Chemoproteomic Selectivity Profiling of PIKK and PI3K Kinase Inhibitors Maria Reinecke Benjamin Ruprecht Sandra Poser Svenja Wiechmann Mathias Wilhelm Stephanie Heinzlmeir Bernhard Kuster Guillaume Médard 10.1021/acschembio.8b01020.s005 https://acs.figshare.com/articles/dataset/Chemoproteomic_Selectivity_Profiling_of_PIKK_and_PI3K_Kinase_Inhibitors/7946567 Chemical proteomic approaches utilizing immobilized, broad-selective kinase inhibitors (Kinobeads) have proven valuable for the elucidation of a compound’s target profile under close-to-physiological conditions and often revealed potentially synergistic or toxic off-targets. Current Kinobeads enrich more than 300 native protein kinases from cell line or tissue lysates but do not systematically cover phosphatidylinositol 3-kinases (PI3Ks) and phosphatidylinositol 3-kinase-related kinases (PIKKs). Some PIKKs and PI3Ks show aberrant activation in many human diseases and are indeed validated drug targets. Here, we report the development of a novel version of Kinobeads that extends kinome coverage to these proteins. This is achieved by inclusion of two affinity probes derived from the clinical PI3K/MTOR inhibitors Omipalisib and BGT226. We demonstrate the utility of the new affinity matrix by the profiling of 13 clinical and preclinical PIKK/PI3K inhibitors. The large discrepancies between the PI3K affinity values obtained and reported results from recombinant assays led us to perform a phosphoproteomic experiment showing that the chemoproteomic assay is the better approximation of PI3K inhibitor action <i>in cellulo</i>. The results further show that NVP-BEZ235 is not a PI3K inhibitor. Surprisingly, the designated ATM inhibitor CP466722 was found to bind strongly to ALK2, identifying a new chemotype for drug discovery to treat <i>fibrodysplasia ossificans progressiva</i>. 2019-03-22 00:00:00 novel version drug targets Chemoproteomic Selectivity Profiling Current Kinobeads ATM inhibitor CP 466722 PIKK drug discovery PI 3K inhibitors Omipalisib chemoproteomic assay ALK PI 3K inhibitor action kinome coverage cell line phosphatidylinositol 3- kinases protein kinases affinity matrix NVP-BEZ 235 close-to-physiological conditions phosphoproteomic experiment PI 3K inhibitor PI 3K Kinase Inhibitors Chemical proteomic approaches broad-selective kinase inhibitors BGT 226. PI 3K affinity values phosphatidylinositol 3- kinase-related kinases PI 3Ks show affinity probes tissue lysates PI 3Ks fibrodysplasia ossificans progressiva