Project Summary Most kinase inhibitors target the ATP binding site, and frequently inhibit numerous kinases in the human kinome. Due to the nature of pan-kinase inhibitors, severe side effects are frequently observed in pre-clinical and clinical studies. Therefore, more specific therapeutics are desirable to achieve safe and effective treatment of human diseases. Allosteric modulators (agonists and antagonists) have greater potential than ATP- competitive inhibitors to achieve selectivity due to the much less conserved allosteric binding sites. High and consistent potency can also be achieved by the allosteric modulators. Furthermore, allosteric modulators can show different efficacies and pharmacological effects in cell and in vivo, compared to ATP competitive inhibitors. These agents can be applied to investigate specific biological and pathological functions of kinases in human diseases. However, identifying allosteric lead compounds and their binding pockets is a great challenge that retards the development of allosteric modulators of kinases. So, in this Award a novel concept and several approaches are proposed to develop allosteric modulators of Phosphatidylinositol 3-Kinase (PI3Kα), which is involved in human cancer, metabolism, innate and adaptive immunity, and Autism spectrum disorder. The peptide fragment trapped at the interface between the kinase domain and the Ras binding domain of PI3Kα was employed as an allosteric starting compound to generate allosteric modulators of PI3Kα. Our early stage pilot studies supported our hypothesis and produced proper starting agents for the development of allosteric agonists and antagonists of PI3Kα. Therefore, we will develop highly potent allosteric modulators of PI3Kα by applying structure-guided drug design techniques such as linker search, scaffold hopping, and virtual synthesis and screening (Aim 1). Solid phase peptide synthesis and solution phase organic synthesis will be applied for generating a peptidomimetic library and a series of small molecule analogs, respectively. X-ray co-crystal structures of PI3Kα in complex with allosteric modulators will be determined and used for the structure-based design. Once low nanomolar inhibition and activation are achieved in biochemical assays, cellular potency of allosteric modulators will be assessed in a series of cell- based proliferation and functional assays (Aim 2). Furthermore, cellular signaling pathways mediated by PI3Kα will be investigated with highly potent allosteric modulators in combination with ATP-competitive inhibitors. In summary, the proposed research will have high impact on biomedical research and drug discovery in terms of new types of chemical entities in kinases and a new concept for the discovery of allosteric modulators. The allosteric modulators developed in this project will facilitate the investigation of new kinase functions mediated by PI3Kα to determine its specific roles and requirement in human diseases. Finally, the...