Inhibition of Toxicity in the β-Amyloid Peptide Fragment β-(25–35) Using N-Methylated Derivatives

Abstract

(25-35) is a synthetic derivative of-amyloid, the peptide that is believed to cause Alzheimer's disease. As it is highly toxic and forms fibrillar aggregates typical of-amyloid, it is suitable as a model for testing inhibitors of aggregation and toxicity. We demonstrate that N-methylated derivatives of-(25-35), which in isolation are soluble and non-toxic, can prevent the aggregation and inhibit the resulting toxicity of the wild type pep-tide. N-Methylation can block hydrogen bonding on the outer edge of the assembling amyloid. The peptides are assayed by Congo red and thioflavin T binding, electron microscopy, and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide (MTT) toxicity assay on PC12 cells. One peptide (Gly 25 N-methylated) has properties similar to the wild type, whereas five have varying effects on prefolded fibrils and fibril assembly. In particular ,-(25-35) with Gly 33 N-methylated is able to completely prevent fibril assembly and to reduce the toxicity of prefolded amyloid. With Leu 34 N-methylated, the fibril morphology is altered and the toxicity reduced. We suggest that the use of N-methylated derivatives of amyloidogenic peptides and proteins could provide a general solution to the problem of amyloid deposition and toxicity. Alzheimer's disease (AD) 1 is the most common form of senile dementia.-Amyloid (A), a 39-43-amino acid-sheet pep-tide, aggregates in the brain to form the major component of characteristic deposits known as senile plaques (1-4). X-ray diffraction data have shown that the conformation of A is characterized by an antiparallel cross-pleated sheet (5), although more recent solid state NMR evidence suggests that the peptide has a parallel-sheet structure (6). Nevertheless, ag-gregation occurs because of hydrogen bonding between-strands, and the resulting fibrils have axes perpendicular to the-strand and parallel to the cross-linking hydrogen bonds (5). Of all of the A derivatives studied so far,-(25-35), sequence GSNKGAIIGLM, is the shortest fragment that exhibits large-sheet fibrils and retains the toxicity of the full-length peptide (2, 7-9). It has been proposed that-(25-35) represents the biologically active region of A. In vitro studies have shown that it does not require aging to aggregate and become toxic (8-10), unlike the full-length peptide. As with A-, toxicity is dependent on the aggregation state of the peptide, because-(25-35) that has been solubilized and unfolded in 35% ace-tonitrile (AcN), 0.1% trifluoroacetic acid is nontoxic (8, 11). In this study,-(25-35) has been chosen as a model for full-length A because it retains both its physical and biological properties , while its short length readily allows derivatives to be synthesized and studied.