Model Construction For Screening Agonist Of Glucagon-like Peptide-1 Receptor

Glucagon-like peptide-1 receptor(GLP-1R)belongs to G-protein-coupled receptor(GPCR),which is one of the important targets for designing type 2 diabetes mellitus.GLP-1R consists of a N-terminal extracellular domain,a seven transmembrane helix domain,and a smaller intracellular C-terminal domain.At present,polypeptides and small molecules developed for GLP-1 analogues and DPP-IV inhibitors have been widely used in the treatment of type 2 diabetes,but the half-life of peptide drugs is relatively short and requires continuous intravenous or subcutaneous injection.difference.However,the existing oral small molecule hypoglycemic drugs have not achieved the desired effects with certain side effects.Therefore,we aimed to find small molecule agonists targeting the target GLP-1R.In this study,Firstly,we constructed a recombinant vector of GLP-1R and GFP,and the vector was transiently transfected into HEK293 cell line.The expression of the target protein was successfully detected by GLP-1R N-terminal antibody and stimulated with a drug that has been proven effective.GLP-1R found that the intracellular cAMP content after stimulation with the drug was significantly higher than that of the control group,and the feasibility of the cell model was successfully verified,and stable cell lines with high expression were obtained by stable transfection.Next,the full-length GLP-1R full-length protein was expressed using a baculovirus expression system,and a small molecule compound bound thereto was selected.In addition,using the Pichia pastoris expression system to express the polypeptide ligase Butelase1 and designing the substrate to detect its activity,try to use the E.coli expression system to express the transmembrane domain in stages and use the polypeptide ligase Butelase1 to segment the transmembrane domain to obtain the full-length cross-section.Membrane domain proteins,screening for small molecule drugs that bind to the transmembrane domain.Finally,the pMFH-His-GLP-1 recombinant vector was constructed to express the GLP-1 polypeptide in the form of inclusion bodies,and the polypeptide purified by expression was used for FITC labeling.The cell model and protein model were constructed,and the high-throughput screening of the natural small molecule compound library was carried out.The selected small molecule compound will be beneficial to the development of a new type 2 diabetes drug.In this study,GLP-1R was expressed in different expression systems and a drug cell and protein screening model was established.The establishment of this drug screening model can provide a theoretical basis for the development of oral small molecule drugs for type 2 diabetes.