First, 6×His-FXR-LBD was purified with a Ni-NTA column followed by AKTA FPLC according to the protocol. Then we used SDS-PAGE to test the purity of 6×His-FXR-LBD. As shown in Figure 1, the 6×His-FXR-LBD was purified successfully.
Figure 1. SDS-PAGE assay
6×His-FXR-LBD was purified with a Ni-NTA column followed by AKTA FPLC. SDS-PAGE results indicated that 6×His-FXR-LBD was purified successfully.
Next, we screened FXR antagonists in Lab in-house compound library by AlphaScreen technology-based assay. As indicated in Figure 2A and 2B, among the compounds, H7 antagonized the GW4064-induced promotion of FXR-LBD binding to its coactivator SRC-1. These results thus implied the antagonistic feature of H7 against FXR.
Figure 2. H7 as an FXR antagonist inhibited FXR activity.
The effect of compounds (20μM) on the combination of FXR-LBD and SRC-1 was detected by AlphaScreen-based protein-peptide interaction assay. A. Among the compounds, H7 was finally selected for its highly antipathogenic activity against FXR. B. H7 antagonized GW4064-induced AlphaScreen signal. All data were presented as mean±S.E.M (*P<0.05, **P< 0.01, ***P< 0.001).
Finally, transactivation assay was further carried out to verify the antagonistic effect of H7 on FXR. As shown in Figure 3, FXR agonist GW4064 efficiently activated reporter gene expression, and H7 antagonized GW4064-induced reporter gene stimulation in transactivation assay. Thus, these results confirmed the antagonism of H7 against FXR transactivation activity.
Figure 3. H7 as an FXR antagonist inhibited transactivation activity.
HEK293T cells were transiently transfected with pcDNA3.1a-FXR, pcDNA3.1a-RXRα, pGL3-FXRE-Luc and pRL-SV40. After 6 h, cells were treated with different concentrations of H7 with GW4064 for 24 h. Then transactivation activity of FXR was detected by luciferase reporter assay. GW4064:FXR agonist, GS: FXR antagonist. All data were presented as mean±S.E.M (*P<0.05, **P< 0.01, ***P< 0.001).
As H7 has been demonstrated a FXR antagonist, we will investigate the regulation of H7 against hepatic glucose production and gluconeogenesis in vitro and in vivo in the future scientific research work.
Hardware&software: our school has built a brand-new bio-lab specifically for this competition, all the equipment follows the standard of universities’ lab. Furthermore, we have cooperation with labs at the Nanjing University of Traditional Medicine, so our hardware is strong and abundant. Our software is even stronger. Our direct teachers are doctors from the Nanjing University of Traditional Medicine, and we also have advisers from well-known universities and institutions such as professors from Cornell, Shanghai Fudan University, and the Chinese Academy of science. So, our software is still the best we can get.
Improvement: As H7 has been demonstrated an FXR antagonist, we will investigate the regulation of H7 against hepatic glucose production and gluconeogenesis in vitro and in vivo in the future scientific research work.
Wechat: Search "2020 IGEM CounterSugar" in WeChat
School Website: https://www.xdfyz.cn/