Type 2 diabetes (T2DM), is caused by multiple reasons, such as high levels of blood sugar, resistance to insulin, and lack of insulin. Instead of inherited defects, it occurs accompanying irregular lifestyles, such as a lack of physical exercise. Due to the inferior quality of and biological resistance to insulin, a larger amount of this chemical is required, therefore, resulting in Hyper-insulinemic syndrome, a high level of insulin in the physical body. In essence, T2D is caused by the improvement of sugar levels.

Our experiment chose Glucokinase (GK), as the target enzyme. GK is a kind of isozyme of hexokinase that expresses in pancreatic b cells and organs that respond to islets, for example, livers, brains, and the intestinal tract. GK takes part in the first step of glycolysis and transfers Glucose into Glucose 6-phosphate(G6P). The appetency of GK, as the receptor of glucose in the cells, to glucose is much lower than the other hexokinases. Meanwhile, it stays unsaturated at physiological glucose concentration and is not inhibited by its catalytic reaction product G6P. Therefore, GK can timely sense the changes in blood glucose in the body, improve the utilization rate of glucose and quickly regulate the blood glucose level to a relatively normal state.

GK, as a glucose receptor in pancreatic islets β cells, is mainly responsible for promoting insulin release and biosynthesis under glucose stimulation, as well as regulating pancreatic islets β cell survival and proliferation. The glycolytic pathway catalyzed by GK produces a large amount of ATP and increases the ratio of ATP / ADP in the cytoplasm, this change of this ratio leads to the closure of ATP sensitive potassium ion channel (KATP), which leads to the depolarization of cell membrane, leading to the increase of intracellular Ca2+ concentration and finally the release of insulin. In addition, the stimulation of glucose leads to the phosphorylation of the S115 position of bad protein on mitochondria, which makes it combine with GK, hindering bad from playing its role of apoptosis, thus promoting the survival of pancreatic islet cells.
So far, dozens of GK agonists have entered clinical trials, among which dorzagliatin developed by Hualing pharmaceutical has entered the stage of drug registration and marketing development in China, and is expected to become the first GK small molecule agonist to be listed. In phase 2 and phase 3 clinical studies, it has a significant hypoglycemic effect, almost no adverse effects of hypoglycemia, and can significantly improve pancreatic islets’ β Cell function, effectively maintaining the stability of blood sugar. Therefore, targeting GK to develop new small molecule agonists has an ideal application prospect.
Our goal of the research is to filter out small molecules compounds that increase the liveness of glucokinase. The final product shall be able to target glucokinase, which is brand-new and resistant to T2DM small molecules.

First, we inserted the DNA fragment of the hGK2 into the BamHI and SalI sites of the pQE-30 vector, and transformed the recombinant plasmid into E. coli M15 competent cells and coated on the LB culture medium plate. For verifying the plasmids, we digested the plasmids with BamHI and SalI and sent the constructed recombinant plasmid to a sequencing company for sequencing.
Then, we inoculate the correct recombinants and induced the hGK2 protein expression by IPTG. We purified the hGK2 protein for glucokinase agonists detection and we screened glucokinase agonists through the hGK2 screen system, and compound 13926 was finally discovered.
Finally, the effects of 13926 on Insulin release and cell viability in INS-832/13 cells were detected by using Insulin Detection Kit and MTT. The results indicated that 13926 effectively promoted insulin secretion and protected it from STZ damage in INS-832/13 cells.

1. Successfully construct pQE-30-hGK2 recombinant plasmid.
2. Express and purify the hGK2 protein.
3. An hGK2 agonist screening platform was established to evaluate the activity of the selecting active compounds.
4. Verify the enhanced effect of the compound screened on Insulin secretion and the protective effect on pancreatic islet cells.

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