5-Nitro-1,2-benzothiazol-3-amine and N-Ethyl-1-[(ethylcarbamoyl)(5-nitro-1,2-benzothiazol-3-yl)amino]formamide Modulate α-Synuclein and Tau Aggregation

Abstract

Protein misfolding results in a plethora of known diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, transthyretin-related amyloidosis, type 2 diabetes, Lewy body dementia, and spongiform encephalopathy. To provide a diverse portfolio of therapeutic small molecules with the ability to reduce protein misfolding, we evaluated a set of 13 compounds: 4-(benzo[d]thiazol-2-yl)aniline (BTA) and its derivatives containing urea (1), thiourea (2), sulfonamide (3), triazole (4), and triazine (5) linker. In addition, we explored small modifications on a very potent antioligomer 5-nitro-1,2-benzothiazol-3-amine (5-NBA) (compounds 6-13). This study aims to define the activity of BTA and its derivatives on a variety of prone-to-aggregate proteins such as transthyretin (TTR_81-127, TTR_101-125), α-synuclein (α-syn), and tau isoform 2N4R (tau 2N4R) through various biophysical methods. Thioflavin T (ThT) fluorescence assay was used to monitor fibril formation of the previously mentioned proteins after treatment with BTA and its derivatives. Antifibrillary activity was confirmed using transmission electron microscopy (TEM). Photoreactive cross-linking assay (PICUP) was utilized to detect antioligomer activity and lead to the identification of 5-NBA (at low micromolar concentration) and compound 13 (at high concentration) as the most promising in reducing oligomerization. 5-NBA and not BTA inhibited the inclusion formation based on the cell-based assay using M17D neuroblastoma cells that express inclusion-prone αS-3K::YFP. 5-NBA abrogated the fibril, oligomer, and inclusion formation in a dose-dependent manner. 5-NBA derivatives could be the key to mitigate protein aggregation. In the future, the results made from this study will provide an initial platform to generate more potent inhibitors of α-syn and tau 2N4R oligomer and fibril formation.

Figure 1

Representative structures of 4-(benzo[d]thiazol-2-yl)aniline (BTA) and its derivatives: urea (1), thiourea (2), sulfonamide (3), and triazole (4). The original compound is designated in blue, and the derivative structural modifications are indicated in red and green.

Figure 2

4-(Benzo[d]thiazol-2-yl)aniline (BTA) is a general inhibitor of fibril formation. (A) A bar graph depicting the arbitrary maximum fluorescence intensity in percentage for fibril type including IAAP, α-syn, and TTR_81–127. Since IAPP, α-syn, and TTR_81–127 had arbitrary percent fluorescence under 40% for both resveratrol BTA treatments, analysis via electron micrograph (EM) was performed. The error bars represent the individual standard error of mean (SEM) for each condition. (B–J) Electron micrograph (EM) of peptides incubated with either 100 μM resveratrol, 100 μM BTA, or 0.1% DMSO control at 37 °C for 1 h (10 μM of IAPP) in PBS, 24 h (2 μM of α-syn in PBS), and 24 h (10 μM of TTR_81–127) in sodium acetate buffer, pH 4. (B) IAPP with ≤0.1% DMSO at 40k magnification. (C) TTR_81–127 with ≤0.1% DMSO at 40k magnification. (D) IAPP with 100 μM BTA at 40k magnification. (E) TTR_81–127 with 100 μM BTA at 40k magnification. (F) IAPP with 100 μM resveratrol at 40k magnification. (G) TTR_81–127 with 100 μM resveratrol at 40k magnification. Scale bars = 200 nm.

Figure 3

Compounds 1–4 failed to substantially abrogate the fibril formation of both TTR fragments (TTR_81–127, TTR_101–125). (A) TTR_81–127 fibril formation of 4-(benzo[d]thiazol-2-yl)aniline (BTA) derivatives 1–4, resveratrol (positive control), and BTA at 100 μM (molar ratio 1:10) assessed by ThT fluorometric assays in a time-dependent manner in the presence of DMSO (0.1%, control; CTRL). (B) TTR_101–125 fibril formation of BTA derivatives 1–4, resveratrol (positive control), and BTA at 100 μM (molar ratio 1:10) by ThT fluorometric assays in a time-dependent manner. Panels (C) and (D) are the same as panel (B) but evaluate compounds at a final concentration of 50 μM (molar ratio 1:5) and 25 μM (molar ratio 1:2.5).

Figure 4

Antifibrillary effect of 5-nitro-1,2-benzothiazol-3-amine (5-NBA) and several derivatives on different prone-to-aggregate proteins with special emphasis on α-syn and tau isoform 2N4R. (A) Histogram representing thioflavin T fluorescence intensity of prone-to-aggregate proteins incubated with compound 5 (negative control, BTA derivatives) and 5-NBA. IAPP, TTR_81–127, and TTR_101–125 were tested at 10 μM. The final compound concentration corresponded to 100 μM. (B) Tau isoform 2N4R kinetics of fibril formation in the presence or absence of 5-NBA, compound 10 (N-(5-nitro-1,2-benzothiazol-3-yl)acetamide), and compound 13 (N-ethyl-1-[(ethylcarbamoyl)(5-nitro-1,2-benzothiazol-3-yl)amino]formamide). 5-NBA and compound 13 delayed the lag time. (C) Dose dependency of 5-NBA on the inhibition of α-syn fibril formation. (D) Similar dose dependency response but applied tau isoform 2N4R with 5-NBA. (E) Dose dependency of N-ethyl-1-[(ethylcarbamoyl)(5-nitro-1,2-benzothiazol-3-yl)amino]formamide (compound 13) on the inhibition of α-syn fibril formation. Log(agonist) vs. normalized response (variable slope) was applied using Prism software and resulted in a LogIC50 of 48.00 ± 7.96 μM. (F) Dose dependency of N-ethyl-1-[(ethylcarbamoyl)(5-nitro-1,2-benzothiazol-3-yl)amino]formamide (compound 13) on the inhibition of tau 2N4R fibril formation. For each concentration (3.125, 6.25, 12.5, 25, 50, 100 μM), triplicate data were collected at the plateau phase for α-syn and at 15 h for tau. For all ThT assays, α-syn was tested at 2 μM (A, C, E) and tau was utilized at 6 μM (B, D, F). The error bars represent the individual standard error of mean (SEM) for each condition evaluated in triplicate.

Figure 5

5-Nitro-1,2-benzothiazol-3-amine (5-NBA) reduced α-syn and tau isoform 2N4R oligomer formation by photoinduced cross-linking of unmodified proteins (PICUP). (A) α-Syn (60 μM) was cross-linked (PICUP assay) with 4-(benzo[d]thiazol-2-yl)aniline (BTA), 5-NBA, and compound 5 at 50 μM. DMSO, BTA, and compound 5 (BTA derivative) failed to prevent the formation of high molecular bands located between 35 and 45 kDa and corresponding to oligomers. Additional controls consist of no light and no cross-linking agent (no Ru(bpy)₃), which resulted in no high molecular bands. (B) Tau isoform 2N4R (6 μM) was cross-linked (PICUP assay) with different compounds at 50 μM. (C) Dose-dependent inhibitory activity of 5-NBA on α-syn oligomerization. The protein (60 μM) was incubated with 5-NBA: 50 μM, 25 μM , 12.5 μM, 6.25 μM, and 3.125 μM. (D) Similar conditions were applied to evaluate the dose-dependent inhibitory activity on tau isoform 2N4R (6 μM). 5-NBA stopped the formation of α-syn and tau oligomers (un-cross-linked) in a dose-dependent manner. A lower concentration of 5-NBA showed a higher prominence of oligomer formation. Coomassie blue-stained polyacrylamide gels showed high-molecular-weight α-syn oligomers with control (0.125% DMSO).

Figure 6

Dose-dependent inhibitory activity of compound 13 on α-syn oligomerization by PICUP. Compound 13 reduced the oligomer formation at a high concentration, i.e., 200 μM. 5-NBA was used as a positive control and tested at 200 μM. The protein was tested at 60 μM. High-molecular-weight α-syn oligomer results from control condition (0.125% DMSO). Additional controls consist of no light and no cross-linking agent (no Ru(bpy)₃), which resulted in no cross-linking protein. The pixel density of the high-molecular-weight bands labeled as oligomers and the low molecular bands identified as monomers has been measured using image J. The pixel density of the higher molecular bands has been divided by the pixel density of the band corresponding to the monomeric state for each condition. The ratio is indicated as RPD (for relative pixel density) below the Coomassie blue-stained polyacrylamide gel.

Figure 7

5-Nitro-1,2-benzothiazol-3-amine (5-NBA) and its derivative, compound 13, reduced α-syn fibril formation as validated by transmission electron microscopy (TEM). α-Syn (2 μM) was incubated with DMSO (0.25%; “CTRL”), 5-NBA (100 μM), or compound 13 (at 100 μM) for ∼48 h prior to TEM visualization. High magnifications (40K) showed fewer fibrils in protein samples supplemented with 5-NBA and compound 13 in comparison with DMSO control. TEM results validate the reduction in fibrils monitored by ThT assays. Scale bars, 200 nm.

Figure 8

5-Nitro-1,2-benzothiazol-3-amine (5-NBA) reduced tau fibril formation as validated by transmission electron microscopy (TEM). Tau isoform 2N4R (6 μM) was incubated with DMSO (0.25%; “CTRL”) or 5-NBA (100 μM) for ∼50 h (i.e., in previously described experiments aimed at monitoring fibril formation by ThT fluorescence) prior to TEM visualization. Scale bars, 200 nm.

Figure 9

5-Nitro-1,2-benzothiazol-3-amine (5-NBA) abrogated the inclusion formation in M17D neuroblastoma cells that express inclusion-prone αS-3K::YFP. (A) Incucyte-based analysis of punctate YFP signals at t = 96 h, normalized to 0.1% DMSO. 8 independent experiments (N = 8) were performed. Student’s t-test, ****, p < 0.0001, ***, p < 0.001, **, p < 0.01. (B) Same as panel A, but confluence was plotted. (C) M17D cells that express an αS-3K::YFP fusion protein (doxycycline (dox)-inducible) were treated with 0.1% DMSO (vehicle control) or different concentrations of 5-NBA at t = 24 h. Cells were induced with dox at t = 48 h. Representative images (YFP, top; bright field, bottom).

Figure 10

4-(Benzo[d]thiazol-2-yl)aniline (BTA) reduced the confluence but not the inclusion formation in M17D neuroblastoma cells that express inclusion-prone αS-3K::YFP. (A) M17D cells that express an αS-3K::YFP fusion protein (dox-inducible) were treated with 0.1% DMSO (vehicle control) or 40 μM BTA at t = 24 h. Cells were induced with doxycycline (dox) at t = 48 h. Representative images (YFP, left; bright field, right); scale bar, 25 μm. (B) Incucyte-based analysis of punctate YFP signals at t = 96 h, normalized to 0.1% DMSO. 8 independent experiments (N = 8) were performed. Student’s t-test, **, p < 0.01. (C) Same as panel B, but confluence was plotted.