Abstract 291: Development of optimized chemical probes targeting PI3Ka to deconvolute the role of class I PI3Ks isoforms in insulin signaling
Authors: De Pascale Martina , Borsari Chiara , Keles Erhan , McPhail Jacob , Schäfer Alexander , Sriramaratnam Rohitha , Gstaiger Matthias , Burke John , Wymann Matthias 
Published: July 2021

Abstract
Phosphatidylinositol-3-kinase (PI3K) activity is aberrant in tumors, and PI3K inhibitors are investigated as cancer therapeutics.[1-5] The major obstacles to the successful implementation of PI3K-targeted cancer therapy are on-target adverse effects on insulin signaling. Isotype selective PI3K inhibitors have been exploited to answer fundamental questions regarding the role of PI3K isoforms in cell biology. However, the availability of claimed isoform-selective PI3Kα inhibitors is limited to BYL719 (Alpelisib)[6] and GDC0032 (Taselisib)[7], which do not maintain PI3Kα selectivity at a concentration required in cellular experimental settings and clinical applications. Highly selective PI3Kα inhibitors are expected to represent ideal tools to elucidate the role of PI3Kα isoform in tumor development and insulin signaling. As the systemic inactivation of PI3Kα is embryonic lethal, genetic approaches are currently limited to organ-specific targeting, and a specific inactivation of PI3Kα in an adult organism has not been achieved up-to-date. Herein, we generate high-quality PI3Kα chemical probes to dissect the role of PI3Kα in cancer and metabolism. We exploit covalent inhibitors, permanently blocking target functions, as a strategy to enhance the ligand binding selectivity for proteins in the same family. The non-conserved nucleophilic amino acid Cys862 in PI3Kα represents a promising target for covalent modifiers. We converted the reversible scaffold of PQR514[5] into irreversible compounds. An extensive Structure Activity Relationship (SAR) study was performed using CNX-1351[8] reacting group and introducing different heteroaliphatic rings in the linker. X-ray crystallography and bottom-up LC-MS/MS based proteomics validated the covalent modification of Cys862. Our pilot chemical probes exceeded in vitro and cellular potency over CNX-1351. The generation of a novel class of covalent PI3Kα-specific inhibitors with improved selectivity and persistency of PI3Kα-inhibition will shed light on the role of PI3Kα in cancer and metabolism. Our results will pave the way for the dissociation of PI3Ki antitumor activity from adverse effects on insulin action. [1] Beaufils F et al. J Med Chem. 2017, 60 (17), 7524-7538. [2] Rageot R et al. J Med Chem. 2019, 62 (13), 6241-6261. [3] Wymann MP et al. Nat Rev Mol Cell Biol. 2008, 9 (2), 62-176. [4] Marone R et al. Biochim Biophys Acta. 2008, 1784 (1), 159-185. [5] Borsari C et al. ACS Med Chem Lett. 2019, 10 (10), 1473-1479. [6] Markham A Drugs 2019, 79 (11), 1249-1253. [7] Zumsteg ZS et al. Clin Cancer Res. 2019, 22 (8), 2009-2019. [8] Nacht M et al. J Med Chem. 2013, 56 (3), 712-721.
Citation Format: Martina De Pascale, Chiara Borsari, Erhan Keles, Jacob McPhail, Alexander Schäfer, Rohitha Sriramaratnam, Matthias Gstaiger, John Burke, Matthias Wymann. Development of optimized chemical probes targeting PI3Ka to deconvolute the role of class I PI3Ks isoforms in insulin signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 291.