Rapid, potent, and persistent covalent chemical probes to deconvolute PI3Kα signaling

Chemical probes have gained importance in the elucidation of signal transduction in biology. Insufficient selectivity and potency, lack of cellular activity and inappropriate use of chemical probes has major consequences on interpretation of biological results. The catalytic subunit of phosphoinositide 3-kinase α (PI3Kα) is one of the most frequently mutated genes in cancer, but fast-acting, high-quality probes to define PI3Kα's specific function to clearly separate it from other class I PI3K isoforms, are not available. Here, we present a series of novel covalent PI3Kα-targeting probes with optimized intracellular target access and kinetic parameters. On-target TR-FRET and off-target assays provided relevant kinetic parameters (<i>k</i> _chem, <i>k</i> _inact and <i>K</i> _i) to validate our chemical probes. Additional intracellular nanoBRET tracer displacement measurements showed rapid diffusion across the cell membrane and extremely fast target engagement, while investigations of signaling downstream of PI3Kα <i>via</i> protein kinase B (PKB/Akt) and forkhead box O (FOXO) revealed blunted pathway activity in cancer cell lines with constitutively activated PI3Kα lasting for several days. In contrast, persistent PI3Kα inhibition was rapidly bypassed by other class I PI3K isoforms in cells lacking functional phosphatase and tensin homolog (PTEN). Comparing the rapidly-diffusing, fast target-engaging chemical probe 9 to clinical reversible PI3Kα-selective inhibitors alpelisib, inavolisib and 9r, a reversible analogue of 9, revealed 9's superior potency to inhibit growth (up to 600-fold) associated with sustained suppression of PI3Kα signaling in breast cancer cell lines. Finally, using a simple washout protocol, the utility of the highly-selective covalent PI3Kα probe 9 was demonstrated by the quantification of the coupling of insulin, EGF and CXCL12 receptors to distinct PI3K isoforms for signal transduction in response to ligand-dependent activation. Collectively, these findings along with the novel covalent chemical probes against PI3Kα provide insights into isoform-specific functions in cancer cells and highlight opportunities to achieve improved selectivity and long-lasting efficacy.