In Vitro Evaluation of Amide-Linked Coumarin Scaffolds for the Inhibition of α‑Synuclein and Tau Aggregation

Abstract

Alzheimer's disease (AD) and Parkinson's disease (PD) are the most prevalent neurodegenerative disorders characterized by continuous loss of functional neurons. The numbers of AD and PD patients will likely double by 2060 and 2040, reaching 13.9 and 1.2 million, respectively, in the US alone. Although both AD and PD are multifactorial in origin, the accumulation of misfolded proteins such as α-synuclein (α-syn) and tau contribute to nerve function disruption. Therefore, inhibition of α-syn and tau aggregation via small-molecule disruptors of oligomer and fibril formation presents a promising method for treating AD and PD. Coumarin scaffolds possess a wide range of bioactivities, particularly their antiamyloidogenic potential, which was explored in this study. Our previous work demonstrated that amide linkers and amino indole moieties have antioligomer and antifibrillar effects. This study involves coupling the coumarin scaffold with various aromatic moieties, including aminoindoles, methoxy-substituted phenyl, and polyhydroxy aromatic functionalities, via an amide linker for establishing the structural activity relationship (SAR) for the inhibition of oligomer and fibril formation. In total, 38 coumarin-based amide compounds were prepared to first explore the antifibrillar activity on recombinant α-syn. The best compounds were then tested to assess the antioligomer effects, tau aggregation activity, inclusion inhibition, and dimerization in cells. Biophysical methods such as thioflavin T (ThT) fluorescence assays, photoinduced cross-linking of unmodified proteins (PICUP), survival assays, and electron microscopic observations were used to evaluate the inhibitory effects of analogs on α-syn and tau aggregation. The coumarin-amide-dihydroxybenzene derivatives demonstrated superior effects on the inhibition of α-syn aggregation when compared with the coumarin-amide-indole derivatives. The methoxy (nondemethylated) counterparts of compounds 13 and 17 failed at reducing α-syn fibril formation. The coumarin-amide-dihydroxybenzene derivatives 13 and 17, exhibited different degrees of inhibition on the α-syn oligomer and inclusion formation. Compound 13 inhibited tau (2N4R isoform) oligomer formation and reduced tau dimerization in a cell-based assay. In conclusion, the results presented herein will guide future optimization of molecules with inhibitory effects on prone-to-aggregate proteins and may pave the way for disease-modifying treatments for neurodegenerative disorders.

1. Synthesis of Coumarin-Based-Amide Compounds (1–38) to Generate the Final Products Presented in Table

1

Compounds 13, 17, and 19 reduced α-syn fibril formation. The ThT fibrillation kinetics of α-syn tested at 6 μM final concentration were measured under two conditions: in the absence of compounds (control, 0.25% DMSO) and in the presence of 100 μM test compounds. Each curve represents the average data from three replicates conducted within the same experiment.

2

Compounds 13 and 17 dose-dependently abrogated α-syn fibril formation. For compounds 13 (A) and 17 (B), the curves display a dose-dependent response at concentrations of 6.25, 12.5, 25, 50, and 100 μM after a 27 h incubation with α-syn at 6 μM.

3

Compound 13 was best at reducing the aggregation of tau isoform 2N4R compared with compound 17. ThS fluorescence curves illustrate the aggregation kinetics of tau isoform 2N4R at a concentration of 12 μM. The PBS buffer was supplemented with 150 μM heparin, 5 mM dithiothreitol (DTT), 40 μM ThS, and 10 mg/mL arachidonic acid in PBS buffer treated with Chelex beads. The control sample consisted of 0.25% DMSO, while compounds were tested at 100 μM. Each curve represents the average data obtained from three replicates.

4

Compound 13 effectively reduced the aggregation of tau isoform 0N4R, as shown by the ThS fluorescence kinetics. Tau 0N4R was used at a concentration of 12 μM, and its aggregation was tracked using ThS fluorescence. The assay buffer (10 mM PBS treated with Chelex beads) was supplemented with 150 μM heparin, 5 mM DTT, 40 μM ThS, and 10 mg/mL arachidonic acid to facilitate aggregation. Compounds 13 and 17 were tested at 100 μM, while the control samples were treated with 0.25% DMSO. Compared to the control and compound 17, tau 0N4R treated with compound 13 markedly reduced fluorescence, indicating a strong inhibitory effect on tau aggregation. Each curve represents the average data obtained from three replicates.

5

Compound 13 successfully inhibited the formation of α-syn oligomers. Compound 9 (noninhibitor of α-syn fibrils) and compound 19 (intermediate inhibitor of α-syn fibril formation) partially reduced the formation of oligomers. The oligomers were generated by exposing the different treated α-syn monomers to tris­(bipyridyl)­ruthenium­(II)­chloride [Ru­(bpy)₃]³⁺ (RTD) and ammonium persulfate under brief light exposure (1 s) in the PICUP cross-linking assay. No incubation or pretreatment was necessary. The protein was used at a concentration of 30 μM and readily cross-linked in the absence or presence of compounds at 100 μM. The negative control consisted of 0.25% DMSO (no compound treatment). The α-syn monomer has a molecular mass of 15 kDa. The oligomers of higher molecular weight are visible between 35 and 40 kDa as well as between 55 and 180 kDa on Coomassie-blue-stained 16% polyacrylamide gels. Additional control samples consisted of the protein not exposed to the light or the cross-linking agent (RTD), resulting in no detectable cross-linked products. Experiments were repeated three times.

6

Compound 13, a coumarin-amide-4,5-dihydroxy derivative, reduced the α-syn oligomer formation in a concentration-dependent manner, as evidenced by the decreased intensity of the bands between the 35 and 40 kDa markers. The antioligomer effect disappeared with the utilization of 12.5 μM and lower concentrations of compound 13. The protein α-syn (6 μM) was incubated with different concentrations of the compound, and the PICUP assay was performed immediately to induce oligomer formation. DMSO (0.125%) served as the control. Experiments were repeated twice.

7

Western blot analysis of treated α-syn protein was used to monitor oligomer formation. (A) The formation of α-syn oligomers over time at varying protein concentrations (30 and 50 μM) and collected at various time points (3, 5, and 7 days) to monitor the progression of oligomer formation, and the most intense oligomer band was observed at 50 μM after 7 days of incubation. (B) α-Syn incubation with compounds 13 and 17 (100 μM) showed a reduction in oligomer formation when compared to the DMSO (0.25%) control. Compounds 12 and 16 were used as negative control. An anti-α-syn syn-33 antibody was used for the detection of α-syn monomeric and oligomeric species. The pixel densities of the oligomer bands located between 35 and 40 kDa were determined using ImageJ.

8

Compounds 12, 13, 16, and 17 failed at curtailing tau 0N4R (6 μM) oligomerization, as assessed by the PICUP assay. Higher molecular weight oligomers (at ≥180 kDa) were generated with the control (0.25% DMSO) and remained with the compound treatments. The monomeric band present at 55 kDa was reduced for all conditions except for the control, no light, and no RTD (cross-linking reagent: Tris­(bipyridyl)­ruthenium­(II)­chloride [Ru­(bpy)₃]³⁺). Compounds 12 and 16 (the methoxy counterparts of compounds 13 and 17) were used as negative controls, as they failed to inhibit α-syn oligomer and fibril formation. All compounds were tested at 100 μM. Compounds with the protein were subjected immediately (i.e., without incubation or pretreatment) to light exposure for 15 s. Experiments were repeated twice.

9

Compound 13 reduced tau 2N4R oligomer formation, while compounds 12, 16, and 17 showed no effect. Oligomers with molecular weights of ≥130 kDa were generated by using the PICUP assay. Control samples consisted of 0.25% DMSO and were not exposed to light or the cross-linking agent (no RTD: Tris­(bipyridyl)­ruthenium­(II)­chloride [Ru­(bpy)₃]³⁺). All test compounds were evaluated at a concentration of 100 μM. The pixel density of each band located between 130 and 180 kDa, corresponding to high molecular weight oligomers, was determined by using ImageJ.

10

Antioligomer effect of compound 13 on tau isoform 1N4R. Tau 1N4R protein (6 μM) was incubated in 10 mM PBS (treated with Chelex beads) along with 150 μM heparin, 5 mM DTT, 40 μM ThS, and 10 mg/mL arachidonic acid. The mixture was treated with either 0.25% DMSO (control) or 100 μM of compounds 13 and 16 and incubated at 37 °C for 3 days. After incubation, samples were run on a 16% SDS-PAGE gel, followed by Western blotting using the Tau-5 antibody to detect both monomeric and oligomeric forms of tau 1N4R. The highest oligomer bands indicated by the asterisk were quantified (pixel intensity) with ImageJ.

11

Treatment with compounds 13 and 17 led to a decrease in the quantity and length of α-syn fibrils, as observed via transmission electron microscopy (TEM). After completing the ThT fibril kinetic formation assay, which was carried out over approximately 28 h, three replicate samples containing 6 μM α-syn and 100 μM compounds 8, 12, 13, 16 to 19 were retrieved from the 96-well plate. Control consisted of 6 μM α-syn with 0.25% DMSO. Subsequently, these samples were applied onto copper grids and examined using TEM. Scale bars = 200 nm.

12

Transmission electron microscopy (TEM) images showing the antiaggregation effect of compounds 13 and 17 on tau isoform 2N4R. DMSO (0.25%; “CTRL”) and the compounds (tested at 100 μM) were incubated with 12 μM tau for 5 days at 37 °C in the presence of 0.15 mM heparin, 5 mM dithiothreitol (DTT), 40 μM ThS, and 0.092 μg/mL arachidonic acid. Negative control consisted of the vehicle (0.25% DMSO). Compound 12, a 4,5-dimethoxybenzene derivative and an inefficacious tau antioligomer, was also tested to confirm weak antiaggregation activity. TEM images (scale bar: 200 nm) showed fewer tau fibrils in the samples treated with compounds 13 and 17 compared to the controls.

13

Treatment with compounds 13 and 17 led to a reduction in the β-sheet content in α-syn. Wildtype recombinant α-syn was incubated for: (A) 0 h with 0.25% DMSO, and (B) 72 h with 0.25% DMSO, compounds 13 and 17. Structural changes were measured using circular dichroism (CD) spectroscopy.

14

Secondary structure changes in tau isoform 2N4R (6 μM) treated with 0.25% DMSO were analyzed by using circular dichroism (CD) spectroscopy. Tau aggregation was induced using heparin. Representative spectra are shown for: (A) 0 h without heparin, (B) 0 h with heparin, and (C) 72 h with heparin, illustrating the typical transition from a random coil to β-sheet conformation.

15

Compound 13 induced a spectral shift toward increased helical content of tau isoform 2N4R. Circular dichroism (CD) spectra were recorded to assess secondary structure changes in tau 2N4R (6 μM) treated with 0.25% DMSO (control) or 100 μM of compounds 12, 13, and 17 for 72 h, with heparin used to induce aggregation. Treatment with compound 13 induced a spectral shift, suggesting interference with β-sheet formation and a potential delay in the formation of β-sheet-rich aggregates.

16

Compound 17 prevented αS inclusion formation. M17D cells expressing the inclusion-prone αS-3K:YFP fusion protein (dox-inducible) were treated with 0.1% DMSO (vehicle; “0 μM”) as well as 5, 10, 20, and 40 μM compounds 13, 15, and 17 at t = 24 h after plating. Cells were induced with doxycycline at t = 48 h. (A) Incucyte-based analysis of punctate YFP signals relative to 0.1% DMSO was done at t = 96 h (N = 3 independent experiments, n = 6–18 individual wells total) (0 μM, n = 18; 5 μM, n = 12; all other concentrations, n = 6). (B) The same experiment as in panel A, but confluence fold changes relative to the DMSO vehicle (0 μM) were plotted. (C) Representative IncuCyte images of reporter cells treated with vehicle vs 40 μM compounds 13, 15, or 17 (t = 96 h), green channel. Arrows indicate αS-rich YFP-positive inclusions, exemplified for the DMSO control. The images presented in panel C were acquired at the same magnification, and all images were taken at the same magnification with a scale bar of 50 μm. All data are presented as fold changes relative to DMSO control ± standard deviation. One-way ANOVA, Dunnett’s posthoc test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns = nonsignificant.

17

Compound 13 reduced tau aggregation intracellularly using a cell-based assay. Quantification of GFP-positive cell populations in the tau aggregation assay. ExpiCHO cells were transfected with split GFP-Tau fusion constructs (pmGFP11C-Tau and pmGFP10C-Tau) and treated with compounds (20 μM) or vehicle control (0.1% DMSO) for 72 h. Flow cytometry analysis was performed to assess GFP fluorescence intensity, indicative of tau aggregation, and cell viability was confirmed by ethidium homodimer-1 exclusion. Statistical analysis was conducted using GraphPad Prism. Data represent the mean ± standard deviation (SD) from three independent replicates. Significant differences between groups are indicated (****, p < 0.0001).

18

Isothermal titration calorimetry (ITC) experiment of α-syn (at 0.05 mM) titrated with 0.5 mM compound 13 (20% DMSO in 10 mM PBS, pH 7.4) was performed using MicroCal PEAQ-ITC at 25 °C. (A) Binding curve obtained by integrating the solvent signal and the fitting of a binding site model called sequential three site (solid line). (B) Thermodynamic parameters resulting from the fitting of the binding curve with the confidence intervals.