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

磷酸肌苷（PIPs）在细胞生理学中起着至关重要的作用，从细胞信号传导到膜运输。磷脂酰肌醇- 4,5-二磷酸[PI(4,5)P2]集中在质膜上，在质膜上，除其他功能外，它是内吞网格蛋白包覆凹坑（CCPs）成核所必需的。由于内吞途径的后续内体阶段由磷脂酰肌醇3-磷酸（即PI(3)P）主导，因此内吞囊泡从质膜分裂并随后与早期内体融合必须伴随着PI(4,5)P2到PI(3)P的PIP转化。这一过程是如何精确完成的尚不完全清楚，但我们自己最近的数据表明，通过获取磷脂酰肌醇-3,4-二磷酸，部分转化可能已经在细胞表面发生[PI(3,4)P2]。具体来说，我们已经证明II类磷脂酰肌醇-3-激酶C2alpha （PI3K - C2a）形成PI(3,4)P2在时空上控制网格蛋白介导的内吞作用（CME）。PI(3,4)P2或PI3K - C2a的耗竭会损害裂变前晚期ccp的成熟。PI3K C2a定时形成PI(3,4)P2是选择性富集内吞中间体中BAR结构域蛋白SNX9所必需的。除了定位于质膜ccp外，PI3K - C2a也存在于核周围。与Emilio Hirsch博士一起，我们证明了PI3K - C2a在初级纤毛基部的中心周围循环内胞室（PRE）中富集。在PRE PI3K中，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通路激活所需的PI(3)P池的形成。最后，越来越多的证据表明，PI3K - C2a等II类PI3K - 3激酶在控制细胞信号传导、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。在本研究中，我们将(i)利用高通量筛选结合药物化学方法和生化研究，开发和表征PI3K - C2a的新型异构体特异性抑制剂；（ii）分析活细胞中PI3K - C2a功能急性扰动的影响，从而从机制上剖析PI3K - C2a在CME、内体膜交通、PIP代谢以及细胞信号传导和增殖中的作用；（iii）我们的目标是通过蛋白质x射线晶体学确定PI3K - C2a与特定抑制剂复合物的三维结构。这些研究有望对PI3K - C2a的细胞功能产生新的重要见解，并可能为开发新的抗癌药物铺平道路。

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.