HTS for enhancers of neutrophil function that specifically augment PIP3 signal

说明(申请人提供)：中性粒细胞是循环白细胞中含量最丰富的细胞类型，是宿主抵御入侵病原体的第一道防线。PtdIns(3，4，5)P3是一种位于细胞膜上的肌醇磷脂，可以调节中性粒细胞的趋化、吞噬、超氧化物生成、死亡/存活和杀菌等功能。PtdIns(3，4，5)P3通过与PtdIns(3，4，5)P3的PtdIns(3，4，5)P3结合，通过与PtdIns(3，4，5)P3的PtdIns(3，4，5)P3的PtdIns(3，4，5)P3结合来调节蛋白质的转位。因此，PH结构域易位为开发中性粒细胞功能调节剂提供了一个有吸引力的靶点。本研究的最终目的是通过高通量的化学遗传筛选，寻找针对PH域质膜转位的PtdIns(3，4，5)P3途径激活物。我们最近建立了一个实验系统，用于在活细胞中可视化这一过程。我们利用Akt的PHAkt结构域(PHAkt)与绿色荧光蛋白(PHAkt-GFP)融合作为这一事件的标志。建立了稳定表达PHAkt-GFP的HL60细胞株。HL60细胞可被诱导分化为形态成熟的中性粒细胞。诱导分化的HL60细胞经化学诱导剂刺激后，可检测到PHAkt-GFP从胞浆向质膜的转位。当PHAkt-GFP在趋化梯度中转移到趋化细胞的前沿时，在化学诱导剂的均匀处理过程中，它会暂时从胞浆转移到质膜。上调PtdIns(3，4，5)P3信号不仅增加了PHAkt-GFP最初的细胞质到质膜的转位，而且显著推迟了随后的从质膜到胞浆的“反向转位”，为高通量筛选(HTS)提供了良好的读数。建立了PHAkt-GFP质膜转位的定量检测方法。此外，我们的初步数据证明了我们的方法用于检测化学诱导剂诱导的PH域膜转位的选择性和重复性。在这项拟议的研究中，我们将使这种基于细胞的系统适应高通量格式，并检查是否可以实现同样的选择性和重复性(目标I)。此外，还将建立几种二次筛查试验，以确认从初筛中确定的每一种阳性化合物的效果，并确定最特异和最有效的化合物，以供未来鉴定(AIM II)。最后，提出了评价已鉴定化合物生物活性的方案。我们将调查已确定的化合物提高PH域膜转位是否能够增强各种中性粒细胞功能(目标III)。公共卫生相关性：项目简介我们研究的最终目标是通过对小分子文库进行高通量筛选，确定PtdIns(3，4，5)P3途径激活剂，这些激活剂专门针对PH域质膜转位。这些激活物的发现将极大地促进我们对PtdIns(3，4，5)P3信号在中性粒细胞功能和天然免疫中的研究。此外，已鉴定的激活剂可直接用作新型免疫增强剂开发的起始化合物，特别是对于由于缺乏足够的病原体杀灭能力而导致的严重感染。

DESCRIPTION (provided by applicant): Neutrophils are the most abundant cell type among circulating white cells and constitute the first line of host defense against invading pathogens. Various neutrophil functions such as chemotaxis, phagocytosis, superoxide production, death/survival, and bacteria killing, can be regulated by PtdIns(3,4,5)P3, an inositol phospholipid localized on the plasma membrane. PtdIns(3,4,5)P3 exerts its function by mediating protein translocation via binding to their pleckstrin homolog (PH)-domains. Thus, PH domain translocation provides an attractive target for developing modulators of neutrophil function. The ultimate goal of this study is to identify PtdIns(3,4,5)P3 pathway activators that specifically target PH domain plasma membrane translocation via conducting a high throughput chemical genetic screening. We have recently established an experimental system for visualizing this process in live cells. We utilized the PH-domain of Akt (PHAkt) fused with green fluorescent protein (PHAkt-GFP) as a marker for this event. A HL60 cell line stably expressing PHAkt-GFP has been generated. HL60 cells can be induced to differentiate towards morphologically mature neutrophils. PHAkt-GFP translocation from cytosol to the plasma membrane could be easily detected in differentiated HL60 cells after chemoattractant stimulation. While PHAkt-GFP translocates to the leading edge of chemotaxing cells in chemotactic gradient, during uniform treatment with chemoattractant it transiently translocates from cytosol to the plasma membrane. Elevating PtdIns(3,4,5)P3 signal not only augments the initial cytoplasm-to-plasma membrane translocation of PHAkt-GFP, but also dramatically delays its subsequent "reverse translocation" from the plasma membrane to cytosol, providing an excellent readout for high throughput screening (HTS). A method for quantifying PHAkt-GFP plasma membrane translocation has also been established. In addition, our preliminary data demonstrated the selectivity and reproducibility of our assay for detecting chemoattractant- elicited PH domain membrane translocation. In this proposed research, we will adapt this cell-based system to a high-throughput format and examine whether the same selectivity and reproducibility can be achieved (Aim I). In addition, several secondary screening assays will be established to confirm the effect of each positive hit compound identified from the primary screening and to identify the most specific and potent ones for future characterization (Aim II). Finally, a plan to evaluate the biological activities of identified compounds is proposed. We will investigate whether elevating PH domain membrane translocation by identified compounds is able to enhance various neutrophil functions (Aim III). PUBLIC HEALTH RELEVANCE: Project Narrative The ultimate goal of our research is to identify PtdIns(3,4,5)P3 pathway activators that specifically target PH domain plasma membrane translocation via performing a high throughput screening of small molecule libraries. Discovery of these activators will greatly facilitate our research on PtdIns(3,4,5)P3 signaling in neutrophil function and innate immunity. Moreover, the identified activators can be directly utilized as starting chemical compounds for novel immune enhancer drug development, particularly for severe infections due to lack of enough pathogen killing capability.