ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTThymidylate synthetase inhibitors. Synthesis of N-substituted 5-aminomethyl-2'-deoxyuridine 5'-phosphatesMarshall S. Edelman, Roxane L. Barfknecht, Rocio Huet-Rose, Sophie Boguslawski, and Mathias P. MertesCite this: J. Med. Chem. 1977, 20, 5, 669–673Publication Date (Print):May 1, 1977Publication History Published online1 May 2002Published inissue 1 May 1977https://pubs.acs.org/doi/10.1021/jm00215a010https://doi.org/10.1021/jm00215a010research-articleACS PublicationsRequest reuse permissionsArticle Views64Altmetric-Citations12LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
288 The PI3 Kinase/AKT pathway plays a critical role in cell survival signaling and the activation of this pathway has been linked to tumorigenesis. Upregulation of AKT (PKB) and PI3 Kinase is seen in many tumor types and the negative regulator of this pathway PTEN is deleted in a number of tumor types. Therefore, this pathway remains an attractive target for cancer drug discovery. Glycogen Synthase Kinase-beta (GSK3b) is characterized to be phosphorylated at its Ser 9 site by AKT. Phosphorylation of GSK3b by AKT results in inactivation of the kinase activity of GSK3b and downstream activation of glycogen synthesis and metabolic events leading to and promoting cell proliferation. Therefore, phosphorylation of GSK3b at the Ser 9 site can be utilized as a pharmacodynamic marker for evaluating the in vivo PK/PD effects of AKT/PI3K inhibitors. We developed a novel in cell ELISA to measure the phosphorylation of GSK3b in U87MG cells. In order to link cellular target inhibition to in vivo target inhibition, we utilized a commercially available sandwich ELISA to measure the in vivo activity of PI3 Kinase/AKT pathway inhibitors quantitating the phosphorylation level of GSK3b in native U87MG glioblastoma cells. We treated these cells and mice bearing U87MG xenografts with the PI3 Kinase inhibitor wortmannin and an Isoquinoline Sulfonamide (IS) AKT inhibitor. The ELISA analysis showed that PI3 Kinase inhibitor Wortmannin and IS AKT inhibitor inhibits GSK3b phosphorylation U87MG cells potently in vitro and in vivo. The inhibition correlates well with the inhibition of phosphorylation of other known AKT substrates such as phospho-Forkhead and phospho-TSC2. In vivo pGSK3b inhibition shows excellent correlation with serum exposure of compound. These results show that phospho-GSK3b can be utilized as a in vitro and in vivo PD marker to develop inhibitors of AKT and PI3 Kinase as potential targeted cancer therapeutic agents.
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTPolynucleotide analogs. Copolymers of vinyl bases with acrylic acid, methylacrylic acid, acrylamide, and 1-vinylpyrrolidoneLinda Maggiora, Sophie Boguslawski, and Mathias P. MertesCite this: J. Med. Chem. 1977, 20, 10, 1283–1287Publication Date (Print):October 1, 1977Publication History Published online1 May 2002Published inissue 1 October 1977https://pubs.acs.org/doi/10.1021/jm00220a011https://doi.org/10.1021/jm00220a011research-articleACS PublicationsRequest reuse permissionsArticle Views87Altmetric-Citations10LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
Ricin A chain was radioactively labeled using reductive alkylation, lactoperoxidase catalyzed iodination, and reaction with iodoacetamide or N-ethylmaleimide (NEM). The inhibition of cell-free rat liver protein synthesis by the modified A chains and the ribosome binding characteristics of each of the labeled derivatives was examined. [3H] NEW was found to quantitatively react with the A chain sulfhydryl group normally involved in a disulfide bond with the B chain in intact ricin. Labeling the protein with [3H] NEM had no effect on the in vitro inhibition of protein synthesis by the A chain. [3H] NEM-labeled A chain binds to rat liver ribosomes in a manner which is dependent on the concentrations of NaCl and Mg2+. At optimal Mg2+ concentration (5.5 mM), A chain binding to ribosomes is saturable and fully reversible either by dilution of the reaction mixture or by addition of unlabeled A chain. At 5.5 mM Mg2+, A chain was found to bind to a single site on rat liver ribosomes with a dissociation constant of 6.2 x 10(-8) M. [3H] NEM-labeled A chain did not bind to isolated 40S ribosomal subunits and bound to 60S ribosomal subunits with a 1 : 1 molar stoichiometry and a dissociation constant of 2.2 x 10(-7) M. The relationship between ribosome binding and A chain inhibition of eucaryotic protein synthesis is discussed.
2560 Phosphoinositide 3-OH kinase (PI3-K) and its downstream target, Akt mediate one of the most important survival signaling pathways in cancer cells. The mechanism by which PI3-K/Akt pathway affects cell survival is through its downstream target proteins such as BAD, Caspase-9, Forkhead transcription factors, IKKα and TSC1/2. Therefore, inhibition of Akt activity is expected to induce apoptotic cell death at multiple levels. In addition, down-regulation of Akt signaling pathway may sensitize cancer cells to cytotoxic oncolytics. There are enormous clinical advantages in exploring this cell survival pathway. This makes Akt among the most validated and attractive targets for the discovery of efficacious therapeutics for the treatment of human cancers. Here we report that isoquinoline sulfonamide-based Akt kinase inhibitors exert potent anti-proliferative effect on U87MG human glioblastoma cells and Jurkat human T-cell leukemia cells. Both cell lines are PTEN-null with constitutively activated Akt. We further demonstrate that these AKT inhibitors induce a robust Caspases activation and rapid phosphatidyl serine (PS) exposure on cell surface, characteristic of cells undergoing rapid apoptosis. Data will also be presented to show that down-regulation of Akt activity either by Akt-specific siRNA or small molecule inhibitors sensitize cancer cells to various oncolytics such as Gemcitabine, Camptothecin and Dexamethasone, as well as TRAIL. We conclude that treatment of U87MG and Jurkat cells with isoquinoline sulfonamide class of Akt inhibitor results in apoptotic cell death. And these results support the notion that synergistic anticancer action can be achieved by combination therapy using Akt inhibitors and conventional cytotoxic cancer therapeutics.
In a very active cell-free system containing polysomes derived from human placenta and a cell-sap fraction prepared from ascites tumor cells, the synthesis of the hormone human placental lactogen (HPL) was detected. The identification was based on the following: ( a ) The in vitro synthesized protein labeled with [ 35 S]methionine migrated at the same rate as authentic HPL on sodium dodecyl sulfate-polyacrylamide gels and ( b ) tryptic fingerprint analysis of the labeled protein yielded peptides having the same mobilities as seen with the same analysis of purified HPL. The amount of HPL synthesized in a cell-free system containing polysomes derived from term placenta was about 10% of the total proteins synthesized and in a comparable system containing first trimester ribosomes the level of synthesis was about 5%. These data suggest the potential for quantitating the HPL mRNA activity as a function of the period of gestation and for isolating the mRNA itself.
