Bromotyrosine derivatives for use in the treatment of protein misfolding diseases

Abstract

The invention relates to bromotyrosine derivatives compounds for use in treating protein misfolding diseases.

Description

Bromotyrosine derivatives for use in the treatment of protein misfolding diseasesTechnical Field

[0001] This disclosure pertains to the field of bromotyrosine derivatives and their use for the treatment of protein misfolding diseases.Background Art

[0002] Unlike traditional infectious agents such as viruses, bacteria, parasites or fungi, prions are unconventional infectious agents composed solely of a protein that can adopt alternative three- dimensional conformations and do not contain genetic material such as DNA or RNA.

[0003] The physiological form of the prion protein (PrPc) is found in healthy cells, particularly in the nervous system. However, in prion diseases, PrPcundergoes a conformational change, folding into a pathogenic conformation known as PrPSc. The unique hallmark of PrPScis its ability to act as a template, inducing PrPcprion proteins to misfold into PrPScpathological conformation and aggregate. PrPSccapacity of templating and conversion allows its self-propagation and spreading within an organism as well as between individuals (Scheckel & Aguzzi. Nat. Rev. Genet. 2018, 19, 405-418; Baral et al. Protein Sci. 2019, 28, 2055-2063). The accumulation of PrPScoligomers and amyloid aggregates, which disrupts cellular function over time, induces neuronal loss and vacuolation and ultimately leads to neurodegenerative disorders. PrPScpropagation contributes to the spongiform appearance of brain architecture observed in prion-affected people, which is a hallmark of prion diseases.

[0004] Prion diseases, also called transmissible spongiform encephalopathies (TSEs), are a group of rare and fatal neurological disorders that affect both humans and animals. Human Prion diseases (Creutzfeldt-Jakob Disease (CJD), variant Creutzfeldt-Jakob Disease (vCJD), Gerstmann- Straussler-Scheinker Disease (GSS), Fatal Familial Insomnia (FFI), Kuru and variably proteasesensitive prionopathy (VPSPr) are rare with an annual incidence between 1.5-2 cases per million worldwide for CJD according to the National Public Health Agency. They can occur in sporadic forms and can also be transmitted through both genetic mutations (inherited forms) and exposure to prion- contaminated tissues or products. The misfolded prion protein is remarkably resilient and can withstand standard sterilization methods, making these diseases difficult to control.

[0005] There is no cure for prion diseases (Baiardi et al. Expert Opin. The Targets. 2023, 1-14). The unique nature of prions and their ability to self-propagate through the conversion of PrPccellular proteins into misfolded, disease-causing PrPScforms and to transmit disease without involving traditional genetic material makes them particularly challenging to target with traditional therapeutic approaches. While there is currently no approved cure for prion diseases, various approaches to develop potential therapies that could target prion proteins or the underlying mechanisms of prion propagation and aggregation have been actively explored (Chen et al. Biosafety and Health ,2021 , 92-100:2590-0536; Mustazza et al. Curr Med Chem. 2020;27(33):5446-5479). A large variety of natural compounds derived from plants, animals, and other sources has been investigated for their ability to inhibit prion propagation, reduce aggregation of misfolded prion proteins, or mitigate theeffects of prion-related neurodegeneration. The anti-prion properties of some natural products among which curcumin (turmeric spice), resveratrol (grapes and certain berries), polyphenol epigallocatechin gallate (EGCG, green tea) and flavonoids (fruits, vegetables, and other plants) have thus been described. It is important to emphasize that while these natural products have shown promise in in vitro studies and in animal models, their effectiveness and safety in treating prion diseases in humans are not yet established.

[0006] In this context, the chemodiversity of marine organisms represents a formidable potential for the development of new therapies. Marine sponges are known to produce a wide range of bioactive compounds and these organisms have already been studied as potential sources of anti-prion compounds (Jennings et al. Prion. 2018, 12(3-4):234-244; Jennings et al. J Nat Prod. 2020, 24;83(12):3751-3757). Marine sponges have therefore been explored for their potential content of anti-prion compounds, focusing on Polynesian sponges. Of the 131 sponge extracts tested, two extracts from Aplysinellidae sponges and one extract from a Pseudoceratinidae sponge were active against both [PS / +] and [URE3] yeast prions. Based on these results, Suberea laboutei (P562- WLF04) was used to isolate and identify the metabolites responsible for anti-prion activity. The six anti-prion compounds identified were bromotyrosine derivatives. Bromotyrosine and its derivatives have attracted interest in the field of natural products research due to their wide biological activities, including notably antimalarial, biocidal antifouling, antimicrobial, antimycobacterial, antiviral, and antifungal activities, cytotoxicity, specific histamine H-3 receptor antagonist capacities, or inhibition of enzymes. Several bromotyrosine derivatives were also able to inhibit cancer proliferation, invasion, and migration.

[0007] In the context of prion diseases, some studies have explored the potential of bromotyrosine derivatives as inhibitors of prion protein aggregation and propagation. In particular, Aplysamine-1 , Aplysamine-2 and Purealidine-Q, isolated from the Verongid sponge Suberites ianthelliformis, have already been described as active against yeast prions [PS / +] and [URE3] (Jennings et al. Prion. 2018, 12(3-4):234-244). Psammaplysene A has recently been shown to reduce prion levels in prion- infected cells and cerebellar organotypic slices and to alleviate locomotor deficits in prion-infected Drosophila expressing ovine PrPc(Avar et al. The EMBO Journal. 2022, 47(23)).Description of the invention

[0008] The inventors have discovered that bromotyrosine derivatives are compounds having biological effects on protein misfolding diseases (PMDs).

[0009] It is to the credit of the inventors to have identified bromotyrosine derivatives with anti-prion activity. Since prion-like protein aggregation is one of the hallmarks of protein misfolding diseases, those bromotyrosine compounds should be able to alleviate the cellular consequences of misfolded proteins aggregation, and as such are useful drug candidates in the context of protein misfolding diseases.

[0010] A first object of the invention is a bromotyrosine compound of formula (I)a pharmaceutically acceptable salt or a solvate thereof, or salt of a solvate thereof, wherein:R3= H, N(CH3)2for use in treating a protein misfolding disease.

[0011] Another object of the invention is a bromotyrosine compound of formula (la)a pharmaceutically acceptable salt or a solvate thereof, or salt of a solvate thereof, for use in treating a protein misfolding disease.

[0012] The bromotyrosine compound (la) is also named Psammaplysene D.

[0013] Another object of the invention is a bromotyrosine compound of formula (lb)(lb)a pharmaceutically acceptable salt or a solvate thereof, or salt of a solvate thereof, for use in treating a protein misfolding disease.

[0014] The bromotyrosine compound (lb) is also named Anomoian F.

[0015] Another object of the invention is a bromotyrosine compound of formula (Ic)a pharmaceutically acceptable salt or a solvate thereof, or salt of a solvate thereof, for use in treating a protein misfolding disease.

[0016] The bromotyrosine compound (Ic) is also named Aplyzanzine C.

[0017] The bromotyrosine compounds of formula (I), (la), (lb) and (Ic) can be in the form of pharmaceutically acceptable salts. Pharmaceutically acceptable salts include the acid and base addition salts thereof.

[0018] The compounds used in the context of the present invention may exist in both unsolvated and solvated forms. The term 'solvate' describes a molecular complex comprising the bromotyrosine compound and a stoichiometric amount of one or more pharmaceutically acceptable solvent molecules. The term 'hydrate' is employed when said solvent is water.

[0019] The compound of formula (I) thus includes references to salts and solvates thereof and to solvates of salts thereof. The compound of formula (I) includes all polymorphs and crystal habits thereof, prodrugs and isomers thereof (including optical, geometric and tautomeric isomers) and isotopically-labelled compounds of formula (I).

[0020] As used herein, the terms “treating” or "treatment" include references to curative, palliative and prophylactic treating or treatment. A “treatment” aims at reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of the disorder or condition to which such term applies.

[0021] As used herein, the term “protein misfolding diseases” (PMDs) refers to a class of diseases in which certain proteins acquire alternative conformations and become pathogenic, and therebydisrupt the function of cells, tissues and organs of the body. In particular, the proteins can fail to fold into their physiological configuration; and in this misfolded state, the proteins can become toxic or they can lose their physiological function.

[0022] In particular, compounds of the invention are claimed for use in treating protein misfolding diseases wherein the protein misfolding disease (PMD) is a prion disease.

[0023] Said prion disease may be Creutzfeldt-Jakob disease, Variant CJD, variably proteasesensitive prionopathy, Gerstmann-Straussler-Scheinker disease, Fatal familial insomnia, Kuru, prion disease associated with diarrhea and autonomic neuropathy bovine spongiform encephalopathy, chronic wasting disease and scrapie or camel prion disease in non-human mammals.

[0024] Protein misfolding diseases include neurodegenerative, neuromuscular, muscular, metabolic, systemic and neurodevelopmental protein misfolding diseases in which protein aggregates are present.

[0025] Examples of such diseases comprise Alzheimer's disease, Parkinson's disease, Huntington’s disease, amyotrophic lateral sclerosis, oculopharyngeal muscular dystrophy (OPMD), inclusion body myositis (IBM), Duchenne muscular dystrophy (DMD), type II diabetes, Partington syndrome / X-linked intellectual disabilities (XLID) linked to the dup24 duplication in the ARX (aristaless related homeobox) gene.

[0026] According to the present invention, the compound of the invention may be administered in a therapeutically effective amount by any of the accepted modes of administration, for example by oral route or intravenously, intraarterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracranially, intramuscularly or subcutaneously; and / or by using infusion techniques, preferably by oral or intravenous route.

[0027] Dosage levels and requirements are well-recognized in the art and may be selected by those of ordinary skill in the art from available methods and techniques suitable for a particular patient. In some embodiments, dosage levels range from about 10-1000 mg / dose for a 70 kg patient. Although one dose per day may be sufficient, up to 5 doses per day may be given. For oral doses, up to 2000 mg / day may be required. As the skilled artisan will appreciate, lower or higher doses may be required depending on particular factors. For instance, specific dosage and treatment regimens will depend on factors such as the patient's general health profile, the severity and course of the patient's disorder or disposition thereto, the patient's age, and the judgment of the treating physician.

[0028] According to one embodiment, the compounds of the invention, their pharmaceutical acceptable salts or solvates may be administered as part of a combination therapy. Thus, are included within the scope of the present invention embodiments comprising co-ad ministration of, and compositions and medicaments which contain, in addition to a compound of the present invention, a pharmaceutically acceptable salt or solvate thereof as active ingredient, additional therapeutic agents and / or active ingredients. Such multiple drug regimens, often referred to as combination therapy, may be used in the treatment or prevention of any disease or disorder associated with proteinmisfolding, particularly those defined above. In particular, gene or cell therapy may be combined to a pharmacological therapy involving the compounds of the invention.

[0029] Thus, the methods of treatment and pharmaceutical compositions of the present invention may employ the compounds of the invention or their pharmaceutical acceptable salts or solvates thereof in the form of monotherapy but said methods and compositions may also be used in the form of multiple therapy in which one or more compounds of Formula I or their pharmaceutically acceptable salts or solvates are co-administered in combination with one or more other therapeutic agents, or administered in combination with one or more other therapeutic agents by a different route or with a different timeframe.

[0030] Generally, for pharmaceutical use, the compounds for use of the invention may be formulated as a pharmaceutical preparation comprising at least one compound for use of the invention and at least one pharmaceutically acceptable carrier, diluent, excipient and / or adjuvant, and optionally one or more further pharmaceutically active compounds.Brief Description of Drawings

[0031] Figure 1 : Structures of the isolated compounds 1-6 from Suberea laboutei.

[0032] Figure 2: Structures of the tested bromotyrosine and bromophenol derivatives 7-14.Examples

[0033] 1. Materials and methods

[0034] 1.1. Biological material

[0035] Sponges were collected by hand using SCUBA in French Polynesia and Wallis & Futuna, respectively during the oceanographic cruises BSMPF-1 (DEBITUS Cecile (2009), BSMPF-1 cruise, RV Alis), Tuam’2011 (DEBITUS Cecile (2011 ), TUAM 2011 cruise, RV Alis), and Wallis 2018 (PETEK Sylvain (2018), WALLIS 2018 cruise, RV Alis) aboard the R / V Alis. Samples were deep-frozen on board until work up. They were then freeze-dried, grounded and extracted as previously described (Caudal et al. Microorganisms. 2023, 11 , 1762). The sponge Suberea ianthelliformis was collected in the Marquesas Archipelago, off the coast of Nuku Hiva (8°55.97’S, 140°01.17’W) between 5 and 42 m on the 28 August 2009. It was identified by Dr. Patricia Sutcliffe and a reference specimen is deposited at the Queensland Museum (Brisbane, Australia) under the accessing number G331075. The sponge Pseudoceratina sp. (OTU QM2081 ) was collected in the Tuamotu Archipelago, off the coast of Raroia (16°00.551 'S, 142°27.243'W) between 45 and 55 m deep on the 13 May 2011. It was identified by John N A Hooper and a reference specimen is deposited at the Queensland Museum (Brisbane, Australia) under the accession number QM G333336. The sponge Suberea laboutei was collected in the Wallis Island lagoon (13°20.308'S, 176°10.392'W) at a depth of 15 m on July 2018. A voucher sample has been deposited at the Queensland Museum (Brisbane, Australia) under the access number G339007 and was identified by Dr. Merrick Ekins.

[0036] 1.2. Yeast-based anti-prion screening assay

[0037] The yeast-based screening assay was performed as previously described (Bach et al. Nat Biotechnol. 2003, 21 (9): 1075-81 ). The yeast strains used in this study were STRg6 (74-D694, Mata, erg6 TRP1, ade1-14, trp1-289, his3£200, ura3-52, Ieu2-3, 112,) containing the strong [PS / +] prion strain and SB34 (Mata, erg6 TRP1, dal5.ADE2, ade2-1, trp1-1, Ieu2-3, 112, his3-11, 15, ura2.HIS3) containing [URE3] prion (Bach et al. Nat Biotechnol. 2003, 21 (9):1075-81 ). Yeasts containing [PS / +] and [URE3] prions form white colonies, while yeasts without [PS / +] or [URE3] prions ([ps / -] or [ur3- 0)] form red colonies (Liebman et al. Genetics. 2012, 191 , 1041-1072). The screening assay for the identification of compounds active against Saccharomyces cerevisiae [PS / +] and [URE3] prions takes advantage of this colorimetric reporter system. Aliquots of 360 pL (STRg6 strain) or 170 pL (SB34 strain) of 0.55 ODeoo exponentially growing cultures were spread using sterile glass beads on square (12 cm x 12 cm) petri plates containing YPD rich medium supplemented with 200 pM for [PS / +] or 800 pM for [URE3] of guanidine hydrochloride (GuHCI, Sigma Aldrich). Small sterile filters (Thermo Fisher Scientific) were then placed on the agar surface and 2 pL of each extract, fraction or purified molecule were applied individually to each filter. 2 pL DMSO (PAN Biotech) and 2 pL GuHCI 1 M were used as negative and positive controls, respectively. The screening plates were incubated for seven to ten days at 25°C until the appearance of the red coloration around the filter on which the positive control has been loaded. Colonies form a red halo if the product spotted on a filter has antiprion activity, while the halo of white colonies remains intact if the molecule is inactive against yeast prions.

[0038] 1.3. Extraction and isolation

[0039] The freeze-dried sponge sample (960 g) of Suberea ianthelliformis was extracted three times at room temperature with a 1 :1 mixture of CH2Cl2 / MeOH (1.5 L). The extracts were combined and dried under reduced pressure to afford a brown residue (50 g). Forty-five grams of this residue were partitioned between CH2CI2 and H2O. The resulting organic layer was collected and dried under reduced pressure to afford a brown material (10.5 g). The aqueous layer was further extracted with n-BuOH to afford the butanolic extract (13 g). The remaining aqueous fraction was dried to afford 21 .2 g material. The CH2CI2 extract was then subjected to normal phase silica-gel MPLC. A gradient composed of CH2Cl2 / MeOH (1 :0 to 0:1 ), followed by a mixture of EtOAc / Acetone / H2O / Formic Acid (5 / 3 / 0.5 / 0.5) were used to afford 8 fractions F1 to F8. The n-BuOH extract was also subjected to normal phase silica gel MPLC, using the same gradient as before to afford 6 fractions F1 to F6. Promising fractions were selected based on the LC-MS analytical profile, and then each fraction was submitted for purification by preparative reversed phase HPLC (column: Waters Sunfire C18 19 mm x 150 mm, 5 p, H2O + 0.1% formic acid / CHsCN + 0.1% formic acid). Some sub-fractions were further purified using semi-preparative reversed phase column (Waters Sunfire C18, 10 mm x 150 mm, 5 p, H2O + 0.1%formic acid / CHsCN + 0.1% formic acid), to deliver the psammaplysenes D, F, G, anomoian F, and N,N-dimethyldibromotyramine. This procedure has been described by El- Demerdash et al. (El-Demerdash et al. Marine Drugs. 2018 Apr 27;16(5):146).

[0040] The freeze-dried sponge sample of Pseudoceratina sp. (OTU QM2081 , 30 g) was extracted by maceration at room temperature with 150 mL of EtOH / H2O (80:20) for 90 minutes, and then twicewith 150 mL of EtOH (96%) for 45 minutes. The extracts were combined and the ethanol was evaporated under reduced pressure. A liquid-liquid partition between CH2CI2 and H2O was done on the resulting residue. After drying of the organic layer upon MgSC , solvents were evaporated under reduced pressure to offer the extract C (1.49 g, 4.9%). A precipitate at the interface between the organic and the aqueous layer was saved and dried (extract P, 1.28 g, 4.21 %). The aqueous layer was partitioned against n-BuOH, to deliver the organic extract B (3.62 g, 12%) after drying and evaporation of the solvents. The precipitate P (150 mg) was fractionated on normal-phase silica SPE (Strata, 1 g, 55 pm, 70 A, Phenomenex, Le Pecq, France), using the gradient from 1 :0 to 0:1 mixture of cyclohexane / AcOEt then 1 :0 to 0:1 mixture of CFECh / MeOH to deliver finally six fractions. The second fraction (34 mg; 10% MeOH), the third (21 mg; 10%— 12% MeOH) and the fourth (24 mg: 14%-20% MeOH) were submitted to further purification by semi-preparative reversed-phase HPLC (H2O / CH3CN / 0.1%TFA). Aplyzanzine C was isolated from each fraction but aplyzanzine E only from the second fraction and 6 from the third one. Extract C (500 mg) was subjected to normal-phase silica-gel chromatography, using the gradient from 1 :0 to 0:1 mixture of cyclohexane / AcOEt then 1 :0 to 0:1 mixture of CH2Cl2 / MeOH successively to produce 13 fractions. After QSi evaluation on V. harveyi, only the third fraction (83 mg; 50% AcOEt) and the fourth (51 mg; 80% AcOEt) showed a biological activity. Respectively, 20 and 10 mg of these two fractions were further purified by semipreparative reversed-phase HPLC (H2O / CH3CN / 0.1%TFA). The aplyzanzine D was the main product isolated, but aplyzanzine F was obtained as a minor compound only from the 50% AcOEt fraction. The extract B (500 mg) was subjected to gel-permeation chromatography on Sephadex LH20, using MeOH as solvent to finally deliver nine fractions. A portion (25 mg) of the sixth fraction (135 mg) was further purified by semi-preparative reversed-phase HPLC (H2O / CH3CN / 0.1%TFA) to offer aplyzanzine C, aplyzanzine E, and purealidin A. This procedure has been described by Tintillier et al. (Tintillier et al. Marine Drugs. 2020; 18(5):272).

[0041] The freeze-dried sponge sample of Suberea laboutei (185.7 g) was extracted by maceration at room temperature with 3 x 500 mL of a mixture of CH2Cl2 / MeOH (1 :1 ) for respectively 18 and 2 x 2 hours and then by ultrasound assisted extraction (UAE) with 2 x 250 mL of the same solvent for 2 x 30 min. The filtered extracts were combined and dried under reduced pressure to afford a brown residue (13.43 g), which was partitioned between CH2CI2 and H2O. A dry organic extract C (1.9 g) and a precipitate noted P (5.2 g) were obtained after evaporation. The aqueous layer was partitioned with n-BuOH to deliver, after drying, the crude extract B (2.1 g). The organic extract C (1.89 g) was submitted to normal-phase silica-gel flash chromatography with a gradient from 1 :0 to 0:1 mixture of cyclohexane / AcOEt then 1 :0 to 1 :1 mixture of CH2Cl2 / MeOH to afford fractions C-F1 to F33. Only C- F32 (15.3 mg, 20% MeOH) and C-F33 (292.4 mg, 50% MeOH) showed anti-prion activities against yeast prions [PS / +] and [URE3]. Fraction C-F32 (11.25 mg) was purified by semi-preparative reversed-phase HPLC (column: Phenomenex Kinetex EVO C18, 10 x 150 mm, 5 pm, H2O + 0.1% formic acid (FA) / CHsCN + 0.1% FA) to deliver purealidin Q (1.9 mg, 1 ) and aplysamine-2 (1.1 mg, 2).

[0042] The butanolic extract B (1.61 g) was fractionated by reversed-phase C18 flash chromatography with a gradient from 1 :0 to 0:1 mixture of H2O / MeOH then EtOH, CH3CN, AcOEtand finally CH2CI2 to deliver 40 fractions B-F1 to F40. Numerous fractions showed anti-prion activities against [PS / +], but only B-F1 (57 mg, 5%MeOH), B-F3 (50.7 mg, 20% MeOH), B-F21 (21 mg), B- F22 (18.4 mg) and B-F23 (15.3 mg) with 100% MeOH, were active against both [PS / +] and [URE3] yeast prions. B-F1 (57 mg) and B-F3 (20 mg) were purified by semi-preparative reversed-phase HPLC (column: Phenomenex Kinetex EVO C18, 10 x 150 mm, 5 pm, H2O + 0.1% FA / CH3CN + 0.1% FA) to deliver pseudoceratinine A (4.2 mg, 3) and aerophobin-2 (3.3 mg, 4) from B-F3, aplysamine- 1 (1 .9 mg, 5) and pseudoceratinine B (2.6 mg, 6) from B-F1 .

[0043] 1.4. PrPScclearance assay and Immunoblots.

[0044] PrPScclearance assay in MovS6 cells were performed as previously described (Oumata et al. PLoS One. 2013, 8, e72112 ; Nguyen et al. Eur. J. Med. Chem. 2014, 82, 363-371 ; Bamia et al. Neurotherapeutics. 2021 , 18, 1137-1150). Briefly, MovS6 cells were seeded in 6-well plates (Starlab) at a density of 5.5.105cells per well in 3 mL DMEM F12 medium (Gibco, #31331 -028) supplemented with 10% fetal calf serum (Eurobio, #SVF001 -01 ). Cells were treated with a range of concentration of molecules solubilized in absolute ethanol and absolute ethanol was used as negative control. MovS6 cells were than maintained at 37°C in a humidified atmosphere containing 5% CO2. After 6 days of culture, cells were washed with PBS 1X (Gibco, #CS1 PBS501 -01 ) and lysed with 1 mL of TL1 lysis buffer / well (0.5% sodium deoxycholate, 0.5% Triton-X100, 5 mM Tris-HCI pH 7.5). Cell lysates were collected and protein concentration was quantified using the BCA Protein Assay kit (Thermo Fisher Scientific, #23235) according to the supplier's instructions.

[0045] In order to determine whether the amount of PrPScis modified by the treatment with the molecules, cell lysates (250 pg) were digested by 20 pg / mL of Proteinase K (PK, Thermo Fisher Scientific, #EO0491 ). PK digestion was stopped after 30 minutes by the addition of 4 mM of the antiprotease Pefabloc (Sigma Aldrich, #30827-99-7). PK-treated lysates were then centrifuged at 16,000 g for 30 minutes and pellets were solubilized in 15 pL of SB2X buffer (4% SDS, 33% glycerol, 0.1 % bromophenol blue, 1X Tris-glycine pH 8.3, 130 mM DTT). The amount of basal PrPcwas also quantified using 25 pg of untreated proteins in CB1X buffer (5 mM Tris-HCI pH 6.8, 10% glycerol, 2% SDS, 0.005% bromophenol blue, 1.25% p-mercapto-ethanol). PK-treated and untreated cell lysates were denatured for 10 min at 95°C. Proteins were separated in NUPAGE 10% Bis-Tris gels (Thermo Fisher Scientific, #NW00100BGX). The migration was carried out for 1 h at 140 V in 1X MES migration buffer (Invitrogen, #NP0002-02). Proteins were then transferred to a 0.45 pm nitrocellulose membrane (GE Healthcare, #10600002) for 1 hour at 0.5 A in transfer buffer (25 mM Tris-HCI pH 8.3, 192 mM glycine, 20% ethanol, 0.1% SDS). Membranes were then incubated overnight at 4°C with 1 / 40,000 Sha31 primary antibody (Berlin pharma, #A03213) in TBS-T (10 mM Tris-HCI pH 7.5, 150 mM NaCI, 0.05% Tween 20). Membranes were washed 5 times with TBS-T, then incubated with 1 / 3,000 HRP-conjugated anti-mouse antibody (Abeam, #ab6789) for 1 hour at room temperature. Membranes corresponding to proteins not treated with PK were also incubated with 1 / 3,000 anti-tubulin antibody (Abeam, #ab6161 ) as a loading control, followed by an incubation with 1 / 3,000 HRP-conjugated anti-rat antibody (Calbiochem, #401416). After 5 new washes with TBS-T, the immunoreactivity of PK-treated and PK-untreated proteins was revealed bychemiluminescence (SuperSignalTM West Pico Plus Chemiluminescent Substrate #34577, Thermo Fisher Scientific), imaged via the Vilbert Lourmat imaging system and quantified using Image J software.

[0046] 2. Results

[0047] 2.1 . Primary screening of various marine organisms’ extracts against [PS / +1 and [URE31 yeast prions.

[0048] To explore the potential of marine organisms' chemodiversity, the inventors studied herein, organic extracts of tropical invertebrates from French Polynesia and Wallis & Futuna.

[0049] In order to select extracts that contain molecules with anti-prion activity, the inventors used a yeast-based screening method (Bach et al. Nat Biotechnol. 2003, 21 (9):1075-81 ) whose effectiveness has already been widely proven (Oumata et al. PLoS One. 2013, 8, e72112 ; Nguyen etal. Eur. J. Med. Chem. 2014, 82, 363-371 ; Bamia et al. Neurotherapeutics. 2021 , 18, 1137-1150). This two-step assay starts with the screening of crude extracts against [PS / +] yeast prion (primary screening). Extracts able to cure [PS / +] prion are then assayed against a second yeast prion called [URE3] (secondary screening). Extracts displaying an anti-prion activity against both [PS / +] and [URE3] yeast prions are then selected for further analysis. Briefly, [PS / +] or [URE3] yeast cells were spread on Petri dishes containing solid agar-based rich medium. Extracts were loaded onto individual filters placed on top of agar surface and dishes were further incubated for seven to ten days at 25°C. Yeast strains containing [PS / +] or [URE3] prions form white colonies whereas they form red [ps / -] or [ure3-0] colonies when the prion is eliminated. Upon the addition of an extract to a filter, its anti-prion activity is detected when a halo of red colonies appears around the filter. Screening scores were attributed according to Table 1. 131 sponge extracts were first assayed for their capacity to cure [PS / +] yeast prion. From the sponge extracts displaying anti-prion activity against [PS / +], two extracts from Aplysinellidae sponges and one extract from a Pseudoceratinidae sponge were active against both [PS / +] and [URE3] yeast prions (screening scored 3, Table 2).

[0050] Table 1Score [PS / +] [URE3]0 - not tested1 + or + / -2 + or + / - + / -3 + or + / - +

[0051] Table 1. Score of tested samples related to their activity against [PS / +] and [URE3] yeast prions. Extracts or compounds are considered as positive when scored 2 or 3.

[0052] Table 2

[0053] Table 2. Description of screened sponge extracts displaying anti-prion activity. Screening scores were attributed according to Table 1 .1PYF for French Polynesia,2WLF for Wallis & Futuna, Queensland Museum’s operational taxonomic unit.

[0054] 2.2. Bioquided isolation and identification of bioactive compounds from Suberea laboutei

[0055] The freeze-dried sponge Suberea laboutei was extracted with a mixture of CFECh / MeOH (1 / 1 ). The resulted crude extract was partitioned between CH2CI2 and water to give an organic extract C, a precipitate P and an aqueous layer. The aqueous layer was further partitioned against n-butanol to furnish the crude butanolic extract B. Bioguided fractionation of the extracts, using normal-phase and / or reversed-phase chromatography led to the isolation of the known compounds: purealidin Q (1 ) and aplysamine-2 (2) from extract C, pseudoceratinine A (3), aerophobin-2 (4), aplysamine-1 (5) and pseudoceratinine B (6) from extract B (Figure 1 ), identified after extensive spectroscopic analyses and comparison with data from the literature. To the best of the inventors’ knowledge, metabolites from the Suberea laboutei species are reported here for the first time.

[0056] The six molecules (1-6) were first tested against [PS / +] and [URE3] yeast prions. Purealidine Q (1 ), aplysamine-2 (2), pseudoceratinine A (3), aplysamine-1 (5) and pseudoceratinine B (6) were able to cure both [PS / +] and [URE3] prions (scored 2 or 3) whereas aerophobin-2 (4) only cured [PS / +] (scored 1 ). Based on these results, molecules 1 , 2, 3, 5 and 6 were further tested for their ability to decrease the propagation of the mammalian prion protein PrPScin a cell-based assay using chronically prion-infected MovS6 cells (Archer et al. J. Virol. 2004, 78, 482-490). MovS6 is a murine peripheral neuroglial cell line expressing the ovine PRNP gene under the control of its endogenous promoter. These cells are chronically infected by the 127S sheep scrapie prion strain (Archer et al. J. Virol. 2004, 78, 482-490; Vilotte et al. J. Virol. 2001 , 75, 5977-5984) and allow rapid testing of molecules against mammalian prions. After 6 days of treatment of prion-infected MovS6 cells with bromotyrosine derivatives 1-6, PrPScwas detected on the basis of its proteinase K (PK) resistance. For this purpose, a fraction of cell lysates was treated by PK to discriminate PrPScproteins that are partially resistant to PK treatment from total PrP proteins (PrPtot). PK-treated and -untreated cell lysates were separated by SDS-PAGE and immunostained using specific anti-PrP antibodies. Purealidin Q (1 ), aplysamine-2 (2) and aplysamine-1 (5) were able to reduce PrPScpropagation with IC50 of 3.3, 5.5 and 6 pM, respectively.

[0057] Pseudoceratinine A (3) and pseudoceratinine B (6) showed no activity against PrPScand aerophobin-2 (4), that has been used as a negative control as it was inactive against [PS / +] and [URE3] yeast prions, was not active either. Purealidine Q (1 ), aplysamine-2 (2) and aplysamine-1 (5), were previously described as active against both [PS / +] and [URE3] yeast prions (Jennings et al. Prion. 2018, 12(3-4):234-244). The inventors have here confirmed these three bromotyrosinederivatives are potent anti-prion compounds as the inventors showed they are also able to reduce the propagation of the mammalian prion PrPScin vitro.

[0058] 2.3 Anti-prion activity of bromotyrosine derivatives from sponges of the order Veronqiida

[0059] The inventors identified two bromotyrosine derivatives with anti-prion activity among the six molecules purified from Suberea laboutei extract (P562-WLF04). Concerning the samples of Suberea ianthelliformis (P102-MNH2) and Pseudoceratina sp (2081) (P281-TRAR04), which also showed anti-prion activity (Table 2), their previously isolated bromotyrosine and bromophenol derivatives were directly selected for screening. Among the molecules tested were the aplyzanzines C (7), D (8), F (9), purealidin A (10) from Pseudoceratina sp (2081), the psammaplysenes D (11 ), F (12), G (13) and anomoian F (14) from Suberea ianthelliformis (Figure 2).The inventors tested the capacity of these 8 additional bromotyrosine derivatives (Figure 2) against [PS / +] and found five active molecules (7, 10, 11 , 12, 14) which were also active against [URE3]. Molecules 8, 9 and 13 were negative against [PS / +] prion. The inventors have demonstrated that when challenged against PrPSc, aplyzanzine C (7), psammaplysene D (11 ) and anomoian F (14) were able to reduce PrPScpropagation in a dose-dependent manner, with IC50 of 4.6, 0.2 and 1 pM, respectively. Purealidin A (10) and psammaplysene F (12) were not able to reduce PrPScpropagation. To the inventors’ knowledge, this is the first report of the anti-prion activity of aplyzanzine C, psammaplysene D and anomoian F. Lately, the anti-prion activity of psammaplysene A, a close analogue of psammaplysene D, has indirectly been found through an arrayed genome-wide RNA interference (RNAi) screening aimed at identifying cellular host-factors that can modify prion propagation (Avar et al. The EMBO Journal. 2022, 47(23)). This screening identified the heterogeneous nuclear ribonucleoprotein Hnrnpk as a prominent limiter of prion propagation. Psammaplysene A, which binds Hnrnpk (Boccitto M et al. Mar. Drugs. 2017; 15(8):246), has been further shown to reduce PrPScprion levels in prion-infected cells and cerebellar organotypic slices and to alleviate locomotor deficits in prion-infected Drosophila expressing ovine PrPc(Avar et a / . The EMBO Journal. 2022, 47(23)). The inventors of the present invention have demonstrated that psammaplysene A is active against [PS / +] and [URE3] yeast prions and displays an activity against PrPScprion (IC50 = 0.3 pM).

Claims

Claims

1. Bromotyrosine compound of formula (I):a pharmaceutically acceptable salt or a solvate thereof, or salt of a solvate thereof, wherein:R3= H, N(CH3)2. for use in treating a protein misfolding disease.

2. Bromotyrosine compound for use according to claim 1 wherein the compound isa pharmaceutically acceptable salt or a solvate thereof, or salt of a solvate thereof.

3. Bromotyrosine compound for use according to claim 1 wherein the compound isa pharmaceutically acceptable salt or a solvate thereof, or salt of a solvate thereof.

4. Bromotyrosine compound for use according to claim 1 wherein the compound isa pharmaceutically acceptable salt or a solvate thereof, or salt of a solvate thereof.

5. Bromotyrosine compound for use according to any one of the preceding claims wherein the protein misfolding disease is a prion disease.

6. Bromotyrosine compound for use according to claim 5 wherein the prion disease is selected from the list consisting of Creutzfeldt-Jakob disease, Variant CJD, variably proteasesensitive prionopathy, Gerstmann-Straussler-Scheinker disease, Fatal familial insomnia, Kuru, prion disease associated with diarrhea and autonomic neuropathy bovine spongiform encephalopathy, chronic wasting disease and scrapie and camel prion disease in non-human mammals.

7. Bromotyrosine compound for use according to any one of claims 1 to 4 wherein the protein misfolding disease is selected from neurodegenerative, neuromuscular, muscular, metabolic and neurodevelopmental protein misfolding diseases.

8. Bromotyrosine compound for use according to claim 7 wherein the protein misfolding disease is selected from Alzheimer's disease, Parkinson's disease, Huntington disease, amyotrophic lateral sclerosis, oculopharyngeal muscular dystrophy (OPMD), inclusion body myositis (IBM), Duchenne muscular dystrophy (DMD), type II diabetes and Partington syndrome / X-linked intellectual disabilities (XLID) linked to the dup24 duplication in the ARX (aristaless related homeobox) gene.

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