Development and characterization of specific small molecule inhibitors of class II phosphatidylinositol 3-kinase C2alpha function

• Applicant: Professor Dr. Marc Nazare
• Subject Area: Biochemistry ; Cell Biology
• Term: from 2015 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 278189589

Phosphoinositides (PIPs) serve crucial roles in cell physiology, ranging from cell signalling to membrane traffic. Phosphatidylinositol- 4,5-bisphosphate [PI(4,5)P2] is concentrated at the plasma membrane where, among other functions, it is required for the nucleation of endocytic clathrin-coated pits (CCPs). As subsequent endosomal stages of the endocytic pathway are dominated by phosphatidylinositol 3-phosphates (i.e. PI(3)P) fission of endocytic vesicles from the plasma membrane and subsequent fusion with early endosomes must be accompanied by PIP conversion from PI(4,5)P2 to PI(3)P. How this is accomplished precisely is not completely understood but our own recent data indicate that part of this conversion may already occur at the cell surface through acquisition of phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2]. Specifically, we have shown that formation of PI(3,4)P2 by class II phosphatidylinositol-3-kinase C2alpha (PI3K C2a) spatiotemporally controls clathrin-mediated endocytosis (CME). Depletion of PI(3,4)P2 or PI3K C2a impairs the maturation of late-stage CCPs before fission. Timed formation of PI(3,4)P2 by PI3K C2a is required for selective enrichment of the BAR domain protein SNX9 at endocytic intermediates. In addition to its localization at plasma membrane CCPs, PI3K C2a is also found perinuclearly. Together with Dr. Emilio Hirsch, we demonstrated that PI3K C2a is enriched in the pericentriolar recycling endocytic compartment (PRE) at the base of the primary cilium. At the PRE PI3K C2a directly or indirectly (i.e. via synthesis of PI(3,4)P2 followed by enzymatic hydrolysis to PI(3)P) regulates the formation of a PI(3)P pool required for Rab11 and Sonic Hedgehog pathway activation. Finally, accumulating evidence suggests important roles for class II PI 3-kinases including PI3K C2a in the control of cell signalling, proliferation, survival, and angiogenesis, thereby identifying PI3K C2a as target for anticancer therapies. In the proposed research we will (i) develop and characterize novel isoform-specific inhibitors of PI3K C2a using high throughput screening combined with medicinal chemistry approaches and biochemical studies, (ii) analyze the effects of acute perturbation of PI3K C2a function in living cells to mechanistically dissect the role of PI3K C2a in CME, endosomal membrane traffic, PIP metabolism, as well as cell signaling and proliferation, and, finally, (iii) we aim to determine the 3-dimensional structure of PI3K C2a in complex with specific inhibitors by protein X-ray crystallography. The proposed studies are expected to yield novel important insights into the cellular function of PI3K C2a and may pave the way for the development of novel anti-cancer agents.

• DFG Programme: Research Grants