PRODUCTION AND CHARACTERIZATION OF ACYLATED AMYLIN RECEPTOR AGONISTS (AMY-RA)

Amylin is a pancreatic beta-peptide cosecreted with insulina and responsible for regulation of glucagon, satiety, gastric emptying, also resulting in decreased exogenous insulin requirements in diabetic patients. Restoring the beta-cell physiology in diabetic patients ideally would require using post-prandial and basal amylin, which is hampered by physicochemical properties, such as low aqueous solubility, prompt amyloid aggregation, and incompatibility in solution when coformulated with insulin. Conjugated molecules in bioactive peptides is a chemical strategy to bypass some stability issues of future biopharmaceutical products and their pharmacokinetics characteristics. In this work we envisioned the design of a protracted amylin analogue as a long fatty acid acylated form aimed to binding circulating albumin and increasing its half-life, which could be used in the restoring of basal levels. We conjugated amylin in solution with a C16 fatty acid, resulting in two chromatographically dissimilar monoacylated products as judged by mass spectrometry. Tryptic fragments of monoacylated amylin were sequenced by mass spectrometry, confirming the acylation at Lys1 for both products. Homo and heteronuclear NMR measurements allowed unambigous identification of the Lys1-Nα and Lys1-Nζ monoacylated isomers. We discuss these results at light of implications for amylin conformation upon acylation, and the interaction with receptor and albumin.

This work was supported by FAPERJ, CNPq, CAPES, INBEBB.