Amphiphilic stilbene derivatives attenuate the neurotoxicity of soluble Aβ42 oligomers by controlling their interactions with cell membranes

The misfolded proteins or polypeptides commonly observed in neurodegenerative diseases, including Alzheimer's disease (AD), are promising drug targets for developing therapeutic agents. To target the amyloid-β (Aβ) peptide plaques and oligomers, the hallmarks of AD, we have developed twelve amphiphilic small molecules with different hydrophobic and hydrophilic fragments. In vitro fluorescence binding assays demonstrate that these amphiphilic compounds show high binding affinity to both Aβ plaques and oligomers, and six of them exhibit selective binding toward Aβ oligomers. These amphiphilic compounds can also label the Aβ species in the brain sections of transgenic AD mice, as shown by immunostaining with an Aβ antibody. Molecular docking studies were performed to obtain structure–affinity relationships. To our delight, four amphiphilic compounds can alleviate the Cu2+–Aβ induced toxicity in cell viability assays. In addition, confocal fluorescence imaging studies provide evidence that two compounds, ZY-15-MT and ZY-15-OMe, can disrupt the interactions between Aβ oligomers and human neuroblastoma SH-SY5Y cell membranes. Overall, these studies strongly suggest that developing compounds with amphiphilic properties that target Aβ oligomers and modulate the Aβ oligomer–cell membrane interactions can be an effective strategy for the development of small molecule AD therapeutics.


Experimental details Preparation of the Aβ 42 Fibrils and Aβ 42 Oligomers
Aβ42 fibrils: 1 mg of commercial Aβ42 monomer powder (from GL biochem) was dissolved in 1 mL 1,1,1,3,3, at room temperature and incubated for 1h. The resulting solution was divided equally into two Eppendorf tubes. The solution in each Eppendorf tube containing 0.5 mg Aβ42 monomer was then evaporated overnight and dried by vacuum centrifuge to generate monomeric films. 100 μM Aβ42 fibrils solution was prepared by dissolving the 0.5 mg monomeric films in 1.1 mL PBS buffer (with final DMSO concentration less than 1%) and stiring at 37 °C for 3 days.
Aβ42 oligomers: The 0.5 mg monomeric films were dissolved in 1.1 mL PBS buffer (with final DMSO concentration less than 1%) and incubated at 4 °C overnight. The resulting solution was then centrifuged at 3000 rpm for 15 mins to remove the insoluble aggregates. The clear supernatant was then used for the following experiments. Aβ42 oligomers concentration was checked by measuring the absorbance of the supernatant at 280 nm with the molar extinction coefficient of 1480 M -1 cm -1 .

Fluorescence Spectral Testing of Compounds with Aβ 42 Fibrils and Aβ 42 Oligomers
The formation of Aβ42 fibrils and oligomers was confirmed by the ThT fluorescence turn-on assay. The fluorescence spectra of ThT in PBS (100 μL PBS, 10.0 µM) were recorded as the baseline. After that, either Aβ42 fibrils or oligomers were added to ThT in PBS. The final volume of the solution mixture should be 100 μL, in which the final concentrations of ThT and Aβ42 species were10.0 µM and 25 µM, respectively.
For the interactions between Aβs and compounds, the fluorescence spectra of the compounds' solution (100 μL PBS, 5.0 µM) were recorded as the baseline without adding various Aβ42 species. After that, either Aβ42 fibrils or oligomers were added to the compounds' solutions in PBS. The final volume of the solution mixture should be 100 μL, in which the final concentrations of the compound and Aβ42 species were 5.0 µM and 25 µM, respectively.

In Vitro Saturation Binding Studies with Aβ 42 Fibrils and Aβ 42 Oligomers
To a solution of increasing concentrations of compounds, a fixed concentration of the generated aggregated Aβ42 fibrils or Aβ42 oligomers solutions (5 to 15 μM) was added to yield a total volume of 100 μL. Nonspecific binding was determined without compounds. The mixture was incubated for 15 min at room temperature. All fluorescence data were obtained on a SpectraMax M2e plate reader (Molecular Devices). The fluorescent intensity was measured at the corresponding emission wavelength of each compound, and the Kd binding curves were generated by GraphPad Prism 8.0 with one site-specific binding model. Equation: Y = Bmax*X/ (Kd +X). For the binding ratio analysis, the concentration of bound compounds were calculated from the cross point between the plateau and the initial slope. The binding ratio = [bound compounds]/[Aβ].

Cytotoxicity Studies
Alamar Blue assay was chosen for the cytotoxicity studies. 3 Mouse neuroblastoma Neuro2A (N2A) cells were grown with DMEM/10% FBS in a petri dish at 37 °C in a humidified atmosphere with 5% CO2. Then N2A cells were seeded in a 96-well plate (1.0 × 10 4 /well). After 24 h incubation, cell media was changed to DMEM/N-2, and N2A cells were incubated for another 1 h. Then the cells were treated with different concentrations of compounds for 40 h, Alamar Blue solution (10 μL) was added, and the cells were incubated for another 90 min at 37 °C. The fluorescence intensity of each well was measured at 590 nm (excitation wavelength = 560 nm).

Histological Staining of 5×FAD Mice Brain Sections
5xFAD transgenic mice brain sections were blocked with bovine serum albumin (2% BSA in PBS, pH 7.4, 10 min). Then the mice brain sections were transferred to a PBS solution of the compound and incubated for 1 h. After which, the brain sections were transferred to a PBS solution of Congo Red or antibody HJ 3.4 (Professor David Holtzman, 1 μg/ml) and incubated for another 1 h. Then, the brain sections were treated with BSA again (5 min) followed by washing with PBS (3 × 2 min), DI water (3 × 2 min). Finally, the mice brain sections were mounted with non-fluorescent mounting media, and the images were obtained by using EVOS FL Auto 2. ImageJ Fiji program was used for colocalization analysis and determination of the Pearson's correlation coefficient. The primary antibodies were labeled with dye CF594 via Mix-n-Stain ™ CF ™ 594 Antibody Labeling Kit (Sigma Aldrich).

Log D measurements
The Log D value was measured following a published procedure using slight modifications. 4 In general, compounds in 0.5 mL octanol was subjected to partition with 0.5 mL octanol-saturated PBS. The whole mixture was stirred vigorously for 5 min, and centrifuged at 2,000 rpm for 5 min. The top octanol layer was separated for later fluorescence measurement. The remaining PBS layer was partitioned with 0.5 mL PBS-saturated octanol, and the second top octanol layer was then separated after vigorous stirring and centrifuging at 2,000 rpm for another 5 min. The two octanol layers' fluorescence spectra were recorded. The log D value was calculated by the fluorescence intensity ratio at the compounds' emission wavelength for the above two octanol extractions.

Molecular Docking
Molecular docking studies were performed with the Schrödinger Suite software. Protein structure Aβ42 tetramers (PDB ID: 6RHY) and the Aβ42 fibrils (PDB ID: 5OQV) were imported from the S5 RCSB database and optimized by minimal minimization with the OPLS3 force field using the Protein Preparation Wizard program. The amphiphilic molecules were prepared using Ligprep, and the pH was set as 7.0 ± 2.0 using Epik. The resulting different protonation states of the compounds were obtained and used for the docking studies. The grid size was set to include the whole optimized protein structure in each direction. The final molecular docking was performed using Glide. The calculated poses were ranked by both the docking score and Glide e-model energy, and the structures with the best docking scores and Glide e-model energies were rendered in PyMol. 5

Cell Imaging Procedures
For oligomers and fibrils imaging: Human neuroblastoma SH-SY5Y cells were grown with DMEM/10% FBS at 37 °C in a humidified atmosphere with 5% CO2, and an 8-well μ-slides (ibidi) chambered coverslip was seeded the SH-SY5Y cells. At about 80% confluency (24 h after seeding), the cells were treated with DMSO as control group, 5 μM oligomers/fibrils and 5 μM (oligomers/fibrils + compounds), respectively. After 24 h incubation, the media was removed from each well. The cells were then fixed with 200 μL 3.7% formaldehyde for 15 min and blocked with 200 μL 3% BSA for 10 min. 3% BSA was then removed from the cells and replaced with 100 μL CF594-HJ 3.4 (1 µg/mL in 3% BSA). The cells were incubated with CF594-HJ 3.4 for 2 h at room temperature and were washed with PBS (5 x 100 μL). Before imaging, the cells in each well were stained with 100 μL NucBlue reagent (Thermo Fisher Scientific) for 15 min and mounted with non-fluorescent mounting media. 5-10 images of each well were taken, and the images were processed and analyzed by Fiji and Zeiss Zen lite software. Three individual replicates were conducted and subjected to the statistical analysis.
For cell membrane staining: BioTracker 490 green cytoplasmic membrane dye (Millipore Sigma, USA) was used according to the manufacture procedures.