Switch‐Peptides: Design and Characterization of Controllable Super‐Amyloid‐Forming Host–Guest Peptides as Tools for Identifying Anti‐Amyloid Agents

Abstract

Several amyloid‐forming proteins are characterized by the presence of hydrophobic and highly amyloidogenic core sequences that play critical roles in the initiation and progression of amyloid fibril formation. Therefore targeting these sequences represents a viable strategy for identifying candidate molecules that could interfere with amyloid formation and toxicity of the parent proteins. However, the highly amyloidogenic and insoluble nature of these sequences has hampered efforts to develop high‐throughput fibrillization assays. Here we describe the design and characterization of host–guest switch peptides that can be used for in vitro mechanistic and screening studies that are aimed at discovering aggregation inhibitors that target highly amyloidogenic sequences. These model systems are based on a host–guest system where the amyloidogenic sequence (guest peptide) is flanked by two β‐sheet‐promoting (Leu‐Ser)n oligomers as host sequences. Two host–guest peptides were prepared by using the hydrophobic core of Aβ comprising residues 14–24 (HQKLVFFAEDV) as the guest peptide with switch elements inserted within (peptide 1) or at the N and C termini of the guest peptide (peptide 2). Both model peptides can be triggered to undergo rapid self‐assembly and amyloid formation in a highly controllable manner and their fibrillization kinetics is tuneable by manipulating solution conditions (for example, peptide concentration and pH). The fibrillization of both peptides reproduces many features of the full‐length Aβ peptides and can be inhibited by known inhibitors of Aβ fibril formation. Our results suggest that this approach can be extended to other amyloid proteins and should facilitate the discovery of small‐molecule aggregation inhibitors and the development of more efficacious anti‐amyloid agents to treat and/or reverse the pathogenesis of neurodegenerative and systemic amyloid diseases.

Methods for the Peptide Synthesis and Analysis

“…In fact, in circular dichroism (CD) studies of O-acyl isopeptides or switch peptides, transitions to their peptide counterparts, controlled induction or reversal of secondary structure, and self-assembly of small peptides have been observed [148,151,152]. These studies have provided a tool to disrupt amyloid-derived peptide assemblies [153] and to identify antiamyloid agents [154].…”

Photocontrol of Reversible Amyloid Formation with a Minimal-Design Peptide

“…Many groups have found solutions to these problems. For example, by modifying amyloid peptide sequences through the introduction of a "host-guest switch", aggregation can be initiated reliably upon the addition of an enzyme or change in pH 38 .…”

Chemical Strategies for Controlling Protein Folding and Elucidating the Molecular Mechanisms of Amyloid Formation and Toxicity

“…Switch peptides, which typically exhibit enhanced synthetic accessibility and solubility compared with their native parent peptides, have been adapted as model systems for the identification of viable aggregation inhibitors of Aβ target sequences. 31 Mutter and co-workers introduced two switch elements at Ser residues flanking peptide Aβ (14-24), a known fibril-forming Aβ fragment (Fig. 4b).…”

“…33 Investigations using this approach have already contributed valuable insight into the initial kinetics of amyloid formation 22 and allowed the identification of specific aggregation "hot spots" 27 and antiamyloid agents. 31 Remarkably, switch peptides have also been adapted to control the reverse process of amyloid formation, namely, the conversion of developed fibrillar aggregates into monomeric units. 24 Here, we review recent results using switch peptides to control and understand the mechanisms of amyloid fibril formation, disassembly, and inhibition.…”

“…The semisynthetic strategy had been designed to omit Met35 in the recombinant fragment, not only to avoid oxidative modifications but also primarily because following thiolysis of the fusion protein, the N-terminal methionine, which is not in the native Aβ sequence, could be selectively removed by CNBr-mediated cleavage. The resulting Aβ 1-29 -SR was then ligated with synthetic A30C-Aβ [30][31][32][33][34][35][36][37][38][39][40] (prepared by Fmoc-SPPS). Following Raney-nickelbased desulfurization the native, full-length Aβ was obtained with yields up to 8.5 mg per liter of expression medium.…”