Structure, Self-Association, and Aggregation Properties of a Long-Acting Amylin Receptor Agonist.

Pharmacokinetic (PK) properties of therapeutic peptides can be extended through covalent conjugation to albumin-binding fatty acids. The present study examines the effects of fatty acid conjugation and vehicle composition on structure, self-association pattern, and aggregation propensity of an amylin analogue. Small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and circular dichroism (CD) were applied to elucidate structural features and self-association patterns, whereas the propensity toward formation of amyloid fibrils under extreme stressful conditions was assessed based on thioflavin T fluorescence. Fatty acid conjugation of the unstructured amylin analogue induced a transition from a flexible and disordered state to a helix-enriched globular core-shell self-associate consistent with a size average of five monomers (pentamer). These structural changes did not increase the propensity toward formation of amyloid fibrils, suggesting that the self-associated species are not on the pathway to amyloid fibril formation. Increasing concentration of the fatty acid-conjugated amylin analogue 1) induced self-association to an average size corresponding to a pentamer around 30 μM, 2) increased repulsion between self-associated species, and 3) increased α-helix content which leveled off around 0.25 mM. Only at significantly higher peptide concentrations (2.2 mM), amyloid fibril formation was observed following 24 h continuous exposure to extreme mechanical stress conditions. The extent of electrostatic repulsion between the self-associated species was reduced by increasing NaCl concentration (up to 50 mM) or by raising pH (from pH 3 to pH 5). However, under conditions with the least electrostatic repulsion, 4 h of continuous exposure to extreme mechanical stress was needed to induce formation of amyloid fibrils of this inherently stable molecule.