Abstract 1954: Development of highly potent, covalent, and selective inhibitors to target PI3Kα in cancer
Authors: Raguž Luka , Constantin Theodora A. , Bissegger Lukas , Keleş Erhan , Orbegozo Clara , Schäfer Thorsten , Sriramaratnam Rohitha , Borsari Chiara , Wymann Matthias P. 
Published: March 2024

Abstract
Aberrant PI3K signaling is linked to various forms of cancer and proliferative disease. Approved clinical therapies targeting overactive PI3K are scarce and often accompanied by severe adverse effects due to lack of PI3K isoform selectivity. We recently reported a proprietary covalent proximity scanning (CoPS) approach to selectively target distant cysteines outside of the ATP-binding pocket in kinases. Using CoPS, we developed isoform specific, irreversible covalent PI3Kα inhibitors (cPI3Kαi) with outstanding target engagement profiles. Here, we report structure-guided scaffold and linker-optimizations resulting in cPI3Kαi with long-term and rapid inhibition of PI3Kα. Cellular activity was improved exploiting additional protein-inhibitor interactions inspired by X-ray crystallographic data, and introduction of structural elements to increase cell permeability. The cPI3Kαi series showed enhanced cellular potency and excellent on-target engagement. Kinetic parameters and covalent bond formation were determined using TR-FRET, nanoBRET tracer displacement assays, and LCMS/MS based proteomics. With our new cPI3Kαi, IC50s of PKB/Akt phosphorylation, pPKB/Akt recovery after drug washout, and growth inhibition experiments were performed in cancer cell lines harboring activating PIK3CA or inactivating PTEN mutations. Efficient and long-lasting inhibition of PKB/Akt phosphorylation was observed in PIK3CA mutant cell lines, while loss of PTEN was associated with partial responses, consistent with contributions of other class I PI3K isoforms to signal reactivation. A dynamic FOXO cytosolic/nuclear translocation assay was used to monitor distal PI3K signaling: cPI3Kαi blunted PI3K signaling rapidly with rates identical to the reversible BYL719/alpelisib. After drug washout, inhibition by BYL719/alpelisib was relieved within minutes, while a complete and prolonged shutdown of downstream signaling events was observed in PIK3CA mutant cell lines but not in cell lines where PTEN function was lost. This relapse in downstream signaling explains the lack of efficacy of PI3Kα selective inhibitors in models with PTEN loss and demonstrates that persistent inhibition across the PI3K pathway is required for efficacy. In summary, the new cPI3Kαi display outstanding specificity and cellular activity in PIK3CA-mutated cancer cells, and support the further development of cPI3Kαi as future clinical candidates and an alternative to established reversible inhibitors.
Citation Format: Luka Raguž, Theodora A. Constantin, Lukas Bissegger, Erhan Keleş, Clara Orbegozo, Thorsten Schäfer, Rohitha Sriramaratnam, Chiara Borsari, Matthias P. Wymann. Development of highly potent, covalent, and selective inhibitors to target PI3Kα 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 1954.