Insulin has been widely used as a peptide drug to treat diabetes by facilitating the uptake of glucose from the blood. Despite the growing interest in the development of oral insulin drugs to address the low patient compliance caused by frequent subcutaneous injections, there have been unresolved challenges that oral insulin could be precluded by proteolytic digestion and low permeability across the intestinal epithelium upon oligomerization. Recent experimental studies have revealed that certain O-linked glycosylation patterns could reduce insulin’s proteolytic susceptibility and dimerization propensity, which is promising for tackling this issue. However, there is a lack of understanding of how different O-linked glycosylation patterns influence the proteolytic stability and dimerization propensity of insulin at the molecular level, which impedes the design of insulin glyco-variants with better properties for oral delivery. In light of this, we propose and test several metrics based on the structural characteristics of insulin and its interaction with alpha-chymotrypsin, one of the proteases found able to degrade insulin. We targeted 12 insulin glycoforms that were previously studied in experimental work. With glycoforms built upon five different insulin wildtype models, we performed 2 microsecond molecular dynamics (MD) simulations for each of them. All the computational metrics were then measured from the MD trajectories and they were compared with the experimental results for assessing the efficacy. As a result, we found that metrics such as solvent accessible surface area of the cleavage/binding sites, tail region helicity, and beta-sheet propensity of the P1-P3 region did not strongly correlate with the two insulin properties of interest. However, we discovered that glycan-involved hydrogen bonds and glycan-dimer occlusion were respectively the key factors regulating the proteolytic stability and dimerization propensity of insulin, which illuminates the path of rational molecular design of insulin glycoforms.

Investigating the influence of O-linked glycosylation on the proteolytic stability and dimerization propensity of insulin using molecular dynamics

ACS Spring 2022

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