Chemoproteomic Selectivity Profiling of PIKK and PI3K Kinase Inhibitors

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.