Construction Of The Drug Screening Model For GLP1 Receptor Agonists

According to the international diabetes federation (IDF) statistics:by 2014 global almost 3.87 million people have diabetes, the prevalence of 8.3%and also in increasing spiraled. Glucagon-like peptide 1 (GLP1) activates GLP1 receptor (GLP1R) to stimulate insulin secretion and inhibit glucagon secretion, and therefore are potential as targets for the treatment of type 2 diabetes. Existing GLP1R agonists are macromolecular peptides (such as Exenatide and Liraglutide), only subcutaneous injection, difficult to preserve and transport. Therefore, it is important to develop GLP1 analogue with small molecule for treating type II diabete. However, so far it is short of effective evaluation and screening model for GLP1R agoist, blocking the development of GLP1 analogue with small molecule. This study established a high-throughput screening model to identity and evaluate GLP1 analogue effectively. In this model, sample is simple to prepare and it is suitable for mixture screening. The false negative rate and false positive rate were greatly reduced with high sensitivity and repeatability. This study will be a basis for GLP1R agoist screening. The results were shown below:Eukaryotic expression vector pCMV6-AC-GFP-GLPlR was constructed. In this vector, the C terminal of GLP1R was tagged by green fluorescent protein (GFP) directly. Vector pCMV6-AC-GFP-GLPlR was transfected into Rin-m5F cells by lipofection, and then monoclonal cell line Rin-m5F/GLPlR was obtained by G418 screening method. This cell could passage more than 40 generations stably, in which fluorescent could be observed even distribution. The expression of GLP1R protein was greatly increased by Western bloting. All these results showed that GLP1R protein with GFP label has been successfully and stably expressed in Rin-m5F cells.2) Available medicine verified the screening model:Green fluorescent distributed evenly in cells in the control group, treatment with glyburide (which is proved as a drug not targeted GLP1R) appeared without obvious fluorescence spots inside, while treatment with exenatide (GLP1R agonist) presented with fluorescence spots and the fluorescence reached the maximum at 40 min. These results showed that screening model Rin-m5F/GLP1R-GFP for GLP1R agonist was constructed successfully.3) This model was applied to evaluate the Liraglutide and other twelve kinds of Proprietary Chinese medicine. The results showed that Liraglutide could active the model, while the water extract or alcohol extract of these Proprietary Chinese medicine could not. The results showed that GLP1 agonist Liraglutide can be specifically recognized by this model while the other twelve kinds of Proprietary Chinese medicine were not GLP1 analogues, inactivating GLP1R targets.