tgx-221 and lysophosphatidic-acid

Phosphatidylinositol-3 kinase (PI3K) pathway regulates multiple cellular functions involving cell survival, growth, motility proliferation, apoptosis, and adhesion. These are deregulated in various diseases such as cancer, atherosclerosis, and inflammation. PI3Ks phosphorylate phosphatidylinositol 4,5-biphosphate (PIP2) yielding phosphatidylinositol 3, 4, 5 triphosphate (PIP3) which in turn activate AKT kinase (serine/threonine kinase), the central enzyme in regulation of metabolic functions. Due to their implications in disease pathophysiology, PI3K/AKT inhibitors became attractive targets for pharmaceutical industries. In order to assess the functional response generated by PI3K inhibitors, an appropriate cell-based screening system is essential in any screening cascade. Here we report the development of highly sensitive in-vitro cell-based kinase ELISA which quantifies the phosphorylated AKT kinase (serine 473) and total AKT kinase directly within the cells upon compound treatment. PI3Kβ overexpressing NIH3T3 cells stimulated by lysophosphatidic acid was used for PI3K/Akt pathway activation. Assay performance reliability and robustness were determined by percentage coefficient of variation (%CV) and Z factor which demonstrated an excellent agreement with assay guidelines. This 96-well plate medium throughput assay methodology was used to screen novel molecules and proved a commendable tool to study the mechanism of action property and target engagement of novel PI3K inhibitors in drug discovery.

Topics: Animals; Chromones; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Lysophospholipids; Mice; Morpholines; NIH 3T3 Cells; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Pyrimidinones; Structure-Activity Relationship