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

肌醇磷脂(PIP)在细胞生理中起着重要作用，从细胞信号转导到细胞膜运输。磷脂酰肌醇-4，5-二磷酸[PI(4，5)P2]集中在质膜上，除了其他功能外，它还是胞内胞质蛋白包裹的核(CCP)成核所必需的。由于内吞途径的后续阶段是由磷脂酰肌醇3-磷酸盐(即PI(3)P)主导的，因此从质膜上分裂内吞小泡并随后与早期内容体融合必须伴随着从PI(4，5)P2到PI(3)P的PIP转换。这一转换的精确实现方式尚不完全清楚，但我们最近的数据表明，这种转换的一部分可能已经发生在细胞表面，通过获得磷脂酰肌醇-3，4-二磷酸[PI(3，4)P2]。具体地说，我们已经证明了由II类磷脂酰肌醇-3-激酶C2a(PI3K C2a)形成的PI(3，4)P2在时空上控制了笼蛋白介导的内吞作用(CME)。PI(3，4)P2或PI3K C2a的缺失会损害晚期CCP在分裂前的成熟。PI3K C2a需要定时形成PI(3，4)P2，以选择性地在胞内中间体富集杆状结构域蛋白SNX9。PI3K C2a除定位于质膜CCP外，在核周也有分布。与埃米利奥·赫希博士一起，我们证明了PI3K C2a在初级纤毛底部的中心周围循环内细胞室(PRE)中富含。在PI3K前，C2a直接或间接(即通过合成PI(3，4)P2，然后酶解成PI(3)P)调节Rab11和Sonic Hedgehog途径激活所需的PI(3)P池的形成。最后，越来越多的证据表明，包括PI3K C2a在内的II类PI 3-Kinase在控制细胞信号、增殖、存活和血管生成方面发挥着重要作用，从而确定PI3K C2a是抗癌治疗的靶点。在拟议的研究中，我们将(I)利用高通量筛选结合药物化学方法和生化研究来开发和表征新型的PI3K C2a异构体特异性抑制剂，(Ii)分析活细胞中PI3K C2a功能的急性扰动的影响，从机制上剖析PI3K C2a在CME、内膜运输、PIP代谢以及细胞信号和增殖中的作用，最后，(Iii)我们的目标是通过蛋白质X-射线结晶学确定PI3K C2a与特定抑制剂形成的复合体的三维结构。这些研究有望对PI3K C2a的细胞功能产生新的重要见解，并可能为新型抗癌药物的开发铺平道路。

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.