Characterization of signal bias at the GLP-1 receptor induced by backbone modification of GLP-1

Abstract

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor that is a major therapeutic target for the treatment of type 2 diabetes. Activation of this receptor promotes insulin secretion and blood glucose regulation. The GLP-1R can initiate signaling through several intracellular pathways upon activation by GLP-1. GLP-1R ligands that preferentially stimulate subsets among the natural signaling pathways (“biased agonists”) could be useful as tools for elucidating the consequences of specific pathways and might engender therapeutic agents with tailored effects. Using HEK-293 cells recombinantly expressing human GLP-1R, we have previously reported that backbone modification of GLP-1, via replacement of selected α-amino acid residues with β-amino acid residues, generates GLP-1 analogues with distinctive preferences for promoting G protein activation versus β-arrestin recruitment. Here, we have explored the influence of cell background across these two parameters and expanded our analysis to include affinity and other key signaling pathways (intracellular calcium mobilization and ERK phosphorylation) using recombinant human GLP-1R expressed in a CHO cell background, which has been used extensively to demonstrate biased agonism of GLP-1R ligands. The new data indicate that α/β-peptide analogues of GLP-1 exhibit a range of distinct bias profiles relative to GLP-1 and that broad assessment of signaling endpoints is required to reveal the spectrum of behavior of modified peptides. These results support the view that backbone modification via α→β amino acid replacement can enable rapid discovery of peptide hormone analogues that display substantial signal bias at a cognate GPCR.

Xenopus‐derived glucagon‐like peptide‐1 and polyethylene‐glycosylated glucagon‐like peptide‐1 receptor agonists: long‐acting hypoglycaemic and insulinotropic activities with potential therapeutic utilities

“…We initially conducted alanine scanning to investigate the SAR of xGLP‐1B. Considering that a conservative N‐terminal sequence is critical for the ability of GLP‐1 analogues to activate the GLP‐1 receptor (Hager et al, ), alanine scanning was started from position 11. Amino acids at positions 11–17 and 20–30 were serially replaced by L‐alanine, giving peptides 1 – 7 and 10 – 20 .…”

Receptor selectivity from minimal backbone modification of a polypeptide agonist

“…This practical distinction has skewed the functional evaluation of peptides containing β-amino-acid residues toward β 3 -residues. Most prior work on peptides that contain α→β replacements (α/β-peptides) has focused on β 3 -residues that maintain the configuration of L-α-amino acids (18,19,(30)(31)(32)(33)(34)(35), which means S for most β 3 -residues but R in a few cases, such as β 3 -hSer or β 3 -hThr. Residues with this absolute stereochemistry, which we designate S* here [in other words, (R)-β 3 -hSer is designated (S*)-β 3 -hSer] can participate in right-handed α-helix-like secondary structures, as demonstrated crystallographically for numerous α/β-peptides containing 25-33% β-residues distributed among L-α residues (36)(37)(38).…”

Evaluation of β‐Amino Acid Replacements in Protein Loops: Effects on Conformational Stability and Structure

“…Flexible loops can be sites for enzymatic cleavage of proteins, and a single β residue is expected to confer significant resistance to proteolysis for a few peptide bonds on either side of the substitution site. Protein loops are commonly found at protein–protein interaction interfaces, and β residue substitutions in these contexts might prove useful for modulating signaling pathways . Recent results suggest that it might ultimately be possible to generate protein analogues with occasional α→β 3 substitution through ribosomal synthesis, which raises the prospect of employing targeted replacements in flexible loops to generate metabolically stabilized variants of full‐fledged proteins.…”

“…Helix stability often declines as a result of α→β 3 replacements, but β 3 →cyclic β replacements usually stabilize α/β‐peptide helices . Helical α/β‐peptides can display useful activities, such as blocking pathogenic protein–protein interactions while resisting proteolytic degradation, or modulating the signaling profile, relative to a natural agonist, caused by activation of a cell‐surface receptor …”