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.
Chen
Jiaqi
J
Department of Microbiology, College of Life Sciences, Nankai University, Tianjin 30071, China.
Bai
Gang
G
Yang
Yang
Y
Geng
Peng
P
Cao
Yu
Y
Zhu
Yuanyuan
Y
D000595:Amino Acid Sequence; D000818:Animals; D001786:Blood Glucose / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D052216:Glucagon-Like Peptide 1 / Q000737:chemistry*; D000067757:Glucagon-Like Peptide-1 Receptor; D005951:Glucose Tolerance Test; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D007004:Hypoglycemic Agents / Q000737:chemistry / Q000494:pharmacology; D007274:Injections, Intraperitoneal; D051379:Mice; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D051381:Rats; D018027:Receptors, Glucagon / Q000037:antagonists & inhibitors / Q000235:genetics; D014162:Transfection
D001786:Blood Glucose; C000593834:Glp1r protein, mouse; C000593835:Glp1r protein, rat; D000067757:Glucagon-Like Peptide-1 Receptor; D007004:Hypoglycemic Agents; D019151:Peptide Library; D010455:Peptides; D018027:Receptors, Glucagon; D049452:Green Fluorescent Proteins; D052216:Glucagon-Like Peptide 1