A structural basis for amylin receptor phenotype.

Amylin receptors (AMYRs) are heterodimers of the calcitonin (CT) receptor (CTR) and one of three receptor activity-modifying proteins (RAMPs), AMY1R, AMY2R, and AMY3R. Selective AMYR agonists and dual AMYR/CTR agonists are being developed as obesity treatments; however, the molecular basis for peptide binding and selectivity is unknown. We determined the structure and dynamics of active AMYRs with amylin, AMY1R with salmon CT (sCT), AMY2R with sCT or human CT (hCT), and CTR with amylin, sCT, or hCT. The conformation of amylin-bound complexes was similar for all AMYRs, constrained by the RAMP, and an ordered midpeptide motif that we call the bypass motif. The CT-bound AMYR complexes were distinct, overlapping the CT-bound CTR complexes. Our findings indicate that activation of AMYRs by CT-based peptides is distinct from their activation by amylin-based peptides. This has important implications for the development of AMYR therapeutics.

KBP-066A, a long-acting dual amylin and calcitonin receptor agonist, induces weight loss and improves glycemic control in obese and diabetic rats.

OBJECTIVE: Dual amylin and calcitonin receptor agonists (DACRAs) are novel therapeutic agents that not only improve insulin sensitivity but also work as an adjunct to established T2DM therapies. DACRAs are currently administered once daily, though it is unknown whether DACRAs with increased plasma half-life can be developed as a once-weekly therapy. METHODS: The in vitro potencies of the KBP-066A and KBP-066 (non-acylated) were assessed using reporter assays. Acylation functionality was investigated by a combination of pharmacokinetics and acute food intake in rats. in vivo efficacies were investigated head-to-head in obese (HFD) and T2D (ZDF) models. RESULTS: In in vitro, KBP-066A activated the CTR and AMY-R potently, with no off-target activity. Acylation functionality was confirmed by acute tests, as KBP-066A demonstrated a prolonged PK and PD response compared to KBP-066. Both compounds induced potent and dose-dependent weight loss in the HFD rat model. In ZDF rats, fasting blood glucose/fasting insulin levels (tAUC) were reduced by 39%/50% and 36%/47% for KBP-066 and KBP-066A, respectively. This effect resulted in a 31% and 46% vehicle-corrected reduction in HbA1c at the end of the study for KBP-066 and KBP-066A, respectively. CONCLUSIONS: Here, we present pre-clinical data on an acylated DACRA, KBP-066A. The in vivo efficacy of KBP-066A is significantly improved compared to its non-acylated variant regarding weight loss and glycemic control in obese (HFD) and obese diabetic rats (ZDF). This compendium of pre-clinical studies highlights KBP-066A as a promising, once-weekly therapeutic agent for treating T2DM and obesity.

Nanoscale Surface Topography Modulates hIAPP Aggregation Pathways at Solid-Liquid Interfaces.

The effects that solid-liquid interfaces exert on the aggregation of proteins and peptides are of high relevance for various fields of basic and applied research, ranging from molecular biology and biomedicine to nanotechnology. While the influence of surface chemistry has received a lot of attention in this context, the role of surface topography has mostly been neglected so far. In this work, therefore, we investigate the aggregation of the type 2 diabetes-associated peptide hormone hIAPP in contact with flat and nanopatterned silicon oxide surfaces. The nanopatterned surfaces are produced by ion beam irradiation, resulting in well-defined anisotropic ripple patterns with heights and periodicities of about 1.5 and 30 nm, respectively. Using time-lapse atomic force microscopy, the morphology of the hIAPP aggregates is characterized quantitatively. Aggregation results in both amorphous aggregates and amyloid fibrils, with the presence of the nanopatterns leading to retarded fibrillization and stronger amorphous aggregation. This is attributed to structural differences in the amorphous aggregates formed at the nanopatterned surface, which result in a lower propensity for nucleating amyloid fibrillization. Our results demonstrate that nanoscale surface topography may modulate peptide and protein aggregation pathways in complex and intricate ways.

Structural Polymorphs Suggest Competing Pathways for the Formation of Amyloid Fibrils That Diverge from a Common Intermediate Species.

It is now recognized that many amyloid-forming proteins can associate into multiple fibril structures. Here, we use two-dimensional infrared spectroscopy to study two fibril polymorphs formed by human islet amyloid polypeptide (hIAPP or amylin), which is associated with type 2 diabetes. The polymorphs exhibit different degrees of structural organization near the loop region of hIAPP fibrils. The relative populations of these polymorphs are systematically altered by the presence of macrocyclic peptides which template ß-sheet formation at specific sections of the hIAPP sequence. These experiments are consistent with polymorphs that result from competing pathways for fibril formation and that the macrocycles bias hIAPP aggregation toward one pathway or the other. Another macrocyclic peptide that matches the loop region but extends the lag time leaves the relative populations of the polymorphs unaltered, suggesting that the branching point for structural divergence occurs after the lag phase, when the oligomers convert into seeds that template fibril formation. Thus, we conclude that the structures of the polymorphs stem from restricting oligomers along diverging folding pathways, which has implications for drug inhibition, cytotoxicity, and the free energy landscape of hIAPP aggregation.

Amylin structure-function relationships and receptor pharmacology: implications for amylin mimetic drug development.

Amylin is an important, but poorly understood, 37 amino acid glucoregulatory hormone with great potential to target metabolic diseases. A working example that the amylin system is one worth developing is the FDA-approved drug used in insulin-requiring diabetic patients, pramlintide. However, certain characteristics of pramlintide pharmacokinetics and formulation leave considerable room for further development of amylin-mimetic compounds. Given that amylin-mimetic drug design and development is an active area of research, surprisingly little is known about the structure/function relationships of amylin. This is largely due to the unfavourable aggregative and solubility properties of the native peptide sequence, which are further complicated by the composition of amylin receptors. These are complexes of the calcitonin receptor with receptor activity-modifying proteins. This review explores what is known of the structure-function relationships of amylin and provides insights that can be drawn from the closely related peptide, CGRP. We also describe how this information is aiding the development of more potent and stable amylin mimetics, including peptide hybrids.

Convergent chemoenzymatic synthesis of a library of glycosylated analogues of pramlintide: structure-activity relationships for amylin receptor agonism.

Pramlintide (Symlin®), a synthetic analogue of the naturally occurring pancreatic hormone amylin, is currently used with insulin in adjunctive therapy for type 1 and type 2 diabetes mellitus. Herein we report a systematic study into the effect that N-glycosylation of pramlintide has on activation of amylin receptors. A highly efficient convergent synthetic route, involving a combination of solid phase peptide synthesis and enzymatic glycosylation, delivered a library of N-glycosylated variants of pramlintide bearing either GlcNAc, the core N-glycan pentasaccharide [Man3(GlcNAc)2] or a complex biantennary glycan [(NeuAcGalGlcNAcMan)2Man(GlcNAc)2] at each of its six asparagine residues. The majority of glycosylated versions of pramlintide were potent receptor agonists, suggesting that N-glycosylation may be used as a tool to optimise the pharmacokinetic properties of pramlintide and so deliver improved therapeutic agents for the treatment of diabetes and obesity.