Abstract 1955: Novel long-acting covalent PI3Kα inhibitors boost potential action in cancer
Authors: Constantin Theodora A. , Bissegger Lukas , Keleş Erhan , Raguž Luka , Orbegozo Clara , Schaefer Thorsten , Barlow-Busch Isobel , Burke John E. , Borsari Chiara , Wymann Matthias P. 
Published: March 2024

Abstract
Phosphoinositide 3-kinase (PI3K) is a key regulator of cell proliferation, survival, and metabolism. Hotspot mutations in the PIK3CA gene constitutively activate the catalytic subunit of the PI3Kα isoform and are found in 10-30% of solid tumors. The development of PI3K pathway inhibitors has been hampered by poor drug tolerance and release of negative feedback loops, leading to rapid reactivation of signaling in response to reversible inhibitors. To remedy these challenges, we developed highly selective covalent PI3Kα inhibitors that irreversibly bind to a non-conserved cysteine (C862) located 11 Å outside the ATP-binding site. Here we present the properties of an optimized drug-like covalent lead, dubbed compound 9, and describe the generation of a biotinylated probe developed to be used in conjunction with compound 9 to monitor PI3Kα target occupancy in relation to downstream PI3K signaling outputs. Biochemical investigations of compound 9 using TR-FRET assays showed high affinity binding (Ki), high reaction rates (kinact) for covalent bond formation with PI3Kα, and negligible off-target reactivity. Cellular NanoBRET assays revealed that compound 9 diffuses three times more rapidly into cells as compared to BYL719 (alpelisib), and leads to rapid and potent on-target engagement. Covalent bond formation to residue C862 of PI3Kα was further confirmed by X-ray crystallography. In PIK3CA mutant cancer cell lines, covalent binding of PI3Kα occurred at low nanomolar concentrations and led to persistent inhibition of Akt/PKB phosphorylation for more than 72 hours after drug removal. Notably, investigation of PI3Kα re-synthesis after labeling with compound 9 showed a negligible reappearance in cancer cell lines, which indicates that persistent suppression of PI3Kα signaling by compound 9 results in a much longer half-life for PI3Kα than previously reported. The irreversible inactivation of PI3Kα by compound 9 provides a marked gain in growth inhibition potency (10-600-fold) in PIK3CA mutant cancer cell lines in comparison to clinical reversible PI3Kα inhibitors and allows for intermittent dosing without compromising efficacy. These data collectively demonstrate that compound 9 enables precise and selective targeting of PI3Kα with high potency and long-lasting efficacy in cancer models. The new class of irreversible PI3Kα inhibitors have a unique pharmacology among PI3K inhibitors characterized by strong decoupling of drug exposure from efficacy which may provide opportunities to lower treatment burden in patients.
Citation Format: Theodora A. Constantin, Lukas Bissegger, Erhan Keleş, Luka Raguž, Clara Orbegozo, Thorsten Schaefer, Isobel Barlow-Busch, John E. Burke, Chiara Borsari, Matthias P. Wymann. Novel long-acting covalent PI3Kα inhibitors boost potential action in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1955.