Small molecule targeting of fused in sarcoma (FUS) and uses thereof

Abstract

Provided is a method of modulating FUS and treating a FUS-associated neurodegenerative disease or disorder with the small molecule MC16.

Description

[0001] PCT APPLICATION 112746-111725 (P25015)

[0002] SMALL MOLECULE TARGETING OF FUSED IN SARCOMA (FUS) AND USES THEREOF

[0003] FIELD OF THE INVENTION

[0004] The invention relates to a first-in-class small molecule targeting RNA binding protein Fused in Sarcoma (FUS) implicated in rare and challenging diseases. The mechanism of action inhibits FUS’s toxic gain of function in the cytoplasm and increases its protective function in the nucleus.

[0005] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes and to the same extent as if each individual publication, patent, patent application or other reference was specifically and individually indicated to be incorporated by reference in its entirety' for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present invention.

[0006] BACKGROUND OF THE INVENTION

[0007] Fused in Sarcoma (FUS), also known as translated in liposarcoma (TLS), is a ubiquitously expressed nuclear RNA / DNA binding protein with diverse roles in DNA repair and RNA metabolism (Renton et al., 2014). Extensively studied in amyotrophic lateral sclerosis (ALS), where FUS mutations contribute to a subset of severe cases (ALS-FUS), dysregulation of wild-type FUS has emerged as a contributing factor in a broader spectrum of neurodegenerative and neuropsychiatric disorders. In addition to frontotemporal dementia (FTD). FUS has been implicated in essential tremor, early-onset atypical Parkinsonism, and. in some reports, certain psychiatric disorders. In the majority of ALS-FUS, multiple pathogenic variants can drive aberrant cytoplasmic accumulation, seeding insoluble aggregates that both deplete nuclear FUS activity and exert toxic gain-of-function in the cytoplasm and mitochondria (Deng et al.. 2015; Zhang et al., 2021). In sporadic ALS, FTD, and a diverse set of neurodegenerative diseases, wildtype FUS is also enriched in non-nuclear fractions, with the level of mislocalization correlated with disease severity7, suggesting that imbalances in FUS distribution may reflect or contribute to disease (Shelkovnikova et al., 2014).

[0008] 1

[0009] 3234573.1 PCT APPLICATION 112746-111725 (P25015)

[0010] The broad disease relevance reflects the involvement of FUS in numerous physiological processes, a consequence of its largely intrinsically disordered structure and modular functional domains. The 526-amino acid human FUS protein comprises five key functional domains: an N- terminal prion- like domain rich in glutamine, glycine, serine, and tyrosine residues (QGSY region), an RNA recognition motif (RRM), three RGG (arginine- glycineglycine) repeat domains implicated in RNA binding via a C2 / C2 zinc finger motif, and non- classical nuclear localization signal (PY-NLS) located in the extreme C-terminal region. These domains enable FUS to interact with a wide range of RNA and protein partners, mediating its involvement in critical cellular processes such as transcriptional regulation, pre- mRNA transport and stability, DNA repair, and microRNA biogenesis. FUS C-terminal domain regulate its localization. Under physiological conditions, FUS predominantly resides in the nucleus, where it is imported by interaction with transporting proteins and the PY -NLS domain (Lagier et al., 2010). While mutations in the PY-NLS domain disrupt FUS nuclear import, cellular stress can result in nuclear egress, both mechanisms ultimately result in aberrant cytoplasmic accumulation of FUS- a pathological hallmark associated with toxicity in ALS and related neurodegenerative diseases. Although FUS contains a predicted nuclear export sequence within its RRM domain, nuclear exit is primarily driven by passive diffusion, with nuclear retention largely mediated by RNA binding to the RRM and RGG domains. Both mechanisms, lack of import and increased egress ultimately result in aberrant accumulation of FUS in the cytoplasm, contributing to its toxic gain-of-function effects in ALS and related disorders. While pathogenic FUS mislocalization is well documented, and the molecular mechanisms regulating its localization, solubility, and RNA-binding dynamics are somewhat understood, it is unclear how to apply this knowledge to rationally design a therapeutic.

[0011] A need exists in the art to modulate FUS to treat neurodegenerative diseases and disorders such as ALS.

[0012] SUMMARY OF THE INVENTION

[0013] In an embodiment of the invention, provided is a method of modulating FUS, comprising the step of administering MCI 6, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

[0014] 2

[0015] 3234573.1 PCT APPLICATION 112746-111725 (P25015)

[0016] In another embodiment of the invention, provided is a method of treating ALS, comprising the step of administering MCI 6, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

[0017] DETAILED DESCRIPTION OF THE INVENTION

[0018] Under normal conditions, FUS resides predominately in the nucleus, where it regulates gene expression by modulating transcription, pre-mRNA splicing, and DNA damage repair to maintain genomic integrity (Lagier et al., 2010). FUS regularly cycles between the nucleus and cytoplasm, however, cellular stress or pathological states are associated with decreased nuclear localization. Here, the inventors observed that MCI 6 increases FUS in nuclear fractions and decreases its cytoplasmic localization for both wildtype and C-terminal variants. The data suggests that this effect is due to interactions with the RNA recognition motif (RRM), a central domain exhibiting stable structure in the largely intrinsically disordered FUS protein, serving as an unsurprising target for small molecule interactions. Interactions with this region may prevent nuclear egress and / or increase nuclear import to either reduce cytotoxicity or improve nuclear function.

[0019] Although containing a predicted nuclear export sequence within the RRM domain, FUS egress out of the nucleus is regulated by passive diffusion rather than active or facilitated transport (Ederle et al., 2018). On the other hand, nuclear import is mediated by karyopherin- family interactions with the C-terminal PY-type nuclear localization signal (PY-NLS) and adjacent arginine-rich (RGG) segments. FUS variants most relevant to neurodegenerative phenotypes, and investigated in this study, are located in the C-terminus, resulting in reduced nuclear import and increased cytoplasmic aggregation. Indeed, P525L and R495X mutants retain partial localization to the nucleus, despite lacking a functional nuclear localization sequence, a process mediated by interactions between RGG regions and Karyopherin-P2 (Gonzalez et al 2021). In full-length FUS, the RRM dynamically interacts with neighboring disordered RGG regions, modulating interactions with other macromolecules (Bonucci et al., 2020). This autoinhibitory interplay might regulate overall conformational sampling, with conformations that expose RGG or C-terminal domains more likely to allow for nuclear import.

[0020] 3

[0021] 3234573.1 PCT APPLICATION 112746-111725 (P25015)

[0022] The RRM domain, along with adjacent RGG / ZnF motifs, enables FUS to bind a broad array of RNA sequences. In the nucleus, nucleic acid interactions are required for FUS to exert its physiological functions. Because FUS nuclear export is passive, it would therefore be reduced through binding large RNA molecules (z.e., physiological processes retain normal cellular distribution of FUS). In the cytoplasm, FUS can seed stress granules, a process that requires RNA binding to the RRM / RGG regions and reduces nuclear imports. For example, disrupting key aromatic residues in the RRM (F305 / 341 / 359 / 368L) increases nuclear FUS and blocks neurodegenerative phenotypes in Drosophila ALS models with C-terminal mutations, whereas RNA-binding competent mutant FUS redistributes to the cytoplasm and seeds stress granules (Daigle et al., 2013). Likewise, mutant FUS recruits wildtype copies into stress granules via RNA interactions (Wang et al., 2015). Interestingly, while FUS has low nucleic acid-binding specificity (Mariani et al., 2024), the composition of RNA inclusions is altered in mutant stress granules, and these alterations can be linked to transcriptomic compositions characteristic of neurodegeneration.

[0023] While stress granules themselves often exert protective functions through coalition and sequestration of several macromolecules, maturation to irreversible aggregates is linked to FUS toxicity. The RRM domain has been implicated in converting phase-separated condensates into irreversible amyloid-like fibril aggregates (Lu et al., 2017). Previous in-vivo studies demonstrate that the RRM domain is required for manifesting FUS cytotoxicity (Sun et al., 2011), underscoring that RRM-mediated interactions (with nucleic acids or other partners) are essential for FUS-driven neurodegeneration. Indeed, ATP binds at physiologically relevant concentrations and inhibits RRM fibrilization required for gain of toxic function in FUS-related diseases.

[0024] Taken together, FUS RRM interactions have important functional consequences: FUS may be retained in the nucleus or in stress granules based on its interaction with RNA molecules and RGG regions through its RRM domain; and irreversible unfolding of the metastable RRM domain, reduced by ligand binding, is required for FUS toxicity. The inventors believe that targeting the RRM could suppress FUS toxicity by preventing aberrant RRM-RNA interactions, and / or by stabilizing the RRM to reduce fibrillar aggregation. A small molecule binding the RRM is likely to sterically occlude RNA-binding surfaces and / or allosterically

[0025] 4

[0026] 3234573.1 PCT APPLICATION

[0027] 112746-111725 (P25015) stabilize the domain, thereby reducing the propensity for FUS to form toxic inclusions, and maintain FUS in a soluble, import-competent state (Daigle et al., 2013).

[0028] The Drosophila eye is a sensitive and genetically tractable system but powerful model to investigate neurodevelopmental toxicity associated with ALS-linked protein like FUS, as it integrates developmental signaling and cellular stress responses. Its development depends on well-orchestrated signaling pathways, including EGFR, Notch, Hedgehog and Wingless (Wg / Wnt) which regulates ommatidial arrangement, pigmentation and cell survival (Malartre, 2016). These pathways are particularly vulnerable to oxidative stress and mitochondrial dysfunction, both of which are induced by mutant forms of FUS during development (Malartre, 2016; Shimamura et al., 2014). The drosophila compound eye can be targeted specifically using the GAL4 / UAS system, driving eye specific delivery' of GLA4 under the GMR promoter coupled with the GAL4 specific promoter USA driving FUS expression. Using this system, it has been shown that flies expressing mutant human FUS (R518K, R521C, and R521H) under GMR-Gal4 in the eye show severe neurodegeneration. The eye phenotypes include disorganized ommatidia and bristle loss also called the rough eye phenotype, as well as loss of eye depigmentation. The eye phenotypes of the WT human FUS were less severe.

[0029] Here, the inventors took advantage of a small molecule developed based on a pharmacophore identified through a Chembridge 50.000 compound library screen designed to screen for compounds that protect stressed cells against cell death in a mitochondria-dependent manner. It was shown to promote mitochondrial biogenesis and alter levels of mitochondrial- dynamics related proteins enhancing mitochondrial respiratory capacity and protecting against energy failure in in primary kidney tubular cells. In vivo, 1 mg / kg of MCI 6 delivered intraperitoneally was found to accelerate renal recovery, reduce vascular permeability, and mitigate fibrosis in models of acute kidney injury and diabetic nephropathy, supporting its systemic mitochondrial protective effects [28, 34], An unpublished click-chemistry based pulldown approach using cell lysates from multiple mouse and human cell lines identified FUS and some of its binding partners as a potential target of MCI 6. To confirm direct binding between FUS and MC16, the inventors performed MST assays using purified FUS and MC16 only, which confirmed the direct interaction of FUS with the ordered RRM domain of FUS.

[0030] 5

[0031] 3234573.1 PCT APPLICATION

[0032] 112746-111725 (P25015)

[0033] After confirming MCI 6 as a direct binding partner of FUS, and to query functional consequences of MC16 treatment on properties of FUS, drosophila as well as multiple cell lines all expressing wild type and mutant human FUS with C-terminal mutations were examined.

[0034] Expression of wild ty pe and mutant human FUS (R518K and R521C) under the GMR-Gal4 driver led to pronounced defects in eye morphology’ and pigmentation. These phenotypes are likely due to disruption of critical eye patterning pathways, reflect interference with development signaling, which are sensitive to oxidative stress and redox imbalance during development. Prior studies have shown that mutant FUS mislocalizes to the cytoplasm and mitochondria, where it impairs mitochondrial dynamics, elevates ROS production and disrupts transcriptional and RNA-binding functions critical for tissue homeostasis (Wang et al., 2019; Machamer et al., 2014). MC 16 treatment was found to increase eye pigmentation in a dose-dependent manner and improve bristle morphology7. Improved morphology might be due to improved mitochondrial structure and function, leading to a restoration of the ROS balance, ultimately preserving downstream signaling required for eye patterning and pigment cell survival (Chen et al., 2011). For example, pigment-producing cells rely on redoxsensitive transcriptional regulators like Mitf (Microphthalmia-associated transcription factor) and Pnt (Pntl, a member of drosophilia Pnt / Nek8 family) which are downstream of EGFR and are vulnerable to oxidative imbalance during eye development (Wang & Montell, 2007; Cook et al., 2009). However, additional experiments are needed to further investigate the potential mechanism.

[0035] As FUS is primarily located in the cell nucleus and the ALS-associated mutations in FUS affecting the nuclear localization signal, lead to FUS accumulation in the cytoplasm, the inventors assessed whether the partial rescue of the fly phenotype is correlated with the nuclear to cytoplasmic FUS ratio. MC16 reduces cytoplasmic levels of mutant R518K and R521C FUS in drosophila, and concomitantly increased nuclear levels. WT FUS levels exhibited a trend towards normalization.

[0036] Further examination of mechanism was performed in transiently and stably transfected cell lines. Specifically, the inventors showed that in an epithelial cell line (B2B) stably transfected

[0037] 6

[0038] 3234573.1 PCT APPLICATION

[0039] 112746-111725 (P25015) with doxycycline-inducible wildtype and mutant FUS (R495X and R521 G) tagged with GFP, that MC16 reduces cytoplasmic levels of mutant R495X and R521C FUS, and concomitantly increased nuclear levels, improving the nuclear to cytoplasmic ratio. WT FUS levels were not affected. Interestingly, in addition to mutant GFP -tagged FUS, endogenous FUS moved in parallel. In mutant FUS expressing cells, irrespective of the mutation, MC16 reduces cytoplasmic levels of endogenous FUS and concomitantly increased nuclear levels. Whereas no effect on endogenous FUS was observed in cells overexpressing WT GFP-tagged FUS.

[0040] This parallel movement of mutant exogenous and wildtype endogenous FUS led us to further explore the potential mechanisms whereby which MCI 6 might restore FUS balance. As MCI 6 has been shown to increase ATP production and proeostasis heavily depends on ATP as the ubiquitin-proteasome system and autophagy / lysosome pathways are ATP-dependent, the inventors asked whether a reduction in cytoplasmic FUS levels might be proteasome dependent. However, blocking the proteasome with the highly specific inhibitor MG132 did not alter cytoplasmic FUS protein levels when compared to MC16+MG132 treatment, supporting the notion that MCI 6 treatment did increase general or specific protein degradation. Many RNA binding proteins, including FUS, are retained in the nucleus by bound nascent pre-mRNA and mature mRNAs. FUS in particular has been shown to bind its own pre-mRNA, and loss of this regulation leads to increased FUS expression and mislocalization (Zhou et al., 2013). In ARPE-19 cells, the inventors showed that endogenous FUS protein binds increased amounts of FUS mRNA in the presence of MC16, as upon FUS immunoprecipitation, increased levels of FUS mRNA could be pulled down that could be amplified by QRT-PCR. This autoregulatory loop might contribute to a long-term reduction of cytotoxic cytoplasmic FUS accumulation. Finally, the parallel movement of mutant and endogenous FUS suggests that the two proteins have the ability to dimerize in response to MCI 6 binding, or by interactions involving additional protein partners, facilitated by MCI 6. Co-immunoprecipitation using GFP-antibody coated agarose beads resulted in increased pulldown of endogenous FUS in the presence of MC 16. As the inventors showed that MC16 binds uniquely to the RRM domain, which in addition to RNA can also bind protein, it is plausible that MCI 6 might contribute to FUS protein dimerization or complex formation with other RNA-binding proteins. This dimerization with a wildtype FUS protein, which under physiological conditions is imported into the nucleus via a proline-tyrosine nuclear localization signal (PY-NLS) located at its C-terminus might facilitate import of the FUS

[0041] 7

[0042] 3234573.1 PCT APPLICATION 112746-111725 (P25015) heteromer. The PY-NLS motif is specifically recognized by transportin- 1 (TNPO1), a member of the Karyopherin-beta family, which facilitates nuclear import of several RNA- binding proteins (Neumann et al., 2012). Various studies have shown that TNPO1 can act as a chaperone-like factor, suppressing FUS phase separation but its effectiveness is influenced by the conformational state of FUS and post-translational modifications, particularly in the RGG domains (Hofweber et al., 2018; Yoshizawa et al., 2018). Taken together, MC16 might affect the FUS nuclear to cytoplasmic ratio in various ways, in particular by retaining FUS in the nucleus by increasing FUS-mRNA complex formation, and by FUS heterodimer formation, increasing FUS transport via the functional endogenous FUS PY-NLS motif.

[0043] Finally, in neuronal N2a cells transiently transfected with wildtype and mutant FUS-GFP plasmids, treated over night with MC16, cytoplasmic levels of R495X-GFP and R521G were both reduced. Specifically, in R495X-GFP cells, about 50% of the signal is present in the cytoplasm, and MC16 reduces that by about half, whereas in R521G-GFP cells about 25% of the signal is in the cytoplasm, and MC16 restores it to WT level. Niu and colleagues (Niu et al., 2012) and others established the correlation between affinity of FUS to TNPO1, localization of FUS, and disease severity or lifespan after diagnosis. Specifically, a variant of FUS (R521L), that like R495X protein does not bind to TNPO1 results in a lifespan of about 13 months after their diagnosis. In contrast, R521G patients live about 2-3 times longer. The imaging data shows that based on localization of FUS MC16 treatment can convert a R495X cell to a R525G cell. If localization of FUS is a predictor of lifespan after diagnosis, this suggests that MCI 6 has the potential to extend life expectancy in ALS patients.

[0044] Collectively, the results provide mechanistic insight into how MCI 6 confers neuroprotective effects in cells and in vivo. By reducing cytoplasmic FUS accumulation and enhancing its nuclear retention, MC16 addresses a central pathological feature of FUS-associated ALS, offering a promising strategy for targeted therapeutic development.

[0045] Thus, FUS is involved in various RNA processes, including transcription, alternative splicing, and mRNA transport. It binds to both single-stranded and double-stranded DNA, promoting RNA polymerase II transcription and pre-mRNA splicing, which is essential for proper gene expression and cellular function.

[0046] 8

[0047] 3234573.1 PCT APPLICATION 112746-111725 (P25015)

[0048] FUS is crucial for DNA repair mechanisms. It participates in the response to DNA damage, particularly in homologous recombination and the repair of double-strand breaks, thereby maintaining genomic stability. Mutations in FUS have been associated with mitochondrial dysfunction, which is often observed in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS). FUS influences mitochondrial dynamics, including fission and fusion processes, and affects mitochondrial DNA repair and replication.

[0049] Studies have shown that FUS mutations can lead to disrupted mitochondrial metabolism, increased oxidative stress, and altered energy production in cells, contributing to the pathophysiology of metabolic and neurodegenerative disorders. FUS is directly implicated in the pathogenesis of ALS, where mutations in the FUS gene lead to the formation of mislocalized FUS protein aggregates in motor neurons. These aggregates disrupt RNA metabolism and cellular stress responses, contributing to neuronal death. FUS pathologies (i.e., mislocalization) are associated with both familial and sporadic ALS cases, highlighting the importance of RNA-binding proteins in ALS pathophysiology'.

[0050] The inventors have found that the compound MC I 6 as shown below binds to FUS protein in the RNA binding domain (RRM) with a nanomolar Kd using nuclear magnetic resonance imaging and microscale thermophoresis. The specific amino acids required for binding were identified.

[0051] MC16 can be synthesized and obtained by the methods outlined in US 10,370,328. which is expressly incorporated herein by reference. The structure of MCI 6 is shown below:

[0052] 9

[0053] 3234573.1 PCT APPLICATION 112746-111725 (P25015)

[0054] Transcriptional regulation: FUS binds its own mRNA in the RRM domain. MCI 6 was found to increase mRNA binding to the FUS protein, leading to increased FUS mRNA transcription. Also, MCI 6 regulated gene and protein expression of genes involved in various cellular processes, in particular mitochondrial biogenesis leading to increased mitochondrial function.

[0055] Mitochondrial dysfunction: Under stress conditions, FUS is associated with mitochondria via its interaction with Hsp60. leading to a reduction in electron transport chain function and mitochondrial fission. MCI 6 was found to decrease FUS binding to mitochondria via Hsp60 either through pretreatment prior to stress or post-treatment, resulting in improved mitochondrial dynamics.

[0056] Stress granule formation: FUS is associated with stress granules, which are formed in response to various cellular stresses like oxidative stress and heat shock. MCI 6 was found to decrease FUS association with granules.

[0057] Mutant FUS: ALS and FTD are associated with mutant FUS. The majority of FUS mutations leading to disease are in the nuclear translocation signal in the C-terminus. Cytoplasmic localization of FUS protein with four independent mutations can be reversed or partially reversed resulting in increased localization to the nucleus and less in the cytoplasm in the presence of MCI 6.

[0058] A “subject” is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate, such as a monkey, chimpanzee, baboon or rhesus, and the terms “subject” and “patient” are used interchangeably herein.

[0059] 10

[0060] 3234573.1 PCT APPLICATION 112746-111725 (P25015)

[0061] The invention also includes pharmaceutical compositions comprising an effective amount of the a FUS modulator of the invention and a pharmaceutically acceptable carrier. The invention includes the FUS modulator of the invention provided as a pharmaceutically acceptable prodrug, hydrate, salt, such as a pharmaceutically acceptable salt, enantiomers, stereoisomers, or mixtures thereof.

[0062] Representative “pharmaceutically acceptable salts'’ include, e.g., water-soluble and waterinsoluble salts, such as the acetate, amsonate (4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate, benzonate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium, calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, gly colly larsanilate. hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate. iodide, isothionate, lactate, lactobionate. laurate, magnesium, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, JV-methylglucamine ammonium salt, 3-hydroxy-2- naphthoate, oleate, oxalate, palmitate, pamoate (l.l-methene-bis-2-hydroxy-3-naphthoate, einbonate). pantothenate, phosphate / diphosphate, picrate, polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate, sulfosalicylate, suramate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate salts.

[0063] The term “carrier,” as used in this disclosure, encompasses carriers, excipients, and diluents and means a material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a pharmaceutical agent from one organ, or portion of the body, to another organ, or portion of the body.

[0064] The term “treating,” with regard to a subject, refers to improving at least one symptom of the subject's disorder. Treating can be curing, improving, or at least partially ameliorating the disorder.

[0065] The term “disorder” is used in this disclosure to mean, and is used interchangeably with, the terms disease, condition, or illness, unless otherwise indicated.

[0066] The term “administer.” “administering.” or “administration” as used in this disclosure refers to either directly administering a compound or pharmaceutically acceptable salt of the

[0067] 11

[0068] 3234573.1 PCT APPLICATION

[0069] 112746-111725 (P25015) compound or a composition to a subject, or administering a prodrug derivative or analog of the compound or pharmaceutically acceptable salt of the compound or composition to the subject, which can form an equivalent amount of active compound within the subject’s body.

[0070] In one embodiment, the FUS modulators of the invention can each be administered in amounts that are sufficient to treat ALS. Administration of the FUS modulators of the invention can be accomplished via any mode of administration for therapeutic agents. These modes include systemic or local administration such as oral, nasal, parenteral (intravenous), intramuscular, intrathecal, intra-vitreal, transdermal, subcutaneous, vaginal, buccal, rectal, topical administration modes or as a drug-eluting stent.

[0071] Depending on the intended mode of administration, the compositions can be in solid, semisolid or liquid dosage form, such as, by way of non-limiting examples, injectables, tablets, suppositories, pills, time-release capsules, elixirs, tinctures, emulsions, syrups, powders, liquids, suspensions, nanoparticles or the like, sometimes in unit dosages and consistent with conventional pharmaceutical practices. Likewise, they can also be administered in intravenous (non-limiting examples include bolus and infusion), intraperitoneal, intrathecal, intra-vitreal injection, subcutaneous or intramuscular form, all using forms well known to those skilled in the pharmaceutical arts.

[0072] Non-limiting illustrative pharmaceutical compositions are tablets and gelatin capsules comprising the FUS modulators of the invention and a pharmaceutically acceptable carrier, such as: a) a diluent, e.g, purified water, triglyceride oils, such as hydrogenated or partially hydrogenated vegetable oil, or mixtures thereof, com oil, olive oil, sunflower oil, safflower oil, fish oils, such as EPA or DHA, or their esters or triglycerides or mixtures thereof, omega- 3 fatty acids or derivatives thereof, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, sodium, saccharin, glucose and / or glycine; b) a lubricant, e.g., silica, talcum, stearic acid, its magnesium or calcium salt, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and / or polyethylene glycol; for tablets also; c) a binder, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, magnesium carbonate, natural sugars such as glucose or beta-lactose, com sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, waxes and / or polyvinylpyrrolidone, if desired; d) a disintegrant, e.g., starches, agar, methyl cellulose, bentonite, xanthan gum, alginic acid or its sodium salt, or

[0073] 12

[0074] 3234573.1 PCT APPLICATION 112746-111725 (P25015) effervescent mixtures; e) absorbent, colorant, flavorant and sweetener; f) an emulsifier or dispersing agent, such as Tween 80, Labrasol, HPMC, DOSS, caproyl 909, labrafac, labrafil, peceol, transcutol, capmul MCM, capmul PG-12, captex 355, gelucire, vitamin E TGPS or other acceptable emulsifier; and / or g) an agent that enhances absorption of the compound such as cyclodextrin, hydroxypropyl-cyclodextrin, PEG400, PEG200.

[0075] Liquid, particularly injectable compositions can, for example, be prepared by dissolution, dispersion, etc. For example, the FUS modulators of the invention are dissolved in or mixed with a pharmaceutically acceptable solvent such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form an injectable isotonic solution or suspension. Proteins such as albumin, chylomicron particles, or serum proteins can be used to solubilize the FUS modulators of the invention.

[0076] The FUS modulators of the invention can be also formulated as a suppository that can be prepared from fatty7emulsions or suspensions; using polyalkylene glycols such as propylene glycol, as the carrier.

[0077] In further embodiments, the pharmaceutical formulations described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations

[0078] The FUS modulators of the invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, containing cholesterol, stearylamine or phosphatidylcholines. In some embodiments, a fdm of lipid components is hydrated with an aqueous solution of drug to a form lipid layer encapsulating the drug, as described in United States Patent No. 5,262,564, the contents of which are herein incorporated by reference in their entirety.

[0079] Parenteral injectable administration is generally used for subcutaneous, intramuscular or intravenous injections and infusions. Injectables can be prepared in conventional forms.

[0080] 13

[0081] 3234573.1 PCT APPLICATION

[0082] 112746-111725 (P25015) either as liquid solutions or suspensions or solid forms suitable for dissolving in liquid prior to injection.

[0083] Compositions can be prepared according to conventional mixing, granulating or coating methods, respectively, and the present pharmaceutical compositions can contain from about 0. 1 % to about 80 %, from about 5 % to about 60 %, or from about 1 % to about 20 % of the FUS modulators of the invention by weight or volume.

[0084] A “therapeutically effective amount” when used in connection with the FUS modulators of the invention is an amount effective for treating or preventing a FUS-associated disease or disorder such as ALS. The dosage regimen utilizing the FUS modulators of the invention is selected in accordance with a variety' of factors including type, species, age, weight, sex. race, diet, concomitant medications, and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal or hepatic function of the patient; and the particular the FUS modulators of the invention employed. A physician or veterinarian of ordinary skill in the art can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.

[0085] Therapeutically effective amounts of the present invention, when used for the indicated effects, range from about 0.1 mg to about 5000 mg of the active ingredient per unit dose which could be administered. In one embodiment, the compositions are in the form of a tablet that can be scored. Appropriate dosages of the FUS modulators of the invention can be determined as set forth in Goodman, L. S.; Gilman, A. The Pharmacological Basis of Therapeutics, 5th ed.; MacMillan: New York, 1975, pp. 201-226, the contents of which are hereby incorporated by reference.

[0086] The FUS modulators of the invention can also be administered in a single daily dose, or the total daily dosage can be administered in divided doses of two, three or four times daily. Furthermore, the FUS modulators of the invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery’ system, the dosage administration can be continuous rather than intermittent throughout the dosage regimen. Other illustrative topical preparations include creams, ointments, lotions, aerosol sprays and gels, wherein the concentration of the FUS modulators of the invention ranges from about 0. 1 % to about 15 %, w / w or w / v.

[0087] 14

[0088] 3234573.1 PCT APPLICATION 112746-111725 (P25015)

[0089] EXAMPLES

[0090] The disclosure is further illustrated by the following examples, which are not to be construed as limiting this disclosure in scope or spirit to the specific procedures herein described. It is to be understood that the examples are provided to illustrate certain embodiments and that no limitation to the scope of the disclosure is intended thereby. It is to be further understood that resort may be had to various other embodiments, modifications, and equivalents thereof which may suggest themselves to those skilled in the art without departing from the spirit of the present disclosure and / or scope of the appended claims.

[0091] The inventors examined cytoplasmic mislocalization of wildtype, R495X, R518K and R521C / G FUS and assessed whether MC16 could restore proper FUS localization and mitigate its toxic effects. Biophysical characterization through microscale thermophoresis demonstrated that MC16 binds directly to purified FUS RNA-recognition motif (RRM), confirming target engagement and providing the dissociation constant. Imaging of mouse neuroblastoma N2a cells, as well as western blot analysis of human B2B epithelial cell and Drosophila melanogaster extracts showed that MCI 6 decreased FUS in cytoplasmic fractions while increasing its nuclear accumulation in both wild-type and mutant forms. In flies expressing mutant FUS using the GAL4 / UAS binary' system, driving gene expression in the developing eye (GRM), MCI 6 improved ommatidial regularity and arrangement, as shown by digital stereomicroscopy and scanning electron microscopy, and restored eye pigmentation as measured by absorbance measurements in head extracts. Together, these data suggest that MC16 directly engages FUS to correct its subcellular distribution and alleviate cellular and organismal pathology, highlighting MCI 6 as a potential therapeutic modulator of FUS in neurodegenerative disease such as ALS and FTD.

[0092] Example 1

[0093] Synthesis of MC-16

[0094] The synthesis of MC16 is described, starting from the commercially available 5-chloro-isatin and 3-methoxy-2hydroxybenzaldehyde. A list of reagents and representative HPLC traces of all intermediates is included.

[0095] Synthesis:

[0096] 15

[0097] 3234573.1 PCT APPLICATION

[0098] 112746-111725 (P25015)

[0099] List of reagents, grades and vendors

[0100] 16

[0101] 3234573.1 PCT APPLICATION 112746-111725 (P25015)

[0102] 5-chloro-l-ethylindoline-2, 3-dione. 5-chloroisatin (18.16 g, 100 mmol) and potassium carbonate (17.95 g, 130 mmol) was added to a round bottom flask that was stored in the oven at 150 °C for at least one hour, then cooled under argon, equipped with a stir bar. The mixture was taken up in anhydrous DMF (500 mL). While stirring at room temperature, iodoethane (10.4 mL, 130 mmol) was added. The reaction continued to stir overnight at room temperature. The next day an LCMS was taken to confirm reaction completion. Once the reaction was completed, the solvent was removed using a rotary evaporator. The residue was then taken up in methyl-tert-butyl ether (MTBE), and celite was added (approximately 30 grams). The mixture was then filtered through a plug of celite. The plug was then rinsed with MTBE until the solvent ran clear. The organic material was then concentrated and used without further purification.

[0103] 5-chloro-l-ethyl-3-hydroxyindolin-2-one. Tetrahydrofuran (120 mL) was added to a flask charged with the alkylated isatin (5 g, 23.85 mmol) and a stir bar. The stirring solution was then cooled to 0 °C. While stirring at this temperature, a solution of sodium borohydride

[0104] 17

[0105] 3234573.1 PCT APPLICATION 112746-111725 (P25015)

[0106] (993 mg, 26.24 mmol, 0.67 M in deionized water) was added. After two minutes, the reaction was quenched using 1 M hydrochloric acid (100 mL). The mixture was salted out using sodium chloride, and the organic layer removed. The aqueous layer was washed twice with ethyl acetate. The combined organic material was dried with sodium sulfate, filtered, and concentrated. The crude material was used for the next step without any further purification.

[0107] 5-chloro-l-ethyl-2-oxoindolin-3-yl dimethylcarbamate. Carbonyl diimidazole (5.8 g, 35.78 mmol). 4-dimethylaminopyridine (291 mg. 2.38 mmol) and anhydrous tetrahydrofuran (240 mL, stored over 4 angstrom molecular sieves) was added to a round bottom flask, charged with the reduced isatin (23.85 mmol) equipped with a stir bar. The reaction stirred at room temperature for 2 hours. A solution of dimethylamine (6 mL, 40% in water) was then added. The reaction continued to stir for one hour. The solution was then diluted with water and salted out using sodium chloride. The aqueous layer was washed two times with ethyl acetate, and the organic material combined. The organic material was then washed with brine, dried with sodium sulfate, filtered and concentrated. tert-butyl (2-formyl-6-methoxyphenyl) carbonate. 2-hydroxy-3-methoxybenzaldehyde (1.853 grams, 12 mmol) and 4-dimethylaminopyridine (73.3 grams, 0.6 mmol) was added to a round bottom flask that was stored in the oven at 150 °C for at least one hour, then cooled under argon, equipped with a stir bar. A solution of di-tert-butyl dicarbonate (12.6 mL. 12.6 mmol, 1 M in THF) was added. The reaction continued to stir until there was no more observable starting material by HPLC. The reaction was then concentrated and used without further purification. tert-butyl (2-(hydroxymethyl)-6-methoxyphenyl) carbonate. Tetrahydro furan (45.0 mL) was added to a clean flask equipped with a stir bar, charged with the aldehyde. The stirring solution was cooled to 0 °C. While stirring at this temperature, a solution of sodium borohydride (0.5 grams, 13.2 mmol, IM in deionized water) was added. After 30 seconds, the reaction was quenched with 1 M hydrochloric acid (60 mL). The solution was salted out using sodium chlonde, and the organic material separated. The aqueous layer was washed twice using ethyl acetate. The combined organic material was dried using sodium sulfate, filtered, and concentrated. The material was then used without further purification.

[0108] 18

[0109] 3234573.1 PCT APPLICATION

[0110] 112746-111725 (P25015)

[0111] 5-chloro-l-ethyl-3-(2-hydroxy-3-methoxybenzyl)-2-oxoindolin-3-yl dimethylcarbamate.

[0112] Anhydrous toluene (48 mL) was added to a clean flask charged with the carbamate (24 mmol). While stirring at room temperature, a solution of lithium bis(trimethylsilyl)amide (29 mL, 29 mmol, 1 M solution in ethyl benzene and THF) was added. After ten minutes of stirring at room temperature, a solution of the alcohol (12 mmol, 0.3 M in THF) was added. The reaction continued to stir at room temperature for 2 hours. Once complete by HPLC, the reaction was quenched using 1 M HC1 (36 mL). The solution was salted out using sodium chloride, and the organic material separated. The aqueous layer was washed twice using ethyl acetate. The combined organic material was dried using sodium sulfate, filtered, and concentrated. The residue was then taken up in ethanol and placed in a 4 °C refrigerator where it sat overnight. The next day, a precipitate was filtered off. This affords MCI 6 in -83% purity. Greater purification was accomplished using a Teledyne ISCO Combiflash under reverse phase conditions to afford >98% pure MC16 in approximately 35% yield from 5-chloro-isatin.

[0113] Example 2 Biological Examples

[0114] Materials and Methods

[0115] Protein expression and purification

[0116] Wildtype human FUS RRM domain (UniProt ID: P35637; residues 285-371) cDNA was cloned into a pET-28a(+) vector purchased from GenScript® containing an N-terminal 6xHis tag and kanamycin resistance gene. The expression construct was transformed into E. coli strain BL21 (LEMO21-DE3) by heat shock. Transformed colonies were selected by growth in the presence of kanamycin and expanded at 37°C in 2X M9 minimal media. Expression was induced with 0.4 mM IPTG at an OD600 of 0.7, and bacteria were grown overnight at 20°C. Cells were harvested by centrifugation at 5,000xg for 18 min, lysed by sonication in low-imidazole binding buffer (200 mM NaCl, 20 mM HEPES, 20 mM imidazole, 1 mM EGTA, 1 mM NaNs. 1 mM MgCh, pH 7.3) supplemented with 1 p.M PMSF, and cleared by centrifugation at 14,000xg for 45 min. The lysate was loaded on a HisTrap FF column (Cytiva) in low-imidazole binding buffer and the targeted protein was eluted in high- imidazole buffer (500 mM imidazole, 50 mM Tris-base, 300 mM NaCl, pH 8.0). The eluted protein was further purified on a size-exclusion Superdex HiLoad 16 / 600 75 pg (Cytiva)

[0117] 19

[0118] 3234573.1 PCT APPLICATION 112746-111725 (P25015) column in storage buffer (100 mM NaCl, 20 mM MES, 10 mM DTT, 5 mM CaCh, 0.02% NaNs, pH 6.5). Protein purity was analyzed by gel electrophoresis.

[0119] Microscale thermophoresis

[0120] Microscale thermophoresis (MST) was performed at 25°C using auto-detect Pico Red in the NanoTemper Monolith NT.115 Pico instrument. 6xHis::RRM was labeled by Red-tris-NTA 2ndgeneration dye per manufacturer instructions (NanoTemper Technologies GmbH). The reaction mixture was centrifuged for 10 min at 15,000xg, and 16 two-fold dilution series were performed for MC16 (22 nM - 730 pM). in which DMSO (25%) and protein (160 nM) concentrations were held constant. To reduce adsorption, the assay buffer (5 mM sodium phosphate, 10 mM NaCl, pH 6.0) was supplemented with 0.5% Tween-20, and Monolith NT.115 premium capillaries were used. To derive KD values, binding curves were fitted using the PALMIST (v 1.5.1) analysis software using the T-Jump preset and a 1: 1 binding mode (Scheuermann et al., 2016). Traces were removed if they exhibited irregular fluorescence profiles or failed to yield fully bound 68% confidence intervals, estimated by bootstrapping (5,000 iterations). Plots were generated in GUSSI (v 2.1.6).

[0121] Cytoplasmic and Nuclear Fractionation

[0122] Cytoplasmic and nuclear extraction from B2B and N2a cells. Cells were harvested by tr psmization and centrifuged at 500xg for 5 minutes. The pellet was washed once with ice- cold PBS, and approximately 2x106cells were resuspended in ice-cold cytoplasmic an extraction reagent I (CER I). After vortexing for 15 seconds, samples were incubated on ice for 10 minutes, followed by the addition of CER II. The mixture was vortexed for 5 seconds, incubated on ice for 1 minute, then vortexed again for 5 seconds before centrifugation at 16,00xg for 5 minutes at 4°C. The resulting supernatant (cytoplasmic fraction) was transferred to a clean tube. The pellet (nuclear fraction) was resuspended in ice-cold nuclear extraction reagent (NER) and vortexed every 10 minutes, for a total of 40 minutes, while on ice. After a final centrifugation at 16,000xg for 10 minutes, the nuclear extract was collected.

[0123] Cytoplasmic and nuclear extraction from Drosophila. Approximately 30 adult Drosophila heads were collected, washed once with ice-cold PBS and homogenized on ice in CER I using a Dounce tissue homogenizer. After 10 minutes of incubation on ice, CER II was added. Sample were briefly vortexed and kept on ice for 1 minute, followed by centrifugation

[0124] 20

[0125] 3234573.1 PCT APPLICATION 112746-111725 (P25015) at 16,000xg for 5 minutes. The cytoplasmic supernatant was carefully collected. The nuclear pellet was resuspended in NER, vortexed every’ 10 minutes over 40 minutes on ice, and centrifuged at 16000xg for 10 minutes to obtain the nuclear fraction.

[0126] Western blotting

[0127] Western blotting was performed using lysates obtained from cytoplasmic and nuclear fractions of Drosophila melanogaster heads as well as B2B and N2a. All lysates were denatured at 95°C for 5 minutes in Laemmli buffer (Bio-Rad) and separated on 4-20% TGX precast gradient gels (Bio-Rad). Proteins were transferred to 0.45 pm PVDF membrane (Amersham™ Hybond) using standard semi-dty transfer conditions. Membranes were blocked in 5% non-fat dry milk prepared in TBS-T for 1 hours at room temperature, followed by overnight incubation at 4°C with mouse anti-FUS / TLS monoclonal antibody (4H11, Santa Cruz Biotechnology, sc-47711) at a 1 :2000 dilution. After three washes with TBST, membranes were incubated with HRP-conjugated anti-mouse IgG secondary antibody (Cell signaling technology, #7076) at a 1:3000 dilution for 1 hour at room temperature. Signal detection was carried out using enhanced chemiluminescence (ECL) (Bio-Rad. #32106).

[0128] To verify the integrity and enrichment of the subcellular fraction, membranes were stripped and re-probed with rabbit anti-HSP90 for cytoplasmic fractions, and rabbit anti-Histone H3 for nuclear fraction, each used at a 1 :2000 dilution. After incubation with appropriate HRP- conjugated secondary antibodies, blot was developed using the same ECL detection protocol. Blot images in TIF format were quantified using the ImageJ gel analyzer tool. All Western blots were performed on three independent biological replicates for each sample type.

[0129] Co-immunoprecipitation (Co-Ip)

[0130] Co-immunoprecipitation was performed to examine the interaction between wildtype and mutant FUS. The whole-cell lysate was prepared in RIPA buffer supplemented with protease cocktail inhibitor and clarified by centrifugation at 20,000xg for 15 minutes at 4°C. The ly sate was incubated overnight at 4°C with rabbit anti-GFP antibody, followed by incubation with Protein A agarose beads for 2-4 hours at 4°C. Beads were washed thoroughly with RIPA buffer to eliminate non-specific binding. Protein complexes were eluted by boiling the beads in Laemmli buffer and resolved by SDS-PAGE. Western blotting was performed using anti-

[0131] 21

[0132] 3234573.1 PCT APPLICATION 112746-111725 (P25015)

[0133] FUS antibody (4H1 1 , Santa Cruz Biotechnology, sc-47711 ) to assess dimer formation between wildtype and mutant FUS.

[0134] N2A cells for FUS imaging and quantification

[0135] Cells: Neuro-2a (N2A) cells were initially procured from the American Type Culture Collection (ATCC, CCL-131). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma- Aldrich, D5796) supplemented with 10% fetal bovine serum (FBS, Genesee Scientific, 25-550), penicillin (100 lU / mL) and streptomycin (100 pg / mL) (Sigma-Aldrich, P4333). Cells were maintained under standard cell culture conditions of 37°C in a humidified incubator with a 5% CO2 environment.

[0136] Transfection: Plasmids were transfected into N2A cells at 2 pg per well in a 6-well plate using 4 pL of JetPRIME® transfection reagent (VWR. 89129-924), following the manufacturer’s instructions. Forty-eight hours post-transfection, the cells were treated with indicated concentration of MCI 6 overnight. The plasmids used for transfection including pEGFP-3C-hFUS-WT, pEGFP-3C-hFUS-R495X, and pEGFP-3C-hFUS-R521G.

[0137] Immunofluorescence microscopy and Image analysis: Cells grown on coverslips were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 10 minutes. Nuclei were stained with Hoechst 33342 (Fisher Scientific, PI62249), and cells were mounted using Invitrogen ProLong Glass Antifade Mountant (Fisher Scientific, P36984). Imaging was performed using a Nikon NIS-Elements AR live-cell microscope equipped with a 60 x oil objective.

[0138] Image analysis was conducted using ImageJ-win64 software. Briefly, image color channels were split, and green fluorescence intensity (GFP-FUS) was measured in both the total cell area (visible) and the nuclear area (blue channel). The nuclear-to-total intensity' ratio was then calculated. For each treatment, at least 20 individual cells were analyzed.

[0139] Drosophila lines, Crosses and drug treatment

[0140] The FUS-WT, FUS-R518K and FUS-R521C transgenic Drosophila lines were obtained from the University of Pittsburgh. These lines had been generated by site specific insertion of the transgene into genetic background using standard transgenesis techniques. All drosophila

[0141] 22

[0142] 3234573.1 PCT APPLICATION 112746-111725 (P25015) stocks were maintained on standard Bloomington medium at 25 °C under light / dark controlled incubator conditions.

[0143] To induce transgene expression specifically in the eye, virgin female flies from the FUS-WT, FUS-518K and FUS-521C lines were crossed with GMR-GAL4 driver flies. Each cross was established in an individual vial containing 4-5 virgin female and an equal number of GMR- GAL4 males. The vials were kept at 28 °C on instant Drosophila food supplemented with MC16 of three different concentrations: 10, 30 and 60 pg / ml. This set ensured consistent exposure to M-1 throughout the development of the Fl generation. Luciferase flies were used as control flies and crossed with the GMR-GAL4 driver to compare the effects of MCI 6 in a non-FUS expressing background.

[0144] Eve Phenotype analysis: For eye phenotype evaluation, 2-day old CO2 anesthetized Fl flies were examined using a Leica stereomicroscope and images were captured with a Leica digital camera. A total of 20 to 50 flies per genoty pe and condition were evaluated.

[0145] Scanning electron microscopy: Scanning electron microscopy (SEM) of adult Drosophila melanogaster eyes was performed using a modified version of the protocol described by Koon et al., 2019. Briefly, 10-30 flies per condition were anesthetized by CO2 and fixed in 2.5% glutaraldehyde (0.1 M phosphate buffer, pH 7.2) at 4°C overnight. To ensure complete submersion, 2.5% polyethylene glycol tert-octyphenyl ether was added when necessary. Fixed samples were washed four times in phosphate buffer and dehydrated through a graded ethanol series (25% to 100%, for 10 min each). Ethanol was gradually replaced with hexamethyldisilazane (HMDS): 1:2 and 2: 1 HMDS: Ethanol (20 min each), followed by two 100% HMDS incubation (20 min each). Samples were transferred to glass dishes, covered with fresh HMDS, and air dried under a vacuum desiccator for 12-24 hours. Dried flies were sputter coated with gold at ~5.0 xl0'2torr prior to imaging (Denton Vacuum, USA). Sputter coated samples were imaged using the Hitachi S-3700N Scanning Electron Microscope. I was performed with secondary electron detector at an accelerating voltage of 4-5kV and working distance of 5-6 mm. Images were acquired at 250x magnification, focusing on capturing the overall compound eye morphology and all the images were saved in high resolution.

[0146] 23

[0147] 3234573.1 PCT APPLICATION 112746-111725 (P25015)

[0148] Index of Regularity (IREG) by using FLEYE PLUG calculator

[0149] To quantify ommatidial organization in Drosophila eyes, high resolution images acquired using digital stereomicroscopy were analyzed with the FLEYE plugin for FIJI (2.14.0 Jave 1.8.0-322, 32 bit). This tool computes an index of regularity (IREG) score based on eye morphology, classifying images into five degeneration categories (PP0-PP4). These classes correspond to increasing severity of eye disorganization: PPO (green) highly regular, PPI (blue) mild disorganization, PP2 (yellow) moderate disorganization, PP3 (orange) high disorganization, and PP4 (red) severe degeneration. Each eye receives a probability distribution across these classes, visualized as stacked bars in the plot. The IREG value is calculated as a weighted average of these probabilities, with lower score reflecting greater degeneration. These values were used for genotype and treatment.

[0150] Pigmentation assay

[0151] Pigmentation was quantified by extracting eye pigments from adult Drosophila melanogaster . Briefly, 30 adult fly heads per genotype were collected and homogenized in 400 pl acidified methanol (0.1% HC1 in methanol). The homogenates were vortexed for 30 minutes at room temperature to facilitate pigment extraction. Following incubation, samples were centrifuged at 13000xg for 10 minutes at room temperature. To enhance pigment clarify, 20 pl of 0.5% hydrogen peroxide (H2O2) was added to the supernatant. After a brief vortex and additional centrifugation, the clarified supernatant was transferred to a 96 well plate. Absorbance was measured at 480 nm using a microplate reader (Synergy Hl). Pigment intensity w as normalized to the number of fly heads per sample. Each condition was analyzed in three independent biological replicates.

[0152] Statistical analysis

[0153] All data are presented as mean ± standard error of the mean (SEM) from at least three independent biological replicates unless otherwise stated. Statistical comparisons between two groups were performed using unpaired two-tailed student’s t-test. A p-value less than 0.05 was considered statistically significant. Graphs and analyses were generated using GraphPad Prism. Significance was indicated as follows: *p<0.05, **p<0.001, ***p<0.0001.

[0154] RESULTS

[0155] MC-16 Directly Interacts with FUS RRM Domain

[0156] 24

[0157] 3234573.1 PCT APPLICATION 112746-111725 (P25015)

[0158] Although FUS was previously identified as a likely protein target of MC I 6 through click chemi stry and pull-down assays, these studies did not demonstrate direct binding. In the largely intrinsically disordered protein, only the RRM domain exhibits stable structure, complicating assessments of direct interactions, as they are likely to be transient. Here, the inventors provide new evidence of physical interaction between FUS and MC16 by using microscale thermophoresis (MST) supporting a direct association. MST was performed with purified 6xHis::RRM (residues 285-371) and MC16. As FUS-MC16 interactions could likely occur in two different cellular compartments (cytoplasm and nucleus) with different ionic composition. MST was performed at both cytoplasmic NaCl (150 mM) and nuclear levels (50 mM), yielding a KD value of 500 + / 0 X nM (68.3% Confidence Interval [300, 900]) and 140 + / - 40 nM for the two conditions. Similar values w ere obtained with full-length FUS (data not shown).

[0159] MC16 treatment mitigates cytoplasmic mislocalization and promotes partial restoration of nuclear localization of ALS- and FTD-associated FUS mutant in N2a cells using imaging

[0160] Fluorescence imaging of N2a cells transiently expressing wild-type or ALS- or FTD- associated FUS mutants (R495X, R521G) tagged with GFP were imaged. The two FUS mutants show significant cytoplasmic mislocalization of the GFP signal. After overnight treatment with MC16 (100 nM-10 pM), nuclear FUS localization was significantly increased. Specifically, FUS-GFP fluorescence imaging showed that wild-type FUS remains confined to the nucleus, while the R495X and R521G mutant FUS levels were markedly reduced in the cytoplasm in both mutants. Quantification of cytoplasmic-to-total fluorescence in 30 cells per condition revealed that cytoplasmic FUS in R495X-expressing cells dropped from -50% to -25%, while in R521G cells it was reduced from -25% to nearly 0% upon MCI 6 treatment, and a dose-dependent effect was established. This study was followed up examining a dose response curve around the efficacious dose, using lOx steps, with 30 cells per population, 3 doses showing a dose related effect.

[0161] MC16 treatment mitigates cytoplasmic mislocalization and promotes partial restoration of nuclear localization of ALS- and FTD-associated FUS mutant in BEAS-2B Cells (B2B Cells)

[0162] 25

[0163] 3234573.1 PCT APPLICATION 112746-111725 (P25015)

[0164] To quantify the effect of MCI 6 on the subcellular distribution of FUS protein, the inventors analyzed the cytoplasmic expression levels of wild-type (WT) and ALS- and FTD-associated mutant FUS (R495X and R521G) in B2B cells. These cells were chosen for the quantitative analysis, as they are a stable cell line using a doxycycline-inducible promoter to activate FUS expression. In addition, since the exogenous FUS proteins are GFP tagged, the effects of MC16 can be examined for both endogenous (~75 kDa) and exogenous FUS (-100 kDa).

[0165] Western blot analysis of the cytoplasmic fractions showed that overexpression of mutant as well as wildtype FUS leads to significant retention of FUS proteins in the cytoplasm. In the presence of MCI 6, a notable reduction in cytoplasmic FUS-GFP was observed for both mutant constructs when compared to the untreated, vehicle condition. The reduction in cytoplasmic exogenous FUS-GFP was paralleled by a reduction in wildtype endogenous FUS for both mutant lines. In the cell line expressing exogenous WT FUS-GFP, both exogenous and endogenous FUS levels remained unchanged upon MCI 6 treatment, indicating that the compound selectively affects the aberrant localization of the mutant forms.

[0166] To assess whether MCI 6 promotes corresponding nuclear retention or import into the nucleus of FUS, the nuclear fraction of the cells was analyzed. Comparing vehicle with MC16 treatment conditions, both R495X and R521C mutant FUS showed a notable increase in nuclear FUS levels in the presence of MC16. As for the concomitant change in cytoplasmic levels for exogenous mutant and endogenous wildtype FUS in the presence of MCI 6, the increase in nuclear exogenous FUS-GFP was paralleled by an increase in wildtype endogenous FUS for both mutant lines. No significant change was observed in WT-FUS levels with MC-16.

[0167] When expressing the obtained data as nuclear to cytoplasmic ratio following MCI 6 treatment, a significant increase was observed, consistent with enhanced nuclear retention of FUS. Notably, the R495X mutant displayed a larger shift in response to MC16 when compared to the milder mutation of R521G.

[0168] These results indicate that MCI 6 effectively enhances nuclear localization of FUS, particularly in disease-associated mutants, most likely by translocating together with wildtype endogenous FUS, suggesting its potential as a therapeutic modulator of FUS mislocalization.

[0169] 26

[0170] 3234573.1 PCT APPLICATION

[0171] 112746-111725 (P25015)

[0172] MC-16 reduces FUS-mediated neurodegeneration in the Drosophila eye

[0173] To evaluate the effect of MC16 on a FUS mediated toxicity in vivo, the inventors employed a Drosophila ocular degeneration model by expressing human FUS and its disease associated mutants in the developing compound eye using the GMR-GAL4 driver system. When assessing the eye phenoty pes based on SEM and digital microscopy images, expression of wild type FUS led to a moderate disruption in eye morphology7characterized by a moderate irregularity in ommatidial organization. FUS mutants R518K and R521C resulted in a more severe degenerative phenotype, with pronounced roughness, disorganized or missing ommatidia, and partial collapse of the compound eye with loss of pigmentation, consistent with enhanced toxicity7, confirming results previously reported published by Lanson et al., (2011).

[0174] SEM imaging revealed distinct differences in the ommatidial arrangement and surface architecture across genotypes and treatment conditions. The Luciferase (control) eyes exhibited a highly regular and well-organized ommatidial array with distinct inter-ommatidial bristles and a smooth surface, indicative of normal eye morphology. In contrast, eyes expressing FUS WT, R518K and R521C displayed moderate to severe disruption of ommatidial patterning with significant surface roughness, fused ommatidia, and loss of defined inter-ommatidial boundaries, consistent with a rough or collapsed eye phenotype. Treatment with 30 and 60 ug / mL MC-16 partially prevented the degenerative phenotypes across the three genotypes.

[0175] To quantify the eye phenoty pe, the high resolution images acquired using digital stereomicroscopy were analyzed with the FLEYE plugin for FIJI. Without treatment, the WT-FUS eyes exhibited a score that consisted in about equal proportions of PP2 (yellow) moderate disorganization, PP3 (orange) high disorganization, and PP4 (red) severe degeneration ommatidia. In contrast, both R518K and R521C FUS flies scored as consisting of -20% PP3 (orange) high disorganization, and 80% PP4 (red) severe degeneration ommatidia. Upon treatment with increasing doses of MCI 6, the scores shifted in all three groups to in favor of a healthier pattern. In WT FUS eyes, most of the severely degenerated ommatidia (PP4, red) were avoided, and in R518K and R521C FUS flies the ratio of PP3:PP4 shifted to 0.5. When analyzing the regularity index, which is calculated as a weighted

[0176] 27

[0177] 3234573.1 PCT APPLICATION 112746-111725 (P25015) average of the PP0-PP4 probabilities, showed a significant increase for the R518K and R521C FUS flies and a trend for the WT FUS flies.

[0178] To quantitatively assess the protective effect of MC-16 on FUS induced pigment loss, pigment was quantified by measuring absorbance at 480 nm from homogenized fly heads. Flies expressing luciferase exhibited robust pigmentation, whereas expression of human FUS wild type (WT) or the disease associated mutants R518K and R521C led to a significant reduction in pigmentation to -15% of wildlype level, consistent with degeneration and pigment cell loss (Lanson et al.. 201 1). Treatment with an increasing concentration of MC-16 (10, 30, and 60 pg / mL) led to a significant, dose dependent increase of pigmentation by -100% in all FUS-expressing lines. Together, these findings demonstrate that MC16 reduces FUS-mediated neurotoxicity in vivo by partially restoring pigmentation and improving eye morphology, highlighting its potential as therapeutic modulator of FUS toxicity.

[0179] MC16 treatment reduced cytoplasmic accumulation and enhances nuclear localization of FUS in drosophila

[0180] To investigate whether the molecular effect of MC16 that is underlying the phenotypic rescue in drosophila is the same as in B2B cells, Western blot analysis was performed on cytoplasmic and nuclear extracts from Drosophila melanogaster expressing human FUS wildtype (WT), R518K and R521C mutant proteins in the presence and absence of MC16.

[0181] Representative blots show consistent expression of HSP90 (cytoplasmic loading control) across all conditions. In both R518K and R521C mutant flies, cytoplasmic FUS levels dropped with MCI 6 treatment, reaching significance in the two highest doses, while only a trend was observed for WT-FUS flies. Correspondingly. MC-16 treatment led to a dosedependent increase in nuclear FUS levels in flies expressing mutant FUS, reaching significance by the highest dose investigated. For the wildtype FUS flies, nuclear FUS levels showed a trend for increase. These findings suggest that MC 16 might reduce the cytoplasmic burden of toxic FUS mutant in drosophila which may underlie its protective effect on eye morphology.

[0182] Potential mechanisms improving the FUS nuclear / cytoplasmic ratio

[0183] 28

[0184] 3234573.1 PCT APPLICATION 112746-111725 (P25015)

[0185] Improving FUS nuclear to cytoplasmic ratio could potentially be achieved in multiple ways. MC16 could increase FUS protein degradation in the nucleus; retain FUS in the nucleus and prevent egress or increase nuclear transport.

[0186] To exclude the possibility that MCI 6 triggers protein degradation, cytoplasmic FUS levels were examined in cells treated with the proteasome inhibitor MG132. As expected, cytoplasmic FUS protein levels increased in the presence of the MG132, but did not alter the MC16+MG132 treatment to the level produced by MG132 alone, supporting that MCI 6 treatment did not cause degradation of cytoplasmic FUS.

[0187] Retention of FUS in the nucleus can be regulated by mRNA binding. As mentioned in the Introduction, FUS exhibits multiple RNA-binding domains, including the RRM and the RGG domains. It is hypothesized that binding of FUS to specific RNAs or mRNAs in the nucleus may stabilize its nuclear localization, possibly by masking nuclear export signals, or by anchoring FUS to chromatin or nuclear substructures. To investigate specific mRNA binding to FUS protein, ARPE-19 cells treated with MC16 were lysed and used for immunoprecipitation for FUS protein, followed by QRT-PCR for FUS mRNA. MCI 6 treatment increased FUS mRNA levels bound to FUS protein 7-fold, while maintaining normal levels of FUS.

[0188] The combined results, demonstrating parallel movement of endogenous wildtype and exogenous mutant FUS from the cytoplasm to the nucleus suggests that wildtype and mutant FUS heterodimerize in the presence of MC I 6 and potentially utilize the nuclear localization signal of wildtype FUS for nuclear transport. Immunoprecipitation of FUS-GFP using agarose beads pre-coated with anti-GFP antibodies pulled down FUS-GFP and endogenous wildtype-FUS. MC16 increased the apparent heterodimerization of the two proteins.

[0189] Taken together, the mutant FUS nuclear to cytoplasmic ratio is presumably achieved by increasing FUS-mRNA interaction, retaining FUS in the nucleus together with increased nuclear transport of mutant FUS binding to wildtype FUS, utilizing the nuclear translocation signal of wildtype FUS. Overall, this results in a loss of toxic gain of function in the cytoplasm and a gain of protective function in the nucleus.

[0190] 29

[0191] 3234573.1 PCT APPLICATION 112746-111725 (P25015)

[0192] Accordingly, the evidence supporting MCI 6 as a uniquely capable therapeutic for ALS and FTD is compelling. The preclinical development of MCI 6 is grounded in established scientific methodologies and validated ALS and FTD model systems. The pathological role of FUS and the mechanism by which MC16 reverses FUS-associated pathology have been rigorously defined and consistently reproduced across multiple in vitro cell line models. Furthermore, the in vivo demonstration of FUS pathology7reversal in Drosophila, a widely employed model for testing functional consequences of ALS- or FTD-causing genes, provides substantial translational support for the therapeutic potential of MCI 6.

[0193] Collectively, these findings provide mechanistic insight into the neuroprotective activity of MC16 in both cellular and in vivo models. By decreasing cytoplasmic FUS accumulation and promoting its nuclear localization, MCI 6 directly mitigates a central pathological hallmark of FUS-associated ALS and FTD. thereby representing a well-founded strategy for targeted therapeutic development.

[0194] REFERENCES

[0195] 1) Wang, T.; Cheng, J.; Wang, S.: Wang, X.; Jiang, H.; Yang, Y.; Wang, Y.; Zhang, C.; Liang, W.; Feng, H. a-Lipoic acid attenuates oxidative stress and neurotoxicity via the ERK / Akt-dependent pathway in the mutant hSODl related Drosophila model and the NSC34 cell line of amyotrophic lateral sclerosis. Brain Res. Bull. 2018. 140, 299- 310.

[0196] 2) Hofmann JW, Seeley WW, Huang EJ. 2018. RNA binding proteins and the pathogenesis of frontotemporal lobar degeneration. Annu Rev Pathol 14: 469- 495.doi: 10. 1146 / annurev-pathmechdis-012418-012955

[0197] 3) Tsai, Y.L., Coady. T.H.. Lu, L., Zheng, D., Alland, L. Tian, B., Shneider, N.A. and Manley, J.L., 2020. ALS / FTD-associated protein FUS induces mitochondrial dysfunction by preferentially sequestering respiratory chain complex mRNAs. Genes & Development, 34(11-12), pp.785-805.

[0198] 4) Dormann, D., Rodde, R., Edbauer, D., Bentmann, E., Fischer, L, Hruscha, A., Than, M.E.. Mackenzie. I.R., Capell, A.. Schmid, B. and Neumann. M., 2010. ALS-associated fused in sarcoma (FUS) mutations disrupt Transportin-mediated nuclear import. The EMBO journal, 29(16), pp.2841-2857.

[0199] 5) Wang, T., Jiao, Y. and Montell, C., 2007. Dissection of the pathway required for generation of vitamin A and for Drosophila phototransduction. Journal of Cell Biology, 177(2), pp.305-316.

[0200] 6) Chen, Y., Yang, M., Deng, J., Chen, X., Ye, Y., Zhu, L., Liu, J., Ye, H., Shen, Y., Li, Y. and Rao, E.J., 2011. Expression of human FUS protein in Drosophila leads to progressive neurodegeneration. Protein & cell, 2(6). pp.477-486.

[0201] 7) Shimamura, M., Kyotani, A., Azuma, Y., Yoshida, H., Nguyen, T.B., Mizuta, L, Yoshida, T., Mizuno, T.. Nakagawa, M., Tokuda, T. and Yamaguchi, M., 2014.

[0202] 30

[0203] 3234573.1 PCT APPLICATION

[0204] 112746-111725 (P25015)

[0205] Genetic link between Cabeza, a Drosophila homologue of Fused in Sarcoma (FUS), and the EGFR signaling pathway. Experimental cell research, 326(1), pp.36-45.) Malartre, M.. 2016. Regulatory mechanisms of EGFR signalling during Drosophila eye development. Cellular and Molecular Life Sciences, 73(9), pp. 1825-1843. ) Tsai, Y.L., Mu. Y.C. and Manley, J.L., 2022. Nuclear RNA transcript levels modulate nucleocytoplasmic distribution of ALS / FTD-associated protein FUS. Scientific reports, 12(1), p.8180. 0) Scheuermann, T.H., Padrick, S.B., Gardner, K.H. and Brautigam, C.A., 2016. On the acquisition and analysis of microscale thermophoresis data. Analytical biochemistry, 496, pp.79-93. l) Lagier-Tourenne, C., Polymenidou, M. and Cleveland, D.W., 2010. TDP-43 and FUS / TLS: emerging roles in RNA processing and neurodegeneration. Human molecular genetics , 79(R1), pp.R46-R64. 2) Ederle. H.. Funk, C., Abou-Ajram. C., Hutten, S., Funk. E.B., Kehlenbach, R.H.. Bailer, S.M. and Dormann, D., 2018. Nuclear egress of TDP-43 and FUS occurs independently of Exportin- 1 / CRM 1. Scientific reports, 3(1), p.7084. 3) Gonzalez, A., Mannen, T., Cagatay. T., Fujiwara, A., Matsumura, H., Niesman, A.B., Brautigam, C.A., Chook, Y.M. and Yoshizawa, T., 2021. Mechanism of karyopherin- P2 binding and nuclear import of ALS variants FUS (P525L) and FUS (R495X). Scientific reports, 11(1), p.3754. 4) Bonucci, A., Murrali, M.G., Banci, L. and Pierattelli, R., 2020. A combined NMR and EPR investigation on the effect of the disordered RGG regions in the structure and the activity of the RRM domain of FUS. Scientific Reports, 10(1), p.20956. 5) Daigle, J.G., Lanson Jr, N.A., Smith, R.B., Casci, I., Maltare, A., Monaghan, J., Nichols, C.D., Kryndushkin, D., Shewmaker, F. and Pandey, U.B., 2013. RNA- binding ability of FUS regulates neurodegeneration, cytoplasmic mislocalization and incorporation into stress granules associated with FUS carrying ALS-linked mutations. Human molecular genetics, 22(6), pp.l 193-1205. 6) Wang, X., Schwartz, J.C. and Cech, T.R., 2015. Nucleic acid-binding specificity’ of human FUS protein. Nucleic acids research. 43(15), pp.7535-7543. 7) Mariani, D., Setti, A., Castagnetti, F., Vitiello, E., Stufera Mecarelli, L., Di Timoteo, G., Giuliani, A., D'Angelo, A., Santini. T., Perego, E. and Zappone, S.. 2024. ALS- associated FUS mutation reshapes the RNA and protein composition of stress granules. Nucleic Acids Research, 52(21), pp. 13269-13289. 8) Lanson Jr, N.A., Maltare, A., King, H., Smith, R.. Kim, J.H., Taylor, J.P., Lloyd, T.E. and Pandey, U.B., 2011. A Drosophila model of FUS-related neurodegeneration reveals genetic interaction between FUS and TDP-43. Human molecular genetics, 20(13), pp.2510-2523. 9) Layalle, S., They, L., Ourghani, S., Raoul, C. and Soustelle, L., 2021. Amyotrophic lateral sclerosis genes in Drosophila melanogaster. International Journal of Molecular Sciences, 22(2), p.904 0) Renton, A.E., Chid, A. and Traynor, B.J., 2014. State of play in amyotrophic lateral sclerosis genetics. Nature neuroscience, 17(1), pp. 17-23. 1)Zhang, Y., Gu, J. and Sun, Q., 2021. Aberrant stress granule dynamics and aggrephagy in ALS pathogenesis. Cells, 10(9), p.2247.

[0206] 31

[0207] 3234573.1 PCT APPLICATION

[0208] 112746-111725 (P25015)) Shelkovnikova, T.A., Robinson, H.K., Troakes, C ., Ninkina, N. and Buchman, V.L., 2014. Compromised paraspeckle formation as a pathogenic factor in FUSopathies. Human molecular genetics, 23(9), pp.2298-2312. ) Chen. Y.: Yang, M.; Deng, J.; Chen. X.; Ye, Y.; Zhu. L.; Liu. J.; Ye. H.; Shen. Y.: Li. Y.; et al. Expression of human FUS protein in Drosophila leads to progressive neurodegeneration. Protein Cell 2011, 2, 477-486. )Boeynaems, S.; Bogaert, E.; Michiels, E.; Gijselinck, L: Sieben, A.; Jovicic. A.; de Baets, G.; Scheveneels, W.; Steyaert, J.; Cuijt, I.; et al. Drosophila screen connects nuclear transport genes to DPR pathology in C9ALS / FTD. Sci. Rep. 2016, 6. 20877.)Ederle, H., Funk, C., Abou-Ajram. C., Hutten, S., Funk, E.B., Kehlenbach, R.H., Bailer, S.M. and Dormann, D , 2018. Nuclear egress of TDP-43 and FUS occurs independently of Exportin-l / CRMl. Scientific reports, 5(1), p.7084. ) Tsai, Y.L., Mu, Y.C. and Manley, J.L., 2022. Nuclear RNA transcript levels modulate nucleocytoplasmic distribution of ALS / FTD-associated protein FUS. Scientific reports, 12()), p.8180. ) Scheuermann, T H., Padrick, S B., Gardner, K.H. and Brautigam, C.A., 2016. On the acquisition and analysis of microscale thermophoresis data. Analytical biochemistry, 496, pp.79-93. ) Wang, T. and Montell, C., 2007. Phototransduction and retinal degeneration in Drosophila. Pflugers Archiv-European Journal of Physiology, 454, pp.821 -847. ) Lu, Y., Lim, L. and Song, J., 2017. RRM domain of ALS / FTD-causing FUS characteristic of irreversible unfolding spontaneously self-assembles into amyloid fibrils. Scientific reports, 7(1), p.1043. ) Sun, Z., Diaz, Z., Fang, X., Hart, M.P., Chesi, A., Shorter, J. and Gitler, A.D., 2011. Molecular determinants and genetic modifiers of aggregation and toxicity for the ALS disease protein FUS / TLS. PLoS biology, 9(4), p.el000614. ) Yoshizawa, T., Ali, R.. Jiou, J.. Fung, H.Y.J.. Burke. K.A., Kim, S.J., Lin, Y., Peeples, W.B., Saltzberg, D., Soniat, M. and Baumhardt, J.M., 2018. Nuclear import receptor inhibits phase separation of FUS through binding to multiple sites. Cell, 173(3), pp.693-705. )Hofweber, M., Hutten, S., Bourgeois, B., Spreitzer, E., Niedner-Boblenz, A., Schifferer, M., Ruepp. M.D., Simons, M., Niessing, D., Madl, T. and Dormann, D., 2018. Phase separation of FUS is suppressed by its nuclear import receptor and arginine methylation. Cell, 173(3), pp.706-719. )Neumann, M., Valori, C.F., Ansorge, O., Kretzschmar, H.A., Munoz, D.G., Kusaka, H., Yokota, O., Ishihara, K., Ang, L.C., Bilbao, J.M. and Mackenzie, I.R., 2012. Transportin 1 accumulates specifically with FET proteins but no other transportin cargos in FTLD-FUS and is absent in FUS inclusions in ALS with FUS mutations. Acta neuropathologica, 124, pp.705-716. ) Smith, E.F., Shaw, P.J. and De Vos, K.J., 2019. The role of mitochondria in amyotrophic lateral sclerosis. Neuroscience letters, 710, p.132933. ) Deng, J., Yang, M., Chen, Y., Chen, X.. Liu, J., Sun. S., Cheng. H.. Li. Y.. Bigio, E.H., Mesulam, M. and Xu, Q., 2015. FUS interacts with HSP60 to promote mitochondrial damage. PLoS genetics, 11(9), p.el 005357. ) Stoica, R., Paillusson, S., Gomez-Suaga, P., Mitchell. J.C., Lau, D.H.. Gray, E H., Sancho, R.M., Vizcay-Barrena, G., De Vos, K.J., Shaw, C.E. and Hanger, D.P., 2016.

[0209] 32

[0210] 3234573.1 PCT APPLICATION 112746-111725 (P25015)

[0211] ALS / FTD-associated FUS activates GSK-3P to disrupt the VAPB-PTPTP 51 interaction and ER-mitochondria associations. EMBO reports, 17(9), pp. 1326-1342.

[0212] 37) Wang, W., Li, L., Lin, W.L., Dickson, D.W., Petrucelli, L., Zhang, T. and Wang, X., 2013. The ALS disease-associated mutant TDP-43 impairs mitochondrial dynamics and function in motor neurons. Human molecular genetics, 22(23), pp.4706-4719.

[0213] 38) Payne, B.A. and Chinnery, P.F., 2015. Mitochondrial dysfunction in aging: Much progress but many unresolved questions. Biochimica et Biophysica Acta (BBA)- Bioenergetics, 1847(H), pp. 1347-1353.

[0214] 39) L6pez-Erauskin, J., Tadokoro. T., Baughn, M.W., Myers, B.. McAlonis-Downes, M.. Chillon-Marinas, C., Asiaban, J.N., Artates. J., Bui, A.T., Vetto, A.P. and Lee, S.K., 2020. ALS / FTD-linked mutation in FUS suppresses intra-axonal protein synthesis and drives disease without nuclear loss-of-function of FUS. Neuron, 106(2), p.354.

[0215] EQUIVALENTS

[0216] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

[0217] 33

[0218] 3234573.1

Claims

PCT APPLICATION 112746-111725 (P25015)CLAIMS;1. A method of modulating FUS, comprising the step of administering MC16. or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

2. A method of treating a FUS-associated neurodegenerative disease or disorder, comprising the step of administering a therapeutically effective amount of MC 16, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

3. The method according to claim 2, wherein the neurodegenerative disease or disorder is ALS or FTD.

4. The method according to claim 2, wherein the neurodegenerative disease or disorder is ALS.343234573.1

Images