Effect of C-terminus Conjugation via Different Conjugation Chemistries on In Vivo Activity of Albumin-Conjugated Recombinant GLP-1

Abstract

Glucagon-like peptide-1 (GLP-1) is a peptide hormone with tremendous therapeutic potential for treating type 2 diabetes mellitus. However, the short half-life of its native form is a significant drawback. We previously prolonged the plasma half-life of GLP-1 via site-specific conjugation of human serum albumin (HSA) at position 16 of recombinant GLP-1 using site-specific incorporation of p-azido-phenylalanine (AzF) and strain-promoted azide-alkyne cycloaddition (SPAAC). However, the resulting conjugate GLP1_8G16AzF-HSA showed only moderate in vivo glucose-lowering activity, probably due to perturbed interactions with GLP-1 receptor (GLP-1R) caused by the albumin-linker. To identify albumin-conjugated GLP-1 variants with enhanced in vivo glucose-lowering activity, we investigated the conjugation of HSA to a C-terminal region of GLP-1 to reduce steric hindrance by the albumin-linker using two different conjugation chemistries. GLP-1 variants GLP1_8G37AzF-HSA and GLP1_8G37C-HSA were prepared using SPAAC and Michael addition, respectively. GLP1_8G37C-HSA exhibited a higher glucose-lowering activity in vivo than GLP1_8G16AzF-HSA, while GLP1_8G37AzF-HSA did not. Another GLP-1 variant, GLP1_8A37C-HSA, had a glycine to alanine mutation at position 8 and albumin at its C-terminus and exhibited in vivo glucose-lowering activity comparable to that of GLP1_8G37C-HSA, despite a moderately shorter plasma half-life. These results showed that site-specific HSA conjugation to the C-terminus of GLP-1 via Michael addition could be used to generate GLP-1 variants with enhanced glucose-lowering activity and prolonged plasma half-life in vivo.

Enhanced anti‐tumor activity of arginine decarboxylase through the incorporation of aromatic amino acids at the multimer‐forming interface

“…Fluorescent dye labeling of RDC variants was performed in a manner similar to a previously reported method. [ 27 ] RDC‐WT and RDC‐frTet were reacted with TCO‐PEG4‐Cy3 dye at a 1:5 molar ratio in PBS (pH 7.4) at room temperature for 1 hour. The reaction mixtures were subjected to SDS‐PAGE gel electrophoresis.…”

Improved systemic half-life of glucagon-like peptide-1-loaded carbonate apatite nanoparticles in rats

“…Moreover, systemically administered peptide drugs have very short half-lives owing to renal clearance and their interaction with the host immune system[ 3 ], thus necessitating multiple administration over the course of treatment, which may in turn causes systemic toxicity and undesirable off-target effects. The drawback of GLP1 has also led to the common strategies of conjugating GLP1 or its analogues to polyethylene-glycol (PEG)[ 4 , 5 ] and albumin[ 6 , 7 ] with the aim to extend the peptide’s half-life. However, the efficiency and long-term safety of these conjugates have limited their use[ 6 ].…”