Inhibitors Of PI3Kα Research Articles

Abstract 4482: Discovery of an irreversible PI3Kα-specific Inhibitor

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 < 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

Structure-based virtual screening approach to the discovery of phosphoinositide 3-kinase alpha inhibitors

Phosphoinositide 3-kinase alpha (PI3Kα) has proved to be an attractive target for the development of therapeutics for the treatment of cancer. Herein we report a successful application of the structure-based virtual screening to identify the novel inhibitors of PI3Kα. These inhibitors have desirable physicochemical properties as a drug candidate and reveal a moderate potency with IC(50) values ranging from 20 to 40 μM. Therefore, they deserve a consideration for further development by structure-activity relationship (SAR) studies to optimize the inhibitory activities. Structural features relevant to the stabilization of the newly identified inhibitors in the ATP-binding site of PI3Kα are addressed in detail.

5-Ureidobenzofuranone indoles as potent and efficacious inhibitors of PI3 kinase-α and mTOR for the treatment of breast cancer

A series of 5-ureidobenzofuran-3-one indoles as potent inhibitors of PI3Kα and mTOR has been developed. The best potency in cells was obtained when the urea group was extended to a 4-[2-(dimethylamino)ethyl]methylamino amidophenyl group. A 7-fluoro group on the indole ring also enhanced cellular potency. Compound 18i, incorporating the optimal functional groups, showed high potency in cellular lines and was further studied in vivo. It was able to inhibit the biomarker phosphorylation for 8h when dosed at 25mg/kg iv. In the MDA-MB-361 breast cancer model, it shrank the tumor size remarkably when dosed at 25mg/kg iv on days 1, 5, and 9.

Abstract 5779: The discovery of the potent and selective PI3K/mTOR dual inhibitor PF-04691502 through structure-based drug design

Abstract The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays crucial roles in cell growth, proliferation and survival. Genomic aberrations in the PI3K pathway, such as mutational activation of PI3Kα or loss of function of tumor suppressor PTEN, have been closely linked to the development and progression of a wide range of cancers. Hence, inhibition of the key targets in the pathway, e.g. PI3K, AKT, mTOR, offers great potential for the treatment of cancer. In an effort to discover compounds that inhibit PI3Kα, a high throughput screen was carried out, and 4-methyl-pyrido-pyrimidine (MPP) derivatives were identified as potent and selective inhibitors of PI3Kα. For example, PF-00271897, 8-cyclopentyl-6-[3-(hydroxymethyl)phenyl]-4-methyl-2-(methylamino)pyrido[2,3-d]pyrimidin-7(8H)-one demonstrated PI3Kα Ki of 2.2 nM. Multiple crystal structures of inhibitors bound to PI3K gamma were determined to inform design and optimization of the ADMET properties of this lead series. Crystallographic studies with PI3K gamma protein indicated that the aminopyrimidine moiety forms two hydrogen bonds to the kinase backbone, and the aromatic moiety at the 6 position binds in a hydrophobic pocket. The X-ray structure suggested that the 4-methyl group on the MPP core structure conferred the excellent overall kinase selectivity to the series. The structure and SAR suggested optimization could come from keeping N-R group at 2 position very small and maintaining aromatic moiety at 6 position for hydrophobic interaction. Introduction of polar groups to the 8N side chains that are located in the ribose binding pocket increased both metabolic stability and solubility. Based on the overall properties, PF-04691502, 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxypyridin-3-yl)-4-methylpyrido[2,3-d]pyrimidin-7(8H)-one, was selected as a clinical candidate. PF-04691502 demonstrated Ki's of 1.2-2.2 nM against PI3K α, β, γ and δ isoforms, and Ki of 9.1 nM against recombinant mTOR. PF-04691502 inhibited AKT phosphorylation at S473 in BT20 breast cancer line with IC50 of 12 nM. PF-04691502 is highly selective for inhibition of PI3K family kinases as shown by lack of activity against a panel of >75 protein kinases, including the class III PI3K hVps34. In the in vivo rat PK studies, PF-04691502 demonstrated the following properties: Clearance = 5.2 ml/min/kg, Vdss = 1.4 L/kg, T1/2 = 3.1 h, F% = 63%. The design, synthesis, in vitro potency SAR, selectivity, ADMET of the MPP derivatives will be discussed. The crystal structure of PF-04691502 in PI3K gamma will also be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5779.

Up‐regulated phosphoinositide 3‐kinase γ (PI3Kγ ) promotes breast cancer metastasis

We recently reported that dysregulation of Gi‐coupled receptors promotes breast cancer metastasis via Rac‐dependent pathways (Xie et al., Cancer Res. 69: 5743–5751). The PI3Kγ is selctively activated by Gi‐coupled receptors. The objective of this study was to define the role of PI3Kγ in breast cancer metastasis. Immunohistochemical staining and Western blot analysis showed that PI3Kγ expression was selectively up‐regulated in human metastatic breast cancer cell line and breast cancer specimens. Transwell chamber assays indicated that migration and invasion of metastatic breast cancer cells in response to chemoattractants of NIH‐3T3 conditioned media was inhibited by a selective inhibitor of PI3Kγ but not PI3Kα or β isoforms. Furthermore, blocking PI3Kγ not PI3Kα and β disrupted the Rac‐dependent lamellipodia formation induced by the activation of Gi‐coupledreceptors, such as protease‐activated receptor 1 and CXC chemokinereceptor 4. In contrast, the lamellipodia formation induced by epidermal growth factor (EGF), a process not dependent on G‐proteins, was blocked by inhibitors of PI3Kα and β not by the PI3Kγ Inhibitor. Our data demonstrated that PI3Kγ functions as a key molecule mediating signal flow from Gi‐coupled receptors to Rac‐dependent pathways and suggests that PI3Kγ may be a novel therapeutic target for the treatment of breast cancers. Supported by Nebraska State LB692 (YX).

Abstract C59: Discovery and characterization of selective PI3Kα and PI3Kα/mTOR-dual inhibitors for targeting PI3Kα-activated tumors

Abstract Background: The PI3K pathway regulates cell growth, proliferation and survival, and is dysregulated with high frequency in human tumors. PI3K pathway activation in tumors occurs via multiple mechanisms including prevalent mutation and amplification of the PIK3CA gene (which encodes the p110 subunit of PI3Kα). Downregulation of the lipid phosphatase PTEN also activates PI3K pathway signaling in a subset of tumors, via a mechanism reported to be PI3Kβ dependent. These findings suggest that isoformselective PI3K inhibitors may have utility in distinct, genetically defined tumor populations. Downstream of PI3K, mTOR controls cell growth and proliferation through its two distinct signaling complexes: mTORC1 and mTORC2. We report on the discovery of PI3Kα and dual PI3Kα/mTOR-selective inhibitors intended to target tumors harboring activating mutations or amplifications in PIK3CA. Methods: We identified and optimized a series of PI3Kα and PI3Kα/mTOR inhibitors through high-throughput screening and medicinal chemistry techniques. Biochemical assays employed recombinant class 1 PI3K and mTOR proteins and cellular assays employed ELISA or western blot assays to monitor PI3K pathway signaling. Results: We have identified potent (biochemical IC50s < 20 nM), ATP-competitive inhibitors of PI3Kα or PI3Kα and mTOR with excellent selectivity over other PI3K isoforms and a diverse panel of protein kinases. Cellular assays in the PI3Kα-activated MCF7 cell line demonstrate that these compounds inhibit phosphorylation of targets downstream of both PI3K (Akt) and mTOR (S6 and p70S6 kinase), and also inhibit tumor cell proliferation. However, in PC3 tumor cells lacking PTEN, effects on PI3K pathway readouts are less evident. In vivo pharmacodynamic analyses following oral administration of PI3Kα/mTOR inhibitors to mice bearing xenograft tumors demonstrate dose-dependent inhibition of phosphorylation of both PI3K and mTOR effectors at well-tolerated doses. Conclusions: PI3Kα and PI3Kα/mTOR-selective small molecule inhibitors were discovered that demonstrate potent in vitro and cellular activity and good oral bioavailability. Cellular selectivity for PI3Kα mutant tumor cells versus PTEN deleted tumor cells was demonstrated. These compounds will allow an exploration of the efficacy and tolerability of selective PI3Kα and PI3Kα/mTOR inhibitors in preclinical tumor models. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C59.