Our composite component, BBa_K4516019, is a platform to screen small molecule FoxO1 antagonists that can be used to treat type 2 diabetes.
As early as 2020, the iGEM20_XDFYZ team was committed to developing a drug screening platform (BBa_K1162006) for the treatment of type 2 diabetes. They found candidate drug molecules by screening FXR antagonists. Based on their work, by reading literature and consulting experts in related fields, we found that in addition to the FXR target, there are also a FoxO1 target that plays an important role in the regulation of blood glucose balance. Therefore, we further optimized the diabetes drug screening platform and constructed a new composite part BBa_K4516019 to screen more kinds of drug molecules that can be used to treat type 2 diabetes.
In order to prove the function of our new composite part hFoxO1-F. luciferase-R. luciferase, we transferred the recombinant plasmid into HepG2 to establish a transcriptional activation screening platform, and screened a totally new compound 355, which has an inhibitory effect on the transcriptional activation activity of FoxO1. Then, by detecting the effect of 355 active compounds on hepatic gluconeogenesis and the mRNA levels of key enzymes in hepatic gluconeogenesis, it was further confirmed that 355 compounds have the effect of inhibiting hepatic gluconeogenesis, thereby achieving the effect of lowering blood sugar.
FoxO1, as a transcription factor, plays an important role in the regulation of blood glucose balance. FoxO1 in the liver can promote the expression of key gluconeogenesis enzyme genes, and an important mechanism of insulin regulation of blood sugar is to increase the phosphorylation of FoxO1, promote its nuclear export, and then reduce its transcriptional activation activity. According to this theory, we constructed a human FoxO1 full-length plasmid, used Firefly luciferase and Renilla luciferase as a reporter gene, established a FoxO1 transcriptional activation screening platform, and used this platform to screen out active compounds that inhibit FoxO1 transcriptional activation activity.
We design three plasmids: pcDNA3.1-hFoxO1 which contains the hFoxO1 expression unit, pGL3pro-3IRE which contains Firefly luciferase, and pRL-SV40 which contains Renilla luciferase. shown in Figure 1.
Figure 1. The map of three plasmids pcDNA3.1-hFoxO1 (A), pGL3pro-3IRE (B), and pRL-SV40
(C).
In order to obtain the target fragment, we used PrimeStar high-fidelity polymerase to amplify hFoxO1 DNA products. Then we obtained the target DNA fragments hFoxO1 through agarose electrophoresis and gel purification. Afterward, we cloned the corresponding plasmids and used DNA sanger sequencing to verify the construction. Then, we extracted the recombinant plasmids from E.coli DH5α and transferred them into HepG2 cell, so that can be used as a transcriptional activation screening platform.
To regulate the expression of hFoxO1, we used small molecular compounds from the database to examine their effects on gluconeogenesis (Table1). Compound 335 was found to have a significant role in reducing glucose concentrations in HepG2 cells.
| Compound | Inhibitory rate (%) |
|---|---|
| AS1842856 | 50.13 |
| 177 | 49.54 |
| 222 | 63.2 |
| 240 | 38.12 |
| 340 | 31.91 |
| 355 | 97.17 |
| 389 | 45.31 |
| 391 | 40.28 |
| 392 | 77.57 |
| 396 | 50.06 |
| 460 | 25.27 |
| 548 | 57.52 |
| 550 | 41.47 |
In Fig.2, in the third column of the histogram, the luciferase activity level was low in the presence of the hFoxO1 transcriptional activator under the action of compound 355, so it could be concluded that compound 355 has an obvious inhibitory effect on hFoxO1 transcriptional activation.
Figure 2. Compound 355 inhibits hFoxO1 transcription activation.
After screening for compound 355 by luciferase activity detection (Fig.2), compound 355 was also used to inhibit both glucose concentration in HepG2 cells and mRNA abundance of key enzymes of gluconeogenesis.
In Fig.3A, we can find that with the gradual increase of the concentration of compound 355, the glucose level showed a counter gradient change and was concentration-dependent, indicating that compound 355 could significantly inhibit gluconeogenesis in hepatocytes.
Figure 3. Detection of the 355 compounds on hepatic gluconeogenesis. G6Pase is an enzyme that releases glucose into the blood by hydrolyzing glucose-6-phosphate in liver tissue. PEPCK is a gluconeogenic enzyme that allows hepatic parenchymal cells to produce glucose from pyruvate derived from amino acid metabolism. In Fig. 3B and 3C, when the volume of compound 355 increases gradually, the mRNA levels of G6pase and PEPCK gradually decreased in a counter gradient and were also concentration-dependent, indicating that compound 355 could significantly reduce the mRNA levels of key enzymes in gluconeogenesis.
