Chemically Precise Glycoengineering Improves Human Insulin

Abstract

Diabetes is a leading cause of death worldwide and results in over 3 million annual deaths. While insulin manages the disease well, many patients fail to comply with injection schedules, and despite significant investment, a more convenient oral formulation of insulin is still unavailable. Studies suggest that glycosylation may stabilize peptides for oral delivery, but the demanding production of homogeneously glycosylated peptides has hampered transition into the clinic. We report here the first total synthesis of homogeneously glycosylated insulin. After characterizing a series of insulin glycoforms with systematically varied O-glycosylation sites and structures, we demonstrate that O-mannosylation of insulin B-chain Thr27 reduces the peptide’s susceptibility to proteases and self-association, both critical properties for oral dosing, while maintaining full activity. This work illustrates the promise of glycosylation as a general mechanism for regulating peptide activity and expanding its therapeutic use.

Chemical synthesis of N‐glycosylated insulin‐like androgenic gland factor from the freshwater prawn Macrobrachium rosenbergii

“…Based on it, the unnatural chemical modification of proteins with glycans has been investigated to improve the stability and the biological function of proteins, such as erythropoietin . Glycosylated insulin analogs have also been synthesized, and it was shown that the glycosylation prolonged the biological activity in vivo …”

“…21 Based on it, the unnatural chemical modification of proteins with glycans has been investigated to improve the stability and the biological function of proteins, such as erythropoietin. 22 Glycosylated insulin analogs have also been synthesized, [23][24][25] and it was shown that the glycosylation prolonged the biological activity in vivo. 23 Interestingly, AGH from the terrestrial isopod Armadillidium vulgare originally possesses an N-linked glycan in A chain, and it has been reported that this glycan moiety is essential for conferring the hormonal activity in vivo.…”

Protein Glycoengineering: An Approach for Improving Protein Properties

“…The validity of this guideline was confirmed by the glycoengineering study of a therapeutic protein, human insulin. In this study, they demonstrated that O-mannosylation of insulin B-chain Thr27 reduced its susceptibility to proteases and self-association (Guan et al, 2018 ).…”

Just a spoonful of sugar: Short glycans affect protein properties and functions

“…In particular, the doubly modified interferon showed increased thermal in vitro stability, a 10‐fold longer elimination half‐life and much lower apparent serum clearance than its nonglycosylated counterpart. As for insulin, mannosylation of a specific Thr in the B chain of the peptide reduced its susceptibility to proteases and aggregation, thus doubling insulin half‐life and opening to the possibility of oral delivery …”

“…This ability could be exploited for the development of new therapeutic approaches for neurodegenerative diseases as well as for improving the properties of therapeutic proteins. A representative case is the one of insulin, where the addition of a sugar at a specific position in the B‐chain reduced the aggregation propensity and improved handling and formulation …”