Phosphatidyl Inositol 3 kinases inhibitor development using novel invitro pharmacological assays

Abstract

The phosphatidyl inositol 3-kinases(PI3Ks) are lipid kinases that regulate the newlinecellular signal transduction pathways involved in cell growth, proliferation, newlinesurvival, apoptosis and adhesion. Deregulations of these pathways are common newlinein oncogenesis and are known to be altered in other metabolic disorders as well. In spite of its huge potential as an attractive target in the above diseases, newlinethere is an unmet need for the development of a successful inhibitor. Unlike protein kinase inhibitor, screening for lipid kinase inhibitor has been challenging due to the lipidic nature of PI3K substrates known as phosphoinositides. Here we report for the first time, the development of unique and novel radioactive lipid kinase screening platform using phosphocellulose newlinepaper that involves transfer of adiolabelled [?P32] ATP to phosphatidylinositol 4,5 phosphate forming Phosphatidylinositol-3,4,5 phosphate captured on the phosphocellulose matrix. Enzyme kinetics and inhibitory properties were established in the plate format using standard inhibitors like LY294002, TGX newline221 and Wortmannin having different potencies towards PI3K isoforms. All the newlineenzyme parameters including ATP and Lipid apparent Km for both were determined and IC50 values generated that matched with historical data. We utilized PI3K ??as an enzyme target to demonstrate phosphocellulose based screening as an excellent platform for the identification of novel chemical entities (NCEs) in PI3K drug discovery and later extended in all isoforms ( PI3K ?, PI3K ?, and PI3K ?). This unique assay methodology was further extended to study inhibitor characterization and evaluated the mechanism of action (MOA) of PI3K inhibitors (LY294002 and TGX-221) with respect to ATP competitiveness against PI3 kinase ?? This robust method can be easily newlineadapted in any lipid kinase drug discovery program for compound profiling and in-depth mechanistic applications without having any potential artifacts. newline