The mammalian Target Of Rapamycin (mTOR) is implicated in cancer and other human diseases and is thus an important target for therapeutic intervention. mTOR exists as two multi-protein complexes (mTORC1 and mTORC2) and its integrity is important for kinase activity and downstream signaling. In a preparation isolated with anti-mTOR antibody, we confirmed the presence of both mTOR complexes by western blotting and in vitro kinase assays using 4E-BP1 as a physiological substrate of mTORC1, and both 4E-BP1 and AKT as physiological substrates of mTORC2. These proofs of concept studies allowed us to develop a high throughput DELPHIA-based biochemical assay using immunoprecipitated mTOR complexes from HeLa cell lysates on protein G coated 384-well plates and 4E-BP1 as a substrate to identify both mTORC1 and mTORC2 inhibitors. From >290,000 compounds screened, there were 334 confirmed hits with >50% inhibition at 10 μM which is equal to a hit rate of 0.12%. OXA-01, a potent mTOR kinase inhibitor was thus discovered by lead optimization of a class of imidazopyrazine hits using conventional medicinal chemistry approaches, combined with a computationally driven homology model. The detailed protocol of high throughput screening for mTORC1/mTORC2 inhibitors will be described in the poster.
99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA