Development of a High-Throughput Cell-Based Reporter Assay for Screening of JAK3 Inhibitors

作者：Yin Chang Hong; Bach Erika A; Baeg Gyeong Hun^*

来源：Journal of Biomolecular Screening, 2011, 16(4): 443-449.

DOI：10.1177/1087057111400190

• assay development
• cytokine
• JAK3 inhibitors
• STAT5 reporter
• high-throughput chemical screening

JAK3 is an ideal target for the treatment of immune-related diseases and the prevention of organ allograft rejection. Several JAK3 inhibitors have been identified by biochemical enzymatic assays, but the majority display significant off-target effects on JAK2. Therefore, there is a need to develop new experimental approaches to identify compounds that specifically inhibit JAK3. Here, we show that in 32D/IL-2R beta cells, STAT5 becomes phosphorylated by an IL-3/JAK2- or IL-2/JAK3-dependent pathway. Importantly, the selective JAK3 inhibitor CP-690,550 blocked the phosphorylation and the nuclear translocation of STAT5 following treatment of cells with IL-2 but not with IL-3. In an attempt to use the cells for large-scale chemical screens to identify JAK3 inhibitors, we established a cell line, 32D/IL-2R beta/6xSTAT5, stably expressing a STAT5 reporter gene. Treatment of this cell line with IL-2 or IL-3 dramatically increased the reporter activity in a high-throughput format. As expected, CP-690,550 selectively inhibited the activity of the 6xSTAT5 reporter following treatment with IL-2. By contrast, the pan-JAK inhibitor curcumin inhibited the activity of this reporter following treatment with either IL-2 or IL-3. Thus, this study indicates that the STAT5 reporter cell line can be used as an efficacious cellular model for chemical screens to identify selective JAK3 inhibitors. (Journal of Biomolecular Screening 2011; 16: 443-449)

• 出版日期2011-4