Abstract 1378: A novel, highly potent PI3Kα covalent inhibitor deconvolutes class I PI3K isoforms in cancer cells
Authors: Keles Erhan , Borsari Chiara , Sriramaratnam Rohitha , Schäfer Thorsten , Wymann Matthias P. 
Published: July 2021

Phosphoinositide 3-kinase (PI3K) imposes central regulatory signals that promote cell growth, proliferation, motility, and suppression of apoptosis. PI3K is dysregulated in a broad spectrum of human tumours.[1] Considering the central role attributed to PI3K signaling in cancer biology, the success of clinical trials in the field was below expectations, mainly due to metabolic on-target adverse effects. Here, we present a novel, highly selective PI3Kα-covalent inhibitor (compound 19), which combines the exceptional affinity of the pan-PI3K inhibitor PQR514[2] with a linker moiety, directing a warhead to selectively react via a Michael addition with isoform-specific Cys862 of PI3Kα. Cellular assay platforms were combined to validate 19, specifically, to assess its target selectivity and occupancy, and to translate its in vitro activity to cellular readouts. Overall, 19 exhibited excellent cellular activity with a superior profile compared to CNX-1351[3], a previously reported covalent PI3Kα inhibitor. A NanoBRET (Bioluminescence Resonance Energy Transfer) drug-target engagement assay allowed for an investigation of inhibitory efficacy in HEK293 cells. NanoBRET experiments exploiting the Cys862Ser mutant PI3Kα protein confirmed selective reactivity with Cys862. Various cell lines either constitutively activated in PI3Kα (MCF7, T47D, SKOV3) or deficient in PTEN (PC3 and A2058) were used to resolve target engagement, which confirmed a persistent and prolonged suppression of protein kinase B (PKB/Akt, Ser473) phosphorylation after washout of 19 over non-covalent structural analogs thereof that lack a functional warhead. Furthermore, nucleo-cytoplasmic translocation probes based on the FOXO1 transcription factor enabled the investigation of signal propagation downstream of PKB/Akt. In PI3Kα-dependent cell lines, FOXO1, maintained a predominant nuclear localization even after removal of 19, confirming extended PI3Kα targeting upon covalent attachment of specifically this drug. Importantly, 19 provided a considerable gain in potency over BYL719, a clinically approved non-covalent PI3Kα-selective inhibitor. Taken together, our results present a major step towards an increased local and temporal control of PI3Kα signaling through isoform-selective covalent anchoring. This strategy is a step towards the development of drug-like, isoform-selective, covalent PI3K inhibitor scaffolds, and 19 provides a template for the development of PI3Kα inhibitors with overall reduced adverse side effects in cancer therapy. Moreover, our covalent inhibition strategy will pave the way for a better molecular understanding of PI3K isoform signaling in general. [1] Wymann M. P. and Schneiter R. Nat. Rev. Mol. Cell Biol. 2008, 9, 162-176. [2] Borsari, C et. al. ACS Med. Chem. Lett. 2019, 10 (10), 1473-1479. [3] Nacht M et. al. J. Med. Chem. 2013, 56 (3), 712-721.

Citation Format: Erhan Keles, Chiara Borsari, Rohitha Sriramaratnam, Thorsten Schäfer, Matthias P. Wymann. A novel, highly potent PI3Kα covalent inhibitor deconvolutes class I PI3K isoforms in cancer cells [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 1378.