Identification of ALPK1 inhibitors

本财政年度的项目目标是建立一种高通量筛选（HTS）方法，以评估ALPK1在其配体adp -庚糖刺激下的磷酸化作用。具体来说，该试验利用市售的表达ALPK1的HEK293 NF-kB报告细胞系。候选化合物将在adp -庚糖存在或不存在的情况下评估报告基因激活。随后将评估化合物在tnf - α存在下的“靶标”活性，tnf - α独立于ALPK1激活NF-kB报告基因。此外，将利用表达相同报告基因的ALPK1 KO细胞系来确认化合物对通路的活性。该试验正在优化化合物和配体孵育时间，以及其他参数。

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.