Abstract
Glucagon-like peptide-1 (GLP-1) signaling through the glucagon-like peptide 1 receptor (GLP-1R) is a key regulator of normal glucose metabolism, and exogenous GLP-1R agonist therapy is a promising avenue for the treatment of type 2 diabetes mellitus. To date, the development of therapeutic GLP-1R agonists has focused on producing drugs with an extended serum half-life. This has been achieved by engineering synthetic analogs of GLP-1 or the more stable exogenous GLP-1R agonist exendin-4 (Ex-4). These synthetic peptide hormones share the overall structure of GLP-1 and Ex-4, with a C-terminal helical segment and a flexible N-terminal tail. Although numerous studies have investigated the molecular determinants underpinning GLP-1 and Ex-4 binding and signaling through the GLP-1R, these have primarily focused on the length and composition of the N-terminal tail or on how to modulate the helicity of the full-length peptides. Here, we investigate the effect of C-terminal truncation in GLP-1 and Ex-4 on the cAMP pathway. To ensure helical C-terminal regions in the truncated peptides, we produced a series of chimeric peptides combining the N-terminal portion of GLP-1 or Ex-4 and the C-terminal segment of the helix-promoting peptide α-conotoxin pl14a. The helicity and structures of the chimeric peptides were confirmed using circular dichroism and NMR, respectively. We found no direct correlation between the fractional helicity and potency in signaling via the cAMP pathway. Rather, the most important feature for efficient receptor binding and signaling was the C-terminal helical segment (residues 22-27) directing the binding of Phe(22) into a hydrophobic pocket on the GLP-1R.
Highlights
Secretion of insulin is considerably higher in response to oral administration of glucose compared with intravenous delivery (1)
Because type 2 diabetes mellitus sufferers continue to respond to glucagon like peptide 1 (GLP-1), but not to glucose-dependent insulinotropic polypeptide (5), recent therapeutic supplement strategies have focused on GLP-1 and the development of synthetic analogs (6)
Design of Truncated GLP-1 and Ex-4 Conotoxin Chimeras— previous work suggested that the C-terminal segment of GLP-1 is important for efficient cAMP signaling via the glucagon-like peptide-1 receptor (GLP-1R) (7, 17–19), the underlying mechanism(s) of action is poorly understood and may be a result of loss of either C-terminal helical structure, binding interactions, or both
Summary
Main model; the helical C terminus of the ligand first binds the NTD of the receptor, dictating binding affinity and specificity (Fig. 1A), before the ligand N terminus interacts with the seventransmembrane bundle domain core of the receptor to affect signaling potency and specificity (13). GLP-1 affects receptor binding and signaling (23–26); to date there has been no investigation into the effect of C-terminal truncation of GLP-1 or Ex-4 on peptide helicity nor has there been research into the effect of inducing helicity in truncated analogs on receptor binding and signaling Another approach to stabilizing helices is grafting of these secondary structural motifs into highly constrained helical peptide scaffolds (27). We recently used an ␣-conotoxin (pc16a) to engineer a potent GLP-1R agonist by combining the conotoxin disulfide bond connectivity with features of a previously published 11-amino acid peptidomimetic GLP-1R agonist (28) Another ␣-conotoxin pl14a (Fig. 1C), first isolated from the cone snail Conus planorbis (29), shares several properties with GLP-1 and Ex-4 as both classes of peptides have a flexible N termini followed by an ␣-helix and both target membrane integrated receptors. These findings provide new insights into the mechanism of GLP-1R activation and may guide future development of minimized and disulfide-constrained GLP-1 or Ex-4 analogs for the treatment of type 2 diabetes mellitus
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