Effect of Peptoid Side Chain Aromatics and Chirality on Aggregation and Oligomerization of Alzheimer's Disease Amyloid-β Protein

Introduction: : Amyloid- protein (A) is widely studied due to its key pathological role in Alzheimer’s disease (AD). As A aggregates into monomer, oligomer, and fibril forms, deposition of these species is associated with synapse damage. The inhibition of A protein aggregation is one therapeutic strategy for AD. Peptoids, or poly-N-substituted glycines, are a novel class of biomimetrics that have high membrane permeability, are resistant to proteolytic degradation, have low immunogenic properties, and may be manipulated in secondary structures. With the advantage that peptoids can also cross the blood brain barrier, peptoids possess unique potential as AD therapeutics. Peptoid JPT1 is a mimic and extension of the Aβ hydrophobic core KLVFF (residues 16-20 of Aβ). JPT1 is designed with chiral monomers and four aromatic side chains to stabilize the peptoid helical structure. Additionally, the aromatic side chains are designed to present spacing on two sides of the helix that matches that of -sheets found in the aggregated A structure, thus facilitating peptoid binding to A and preventing fibril formation and growth. Additional peptoids were also designed to test the importance of the helical structure and the placement of aromatics on A aggregation. Toward the former, peptoid JPT1a was designed as an achiral version of JPT1. Toward the latter, JPT1FN was designed with two sides of the helical structure having one aromatic each, JPT1FS was designed with one helical side having two aromatics, and JPT1C was designed with the absence of aromatics.

Materials and

Methods: : Lyophilized A40 was reconstituted in 50mM NaOH and purified via size exclusion chromatography (SEC) to remove pre-existing aggregate seeds. The resulting monomer was used immediately in aggregation assays.
A40 aggregation assays were performed in the presence of thioflavin T (ThT), which facilitated detection of A aggregates via a shifted, enhanced fluorescence upon binding amyloid. Samples containing 10µM A40, either 0 (Control), 10, 50, or 100µM peptoid, 20µM ThT, and 150mM NaCl in 40 mM Tris-HCl, pH 8.0, were prepared in a 96- well plate and agitated to induce aggregation. ThT fluorescence was monitored every 15min. The lag time to aggregate formation and extent of aggregation at equilibrium were determined from fluorescence vs. time curves. Upon achieving a plateau in ThT fluorescence values, aggregation end products were collected and gridded onto copper grids for negative staining with uranyl acetate and imaging via TEM using a JEM-1400Plus TEM.

Results, Conclusions, and Discussions:: When aggregation was monitored via ThT, the most significant effects were observed in the lag time to aggregation formation. JPT1 and JPT1a both significantly reduced lag time (Fig1B). JPT1 had a more significant impact on the lag time than JPT1C (Fig2B), demonstrating that the presence of side chains is critical to inhibition. JPT1FN, presenting one aromatic on each of two faces of the helix, also had a significantly lower impact on the lag time than JPT1 (Fig2B). In contrast, JPT1FS, presenting two aromatics on one face of the helix, did not significantly differ in its impact on the lag time than JPT1 (Fig2B). These results demonstrate that the placement of aromatic rings is also important for inhibition, with two rings on the same side of the helix enhancing inhibitory capabilities.

TEM was utilized to observe aggregate morphology. When monomer was aggregated alone, a network of long, thin fibrils was observed (Fig3A). In contrast, monomer aggregated in the presence of JPT1 yielded short, stacked fibrils with minimal networking (Fig3B). Fibrils formed in the presence of JPT1a exhibit a shorter structure than the control fibrils (Fig3C), but not to the extent of fibrils in the presence of JPT1 (Fig3B). Fibrils formed in the presence of JPT1FN (Fig3E) and JPT1FS (Fig3F) exhibited slightly longer fibrils than those in the presence of JPT1(Fig3B) but were still shorter than fibrils in the absence of peptoids (Fig3A) and had decreased networking. Fibrils formed in the presence of JPT1C (Fig3D) exhibited long, thin fibril networks, comparable to monomer aggregated alone (Fig3A). These results demonstrate that JPT1 alters fibril morphology and further supports that chirality and aromatic placement are important for JPT1 effectiveness.
Together, these results support the design of the JPT1 peptoid as a modulator of A aggregation. Combined with the utility of peptoids to cross the blood-brain barrier and evade proteolytic degradation, these results illustrate the potential of peptoid JPT1 as a novel therapeutic for AD. Future investigation will explore the effect of peptoids on oligomer formation and measure binding affinity between peptoids and A monomer, oligomer, and fibril.

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