A Sensitive Aβ Oligomerization Assay for Identification of Small Molecule Inhibitors

Abstract

Amyloid deposits found in Alzheimer's disease result from aggregation of peptide which leads to loss of synaptic function, chronic microglial activation and cognitive impairment. Because of this, identification of small molecule inhibitors of aggregation as potential therapeutics is a topic of current interest. The majority of inhibitor screening approaches rely on in vitro assays that lack the necessary sensitivity to distinguish low-molecular weight oligomers from larger, more advanced-stage fibrillar structures. Differentiating between these two structures is of vital concern since recent studies indicate that small, early-stage oligomers are the most neurotoxic form of peptide aggregate. To address this limitation, we have explored the adaptability of a recently described ELISA-based assay for discovery of small molecule inhibitors of oligomerization. Results show that this assay is highly sensitive as it is able to quantify oligomers with as little as 80 nM input peptide. In addition, data were obtained re-confirming the function of curcumin as a potent inhibitor of aggregation (IC 50 = 2 μM) and defining its inhibitor:peptide functional stoichiometry. Further examination of other known anti-aggregation compounds showed that this assay is able to discriminate between inhibitors of early-stage, low-molecular weight oligomers and later-stage, high-molecular weight fibrillar structures. These findings indicate that this new ELISA-based assay is capable of identifying novel small molecule inhibitors that function during the initial stages of peptide assembly.

A Chemical Analog of Curcumin as an Improved Inhibitor of Amyloid Abeta Oligomerization

“…Analogs were tested for anti-Aβ aggregation activity at curcumin's IC 50 value (1 µM) for Aβ oligomerization. This IC 50 value for curcumin was previously established [33], [35]. For analog screening, monomeric Aβ peptide (200 nM) was incubated alone or with 1 µM curcumin or with 1 µM of the indicated analog.…”

“…In order to perform large-scale screening of our analog library in a rapid, reproducible and cost-effective manner, we developed a novel ELISA-based assay to quantify oligomeric Aβ peptide [33] . Importantly, this assay clearly distinguishes the oligomeric conformation of the Aβ aggregate from the fibrillar form, which is important since the oligomeric form of Aβ aggregates is receiving increasing attention as a major factor responsible for synaptic dysfunction [34] .…”

Unraveling the Early Events of Amyloid-β Protein (Aβ) Aggregation: Techniques for the Determination of Aβ Aggregate Size

“…The latter format serves to enhance the assay signal. Alternatively, in the sandwich ELISA format, a sequence-specific capture antibody (see Table 1 ) adsorbed onto the surface is used to capture Aβ protein, which is subsequently detected using the same sequence-specific antibody, such that only Aβ species containing multiple monomeric units, and therefore multiple epitopes, are detected [ 130 , 133 ]. Consequently, this sandwich ELISA will recognize only aggregated Aβ, but not Aβ monomer.…”

“…Various researchers have utilized ELISA for the detection of Aβ oligomers [ 128 , 130 , 133 – 135 ]. A study by Englund et al employed a sandwich ELISA with monoclonal antibody 158 for the detection of low molecular weight oligomeric Aβ 1-40 produced by dissolving Aβ 1-40 in 10 mM NaOH with dilution to 50 μM in 2 X PBS and Aβ 1-42 protofibrils produced by dissolving Aβ 1-42 in 10 mM NaOH with dilution to 443 μM in 2 X PBS and incubation overnight at 37 °C [ 130 ].…”

“…A study by Englund et al employed a sandwich ELISA with monoclonal antibody 158 for the detection of low molecular weight oligomeric Aβ 1-40 produced by dissolving Aβ 1-40 in 10 mM NaOH with dilution to 50 μM in 2 X PBS and Aβ 1-42 protofibrils produced by dissolving Aβ 1-42 in 10 mM NaOH with dilution to 443 μM in 2 X PBS and incubation overnight at 37 °C [ 130 ]. Gonzales et al utilized a similar ELISA assay to detect low molecular weight Aβ 1-42 formed by dissolving Aβ 1-42 in HFIP with dilution to 200 nM in PBS (pH 7.2) and incubation at 37 °C for 24 h [ 133 ]. The size of these species was confirmed with PAGE to be tetramer and pentamer; however, the bands were very faint, indicating the superior sensitivity of the ELISA assay for these oligomeric species.…”