Strategy to discover full-length amyloid-beta peptide ligands using high-efficiency microarray technology

Although the formation of β-amyloid (Aβ) fibrils in neuronal tissues is a hallmark of Alzheimer disease (AD), small-sized Aβ oligomers rather than mature fibrils have been identified as the most neurotoxic species. Therefore, the design of new inhibitors, able to prevent the aggregation of Aβ, is believed to be a promising therapeutic approach to AD. Unfortunately, the short-lived intermediate structures that occur in a solution along the Aβ aggregation pathway escape conventional experimental investigations and there is urgent need of new tools aimed at the discovery of agents targeting monomeric Aβ and blocking the early steps of amyloid aggregation. Here, we show the combination of high-efficiency slides (HESs) with peptide microarrays as a promising tool for identifying small peptides that bind Aβ monomers. To this aim, HESs with two immobilized reference peptides, (i.e., KLVFF and Semax) with opposite behavior, were investigated for binding to fluorescently labeled Aβ peptide. Transmission electron microscopy was used to demonstrate Aβ fibrillar aggregates missing. The use of HESs was critical to ensure convenient output of the fluorescent microarrays. The resulting sensitivity, as well as the low sample consumption and the high potential for miniaturization, suggests that the proposed combination of peptide microarrays and highly efficient slides would be a very effective technology for molecule profiling in AD drug discovery.


Quantitative evaluation
To evaluate the sensibility of the amyloid incubation assay the fluorescence of Cy3-Aβ40 spot arrays was calibrated analyzing three Cy3-labeled amyloid solutions spotted on epoxysilanecoated HESs at different amyloid concentrations. For sample preparation, a proper amount of the lyophilized Cy3-Aβ40 product has been dissolved in a phosphate-buffered saline containing dimethyl sulfoxide (0.01 M phosphate, 0.154 M sodium chloride, pH 7.4, 10 vol % of DMSO).
The PBS was added until the amyloid concentration reached a final value of 1 μM. Diluted amyloid solutions were prepared by adding the PBS to fresh aliquots of the 1μM amyloid solution in order to reach 1:10 and 1:100 dilutions. After preparation amyloid solutions were stored at −80 °C until use.
Three sets of 4 × 4 spot arrays, replicated two-times, have been spotted for each of the amyloid solutions by using a PerkinElmer Piezorray non-contact system. The volume of spotted drops was 333 pL ± 5%. A reference solution, consist of dilution buffer was spotted at the same time to assure a negative control.
Florescence was measured on the spotted HES with a PerkinElmer Scanarray instrument immediately after the amyloid-spotting procedure. Various acquisition conditions were tested in order to get all spots visible although below the detector saturation value ( Figure S4B). Under sensor saturation conditions (100% power laser and gain) all the amyloid concentrations clearly appear as white spots ( Figure S4A). In addition, PBS spots are also detected. As discussed in the full paper, this signal derive from secondary luminescence effects due to the salt components. Error bars determined as standard deviation of 16 spots measured intensity.

Study of any interactions between the anchored amyloid and amyloid target
To explore the tendency of full-length amyloid peptides to form molecular aggregates at the HESs surface through amyloid-amyloid molecular interactions, the binding capacity of Aβ40 spots was compared with the binding capacity of the KLVFF amyloid sequence. Cy3-labeled Aβ40 and KLVFF peptides were arrayed on an epoxysilane-coated slide from a 1 μM and 1 mg/mL peptide solution, respectively. Cy3-Aβ40 incubation test has been performed by assaying, at the same time, the KLVFF sequence and amyloid beta peptide, with a 1 μM Cy3labeled Aβ40 solution and the relative fluorescence was measured on the dried slide before and immediately after the amyloid incubation.
The amyloid incubation is detailed in the Experimental section of the main manuscript. Briefly, after the spotting step, peptide and amyloid spot arrays were anchored in a humid chamber and S7 then BSA-blocked. After N 2 -drying, the BSA-blocked slide was scanned in order to check for peptide and amyloid spots still visible despite the extensive washings involving in the procedure.
Finally, fluorescence measurements have been performed after the amyloid incubation to check for fluorescent spots deriving from the binding interaction with the KLVFF and Cy3-Aβ40 spot arrays.
As shown in Figure S5A, BSA-blocked HESs does not show fluorescence for the KLVFF spot array. This is consistent with the absence of a fluorescent cyanine marker for this peptide.
Fluorescent spots have been instead detected for the Cy3-Aβ40 spot array.
However, after amyloid incubation among the Cy3-Aβ40 and KLVFF spot arrays, the last become clearly visible thanks to the appearance of brilliant Cy3-Aβ40 spots. In contrast, Cy3-Aβ40 spots retain the majority of their morphology and brilliance and no fluorescent enhancement is observed after the amyloid incubation (Figure S5 B).
These data suggest that when a KLVFF peptide is anchored at a solid support, it may link a fulllength amyloid beta peptide only if the peptide sequence is well-exposed at the solid-liquid interface where the interaction with the solution occurs, just like in the case of KLVFF peptides spotted on the HESs surface. When the KLVFF sequence is instead embedded into a full-length Aβ40 sequence, its ligand propensity is reduced due to the occurring of steric effects that inhibit the interaction. This hypothesis matches with our incubation data, in fact we observed a positive variation of the fluorescence intensity only when the KLVFF sequence is anchored at the support surface, but no variation of the fluorescence signal takes place when it is a part of a full-length amyloid molecule. As a result no formation of amyloid aggregates can occurs on HESs surface, under the experimental conditions used in this work S8 Figure S5: Fluorescence measurements of KLVFF and Cy3-Aβ40 spot arrays before (A) and after incubation with 1 mM Cy3-Aβ40 solution (B). Scale bar, 100 mm B