(504b) A Novel Transthyretin Peptide Mimic As a Therapeutic for Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common form of dementia and one of many diseases caused by amyloidogenic proteins. While amyloidogenic proteins differ in many ways, they all undergo aggregation in which monomeric protein spontaneously self‑assembles into soluble oligomers and insoluble fibrils. In the case of AD, it is the amyloid-β (Aβ) protein that aggregates, resulting in the formation of extracellular senile plaques in the brain. While Aβ monomer occurs naturally in blood, plasma, and cerebrospinal fluid (CSF), Aβ aggregates, particularly oligomers, induce cellular toxicity. Several CSF proteins may naturally sequester Aβ aggregation, with one of the most promising being transthyretin (TTR). TTR is a homotetrameric protein that functions as a carrier for thyroxine and retinol binding protein. A double mutation (F87M/L110M) produces an engineered protein that is stable as a monomer (mTTR). Both TTR and mTTR bind Aβ, inhibit Aβ aggregation, and reduce Aβ toxicity. This has led our lab to synthesize a series of cyclic conformationally constrained peptide mimics of TTR that can bind to Aβ and inhibit its toxicity in vitro.

In this study, we evaluated the therapeutic potential of one of these cyclic peptides, cG8, based on its selectivity, specificity, and stability. We explored its interaction with specific Aβ aggregates (monomer, oligomer, fibril), evaluated its stability and binding to Aβ under physiologically relevant conditions, and determined its ability to inhibit the aggregation of other amyloidogenic proteins. We also compared cG8 to mTTR, to evaluate the relative advantages of a peptide-based versus protein therapeutic.

To measure selectivity for binding to Aβ monomer, oligomers, or fibrils, cG8 or mTTR was biotinylated and adsorbed to pre-blocked NeutrAvidin ELISA plates. Aβ monomer (fresh), oligomers (24h, RT), or fibrils (72h, 37°C) were added, and binding was assessed with an anti-Aβ 6E10 antibody. cG8 and mTTR both bound significantly more Aβ oligomers and fibrils than Aβ monomer, indicating selectivity for the toxic forms of Aβ. To further evaluate the selectivity under biologically relevant conditions, binding of Aβ to cG8 in the presence of fetal bovine serum (FBS), brain lipids, and mouse brain tissue lysate was evaluated using the same assay. Although binding of Aβ decreased for both cG8 and mTTR in the presence of tissue lysate, cG8 retained Aβ binding capacity significantly better than did mTTR. Repeating the assay but with FBS or lipids indicated that it is largely the proteins in tissue lysate that cause the decreased Aβ binding. Immunoprecipitation and mass spectrometry studies are currently underway to identify the co-adsorbing proteins responsible for decreased Aβ-cG8 or Aβ-mTTR binding. These results indicate that the peptide cG8 suffers less from interference from other proteins than mTTR because it was designed to contain a minimal Aβ-binding domain, whereas the protein mTTR also contains other domains that will bind nonspecifically to serum or CSF proteins.

We used Thioflavin T (ThT) fluorescence to assess the ability of cG8 and mTTR to prevent Aβ aggregation or to disaggregate pre-formed fibrils. When co-incubated with freshly prepared Aβ, both cG8 and mTTR were very effective at inhibiting Aβ aggregation even at sub-stoichiometric concentrations. However, neither cG8 nor mTTR were effective at disaggregating preformed Aβ fibrils (48 h, 37°C) even at equimolar concentrations. Thus, although both cG8 and mTTR are highly effective at preventing the maturation of Aβ monomers into fibrils, neither disaggregate pre-formed fibrils into the more toxic oligomeric species.

Besides Aβ, there are many other proteins that aggregate into amyloid fibrils. Two well-known examples are amylin (islet amyloid polypeptide), associated with type II diabetes, and α-synuclein, associated with Parkinson’s disease. To evaluate whether cG8 and mTTR are selective for Aβ or are general anti-amyloid inhibitors, we incubated amylin or α-synuclein with cG8 or mTTR. Both cG8 and mTTR proved ineffective at inhibiting α-synuclein aggregation, as measured by ThT fluorescence. However, both significantly reduced amylin aggregation even at 20-fold excess amylin. We explain this outcome by hypothesizing that the aggregation mechanism of amylin and Aβ may be similar to each other, and distinct from that of α-synuclein.

The stability of cG8 under biologically relevant conditions was evaluated by incubation in either simulated gastric fluid (SGF, pH 1.2) or simulated intestinal fluid (SIF, pH 6.8) for 24 h at 37°C. After incubation, degradation of cG8 and its ability to inhibit Aβ aggregation was assessed via mass spectrometry (MS) analysis and ThT fluorescence, respectively. No chemical degradation of cG8 was observed after incubation in SGF and SIF. Additionally, cG8 exposed to SGF and SIF not only retained its ability to significantly inhibit fibril formation but did so at a level comparable to mTTR.

This study demonstrates that cG8 binding to Aβ is affected significantly less by the presence of biological materials than is Aβ-mTTR binding. Additionally, cG8 is stable and retains its inhibitory capability when exposed to biological fluids. Although cG8 does bind to Aβ fibrils, it does not cause fibril disaggregation which could potentially lead to a toxic response. cG8 was also able to prevent the aggregation of amylin, which is a common occurrence in type II diabetes. Combined, these results support the use of cG8 as an AD therapeutic and indicate that cG8 may be beneficial in the treatment of other amyloidogenic diseases. In vivo experiments to evaluate cG8 distribution and efficacy in transgenic mice are currently underway.