Lixin Qiao

Selective irreversible inhibition of a protease by targeting a noncatalytic cysteine

Designing selective inhibitors of proteases has proven problematic, in part because pharmacophores that confer potency exploit the conserved catalytic apparatus. We developed a fundamentally different approach by designing irreversible inhibitors that target noncatalytic cysteines that are structurally unique to a target in a protein family. We have successfully applied this approach to the important therapeutic target HCV protease, which has broad implications for the design of other selective protease inhibitors.

Discovery of a potent and isoform-selective targeted covalent inhibitor of the lipid kinase PI3Kα.

PI3Kα has been identified as an oncogene in human tumors. By use of rational drug design, a targeted covalent inhibitor 3 (CNX-1351) was created that potently and specifically inhibits PI3Kα. We demonstrate, using mass spectrometry and X-ray crystallography, that the selective inhibitor covalently modifies PI3Kα on cysteine 862 (C862), an amino acid unique to the α isoform, and that PI3Kβ, -γ, and -δ are not covalently modified. 3 is able to potently (EC(50) < 100 nM) and specifically inhibit signaling in PI3Kα-dependent cancer cell lines, and this leads to a potent antiproliferative effect (GI(50) < 100 nM). A covalent probe, 8 (CNX-1220), which selectively bonds to PI3Kα, was used to investigate the duration of occupancy of 3 with PI3Kα in vivo. This is the first report of a PI3Kα-selective inhibitor, and these data demonstrate the biological impact of selectively targeting PI3Kα.

Abstract 4482: Discovery of an irreversible PI3Kα-specific Inhibitor

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 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