Abstract 4482: Discovery of an irreversible PI3Kα-specific Inhibitor
Authors: Qiao Lixin , Nacht Mariana , Sheets Michael P. , Martin Thia St. , Labenski Matthew , Mazdiyasni Hormoz , Zhu Zhendong , Chaturvedi Prasoon , Bhavsar Deepa , Niu Deqiang , Westlin William , Petter Russell , Singh Juswinder 
Published: June 2012

Abstract
The PI3K pathway, which regulates cell growth, proliferation and survival, is activated in many types of human tumors by mutational activation of PI3Kα, PTEN inactivation, or activation of upstream receptor tyrosine kinases. Several PI3K inhibitors are currently in clinical development, but most are pan-PI3K inhibitors. Tumor biology data suggests that targeting PI3Kα specifically should be efficacious, and there may be advantages to not disrupting other members of the complex PI3K signaling cascade. Using structure-based drug design (SBDD), we have discovered a series of small molecules that selectively inhibit PI3Kα via the formation of an irreversible covalent bond. In addition to the enhanced pharmacodynamics achieved through permanent silencing of targets, covalent inhibitors also exhibit key advantages in terms of isoform selectivity and translational biomarker opportunities.
SBDD was used to design small molecules which bond to an amino acid present uniquely in the α-isoform of PI3K. Mass spectrometry verified covalent bond formation to PI3Kα but not to the other isoforms. PI3K enzyme activity was measured using an HTRF assay. PI3Kα inhibition was evaluated in SKOV-3 cells by measuring P-AktSer473 levels. Washout experiments were performed to assess prolonged duration of action in cells. Effects on cell proliferation were assessed in cell lines bearing different mutations, such as SKOV-3 (PIK3CA H1047R), PC3 (PTEN-null) and others. SKOV-3 xenograft studies were performed in mice to measure P-AktSer473 inhibition and tumor growth inhibition in vivo. A biotinylated covalent probe molecule specific for PI3Kα was used to verify and quantitate target occupancy by the covalent inhibitor, both in vitro and ex vivo.
Using trypsin digestion and MS-MS analysis, we confirmed that the designed inhibitors covalently bonded to the protein at the desired amino acid that is unique to PI3Kα. The covalent bonding resulted in potent inhibition of the PI3Kα enzyme activity and inhibition of P-AktSer473 in cells (EC50 &lt; 100nM). Moreover, the inhibition of PI3Kα activity continued after compound removal, confirming prolonged duration of activity. Only cell lines driven by PI3Kα were potently inhibited by PI3Kα-selective inhibitors. A lead compound also demonstrated in vivo inhibition of PI3Kα and tumor growth inhibition in an SKOV-3 xenograft model, and a biotinylated covalent probe molecule verified that PI3Kα was covalently inhibited by the compound in vivo.
Selective irreversible inhibitors of PI3Kα demonstrate prolonged duration of action and activity in vivo. This targeted approach should yield a first-in-class selective covalent PI3Kα inhibitor with clinical advantages.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4482. doi:10.1158/1538-7445.AM2011-4482