HIGM patients. The AID(R112H) mouse model for HIGM provides a powerful and more precise tool than conventional knockout strategies.
Assuntos
Citidina Desaminase/genética , Modelos Animais de Doenças , Síndrome de Imunodeficiência com Hiper-IgM/genética , Síndrome de Imunodeficiência com Hiper-IgM/imunologia , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Mutação , Hipermutação Somática de Imunoglobulina , Animais , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Citidina Desaminase/metabolismo , Análise Mutacional de DNA , Feminino , Centro Germinativo/imunologia , Síndrome de Imunodeficiência com Hiper-IgM/metabolismo , Imunofenotipagem , Padrões de Herança , Contagem de Linfócitos , Masculino , Camundongos , Linhagem , Fenótipo , Característica Quantitativa HerdávelRESUMO
Decitabine (5-aza-2'-deoxycytidine, 5-azadC) is used in the treatment of Myelodysplatic syndrome (MDS) and Acute Myeloid Leukemia (AML). Its mechanism of action is thought to involve reactivation of genes implicated in differentiation and transformation, as well as induction of DNA damage by trapping DNA methyltranferases (DNMT) to DNA. We demonstrate for the first time that base excision repair (BER) recognizes 5-azadC-induced lesions in DNA and mediates repair. We find that BER (XRCC1) deficient cells are sensitive to 5-azadC and display an increased amount of DNA single- and double-strand breaks. The XRCC1 protein co-localizes with DNMT1 foci after 5-azadC treatment, suggesting a novel and specific role of XRCC1 in the repair of trapped DNMT1. 5-azadC-induced DNMT foci persist in XRCC1 defective cells, demonstrating a role for XRCC1 in repair of 5-azadC-induced DNA lesions. Poly (ADP-ribose) polymerase (PARP) inhibition prevents XRCC1 relocation to DNA damage sites, disrupts XRCC1-DNMT1 co-localization and thereby efficient BER. In a panel of AML cell lines, combining 5-azadC and Olaparib cause synthetic lethality. These data suggest that PARP inhibitors can be used in combination with 5-azadC to improve treatment of MDS and AML.
Assuntos
Antimetabólitos Antineoplásicos/toxicidade , Azacitidina/análogos & derivados , Reparo do DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Azacitidina/toxicidade , Linhagem Celular Tumoral , Cricetinae , DNA (Citosina-5-)-Metiltransferases/análise , Adutos de DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/análise , Decitabina , Humanos , Reparo de DNA por Recombinação , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
The availability of analytical methods for the characterization of lipid nanoparticles (LNPs) for in-vivo intracellular delivery of nucleic acids is critical for the fast development of innovative RNA therapies. In this study, analytical protocols to measure (i) chemical composition, (ii) drug loading, (iii) particle size, concentration, and stability as well as (iv) structure and morphology were evaluated and compared based on a comprehensive characterization strategy linking key physical and chemical properties to in-vitro efficacy and toxicity. Furthermore, the measurement protocols were assessed either by testing the reproducibility and robustness of the same technique in different laboratories, or by a correlative approach, comparing measurement results of the same attribute with orthogonal techniques. The characterization strategy and the analytical measurements described here will have an important role during formulation development and in determining robust quality attributes ultimately supporting the quality assessment of these innovative RNA therapeutics.
Assuntos
Nanopartículas , Ácidos Nucleicos , Reprodutibilidade dos Testes , Lipídeos/química , RNA Interferente Pequeno/genética , Nanopartículas/química , Lipossomos , Tamanho da PartículaRESUMO
Cytotoxicity of 5-fluorouracil (FU) and 5-fluoro-2'-deoxyuridine (FdUrd) due to DNA fragmentation during DNA repair has been proposed as an alternative to effects from thymidylate synthase (TS) inhibition or RNA incorporation. The goal of the present study was to investigate the relative contribution of the proposed mechanisms for cytotoxicity of 5-fluoropyrimidines. We demonstrate that in human cancer cells, base excision repair (BER) initiated by the uracil-DNA glycosylase UNG is the major route for FU-DNA repair in vitro and in vivo. SMUG1, TDG and MBD4 contributed modestly in vitro and not detectably in vivo. Contribution from mismatch repair was limited to FU:G contexts at best. Surprisingly, knockdown of individual uracil-DNA glycosylases or MSH2 did not affect sensitivity to FU or FdUrd. Inhibitors of common steps of BER or DNA damage signalling affected sensitivity to FdUrd and HmdUrd, but not to FU. In support of predominantly RNA-mediated cytotoxicity, FU-treated cells accumulated ~3000- to 15 000-fold more FU in RNA than in DNA. Moreover, FU-cytotoxicity was partially reversed by ribonucleosides, but not deoxyribonucleosides and FU displayed modest TS-inhibition compared to FdUrd. In conclusion, UNG-initiated BER is the major route for FU-DNA repair, but cytotoxicity of FU is predominantly RNA-mediated, while DNA-mediated effects are limited to FdUrd.
Assuntos
Reparo do DNA , Fluoruracila/metabolismo , Uracila-DNA Glicosidase/metabolismo , Animais , Ciclo Celular , Linhagem Celular Tumoral , DNA/química , DNA/metabolismo , Dano ao DNA , Endodesoxirribonucleases/genética , Floxuridina/metabolismo , Floxuridina/toxicidade , Fluoruracila/toxicidade , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Proteína 2 Homóloga a MutS/genética , RNA/metabolismo , Timidina/análogos & derivados , Timidina/metabolismo , Timidina/toxicidade , Timina DNA Glicosilase/genética , Timina DNA Glicosilase/metabolismo , Uracila-DNA Glicosidase/genética , Uridina/análogos & derivados , Uridina/metabolismo , Uridina/toxicidadeRESUMO
While chemotherapy alone or in combination with radiotherapy and surgery are important modalities in the treatment of colorectal cancer, their widespread use is not paired with an abundance of diagnostic tools to match individual patients with the most effective standard-of-care chemo- or radiotherapy regimens. Patient-derived organoids are tumour-derived structures that have been shown to retain certain aspects of the tissue of origin. We present here a systematic review of studies that have tested the performance of patient derived organoids to predict the effect of anti-cancer therapies in colorectal cancer, for chemotherapies, targeted drugs, and radiation therapy, and we found overall a positive predictive value of 68% and a negative predictive value of 78% for organoid informed treatment, which outperforms response rates observed with empirically guided treatment selection.
Assuntos
Neoplasias Colorretais , Humanos , Neoplasias Colorretais/terapia , OrganoidesRESUMO
8-oxo Guanine DNA Glycosylase 1 is the initiating enzyme within base excision repair and removes oxidized guanines from damaged DNA. Since unrepaired 8-oxoG could lead to G : CâT : A transversion, base removal is of utmost importance for cells to ensure genomic integrity. For cells with elevated levels of reactive oxygen species this dependency is further increased. In the past we and others have validated OGG1 as a target for inhibitors to treat cancer and inflammation. Here, we present the optimization campaign that led to the broadly used tool compound TH5487. Based on results from a small molecule screening campaign, we performed hit to lead expansion and arrived at potent and selective substituted N-piperidinyl-benzimidazolones. Using X-ray crystallography data, we describe the surprising binding mode of the most potent member of the class, TH8535. Here, the N-Piperidinyl-linker adopts a chair instead of a boat conformation which was found for weaker analogues. We further demonstrate cellular target engagement and efficacy of TH8535 against a number of cancer cell lines.
Assuntos
DNA Glicosilases , Neoplasias , Humanos , DNA Glicosilases/química , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Guanina/química , Guanina/metabolismo , Reparo do DNA , Benzimidazóis/farmacologia , Dano ao DNARESUMO
Genomic uracil is a DNA lesion but also an essential key intermediate in adaptive immunity. In B cells, activation-induced cytidine deaminase deaminates cytosine to uracil (U:G mispairs) in Ig genes to initiate antibody maturation. Uracil-DNA glycosylases (UDGs) such as uracil N-glycosylase (UNG), single strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1), and thymine-DNA glycosylase remove uracil from DNA. Gene-targeted mouse models are extensively used to investigate the role of these enzymes in DNA repair and Ig diversification. However, possible species differences in uracil processing in humans and mice are yet not established. To address this, we analyzed UDG activities and quantities in human and mouse cell lines and in splenic B cells from Ung(+/+) and Ung(-/-) backcrossed mice. Interestingly, human cells displayed â¼15-fold higher total uracil excision capacity due to higher levels of UNG. In contrast, SMUG1 activity was â¼8-fold higher in mouse cells, constituting â¼50% of the total U:G excision activity compared with less than 1% in human cells. In activated B cells, both UNG and SMUG1 activities were at levels comparable with those measured for mouse cell lines. Moreover, SMUG1 activity per cell was not down-regulated after activation. We therefore suggest that SMUG1 may work as a weak backup activity for UNG2 during class switch recombination in Ung(-/-) mice. Our results reveal significant species differences in genomic uracil processing. These findings should be taken into account when mouse models are used in studies of uracil DNA repair and adaptive immunity.
Assuntos
Uracila-DNA Glicosidase/química , Animais , Reparo do DNA , Humanos , Switching de Imunoglobulina , Imunoglobulinas/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Recombinação Genética , Especificidade da Espécie , Timina DNA Glicosilase/químicaRESUMO
BACKGROUND: Pregnancy, infancy, and childhood are sensitive windows for environmental exposures. Yet the health effects of exposure to nano- and microplastics (NMPs) remain largely uninvestigated or unknown. Although plastic chemicals are a well-established research topic, the impacts of plastic particles are unexplored, especially with regard to early life exposures. OBJECTIVES: This commentary aims to summarize the knowns and unknowns around child- and pregnancy-relevant exposures to NMPs via inhalation, placental transfer, ingestion and breastmilk, and dermal absorption. METHODS: A comprehensive literature search to map the state of the science on NMPs found 37 primary research articles on the health relevance of NMPs during early life and revealed major knowledge gaps in the field. We discuss opportunities and challenges for quantifying child-specific exposures (e.g., NMPs in breastmilk or infant formula) and health effects, in light of global inequalities in baby bottle use, consumption of packaged foods, air pollution, hazardous plastic disposal, and regulatory safeguards. We also summarize research needs for linking child health and NMP exposures and address the unknowns in the context of public health action. DISCUSSION: Few studies have addressed child-specific sources of exposure, and exposure estimates currently rely on generic assumptions rather than empirical measurements. Furthermore, toxicological research on NMPs has not specifically focused on child health, yet children's immature defense mechanisms make them particularly vulnerable. Apart from few studies investigating the placental transfer of NMPs, the physicochemical properties (e.g., polymer, size, shape, charge) driving the absorption, biodistribution, and elimination in early life have yet to be benchmarked. Accordingly, the evidence base regarding the potential health impacts of NMPs in early life remains sparse. Based on the evidence to date, we provide recommendations to fill research gaps, stimulate policymakers and industry to address the safety of NMPs, and point to opportunities for families to reduce early life exposures to plastic. https://doi.org/10.1289/EHP9086.
Assuntos
Microplásticos , Plásticos , Criança , Saúde da Criança , Exposição Ambiental , Feminino , Humanos , Lactente , Placenta , Gravidez , Distribuição TecidualRESUMO
State-of-the-art in vitro test systems for nanomaterial toxicity assessment are based on dyes and several staining steps which can be affected by nanomaterial interference. Digital holographic microscopy (DHM), an interferometry-based variant of quantitative phase imaging (QPI), facilitates reliable proliferation quantification of native cell populations and the extraction of morphological features in a fast and label- and interference-free manner by biophysical parameters. DHM therefore has been identified as versatile tool for cytotoxicity testing in biomedical nanotechnology. In a comparative study performed at two collaborating laboratories, we investigated the interlaboratory variability and performance of DHM in nanomaterial toxicity testing, utilizing complementary standard operating procedures (SOPs). Two identical custom-built off-axis DHM systems, developed for usage in biomedical laboratories, equipped with stage-top incubation chambers were applied at different locations in Europe. Temporal dry mass development, 12-h dry mass increments and morphology changes of A549 human lung epithelial cell populations upon incubation with two variants of poly(alkyl cyanoacrylate) (PACA) nanoparticles were observed in comparison to digitonin and cell culture medium controls. Digitonin as cytotoxicity control, as well as empty and cabazitaxel-loaded PACA nanocarriers, similarly impacted 12-h dry mass development and increments as well as morphology of A549 cells at both participating laboratories. The obtained DHM data reflected the cytotoxic potential of the tested nanomaterials and are in agreement with corresponding literature on biophysical and chemical assays. Our results confirm DHM as label-free cytotoxicity assay for polymeric nanocarriers as well as the repeatability and reproducibility of the technology. In summary, the evaluated DHM assay could be efficiently implemented at different locations and facilitates interlaboratory in vitro toxicity testing of nanoparticles with prospects for application in regulatory science.
Assuntos
Holografia , Microscopia , Digitonina , Holografia/métodos , Humanos , Técnicas In Vitro , Microscopia/métodos , Reprodutibilidade dos TestesRESUMO
Oxidative DNA damage is recognized by 8-oxoguanine (8-oxoG) DNA glycosylase 1 (OGG1), which excises 8-oxoG, leaving a substrate for apurinic endonuclease 1 (APE1) and initiating repair. Here, we describe a small molecule (TH10785) that interacts with the phenylalanine-319 and glycine-42 amino acids of OGG1, increases the enzyme activity 10-fold, and generates a previously undescribed ß,δ-lyase enzymatic function. TH10785 controls the catalytic activity mediated by a nitrogen base within its molecular structure. In cells, TH10785 increases OGG1 recruitment to and repair of oxidative DNA damage. This alters the repair process, which no longer requires APE1 but instead is dependent on polynucleotide kinase phosphatase (PNKP1) activity. The increased repair of oxidative DNA lesions with a small molecule may have therapeutic applications in various diseases and aging.
Assuntos
Dano ao DNA , DNA Glicosilases , Reparo do DNA , Estresse Oxidativo , Biocatálise/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , DNA Glicosilases/química , DNA Glicosilases/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Ativação Enzimática , Glicina/química , Humanos , Ligantes , Estresse Oxidativo/genética , Fenilalanina/química , Especificidade por SubstratoRESUMO
The most common oxidative DNA lesion is 8-oxoguanine which is mainly recognized and excised by the 8-oxoG DNA glycosylase 1 (OGG1), initiating the base excision repair (BER) pathway. Telomeres are particularly sensitive to oxidative stress (OS) which disrupts telomere homeostasis triggering genome instability. In the present study, we have investigated the effects of inactivating BER in OS conditions, by using a specific inhibitor of OGG1 (TH5487). We have found that in OS conditions, TH5487 blocks BER initiation at telomeres causing an accumulation of oxidized bases, that is correlated with telomere losses, micronuclei formation and mild proliferation defects. Moreover, the antimetabolite methotrexate synergizes with TH5487 through induction of intracellular reactive oxygen species (ROS) formation, which potentiates TH5487-mediated telomere and genome instability. Our findings demonstrate that OGG1 is required to protect telomeres from OS and present OGG1 inhibitors as a tool to induce oxidative DNA damage at telomeres, with the potential for developing new combination therapies for cancer treatment.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , DNA Glicosilases/antagonistas & inibidores , Reparo do DNA/efeitos dos fármacos , Metotrexato/farmacologia , Estresse Oxidativo , Piperidinas/farmacologia , Telômero/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , DNA Glicosilases/metabolismo , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Instabilidade Genômica , Humanos , Oxirredução , Espécies Reativas de Oxigênio/metabolismoRESUMO
Acute myeloid leukemia (AML) is an aggressive hematologic malignancy, exhibiting high levels of reactive oxygen species (ROS). ROS levels have been suggested to drive leukemogenesis and is thus a potential novel target for treating AML. MTH1 prevents incorporation of oxidized nucleotides into the DNA to maintain genome integrity and is upregulated in many cancers. Here we demonstrate that hematologic cancers are highly sensitive to MTH1 inhibitor TH1579 (karonudib). A functional precision medicine ex vivo screen in primary AML bone marrow samples demonstrated a broad response profile of TH1579, independent of the genomic alteration of AML, resembling the response profile of the standard-of-care treatments cytarabine and doxorubicin. Furthermore, TH1579 killed primary human AML blast cells (CD45+) as well as chemotherapy resistance leukemic stem cells (CD45+Lin-CD34+CD38-), which are often responsible for AML progression. TH1579 killed AML cells by causing mitotic arrest, elevating intracellular ROS levels, and enhancing oxidative DNA damage. TH1579 showed a significant therapeutic window, was well tolerated in animals, and could be combined with standard-of-care treatments to further improve efficacy. TH1579 significantly improved survival in two different AML disease models in vivo. In conclusion, the preclinical data presented here support that TH1579 is a promising novel anticancer agent for AML, providing a rationale to investigate the clinical usefulness of TH1579 in AML in an ongoing clinical phase I trial. SIGNIFICANCE: The MTH1 inhibitor TH1579 is a potential novel AML treatment, targeting both blasts and the pivotal leukemic stem cells while sparing normal bone marrow cells.
Assuntos
Crise Blástica/tratamento farmacológico , Enzimas Reparadoras do DNA/antagonistas & inibidores , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Mitose , Células-Tronco Neoplásicas/efeitos dos fármacos , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Pirimidinas/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose , Crise Blástica/genética , Crise Blástica/metabolismo , Crise Blástica/patologia , Proliferação de Células , Citarabina/administração & dosagem , Doxorrubicina/administração & dosagem , Feminino , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Prognóstico , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Identification of the molecular mechanism of action (MoA) of bioactive compounds is a crucial step for drug development but remains a challenging task despite recent advances in technology. In this study, we applied multidimensional proteomics, sensitivity correlation analysis, and transcriptomics to identify a common MoA for the anticancer compounds RITA, aminoflavone (AF), and oncrasin-1 (Onc-1). Global thermal proteome profiling revealed that the three compounds target mRNA processing and transcription, thereby attacking a cancer vulnerability, transcriptional addiction. This led to the preferential loss of expression of oncogenes involved in PDGF, EGFR, VEGF, insulin/IGF/MAPKK, FGF, Hedgehog, TGFß, and PI3K signaling pathways. Increased reactive oxygen species level in cancer cells was a prerequisite for targeting the mRNA transcription machinery, thus conferring cancer selectivity to these compounds. Furthermore, DNA repair factors involved in homologous recombination were among the most prominently repressed proteins. In cancer patient samples, RITA, AF, and Onc-1 sensitized to poly(ADP-ribose) polymerase inhibitors both in vitro and ex vivo These findings might pave a way for new synthetic lethal combination therapies.Significance: These findings highlight agents that target transcriptional addiction in cancer cells and suggest combination treatments that target RNA processing and DNA repair pathways simultaneously as effective cancer therapies.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Oncogenes/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Transcrição Gênica/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Feminino , Flavonoides/farmacologia , Flavonoides/uso terapêutico , Furanos/farmacologia , Furanos/uso terapêutico , Perfilação da Expressão Gênica , Humanos , Indóis/farmacologia , Indóis/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Proteoma/genética , Proteômica/métodos , Reparo de DNA por Recombinação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Mutações Sintéticas Letais/efeitos dos fármacosRESUMO
Recent RNA virus outbreaks such as Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Ebola virus (EBOV) have caused worldwide health emergencies highlighting the urgent need for new antiviral strategies. Targeting host cell pathways supporting viral replication is an attractive approach for development of antiviral compounds, especially with new, unexplored viruses where knowledge of virus biology is limited. Here, we present a strategy to identify host-targeted small molecule inhibitors using an image-based phenotypic antiviral screening assay followed by extensive target identification efforts revealing altered cellular pathways upon antiviral compound treatment. The newly discovered antiviral compounds showed broad-range antiviral activity against pathogenic RNA viruses such as SARS-CoV-2, EBOV and Crimean-Congo hemorrhagic fever virus (CCHFV). Target identification of the antiviral compounds by thermal protein profiling revealed major effects on proteostasis pathways and disturbance in interactions between cellular HSP70 complex and viral proteins, illustrating the supportive role of HSP70 on many RNA viruses across virus families. Collectively, this strategy identifies new small molecule inhibitors with broad antiviral activity against pathogenic RNA viruses, but also uncovers novel virus biology urgently needed for design of new antiviral therapies.
Assuntos
Antivirais/farmacologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Vírus de RNA/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Ebolavirus/efeitos dos fármacos , Ebolavirus/fisiologia , Proteínas de Choque Térmico HSP70/metabolismo , Vírus da Febre Hemorrágica da Crimeia-Congo/efeitos dos fármacos , Vírus da Febre Hemorrágica da Crimeia-Congo/fisiologia , Humanos , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica , Proteoma/efeitos dos fármacos , Proteostase/efeitos dos fármacos , Infecções por Vírus de RNA/metabolismo , Infecções por Vírus de RNA/virologia , Vírus de RNA/fisiologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas Virais/metabolismoRESUMO
Uracil in DNA is repaired by base excision repair (BER) initiated by a DNA glycosylase, followed by strand incision, trimming of ends, gap filling and ligation. Uracil in DNA comes in two distinct forms; U:A pairs, typically resulting from replication errors, and mutagenic U:G mismatches, arising from cytosine deamination. To identify proteins critical to the rate of repair of these lesions, we quantified overall repair of U:A pairs, U:G mismatches and repair intermediates (abasic sites and nicked abasic sites) in vitro. For this purpose we used circular DNA substrates and nuclear extracts of eight human cell lines with wide variation in the content of BER proteins. We identified the initiating uracil-DNA glycosylase UNG2 as the major overall rate-limiting factor. UNG2 is apparently the sole glycosylase initiating BER of U:A pairs and generally initiated repair of almost 90% of the U:G mismatches. Surprisingly, TDG contributed at least as much as single-strand selective monofunctional uracil-DNA glycosylase 1 (SMUG1) to BER of U:G mismatches. Furthermore, in a cell line that expressed unusually high amounts of TDG, this glycosylase contributed to initiation of as much as approximately 30% of U:G repair. Repair of U:G mismatches was generally faster than that of U:A pairs, which agrees with the known substrate preference of UNG-type glycosylases. Unexpectedly, repair of abasic sites opposite G was also generally faster than when opposite A, and this could not be explained by the properties of the purified APE1 protein. It may rather reflect differences in substrate recognition or repair by different complex(es). Lig III is apparently a minor rate-regulator for U:G repair. APE1, Pol beta, Pol delta, PCNA, XRCC1 and Lig I did not seem to be rate-limiting for overall repair of any of the substrates. These results identify damaged base removal as the major rate-limiting step in BER of uracil in human cells.
Assuntos
DNA Glicosilases/metabolismo , Reparo do DNA , Uracila/química , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citosina/metabolismo , Humanos , Modelos Biológicos , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese , Análise de Regressão , Uracila/metabolismoRESUMO
Due to a polar or even charged binding interface, DNA-binding proteins are considered extraordinarily difficult targets for development of small-molecule ligands and only a handful of proteins have been targeted successfully to date. Recently, however, it has been shown that development of selective and efficient inhibitors of 8-oxoguanine DNA glycosylase is possible. Here, we describe the initial druggability assessment of DNA glycosylases in a computational setting and experimentally investigate several methods to target endonuclease VIII-like 1 (NEIL1) with small-molecule inhibitors. We find that DNA glycosylases exhibit good predicted druggability in both DNA-bound and -unbound states. Furthermore, we find catalytic sites to be highly flexible, allowing for a range of interactions and binding partners. One flexible catalytic site was rationalized for NEIL1 and further investigated experimentally using both a biochemical assay in the presence of DNA and a thermal shift assay in the absence of DNA.
RESUMO
Base excision repair (BER) repairs mutagenic or genotoxic DNA base lesions, thought to be important for both the etiology and treatment of cancer. Cancer phenotypic stress induces oxidative lesions, and deamination products are responsible for one of the most prevalent mutational signatures in cancer. Chemotherapeutic agents induce genotoxic DNA base damage that are substrates for BER, while synthetic lethal approaches targeting BER-related factors are making their way into the clinic. Thus, there are three strategies by which BER is envisioned to be relevant in cancer chemotherapy: (i) to maintain cellular growth in the presence of endogenous DNA damage in stressed cancer cells, (ii) to maintain viability after exogenous DNA damage is introduced by therapeutic intervention, or (iii) to confer synthetic lethality in cancer cells that have lost one or more additional DNA repair pathways. Here, we discuss the potential treatment strategies, and briefly summarize the progress that has been made in developing inhibitors to core BER-proteins and related factors.
Assuntos
Antineoplásicos/farmacologia , Enzimas Reparadoras do DNA/antagonistas & inibidores , Reparo do DNA/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , DNA/metabolismo , Dano ao DNA , Humanos , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/metabolismoRESUMO
The onset of inflammation is associated with reactive oxygen species and oxidative damage to macromolecules like 7,8-dihydro-8-oxoguanine (8-oxoG) in DNA. Because 8-oxoguanine DNA glycosylase 1 (OGG1) binds 8-oxoG and because Ogg1-deficient mice are resistant to acute and systemic inflammation, we hypothesized that OGG1 inhibition may represent a strategy for the prevention and treatment of inflammation. We developed TH5487, a selective active-site inhibitor of OGG1, which hampers OGG1 binding to and repair of 8-oxoG and which is well tolerated by mice. TH5487 prevents tumor necrosis factor-α-induced OGG1-DNA interactions at guanine-rich promoters of proinflammatory genes. This, in turn, decreases DNA occupancy of nuclear factor κB and proinflammatory gene expression, resulting in decreased immune cell recruitment to mouse lungs. Thus, we present a proof of concept that targeting oxidative DNA repair can alleviate inflammatory conditions in vivo.