Development and characterization of specific small molecule inhibitors of class II phosphatidylinositol 3-kinase C2alpha function
Cell Biology
Final Report Abstract
Accumulating evidence suggests important roles for class II PI 3-kinases including PI3K C2α in the control of cell signaling, proliferation, and survival. Downregulation of PI3K C2α below a critical threshold reduces cell proliferation (i.e. of hepatoma cells), induces apoptotic cell death, and sensitizes cancer cells to chemotherapeutic agents. PI3K C2α is essential for endothelial cell function and migration and, thus, for angiogenesis, a crucial mechanisms enabling tumor survival in vivo. Thus, class II PI 3-kinases, including PI3K C2α represent potential targets for anticancer therapies. Based on these findings we started the project aiming to developing specific small molecule inhibitors for PI3K C2α. A high throughput screening of a library of 37,224 compounds using an ADP-GloTM assay for PI3K C2α Δ1-298 and several back-screening campaigns identified 2-thiopteridinones as the most active (IC50: 0.79 µM) and selective class II PI3K inhibitors. Extensive structure-activity relationship (SAR) of the 2-thiopteridinone hit by systematic single point modifications at the entire molecule perimeter revealed key structural determinants and pharmacophore features essential for affinity towards PI3K C2α as well as for the specificity within the lipid kinase family. Several iterative cycles of design, synthesis and testing of more than 170 compounds was performed and enabled, in the absences of X-ray co-crystal structure, a first ligand-based binding hypothesis. While any modifications at the pteridinone core scaffold, including scaffold hopping strategies, were detrimental for affinity PI3K C2α, the structural modulation at the two existing attached arms at the 2-position and 3-position, representing two extension vectors of the 2-thiopteridinone core, were favorably responsive to structural changes. Crucial for C2α specificity was the revelation of a particular activity and specificity handle by substitution with a thiazolyl-phenyl ring system. The best representative shows nanomolar inhibitory activity against PI3K C2α in the (IC50: 47 nM, ADP- Glo kinase assay; 126 nM, Adapta Assay) and full selectivity against a panel of diverse kinase and all related lipid kinases tested (<20% inhibition at 10µM concentration). Subsequent evaluation in live cells showed no signs of cytotoxicity in four cell lines at high concentrations of 100 µM even in the absence serum. Overall, we identified the first specific PI3K C2α inhibitor and dissected key structural features determining PI3K C2α affinity and selectivity based on a ligand-centered perspective. This probe will further facilitate investigations of PI3K C2α pharmacology in cellular settings in particular clarifying on endocytosis and endosomal membrane traffic as well as on cell signaling. The specific PI3K C2α inhibitor may also assist obtaining a protein crystal structure of PI3K C2α and provide insights into the catalytic mechanism and the regulation of the kinase as well allowing structure-guided drug design.
