Identification of ALPK1 inhibitors

本财政年度该项目的目标是建立一种高通量筛选（HTS）试验，以评估ALPK 1在其配体ADP-庚糖刺激下的磷酸化。具体而言，该试验使用了表达ALPK 1的市售HEK 293 NF-kB报告细胞系。将在存在或不存在ADP-庚糖的情况下评价候选化合物的报告子活化。随后将在TNF-α存在下评价化合物的“靶向”活性，TNF-α独立于ALPK 1激活NF-κ B报告基因。此外，表达相同报告基因的ALPK 1 KO细胞系将用于确认化合物对途径的活性。该测定法正在针对化合物和配体孵育时间以及其他参数进行优化。

The goal of the project during this fiscal year has been to establish a high-throughput screening (HTS) assay to assess phosphorylation by ALPK1 upon stimulation by its ligand ADP-heptose. Specifically, the assay utilizes a commercially available HEK293 NF-kB reporter cell line expressing ALPK1. Candidate compounds will be evaluated for reporter activation in the presence or absence of ADP-heptose. Compounds will be subsequently evaluated for "on target" activity in the presence of TNF-alpha, which activates the NF-kB reporter independently of ALPK1. Furthermore, ALPK1 KO cell lines expressing the same reporter, will be utilized to confirm compound on pathway activity. The assay is being optimized for compound and ligand incubation time, among other parameters.