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  • Since our new compound Fex

    2021-09-26

    Since our new compound Fex-3 could be a new FXR ligand, we try to demonstrate that Fex-3 was an intestinal-restricted FXR agonist which only activated FXR in the intestine not in the liver or other organs. Initially, we investigated this in Caco-2 Ac-Endothelin-1 (16-21), human synthesis with transwell experiments. From and , we found that the accumulative amount of Fex-3 and Fex in cell was nearly the same but the penetrating velocity was different and Fex was faster than Fex-3 obviously. In addition, Fex uptaking amount increased during the time while Fex-3 went to platform at about 3h (). The concentration curves also show that Fex-3 could be absorbed into Caco-2 cells more easily than Fex (). These results indicated that Fex-3 was more intestinal-restricted than Fex, inferred to that Fex-3 could not enter into circulation. With the results obtained from the above experiments we tried to explore the ability of Fex-3 . After one week accommodating, the mice were treated with Fex or Fex-3 (control with vehicle) for 5days (the doses of Fex and Fex-3 are both 100mgkg) and sacrificed 1h after the final treatment with tissues (liver, kidney, duodenum, jejunum and ileum) and serum collected. We measured the concentration of Fex and Fex-3 in jejunum, ileum, liver and plasma by LC-MS/MS. From , we found that Fex accumulated mostly in ileum, then in jejunum, but nearly in liver and serum which was consistent with the reported literature. Interestingly, Fex-3 only accumulated in the ileum and the detectable drug concentration Fex-3 was obviously higher than Fex. This result might demonstrate that Fex-3 could be a better intestinal-restricted FXR agonist than Fex. Next, the mRNA expression of FXR in duodenum, jejunum, ileum, liver and kidney were detected by PCR. The FXR mRNA level in Fex-3 treated mouse ileum was significantly higher than vehicle (). Fex-3 had no influence on the FXR mRNA expression in other organs, including the liver. This was consistent with the results shown in , which means that Fex-3 mainly activated FXR of the ileum. In order to explain that why Fex-3 exhibits better intestinal selectivity, we calculated the of Fex and Fex-3 out as 7.01 and 7.42. Since Fex-3 has the higher value, we supposed that the solubility of Fex-3 in aqueous solution was poorer than Fex. The calculation results implied that Fex-3 would accumulate in the intestinal epithelial cells and does not enter into the blood. However, we found that Fex-3 could not be dissolved in DMSO/mineral oil medium and the drug dissolved out and was adhered to the intestine in i.p. treated mice. Hence, the results from i.p. treated mice could not be used for elucidating our hypothesis. Then in exploring the effects, we discovered that Fex-3 p.o. treated (100mgkg for 5d) produced robust induction of the FXR target gene in the ileum. However, Fex-3 cannot induce the expression of in the liver, kidney and other part of intestine. Additionally, we found that Fex-3 induced FXR downstream genes and significantly than Fex while suppressed the expression of (). We could make a conclusion that Fex-3 works only in the ileum and exhibits better tissue-selectivity than Fex. To investigate the binding mode between Fex-3 and FXR, molecular docking was conducted based on the previously reported co-crystal structure of Fex and FXR (PDB ID: ). The green (Fex) and purple (Fex-3) structures occupy nearly the same cavity of FXR (). However, Fex-3 could stick into the protein inner than Fex as shown in the figure. For further investigation into the binding affinity of Fex and Fex-3 with FXR, the K value was measured by fortebio. As shown in , the binding affinity of Fex-3 with FXR was significantly higher than Fex with nearly two order of magnitude. In summary, we have successfully designed and synthesized a novel intestinal-restricted FXR agonist Fex-3. In comparison with Fex, Fex-3 exhibits slightly higher FXR agonism activity and much better selectivity in the ileum. Intestinally restricted not systemic FXR activation leads to decrease in obesity and insulin resistance and other whole-body benefits. To further investigation into efficacy of Fex-3, animal experiments are being conceived and designed.