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1.
Nat Immunol ; 21(7): 736-745, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32367036

RESUMO

Cytosolic sensing of pathogens and damage by myeloid and barrier epithelial cells assembles large complexes called inflammasomes, which activate inflammatory caspases to process cytokines (IL-1ß) and gasdermin D (GSDMD). Cleaved GSDMD forms membrane pores, leading to cytokine release and inflammatory cell death (pyroptosis). Inhibiting GSDMD is an attractive strategy to curb inflammation. Here we identify disulfiram, a drug for treating alcohol addiction, as an inhibitor of pore formation by GSDMD but not other members of the GSDM family. Disulfiram blocks pyroptosis and cytokine release in cells and lipopolysaccharide-induced septic death in mice. At nanomolar concentration, disulfiram covalently modifies human/mouse Cys191/Cys192 in GSDMD to block pore formation. Disulfiram still allows IL-1ß and GSDMD processing, but abrogates pore formation, thereby preventing IL-1ß release and pyroptosis. The role of disulfiram in inhibiting GSDMD provides new therapeutic indications for repurposing this safe drug to counteract inflammation, which contributes to many human diseases.


Assuntos
Dissulfiram/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas de Ligação a Fosfato/antagonistas & inibidores , Piroptose/efeitos dos fármacos , Sepse/tratamento farmacológico , Animais , Caspase 1/genética , Caspase 1/metabolismo , Inibidores de Caspase/farmacologia , Caspases/metabolismo , Caspases Iniciadoras/genética , Caspases Iniciadoras/metabolismo , Linhagem Celular Tumoral , Dissulfiram/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Feminino , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/imunologia , Lipossomos , Camundongos , Mutagênese Sítio-Dirigida , Proteínas de Ligação a Fosfato/genética , Proteínas de Ligação a Fosfato/metabolismo , Piroptose/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sepse/imunologia , Células Sf9 , Spodoptera
2.
J Lipid Res ; 61(7): 972-982, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32457038

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus (SARS-CoV)-2 has resulted in the death of more than 328,000 persons worldwide in the first 5 months of 2020. Herculean efforts to rapidly design and produce vaccines and other antiviral interventions are ongoing. However, newly evolving viral mutations, the prospect of only temporary immunity, and a long path to regulatory approval pose significant challenges and call for a common, readily available, and inexpensive treatment. Strategic drug repurposing combined with rapid testing of established molecular targets could provide a pause in disease progression. SARS-CoV-2 shares extensive structural and functional conservation with SARS-CoV-1, including engagement of the same host cell receptor (angiotensin-converting enzyme 2) localized in cholesterol-rich microdomains. These lipid-enveloped viruses encounter the endosomal/lysosomal host compartment in a critical step of infection and maturation. Niemann-Pick type C (NP-C) disease is a rare monogenic neurodegenerative disease caused by deficient efflux of lipids from the late endosome/lysosome (LE/L). The NP-C disease-causing gene (NPC1) has been strongly associated with viral infection, both as a filovirus receptor (e.g., Ebola) and through LE/L lipid trafficking. This suggests that NPC1 inhibitors or NP-C disease mimetics could serve as anti-SARS-CoV-2 agents. Fortunately, there are such clinically approved molecules that elicit antiviral activity in preclinical studies, without causing NP-C disease. Inhibition of NPC1 may impair viral SARS-CoV-2 infectivity via several lipid-dependent mechanisms, which disturb the microenvironment optimum for viral infectivity. We suggest that known mechanistic information on NPC1 could be utilized to identify existing and future drugs to treat COVID-19.


Assuntos
Anticolesterolemiantes/uso terapêutico , Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/genética , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Pandemias , Pneumonia Viral/tratamento farmacológico , Androstenos/uso terapêutico , Betacoronavirus/metabolismo , Betacoronavirus/patogenicidade , Colesterol/metabolismo , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/epidemiologia , Reposicionamento de Medicamentos/métodos , Humanos , Hidroxicloroquina/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/virologia , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/metabolismo , Doença de Niemann-Pick Tipo C/patologia , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/diagnóstico , Pneumonia Viral/epidemiologia , Ligação Proteica , Receptores Virais/antagonistas & inibidores , Receptores Virais/genética , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
3.
FASEB J ; 34(6): 7253-7264, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32367579

RESUMO

Drug repurposing is potentially the fastest available option in the race to identify safe and efficacious drugs that can be used to prevent and/or treat COVID-19. By describing the life cycle of the newly emergent coronavirus, SARS-CoV-2, in light of emerging data on the therapeutic efficacy of various repurposed antimicrobials undergoing testing against the virus, we highlight in this review a possible mechanistic convergence between some of these tested compounds. Specifically, we propose that the lysosomotropic effects of hydroxychloroquine and several other drugs undergoing testing may be responsible for their demonstrated in vitro antiviral activities against COVID-19. Moreover, we propose that Niemann-Pick disease type C (NPC), a lysosomal storage disorder, may provide new insights into potential future therapeutic targets for SARS-CoV-2, by highlighting key established features of the disorder that together result in an "unfavorable" host cellular environment that may interfere with viral propagation. Our reasoning evolves from previous biochemical and cell biology findings related to NPC, coupled with the rapidly evolving data on COVID-19. Our overall aim is to suggest that pharmacological interventions targeting lysosomal function in general, and those particularly capable of reversibly inducing transient NPC-like cellular and biochemical phenotypes, constitute plausible mechanisms that could be used to therapeutically target COVID-19.


Assuntos
Antivirais/farmacocinética , Betacoronavirus/fisiologia , Infecções por Coronavirus/tratamento farmacológico , Reposicionamento de Medicamentos , Endossomos/virologia , Hidroxicloroquina/farmacologia , Lisossomos/virologia , Doença de Niemann-Pick Tipo C/patologia , Pneumonia Viral/tratamento farmacológico , Proteína ADAM17/fisiologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Alanina/análogos & derivados , Alanina/farmacologia , Alanina/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Benzilisoquinolinas/farmacologia , Benzilisoquinolinas/uso terapêutico , Transporte Biológico , Catepsina L/fisiologia , Endocitose , Endossomos/efeitos dos fármacos , Endossomos/fisiologia , Glicopeptídeos/farmacologia , Glicopeptídeos/uso terapêutico , Humanos , Hidroxicloroquina/farmacocinética , Hidroxicloroquina/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/fisiologia , Doença de Niemann-Pick Tipo C/metabolismo , Oxisteróis/metabolismo , Pandemias , Peptidil Dipeptidase A/metabolismo , Receptores Virais/metabolismo , Serina Endopeptidases/fisiologia , Triazóis/farmacologia , Triazóis/uso terapêutico , Internalização do Vírus/efeitos dos fármacos
4.
EMBO Mol Med ; 12(8): e12697, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32473600

RESUMO

Baricitinib is an oral Janus kinase (JAK)1/JAK2 inhibitor approved for the treatment of rheumatoid arthritis (RA) that was independently predicted, using artificial intelligence (AI) algorithms, to be useful for COVID-19 infection via proposed anti-cytokine effects and as an inhibitor of host cell viral propagation. We evaluated the in vitro pharmacology of baricitinib across relevant leukocyte subpopulations coupled to its in vivo pharmacokinetics and showed it inhibited signaling of cytokines implicated in COVID-19 infection. We validated the AI-predicted biochemical inhibitory effects of baricitinib on human numb-associated kinase (hNAK) members measuring nanomolar affinities for AAK1, BIKE, and GAK. Inhibition of NAKs led to reduced viral infectivity with baricitinib using human primary liver spheroids. These effects occurred at exposure levels seen clinically. In a case series of patients with bilateral COVID-19 pneumonia, baricitinib treatment was associated with clinical and radiologic recovery, a rapid decline in SARS-CoV-2 viral load, inflammatory markers, and IL-6 levels. Collectively, these data support further evaluation of the anti-cytokine and anti-viral activity of baricitinib and support its assessment in randomized trials in hospitalized COVID-19 patients.


Assuntos
Antivirais/farmacologia , Inteligência Artificial , Azetidinas/farmacologia , Betacoronavirus , Infecções por Coronavirus/tratamento farmacológico , Pandemias , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Sulfonamidas/farmacologia , Adulto , Idoso , Antivirais/farmacocinética , Antivirais/uso terapêutico , Azetidinas/farmacocinética , Azetidinas/uso terapêutico , Citocinas/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Leucócitos/efeitos dos fármacos , Fígado , Masculino , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/virologia , Sulfonamidas/farmacocinética , Sulfonamidas/uso terapêutico
5.
Nat Chem Biol ; 16(6): 635-643, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32251410

RESUMO

Doublecortin like kinase 1 (DCLK1) is an understudied kinase that is upregulated in a wide range of cancers, including pancreatic ductal adenocarcinoma (PDAC). However, little is known about its potential as a therapeutic target. We used chemoproteomic profiling and structure-based design to develop a selective, in vivo-compatible chemical probe of the DCLK1 kinase domain, DCLK1-IN-1. We demonstrate activity of DCLK1-IN-1 against clinically relevant patient-derived PDAC organoid models and use a combination of RNA-sequencing, proteomics and phosphoproteomics analysis to reveal that DCLK1 inhibition modulates proteins and pathways associated with cell motility in this context. DCLK1-IN-1 will serve as a versatile tool to investigate DCLK1 biology and establish its role in cancer.


Assuntos
Carcinoma Ductal Pancreático/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Movimento Celular , Ensaios de Seleção de Medicamentos Antitumorais , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Proteínas Quinases/farmacocinética , Proteômica , Ratos , Relação Estrutura-Atividade , Peixe-Zebra
6.
J Med Chem ; 63(8): 4315-4333, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32223236

RESUMO

The frequent deregulation of MYC and its elevated expression via multiple mechanisms drives cells to a tumorigenic state. Indeed, MYC is overexpressed in up to ∼50% of human cancers and is considered a highly validated anticancer target. Recently, we discovered that WD repeat-containing protein 5 (WDR5) binds to MYC and is a critical cofactor required for the recruitment of MYC to its target genes and reported the first small molecule inhibitors of the WDR5-MYC interaction using structure-based design. These compounds display high binding affinity, but have poor physicochemical properties and are hence not suitable for in vivo studies. Herein, we conducted an NMR-based fragment screening to identify additional chemical matter and, using a structure-based approach, we merged a fragment hit with the previously reported sulfonamide series. Compounds in this series can disrupt the WDR5-MYC interaction in cells, and as a consequence, we observed a reduction of MYC localization to chromatin.


Assuntos
Desenho de Fármacos , Descoberta de Drogas/métodos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Sulfonamidas/síntese química , Sulfonamidas/farmacologia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-myc/metabolismo , Relação Estrutura-Atividade
7.
EBioMedicine ; 52: 102664, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32062360

RESUMO

BACKGROUND: Acute myeloid leukaemia (AML) is a malignant haematological tumour with high heterogeneity and mortality. A reliable prognostic assessment is critical for treatment strategies. However, the current prognostic evaluation system of AML is insufficient. METHODS: Genome-wide univariate Cox regression analysis was performed on three independent AML datasets to screen for the prognostic-related genes. Kaplan-Meier survival analysis was employed to verify the efficacy of FHL1 in evaluating overall survival in 1298 de novo AML patients, 648 non-acute promyelocytic leukaemia AML patients and 407 cytogenetically normal AML patients; the data for some of these patients were also used for EFS and RFS validation. Multivariate Cox regression was performed to validate FHL1 as an independent prognostic indicator. WGCNA, GSEA, and gene correlation analysis were applied to explore the mechanism of FHL1 in AML. The synergistic cytocidal effect of FHL1 knockdown was verified in in vitro experiments. FINDINGS: Comprehensive genome-wide analyses and large-sample validation showed that FHL1 is a powerful prognostic candidate for overall survival, event-free survival, and relapse-free survival in AML and is independent of prognosis-related clinical factors and genetic abnormalities. The molecular mechanism may occur through regulation of FHL1 in leukaemia stem cells, tumour-associated signalling pathways, and transmembrane transport of chemotherapeutic drugs. FHL1-targeted intervention enhances the sensitivity of AML cells to cytarabine. INTERPRETATION: FHL1 may serve as an evaluation factor for clinical strategy selection, and its targeted intervention may be beneficial for chemotherapy in AML patients.


Assuntos
Biomarcadores Tumorais , Estudo de Associação Genômica Ampla , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/genética , Leucemia Mieloide Aguda/genética , Proteínas Musculares/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biologia Computacional/métodos , Feminino , Perfilação da Expressão Gênica , Genômica/métodos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Estimativa de Kaplan-Meier , Proteínas com Domínio LIM/antagonistas & inibidores , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/terapia , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Proteínas Musculares/antagonistas & inibidores , Prognóstico , Curva ROC , Adulto Jovem
8.
Biochem Biophys Res Commun ; 524(4): 839-846, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32046858

RESUMO

Increasing evidences demonstrate that circular RNAs (circRNAs) are extensively implicated in various cancers including colorectal cancer (CRC). In the present study, we found that circRNA HIPK3 (circPIK3) was upregulated in CRC. We identified that circHIPK3 was closely related with unfavorable clinicopathological features in patients with CRC. Functional transwell assay and proliferation assay indicated that circHIPK3 served as an oncogene and promoted CRC cells migration, invasion and proliferation. Meanwhile, we found that formin like 2 (FMNL2) was a key downstream molecule in circHIPK3-induced metastasis and proliferation in CRC cells. We further verified that circHIPK3 was mainly located at cytoplasm through an immunofluorescence assay. An online bioinformatics screening and a GEO datasets analysis showed that microRNA 1207-5p (miR-1207-5p) was downregulated in CRC. Also, we found that miR-1207-5p shared a similar miR-1207-5p response elements (MREs-1207-5p). Meanwhile, we showed that miR-1207-5p suppressed CRC cells migration, invasion and proliferation via directly targeting of FMNL2. Even further, via a constructed luciferase assay, we indicated that circHIPK3 was another target of miR-1207-5p. Functionally, we proved that circHIPK3 enhanced FMNL2 mediated promotion of migration, invasion and proliferation by sponging of miR-1207-5p in CRC cells. In summary, the outcomes of this study illustrated that circHIPK3 promoted CRC cells migration, invasion and proliferation modulating of FMNL2 by sponging of miR-1207-5p. Our findings indicated that circHIPK3/miR-1207-5p/FMNL2 axis might be a new strategy in molecular treatment of CRC.


Assuntos
Neoplasias Colorretais/genética , Forminas/genética , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , MicroRNAs/genética , Proteínas Serina-Treonina Quinases/genética , RNA Circular/genética , Idoso , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Feminino , Forminas/antagonistas & inibidores , Forminas/metabolismo , Células HT29 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metástase Linfática , Masculino , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , RNA Circular/antagonistas & inibidores , RNA Circular/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Análise de Sobrevida
9.
Biochem J ; 477(4): 801-814, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32011652

RESUMO

Autophagy is a critical cellular homeostatic mechanism, the dysfunction of which has been linked to a wide variety of disease states. It is regulated through the activity of specific kinases, in particular Unc-51 like autophagy activating kinase 1 (ULK1) and Phosphatidylinositol 3-kinase vacuolar protein sorting 34 (VPS34), which have both been suggested as potential targets for drug development. To identify new chemical compounds that might provide useful chemical tools or act as starting points for drug development, we screened each protein against the Published Kinase Inhibitor Set (PKIS), a library of known kinase inhibitors. In vitro screening and analysis of the published selectivity profiles of the hits informed the selection of three relatively potent ATP-competitive inhibitors against each target that presented the least number of off-target kinases in common. Cellular assays confirmed potent inhibition of autophagy in response to two of the ULK1 inhibitors and all three of the VPS34 inhibitors. These compounds represent not only a new resource for the study of autophagy but also potential chemical starting points for the validation or invalidation of these two centrally important autophagy kinases in disease models.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/antagonistas & inibidores , Autofagia , Classe III de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Descoberta de Drogas , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Osteossarcoma/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Osteossarcoma/tratamento farmacológico , Osteossarcoma/metabolismo , Fosforilação , Células Tumorais Cultivadas
10.
Nat Commun ; 11(1): 152, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31919352

RESUMO

Niemann-Pick C1 (NPC1), a lysosomal protein of 13 transmembrane helices (TMs) and three lumenal domains, exports low-density-lipoprotein (LDL)-derived cholesterol from lysosomes. TMs 3-7 of NPC1 comprise the Sterol-Sensing Domain (SSD). Previous studies suggest that mutation of the NPC1-SSD or the addition of the anti-fungal drug itraconazole abolishes NPC1 activity in cells. However, the itraconazole binding site and the mechanism of NPC1-mediated cholesterol transport remain unknown. Here, we report a cryo-EM structure of human NPC1 bound to itraconazole, which reveals how this binding site in the center of NPC1 blocks a putative lumenal tunnel linked to the SSD. Functional assays confirm that blocking this tunnel abolishes NPC1-mediated cholesterol egress. Intriguingly, the palmitate anchor of Hedgehog occupies a similar site in the homologous tunnel of Patched, suggesting a conserved mechanism for sterol transport in this family of proteins and establishing a central function of their SSDs.


Assuntos
Antifúngicos/farmacologia , Colesterol/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Itraconazol/farmacologia , Animais , Sítios de Ligação/genética , Transporte Biológico/fisiologia , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Microscopia Crioeletrônica , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Receptor Patched-1/metabolismo , Domínios Proteicos
11.
Intern Med ; 59(2): 271-275, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31941873

RESUMO

Anti-leucine-rich glioma inactivated-1 (anti-LGI1) encephalitis is a subgroup of autoimmune encephalitis. We herein report the case of a 60-year-old man who presented with typical symptoms, including short-term memory loss, mental abnormalities, hyponatremia and seizures characterized by faciobrachial dystonic seizures and who was diagnosed with anti-LGI1 encephalitis. At the same time, he was diagnosed with essential thrombocythemia. A significant improvement was obtained by treatment with corticosteroid, immunoglobulin, mycophenolate mofetil, and hydroxyurea. Autoimmune diseases are associated with a significantly increased risk of developing myeloproliferative neoplasms, which may explain the coexistence of anti-LGI1 encephalitis and essential thrombocythema in this patient; however, but more cases and studies are needed to determine whether there is any correlation between these conditions.


Assuntos
Encefalite/complicações , Glioma/complicações , Doença de Hashimoto/complicações , Encefalite Límbica/complicações , Trombocitemia Essencial/complicações , Corticosteroides/uso terapêutico , Autoanticorpos/imunologia , Encefalite/tratamento farmacológico , Doença de Hashimoto/tratamento farmacológico , Humanos , Hiponatremia/etiologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Leucina , Encefalite Límbica/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Convulsões/etiologia , Trombocitemia Essencial/tratamento farmacológico
12.
PLoS One ; 15(1): e0227727, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31940398

RESUMO

We sought to design ubiquitin-proteasome system inhibitors active against solid cancers by targeting ubiquitin receptor RPN13 within the proteasome's 19S regulatory particle. The prototypic bis-benzylidine piperidone-based inhibitor RA190 is a michael acceptor that adducts Cysteine 88 of RPN13. In probing the pharmacophore, we showed the benefit of the central nitrogen-bearing piperidone ring moiety compared to a cyclohexanone, the importance of the span of the aromatic wings from the central enone-piperidone ring, the contribution of both wings, and that substituents with stronger electron withdrawing groups were more cytotoxic. Potency was further enhanced by coupling of a second warhead to the central nitrogen-bearing piperidone as RA375 exhibited ten-fold greater activity against cancer lines than RA190, reflecting its nitro ring substituents and the addition of a chloroacetamide warhead. Treatment with RA375 caused a rapid and profound accumulation of high molecular weight polyubiquitinated proteins and reduced intracellular glutathione levels, which produce endoplasmic reticulum and oxidative stress, and trigger apoptosis. RA375 was highly active against cell lines of multiple myeloma and diverse solid cancers, and demonstrated a wide therapeutic window against normal cells. For cervical and head and neck cancer cell lines, those associated with human papillomavirus were significantly more sensitive to RA375. While ARID1A-deficiency also enhanced sensitivity 4-fold, RA375 was active against all ovarian cancer cell lines tested. RA375 inhibited proteasome function in muscle for >72h after single i.p. administration to mice, and treatment reduced tumor burden and extended survival in mice carrying an orthotopic human xenograft derived from a clear cell ovarian carcinoma.


Assuntos
Antineoplásicos/farmacologia , Compostos de Benzilideno/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Inibidores de Proteassoma/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Compostos de Benzilideno/química , Compostos de Benzilideno/uso terapêutico , Linhagem Celular Tumoral , Feminino , Humanos , Concentração Inibidora 50 , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Estrutura Molecular , Neoplasias/genética , Neoplasias/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/química , Inibidores de Proteassoma/uso terapêutico , Ligação Proteica , Relação Estrutura-Atividade , Ubiquitina/antagonistas & inibidores , Ubiquitina/metabolismo , Proteínas Ubiquitinadas/metabolismo , Ubiquitinação/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
13.
EBioMedicine ; 52: 102632, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31981983

RESUMO

BACKGROUND: Small cell lung cancer (SCLC) has a poor prognosis, and its treatment options are limited. Delta-like protein 3 (DLL3) is expressed specifically in SCLC and is considered a promising therapeutic target for patients with this disease. Rovalpituzumab tesirine (Rova-T) was the first antibody-drug conjugate targeting DLL3. Although Rova-T development was unfortunately terminated, DLL3 remains an ideal target for SCLC. Near infrared photoimmunotherapy (NIR-PIT) is a new form of cancer treatment that employs an antibody-photosensitiser conjugate followed by NIR light exposure and damage target cells specifically. In this study, we demonstrate DLL3-targeted NIR-PIT to develop a novel molecularly targeted treatment for SCLC. METHODS: The anti-DLL3 monoclonal antibody rovalpituzumab was conjugated to an IR700 photosensitiser (termed 'rova-IR700'). SCLC cells overexpressing DLL3 as well as non-DLL3-expressing controls were incubated with rova-IR700 and then exposed to NIR-light. Next, mice with SCLC xenografts were injected with rova-IR700 and irradiated with NIR-light. FINDINGS: DLL3-overexpressing cells underwent immediate destruction upon NIR-light exposure, whereas the control cells remained intact. The xenograft in mice treated with rova-IR700 and NIR-light shrank markedly, whereas neither rova-IR700 injection nor NIR-light irradiation alone affected tumour size. INTERPRETATION: Our data suggest that targeting of DLL3 using NIR-PIT could be a novel and promising treatment for SCLC. FUNDING: Research supported by grants from the Program for Developing Next-generation Researchers (Japan Science and Technology Agency), KAKEN (18K15923, JSPS), Medical Research Encouragement Prize of The Japan Medical Association, The Nitto Foundation, Kanae Foundation for the Promotion of Medical Science.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Luz , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Fototerapia , Carcinoma de Pequenas Células do Pulmão/metabolismo , Animais , Biomarcadores , Linhagem Celular Tumoral , Modelos Animais de Doenças , Imunofluorescência , Humanos , Imunoconjugados/farmacologia , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Fototerapia/métodos , Ligação Proteica/imunologia , Carcinoma de Pequenas Células do Pulmão/patologia , Carcinoma de Pequenas Células do Pulmão/terapia , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Cancer Biother Radiopharm ; 35(1): 41-49, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31916845

RESUMO

Background: NRAGE (neurotrophin receptor-interacting melanoma antigen-encoding gene homolog) has a complex role and regulates cell growth in different tumor cells. Although NRAGE was been discovered for more than 10 years ago, the function of NRAGE in hepatoblastoma (HB) cells is currently unknown. Materials and Methods: The expression of NRAGE was detected by reverse transcription-quantitative polymerase chain reaction assay or western blotting assay. Cellular apoptosis was analyzed to estimate the effect of NRAGE under radiation. The ability of clonogenic capacity was evaluated to confirm the influence of proliferation for NRAGE by radiation. The immunofluorescence assay was used to further study the expression of NRAGE under radiation. A nude mouse tumor xenograft model was constructed to confirm the effect of NRAGE deficiency under radiation conditions in vivo. Results: The authors determined that deletion of NRAGE significantly inhibited HB cell proliferation in vitro and in vivo, and NRAGE knockdown apparently sensitized HB cells to ionizing radiation (IR). Further mechanistic studies revealed that NRAGE plays a critical role in homologous recombination by inhibiting the expression of RNF8 (ring finger protein 8) and BARD1 (BRCA1 associated RING domain 1) and the recruitment of RAD51. Conclusions: The authors demonstrated that downregulation of NRAGE sensitizes HB cell lines to IR in vitro and in vivo. It provides a promising therapeutic strategy for HB patients by specifically targeting NRAGE.


Assuntos
Antígenos de Neoplasias/biossíntese , Hepatoblastoma/genética , Hepatoblastoma/radioterapia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/radioterapia , Proteínas de Neoplasias/biossíntese , Reparo de DNA por Recombinação , Animais , Antígenos de Neoplasias/genética , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Regulação para Baixo , Técnicas de Silenciamento de Genes , Células HEK293 , Células Hep G2 , Hepatoblastoma/metabolismo , Hepatoblastoma/patologia , Xenoenxertos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Transfecção , Proteínas com Motivo Tripartido/antagonistas & inibidores , Proteínas com Motivo Tripartido/biossíntese , Proteínas com Motivo Tripartido/genética , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitina-Proteína Ligases/genética
15.
Int J Oncol ; 56(2): 430-438, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31894299

RESUMO

The disruption of protein translation machinery is a common feature of cancer initiation and progression, and drugs that target protein translation offer new avenues for therapy. The translation initiation factor, eukaryotic initiation factor 4E (eIF4E), is induced in a number of cancer cell lines and is one such candidate for therapeutic intervention. Friend leukemia integration 1 (FLI1) is a potent oncogenic transcription factor that promotes various types of cancer by promoting several hallmarks of cancer progression. FLI1 has recently been implicated in protein translation through yet unknown mechanisms. This study identified a positive association between FLI1 expression and mitogen­activated protein kinase (MAPK)­interacting serine/threonine kinase1 (MKNK1), the immediate upstream regulator of the eIF4E initiation factor. The short hairpin RNA (shRNA)­mediated silencing or overexpression of FLI1 in leukemic cell lines downregulated or upregulated MKNK1 expression, respectively. Promoter analysis identified a potent FLI1 binding site in the regulatory region of the MKNK1 promoter. In transient transfection experiments, FLI1 increased MKNK1 promoter activity, which was blocked by mutating the FLI1 binding site. FLI1 specifically affected the expression of MKNK1, but not that of MKNK2. The siRNA­mediated downregulation of MKNK1 downregulated the expression of survivin (BIRC5) and significantly suppressed cell proliferation in culture. FLI1 inhibitory compounds were shown to downregulate this oncogene through the suppression of MAPK/extracellular­regulated kinase (ERK) signaling and the subsequent activation of miR­145, leading to a lower MKNK1 expression and the suppression of leukemic growth. These results uncover a critical role for FLI1 in the control of protein translation and the importance of targeting its function and downstream mediators, such as MKNK1, for cancer therapy.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucemia Eritroblástica Aguda/genética , Biossíntese de Proteínas/genética , Proteínas Serina-Treonina Quinases/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Transcrição Genética/genética , Compostos de Anilina , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Fator de Iniciação 4E em Eucariotos/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Eritroblástica Aguda/tratamento farmacológico , Leucemia Eritroblástica Aguda/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , MicroRNAs/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Regiões Promotoras Genéticas/genética , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Proto-Oncogênica c-fli-1/antagonistas & inibidores , Purinas , RNA Interferente Pequeno/metabolismo , Survivina/metabolismo , Transcrição Genética/efeitos dos fármacos
16.
J Med Chem ; 63(2): 621-637, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31910010

RESUMO

Dysregulation of translation initiation factor 4E (eIF4E) activity occurs in various cancers. Mitogen-activated protein kinase (MAPK) interacting kinases 1 and 2 (MNK1 and MNK2) play a fundamental role in activation of eIF4E. Structure-activity relationship-driven expansion of a fragment hit led to discovery of dual MNK1 and MNK2 inhibitors based on a novel pyridine-benzamide scaffold. The compounds possess promising in vitro and in vivo pharmacokinetic profiles and show potent on target inhibition of eIF4E phosphorylation in cells.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Linhagem Celular Tumoral , Cristalografia por Raios X , Descoberta de Drogas , Fator de Iniciação 4E em Eucariotos/antagonistas & inibidores , Humanos , Modelos Moleculares , Fosforilação , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Relação Estrutura-Atividade
17.
Ann Neurol ; 87(3): 405-418, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31900946

RESUMO

OBJECTIVE: Leucine-rich glioma-inactivated 1 (LGI1) encephalitis is the second most common antibody-mediated encephalopathy, but insight into the intrathecal B-cell autoimmune response, including clonal relationships, isotype distribution, frequency, and pathogenic effects of single LGI1 antibodies, has remained limited. METHODS: We cloned, expressed, and tested antibodies from 90 antibody-secreting cells (ASCs) and B cells from the cerebrospinal fluid (CSF) of several patients with LGI1 encephalitis. RESULTS: Eighty-four percent of the ASCs and 21% of the memory B cells encoded LGI1-reactive antibodies, whereas reactivities to other brain epitopes were rare. All LGI1 antibodies were of IgG1, IgG2, or IgG4 isotype and had undergone affinity maturation. Seven of the overall 26 LGI1 antibodies efficiently blocked the interaction of LGI1 with its receptor ADAM22 in vitro, and their mean LGI1 signal on mouse brain sections was weak compared to the remaining, non-ADAM22-competing antibodies. Nevertheless, both types of LGI1 antibodies increased the intrinsic cellular excitability and glutamatergic synaptic transmission of hippocampal CA3 neurons in slice cultures. INTERPRETATION: Our data show that the patients' intrathecal B-cell autoimmune response is dominated by LGI1 antibodies and that LGI1 antibodies alone are sufficient to promote neuronal excitability, a basis of seizure generation. Fundamental differences in target specificity and antibody hypermutations compared to the CSF autoantibody repertoire in N-methyl-D-aspartate receptor encephalitis underline the clinical concept that autoimmune encephalitides are very distinct entities. Ann Neurol 2020;87:405-418.


Assuntos
Anticorpos Monoclonais/farmacologia , Autoanticorpos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Neurônios/fisiologia , Proteínas ADAM/efeitos dos fármacos , Idoso , Animais , Anticorpos Monoclonais/líquido cefalorraquidiano , Autoanticorpos/líquido cefalorraquidiano , Região CA3 Hipocampal/fisiologia , Células Cultivadas , Encefalite/líquido cefalorraquidiano , Encefalite/imunologia , Feminino , Doença de Hashimoto/líquido cefalorraquidiano , Doença de Hashimoto/imunologia , Humanos , Isotipos de Imunoglobulinas , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/efeitos dos fármacos , Ratos , Transmissão Sináptica/efeitos dos fármacos
18.
Eur J Med Chem ; 188: 112027, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31923859

RESUMO

Genetic rearrangements of the mixed lineage leukemia (MLL) leading to oncogenic MLL-fusion proteins (MLL-FPs). MLL-FPs occur in about 10% of acute leukemias and are associated with dismal prognosis and treatment outcomes which emphasized the need for new therapeutic strategies. In present study, by a cell-based screening in-house compound collection, we disclosed that Rabeprazole specially inhibited the proliferation of leukemia cells harboring MLL-FPs with little toxicity to non-MLL cells. Mechanism study showed Rabeprazole down-regulated the transcription of MLL-FPs related Hox and Meis1 genes and effectively inhibited MLL1 H3K4 methyltransferase (HMT) activity in MV4-11 cells bearing MLL-AF4 fusion protein. Displacement of MLL1 probe from WDR5 protein suggested that Rabeprazole may inhibit MLL1 HMT activity through disturbing MLL1-WDR5 protein-protein interaction. Moreover, other proton pump inhibitors (PPIs) also indicated the inhibition activity of MLL1-WDR5. Preliminary SARs showed the structural characteristics of PPIs were also essential for the activities of MLL1-WDR5 inhibition. Our results indicated the drug reposition of PPIs for MLL-rearranged leukemias and provided new insight for further optimization of targeting MLL1 methyltransferase activity, the MLL1-WDR5 interaction or WDR5.


Assuntos
Antineoplásicos/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Leucemia/tratamento farmacológico , Proteína de Leucina Linfoide-Mieloide/antagonistas & inibidores , Inibidores da Bomba de Prótons/farmacologia , Rabeprazol/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia/metabolismo , Leucemia/patologia , Estrutura Molecular , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Ligação Proteica/efeitos dos fármacos , Inibidores da Bomba de Prótons/síntese química , Inibidores da Bomba de Prótons/química , Rabeprazol/síntese química , Rabeprazol/química , Relação Estrutura-Atividade
19.
Life Sci ; 243: 117234, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31887299

RESUMO

PURPOSE: In acute myeloid leukemia (AML), complete remission can be achieved in parts of patients using cytarabine/anthracycline combination-based chemotherapy, however, drug resistance-related recurrence is still a common cause of treatment failure, leading to high mortality among patients. In our research, we revealed the molecular mechanisms that were sufficient to improve sensitivity of AML cells to the anthracycline daunorubicin (DNR). METHODS: We evaluated the effects of autophagy and apoptosis induced by DNR using two AML cell lines HL60 and U937.Western blot was preformed to analyze the apoptotic pathway protein expression and flow cytometric analysis was used to detect the level of apoptosis in AML cells. The levels of autophagy-related proteins were detected by western blotting and autophagic vesicles were observed by electron microscopy. RESULTS: DNR effectively induced autophagy in two AML cell lines HL60 and U937 confirming by upregulation of LC3-II lipidation, formation of autophagosomes. Inhibition of autophagy by pharmacologic inhibitor HCQ promoted apoptosis induced by DNR, suggesting that autophagy played a vital role in pro-survival in AML. Furthermore, ULK1 inhibition by a highly selective kinase inhibitor SBI-0206965 and shRNA enhanced cytotoxicity of DNR against AML cells. Independent of mTOR -ULK1 signaling pathway, activation of autophagy of DNR was proved to be mediated by AMPK (pThr172)/ULK1 pathway. CONCLUSIONS: These results revealed that pro-survival autophagy induced by ULK1 activation was one of the potential mechanisms of AML resistance to DNR. Targeting ULK1 selectively could be a promising therapeutic strategy to enhance sensitivity of DNR for AML therapy.


Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/antagonistas & inibidores , Daunorrubicina/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Antibióticos Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Daunorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/farmacologia , Células HL-60 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/patologia , Fosforilação , Células U937
20.
Cancer Res ; 80(4): 798-810, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31882401

RESUMO

Patients with melanoma resistant to RAF/MEK inhibitors (RMi) are frequently resistant to other therapies, such as immune checkpoint inhibitors (ICI), and individuals succumb to their disease. New drugs that control tumor growth and favorably modulate the immune environment are therefore needed. We report that the small-molecule CX-6258 has potent activity against both RMi-sensitive (RMS) and -resistant (RMR) melanoma cell lines. Haspin kinase (HASPIN) was identified as a target of CX-6258. HASPIN inhibition resulted in reduced proliferation, frequent formation of micronuclei, recruitment of cGAS, and activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. In murine models, CX-6258 induced a potent cGAS-dependent type-I IFN response in tumor cells, increased IFNγ-producing CD8+ T cells, and reduced Treg frequency in vivo. HASPIN was more strongly expressed in malignant compared with healthy tissue and its inhibition by CX-6258 had minimal toxicity in ex vivo-expanded human tumor-infiltrating lymphocytes (TIL), proliferating TILs, and in vitro differentiated neurons, suggesting a potential therapeutic index for anticancer therapy. Furthermore, the activity of CX-6258 was validated in several Ewing sarcoma and multiple myeloma cell lines. Thus, HASPIN inhibition may overcome drug resistance in melanoma, modulate the immune environment, and target a vulnerability in different cancer lineages. SIGNIFICANCE: HASPIN inhibition by CX-6258 is a novel and potent strategy for RAF/MEK inhibitor-resistant melanoma and potentially other tumor types. HASPIN inhibition has direct antitumor activity and induces a favorable immune microenvironment.


Assuntos
Azepinas/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Indóis/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Melanoma/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Neoplasias Cutâneas/tratamento farmacológico , Animais , Azepinas/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/imunologia , Feminino , Técnicas de Silenciamento de Genes , Humanos , Indóis/uso terapêutico , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Melanoma/imunologia , Melanoma/patologia , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases raf/antagonistas & inibidores
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