The N-Methylated Peptide SEN304 Powerfully Inhibits Aβ(1–42) Toxicity by Perturbing Oligomer Formation

Abstract

Oligomeric forms of β-amyloid (Aβ) have potent neurotoxic activity and are the primary cause of neuronal injury and cell death in Alzheimer's disease (AD). Compounds that perturb oligomer formation or structure may therefore be therapeutic for AD. We previously reported that d-[(chGly)-(Tyr)-(chGly)-(chGly)-(mLeu)]-NH(2) (SEN304) is able to inhibit Aβ aggregation and toxicity, shown primarily by thioflavin T fluorescence and MTT (Kokkoni, N. et al. (2006) N-Methylated peptide inhibitors of β-amyloid aggregation and toxicity. Optimisation of inhibitor structure. Biochemistry 45, 9906-9918). Here we extensively characterize how SEN304 affects Aβ(1-42) aggregation and toxicity, using biophysical assays (thioflavin T, circular dichroism, SDS-PAGE, size exclusion chromatography, surface plasmon resonance, traveling wave ion mobility mass spectrometry, electron microscopy, ELISA), toxicity assays in cell culture (MTT and lactate dehydrogenase in human SH-SHY5Y cells, mouse neuronal cell death and synaptophysin) and long-term potentiation in a rat hippocampal brain slice. These data, with dose response curves, show that SEN304 is a powerful inhibitor of Aβ(1-42) toxicity, particularly effective at preventing Aβ inhibition of long-term potentiation. It can bind directly to Aβ(1-42), delay β-sheet formation and promote aggregation of toxic oligomers into a nontoxic form, with a different morphology that cannot bind thioflavin T. SEN304 appears to work by inducing aggregation, and hence removal, of Aβ oligomers. It is therefore a promising lead compound for Alzheimer's disease.

Inhibition of protein aggregation and amyloid formation by small molecules

“…Biophysical assays, toxicity assays in cell culture (MTT and lactate dehydrogenase in human SH-SHY5Y cells, mouse neuronal cell death and synaptophysin) and long-term potentiation (LTP) showed SEN304 binds directly to Ab1-42 and redirects toxic oligomers into non-toxic forms with different morphologies. SEN304 does not work in the manner that was expected (by blocking Ab aggregation) -instead it induced aggregation, and removed toxic oligomers [14 ]. SEN1269, based on RS-0406 and discovered by high-throughput screening was modified to give SEN1576, which can be given orally, binds to monomeric Ab1-42, protects neuronal cells exposed to Ab1-42, reduces deficits in LTP and improves behavior following injection of Ab oligomers to normal rats [15].…”

D-amino acid-based peptide inhibitors as early or preventative therapy in Alzheimer disease

“…They characterized SEN304 further using various biophysical assays along with toxicity assays in SH-SHY5Y cells, neuronal viability and synaptophysin levels in mouse neuronal cultures, and longterm potentiation in rat hippocampal brain slices. 41 The inhibition of long-term potentiation by 1 μM Aβ1-42 was reversed by adding 100 nM SEN304 to the rat hippocampal brain slices. Electron microscopic analysis supported their conclusion, as seen with the other D-peptides, that SEN304 drives Aβ monomer and oligomers to alternative large ThT-negative aggregates that are not cytotoxic.…”

Biotechnologically engineered protein binders for applications in amyloid diseases

“…To avoid such side effects it is possible to add flanking charged residues [64,65] or to replace the backbone amide hydrogen atoms with methyl groups such that the self-assembly of the inhibitor is blocked and b-sheet-breaking activities enhanced [31,[66][67][68][69]. Indeed, feeding an N-methylated peptide fragment from the Ab C-terminus to fly larvae that transgenically expressed Ab(1-42) reduced their locomotive impairments as adult animals and prolonged survival after eclosion [68].…”