Glucagon-like peptide-2 (GLP-2) is secreted postprandially by enteroendocrine L-cells and stimulates growth of the gut and bone. One GLP-2 analogue is approved for short bowel syndrome (SBS). To improve therapeutic efficacy, we developed biased GLP-2 receptor (GLP-2R) agonists through N-terminal modifications.
Variants with Ala and Trp substitutions of the first seven positions of GLP-2(1-33) were studied in vitro for affinity, G protein activation (cAMP accumulation), recruitment of β-arrestin 1 and 2, and internalization of the human and mouse GLP-2R. The intestinotrophic actions of the most efficacious (cAMP) biased variant was examined in mice.
Overall, Ala substitutions had more profound effects than Trp substitutions. For both, alterations at positions 1, 3 and 6 most severely impaired activity. β-arrestin recruitment was more affected than cAMP accumulation. Among the Ala substitutions, [H1A], [D3A] and [F6A] impaired potency for cAMP-accumulation >20-fold and efficacy (E ) to 48-87%, and were unable to recruit arrestins. The Trp substitutions, [A2W], [D3W] and [G4W] were also partial agonists (E of 46-59%) with 1.7-12-fold decreased potencies in cAMP and diminished β-arrestin recruitment. The biased variants, [F6A], [F6W] and [S7W] induced less GLP-2R internalization compared to GLP-2, which induced internalization in a partly arrestin-independent manner. In mice, [S7W] enhanced the gut trophic actions with increased weight of the small intestine, increased villus height and crypt depth compared to GLP-2.
G protein-biased GLP-2R agonists with diminished receptor desensitization have superior intestinotrophic effect and could represent improved treatment of intestinal insufficiency including SBS.

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