Covalent Proximity Scanning of a Distal Cysteine to Target PI3Kα

Covalent protein kinase inhibitors exploit currently noncatalytic cysteines in the adenosine 5'-triphosphate (ATP)-binding site via electrophiles directly appended to a reversible-inhibitor scaffold. Here, we delineate a path to target solvent-exposed cysteines at a distance >10 Å from an ATP-site-directed core module and produce potent covalent phosphoinositide 3-kinase α (PI3Kα) inhibitors. First, reactive warheads are used to reach out to Cys862 on PI3Kα, and second, enones are replaced with druglike warheads while linkers are optimized. The systematic investigation of intrinsic warhead reactivity (<i>k</i>_chem), rate of covalent bond formation and proximity (<i>k</i>_inact and reaction space volume <i>V</i>_r), and integration of structure data, kinetic and structural modeling, led to the guided identification of high-quality, covalent chemical probes. A novel stochastic approach provided direct access to the calculation of overall reaction rates as a function of <i>k</i>_chem, <i>k</i>_inact, <i>K</i>_i, and <i>V</i>_r, which was validated with compounds with varied linker lengths. X-ray crystallography, protein mass spectrometry (MS), and NanoBRET assays confirmed covalent bond formation of the acrylamide warhead and Cys862. In rat liver microsomes, compounds <b>19</b> and <b>22</b> outperformed the rapidly metabolized CNX-1351, the only known PI3Kα irreversible inhibitor. Washout experiments in cancer cell lines with mutated, constitutively activated PI3Kα showed a long-lasting inhibition of PI3Kα. In SKOV3 cells, compounds <b>19</b> and <b>22</b> revealed PI3Kβ-dependent signaling, which was sensitive to TGX221. Compounds <b>19</b> and <b>22</b> thus qualify as specific chemical probes to explore PI3Kα-selective signaling branches. The proposed approach is generally suited to develop covalent tools targeting distal, unexplored Cys residues in biologically active enzymes.