Covalent Proximity Scanning of a Distal Cysteine to Target PI3K alpha

Covalent protein kinase inhibitors exploit currentlynoncatalytic 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-exposedcysteines at a distance >10 A from an ATP-site-directed core moduleand produce potent covalent phosphoinositide 3-kinase alpha(PI3K alpha)inhibitors. First, reactive warheads are used to reach out to Cys862on PI3K alpha, and second, enones are replaced with druglike warheadswhile linkers are optimized. The systematic investigation of intrinsicwarhead reactivity (kchem), rate of covalent bond formation andproximity (kinactand reaction space volumeVr), and integration ofstructure data, kinetic and structural modeling, led to the guidedidentification of high-quality, covalent chemical probes. A novelstochastic approach provided direct access to the calculation of overall reaction rates as a function ofkchem,kinact,Ki, andVr, whichwas validated with compounds with varied linker lengths. X-ray crystallography, protein mass spectrometry (MS), and NanoBRETassays confirmed covalent bond formation of the acrylamide warhead and Cys862. In rat liver microsomes, compounds19and22outperformed the rapidly metabolized CNX-1351, the only known PI3K alpha irreversible inhibitor. Washout experiments in cancer celllines with mutated, constitutively activated PI3K alpha showed a long-lasting inhibition of PI3K alpha. In SKOV3 cells, compounds19and22revealed PI3K beta-dependent signaling, which was sensitive to TGX221. Compounds19and22thus qualify as specific chemicalprobes to explore PI3K alpha-selective signaling branches. The proposed approach is generally suited to develop covalent tools targetingdistal, unexplored Cys residues in biologically active enzymes