Small molecule microarrays enable the discovery of compounds that bind the Alzheimer's Aβ peptide and reduce its cytotoxicity

Abstract

The amyloid-β (Aβ) aggregation pathway is a key target in efforts to discover therapeutics that prevent or delay the onset of Alzheimer's disease. Efforts at rational drug design, however, are hampered by uncertainties about the precise nature of the toxic aggregate. In contrast, high-throughput screening of compound libraries does not require a detailed understanding of the structure of the toxic species, and can provide an unbiased method for the discovery of small molecules that may lead to effective therapeutics. Here, we show that small molecule microarrays (SMMs) represent a particularly promising tool for identifying compounds that bind the Aβ peptide. Microarray slides with thousands of compounds immobilized on their surface were screened for binding to fluorescently labeled Aβ. Seventy-nine compounds were identified by the SMM screen, and then assayed for their ability to inhibit the Aβ-induced killing of PC12 cells. Further experiments focused on exploring the mechanism of rescue for one of these compounds: Electron microscopy and Congo red binding showed that the compound enhances fibril formation, and suggest that it may rescue cells by accelerating Aβ aggregation past an early toxic oligomer. These findings demonstrate that the SMM screen for binding to Aβ is effective at identifying compounds that reduce Aβ toxicity, and can reveal potential therapeutic leads without the biases inherent in methods that focus on inhibitors of aggregation.

Figure 1

10–20% Tris-Tricine SDS-PAGE gels showing the oligomeric state of Aβ under SMM agitation conditions. (a) HiLyte Fluor 488-labeled Aβ42 (lanes 2–5) and HiLyte Fluor 647-labeled Aβ40 (lanes 6–9) after agitation for 30 minutes. (b) Fluorescently labeled Aβ40 after 60 minutes agitation. Monomeric Aβ appears at ~ 4 kDa. Gels were visualized by silver staining.

Figure 2

(a) The SMM binding screen. Compounds are covalently attached in an array of spots on the surface of a slide, and probed with fluorescently-tagged Aβ peptide. Those compounds that bind Aβ and withstand several washes are revealed as fluorescent spots. (b) Fluorescent read-out of the NPC-SMM slide following incubation with fluorescent Aβ40. Enlargement of a grid section shows compound 2002-H20 binding the peptide (false-colored red) as well as fluorescent dyes used in grid alignment (false-colored green and red) and non-fluorescing DMSO control spots. The structure of 2002-H20 is shown with isocyanate-reactive functional groups colored red to indicate the positions available for attachment to the slide. Because two functional groups (an amine and a phenol) are available for cross-linking, the population displayed on the surface is assumed to include molecules displayed in more than one orientation, with some exposing the amine and others exposing the phenol for interaction with Aβ. (c) Three replicate SMM screens of the NPC compound set show that compound 2002-H20 binds fluorescently labeled Aβ40 reproducibly and consistently. (d) Histogram of the composite Z-scores of SMM fluorescence results from 3 replicates of the DIV and NPC slides. Results are divided into 254 bins with compounds shown in blue and DMSO controls in red. The green box surrounds bins for 79 assay positive compounds with composite Z-scores = 3.4.

Figure 3

Relative rescue scores for the top 15 compounds, which (at 100 μM) reduced the toxicity of Aβ42 to PC12 cells. Viability of cells in the absence of Aβ or exogenous compounds was scaled (A_570-670 = 0.716) to 100% and viability of cells exposed to Aβ42 alone (A_570-670 = 0.496) was scaled as 0%. As shown in gray, compound 2002-H20 increased viability by 41%. Cell viability was assayed using the MTT assay. Compound identities and toxicity data for all 79 compounds provided in Supporting Information.

Figure 4

The benzoxazole motif in compound 2002-H20 is similar to the benzothiazole motif in Pittsburgh Compound-B and thioflavin-T, two compounds that bind Aβ and are used routinely to detect amyloid. Another SMM hit compound, 2002-G12, contains similar benzimidozole motifs and reduced Aβ42 toxicity by 76% (Fig. 3).

Figure 5

Dose-dependence of compound 2002-H20’s ability to rescue PC12 cells from Aβ42-induced toxicity. 100% rescue scaled as the absorbance difference of cells incubated in buffer without peptide (A_570–670 = 0.976) and 0% as absorbance difference of cells with 20 μM Aβ42 and no 2002-H20 (A_570–670 = 0.389). An asterisk (*) indicates statistical significance as determined by the two-tailed Student’s T-test (p < 0.05) and error bars reflect standard error from the average of three measurements.

Figure 6

Viability of PC12 cells exposed to compound 2002-H20 at varying concentrations. 100% is scaled as the measured viability of cells not exposed to exogenous compound or peptide, but incubated in buffer with 1% (v/v) DMSO (A_570–670 = 0.976). Average of three measurements with standard error reported.

Figure 7

Compound 2002-H20 increases amyloid fibril formation of synthetic Aβ42. (a) Congo red spectral shift. (b) Transmission electron microscopy. For both experiments, 20 μM of synthetic Aβ42 was incubated with the indicated concentration of compound 2002-H20. Incubations were at 37°C with gentle agitation for 24 hours.