Establishment Of Human DPPIV High-throughput Screening Assay And Discovery Of Novel Inhibitors

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that stimulates insulinsecretion and protects β-cells, inhibits glucagon secretion and gadtric emptying, andreduces appetite and food intake. It is shown that in Type 2 diabetes patientsincreasing the concentration of GLP-1 can decrease plasma glucose level and treatthe disease. However, GLP-1 is rapidly cleavaged in vovo by Dipeptidyl peptidase IV(DPPIV), resulting in inactivation. To inhibit DPPIV activity could effectivelyelevate the GLP-1 level in plasma and treat dibetes mellitus. The aim of this work isto express recombinant DPPIV using baculovirus expression system, and set up thehigh-throughput screening (HTS) assay for DPPIV inhibitors screening. Through thescreening of compound library in National Center for Drug Screening, we hope tofind novel and effecive DPPIV inhibitors for further development of newanti-diabetic drugs.The vecter pFastBac1 was modified and the cDNA of extracellular domain ofDPPIV (i.e. the sequence encoding the 29th to 766th amino acids of DPPIV) wasconstructed into the modified pFastBac1. The constructed plasmid was transformedinto the competent cells of E.coli strain DH10Bac. Through blue-white screening andbacmid extraction we get the bacmid containning DPPIV cDNA. The recombinantbacmid was then transfected into the Sf9 insect cells and several days laterbaculovirus was harvested. When Hi5 insect cells were infected by amplified virus,the DPPIV protein was expressed and secreted into the medium. Active DPPIV waspurified more than 90% homogenity by nickel affinity chromatography from the medium.Enzymatic characterization of DPPIV including pH profile, the kinetics of enzymaticreaction, the inhibition by reported inhibitor, and thermal stability was studied andproved to be similar to those reported. By optimizing the reaction condition andscreening system, we established the standard operation procedure forhigh-throughput screening DPPIV inhibitors. 31,705 compounds were screened and 7hits were found with the hit ratio about 0.009%. The best compound showed an IC50at mircomole level. The modification and SAR (Structure-activity relationship) of thehits were being studied and the most effective inhibitor would be selected andvalidated its anti-diabetic activity in vovo.