ABSTRACT
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.
Subject(s)
14-3-3 Proteins/genetics , Fetal Proteins/genetics , Lymphocytes/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Protein-Tyrosine Kinases/genetics , Ubiquitin/genetics , 14-3-3 Proteins/metabolism , A549 Cells , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Fetal Proteins/antagonists & inhibitors , Fetal Proteins/metabolism , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Gene Expression Regulation, Neoplastic , HEK293 Cells , Humans , Lymphocytes/drug effects , Lymphocytes/pathology , Mice , Phospholipase C gamma/genetics , Phospholipase C gamma/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/mortality , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Protein Binding , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolism , Pyrimidines/pharmacology , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism , STAT5 Transcription Factor/genetics , STAT5 Transcription Factor/metabolism , Signal Transduction , Survival Analysis , Tumor Burden/drug effects , Ubiquitin/metabolism , Xenograft Model Antitumor AssaysABSTRACT
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.
Subject(s)
Cigarette Smoking/epidemiology , Electronic Nicotine Delivery Systems/statistics & numerical data , Health Behavior , Students/statistics & numerical data , Adolescent , Adult , Alcohol Drinking/epidemiology , Binge Drinking/epidemiology , Female , Humans , Male , Marijuana Smoking/epidemiology , Self Report , Surveys and Questionnaires , Universities , Vaping/epidemiology , Young AdultABSTRACT
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.
