Type 2 diabetes mellitus (T2DM) and obesity have been considered epidemics and threats to public health worldwide. Exendin-4 (Ex), a GLP-1R agonist, has potential for treating T2DM and obesity. However, Ex has a half-life of only 2.4 h in humans and needs to be administered twice daily, which hampers its clinical application. In this study, we synthesized four new GLP-1R agonists by genetically fusing Ex to the N-terminus of HSA-binding ankyrin repeat proteins (DARPins) via linkers of different lengths, denoted as Ex-DARPin-GSx fusion proteins (x = 0, 1, 2, and 3). The Ex-DARPin fusion proteins were substantially stable, resulting in incomplete denaturation even at 80 °C. The in vitro bioactivity results demonstrated that Ex-DARPin fusion proteins could bind to HSA and activate GLP-1R. The Ex-DARPin fusion proteins had a comparable half-life (29-32 h), which is much longer than that of native Ex (0.5 h in rats). Subcutaneous injection of 25 nmol/kg Ex-DARPin fusion protein normalized blood glucose (BG) levels for at least 72 h in mice. The Ex-DARPin fusion proteins, injected at 25 nmol/kg every three days, significantly lowered BG, inhibited food consumption, and reduced body weight (BW) for 30 days in STZ-induced diabetic mice. Histological analysis of pancreatic tissues using H&E staining revealed that Ex-DARPin fusion proteins significantly improved the survival of pancreatic islets in diabetic mice. The differences in in vivo bioactivity of fusion proteins with different linker lengths were not significant. According to the findings in this study, long-acting Ex-DARPin fusion proteins designed by us hold promise for further development as antidiabetic and antiobesity therapeutic agents. Our findings also indicate that DARPins are a universal platform for generating long-acting therapeutic proteins via genetic fusion, thus broadening the application scope of DARPins.
Keywords: DARPins; Exendin-4; Half-life; Hypoglycemic effect; Pharmacodynamic property.
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