Small Molecule Modulators of the Actin Cytoskeleton

描述（由申请人提供）：细胞内肌动蛋白丝动力学是多种过程的基础，如神经元轴突引导、吞噬、血小板活化、细胞器运动、细胞运动和癌症转移。在分子水平上了解肌动蛋白动力学将使我们对这些过程有更深入的了解，并可能导致发现抑制肿瘤细胞侵袭和转移的新药物靶点。小分子尤其有助于探测快速过程，如细胞骨架动力学，尽管其中大多数是偶然发现的。这个研究项目的中心是系统地发现小分子工具，用它来研究哺乳动物细胞中的肌动蛋白动力学。本文提出了五个目标：1)确定G72*的分子靶点，G72*是一种抑制磷酸肌醇（4,5）二磷酸（PIP2）诱导的爪蟾卵提取物中肌动蛋白组装的环肽。2)利用面向多样性的合成方法生成细胞可渗透的环状肽和近端盖肽库。3)对海绵提取物进行纯化，形成半纯馏分进行高通量筛选。4)筛选合成和天然产物库中抑制phorbo -12-肉豆蔻酸-13-乙酸酯（PMA）诱导的哺乳动物细胞膜褶皱的化合物。5)结合亲和层析和蛋白质组学分析方法，鉴定在膜褶皱过程中组装的磷酸化蛋白和与f -肌动蛋白特异性相关的蛋白。

DESCRIPTION (provided by applicant): Actin filament dynamics in the cell underlie diverse processes, such as neuronal axon guidance, phagocytosis, platelet activation, organelle movement, cell motility, and cancer metastasis. Understanding actin dynamics at the molecular level will give us insight into these processes and may lead to the identification of new drug targets for the inhibition of tumor cell invasion and metastasis. Small molecules in particular have been instrumental in probing fast process such as cytoskeletal dynamics, although most of these have been discovered by serendipity. This research program centers on the systematic discovery of small molecule tools with which to study actin dynamics in mammalian cells. Five aims are proposed: 1) To identify the molecular target of G72*, a cyclic peptide that inhibits phosphoinositol(4,5) bisphosphate (PIP2)-induced actin assembly in Xenopus egg extracts. 2) To generate libraries of cell-permeable cyclic and near, end-capped peptides using diversity-oriented synthetic methods. 3) To purify marine sponge extracts and format the resulting semi-pure fractions for high-throughput screening. 4) To screen synthetic and natural product libraries for compounds that inhibit phorbol-12-myristate-13-acetate (PMA)-induced membrane ruffling in mammalian cells. 5) To identify phosphoproteins and proteins that associate specifically with F-actin assembled during membrane ruffling using a combination of affinity chromatography and proteomic profiling methods.