High throughput screening for inhibitors of Akt plasma m

Boston Children'S Hospital, Boston MA

Investigators

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Abstract

DESCRIPTION (provided by applicant): A number of diseases, including cancer, are linked to perturbation of intracellular signaling pathways. One such pathway is the Ptdlns(3,4,5)P3/Akt (PKB) signaling pathway which is over activated in a wide range of tumor types. Deactivation of this pathway suppresses the growth of tumor cells and also reduces resistance of tumor cells to chemotherapeutic drug treatment. Akt is a serine/threonine protein kinase with oncogenic and anti-apoptotic activities. Its activation relies on Ptdlns(3,4,5)P3 (an inositol phospholipid localized on the plasma membrane) -mediated translocation of Akt from the cytoplasm to the plasma membrane where Akt gets phosphorylated and activated. Thus, Akt translocation provides an attractive target for anti-cancer drug discovery. Recently, we established an experimental system for visualizing Akt plasma membrane translocation in live cells and plan to utilize this system to conduct a high throughput screening for small- molecule inhibitors of Akt plasma membrane translocation. In this proposed research, we will adapt our cell- based system to a high-throughput format (Aim I). The selectivity and reproducibility of our high throughput screening assay will be examined by performing a pilot screening of a known bioactive compounds library (Aim II). An assay for the secondary screening is proposed to role out artifacts from the primary screen. In this assay, Akt plasma membrane translocation will be assessed by the level of Akt phosphorylation which can be monitored by western blot analysis using phospho-Akt specific antibody (Aim III). Finally, a plan to evaluate the biological activities of identified compounds is proposed. We will investigate whether blocking Akt membrane translocation by identified compounds is able to initiate the death of tumor cells and increase the efficacy of chemotherapeutic anticancer drugs (Aim IV). The ultimate goal of our research is to identify inhibitors of Akt plasma membrane translocation via performing a high through screening of small molecule libraries. Discovery of these inhibitors will greatly facilitate our research on Ptdlns(3,4,5)P3/Akt signaling. Moreover, the identified inhibitors can be directly utilized as starting chemical compounds for novel anti- cancer drug development.