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

II类磷脂酰肌醇3-激酶C2α功能的特异性小分子抑制剂的开发和表征

• 批准号: 278189589
• 负责人: Professor Dr. Marc Nazare
• 金额: --
• 依托单位: Arbeitsgruppe Medizinische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/278189589?language=en
• 关键词: Development; characterization; specific; small; molecule

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.

磷酸肌醇（PIP）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]在质膜处浓缩，在其他功能中，它是内吞网格蛋白包被的小凹（CCP）成核所需的。由于内吞途径的后续内体阶段由磷脂酰肌醇3-磷酸主导，（即PI（3）P）胞吞囊泡从质膜分裂并随后与早期内体融合必须伴随着PIP从PI（4，5）P2至P1（3）P.如何做到这一点是不完全理解，但我们自己最近的数据表明，这种转换的一部分可能已经发生在通过获得磷脂酰肌醇-3，4-二磷酸[PI（3，4）P2]而在细胞表面形成。具体来说，我们已经表明，形成PI（3，4）P2的II类磷脂酰肌醇-3-激酶C2 α（PI 3 K C2 a）时空控制网格蛋白介导的内吞作用（CME）。PI（3，4）P2或PI 3 K C2 a的耗尽损害了后期CCP在裂变前的成熟。通过PI 3 K C2 a定时形成PI（3，4）P2是选择性富集BAR结构域蛋白SNX 9在内吞中间体处所需的。除了定位于质膜CCP外，还发现PI 3 K C2 a在核周。与Emilio Hirsch博士一起，我们证明了PI 3 K C2 a在初级纤毛基部的中心粒周围再循环内吞隔室（PRE）中富集。在PRE时，PI 3 K C2 a直接或间接（即通过合成PI（3，4）P2，然后酶水解为PI（3）P）调节Rab 11和Sonic Hedgehog途径激活所需的PI（3）P库的形成。最后，越来越多的证据表明II类PI 3-激酶包括PI 3 K C2 a在控制细胞信号传导、增殖、存活和血管生成中的重要作用，从而将PI 3 K C2 a鉴定为抗癌疗法的靶标。在拟议的研究中，我们将（i）使用高通量筛选结合药物化学方法和生物化学研究开发和表征PI 3 K C2 a的新型亚型特异性抑制剂，（ii）分析活细胞中PI 3 K C2 a功能的急性扰动的影响，以机械地剖析PI 3 K C2 a在CME、内体膜运输、PIP代谢以及细胞信号传导和增殖中的作用，最后，（iii）我们的目标是通过蛋白质X射线晶体学确定与特异性抑制剂复合的PI 3 K C2 a的三维结构。这些研究有望对PI 3 K C2 a的细胞功能产生新的重要见解，并可能为开发新型抗癌药物铺平道路。