COMPOUNDS FOR USE IN THE TREATMENT OF SYNUCLEINOPATHIES

Abstract

The present invention relates to a compound selected from the group consisting of of N-[(2S)-1-amino-3-(3-fluorophenyl)propan-2-yl]-5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-carboxamide, [2-(2,6-dichloroanilino)-4,5-dihydroimidazol-1-yl]-phenylmethanone, 2-amino-5-bromo-4-phenyl-1H-pyrimidin-6-one, N-(diaminomethylidene)-2-(2,6-dichlorophenyl)acetamide and 2-(1,4-dioxaspiro[4.5]decan-2-ylmethyl)guanidin, or a pharmaceutically acceptable salt thereof and combinations comprising at least one of said compounds, for use in the treatment and/or prevention of synucleinopathies.

Claims

1.-45. (canceled)

46. A method for the treatment and/or prevention of synucleinopathies wherein a compound selected from the group consisting of N-[1-amino-3-(3-fluorophenyl)propan-2-yl]-5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-carboxamide, [2-(2,6-dichloroanilino)-4,5-dihydroimidazol-1-yl]-phenylmethanone, 2-amino-5-bromo-4-phenyl-1H-pyrimidin-6-one, N-(diaminomethylidene)-2-(2,6-dichlorophenyl)acetamide and 2-(1,4-dioxaspiro[4.5]decan-2-ylmethyl)guanidine, or a pharmaceutically acceptable salt thereof, is administered to a patient in need thereof.

47. The method according to claim 46, wherein the compound is N-[1-amino-3-(3-fluorophenyl)propan-2-yl]-5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-carboxamide, or a pharmaceutically acceptable salt thereof.

48. The method according to claim 46, wherein the compound is [2-(2,6-dichloroanilino)-4,5-dihydroimidazol-1-yl]-phenylmethanone.

49. The method according to claim 46, wherein the compound is 2-amino-5-bromo-4-phenyl-1H-pyrimidin-6-one.

50. The method according to claim 46, wherein the compound is N-(diaminomethylidene)-2-(2,6-dichlorophenyl)acetamide, or a pharmaceutically acceptable salt thereof.

51. The method according to claim 46, wherein the compound is 2-(1,4-dioxaspiro[4.5]decan-2-ylmethyl)guanidine, or a pharmaceutically acceptable salt thereof.

52. The method according to claim 46, wherein the synucleinopathy to be treated and/or prevented is selected from the group consisting of Parkinson's disease (PD), Multiple System Atrophy (MSA), and Dementia with Lewy Bodies.

53. The method according to claim 52, wherein the synucleinopathy to be treated and/or prevented is Parkinson's Disease (PD).

54. A method for the treatment and/or prevention of synucleinopathies wherein a combination comprising two or more compounds selected from the group consisting of 1,2-bis[(4-chlorophenyl)methylideneamino]guanidine, N-[1-(1-naphthyl)ethyl]-3-[3-(trifluoromethyl)phenyl]propan-1-amine, N-[(2S)-1-amino-3-(3-fluorophenyl)propan-2-yl]-5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-carboxamide, [2-(2,6-dichloroanilino)-4,5-dihydroimidazol-1-yl]-phenylmethanone, methyl N-(6-propoxy-1H-benzimidazol-2-yl) carbamate, 4-(2-(diaminomethylidene)hydrazinyl)phenyl]iminothiourea, 2-amino-5-bromo-4-phenyl-1H-pyrimidin-6-one, 2-(1,4-dioxaspiro[4.5]decan-2-ylmethyl)guanidine, 5-[2-[[3-(1,3-Benzodioxol-5-yl)-1-methylpropyl]amino]-1-hydroxyethyl]-2-hydroxybenzamide, 2-amino-9-[4-(hydroxymethyl)cyclopent-2-en-1-yl]-1H-purin-6-one, 4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol, 4-amino-N-(4-methylpyrimidin-2-yl)benzenesulfonamide, levodopa, carbidopa, benserazide, entacapone, opicapone, amantadine, pimavanserin, rotigotine, ropinirole, apomorphine, rasagiline, safinamide, istradefylline, KW-6356, donepezil, rivastigmine, liraglutide, prasinezumab, venglustat, dipraglurant, mesdopetam (IRL-790), SEP-363856, cinpanemab, posiphen, MEDI-1341, UCB-0599, ABBV-0805, NPT-20011, PD-03, PD-01, LuAF-82422, NPT-088, UB-312, and Anle138b or a pharmaceutically acceptable salt thereof, wherein at least one of the compounds in the combination is selected from the group consisting of N-[(2S)-1-amino-3-(3-fluorophenyl)propan-2-yl]-5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-carboxamide, [2-(2,6-dichloroanilino)-4,5-dihydroimidazol-1-yl]-phenylmethanone, 2-amino-5-bromo-4-phenyl-1H-pyrimidin-6-one and 2-(1,4-dioxaspiro[4.5]decan-2-ylmethyl)guanidine, or a pharmaceutically acceptable salt thereof, is administered to a patient in need thereof.

55. The method according to claim 54, wherein the combination comprises at least one compound selected from N-[1-amino-3-(3-fluorophenyl)propan-2-yl]-5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-carboxamide or a pharmaceutically acceptable salt thereof, N-[(2S)-1-amino-3-(3-fluorophenyl)propan-2-yl]-5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-carboxamide or a pharmaceutically acceptable salt thereof, N-[1-amino-3-(3-fluorophenyl)propan-2-yl]-5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-carboxamide hydrochloride, N-[(2S)-1-amino-3-(3-fluorophenyl)propan-2-yl]-5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-carboxamide hydrochloride, [2-(2,6-dichloroanilino)-4,5-dihydroimidazol-1-yl]-phenylmethanone, 2-amino-5-bromo-4-phenyl-1H-pyrimidin-6-one, 2-(1,4-dioxaspiro[4.5]decan-2-ylmethyl)guanidine or a pharmaceutically acceptable salt thereof, 2-(1,4-dioxaspiro[4.5]decan-2-ylmethyl)guanidine hydroiodide, or 2-(1,4-dioxaspiro[4.5]decan-2-ylmethyl)guanidine sulfate.

56. A method for the treatment and/or prevention of synucleinopathies wherein a combination comprising two or more compounds selected from the group consisting of 1,2-bis[(4-chlorophenyl)methylideneamino]guanidine, N-[1-(1-naphthyl)ethyl]-3-[3-(trifluoromethyl)phenyl]propan-1-amine, N-[(2S)-1-amino-3-(3-fluorophenyl)propan-2-yl]-5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-carboxamide, [2-(2,6-dichloroanilino)-4,5-dihydroimidazol-1-yl]-phenylmethanone, methyl N-(6-propoxy-1H-benzimidazol-2-yl) carbamate, 4-(2-(diaminomethylidene)hydrazinyl)phenyl]iminothiourea, N-(diaminomethylidene)-2-(2,6-dichlorophenyl)acetamide, 2-amino-5-bromo-4-phenyl-1H-pyrimidin-6-one, 2-(1,4-dioxaspiro[4.5]decan-2-ylmethyl)guanidine, 5-[2-[[3-(1,3-Benzodioxol-5-yl)-1-methylpropyl]amino]-1-hydroxyethyl]-2-hydroxybenzamide, 2-amino-9-[4-(hydroxymethyl)cyclopent-2-en-1-yl]-1H-purin-6-one, 4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol, 4-amino-N-(4-methylpyrimidin-2-yl)benzenesulfonamide, carbidopa, benserazide, entacapone, opicapone, amantadine, pimavanserin, rotigotine, ropinirole, apomorphine, rasagiline, safinamide, istradefylline, KW-6356, donepezil, rivastigmine, liraglutide, prasinezumab, venglustat, dipraglurant, mesdopetam (IRL-790), SEP-363856, cinpanemab, posiphen, MEDI-1341, UCB-0599, ABBV-0805, NPT-20011, PD-03, PD-01, LuAF-82422, NPT-088, UB-312, and Anle138b or a pharmaceutically acceptable salt thereof, wherein at least one of the compounds in the combination is N-(diaminomethylidene)-2-(2,6-dichlorophenyl)acetamide or a pharmaceutically acceptable salt thereof, is administered to a patient in need thereof.

57. The method according to claim 56, wherein the combination comprises N-(diaminomethylidene)-2-(2,6-dichlorophenyl)acetamide hydrochloride.

58. The method according to claim 54, wherein the synucleinopathy to be treated and/or prevented is selected from the group consisting of Parkinson's disease (PD), Multiple System Atrophy (MSA), and Dementia with Lewy Bodies.

59. The method according to claim 56, wherein the synucleinopathy to be treated and/or prevented is selected from the group consisting of Parkinson's disease (PD), Multiple System Atrophy (MSA), and Dementia with Lewy Bodies.

60. The method according to claim 46, wherein the compound is N-[(2S)-1-amino-3-(3-fluorophenyl)propan-2-yl]-5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-carboxamide, or a pharmaceutically acceptable salt thereof.

61. The method according to claim 46, wherein the compound is N-[1-amino-3-(3-fluorophenyl)propan-2-yl]-5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-carboxamide hydrochloride.

62. The method according to claim 46, wherein the compound is N-[(2S)-1-amino-3-(3-fluorophenyl)propan-2-yl]-5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-carboxamide hydrochloride.

63. The method according to claim 46, wherein the compound is N-(diaminomethylidene)-2-(2,6-dichlorophenyl)acetamide hydrochloride.

64. The method according to claim 46, wherein the compound is 2-(1,4-dioxaspiro[4.5]decan-2-ylmethyl)guanidine hydroiodide.

65. The method according to claim 46, wherein the compound is 2-(1,4-dioxaspiro[4.5]decan-2-ylmethyl)guanidine sulfate.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0163] FIG. 1A: shows α-synuclein aggregation kinetics measured by Thioflavin T fluorescence emission, represented as normalised means. Error bars are represented as standard error. Aggregation kinetics in control conditions (filled circle) or in the presence of the tested compounds: Robenidine (filled square); oxibendazole (triangle); guanfacine (inverted triangle); bropirimine (diamond); cinacalcet (empty circle) and ambazone (empty square).

[0164] FIG. 1B: shows α-synuclein aggregation kinetics measured by Thioflavin T fluorescence emission, represented as normalised means. Error bars are represented as standard error. Aggregation kinetics in control conditions (filled circle) or in the presence of the tested compounds: medroxalol (filled square); benclonidine (triangle); sulfamerazine (inverted triangle); afuresertib (diamond); (−)-carbovir (empty circle); guanadrel (empty square).

[0165] FIG. 2A: shows α-synuclein aggregation measured by light scattering at end-point (48 h). Light-scattering measurements at 300 and 340 nm in control conditions and in the presence of the tested compounds: Robenidine; oxibendazole; guanfacine; bropirimine; cinacalcet, and ambazone. Data is presented as mean and standard error.

[0166] FIG. 2B shows α-synuclein aggregation measured by light scattering at end-point (48 h). Light-scattering measurements at 300 and 340 nm in control conditions and in the presence of the tested compounds: medroxalol; benclonidine; sulfamerazine; afuresertib; (−)-carbovir; guanadrel. Data is presented as mean and standard error.

[0167] FIG. 3-9: show α-synuclein aggregation measured by Transmission Electron Microscopy (TEM) at end-point (48 h). Representative TEM images of α-synuclein in the absence (FIG. 3 control) or presence of the tested compounds: FIG. 4 (Robenidine); FIG. 5 (oxibendazole); FIG. 6 (guanfacine); FIG. 7 (bropirimine); FIG. 8 (cinacalcet) and FIG. 9 (ambazone).

[0168] FIG. 10-15: show α-synuclein aggregation measured by TEM at end-point (48 h). Representative TEM images of α-synuclein in the absence (FIG. 10 control) or presence of the tested compounds: FIG. 11 (benclonidine); FIG. 12 (sulfamerazine); FIG. 13 (afuresertib); FIG. 14 ((−)-carbovir); FIG. 15 (guanadrel).

[0169] FIG. 16-20 show neuroprotection in MPP+ treated dopaminergic neurons achieved with Guanfacine (16), Bropirimine (17), Benclonidine (18), Afuresertib (19) and Guanadrel (20).

EXAMPLES

Materials and Methods

A. Thioflavin-T Assay

[0170] Lyophilized α-synuclein was dissolved in PBS (phosphate buffer solution) to a final solution of 210 μM and filtered through a Millipore 0.22 μm filter. α-synuclein aggregation was performed in a 96-well plate (non-treated, black plastic), each well containing a Teflon® polyball ⅛″ diameter (Polysciences Europe GmbH, Eppelheim, Germany), 40 μM Thioflavin-T (Th-T), 70 μM α-synuclein, 100 μM of the compounds to be tested and PBS up to a final volume of 150 μL. The plates were fixed into an orbital culture shaker Max-Q 4000 (Thermo Scientific, Waltham, Mass., USA) to keep the incubation at 37° C., 100 rpm. Every, 2 h, the fluorescence intensity was measured using a Victor 3.0 Multilabel Reader (PerkinElmer, Waltham, Mass., USA), by exciting the mixtures with 430-450 nm filter and collecting the emission intensity with 480-510 nm filter. Each measurement was performed in triplicates.

[0171] Each plate contained three (3) α-synuclein controls in the absence of any of the tested compounds. The averaged Th-T fluorescence obtained for these wells at the end of the experience was normalized to 1, and the kinetic curves in the different wells re-scaled accordingly. After compiling the fluorescence signals, means and standard error of mean (SEM) were used to fit the aggregation kinetic with equation (1),

[00001]$\begin{array}{cc}\alpha =1-\frac{1}{k_b\left(e^{kat}-1\right)+1}& \mathrm{Equation}\left(1\right)\end{array}$

[0172] wherein k.sup.b and k.sup.a indicate the homogeneous nucleation rate constant and the secondary rate constant, accounting for fibril elongation and secondary nucleation, respectively.

[0173] Re-scaled curves were used to compare the controls with the effect of the compounds and to ensure that the controls were reproducible between different experiments.

Light Scattering

[0174] Total aggregate formation at 48 h was measured by light scattering adding 80 μL of pre-aggregated α-synuclein into a quartz cuvette. Samples were previously re-suspended by carefully vortexing and pipetting, and then excited at 300 and 340 nm and 90° scattering collected between 280 to 360 nm. These measurements were performed in a Cary Eclipse Fluorescence Spectrophotometer (Agilent, Santa Clara, Calif., USA).

Transmission Electron Microscopy (TEM)

[0175] α-synuclein samples were diluted 1:10 in PBS, sonicated for 5 minutes and 5 μL of the resulting mixture immediately placed on a carbon-coated copper grid. After 5 minutes, samples were carefully dried with a piece of filter paper to remove the excess of liquid and washed with MilliQ water twice. Then, 5 μL of a 2% (w/v) solution of uranyl acetate was placed on top of the grid for 2 minutes. Uranyl acetate excess was removed with filter paper. Finally, grids were left to air-dry for 10 min. Images were obtained using a TEM microscope Jeol 1400 (Peabody, Mass., USA) operating at an accelerating voltage of 120 kV. A minimum of 30 fields were screened for each sample to obtain representative images.

Test Compounds

[0176] Robenidine hydrochloride, cinacalcet hydrochloride, afuresertib hydrochloride, oxibendazole, guanfacine hydrochloride, and sulfamerazine were obtained from MedChemExpress. Benclonidine was obtained from Angene. Ambazone, and (−)-carbovir were obtained from Toronto Research Chemicals. Bropirimine, and guanadrel hydroiodide were obtained from Enamine. Medroxalol was obtained from Coompo.

Results

[0177]

TABLE-US-00001 TABLE 1 Parameters of alpha-synuclein aggregation kinetics measured by Thioflavin fluorescence. Kb Ka Control 0.049 ± 0.018 0.183 ± 0.027 Robenidine 0.001 ± 0.444 0.053 ± 0.124 Oxibendazole 0.003 ± 0.004 0.369 ± 0.083 Guanfacine 0.014 ± 0.005 0.161 ± 0.027 Bropirimine 0.027 ± 0.006 0.205 ± 0.015 Cinacalcet 0.066 ± 0.029 0.142 ± 0.032 Ambazone nd nd Medroxalol 0.010 ± 0.005 0.312 ± 0.034 Benclonidine 0.007 ± 0.003 0.225 ± 0.024 Sulfamerazine 0.002 ± 0.001 0.248 ± 0.019 Afuresertib 0.056 ± 0.018 0.141 ± 0.022 (−)-Carbovir 0.027 ± 0.004 0.172 ± 0.010 Guanadrel 0.044 ± 0.008 0.150 ± 0.013 Kb: homogeneus nucleation rate constant (fibril elongation). Ka: secondary rate constant (secondary nucleation)

TABLE-US-00002 TABLE 2 Normalized light scattering values at 300 and 340 nm 300 nm 340 nm Control   1 ± 0.22   1 ± 0.22 Robenidine 0.31 ± 0.03 0.31 ± 0.02 Oxibendazole 0.13 ± 0.02 0.51 ± 0.10 Guanfacine 0.65 ± 0.10 0.68 ± 0.10 Bropirimine 0.55 ± 0.07 0.79 ± 0.10 Cinacalcet 0.44 ± 0.17 0.48 ± 0.18 Ambazone 0.61 ± 0.15 0.53 ± 0.15 Medroxalol 0.72 ± 0.03 0.75 ± 0.04 Benclonidine 0.95 ± 0.07 0.98 ± 0.07 Sulfamerazine 0.55 ± 0.10 0.78 ± 0.14 Afuresertib 0.44 ± 0.08 0.62 ± 0.09 (−)-Carbovir 0.72 ± 0.08 0.77 ± 0.09 Guanadrel 0.68 ± 0.06 0.68 ± 0.06
These data are represented in FIGS. 2A and 2B.

[0178] The high-throughput screening protocol uses thioflavin-T (Th-T) as reporter of amyloid formation, completing highly reproducible reactions in less than 48 h. Each compound was tested in 3 independent experiments. Robenidine, oxibendazole and cinacalcet (FIG. 1A) exhibited interesting inhibitory capacity, reducing Th-T-positive species more than 50%. Ambazone interfered with the Th-T fluorescence readout, as it presented significant absorbance at 425 nm. A more modest inhibition was observed with guanfacine. Bropirimine did not cause detectable inhibition of Th-T fluorescence, but it caused a significant effect on aggregation measured by light scattering (see below, FIG. 2A). This apparent discrepancy may be caused by a selective effect of bropirimine on amorphous (not amyloid fibril) aggregates. Other potent inhibitors were benclonidine, afuresertib, and sulfamerazine (FIG. 1B). Other compounds presented more modest inhibition of fibril formation, such as medroxalol, (−)-carbovir, and guanadrel (FIG. 1B).

[0179] Light scattering measurements at end-point confirmed the inhibitory effect of the compounds, with significant reduction of the absorbance at 300 and 340 nm by robenidine, oxibendazole, guanfacine, bropirimine, cinacalcet, and ambazone (FIG. 2A). Additional light scattering measurements also confirmed the inhibitory activity of medroxalol, sulfamerazine, afuresertib, (−)-carbovir and guanadrel, that also reduced absorbance at both length waves (FIG. 2B).

[0180] Finally, Transmission Electronic Microscopy (TEM) experiments confirmed the effects of the compounds on α-syn aggregation. TEM images of untreated samples showed a higher number of fibrils when compared with samples treated with the positive hits identified by Th-T fluorescence and Light Scattering measurements (FIG. 3-9 and FIG. 10-15).

B. Efficacy in a Parkinson Disease Model

[0181] The efficacy of 5 compounds was assessed in a primary culture of dopaminergic neurons injured with a mitochondrial toxin (MPP+), which is often used to experimentally mimic the pathology of Parkinson disease (FIG. 16 to 20).

Primary Culture of Mesencephalic Neurons

[0182] To obtain rat dopaminergic neurons, pregnant female rats (Wistar, Janvier Labs, France) of 15 days of gestation were killed using a deep anesthesia in a CO2 chamber followed by a cervical dislocation. Midbrains obtained from 15-day-old rat embryos were dissected under microscope and placed in ice-cold medium of Leibovitz (L15) containing 2% of Penicillin-Streptomycin (PS) and 1% of bovine serum albumin (BSA). The ventral portion of the mesencephalic flexure, a region of the developing brain rich in dopaminergic neurons, was used for the cell preparations.

[0183] The midbrains were dissociated by trypsinization for 20 min at 37° C. (solution at a final concentration of 0.05% trypsin and 0.02% EDTA). The reaction was stopped by adding Dulbecco's modified Eagle's medium (DMEM) containing DNAase I grade II (0.5 mg/mL) and 10% of fetal calf serum (FCS). Cells were then mechanically dissociated by 3 passages through a 10 ml pipette. Cells were then centrifuged at 180×g for 10 min at +4° C. on a layer of BSA (3.5%) in L15 medium. The supernatant was discarded and the cell pellet was re-suspended in a defined culture serum-free medium consisting of Neurobasal (Invitrogen) supplemented with B27 (2%), L-glutamine (2 mM) and 2% of PS solution and 10 ng/ml of Brain-derived neurotrophic factor (BDNF) and 1 ng/mL of Glial-Derived Neurotrophic Factor (GDNF). Viable cells were counted in a Neubauer cytometer using the trypan blue exclusion test. The cells were seeded at a density of 40.000 cells/well in 96 well-plates (pre-coated with poly-L-lysine) and maintained in a humidified incubator at 37° C. in 5% CO2/95% air atmosphere.

[0184] Half of the medium was changed every 2 days with fresh medium. On 96-wells plates, only 60 wells were used, the first columns and first lines were not used to avoid any edge effect, the empty wells were filled with sterile water.

Test Compound Application and MPP+ Exposure

[0185] Test compounds were tested on culture in 96-well plates (n=6 culture wells per condition).

[0186] Vehicle: Culture medium (0.1% DMSO).

[0187] Pre-incubation: On day 6 of culture, the compounds (from 10 nM to 10 μM) were dissolved in DMSO and then in culture medium, and were pre-incubated with primary dopaminergic neurons for 1 hour or 4 hours before the application of MPP+.

[0188] Injury: One or four hours after the application of the test compounds, MPP+ was added to a final concentration of 4 μM, diluted in control medium still in presence of compounds for 48 hours.

End-Point Evaluation

[0189] 48 hours after the intoxication, the cell culture supernatant was removed, and the cells were fixed by a solution of 4% paraformaldehyde in PBS, pH=7.3 for 20 min at room temperature. The cells were washed twice in PBS, and then permeabilized. Non-specific sites were blocked with a solution of PBS containing 0.1% saponin and 1% FCS for 15 min at room temperature.

Immunostaining: TH and α-syn

[0190] The cultures were incubated with: [0191] A monoclonal anti-Tyrosine Hydroxylase (TH) antibody produced in mouse at dilution of 1/10000 in PBS containing 1% FCS, 0.1% saponin, for 2 hours at room temperature. [0192] a polyclonal anti-alpha synuclein (α-syn) antibody produced in rabbit at dilution of 1/200 in PBS containing 1% FCS, 0.1% saponin, for 2 hours at room temperature.

[0193] These antibodies were revealed with Alexa Fluor 488 goat anti-mouse IgG at the dilution 1/800 and with Alexa Fluor 568 goat anti-rabbit IgG at the dilution 1/400 in PBS containing 1% FCS, 0.1% saponin, for 1 hour at room temperature.

Automatic Computer Analysis

[0194] For each condition, 20 (for the TH survival and α-syn readouts) pictures representing the whole well area, were automatically taken using ImageXpress® (Molecular Devices) at 10× magnification (20 pictures, for TH and α-syn into TH neurons) using the same acquisition parameters. From images, analyses were directly and automatically performed by Custom Module Editor® (Molecular Devices). The following read-outs were measured: [0195] Analysis of total number of THneurons (TH positive neurons), [0196] total length of neurite network of TH positive neurons (in μm), [0197] α-syn aggregation (overlapping between TH and α-syn staining),

Statistical Analysis

[0198] Results are expressed in percentage of control. All values show the mean+/−SEM (standard error of the mean) from 4-6 wells per condition.

Results

[0199] Treatment with MPP+ caused a decrease in the number of dopaminergic neurons (TH number) and a reduction of their neurite network, accompanied with an increase of α-syn aggregation (ratio a-syn area/TH number). Pre-treatment with guanfacine (FIG. 16), bropirimine (FIG. 17), benclonidine (FIG. 18), afuresertib (FIG. 19) or guanadrel (FIG. 20) at different concentrations partially prevented the loss of dopaminergic neurons and decreased α-syn aggregation triggered by the MPP+ insult, confirming that these compounds have a neuroprotective effect on dopaminergic neurons after an injury with MPP+.