Yang-yang He1, Yu Yan1, Chang Zhang2, Peng-yuan Li3, Ping Wu1, Peng Du2, Da-di Zeng2, Jian-song Fang1, Shuang Wang2, Guan-hua Du1. 1. Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China. 2. Institute of Biotechnology, Chinese Academy of Military Medical Sciences, Beijing 100071, China. 3. Drug Manufacturing Room of Pharmaceutical Department, Henan Provincial People's Hospital, Zhengzhou 450003, China.
Abstract
AIM: Blockade of interleukin-6 (IL-6) or its receptor (IL-6R) is effective in preventing the progression of autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. In the present study, we established a novel cell-based assay for identifying small molecule IL-6R antagonists. METHODS: HEK293A cells were transfected with recombinant plasmids pTaglite-SNAP-IL6R and pABhFc-IL6 to obtain membrane-bound IL-6R and recombinant human IL-6 coupled with human Fc fragment (rhIL-6), respectively. A novel screening assay based on the interaction between IL-6R and rhIL-6 was established, optimized and validated. The stability of the assay was also assessed by calculating the Z'-factor. RESULTS: RhIL-6 dose-dependently bound to IL-6R expressed at HEK293A cell surface. The IC50 value of the known antagonist ab47215 was 0.38±0.08 μg/mL, which was consistent with that obtained using the traditional method (0.36±0.14 μg/mL). The value of Z'-factor was 0.68, suggesting that the novel assay was stable for high throughput screening. A total of 474 compounds were screened using the novel screening assay, and 3 compounds exhibited antagonistic activities (IC50=8.73±0.28, 32.32±9.08, 57.83±4.24 μg/mL). Furthermore, the active compounds dose-dependently inhibited IL-6-induced proliferation of 7TD1 cells, and reduced IL-6-induced STAT3 phosphorylation in U937 cells. CONCLUSION: A novel cell-based screening assay for identifying small molecule IL-6R antagonists was established, which simplifies the procedures in traditional cellular ELISA screening and profiling and reduces the costs.
AIM: Blockade of interleukin-6 (IL-6) or its receptor (IL-6R) is effective in preventing the progression of autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. In the present study, we established a novel cell-based assay for identifying small molecule IL-6R antagonists. METHODS: HEK293A cells were transfected with recombinant plasmids pTaglite-SNAP-IL6R and pABhFc-IL6 to obtain membrane-bound IL-6R and recombinant humanIL-6 coupled with human Fc fragment (rhIL-6), respectively. A novel screening assay based on the interaction between IL-6R and rhIL-6 was established, optimized and validated. The stability of the assay was also assessed by calculating the Z'-factor. RESULTS: RhIL-6 dose-dependently bound to IL-6R expressed at HEK293A cell surface. The IC50 value of the known antagonist ab47215 was 0.38±0.08 μg/mL, which was consistent with that obtained using the traditional method (0.36±0.14 μg/mL). The value of Z'-factor was 0.68, suggesting that the novel assay was stable for high throughput screening. A total of 474 compounds were screened using the novel screening assay, and 3 compounds exhibited antagonistic activities (IC50=8.73±0.28, 32.32±9.08, 57.83±4.24 μg/mL). Furthermore, the active compounds dose-dependently inhibited IL-6-induced proliferation of 7TD1 cells, and reduced IL-6-induced STAT3 phosphorylation in U937 cells. CONCLUSION: A novel cell-based screening assay for identifying small molecule IL-6R antagonists was established, which simplifies the procedures in traditional cellular ELISA screening and profiling and reduces the costs.
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