Label-Free High-Throughput Screening Assay for Inhibitors of Alzheimer’s Amyloid-β Peptide Aggregation Based on MALDI MS

Abstract

Aggregation of amyloid-β (Aβ) peptides is causatively linked to Alzheimer's disease (AD); thus, suppression of this process by small molecule inhibitors is a widely accepted therapeutic and preventive strategy for AD. Screening of the inhibitors of Aβ aggregation deserves much attention; however, despite intensive efforts, there are only a few high-throughput screening methods available, all of them having drawbacks related to the application of external fluorescent probes or artificial Aβ derivatives. We have developed a label-free MALDI MS-based screening test for inhibitors of Aβ₄₂ fibrillization that exhibits high sensitivity, speed, and automation possibilities suitable for high-throughput screening. The test was evaluated by transmission electron microscopy and compared with a fluorimetric thioflavin-based assay, where interference of a number of tested compounds with thioflavin T binding and/or fluorescence caused false-positive results. The MALDI MS-based method can significantly speed up in vitro screening of compound libraries for inhibitors of Aβ₄₂ fibrillization.

Hyperspectral Imaging Signatures Detect Amyloidopathy in Alzheimer’s Mouse Retina Well before Onset of Cognitive Decline

“…Mass-spectrometry based screens such as 6 that of Zovo et al are able to identify Aβ fibrillogenesis inhibitors but not those of Aβ 7 oligomerization. 20 Cell-culture and cytotoxicity models still remain the mainstay of Aβ 8 aggregation inhibitor screening. All of the existing models of this type employ altered forms of 9…”

Determination of critical nucleation number for a single nucleation amyloid-β aggregation model

“…Insulin sample were used as internal standards to calculate the absolute concentrations of A β 42 in the sample as previously shown [23]. Typically, 2 μL aliquot of sample from each incubations at different time points (20–100 pmol of samples for 10–50 μM A β incubations) and mixed with 16 pmol of insulin (1 μL sample) for each sample in the matrix.…”

Targeting Assembly and Disassembly of Protein Aggregates

“…The preferred addition of monomeric Ab peptides to growing fibrils reduces the levels of newly formed, soluble Ab oligomers [102]. It must be mentioned that the reliability of thioflavin T (ThT)-based in vitro Ab fibrillation assays is questionable [104,105]. Structurally similar small molecules may displace ThT from its binding sites on Ab fibrils rather than truly inhibiting Ab fibrillation.…”

“…Structurally similar small molecules may displace ThT from its binding sites on Ab fibrils rather than truly inhibiting Ab fibrillation. Using a label-free assay based on matrix assisted laser desorption/ionization (MALDI)-time of flight (TOF) MS, MB is classified as a false positive, without effects on Ab fibrillation, while azure C shows mM rather than nM potency [104].…”

“…Its low mM anti-aggregation potency on Ab peptides is attributed to paninhibition of oligomer formation/elongation/fibril disassembly [155,156], or to an oligomeric species-specific inhibition [101]. The anti-aggregation effects of curcumin are questioned by some research groups [104]. The interference of fluorescent curcumin with thioflavin T and other dyes in aggregation assays in vitro, and the different experimental protocols used in published studies, may explain the controversial observations [157].…”