Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 13 de 13
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Genes Dev ; 35(13-14): 1005-1019, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34168039

RESUMO

N6-methyladenosine (m6A) is an abundant internal RNA modification, influencing transcript fate and function in uninfected and virus-infected cells. Installation of m6A by the nuclear RNA methyltransferase METTL3 occurs cotranscriptionally; however, the genomes of some cytoplasmic RNA viruses are also m6A-modified. How the cellular m6A modification machinery impacts coronavirus replication, which occurs exclusively in the cytoplasm, is unknown. Here we show that replication of SARS-CoV-2, the agent responsible for the COVID-19 pandemic, and a seasonal human ß-coronavirus HCoV-OC43, can be suppressed by depletion of METTL3 or cytoplasmic m6A reader proteins YTHDF1 and YTHDF3 and by a highly specific small molecule METTL3 inhibitor. Reduction of infectious titer correlates with decreased synthesis of viral RNAs and the essential nucleocapsid (N) protein. Sites of m6A modification on genomic and subgenomic RNAs of both viruses were mapped by methylated RNA immunoprecipitation sequencing (meRIP-seq). Levels of host factors involved in m6A installation, removal, and recognition were unchanged by HCoV-OC43 infection; however, nuclear localization of METTL3 and cytoplasmic m6A readers YTHDF1 and YTHDF2 increased. This establishes that coronavirus RNAs are m6A-modified and host m6A pathway components control ß-coronavirus replication. Moreover, it illustrates the therapeutic potential of targeting the m6A pathway to restrict coronavirus reproduction.


Assuntos
Coronavirus Humano OC43/fisiologia , Processamento Pós-Transcricional do RNA/genética , SARS-CoV-2/fisiologia , Replicação Viral/genética , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Linhagem Celular , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Regulação da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Proteínas do Nucleocapsídeo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Replicação Viral/efeitos dos fármacos
2.
Nature ; 593(7860): 597-601, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33902106

RESUMO

N6-methyladenosine (m6A) is an abundant internal RNA modification1,2 that is catalysed predominantly by the METTL3-METTL14 methyltransferase complex3,4. The m6A methyltransferase METTL3 has been linked to the initiation and maintenance of acute myeloid leukaemia (AML), but the potential of therapeutic applications targeting this enzyme remains unknown5-7. Here we present the identification and characterization of STM2457, a highly potent and selective first-in-class catalytic inhibitor of METTL3, and a crystal structure of STM2457 in complex with METTL3-METTL14. Treatment of tumours with STM2457 leads to reduced AML growth and an increase in differentiation and apoptosis. These cellular effects are accompanied by selective reduction of m6A levels on known leukaemogenic mRNAs and a decrease in their expression consistent with a translational defect. We demonstrate that pharmacological inhibition of METTL3 in vivo leads to impaired engraftment and prolonged survival in various mouse models of AML, specifically targeting key stem cell subpopulations of AML. Collectively, these results reveal the inhibition of METTL3 as a potential therapeutic strategy against AML, and provide proof of concept that the targeting of RNA-modifying enzymes represents a promising avenue for anticancer therapy.

4.
Haematologica ; 104(5): 963-972, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30573504

RESUMO

Mitochondrial DNA encodes 13 proteins that comprise components of the respiratory chain that maintain oxidative phosphorylation. The replication of mitochondrial DNA is performed by the sole mitochondrial DNA polymerase γ. As acute myeloid leukemia (AML) cells and stem cells have an increased reliance on oxidative phosphorylation, we sought to evaluate polymerase γ inhibitors in AML. The thymidine dideoxynucleoside analog, alovudine, is an inhibitor of polymerase γ. In AML cells, alovudine depleted mitochondrial DNA, reduced mitochondrial encoded proteins, decreased basal oxygen consumption, and decreased cell proliferation and viability. To evaluate the effects of polymerase γ inhibition with alovudine in vivo, mice were xenografted with OCI-AML2 cells and then treated with alovudine. Systemic administration of alovudine reduced leukemic growth without evidence of toxicity and decreased levels of mitochondrial DNA in the leukemic cells. We also showed that alovudine increased the monocytic differentiation of AML cells. Genetic knockdown and other chemical inhibitors of polymerase γ also promoted AML differentiation, but the effects on AML differentiation were independent of reductions in oxidative phosphorylation or respiratory chain proteins. Thus, we have identified a novel mechanism by which mitochondria regulate AML fate and differentiation independent of oxidative phosphorylation. Moreover, we highlight polymerase γ inhibitors, such as alovudine, as novel therapeutic agents for AML.


Assuntos
Diferenciação Celular/efeitos dos fármacos , DNA Polimerase gama/antagonistas & inibidores , Didesoxinucleosídeos/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Mitocôndrias/patologia , Monócitos/patologia , Fosforilação Oxidativa/efeitos dos fármacos , Animais , Antivirais/farmacologia , Apoptose , Proliferação de Células , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Timidina/química , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Cell Rep ; 6(1): 141-54, 2014 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-24388747

RESUMO

Metastasis is the major cause of death in cancer patients, yet the genetic and epigenetic programs that drive metastasis are poorly understood. Here, we report an epigenetic reprogramming pathway that is required for breast cancer metastasis. Concerted differential DNA methylation is initiated by the activation of the RON receptor tyrosine kinase by its ligand, macrophage stimulating protein (MSP). Through PI3K signaling, RON/MSP promotes expression of the G:T mismatch-specific thymine glycosylase MBD4. RON/MSP and MBD4-dependent aberrant DNA methylation results in the misregulation of a specific set of genes. Knockdown of MBD4 reverses methylation at these specific loci and blocks metastasis. We also show that the MBD4 glycosylase catalytic residue is required for RON/MSP-driven metastasis. Analysis of human breast cancers revealed that this epigenetic program is significantly associated with poor clinical outcome. Furthermore, inhibition of Ron kinase activity with a pharmacological agent blocks metastasis of patient-derived breast tumor grafts in vivo.


Assuntos
Neoplasias da Mama/genética , Metilação de DNA , Endodesoxirribonucleases/metabolismo , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/metabolismo , Carcinogênese/genética , Endodesoxirribonucleases/genética , Feminino , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Células MCF-7 , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Metástase Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/genética
6.
Bioorg Med Chem Lett ; 23(16): 4517-22, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23850198

RESUMO

The discovery and potency optimization of a series of 7-aminofuro[2,3-c]pyridine inhibitors of TAK1 is described. Micromolar hits taken from high-throughput screening were optimized for biochemical and cellular mechanistic potency to ~10nM, as exemplified by compound 12az. Application of structure-based drug design aided by co-crystal structures of TAK1 with inhibitors significantly shortened the number of iterations required for the optimization.


Assuntos
MAP Quinase Quinase Quinases/antagonistas & inibidores , Piridinas , Aminas/síntese química , Aminas/química , Aminas/farmacologia , Animais , Cristalografia por Raios X , Desenho de Fármacos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Furanos/síntese química , Furanos/química , Furanos/farmacologia , Humanos , Concentração Inibidora 50 , Camundongos , Estrutura Molecular , Neoplasias/tratamento farmacológico , Piridinas/síntese química , Piridinas/farmacocinética , Piridinas/farmacologia , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Bioorg Med Chem Lett ; 23(16): 4511-6, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23856049

RESUMO

The kinase selectivity and pharmacokinetic optimization of a series of 7-aminofuro[2,3-c]pyridine inhibitors of TAK1 is described. The intersection of insights from molecular modeling, computational prediction of metabolic sites, and in vitro metabolite identification studies resulted in a simple and unique solution to both of these problems. These efforts culminated in the discovery of compound 13a, a potent, relatively selective inhibitor of TAK1 with good pharmacokinetic properties in mice, which was active in an in vivo model of ovarian cancer.


Assuntos
Inibidores Enzimáticos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Piridinas , Aminas/síntese química , Aminas/química , Aminas/farmacologia , Animais , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Furanos/síntese química , Furanos/química , Furanos/farmacologia , Humanos , Concentração Inibidora 50 , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Estrutura Molecular , Neoplasias/tratamento farmacológico , Fosfotransferases/química , Fosfotransferases/metabolismo , Piridinas/síntese química , Piridinas/farmacocinética , Piridinas/farmacologia , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Bioorg Med Chem Lett ; 23(15): 4381-7, 2013 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-23773865
9.
Mol Cancer Ther ; 8(12): 3266-75, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19996276

RESUMO

AQ4N (banoxantrone) is a prodrug that, under hypoxic conditions, is enzymatically converted to a cytotoxic DNA-binding agent, AQ4. Incorporation of AQ4N into conventional chemoradiation protocols therefore targets both oxygenated and hypoxic regions of tumors, and potentially will increase the effectiveness of therapy. This current pharmacodynamic and efficacy study was designed to quantify tumor exposure to AQ4 following treatment with AQ4N, and to relate exposure to outcome of treatment. A single dose of 60 mg/kg AQ4N enhanced the response of RT112 (bladder) and Calu-6 (lung) xenografts to treatment with cisplatin and radiation therapy. AQ4N was also given to separate cohorts of tumor-bearing mice 24 hours before tumor excision for subsequent analysis of metabolite levels. AQ4 was detected by high performance liquid chromatography/mass spectrometry in all treated samples of RT112 and Calu-6 tumors at mean concentrations of 0.23 and 1.07 microg/g, respectively. These concentrations are comparable with those shown to be cytotoxic in vitro. AQ4-related nuclear fluorescence was observed in all treated tumors by confocal microscopy, which correlated with the high performance liquid chromatography/mass spectrometry data. The presence of the hypoxic marker Glut-1 was shown by immunohistochemistry in both Calu-6 tumors and RT112 tumors, and colocalization of AQ4 fluorescence and Glut-1 staining strongly suggested that AQ4N was activated in these putatively hypoxic areas. This is the first demonstration that AQ4N will increase the efficacy of chemoradiotherapy in preclinical models; the intratumoral levels of AQ4 found in this study are comparable with tumor AQ4 levels found in a recent phase I clinical study, which suggests that these levels could be potentially therapeutic.


Assuntos
Antraquinonas/farmacologia , Neoplasias/terapia , Pró-Fármacos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Antraquinonas/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Cisplatino/farmacologia , Terapia Combinada , Citotoxinas/metabolismo , Citotoxinas/farmacologia , Sinergismo Farmacológico , Feminino , Humanos , Hipóxia , Espectrometria de Massas , Camundongos , Camundongos Nus , Microscopia Confocal , Neoplasias/metabolismo , Neoplasias/patologia , Pró-Fármacos/metabolismo , Radioterapia/métodos , Resultado do Tratamento
10.
Clin Cancer Res ; 14(4): 1096-104, 2008 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-18281542

RESUMO

PURPOSE: AQ4N is a novel bioreductive prodrug under clinical investigation. Preclinical evidence shows that AQ4N penetrates deeply within tumors and undergoes selective activation to form AQ4, a potent topoisomerase II inhibitor, in hypoxic regions of solid tumors. This proof-of-principle, phase I study evaluated the activation, hypoxic selectivity, and safety of AQ4N in patients with advanced solid tumors. EXPERIMENTAL DESIGN: Thirty-two patients with cancer (8 glioblastoma, 9 bladder, 8 head and neck, 6 breast, and 1 cervix) received a single 200 mg/m(2) dose of AQ4N before elective surgery. AQ4 and AQ4N levels in 95 tissues (tumor, healthy tissue) were assessed by liquid chromatography-tandem mass spectrometry. Tissue sections were also analyzed for AQ4 fluorescence using confocal microscopy, and for expression of the hypoxia-regulated glucose transporter, Glut-1. RESULTS: Activated AQ4 was detected in all tumor samples with highest levels present in glioblastoma (mean 1.2 microg/g) and head and neck (mean 0.65 microg/g) tumors; 22 of 32 patients had tumor AQ4 concentrations > or = 0.2 microg/g, levels previously shown to be active in preclinical studies. In 24 of 30 tumor samples, AQ4 was detected at higher concentrations than in adjacent normal tissue (tumor to normal ratio range 1.1-63.6); distant skin samples contained very low concentrations of AQ4 (mean 0.037 microg/g). Microscopic evaluation of tumor sections revealed that AQ4 colocalized within regions of Glut-1+ hypoxic cells. CONCLUSIONS: AQ4N was activated selectively in hypoxic regions in human solid tumors. Intratumoral concentrations of AQ4 exceeded those required for activity in animal models and support the evaluation of AQ4N as a novel tumor-targeting agent in future clinical studies.


Assuntos
Antraquinonas/metabolismo , Antineoplásicos/metabolismo , Neoplasias/tratamento farmacológico , Pró-Fármacos/metabolismo , Antraquinonas/farmacocinética , Antraquinonas/uso terapêutico , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Hipóxia Celular , Transportador 2 de Aminoácido Excitatório/biossíntese , Transportador 2 de Aminoácido Excitatório/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Microscopia Confocal , Pró-Fármacos/farmacocinética , Pró-Fármacos/uso terapêutico , Distribuição Tecidual
11.
Clin Cancer Res ; 13(7): 2216-25, 2007 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-17404106

RESUMO

PURPOSE: The antitumor activities and pharmacokinetics of the hypoxia-activated cytotoxin AQ4N and its metabolites were assessed in several preclinical models of pancreatic cancers. EXPERIMENTAL DESIGN: The cytotoxic effects of AQ4N prodrug and its bioreduced form, AQ4, were tested against multiple human tumor cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Nude mice bearing s.c. or orthotopically implanted human BxPC-3 or Panc-1 tumor cells were treated with AQ4N. Tumor growth inhibition, time to progression/end point, and liver metastasis were evaluated in treatment versus control groups. Plasma and tumor levels of AQ4N and its metabolites were quantitated by liquid chromatography-tandem mass spectrometry. RESULTS: In contrast to AQ4N, the bioreduced AQ4 metabolite displayed potent cytotoxicity in many human tumor lines, including those derived from human pancreatic adenocarcinomas. Single-agent administration of AQ4N significantly delayed tumor growth, progression, and survival in a manner comparable with gemcitabine in multiple pancreatic tumor models in vivo. Survival increases were accompanied by a reduction in incidence and spread of liver metastasis. Quantitation of AQ4N and its metabolites in tumor-bearing mice showed that the prodrug is rapidly cleared from the circulation by 24 h and neither of the bioreduced metabolites was detected in plasma. In contrast, AQ4N readily penetrated BxPC-3 tumors and the cytotoxic AQ4 metabolite rapidly accumulated in tumor tissues at high levels in a dose-dependent fashion. CONCLUSION: AQ4N undergoes rapid and selective conversion into the potent antineoplastic metabolite AQ4 in tumors in vivo and provides proof of principle for the use of hypoxia-activated prodrugs in the treatment against pancreatic cancers.


Assuntos
Antraquinonas/farmacologia , Antineoplásicos/farmacologia , Hipóxia Celular/fisiologia , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Antraquinonas/metabolismo , Antineoplásicos/metabolismo , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Camundongos , Camundongos Nus , Metástase Neoplásica/tratamento farmacológico , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia
12.
Cancer Res ; 65(21): 9799-806, 2005 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16267001

RESUMO

Mutation of the POLH gene encoding DNA polymerase eta (pol eta) causes the UV-sensitivity syndrome xeroderma pigmentosum-variant (XP-V) which is linked to the ability of pol eta to accurately bypass UV-induced cyclobutane pyrimidine dimers during a process termed translesion synthesis. Pol eta can also bypass other DNA damage adducts in vitro, including cisplatin-induced intrastrand adducts, although the physiological relevance of this is unknown. Here, we show that independent XP-V cell lines are dramatically more sensitive to cisplatin than the same cells complemented with functional pol eta. Similar results were obtained with the chemotherapeutic agents, carboplatin and oxaliplatin, thus revealing a general requirement for pol eta expression in providing tolerance to these platinum-based drugs. The level of sensitization observed was comparable to that of XP-A cells deficient in nucleotide excision repair, a recognized and important mechanism for repair of cisplatin adducts. However, unlike in XP-A cells, the absence of pol eta expression resulted in a reduced ability to overcome cisplatin-induced S phase arrest, suggesting that pol eta is involved in translesion synthesis past these replication-blocking adducts. Subcellular localization studies also highlighted an accumulation of nuclei with pol eta foci that correlated with the formation of monoubiquitinated proliferating cell nuclear antigen following treatment with cisplatin, reminiscent of the response to UV irradiation and further indicating a role for pol eta in dealing with cisplatin-induced damage. Together, these data show that pol eta represents an important determinant of cellular responses to cisplatin, which could have implications for acquired or intrinsic resistance to this key chemotherapeutic agent.


Assuntos
Cisplatino/farmacologia , Dano ao DNA/fisiologia , DNA Polimerase Dirigida por DNA/fisiologia , Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , DNA/efeitos dos fármacos , DNA/metabolismo , DNA Polimerase beta/metabolismo , Reparo do DNA/fisiologia , DNA Polimerase Dirigida por DNA/deficiência , DNA Polimerase Dirigida por DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fibroblastos/efeitos dos fármacos , Humanos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ubiquitina/metabolismo , Xeroderma Pigmentoso/patologia , Proteína de Xeroderma Pigmentoso Grupo A/fisiologia
13.
DNA Repair (Amst) ; 4(5): 583-93, 2005 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-15811630

RESUMO

Specialized DNA polymerases are required to bypass DNA damage lesions that would otherwise cause replication arrest and cell death. When operating on non-canonical templates, such as undamaged DNA or on non-cognate lesions, these polymerases exhibit considerably reduced fidelity, resulting in the generation of mutations. Ectopic overexpression of these polymerases can also lead to an increased mutation rate and an enhanced capability of DNA repair, suggesting that they could potentially act as oncogenes if they were overexpressed in cancers. Here, we examine expression patterns of DNA polymerases in matched normal and tumor samples from a diverse range of tissues. As well as investigating the specialized polymerases beta, lambda, iota and kappa, we also investigate the expression of the replicative polymerases alpha, delta and epsilon. The data presented provide evidence for the overexpression of specialized polymerases in tumors, with more than 45% of the 68 tumor samples studied demonstrating greater than two-fold enhanced expression of at least one specialized polymerase. Of particular note, DNA polymerase beta (pol beta) was found to be overexpressed at both the mRNA and protein level in approximately one third of all tumor types studied, with overexpression being particularly frequent in uterus, ovary, prostate and stomach samples. Pols lambda, and iota were also found to be overexpressed to a significant extent in a range of tumor types, albeit less frequently than pol beta. In contrast, pol kappa was rarely found to be overexpressed in tumors but was found to be commonly underexpressed in many samples. Downregulation of pol beta expression by siRNA resulted in an increased sensitivity to the chemotherapeutic agent cisplatin, suggesting a role for this polymerase in providing tolerance to cisplatin-induced damage. These observations suggest that specialised DNA polymerases, and particularly pol beta, could be considered both as caretaker genes altered during tumorigenesis, and as potential drug targets to sensitise tumors to chemotherapy.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Perfilação da Expressão Gênica , Neoplasias/enzimologia , Cisplatino/farmacologia , Dano ao DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/genética , Humanos , Neoplasias/genética , Neoplasias/patologia , Análise de Sequência com Séries de Oligonucleotídeos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...