Amylin (free acid) (human)

Biological drugs, specifically proteins and peptides, are a privileged class of medicinal agents and are characterized with high specificity and high potency of therapeutic activity. However, biologics are fragile and require special care during storage, and are often modified to optimize their pharmacokinetics in terms of proteolytic stability and blood residence half-life. In this review, we showcase glycosylation as a method to optimize biologics for storage and application. Specifically, we focus on chemical glycosylation as an approach to modify biological drugs. We present case studies that illustrate the success of this methodology and specifically address the highly important question: does connectivity within the glycoconjugate have to be native or not? We then present the innovative methods of chemical glycosylation of biologics and specifically highlight the emerging and established protecting group-free methodologies of glycosylation. We discuss thermodynamic origins of protein stabilization via glycosylation, and analyze in detail stabilization in terms of proteolytic stability, aggregation upon storage and/or heat treatment. Finally, we present a case study of protein modification using sialic acid-containing glycans to avoid hepatic clearance of biological drugs. This review aims to spur interest in chemical glycosylation as a facile, powerful tool to optimize proteins and peptides as medicinal agents.

The human islet amylin polypeptide is produced along with insulin by pancreatic islets. Under some circumstances, amylin can aggregate to form amyloid fibrils, whose presence in pancreatic cells is a common pathological feature of Type II diabetes. A growing body of evidence indicates that small, early stage aggregates of amylin are cytotoxic. A better understanding of the early stages of the amylin aggregation process and, in particular, of the nucleation events leading to fibril growth could help identify therapeutic strategies. Recent studies have shown that, in dilute solution, human amylin can adopt an α-helical conformation, a β-hairpin conformation, or an unstructured coil conformation. While such states have comparable free energies, the β-hairpin state exhibits a large propensity towards aggregation. In this work, we present a detailed computational analysis of the folding pathways that arise between the various conformational states of human amylin in water. A free energy surface for amylin in explicit water is first constructed by resorting to advanced sampling techniques. Extensive transition path sampling simulations are then employed to identify the preferred folding mechanisms between distinct minima on that surface. Our results reveal that the α-helical conformer of amylin undergoes a transformation into the β-hairpin monomer through one of two mechanisms. In the first, misfolding begins through formation of specific contacts near the turn region, and proceeds via a zipping mechanism. In the second, misfolding occurs through an unstructured coil intermediate. The transition states for these processes are identified. Taken together, the findings presented in this work suggest that the inter-conversion of amylin between an α-helix and a β-hairpin is an activated process and could constitute the nucleation event for fibril growth.