RESUMO
TNK1 is a non-receptor tyrosine kinase with poorly understood biological function and regulation. Here, we identify TNK1 dependencies in primary human cancers. We also discover a MARK-mediated phosphorylation on TNK1 at S502 that promotes an interaction between TNK1 and 14-3-3, which sequesters TNK1 and inhibits its kinase activity. Conversely, the release of TNK1 from 14-3-3 allows TNK1 to cluster in ubiquitin-rich puncta and become active. Active TNK1 induces growth factor-independent proliferation of lymphoid cells in cell culture and mouse models. One unusual feature of TNK1 is a ubiquitin-association domain (UBA) on its C-terminus. Here, we characterize the TNK1 UBA, which has high affinity for poly-ubiquitin. Point mutations that disrupt ubiquitin binding inhibit TNK1 activity. These data suggest a mechanism in which TNK1 toggles between 14-3-3-bound (inactive) and ubiquitin-bound (active) states. Finally, we identify a TNK1 inhibitor, TP-5801, which shows nanomolar potency against TNK1-transformed cells and suppresses tumor growth in vivo.
Assuntos
Proteínas 14-3-3/genética , Proteínas Fetais/genética , Linfócitos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Proteínas Tirosina Quinases/genética , Ubiquitina/genética , Proteínas 14-3-3/metabolismo , Células A549 , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proteínas Fetais/antagonistas & inibidores , Proteínas Fetais/metabolismo , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/patologia , Camundongos , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Pirimidinas/farmacologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Análise de Sobrevida , Carga Tumoral/efeitos dos fármacos , Ubiquitina/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Objective: E-cigarette use among college students has increased drastically in recent years. This study aims to inform development of a comprehensive measure of e-cigarette consumption for this population. Participants and Methods: This mixed-method study collected both quantitative and qualitative data from a sample of 43 experienced e-cigarette users from two college campuses, using a self-reported questionnaire and a semi-structured interview, in March-September of 2017. Results: College student users found some of the consumption questions in national surveys difficult to answer. Switching nicotine levels, mixing flavors, co-using with alcohol and marijuana, using with others who were vaping or drinking, and vaping in a car or indoor space were all common. The participants defined "regular user" based on ownership of a device rather than on quantity/frequency. Conclusions: A comprehensive e-cigarette consumption measure should cover the complex characteristics that are potentially associated with negative health consequences such as flavorings, co-use and social contexts.
Assuntos
Fumar Cigarros/epidemiologia , Sistemas Eletrônicos de Liberação de Nicotina/estatística & dados numéricos , Comportamentos Relacionados com a Saúde , Estudantes/estatística & dados numéricos , Adolescente , Adulto , Consumo de Bebidas Alcoólicas/epidemiologia , Consumo Excessivo de Bebidas Alcoólicas/epidemiologia , Feminino , Humanos , Masculino , Fumar Maconha/epidemiologia , Autorrelato , Inquéritos e Questionários , Universidades , Vaping/epidemiologia , Adulto JovemRESUMO
We report the application of phosphoramidate pronucleotide (ProTide) technology to the antiviral agent carbocyclic L-d4A (L-Cd4A). The phenyl methyl alaninyl parent ProTide of L-Cd4A was prepared by Grignard-mediated phosphorochloridate reaction and resulted in a compound with significantly improved anti-HIV (2600-fold) and HBV activity. We describe modifications of the aryl, ester, and amino acid regions of the ProTide and how these changes affect antiviral activity and metabolic stability. Separate and distinct SARs were noted for HIV and HBV. Additionally, ProTides were prepared from the D-nucleoside D-Cd4A and the dideoxy analogues L-CddA and D-CddA. These compounds showed more modest potency improvements over the parent drug. In conclusion, the ProTide approach is highly successful when applied to L-Cd4A with potency improvements in vitro as high as 9000-fold against HIV. With a view to preclinical candidate selection we carried out metabolic stability studies using cynomolgus monkey liver and intestinal S9 fractions.
