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
(kchem), rate of covalent bond formation
and proximity (kinact and reaction space
volume Vr), 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 kchem, kinact, Ki, and Vr, 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 19 and 22 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 19 and 22 revealed PI3Kβ-dependent signaling,
which was sensitive to TGX221. Compounds 19 and 22 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.