Exploring the rhodanine universe: Design and synthesis of fluorescent rhodanine-based derivatives as anti-fibrillar and anti-oligomer agents against α-synuclein and 2N4R tau

Abstract

Tau and α-synuclein (α-syn) are prone-to-aggregate proteins that can be responsible for pathological lesions found in the brains of Alzheimer's disease (AD), Lewy body dementia (LBD), and Parkinson's disease (PD) patients. The early-stage oligomers and protofibrils of tau are believed to be strongly linked to human cognitive impairment while the toxic α-syn oligomers are associated with behavioral motor deficits. Therefore, concurrent targeting of both proteinaceous aggregates and oligomers are very challenging. Herein, rhodanine-based compounds were designed and synthesized to target the fibrils and oligomers of tau and α-syn proteins. In particular, the indole-containing rhodanines 5l and 5r displayed significantly high anti-aggregation activity towards α-syn fibrils by reducing of the thioflavin-T (ThT) fluorescence to less than 5 %. Moreover, 5r showed a remarkable decrease in the fluorescence of thioflavin-S (ThS) when incubated with the non-phosphorylated tau 0N4R and 2N4R, as well as the hyperphosphorylated tau isoform 1N4R. Transmission electron microscopy (TEM) analyses validated the powerful anti-fibrillar activity of 5l and 5r towards both protein aggregates. In addition, both 5l and 5r highly suppressed 0N4R tau and α-syn oligomer formation using the photo-induced cross-linking of unmodified protein (PICUP) assay. The fluorescence emission intensity of 5l was quenched to almost half in the presence of both protein fibrils at 510 nm. 5r showed a similar fluorescence response upon binding to 2N4R fibrils while no quenching effect was observed with α-syn aggregates. Ex vivo disaggregation assay using extracted human Aβ plaques was employed to confirm the ability of 5l and 5r to disaggregate the dense fibrils. Both inhibitors reduced the Aβ fibrils isolated from AD brains. 5l and 5r failed to show activity toward the cell-based α-syn inclusion formation. However, another indolyl derivative 5j prevented the α-syn inclusion at 5 µM. Collectively, the indolyl-rhodanine scaffold could act as a building block for further structural optimization to obtain dual targeting disease-modifying candidates for AD, LBD, and PD.

Keywords: Alpha-synuclein; Alzheimer’s disease; Anti-oligomer agents; Parkinson’s disease; Tau isoforms.

Fig. 1.

Design of rhodanine-based compounds as dual anti-fibrillar agents.

Fig. 2.

Compounds 3c, 4c, 5j, 5k, 5l, 5c, 5d, 5m, 5n, 5r were the best inhibitors of α-syn fibril formation. The ThT fluorescence kinetic curves of the synthesized compounds (100 μM) were assessed with α-syn (6 μM) in 10 mM PBS supplemented with 0.5 mM SDS and 300 mM NaCl; A. Compounds 3a-d, 4a-d, and 5a-l; B. Compounds 5m-s. C-D. The dose-dependent inhibition curves of compounds; C. 5l and D. 5r at different concentrations (1.56, 3.13, 6.25, 12.5, 25, 50, 100 μM) on α-syn (6 μM) fibrils. Triplicate data were scored for each concentration from five successive time points at the plateau phase. The error bars represent the SEM for each measurement.

Fig. 3.

Compound 5r reduced the aggregation of tau. Thioflavin S (ThS) fluorescence kinetic curves were obtained with tau isoform 0N4R (6 μM), tau isoform 2N4R (12 μM), and hyperphosphorylated tau (p-tau) isoform 1N4R (6 μM) in 10 mM PBS (pH 7.4) supplemented with 5 mM DTT, 40 μM ThS, 0.15 mM heparin, and 0.92 μg/mL of arachidonic acid. Compound 5r was tested at a concentration of 100 μM.

Fig. 4.

Compounds 5i, 5k, 5l, 5m, 5n, 5q, and 5r inhibited the α-syn oligomer formation. PICUP assays were performed to monitor the ability of rhodanines to reduce oligomer formation. Samples consisting of α-syn (30 μM) were treated with each compound (100 μM), and immediately cross-linked and loaded in 16 % SDS-PAGE gel. Treatment consisted of the following compounds: A. 3a, 3c, 3d, 5c-5h; B. 4a-4d, 5a, 5b, 5i-5l; C. 5m-5s. The controls consisted of no light exposure, no tris(2,2′-bipyridyl)ruthenium(II) chloride (Ru(BPY)₃, a cross-linking agent), and no compound (i.e. 0.25% DMSO). The most significant oligomer generated is represented by a band located between 35 and 40 kDa.

Fig. 5.

Compounds 5k, 5l, 5i, and 5r reduced α-syn oligomer formation in a dose-dependent manner. The α-syn protein (30 μM) was subjected to PICUP assays in the presence of increasing concentrations of the best compounds. Samples were loaded in 16 % SDS-PAGE gels. The oligomer of interest consists of the band located between 35 and 40 kDa. A. The Coomassie stained gel demonstrates the dose dependent inhibitory effect of compounds 5k, 5l, and 5i. B. The second gel presented shows the anti-oligomer dose-dependent relationships of compound 5r.

Fig. 6.

Compounds 5l and 5r exhibited a tau 0N4R anti-oligomer activity at high micromolar concentrations. PICUP assays were performed using tau isoform 0N4R at a concentration of 6 μM in the presence of A. compound 5l and B. compound 5r. PICUP assay resulted in a high molecular weight band (≥180 kDa) representing the oligomer. The reduction of oligomer and increase in monomer concentration occurred at high micromolar concentrations with both compounds 5l and 5r. The pixel density of the oligomer bands was measured with the ImageJ software. The percentage reduction was calculated by dividing the pixel density of the lane with compound treatment by the pixel density of the control lane (without the compound) and then multiplied by 100. The resulting value was subtracted from 100 to determine the percentage of inhibition of oligomer formation.

Fig. 7.

Validation of α-syn anti-oligomer effects provided by compounds 5l and 5r. A solution of α-syn (30 μM) in 10 mM PBS (pH 7.4) was incubated with 0.25 % DMSO or 100 μM of compound 5l, 5r, or III for 3 days at 37 °C. Samples were then loaded in a 16% SDS-PAGE gel. Western blot was performed using anti-syn-33 to detect the α-syn oligomer (between 35 and 40 kDa) and monomer (located at 15 kDa).

Fig. 8.

Optical properties of compounds 5l and 5r. A. Excitation and emission of 5l and 5r (10 μM). B-C. Fluorescence binding profile of B. 5l (1 μM), C. 5r (1 μM) in the presence and absence of α-syn (50 μg/mL) and 2N4R tau (50 μg/mL) fibrils at λ_ex = 450 nm. The buffer consisted of 10 mM PBS (pH 7.4) with 10 % ethylene glycol.

Fig. 9.

Validation of the α-syn and tau anti-fibrillar activity of the best compounds. TEM analyses of the anti-fibril activity of 5l, 5r, 5j, and 3b (100 μM) with α-syn (2 μM, upper row) and 2N4R tau (6 μM, lower row). Both proteins were incubated with: A-B. Control (DMSO, 0.25 %); C-D. 5l; E-F. 5r; G-H. 5j; I-J. 3b. Incubation time was 3 days and 5 days for α-syn and 2N4R tau, respectively, at 37 °C. Scale bars: 200 nm, magnification: 40 K.

Fig. 10.

Compound 5l and 5r inhibited the aggregation of Aβ 1–40. A. Kinetics of aggregation of Aβ 1–40 (21 μM) in the presence of DMSO (0.25 %) and compound (100 μM) in 10 mM PBS supplemented with 0.5 mM SDS and 300 mM NaCl. B. The maximum fluorescence intensity extracted from the plateau phase of the kinetics of aggregation were plotted for control (DMSO) and compounds III, 3b, 5l, and 5r. Difference of treatments in comparison to the control DMSO are significant at p < 0.001 as assessed by one-way ANOVA plus Dunnett’s post-hoc test.

Fig. 11.

Compounds 5l and 5r disaggregated the Aβ-plaques isolated from Alzheimer’s disease (AD) brains. Ex vivo TEM analyses were done after 5 days of incubation at 37 °C of Aβ-plaques (0.37 mg/ml) in 10 mM PBS (pH 7.4) with: (A) DMSO (control vehicle, 0.125 %); (B) 5l (50 μM); (C) 5r (50 μM). Scale bars: 200 nm, magnification: 40 K.

Fig. 12.

Compound 5j prevented αS inclusion formation. M17D cells expressing the inclusion-prone αS-3 K::YFP fusion protein (dox-inducible) were treated with 0.1 % DMSO (vehicle; “0 μM”) as well as 1.25, 2.5 and 5 μM of compounds 3b and 5j 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 performed at t = 96 h. N = 3 independent experiments, n = 12–18 individual wells total (0 μM, n = 18; all other concentrations, n = 12). B) Same as 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 5 and 2.5 μM of compound 3b and 5j (t = 96 h), phase and green channel. Arrows indicate αS-rich YFP-positive inclusions. Scale bar: 50 μm. All data are presented as fold-changes relative to DMSO control + /− standard deviation. Brown-Forsythe and Welch ANOVA plus Dunnett’s T3 post-hoc test (A); one-way ANOVA plus Dunnett’s post-hoc test (B); *, p < 0.05; ****, p < 0.001; ns, non-significant.

Scheme 1.

Synthetic procedure for the target compounds 3a-d, 4a-d, and 5a-l. Reagents and conditions: (a) water, 100 °C, 4–6 h, 62–75 %; (b) DMAP, DCM, r.t., overnight, 63–92 %.

Scheme 2.

Synthetic procedure for the optimized anti-oligomer compounds 5m-s. Reagents and conditions: (a) DMAP, DCM, r.t., overnight, 65–92 %; (b) K₂CO₃, DMF, r.t., 3 h, 94 %; (c) 2c or 2d, DMAP, DCM, r.t., overnight, 82–93 %.