Identifying glucagon-like peptide-1 mimetics using a novel functional reporter gene high-throughput screening assay

Glucagon-like peptide-1 (GLP-1) stimulates insulin and inhibits glucagon secretion and therefore could potentially be used to treat diabetes type II. However, its therapeutic use is limited by its short half-life in vivo, due mainly to enzymatic degradation by dipeptidyl peptidase IV (DPP-IV). Developing GLP-1 analogs with greater bioactivity is therefore an important step toward using them therapeutically. Accordingly, we aimed to identify GLP-1 mimetic peptides by creating a high-throughput screening (HTS) assay of a phage displayed (PhD) peptide library. This assay was functionally based using the GLP-1 receptor (GLP-1R) gene. Rat GLP-1R cDNA was transfected into CHO/enhanced green fluorescent protein (EGFP) cells by lipofection. The resulting stable, recombinant cell line functionally expressed the GLP-1R and a cAMP-responsive EGFP reporter gene, to monitor receptor activation, and was used to screen a PhD dodecapeptide library. After four rounds of selection, 10 positive clones were selected based on functional evaluation and sequenced. Three sequences were obtained, corresponding to three different domains of GLP-1 (Group 1: 22-34; Group 2: 18-29; and Group 3: 6-17). The Group 3 peptide had the highest bioactivity, was synthesized, and designated KS-12. Importantly, KS-12 activated GLP-1R in vitro and reduced blood glucose levels in a dose-dependent manner when administered to Chinese Kunming mice. Although KS-12 was not as effective as GLP-1, it was significantly resistant to DPP-IV both in vitro and in vivo. Thus, this study provides a novel way to screen DPP-IV resistant agonist peptides of GLP-1 from a PhD peptide library using the functional reporter gene HTS assay.