Validation and Characterization of a Novel Peptide That Binds Monomeric and Aggregated β-Amyloid and Inhibits the Formation of Neurotoxic Oligomers

Abstract

Although the formation of ␤-amyloid (A␤) deposits in the brain is a hallmark of Alzheimer disease (AD), the soluble oligomers rather than the mature amyloid fibrils most likely contribute to A␤ toxicity and neurodegeneration. Thus, the discovery of agents targeting soluble A␤ oligomers is highly desirable for early diagnosis prior to the manifestation of a clinical AD phenotype and also more effective therapies. We have previously reported that a novel 15-amino acid peptide (15-mer), isolated via phage display screening, targeted A␤ and attenuated its neurotoxicity (Taddei, K., Laws, S. M., Verdile, G., Munns, S., D'Costa, K., Harvey, A. R., Martins, I. J., Hill, F., Levy, E., Shaw, J. E., and Martins, R. N. (2010) Neurobiol. Aging 31, 203-214). The aim of the current study was to generate and biochemically characterize analogues of this peptide with improved stability and therapeutic potential. We demonstrated that a stable analogue of the 15-amino acid peptide (15M S.A.) retained the activity and potency of the parent peptide and demonstrated improved proteolytic resistance in vitro (stable to t ‫؍‬ 300 min, c.f. t ‫؍‬ 30 min for the parent peptide). This candidate reduced the formation of soluble A␤42 oligomers, with the concurrent generation of non-toxic, insoluble aggregates measuring up to 25-30 nm diameter as determined by atomic force microscopy. The 15M S.A. candidate directly interacted with oligomeric A␤42, as shown by coimmunoprecipitation and surface plasmon resonance/Biacore analysis, with an affinity in the low micromolar range. Furthermore, this peptide bound fibrillar A␤42 and also stained plaques ex vivo in brain tissue from AD model mice. Given its multifaceted ability to target monomeric and aggregated A␤42 species, this candidate holds promise for novel preclinical AD imaging and therapeutic strategies. Alzheimer disease (AD)4 is a progressive neurodegenerative disorder, accounting for 50 -70% of late-onset dementia cases (2). The major biochemical hallmarks of AD are extracellular amyloid plaques and intracellular neurofibrillary tangles. The predominant species present in amyloid plaques is ␤-amyloid (A␤), a 4.5-kDa peptide generated through amyloidogenic processing (sequential cleavage by ␤-and ␥-secretases) of the parent amyloid precursor protein (APP). This produces two forms of A␤, comprising either 40 or 42 amino acids, where the relative amount of the longer 42-amino acid form (A␤42) is especially critical for AD progression, given its higher propensity to aggregate and form neurotoxic species (3, 4).In AD brain, the monomeric A␤42 peptide is known to aggregate and form various ordered assemblies, which precede plaque formation. These include low-n oligomers (dimers to octamers, reviewed in Ref. 5), high molecular weight oligomers such as A␤-derived diffusible ligands (6) and globulomers (7), protofibrils (8), and fibrils (9). Much evidence has indicated that soluble A␤42 oligomers, rather than mature amyloid plaques, correlate with disease severity (10, 11) and contribute to synapti...

Arginine and Arginine-Rich Peptides as Modulators of Protein Aggregation and Cytotoxicity Associated With Alzheimer’s Disease

“…Gibson and Murphy (2005) previously reported that polyarginine-5 (R5) and KLVFF-R5 peptides broadly increased the aggregation of amyloid-beta 1-42 overall, resulting in the formation of larger amyloid-beta 1-42 aggregates with lower cytotoxicity. Barr et al (2016) reported that a 15-residue cationic peptide, with 20% of its sequence comprised of arginine residues, reduced the formation of soluble, cytotoxic amyloid-beta 1-42 oligomers by driving the aggregation pathway toward the formation of larger, amorphous aggregates; these amorphous aggregates were also observed to have lower cytotoxicity than the oligomers formed by amyloid-beta 1-42 alone. A potential therapeutic mechanism for peptides targeting amyloid-beta could therefore involve the reduction of cytotoxic, soluble oligomeric amyloid-beta species by driving the aggregation pathway toward the formation of non-toxic insoluble aggregate species, rather than aiming to inhibit aggregation.…”

Evidence that the Human Innate Immune Peptide LL-37 may be a Binding Partner of Amyloid-β and Inhibitor of Fibril Assembly

“…Equilibrium binding studies yield K D values of 69 and 535 μM for Aβ 42 monomers and oligomers, respectively. In a similar study, Barr et al verified the inhibition of Aβ fibril formation by a 15-amino acid peptide [ 41 ]. SPR surfaces functionalized with monomeric, oligomeric, and fibrillar Aβ 42 revealed a stronger affinity of the polypeptide toward the fibrillar form, although only a K D value of 11 μM was measured by injecting Aβ 42 onto immobilized peptides and no affinity constant was given.…”

Modulation of Amyloid β-Protein (Aβ) Assembly by Homologous C-Terminal Fragments as a Strategy for Inhibiting Aβ Toxicity

“…As discussed earlier, CTFs likely coassemble with Aβ42 and form hetero-oligomers that are not toxic. Other inhibitors, including various peptides and small molecules, have been reported to use similar mechanisms for inhibition of Aβ toxicity, that is, binding to and co-oligomerizing with Aβ42 to form nontoxic structures. ,− Due to the complexity of the relationship between assembly and toxicity inhibition, using assembly inhibition as a strategy for drug design should be considered with caution. Though peptide-based Aβ aggregation inhibitors have been studied for about 20 years now, several key unsolved questions can hinder the drug discovery processes: (1) Why are Aβ oligomers toxic?…”