Considerable genetic and biochemical evidence indicates that aggregation of the amyloid-β peptide (Aβ) plays a causative role in the neurodegeneration, memory loss, and dementia associated with Alzheimer’s disease (AD).1–5
Although the precise structure of the toxic aggregate is still under investigation, the major features of the “amyloid cascade” pathway are understood: Proteolytic processing of the amyloid precursor protein (APP) results in the extracellular release of the 4 kDa Aβ peptide. Differential cleavage by secretase enzymes leads to the formation of Aβ variants ranging in size from 39 to 43 amino acids, with the 40-residue Aβ40 and 42-residue Aβ42 peptides being the most prevalent.6–8
Compared to Aβ40, Aβ42 is more prone to aggregation,9
and more closely correlated with symptomatic disease.2,11
Aβ42 is also the predominant component of the extracellular plaque that has long been viewed as the pathological hallmark of AD.12,13
While this insoluble plaque provides posthumous evidence of the disease, numerous findings over the past decade implicate earlier soluble intermediates as the neurotoxic agents. Aβ-derived diffusible ligands (ADDLs) have been observed to induce neuronal death in cell culture,14
and soluble Aβ oligomeric species are more closely associated with cognitive decline in AD patients and synapse loss in transgenic mice than fibril or plaque load.15–17
Most recently, a murine model in which ADDLs were endogenously expressed with sequence mutations that prevented subsequent plaque formation showed that cognitive deficits are dependent on soluble Aβ oligomers rather than end-stage aggregates.18
With the prevalence of Alzheimer’s disease projected to rise dramatically in the coming decades,19
there is increasing urgency to develop novel therapeutics to prevent and/or treat this debilitating disease. The pharmaceuticals currently used to treat AD include cholinesterase inhibitors and memantine, a modulator of glutamate receptors.20,21
These drugs provide symptomatic relief and may slow cognitive decline; however, they do not target the underlying molecular cause of AD. Therefore, currently approved drugs neither halt, nor reverse progression of the disease. In contrast to these existing drugs, novel compounds that interfere with Aβ aggregation, and thereby block the molecular events that cause AD, represent a promising approach to the prevention and/or cure of Alzheimer’s disease.
In principle, compounds that interfere with Aβ aggregation could be discovered by either of two approaches: Structure-based rational drug design or high-throughput screening. Since a range of intermediates along the Aβ aggregation pathway have been implicated as potential toxic species,22–25
and none of their precise structures are known, structure-based drug design is not yet possible. For the time being, high-throughput screening remains a more promising approach.
A number of high-throughput methods have been developed to screen for compounds that interfere with Aβ aggregation. Most screens use synthetic Aβ peptide and monitor aggregation by following the fluorescence of thioflavin-T or another dye that binds to fibrillar aggregates.26–28
Although relatively convenient to perform, these screens have limitations: First, they are hampered by the need for substantial quantities of synthetic peptide, which is expensive and hard to produce in a form that is free of pre-aggregated seeds. A second and more significant drawback of dye-binding assays is their reliance on a reporter that detects amyloid fibrils or protofibrils, rather than the early intermediates that are now thought to be the neurotoxic agent. The limitations of assays that screen for inhibitors of fibrillization are highlighted by recent reports that accelerating
fibril formation may, in fact, be advantageous as a way to decrease the presence of toxic soluble Aβ oligomers.29,30
As an alternative to traditional assays using synthetic Aβ peptide, our lab previously reported the development of a high-throughput screen using an Aβ42-GFP fusion protein expressed in E. coli
In that system, aggregation of the Aβ42 sequence drags the entire fusion protein into misfolded insoluble aggregates, thereby preventing the folding and fluorescence of GFP. Compounds that inhibit the aggregation of Aβ42 enable the GFP part of the fusion to fold into its native structure, and thereby give rise to green fluorescent colonies (or liquid cultures) of E. coli
. The Aβ42-GFP screen overcame two of the limitations of the dye binding assays: (i) Synthetic Aβ peptide was not required, and (ii) the assay reported Aβ misfolding and aggregation without requiring the formation of amyloid fibrils.
Application of the GFP-based screen enabled the identification of several aggregation inhibitors from a library of triazine derivatives,32
and from other libraries screened subsequently. Some of these compounds look promising and are being evaluated in a Drosophila
model of AD. Nonetheless, the GFP-based screen also has several limitations: (i) Because the Aβ42-GFP fusion protein was expressed in E. coli,
compounds that fail to cross the cell wall and membrane of E. coli,
or which were toxic to E. coli,
may have been missed. (ii) Because the folding and fluorescence of the GFP reporter is based on the solubility of the upstream sequence,33
the screen may not distinguish between compounds that block all aggregation (by favoring monomeric Aβ) and those that block fibrillization by favoring the toxic soluble oligomers. Thus, compounds that allow the Aβ42-GFP fusion protein to fold and fluoresce by enhancing the stability of soluble oligomers would be mistakenly identified as inhibitors of aggregation (hits) rather than as enhancers of the toxic species. (iii) Most importantly, because the Aβ42-GFP screen was designed to identify compounds that inhibit aggregation, it would miss potential therapeutics that remove toxic oligomers by enhancing their aggregation into insoluble fibrils.
These considerations motivated us to develop a novel method to screen compounds based solely on their ability to bind to monomeric Aβ. Ultimately, whether a small molecule reduces the toxicity of Aβ by inhibiting aggregation, accelerating it past the toxic species, or redirecting it to an alternate pathway, the compound must bind the Aβ peptide.
High-throughput identification of compounds that bind to specific proteins has been accomplished previously through the use of small molecule microarrays (SMMs). SMMs are glass slides on which libraries of small molecules are covalently immobilized in an array of microscopic spots.34
Compounds can be attached to the surface by any of several mild coupling reactions. For the current study, SMMs were prepared by installing an isocyanate on the surface of the slides and then attaching the small molecules to this group.35–37
Because isocyanate reacts with amines (primary and secondary), thiols, alcohols (primary and secondary), and several other groups, a compound possessing several different functional groups can attach to the surface in a range of orientations with various functional groups available to interact with the probe. To ensure that the small molecules are presented at a distance from the surface sufficient to enable interactions with a soluble probe (Aβ peptide in the current study), the isocyanate is separated from the surface by a polyethylene glycol spacer.
A single SMM slide typically displays 10,800 compounds, allowing for rapid and efficient screening of diverse libraries of small molecules. SMM slides are probed with a fluorophore- or epitope-tagged protein (in this case, fluorescently-labeled Aβ peptide), and compounds that bind the protein are detected by automated fluorescence read-out (a schematic is shown in ).35–37
SMMs have been used successfully to identify a wide range of protein-ligand interactions, including calmodulin and human immunoglobin G ligands,38,39
and inhibitors of histone deacetylases.42
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 (more ...)
The SMM technology has several features that make it ideal as a high-throughput screen for compounds that bind Aβ. First, it does not require knowledge of the protein structure or binding pocket. This unbiased approach is crucial since the structure of the toxic Aβ aggregate is not known, and a traditional active site pocket is not likely to exist. Second, the high sensitivity of the SMM assay allows the Aβ peptide to be used at very low concentrations. This is important because Aβ aggregation occurs via a nucleation-dependent mechanism,43
and keeping the peptide below the critical concentration favors the monomeric form, thereby facilitating the identification of compounds that interact with Aβ at or before the earliest steps of aggregation. An added benefit is that low concentrations of peptide make the assay relatively inexpensive.
Here we describe the development of SMM technology to screen libraries of small molecules for compounds that bind the Alzheimer’s Aβ peptide. As an initial step toward SMM assay development, we identified conditions that favor the majority of the Aβ probe being present as monomer. Next, two SMM slide sets – one with natural products and synthetic commercial compounds (NPC), and the other with diversity-oriented synthesis compounds (DIV) – were screened with fluorescently labeled Aβ40. Compounds in printed features that bound the peptide were identified as hits, as described previously.37,44
These compounds were subsequently assayed for their ability to rescue PC12 cells from Aβ42-induced toxicity. Further examination of a commercially available compound revealed dose-dependent rescue of PC12 cells. Finally, we demonstrated that this compound actually promotes,
rather than inhibits, fibrillogenesis. These results validate the ability of the SMM screen to reveal compounds that interact with the Aβ peptide, and may provide leads for therapeutics that prevent or cure Alzheimer’s disease.