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The phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases that regulate the cellular signal transduction pathways involved in cell growth, proliferation, survival, apoptosis, and adhesion. Deregulation of these pathways are common in oncogenesis, and they are known to be altered in other metabolic disorders as well. Despite its huge potential as an attractive target in these diseases, there is an unmet need for the development of a successful inhibitor. Unlike protein kinase inhibitors, screening for lipid kinase inhibitors has been challenging. Here we report, for the first time, the development of a radioactive lipid kinase screening platform using a phosphocellulose plate that involves transfer of radiolabeled [γ-(32)P]ATP to phosphatidylinositol 4,5-phosphate forming phosphatidylinositol 3,4,5-phosphate, captured on the phosphocellulose plate. Enzyme kinetics and inhibitory properties were established in the plate format using standard inhibitors, such as LY294002, TGX-221, and wortmannin, having different potencies toward PI3K isoforms. ATP and lipid apparent Km for both were determined and IC50 values generated that matched the historical data. Here we report the use of a phosphocellulose plate for a lipid kinase assay (PI3Kβ as the target) as an excellent platform for the identification of novel chemical entities in PI3K drug discovery.

Using in vitrotooth germ cultures and analysis by confocal microscopy, ameloblasts treated with sodium fluoride were found to have elevated amounts of filamentous actin. Because this response is reduced by inhibitors of the Rho/ROCK signaling pathway, we generated mice that express dominant negative RhoA (RhoADN) in ameloblasts for in vivo analysis. Expression of the EGFP-RhoADN fusion protein was evaluated by RT-PCR and immunohistochemistry, and teeth were analyzed by scanning electron microscopy. The 3 strains expressed at either low (TgEGFP-RhoADN-8), intermediate (TgEGFP-RhoADN-2), or high (TgEGFP-RhoADN-13) levels, and the molar teeth from the 3 strains had enamel hypoplasia and surface defects. We conclude that RhoADN expressed in ameloblasts interferes with normal enamel development through the pathway that is induced by sodium fluoride.