Chemoproteomic Selectivity Profiling of PIKK and PI3K Kinase Inhibitors

Reinecke, M., Ruprecht, B., Poser, S., Wiechmann, S., Wilhelm, M., Heinzlmeir, S., Kuster, B., and Medard, G. (2019). ACS Chem Biol 14, 655-664. doi: 10.1021/acschembio.8b01020


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 in cellulo. 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 fibrodysplasia ossificans progressiva.