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.
Highlights
Diabetes mellitus is one of the most common chronic diseases worldwide, involving the loss of control of blood glucose levels, which results in a continuously elevated glucose concentration
We previously reported the site-specific human serum albumin (HSA) conjugation to Glucagon-like peptide-1 (GLP-1) by expressing recombinant GLP-1 variants bearing an non-natural amino acids (NNAAs) as an albumin conjugation site fused to superfolder green fluorescent protein in genetically engineered Escherichia coli [24]
We investigated the effects of albumin conjugation sites and conjugation chemistry on in vivo blood-lowering activities with the ultimate goal of designing albumin-GLP1 conjugates with enhanced therapeutic potency
Summary
Diabetes mellitus is one of the most common chronic diseases worldwide, involving the loss of control of blood glucose levels, which results in a continuously elevated glucose concentration. In 2016, an estimated 1.6 million deaths were directly caused by diabetes [2]. To prevent these complications, controlling the blood glucose levels of patients with diabetes is of great importance. (GLP-1) is an essential hormone that contributes to the regulation of blood glucose levels. After secretion from L-cells of the intestine [3], GLP-1 is directed toward many organs to reduce the blood glucose levels, including the pancreas, heart, muscles, kidneys, liver, and even the brain [4]. Theresulted in vitro activity of GLP1-8G37C-HSA was comparable activity, consistent with previous findings [23,25]. The in vitro activity of of GLP1-8G37Cto that of GLP1-8G16AzF-HSA. Despite the low EC50 value of lowering activity was inferior to those. 7B)lowering due to itsactivity very short plasmawas half-life (only a fewGLP1-8G37Cminutes in animal) [32,33].
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