Towards Better Diabetes Therapeutics: Designing a More Stable Insulin Analog

Abstract

Insulin is a hormone that plays a central role in the regulation of human metabolism, and as a drug, is used in the treatment of diabetes mellitus. Hyperglycemia characterizes this condition due to a range of reasons from impaired insulin production by pancreatic beta cells to abnormalities resulting in resistance to insulin action. Depending on time and mechanism of action, the main types of insulin analogs are basal and prandial. Basal insulin analogs are slow-acting insulins that maintain a continuous basal level of insulin in the bloodstream. Prandial insulin analogs are fast-acting and their therapeutic goal is to avoid immediate and late post-prandial hyperglycemia. Most analogs face the problem of chemical degradation and amyloid-like fibril formation (fibrillation) in delivery devices. Thus, many modifications have been made to insulin in the effort to make it more stable and faster-acting. This thesis aims to study the effects of modifications that could be used to design an insulin analog with improved chemical and physical properties, while maintaining biological activity. We studied six amino-acid substitutions to native human insulin in different combinations: desB1 , AB2 , EB3, EA8 , EA14, and EB29. Analogs of the protein were chemically synthesized. Then, fibrillation and circular dichroism assays were performed using purified proteins. The results suggested that EB3 and EA14 are stabilizing modifications that prevent fibril formation, whereas EA8 and EA14 increase the structural stability of an analog. Our findings also suggested that certain modifications in isolation may not impact overall stability, but when combined with others, may show detectable effects, which is why EA8 and EA14 became the focus of further experiments. Cell-based activity assays indicated that all the analogs had similar biological activities. Future work will assess chemical degradation, solubility, amide proton exchange (as monitored by NMR), and mitogenicity.