Enzyme-Linked Immunosorbent Assay-Based Method to Quantify the Association of Small Molecules with Aggregated Amyloid Peptides

Abstract

This paper describes a simple ELISA protocol for quantifying the binding of small molecules to aggregated Amyloid-β (Aβ) peptides. Amyloid-targeting small molecules have attracted wide interest as potential agents for the treatment or diagnosis of neurodegenerative disorders such as Alzheimer’s disease. The lack of general methods to evaluate small molecule-amyloid binding interactions, however, has significantly limited the number of amyloid-targeting molecules that have been studied to date. Here, we demonstrate a general method to quantify small molecule-amyloid binding interactions via a modified quantitative ELISA protocol. A key feature of this protocol is the treatment of commercial ELISA plates with an air plasma to help maintain the desired β-sheet content of the aggregated Aβ upon immobilization of these peptides on to the polystyrene surface. We developed an ELISA-based competition assay on these air plasma-treated plates and evaluated the binding of five previously known amyloid-binding small molecules to aggregated Aβ. We show that this general ELISA-based competition assay can be used to quantify small molecule-amyloid binding interactions in the low nanomolar to low micromolar range, which is the typical range of affinities for many amyloid-targeting diagnostic agents under current development. This simple protocol for quantifying the interaction of small molecules with aggregated Aβ peptides overcomes many limitations of previously reported spectroscopic or radioactivity assays, and may, therefore, facilitate the screening and evaluation of a more structurally diverse set of amyloid-targeting agents than had previously been possible.

A Tetra(Ethylene Glycol) Derivative of Benzothiazole Aniline Enhances Ras-Mediated Spinogenesis

“…Several studies have shown that A␤ accumulation contributes to cognitive deficits (Chang et al, 2011;Chételat et al, 2012). Since we observed that BTA-EG 4 decreases A␤ levels both in vitro and in vivo (Fig.…”

“…Several recent studies have shown that A␤ aggregation is correlated with deficits in learning and memory, and therapies that decrease A␤ can rescue these deficits (Loane et al, 2009;Chang et al, 2011). For instance, ␥-secretase inhibitor (DAPT)-injected mice had decreased A␤ levels and improved behavioral performance after traumatic brain injury (Loane et al, 2009).…”

“…For instance, ␥-secretase inhibitor (DAPT)-injected mice had decreased A␤ levels and improved behavioral performance after traumatic brain injury (Loane et al, 2009). Other studies using mouse models of AD showed reduced A␤ levels after treatment with either ␤-secretase or histone deacetylase (HDAC) inhibitors (Chang et al, 2011;Ricobaraza et al, 2011). These therapies were also able to prevent or improve memory deficits in AD mice.…”

“…Members of the benzothiazole aniline (BTA) family of compounds are characterized as a class of small molecules that have shown great promise in preventing A␤-protein interactions. Yang et al previously reported that a tetra-ethylene glycol derivative of BTA (BTA-EG 4 ) inhibits A␤-induced cellular damage by surrounding aggregated A␤ with a bioresistive coating, preventing its association with cellular proteins (Inbar et al, 2006;Habib et al, 2010;Capule and Yang, 2012). BTA-EG 4 is able to adhere to aggregated forms of A␤ with high affinity (20 nM), cross the blood-brain barrier, and has adequate solubility in aqueous solutions (Inbar et al, 2006).…”

Screening of the Binding of Small Molecules to Proteins by Desorption Electrospray Ionization Mass Spectrometry Combined with Protein Microarray

“…Many high-throughput screening (HTS) methods have been applied for the analysis of the binding of protein ligands, such as enzyme linked immunosorbent assays (ELISA) [5,6] and yeast two-hybrid assays [7,8]. However, these methods are not so convenient and often require expensive experimental setups.…”