Low-dose dopaminergic drugs to delay the progression of spinocerebellar ataxia type 3 and alzheimer's disease

Abstract

The present invention refers to dopaminergic drugs such as dopamine precursors and aromatic L-amino acid decarboxylase (AADC) inhibitors for use in a treatment to delay the progression of pathologies in which abnormal amyloid deposits spread to different brain regions, such as Spinocerebellar Ataxia Type 3 and Alzheimer's Disease, administered in levodopa-equivalent doses below 300 mg / day. The said dopaminergic drugs are administered by one of the commonly adopted routes to ensure sufficiently high levels of brain dopamine. Example of compounds, dose effect. The present invention further refers to pharmaceutical formulations comprising the said dopaminergic drugs. The present invention's compounds pharmaceutical formulations and uses may be advantageously employed in a treatment to prevent further accumulation of amyloid deposits in neurons, and minimize adverse effects associated with the prolonged use of dopamine precursors and / or their peripheral degradation.

Description

[0001] DESCRIPTION

[0002] LOW-DOSE DOPAMINERGIC DRUGS TO DELAY THE PROGRESSION OF

[0003]

[0004] Technical field of the invention

[0005] The present invention relates to the technical field of human health; medical science; preparations for medical purposes; in particular, medicinal preparations containing organic active ingredients .

[0006] Thiophene containing compounds are well known to exhibit various biological effects . Heterocycles containing the thienopyrimidine moiety

[0007] Thiophene containing compounds are well known to exhibit various biological effects . Heterocycles containing the thienopyrimidine moiety

[0008] State of the art

[0009] Spinocerebellar Ataxia Type 3 (SCA3) and Alzheimer' s Disease (AD) are two neurodegenerative disorders characterized by the deposition in different brain regions of amyloid aggregates containing disease-specific proteins . SCA3, or Machado- Joseph Disease, is a rare disease that was first recognized in the 1970s in families of Azorean descent . SCA3 symptoms start to manifest on average between the third to fifth decade of life and include progressive lack of coordination / accuracy of movement (cerebellar ataxia) , abnormal gait and impaired balance, involuntary muscle contractions (dystonia) , stiffness of muscles (spasticity) , difficulties of speech (dysarthria) , swallowing disorders, and double vision. Healthy individuals harbour CAG repeat lengths between 12 and 44, whereas in SCA3-af f ected individuals this length is consensually above 61. The expansion of the unstable CAG repeat is translated into an expanded polyglutamine (polyQ) tract of ataxin-3, a protein whose accumulation in neuronal nuclearinclusions is a pathological hallmark of SCA3 . SCA3 belongs to the group of polyQ diseases that includes five additional SCA types and Huntington' s disease (HD) , among others . SCA3 also belongs to the large group of protein-misfolding diseases that include Alzheimer' s disease (AD) and are characterized by the aggregation and accumulation of misfolded proteins in cells . AD, in particular, is characterized by the accumulation of two types of protein aggregates consisting of (i) extracellular senile plaques containing the peptide amyloid-beta (Abeta) and (ii) intracellular neurofibrillary tangles composed of the protein tau. The accumulation of Abeta plaques affects the cerebral cortex and hippocampus areas responsible for cognition language processing, reasoning and memory consolidation, whereas tau tangles typically spread across the medial temporal lobe, followed by the association cortices and eventually reaching the isocortical regions [5] . Familial and sporadic forms of AD have been described. Approximately 1-2% of the total AD cases are inherited in an autosomal dominant fashion, showing alterations in specific genes such as the amyloid precursor protein gene

[0021] . AD is the most common neurodegenerative disease and represents 60 to 80% of all cases of dementia .

[0010] While the onset of amyloid deposition can be ascribed to a combination of multiple factors, it is well known that the occurrence of amyloid deposits of ataxin-3 in SCA3, or Abeta and tau in AD, can accelerate disease progression due to the formation of new (and smaller) aggregates at the surface of pre-existing ones . Among the different therapeutic strategies to tackle protein misfolding diseases, abnormal protein aggregation can be targeted by delaying or preventing (i) the de novo formation of new assemblies, (ii) the growth or elongation of existing aggregates and (iii) the templated formation of new assemblies . The simultaneous inhibition of the three steps is also possible through upstream strategies that prevent the protein misfolding events preceding protein self-assembly.No disease-modifying treatments are available for SCA3 . Phase I clinical trials have addressed the safety of using antisense oligonucleotides to lower ataxin-3 levels in SCA3 patients (NCT05160558 ) . This approach relies on the hypothesis that ataxin-3 is not an essential protein for cell function; conversely, protein aggregation inhibition strategies aim at preventing toxic gain-of-function without affecting the functional roles of the (non-aggregated) protein. Biohaven reported in May 2022 no effect on the primary outcome of a Phase 3 trial of troriluzole for SCA3 . Repurposing trehalose for the treatment of SCA3 is under investigation by Seelos Therapeutics (NCT05490563) .

[0011] On the other hand, anti-amyloid strategies have been approved for the treatment of AD using monoclonal antibodies directed against Abeta . Examples of these immunotherapies include Aducanumab (Aduhelm, Biogen) and Lecanemab-irmb (Leqembi, Eisai / Biogen) . These drugs lower the amount of Abeta in the brain but it is not consensual whether this effect improves people' s symptoms and quality of life . Potential side effects associated with Aducanumab and Lecanemab-irmb require regular brain imaging tests to monitor e . g. brain swelling and bleeding spots . In a statement dated January 2024, Biogen discontinued Aducanumab 'to reprioritize its resources in AD' and not for reasons related to safety or efficacy. Dopamine is a catecholamine neurotransmitter mainly produced in the substantia nigra and ventral tegmental area of the midbrain, but also in the hypothalamus, amygdala, and cortex regions of the brain. In Parkinson' s disease (PD) , the death of dopaminergic levels leads to reduced levels of dopamine and the consequential loss of controlled body movements, tremors, slowness of movements (bradykinesia) , muscle rigidity and balance problems . While there is no cure for PD, dopamine replacement therapies, such as the use of dopaminergic drugs, help to manage PD symptoms . By the time the motor symptoms manifest and PD is diagnosed, widespread loss of dopamine-producing cells has already occurred.

[0012] Dopamine-like agents are known to have an anti-amyloid activity, manifested, for example, by the destabilization / disaggregation of Abeta fibrils in the presence of high concentrations of dopamine(above 100 mM)

[0019] ; the exact mechanism behind this anti-amyloid activity is, however, unclear . Dopamine also prevents the formation of amyloid fibrils of a-synuclein (asyn) , a protein whose misfolding and aggregation are associated with PD. In this case, the inhibitory effect of dopamine results from the formation of dopamin-asyn adduct and the consequential aggregation into non-fibrillar asyn oligomers [ 8, 20] . Nevertheless, to face the progressive dopamine deficiency in PD, dopaminergic drugs are administered at gradually increasing doses to PD patients . For example, the dopamine precursor levodopa is typically dosed 3 times daily but, as PD progresses, higher and more frequent doses are required to combat the slowed response to medication

[0002] . Levodopa is frequently combined with aromatic L-amino acid decarboxylase (AADC) inhibitors, such as carbidopa, which prevent peripheral dopamine synthesis, thereby increasing the amount of levodopa that reaches the brain. However, dopamine overproduction by the peripheral metabolism is on the basis of adverse effects of levodopa, such as nausea and vomiting. High doses of levodopa are also associated with severe dyskinesia symptoms ( fast, unpredictable, and involuntary movements) . As such, despite antiaggregation effects of dopamine and dopamine-like agents are documented at molecular level, the effective clinical utility of dopaminergic drugs in the setting of protein aggregation diseases other than PD is totally lacking in prior art . Furthermore, clinical evidence supporting the administration dose and formulation parameters to reach effectiveness while minimizing adverse effects in conditions other than PD is totally lacking.

[0013] Summary of the Invention

[0014] The analysis of the interaction between the AlphaFold2 model of full-length Ataxin-3 and dopamine, predicted using the software AutoDock4 and the graphical user interface AutoDockTools , led to the identification of two main "dopamine docking sites" which, importantly, are in regions predicted to be involved in ataxin-3 aggregation in the globular Josephin domain. Docking site 2 is particularly interesting, as it is delineated by residues from theaggregation-prone region of ataxin-3 which may establish n-n stacking interactions with the aromatic moiety of dopamine . The characteristics of the binding site suggest that it may accommodate larger molecules with additional benzene- or indol-derived substructures . As such, a number of other molecules are predicted to bind to the said "dopamine docking site 2" and this is confirmed by ThT binding assays .

[0015] As such, the present invention refers to compounds for use in a treatment to delay or halt progression of Machado- Joseph Disease through binding at a "Dopamine docking site" in Ataxin 3 comprising one or more hydroxyl group attached to structures selected from the list consisting of :

[0016] a) a benzene ring (hydroxybenzene) , further comprising substituents such as :

[0017] Amine and propyl side chains (as exemplified by Dopamine) ; Carboxylic acid side chain (as exemplified by Levodopa) ; Hydrazine group and propionic acid side chain (as exemplified by Carbidopa) ;

[0018] Amine group and propionic acid side chain (as exemplified by Methyldopa) ;

[0019] Amine and methyl groups (as exemplified by Methyldopate ) ; Amide group and acetic acid side chain (as exemplified by Benserazide) ;

[0020] Ketone group, tetrahydronaphthalene ring, and carboxylic acid side chain (as exemplified by Entacapone) ;

[0021] Amine groups and propyl side chains (as exemplified by Isoproterenol) ;

[0022] Amine groups and phenyl side chain (as exemplified by Levonordef rin) ;

[0023] Methoxy group, amine group, and hydroxypropyl side chain (as exemplified by Isoetharine Mesylate) ;

[0024] Amine groups and aliphatic side chains (as exemplified by Dobutamine) ;

[0025] Amine groups, and phenyl side chain (as exemplefied by Racepinephrin) ;Methoxy groups, ketone group, tetrahydroisoquinoline ring, and a carboxamide side chain (as exemplified by Tolcapone) ; Chlorine atoms (as exemplified by Hexachlorophene)

[0026] b) more complex aromatic rings consisting of :

[0027] Flavone ring (as exemplified by Luteolin) ;

[0028] Tetracyclic rings with methoxy groups (as exemplified by Apomorphine) ;

[0029] Pyridine ring (as exemplified by Ciclopirox) ;

[0030] Quinolines and pyridine ring (as exemplified by Clioquinol) ; Bicyclic ring with chloride (as exemplified by Fenoldopan) ;

[0031] c) a carbonyl group (carboxyl) , further comprising:

[0032] Amines and barbiturate ring, (as exemplified by Etifenin) ; Amines and nitrogen-containing heterocyclic rings (as exemplified by Pentetic acid) ;

[0033] d) a carboxamide group (as exemplified by Deferoxamine) ; and combinations thereof, according to claim 1.

[0034] The present invention further refers to compounds for use in a treatment to delay or halt progression of Machado- Joseph Disease through binding at a "Dopamine docking site" in Ataxin 3 comprising one or more methoxy groups, attached to a ring structure selected from the list consisting of :

[0035] a) Two or more phenyl rings connected by a carbonyl group (as exemplified by Avobenzone) ;

[0036] b) Tetrahydroisoquinoline ring (as exemplified by Tetrabenazine and Benzquinamide ) ;

[0037] and combinations thereof, according to claim 2.

[0038] In another embodiment, the present invention refers to compounds for use in a treatment to delay or halt progression of Machado- Joseph Disease through binding at a "Dopamine" docking site in Ataxin 3, the said compounds being selected from the list consisting of Dopamine, Levodopa, Carbidopa, Methyldopa,Benserazide, Entacapone, Isoproterenol, Levonordef rin, Isoetharine Mesylate, Dobutamine, Racepinephrin, Tolcapone, Ciclopirox, Apomorphine, Deferoxamine, Etifenin, Pentetic acid, Clioquinol, Luteolin, Hexachlorophene, Avobenzone, Fenoldopam, Methyldopate, Tetrabenazine, Benzquinamide and combinations thereof, according to claim 3.

[0039] In other embodiments of the present invention, a pharmaceutical composition of a medicament to delay or halt progression of Machado- Joseph Disease comprises compounds with one or more hydroxyl group attached to structures selected from the list consisting of :

[0040] a) a benzene ring, further comprising substituents such as : Amine and propyl side chains (as exemplified by Dopamine) ; Carboxylic acid side chain (as exemplified by Levodopa) ; Hydrazine group and propionic acid side chain (as exemplified by Carbidopa) ;

[0041] Amine group and propionic acid side chain (as exemplified by Methyldopa) ;

[0042] Amine and methyl groups (as exemplified by Methyldopate) ; Amide group and acetic acid side chain (as exemplified by Benserazide) ;

[0043] Ketone group, tetrahydronaphthalene ring, and carboxylic acid side chain (as exemplified by Entacapone) ;

[0044] Amine groups and propyl side chains (as exemplified by Isoproterenol) ;

[0045] Amine groups and phenyl side chain (as exemplified by Levonordef rin) ;

[0046] Methoxy group, amine group, and hydroxypropyl side chain (as exemplified by Isoetharine Mesylate) ;

[0047] Amine groups and aliphatic side chains (as exemplified by Dobutamine) ;

[0048] Amine groups, and phenyl side chain (as exemplefied by Racepinephrin) ;

[0049] Methoxy groups, ketone group, tetrahydroisoquinoline ring, and a carboxamide side chain (as exemplified by Tolcapone) ; Chlorine atoms (as exemplified by Hexachlorophene)b) more complex aromatic rings consisting of :

[0050] Flavone ring (as exemplified by Luteolin) ;

[0051] Tetracyclic rings with methoxy groups (as exemplified by Apomorphine) ;

[0052] Pyridine ring (as exemplified by Ciclopirox) ;

[0053] Quinolines and pyridine ring (as exemplified by Clioquinol) ; Bicyclic ring with chloride (as exemplified by Fenoldopan) ;

[0054] c) a carbonyl group, further comprising:

[0055] Amines and barbiturate ring, (as exemplified by Etifenin) ; Amines and nitrogen-containing heterocyclic rings (as exemplified by Pentetic acid) ;

[0056] d) a carboxamide group (as exemplified by Deferoxamine) ; and combinations thereof, according to claim 4.

[0057] In another embodiment, a pharmaceutical composition of a medicament to delay or halt progression of Machado- Joseph Disease comprises compounds with one or more methoxy groups, attached to a ring structure selected from the list consisting of :

[0058] a) Phenyl rings connected by carbonyl group (as exemplified by Avobenzone) ;

[0059] b) Tetrahydroisoquinoline ring (as exemplified by Tetrabenazine and Benzquinamide ) ;

[0060] and combinations thereof, according to claim 5.

[0061] In another embodiment, a pharmaceutical composition of a medicament to delay or halt progression of Machado- Joseph Disease comprises compounds selected from the list consisting of Dopamine, Levodopa, Carbidopa, Methyldopa, Benserazide, Entacapone, Isoproterenol, Levonordef rin, Isoetharine Mesylate, Dobutamine, Racepinephrin, Tolcapone, Ciclopirox, Apomorphine, Deferoxamine, Etifenin, Pentetic acid, Clioquinol, Luteolin, Hexachlorophene, Avobenzone, Fenoldopam, Methyldopate, Tetrabenazine, Benzquinamide and combinations thereof, according to claim 6.Detailed description of the Invention

[0062] The analysis of the interaction between the AlphaFold2 model (20) of full-length Ataxin-3 and dopamine is predicted using the software AutoDock4 and the graphical user interface AutoDockTools . Two main docking sites in the globular Josephin domain are identified. The docking site 1 (Dockl) is located in the alphahelical hairpin (Figure 1A) and the binding pocket is bordered by residues Phe61, Glu44, Asn68, Pro65, and Gln64 . In this docking pose, the dopamine molecule is establishing hydrogen bonds with the side chains from Glu44 and Asn68, and with the main-chain atoms of Gln64 and Phe61, while stacking with the side chain of Phe61 (Figure IB) . The second docking site 2 (Dock 2 ) forms a hydrophobic pocket delineated by the side chains of Phe94, Tyr99, and the main chain of residues Argl01-Ilel04, which is further complemented by the side chain of Glnl57.

[0063] Docking site 2 is particularly interesting, as the predicted dopamine binding cavity is delineated by residues from the aggregation-prone region of ataxin-3, including Phe94 which may establish n-n stacking interactions with the aromatic moiety of dopamine .

[0064] The predicted dopamine docking site 2, displays properties compatible with a bona-fide ligand binding site . The binding pocket has an overall volume of 322 A3 (Figure 2A) , within a patch of positive electrostatic potential close to an exposed surface with high relative hydrophobicity (Figure 2B) .

[0065] The analysis of the predicted binding mode of dopamine and the characteristics of the binding site suggest that it may accommodate larger molecules with additional benzene-derived (e . g. biphenyl, benzophenone, dibenzoylmethane) or indol-based substructures . As such, a number of other molecules are predicted to bind to the said "dopamine docking site 2" ( Figure 3) . These include molecules such as Carbidopa, Methyldopa, Benserazide, Entacapone, Isoproterenol, Levonordef rin, Isoetharine Mesylate, Dobutamine, Racepinephrin, Tolcapone, Ciclopirox, Apomorphine, Deferoxamine, Etifenin, Pentetic acid, Clioquinol, Luteolin, Hexachlorophene, Racepinephrin, Avobenzone, Fenoldopam and Methyldopate (Figure 3) .Since the docking site is extended by an adj acent region with high relative hydrophobicity, there is a potential for binding of the benzoquinolizine Tetrabenazine, used for symptomatic treatment of chorea associated with Huntington' s disease, and of Benzquinamide, and anti-hemetic . Both molecules were also predicted to dock preferentially to the same site (Figure 3) .

[0066] In confirmation, results show that carbidopa, methyldopa, benserazide, entacapone, isoproterenol, levordefrin, and isoetharine mesylate inhibit or delay ataxin-3 aggregation, as evaluated by thioflavin-T binding assays (Figure 4A and B) .

[0067] Interestingly, experimental data also showed that tolcapone, a benzophenone, was also able to interfere with ataxin-3 selfassembly. Tolcapone is predicted to bind to the same pocked. The same is observed for ciclopirox, a hydroxypyridine with two of the hydrogens substituted by methyl and cyclohexyl groups .

[0068] Furthermore, Apomorphine, Dobutamine, Carbidopa, Methyldopa, Deferoxamine, Levonordef rin, Isoetharine Mesylate, Ciclopirox, Etifenin, Isoproterenol, Pentetic acid, Clioquinol, Luteolin, Hexachlorophene, Racepinephrin, Avobenzone, Fenoldopam, Tolcapone, Entacapone, Methyldopate and Isoproterenol all showed to inhibit ataxin-3 aggregation (Figure 4C and 4D) .

[0069] By binding to this site, the above-mentioned molecules can limit the local conformational fluctuations required to expose the aggregation-prone site of the Josephin domain that subsequentially primes the ataxin-3 self-assembly into amyloid fibrils . Therefore, molecules with the above-mentioned properties act as "molecular staples", and restrict the motion required for the initial ataxin-3 aggregation.

[0070] In conclusion, the present invention refers to compounds for use in a treatment to delay or halt progression of Machado- Joseph Disease through binding at a "Dopamine" docking site in Ataxin 3, the said compounds comprising chemical entities structurally related to Dopamine, Levodopa, Carbidopa, Methyldopa, Benserazide, Entacapone, Isoproterenol, Levonordef rin, Isoetharine Mesylate, Dobutamine, Racepinephrin, Tolcapone, Ciclopirox, Apomorphine, Deferoxamine, Etifenin, Pentetic acid, Clioquinol, Luteolin,Hexachlorophene, Avobenzone, Fenoldopam, Methyldopate, Tetrabenazine and Benzquinamide .

[0071] In another embodiment, the present invention refers to pharmaceutical composition of a medicament comprising the above-mentioned compounds .

[0072] Brief description of the Figures

[0073] Figure 1. Overview of Dopamine docking sites 1 and 2 on ataxin-3 Josephin domain. A) and B) - Overview of Dopamine docking site 1. A) Sphere representation of dopamine (blue) docked on the flexible helical hairpin of the Josephin domain (cartoon representation, red) ; the aggregation-prone region of the Josephin domain (residues 76-96) is colored yellow. B) Close-up view of the dopamine (blue sticks) binding pocket 1. Black dashes represent predicted hydrogen bonds . C) and D) - Overview of Dopamine docking site 2. C) Sphere representation of dopamine (blue) docked on the hydrophobic surface pocket of the Josephin domain (cartoon representation, red) ; the aggregation-prone region of the Josephin domain (residues 76-96) is colored yellow. D) Close-up view of the dopamine (blue sticks) binding pocket 2. Yellow dashes represent putative hydrogen bonds .

[0074] For protein preparation, the three-dimensional AlphaFold model of full-length Ataxin-3, is obtained through Uniprot (accession P54252 ) . Using AutodockTools the protein structure was processed and charges were assigned to the protein using the AutoDock force field in AutodockTools . Additional docking studies use the experimental three dimensional structure of the Josephin domain (PDB accession 1YZB) . For ligand preparation, the three-dimensional structure of the dopamine, is obtained from PubChem with the accession code CID 681. The other molecules used for docking are Carbidopa (CID34359) ; Methyldopa (CID38853) ; Levonordef rin (CID164739) ; Isoetharine Mesylate (CID23702 ) ; Isoproterenol (CID3779) ; Luteolin (CID5280445) ; Fenoldopam (CID3341 ) ; Tolcapone (CID4659569) ; Entacapone (CID5281081 ) ; Isoproterenol (CID3779) ; Benserazide (CID2327 ) ; Benzoquinamide (CID2342 ) ; Tetrabenazine (CID6018 ) . The files are prepared andconverted to mol2 format with Avogadro . AutodockTools is used to convert the ligand structures into the PDBQT file format . Charges and atom types were assigned to the ligand using the AutoDock force field in AutodockTools . For grid box preparation, a grid box is defined within the protein structure to encompass the whole protein model . AutodockTools was used to define the size and coordinates of the grid box . For generation of input files, the docking parameter file (DPF) is generated using AutodockTools . The DPF contains settings for the docking algorithm, search parameters, and scoring function. The number of docking runs is set to 100. For all other parameters, such as the number of energy evaluations and maximum number of generations, the default parameters are used. For running the docking experiment, the Autodock4 program is executed with the DPF file as input . The docking simulation is performed using a Lamarckian genetic algorithm to explore different orientations and conformations of the ligand within the grid box . The program iteratively searches for the best docking solution based on the specified parameters . For analysis of docking results, the output files generated by Autodock4 are analyzed to examine the docked poses and their corresponding binding energies . The results are visualized using AutodockTools or other molecular visualization software . The docked poses are ranked based on their binding energies or other scoring functions . The docking results are interpreted to gain insights into the ligand-protein interactions and potential binding modes . For calculation of the electrostatic and surface properties, the electrostatic and relative hydrophobicity of the protein are calculated as described in ref . 5 using the webserver https : / / proteinsol .manchest er . ac .uk / .

[0075] Figure 2. Main features of the Dopamine binding pocket 2 in the Josephin domain. A) Overview of ligand binding pocket 2 on the Josephin domain of Ataxin-3. The bottom of the pocket is hydrophobic and flanked by the aromatic residues Phe94, Leul52 and Ilel62, in close contact to Vall64, Phell2, Ilel04, Glul09 and Prol02. B) Representation of the electrostatic potential and relative hydrophobicity ratio of the binding pocket 2.Figure 3. Compounds showing structural interactions with the "Dopamine docking site" (docking site 2 ) in Ataxin 3. CID represents compound ID in the Pubchem database .

[0076] Figure 4. Dopamine structurally related compounds that can delay the aggregation of Atx3, as assessed by the ThT assay. A) ThT assay results; B) Structures of compounds able to delay aggregation of Atx3 . C) ThT assay results of compounds able to inhibit Atx3 aggregation. D) Structures of compounds able to inhibit aggregation of Atx3 . CID represents compound ID in the Pubchem database .

[0077] For protein expression and purification, both Atx3 isoforms were expressed and purified as previously described ( 16) . Briefly, the pDEST17ATX3 ( 13Q) plasmid was transformed into E . coli BL21 (DE3) -SI cells (Life Technologies) . Cells were grown at 37 °C, 180 rpm, in Luria Broth (LB) medium without NaCl supplemented with 100 pg.mL-1 of ampicillin and 0.4 % (w / v) glucose until CD600nm reached 0.8. The culture was then cooled to 30 °C and protein expression was induced with 300 mM NaCl . After 3 hours of expression, the cells were harvested by centrifugation and resuspended in buffer A (20 mM sodium phosphate pH 7.5, 600 mM NaCl, 2.5 % (v / v) glycerol, 20 mM imidazole) containing 100 pg. L-1 lysozyme . Cells were disrupted by stirring 1 hour on ice in the presence of 0.02 mg.mL-1 DNase, 0.02 mg.mL-1 RNase, ImM MgC12 and 1 mM PMSF. After centrifugation supernatant was loaded into a pre-equilibrated Ni2+ charged IMAC column (GE Healthcare Life Sciences) and protein was eluted with 250mM of imidazole . Then, loaded into a HiPrep 26 / 60 Sephacryl S-300 HR column (GE Healthcare Life Sciences) equilibrated with purification buffer (20 mM sodium phosphate pH 7.5, 200 mM NaCl, 5 % (v / v) glycerol, 2 mM EDTA, 1 mM DTT) . After SDS-PAGE analysis, fractions corresponding to Atx3 were pooled and concentrated on an Amicon Ultra-15 centrifuge filter (Millipore) to 20-30 mg.mL-1, frozen in liquid nitrogen and stored at -80 °C . Before each aggregation assay, purified Atx3 aliquots were applied to a Superose 12 10 / 300 GL column (GE Healthcare Life Sciences) pre-equilibrated with aggregation buffer (20 mM HEPES pH 7.5, 150 mM NaCl and 1 mM DTT) . Protein elution was monitored at 280 nm andthe concentration of the fractions corresponding to monomeric protein was determined by measuring the absorbance at 280 nM and using protein' s coefficients, 31650 M-l . cm-1.

[0078] To perform the ThT Assay, Atx3 aggregation was monitored by following the increase in thioflavin T (ThT) fluorescence at 480 nm (440 nm excitation) on a CHAMELEON V plate reader (HIDEX) using a 384-well microplate (low flange, black, flat bottom, polystyrene; Corning, Kennebunk, ME) . Briefly, 50 pL samples of Atx3 at 3 pM in aggregation buffer containing 15 pM ThT were incubated at 37 °C and ThT fluorescence was measured every 30 min for approximately 48h. To prevent evaporation, 20 pL of paraffin oil was put in each reaction well with an automatic multichannel pipette (Eppendorf Xplorer, ref 4861000120) .

[0079] For compound screening, a commercial chemical library (Prestwick, Illkirch) containing 1280 drug repurposing compounds was screened. Atx3 13Q was dispensed into a 384-well microplate with an automatic multichannel pipette (Eppendorf Xplorer, ref 4861000120, Eppendorf, Germany) . Then an automated liquid handler (JANUS Automated Workstation; PerkinElmer) equipped with pin tool replicators (V&P Scientific) coupled to a Modular Dispense Technology (MDT) head was used to add 0.1 pL of test compounds ( from a 1 mM stock) or DMSO ( for controls) . A total of four 384-well microplates were filled, each plate testing 320 chemical compounds ( 1 compound per well) and running 32 control reactions in the presence of DMSO. The final reaction mixture contained 3 pM of Atx3 13Q and 2 pM of test compound / DMSO .

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[0105] Lisbon, 15thJanuary 2025

Claims

CLAIMS1. Compounds for use in a treatment to delay or halt progression of Machado- Joseph Disease through binding at a "Dopamine docking site" in Ataxin 3 comprising one or more hydroxyl group attached to structures selected from the list consisting of :a) a benzene ring (hydroxybenzene) , further comprising substituents such as :Amine and propyl side chains (as exemplified by Dopamine) ; Carboxylic acid side chain (as exemplified by Levodopa) ; Hydrazine group and propionic acid side chain (as exemplified by Carbidopa) ;Amine group and propionic acid side chain (as exemplified by Methyldopa) ;Amine and methyl groups (as exemplified by Methyldopate ) ; Amide group and acetic acid side chain (as exemplified by Benserazide) ;Ketone group, tetrahydronaphthalene ring, and carboxylic acid side chain (as exemplified by Entacapone) ;Amine groups and propyl side chains (as exemplified by Isoproterenol) ;Amine groups and phenyl side chain (as exemplified by Levonordef rin) ;Methoxy group, amine group, and hydroxypropyl side chain (as exemplified by Isoetharine Mesylate) ;Amine groups and aliphatic side chains (as exemplified by Dobutamine) ;Amine groups, and phenyl side chain (as exemplefied by Racepinephrin) ;Methoxy groups, ketone group, tetrahydroisoquinoline ring, and a carboxamide side chain (as exemplified by Tolcapone) ; Chlorine atoms (as exemplified by Hexachlorophene)b) more complex aromatic rings consisting of :Flavone ring (as exemplified by Luteolin) ;Tetracyclic rings with methoxy groups (as exemplified by Apomorphine) ;Pyridine ring (as exemplified by Ciclopirox) ;Quinolines and pyridine ring (as exemplified by Clioquinol) ; Bicyclic ring with chloride (as exemplified by Fenoldopan) ;c) a carbonyl group (carboxyl) , further comprising:Amines and barbiturate ring, (as exemplified by Etifenin) ; Amines and nitrogen-containing heterocyclic rings (as exemplified by Pentetic acid) ;d) a carboxamide group (as exemplified by Deferoxamine) ;and combinations thereof .2 . Compounds for use in a treatment to delay or halt progression of Machado- Joseph Disease through binding at a "Dopamine docking site" in Ataxin 3 comprising one or more methoxy groups, attached to a ring structure selected from the list cons i s ting o f :a) two or more phenyl rings connected by a carbonyl group (as exemplified by Avobenzone) ;b) Tetrahydroisoquinoline ring (as exemplified by Tetrabenazine and Benzquinamide) ;and combinations thereof .

3. Compounds for use in a treatment to delay or halt progression of Machado- Joseph Disease through binding at a "Dopamine" docking site in Ataxin 3, the said compounds being selected from the list consisting of Dopamine, Levodopa, Carbidopa, Methyldopa, Benserazide, Entacapone, Isoproterenol, Levonordef rin, Isoetharine Mesylate, Dobutamine, Racepinephrin, Tolcapone, Ciclopirox, Apomorphine, Deferoxamine, Etifenin, Pentetic acid, Clioquinol, Luteolin, Hexachlorophene, Avobenzone, Fenoldopam, Methyldopate, Tetrabenazine, Benzquinamide and combinations thereof .Pharmaceutical composition of a medicament to delay or halt progression of Machado- Joseph Disease comprising compounds with one or more hydroxyl group attached to structures selected from the list consisting of :a) a benzene ring, further comprising substituents such as : Amine and propyl side chains (as exemplified by Dopamine) ; Carboxylic acid side chain (as exemplified by Levodopa) ; Hydrazine group and propionic acid side chain (as exemplified by Carbidopa) ;Amine group and propionic acid side chain (as exemplified by Methyldopa) ;Amine and methyl groups (as exemplified by Methyldopate ) ; Amide group and acetic acid side chain (as exemplified by Benserazide) ;Ketone group, tetrahydronaphthalene ring, and carboxylic acid side chain (as exemplified by Entacapone) ;Amine groups and propyl side chains (as exemplified by Isoproterenol) ;Amine groups and phenyl side chain (as exemplified by Levonordef rin) ;Methoxy group, amine group, and hydroxypropyl side chain (as exemplified by Isoetharine Mesylate) ;Amine groups and aliphatic side chains (as exemplified by Dobutamine) ;Amine groups, and phenyl side chain (as exemplefied by Racepinephrin) ;Methoxy groups, ketone group, tetrahydroisoquinoline ring, and a carboxamide side chain (as exemplified by Tolcapone) ; Chlorine atoms (as exemplified by Hexachlorophene)b) more complex aromatic rings consisting of :Flavone ring (as exemplified by Luteolin) ;Tetracyclic rings with methoxy groups (as exemplified by Apomorphine) ;Pyridine ring (as exemplified by Ciclopirox) ;Quinolines and pyridine ring (as exemplified by Clioquinol) ;Bicyclic ring with chloride (as exemplified by Fenoldopan) ;c) a carbonyl group, further comprising:Amines and barbiturate ring, (as exemplified by Etifenin) ; Amines and nitrogen-containing heterocyclic rings (as exemplified by Pentetic acid) ;d) a carboxamide group (as exemplified by Deferoxamine) ;and combinations thereof .

5. Pharmaceutical composition of a medicament to delay or halt progression of Machado- Joseph Disease comprising compounds with one or more methoxy groups, attached to a ring structure selected from the list consisting of :a) Phenyl rings connected by carbonyl group (as exemplified by Avobenzone) ;b) Tetrahydroisoquinoline ring (as exemplified by Tetrabenazine and Benzquinamide) ;and combinations thereof .

6. Pharmaceutical composition of a medicament to delay or halt progression of Machado- Joseph Disease comprising compounds selected from the list consisting of Dopamine, Levodopa, Carbidopa, Methyldopa, Benserazide, Entacapone, Isoproterenol, Levonordef rin, Isoetharine Mesylate, Dobutamine, Racepinephrin, Tolcapone, Ciclopirox, Apomorphine, Deferoxamine, Etifenin, Pentetic acid, Clioquinol, Luteolin, Hexachlorophene, Avobenzone, Fenoldopam, Methyldopate, Tetrabenazine, Benzquinamide and combinations thereof .Lisbon, 15thJanuary 2025

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