Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Ca-Ca distances, solvent exposure, and side-chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C-terminus of the Bchain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild-type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R-state conformation and thus showing that the R-state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function.

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An ingenious system evolved to facilitate insulin binding to the insulin receptor as a monomer and at the same time ensure sufficient stability of insulin during storage. Insulin dimer is the cornerstone of this system. Insulin dimer is relatively weak, which ensures dissociation into monomers in the circulation, and it is stabilized by hexamer formation in the presence of zinc ions during storage in the pancreatic β-cell. Due to the transient nature of insulin dimer, direct investigation of this important form is inherently difficult. To address the relationship between insulin oligomerization and insulin stability and function, we engineered a covalently linked insulin dimer in which two monomers were linked by a disulfide bond. The structure of this covalent dimer was identical to the self-association dimer of human insulin. Importantly, this covalent dimer was capable of further oligomerization to form the structural equivalent of the classical hexamer. The covalently linked dimer neither bound to the insulin receptor, nor induced a metabolic response in vitro. However, it was extremely thermodynamically stable and did not form amyloid fibrils when subjected to mechanical stress, underlining the importance of oligomerization for insulin stability.

Small molecules with insulin mimetic effects and oral availability are of interest for potential substitution of insulin injections in the treatment of diabetes. We have searched databases for compounds capable of mimicking one epitope of the insulin molecule known to be involved in binding to the insulin receptor (IR). This approach identifies thymolphthalein, which is an apparent weak agonist that displaces insulin from its receptor, stimulates auto- and substrate phosphorylation of IR, and potentiates lipogenesis in adipocytes in the presence of submaximal concentrations of insulin. The various effects are observed in the 10(-5)-10(-3) M range of ligand concentration and result in partial insulin activity. Furthermore, analogues of the related phenol red and fluorescein molecules fully displace insulin from the IR ectodomain, however, without insulin agonistic effects. The interactions are further characterized by NMR, UV-vis, and fluorescence spectroscopies. It is shown that both fluorescence and UV-vis changes in the ligand spectra induced by IR fragments occur with Kd values similar to those obtained in the displacement assay. Nevertheless, insulin itself cannot completely abolish binding of the small molecules. Determination of the binding stoichiometry reveals multiple binding sites for ligands of which one overlaps with the insulin binding site on the receptor.

A hallmark of the pancreatic hormone amylin is its high propensity toward the formation of amyloid fibrils, which makes it a challenging drug design effort. The amylin analogue pramlintide is commercially available for diabetes treatment as an adjunct to insulin therapy but requires three daily injections due to its short half-life. We report here the development of the stable, lipidated long-acting amylin analogue cagrilintide (23) and some of the structure–activity efforts that led to the selection of this analogue for clinical development with obesity as an indication. Cagrilintide is currently in clinical trial and has induced significant weight loss when dosed alone or in combination with the GLP-1 analogue semaglutide.