RESUMO
Huntington's disease (HD) is a hereditary neurodegenerative disorder of the basal ganglia for which disease-modifying treatments are not yet available. Although gene-silencing therapies are currently being tested, further molecular mechanisms must be explored to identify druggable targets for HD. Cytoplasmic polyadenylation element binding proteins 1 to 4 (CPEB1 to CPEB4) are RNA binding proteins that repress or activate translation of CPE-containing transcripts by shortening or elongating their poly(A) tail. Here, we found increased CPEB1 and decreased CPEB4 protein in the striatum of patients and mouse models with HD. This correlated with a reprogramming of polyadenylation in 17.3% of the transcriptome, markedly affecting neurodegeneration-associated genes including PSEN1, MAPT, SNCA, LRRK2, PINK1, DJ1, SOD1, TARDBP, FUS, and HTT and suggesting a new molecular mechanism in neurodegenerative disease etiology. We found decreased protein content of top deadenylated transcripts, including striatal atrophylinked genes not previously related to HD, such as KTN1 and the easily druggable SLC19A3 (the ThTr2 thiamine transporter). Mutations in SLC19A3 cause biotin-thiamineresponsive basal ganglia disease (BTBGD), a striatal disorder that can be treated with a combination of biotin and thiamine. Similar to patients with BTBGD, patients with HD demonstrated decreased thiamine in the cerebrospinal fluid. Furthermore, patients and mice with HD showed decreased striatal concentrations of thiamine pyrophosphate (TPP), the metabolically active form of thiamine. High-dose biotin and thiamine treatment prevented TPP deficiency in HD mice and attenuated the radiological, neuropathological, and motor HD-like phenotypes, revealing an easily implementable therapy that might benefit patients with HD.
Assuntos
Doença de Huntington , Poliadenilação , Fatores de Transcrição/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Humanos , Doença de Huntington/genética , Doença de Huntington/terapia , Proteínas de Membrana Transportadoras , TranscriptomaRESUMO
HIV capsid protein is an important target for antiviral drug design. High-throughput screening campaigns have identified two classes of compounds (PF74 and BI64) that directly target HIV capsid, resulting in antiviral activity against HIV-1 and HIV-2 laboratory strains. Using recombinant proteins, we developed a suite of label-free assays to mechanistically understand how these compounds modulate capsid activity. PF74 preferentially binds to the preassembled hexameric capsid form and prevents disruption of higher-order capsid structures by stabilizing capsid intersubunit interactions. BI64 binds only the monomeric capsid and locks the protein in the assembly incompetent monomeric form by disrupting capsid intersubunit interactions. We also used these assays to characterize the interaction between capsid and the host protein cleavage and polyadenylation specific factor 6 (CPSF6). Consistent with recently published results, our assays revealed CPSF6 activates capsid polymerization and preferentially binds to the preassembled hexameric capsid form similar to the small molecule compound, PF74. Furthermore, these label-free assays provide a robust method for facilitating the identification of a different class of small molecule modulators of capsid function.
Assuntos
Fármacos Anti-HIV/farmacologia , Técnicas Biossensoriais/métodos , Capsídeo/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Sequência de Aminoácidos , Fármacos Anti-HIV/química , Fármacos Anti-HIV/metabolismo , Benzimidazóis/farmacologia , Capsídeo/química , HIV-1 , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Indóis/química , Indóis/metabolismo , Indóis/farmacologia , Dados de Sequência Molecular , Fenilalanina/análogos & derivados , Fenilalanina/química , Fenilalanina/metabolismo , Fenilalanina/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Poliadenilação e Clivagem de mRNA/genéticaAssuntos
Antineoplásicos/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Mutação , Niacinamida/análogos & derivados , Compostos de Fenilureia/uso terapêutico , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Benzamidas/farmacologia , Benzamidas/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Mesilato de Imatinib , Leucemia Mieloide Aguda/complicações , Niacinamida/uso terapêutico , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Sorafenibe , Resultado do TratamentoAssuntos
Antineoplásicos/uso terapêutico , Benzenossulfonatos/uso terapêutico , Síndrome Hipereosinofílica/tratamento farmacológico , Mutação , Piperazinas/uso terapêutico , Piridinas/uso terapêutico , Pirimidinas/uso terapêutico , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Adolescente , Adulto , Idoso , Benzamidas , Doença Crônica , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Síndrome Hipereosinofílica/genética , Mesilato de Imatinib , Masculino , Pessoa de Meia-Idade , Niacinamida/análogos & derivados , Compostos de Fenilureia , SorafenibeRESUMO
Chronic eosinophilic leukemia (CEL) is a rare myeloproliferative neoplasm characterized by the FIP1L1-PDGFRA fusion gene, variant PDGFRA fusions or other genetic lesions. Most FIP1L1-PDGFRA positive patients enjoy durable and complete molecular responses to low-dose imatinib (Glivec/Gleevec). However, resistance mediated by a T674I mutation in the ATP-binding pocket of PDGFRA has been reported in advanced disease, and sorafenib, a potent inhibitor of RAF-1, B-RAF, VEGFR and PDGFR, is active against this mutant in vitro. We describe a case of FIP1L1-PDGFRalpha T674I CEL in blast crisis that responded to sorafenib (Nexavar). However, this clinical response was short-lived because of the rapid emergence of a FIP1L1-PDGFRalpha D842V mutant. An N-Nitroso-N-ethylurea-mutagenesis screen indeed identified this mutant as a major sorafenib-resistant mutant. In vitro, the novel FIP1L1-PDGFRalpha D842V mutant is highly resistant to sorafenib, imatinib, dasatinib (Sprycell) and PKC412 (Midostaurin). Thus, sorafenib is clinically active in imatinib-resistant FIP1L1-PDGFRalpha T674I CEL, but the rapid emergence of other mutants may limit the response duration. The identification of new PDGFR inhibitors will be required to overcome resistance by this D842V mutant.
Assuntos
Benzenossulfonatos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Síndrome Hipereosinofílica/tratamento farmacológico , Síndrome Hipereosinofílica/genética , Mutação/genética , Proteínas de Fusão Oncogênica/genética , Inibidores de Proteínas Quinases/uso terapêutico , Piridinas/uso terapêutico , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Idoso , Animais , Crise Blástica , Western Blotting , Células Cultivadas , Doença Crônica , Humanos , Síndrome Hipereosinofílica/metabolismo , Masculino , Camundongos , Niacinamida/análogos & derivados , Compostos de Fenilureia , Fosforilação/efeitos dos fármacos , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/efeitos dos fármacos , Células Precursoras de Linfócitos B/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Terapia de Salvação , SorafenibeRESUMO
The FIP1L1-PDGFRA oncogene is a common cause of chronic eosinophilic leukemia (CEL), and encodes an activated tyrosine kinase that is inhibited by imatinib. FIP1L1-PDGFRA-positive patients with CEL respond to low-dose imatinib therapy, but resistance due to acquired T674I mutation has been observed. We report here the identification of sorafenib as a potent inhibitor of the FIP1 like 1-platelet-derived growth factor receptor alpha (FIP1L1-PDGFRalpha) (T674I) mutant. Sorafenib inhibited the proliferation of FIP1L1-PDGFRalpha and FIP1L1-PDGFRalpha(T674I)-transformed Ba/F3 cells and induced apoptosis of the EOL-1 cell line at a low nanomolar concentration. Western blot analysis confirmed that these effects were due to a direct effect on FIP1L1-PDGFRalpha and FIP1L1-PDGFRalpha(T674I). Sorafenib was recently approved for the treatment of renal cell carcinoma. Our data suggest that low doses of sorafenib could be efficient for the treatment of FIP1L1-PDGFRA-positive CEL and could be used to overcome resistance to imatinib associated with the T674I mutation.
Assuntos
Benzenossulfonatos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Síndrome Hipereosinofílica/metabolismo , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Piperazinas/farmacologia , Mutação Puntual , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Fatores de Poliadenilação e Clivagem de mRNA/antagonistas & inibidores , Benzamidas , Benzenossulfonatos/uso terapêutico , Carcinoma/tratamento farmacológico , Carcinoma/genética , Carcinoma/metabolismo , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Síndrome Hipereosinofílica/tratamento farmacológico , Síndrome Hipereosinofílica/genética , Mesilato de Imatinib , Células K562 , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Mutação , Niacinamida/análogos & derivados , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Compostos de Fenilureia , Inibidores de Proteínas Quinases/uso terapêutico , Piridinas/uso terapêutico , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Sorafenibe , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
Eosinophil-associated conditions, such as asthma and eosinophilic bronchitis, have been associated with chronic persistent cough, usually responding to corticosteroid therapy. This case study reports a case of persistent cough associated with gastro-oesophageal reflux (GOR) and hypereosinophilia. Treatment of GOR with proton pump inhibitors and fundoplication did not control the cough. However, high dose prednisolone, but not inhaled corticosteroids, did. The presence of the FIP1L1-PDGFRA fusion gene in myeloid cells was confirmed by fluorescence in situ hybridisation analysis using CHIC2 deletion as a surrogate marker. The cough and other disease features were subsequently suppressed by the tyrosine kinase inhibitor, imatinib. This is the first case of persistent cough caused by hypereosinophilic syndrome characterised by FIP1L1-PDGFRA fusion gene and aberrant tyrosine kinase activity.