Both single mutation and molecular conjugation on the mutant prevent calcitonin from forming amyloid fibrils.

Human calcitonin (hCT) is a peptide hormone made up of 32 amino acids, produced by the parafollicular cells of the thyroid gland. Its main role is to decrease blood calcium levels by inhibiting osteoclast activity and reducing calcium reabsorption in the kidneys and intestines. This characteristic positions hCT as a potential treatment for bone-related conditions, such as osteoporosis and Paget's disease. However, hCT has a strong tendency to form amyloid fibrils, which can lead to a loss of its biological function. This research aims to stabilize hCT's conformation through molecular conjugation to prevent its aggregation. We used a cross-linking tool with a boronic ester structure (NPC) to react with the side-chain amino groups of two residues in hCT, effectively "locking" its structure. hCT can be "unlocked" using hydrogen peroxide, returning it to its original form. To support this experimental design, we synthesized two hCT variants, hCT-Q14K and hCT-F22K. Our results showed that both variants successfully conjugated with NPC, and the conjugation could be easily removed with hydrogen peroxide within 30 min. NPC-conjugated hCT did not form amyloid as revealed by transmission electron microscopy. Moreover, thioflavin-T fluorescence kinetic studies and dynamic light scattering measurement demonstrated that the aggregation tendencies of hCT-F22K were significantly lower than those of hCT, while still maintaining their biological activity, suggesting this design is feasible. This approach may significantly aid in the development of calcitonin drugs.