Inhibition of dipeptide repeat proteins

Abstract

To provide a novel approach for treating neurodegenerative disorders by providing a method for inhibiting or reducing cellular toxicity caused by dipeptide repeat proteins (DRPs).SOLUTION: Methods are disclosed for treating neurodegenerative disorders, such as ALS and FTD, by using a type I protein arginine methyltransferase (Type I PRMT) inhibitor to reduce cellular toxicity caused by DRPs.SELECTED DRAWING: None

Description

Related Applications

[0001] This application claims priority to U.S. Provisional Application No. 62 / 868,267, filed Jun. 28, 2019, and U.S. Provisional Application No. 63 / 028,753, filed May 22, 2020. The contents of both applications are incorporated herein by reference.

Technical Field

[0002] The present invention relates to the treatment of neurological diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).

Background Art

[0003] ALS is a progressive neurological disease characterized by muscle fiber atrophy caused by the degeneration of motor neurons in the spinal cord and brain. FTD is the second most common early-onset dementia (after Alzheimer's disease) in people under 65 years of age.

[0004] In recent years, it has been reported that the expansion of the hexanucleotide (GGGGCC) repeat of the gene C9 open reading frame 72 (C9ORF72) on chromosome 9 may contribute to the development of both ALS and FTD. See Jovicic et al., "Modifiers of C9orf72 DRP toxicity implicate nucleocytoplasmic transport impairments in c9FTD / ALS," Nat Neurosci 2015 Sep 18(9):1226-1229. RNA transcribed from the mutant C9ORF72 gene containing the expanded GGGGCC repeat is translated via a non-ATG initiated mechanism. ​​​​​​​​​ This promotes the formation and intracellular accumulation of dipeptide repeat proteins (DRPs). DRPs translated from all six reading frames in either the sense or antisense direction of the hexanucleotide repeat result in the expression of five DRPs: glycine-alanine (GA), glycine-arginine (GR), proline-alanine (PA), proline- arginine (PR), and glycine-proline (GP) (GP can be produced from both sense and antisense reading frames in particular). However, the mechanism of action and the contribution of each DRP to neurodegeneration remain unclear. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0005] An object of the present invention is to provide a new approach for treating neurodegenerative diseases by inhibiting or reducing the cytotoxicity caused by DRPs.

[0006] Methods for reducing the cytotoxicity caused by DRPs are disclosed. Two types of DRPs, glycine-arginine (GR) and proline-arginine (PR), are particularly harmful to cell viability and, if unchecked, can cause cell dysfunction or cell death, especially in neurons. Without being bound by theory, in some embodiments, the toxic effects caused by DRPs are more likely caused by the asymmetric methylation of arginine substrates within the dipeptide repeat rather than by abnormal methylation of endogenous proteins.

[0007] ​​​​​In one aspect of the present invention, an effective amount of type I protein arginine methyltransferase (type I PRMT) inhibitor is administered to reduce the toxicity of DRP in cells, for example , to nullify the toxicity caused by asymmetric methylation of arginine substrates within the dipeptide repeat . A method is disclosed that includes this. Useful type I PRMT inhibitors are those that can inhibit at least one of PRMT 1, PRMT3, PRMT4, PRMT6, and / or PRMT8 . Examples of type I PRMT inhibitors include, but are not limited to, M S023, MS049, EPZ020411, GSK715 (also known as GSK3368715 or EPZ019997), and / or TP064 (described in more detail below

[0008] . Type I PRMT inhibitors are particularly useful for reducing the cytotoxicity caused by DRPs that contain the amino acid arginine (R), such as polyglycine-arginine (GR) and / or polyproline-arginine (PR) dipeptide repeats. The present invention can be particularly useful for protecting nerve cells, such as sensory neurons, motor neurons, and / or interneurons, from DRP toxicity. In particular, the above method can be useful for treating such diseases when neurodegenerative diseases are associated with the expression of DRP in the nerve cells of the subject. For example, the above method can be used for the treatment of C9ORF 72-linked ALS or C9ORF72-linked FTD.

[0009] In yet another aspect, the treatment method includes a second therapeutic agent, such as riluzole and / or .

[0010] In yet another aspect, the treatment method includes a second therapeutic agent, such as riluzole and / or or further comprises administration of edarabone. Alternatively, the second therapeutic agent is an antibody or an antigen-binding site thereof, for example, an antibody that blocks the interaction between human CD40 and human CD40L.

[0011] Other features and advantages of the present disclosure will become apparent from the following detailed description and examples, which should not be construed as limiting. The contents of all references, GenBank entries, patents, and published patent applications cited throughout this application are hereby expressly incorporated by reference into this specification.

Brief Description of the Drawings

[0012]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 27

Figure 28

Figure 29

Figure 30

Figure 31

Figure 32

Figure 33

Figure 34

DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention inhibits DRP-induced toxicity in cells by contacting the cells with an effective amount of a type I PRMT inhibitor (e.g., MS023, MS049, E PZ020411, GSK715, TP064, and / or derivatives thereof, etc., a drug that inhibits at least one of PRMT1, PRMT3, PRMT4, PRMT6, or PRMT8), thereby abrogating toxicity caused by asymmetric methylation of arginine substrates within dipeptide repeats such as GR and / or PR, and providing a method in which cell dysfunction or cell death is suppressed. Further provided herein is a method of treating neurodegenerative diseases associated with the expression of DRP (e.g., ALS or FTD) by administering an effective amount of a type I PRMT inhibitor, and the use of a type I PRMT inhibitor for the manufacture of a medicament for use in the treatment of neurodegenerative diseases or for use in the treatment of neurodegenerative diseases.

[0014] [I. DEFINITIONS] The following abbreviations are used throughout this specification and are known to those skilled in the art: ALS (amyotrophic lateral sclerosis); FTD (frontotemporal dementia); C9ORF72 (chromosome 9 open reading frame 72); SOD1 (superoxide dismutase-1); and PRMT (protein arginine methyltransferase).

[0015] In the following description, and in the documents incorporated by reference, several terms are used extensively. The following definitions are provided to facilitate the understanding of the methods and compositions disclosed herein.

[0016] As used herein, the term "dipeptide repeat protein" (DRP) refers to a peptide consisting of repeating units of two amino acids. As an example of a DRP, RNA transcribed from the mutant C9ORF72 gene (containing an expanded GGGGCC repeat) is translated via a non-AUG initiated mechanism to form a DRP. DRPs translated from all six reading frames in either the sense or antisense direction of a hexanucleotide repeat result in the expression of five DRPs: glycine-alanine (GA), glycine-arginine (GR), proline- alanine (PA), proline-arginine (PR), and glycine-proline (GP; GP can be generated from both sense and antisense reading frames in particular). DRPs have been shown to be "toxic" to cells by, for example, interfering with the

[0017] normal function of genes (such as the C9orf72 gene) and by interfering with cellular proteins, and thus causing cell dysfunction, degeneration, and death. Accordingly, the terms "toxic" and "toxicity" are used interchangeably, and a substance (e.g., a DRP or a mixture of DRPs) that is toxic to cells (e.g., neurons) and ​​So much so that it can damage organisms such as humans, animals, bacteria, etc. that call or constitute its cells Degree refers to. Such toxic effects include, for example, loss of cell function and / or cell death .

[0018] As used herein, the term "methyltransferase" refers to a class of transferases that can transfer a methyl group from a donor molecule to a receptor molecule, such as an amino acid residue of a protein or a nucleic acid base of a DNA molecule . Methyltransferases usually , using the reactive methyl group attached to the sulfur of S-adenosylmethionine (SAM) as a methyl donor . As an example of a type of methyltransferase, "protein arginine methyltransferase" (PRMT) can be mentioned . PRMT catalyzes the methylation of arginine residues (a common post-translational modification of proteins) . The dimethylation of arginine proceeds via the intermediate ω-NG-monomethylarginine, resulting in either symmetric ω-NG,N‘G-dimethylarginine or asymmetric ω-NG,NG-dimethylarginine . PRMT is classified into type I enzymes and type II enzymes according to the final product . Both classes Catalyze the formation of monomethylated arginine. Type I PRMT converts the intermediate ω-NG-monomethylarginine to asymmetric dimethylarginine, while type II PRMT converts the intermediate ω-NG-monomethylarginine to symmetric dimethylarginine . In mammals, Type I PRMT includes PRMT1, PRMT3, PRMT4, PRMT6, and PRMT8 . . .

[0019] An "inhibitor" of PRMT (e.g., a type I PRMT inhibitor) is a PRMT (e.g., a type I P ​A molecule that "inhibits" or "blocks" the activity of RMT. The terms "inhibit" or "block" are used interchangeably and include, for example, at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of both partial and complete inhibition / blockade as determined by, for example, the methods described herein. Alternatively, inhibition / blockade by an inhibitor (e.g., a type I PRMT inhibitor) results in, for example, an increase in cell activity and / or cell viability of at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% as determined by, for example, the methods described herein. Inhibitors of PRMT include, for example, MS023, MS049, EPZ020411 , GSK715, and / or TP064. The term "effective amount" refers to the amount of a drug that provides a desired biological, therapeutic, and / or prophylactic result. The above results include a reduction, improvement, remission, alleviation, delay, and / or mitigation of one or more of the symptoms, signs, or causes of a disease, or other desirable changes in a biological system. The therapeutically effective amount of a composition can vary according to factors such as the medical condition, age, gender, and weight of the individual, as well as the ability of the agent to elicit a desired response in the individual. The therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or detrimental effects of the agent. With respect to a decrease in cell toxicity, an effective amount includes, for example, an amount sufficient to equalize the rate of cell proliferation to that of healthy, untreated cells that are not abnormally proliferating. For neurodegenerative diseases (e.g.,

[0020]

[0021] ​​​​​​​​​ For the treatment of ALS or FTD, an effective amount is an amount sufficient to prevent or delay symptoms associated with the disease (e.g., muscle weakness and / or cognitive impairment). The effective amount can be administered in one or multiple doses. In one example, an "effective amount" is an amount of type I PRMT that has been clinically proven to affect a significant improvement in symptoms associated with the disease (e.g., ALS or FTD).

[0022] As used herein, the terms "fixed dose," "flat dose" and "flat-fixed dose" are used interchangeably and refer to a constant dose administered to a patient regardless of the patient's body weight or body surface area (BSA). Thus, a fixed dose or flat dose is provided as the absolute amount of the drug rather than as an mg / kg dose (e.g., for type I PRMT).

[0023] The term "neuron" refers to the functional unit of the nervous system, a specialized impulse-conducting cell consisting of a cell body and its processes, axons, and dendrites. Neurons include sensory neurons, motor neurons, and interneurons.

[0024] The term "neural stem cell" or "neural progenitor cell" or "neural precursor cell" refers to a cell that can generate progeny that are either neurons (such as neural progenitor cells or mature neurons) or glial cells (such as glial progenitor cells, mature astrocytes, or mature oligodendrocytes). Usually, the cell expresses some of the phenotypic markers characteristic of the neural lineage.

[0025] ​​​​​​​​​As used herein, the term "subject" refers to a human or other animal who is, for example, a human having a neurological disorder. In some embodiments, the subject is a mammal. Examples of subjects include, but are not limited to, humans, horses, monkeys, dogs, cats, mice, rats, cows, pigs, goats, and sheep. In some embodiments, the "subject" is generally a human patient diagnosed with ALS or FTD.

[0026] The terms "C9ORF72-linked ALS" and "C9ORF72-linked FTD" refer to forms of ALS and FTD that afflict individuals having an expanded hexanucleotide (GGGGCC) repeat mutation, such as the C9 ORF72 mutation described above. In the general population (not affected by ALS or FTD), the open reading frame 72 on chromosome 9 typically shows a tract of 3 to 10 GGGGCC hexanucleotide repeats, and in almost every case, fewer than 20 repeats. Thus, individuals afflicted with ALS or FTD having more than 20 hexanucleotide repeats or more than 30 hexanucleotide repeats in the open reading frame 72 on chromosome 9 may have "C9ORF72-linked ALS" or "C9ORF72-linked FTD".

[0027] As used herein, the terms "treatment" or "treating" are intended to encompass the prevention of the onset, delay of the progression, regression, or other improvement of neurological disorders such as neurodegenerative and / or neuromuscular diseases.

[0028] ​​​​​​As used herein, the term "neurodegenerative disease" refers to a condition characterized by progressive functional impairment, degeneration, and death of a specific population of neurons. Examples of neurodegenerative diseases include, for example, ALS and FTD, which can be inherited in an autosomal dominant manner with age-dependent penetrance, and include "C9ORF72-linked ALS" and "C9ORF72-linked FTD".

[0029] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a molecule" includes one or more such molecules, and reference to "the method" includes reference to equivalent steps and methods known to those skilled in the art that can be modified or substituted for the methods described herein.

[0030] The terms "about" or "approximately" mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, for example, the limitations of the measurement system, or the degree of accuracy required for a particular purpose. For example, "about" can mean within one standard deviation, or more than one standard deviation, of a value according to the practice in the art. Alternatively, "about" can mean within a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. When a particular value is recited in the present application and claims, the term "about" is assumed to mean within an acceptable error range for the particular value unless otherwise specified. ​

[0031] [II. Composition] Furthermore, a composition used in the method of the present invention, such as a pharmaceutical composition, is provided. Such a composition contains a type I PRMT inhibitor formulated together with a pharmaceutically acceptable carrier . The type I PRMT inhibitor is, for example, a drug that inhibits PRMT1, PRMT3, PRMT4, PRMT6, and / or PRMT8, and includes, for example, MS023, MS049, EP Z020411, GSK715, and / or TP064.

[0032] One exemplary type I PRMT inhibitor is the compound known as MS023 having the following chemical formula: : [Chemical formula]

[0033] Another exemplary type I PRMT inhibitor is the compound known as MS049 having the following chemical formula: : [Chemical formula]

[0034] Another exemplary type I PRMT inhibitor is the compound known as EPZ020411 having the following chemical formula: : [Chemical formula]

[0035] Another exemplary type I PRMT inhibitor is the compound known as GSK715 having the following chemical formula: : [Chemical formula]

[0036] Another exemplary type I PRMT inhibitor is TP064, which has the following chemical formula and is a compound:

Chemical formula

[0037] More generally, type I PRMT inhibitors may also include, for example, the compounds described in U.S. Patent Application Publication Nos. US20 16 / 0137609 and US2019 / 0077795, both of which are assigned to Epizyme, Inc. of Cambridge, Massachusetts, or US20100151506, which is assigned to the University of South Carolina, the disclosures of which are hereby incorporated by reference in their entireties. The term "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition, or vehicle such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material involved in the transport or

[0038] transportation of a chemical agent. Examples of pharmaceutically acceptable carriers are solvents, diluents, dispersion media, suspending aids, surfactants, preservatives, solid binders, stabilizers, fillers, binders, lubricants, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc., which are physiologically compatible. The various vehicles and carriers used in the formulation of pharmaceutical compositions, as well as the known techniques for preparing them, are disclosed in Remington's Pharmaceutical Sciences (A. Osol et al. eds., 15 th ed. 1975). Pharmaceutically acceptable carriers may include adjunct substances such as wetting or emulsifying agents, preservatives or buffers. th ed. 1975). th Pharmaceutically acceptable carriers may include adjunct substances such as wetting or emulsifying agents, preservatives or buffers. ​​​​​​They may further contain a small amount of the substance, which enhances the storage life or effectiveness of the agent.

[0039] Pharmaceutically acceptable carriers also include pharmaceutically acceptable salts, and the term "pharmaceutically acceptable salts" includes salts of the active compounds prepared with relatively non-toxic acids or bases, depending on the specific substituents found in the compounds described herein. Pharmaceutically acceptable salts include, but are not limited to, salts of acidic or basic groups. Compounds that are essentially basic can form various salts with various inorganic and organic acids. Acids that can be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are non-toxic acid addition salts, i.e., including, but not limited to, sulfuric acid, thiosulfuric acid, citric acid, maleic acid acetic acid, oxalic acid, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate bisulfite, phosphate, acid phosphate, isonicotinate, borate, acetate, lactate salicylate, citrate, acid citrate, tartrate, oleate, tannate pantothenate, tartrate, ascorbate, succinate, maleate, gentisate fumarate, gluconate, glucuronate, saccharate, formate, benzoate glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate p-toluenesulfonate, bicarbonate, malonate, mesylate, esylate, napsidicate, tosylate, besylate, orthophosphate, trifluoroacetate and salts containing pharmaceutically acceptable anions including pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy- 3-naphthoate)) salts. That is. Compounds containing an amino moiety can form pharmaceutically acceptable salts with various amino acids in addition to the acids described above. Compounds that are essentially acidic can form basic salts with various pharmaceutically acceptable cations. Examples of such salts include, but are not limited to, alkali metal or alkaline earth metal salts, particularly calcium, magnesium, ammonium, sodium, lithium, zinc, potassium, and iron salts. The present invention also includes quaternary ammonium salts of the compounds described herein, which have one or more tertiary amine moieties. The above carrier enables the compound of the present invention to be formulated for oral ingestion by a patient to be treated as tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, etc. Suitable excipients are, in particular, sugars including lactose, sucrose, mannitol, or sorbitol; fillers such as cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrants such as cross-linked polyvinylpyrrolidone, agar, or alginic acid, or salts thereof such as sodium alginate may be added. In one embodiment, mannitol and magnesium stearate are used as pharmaceutically acceptable carriers. In some preferred embodiments, the compounds of the present invention are administered together with an adjuvant. are exemplified. The present invention also includes quaternary ammonium salts of the compounds described herein, which have one or more tertiary amine moieties.

[0040] The above carrier enables the compound of the present invention to be formulated for oral ingestion by a patient to be treated as tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, etc. Suitable excipients are, in particular, sugars including lactose, sucrose, mannitol, or sorbitol; fillers such as cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrants such as cross-linked polyvinylpyrrolidone, agar, or alginic acid, or salts thereof such as sodium alginate may be added. In one embodiment, mannitol and magnesium stearate are used as pharmaceutically acceptable carriers. to make it possible. Suitable excipients are, in particular, sugars including lactose, sucrose, mannitol, or sorbitol; fillers such as cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrants such as cross-linked polyvinylpyrrolidone, agar, or alginic acid, or salts thereof such as sodium alginate may be added. In one embodiment, mannitol and magnesium stearate are used as pharmaceutically acceptable carriers. sugars; for example, starches such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrants such as cross-linked polyvinylpyrrolidone, agar, or alginic acid, or salts thereof such as sodium alginate may be added. In one embodiment, mannitol and magnesium stearate are used as pharmaceutically acceptable carriers. starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrants such as cross-linked polyvinylpyrrolidone, agar, or alginic acid, or salts thereof such as sodium alginate may be added. In one embodiment, mannitol and magnesium stearate are used as pharmaceutically acceptable carriers. cellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrants such as cross-linked polyvinylpyrrolidone, agar, or alginic acid, or salts thereof such as sodium alginate may be added. In one embodiment, mannitol and magnesium stearate are used as pharmaceutically acceptable carriers. pyrrolidone, agar, or alginic acid, or salts thereof such as sodium alginate may be added. In one embodiment, mannitol and magnesium stearate are used as pharmaceutically acceptable carriers. pyrrolidone, agar, or alginic acid, or salts thereof such as sodium alginate may be added. In one embodiment, mannitol and magnesium stearate are used as pharmaceutically acceptable carriers. salts thereof such as sodium alginate may be added. In one embodiment, mannitol and magnesium stearate are used as pharmaceutically acceptable carriers. In one embodiment, mannitol and magnesium stearate are used as pharmaceutically acceptable carriers.

[0041] In some preferred embodiments, the compounds of the present invention are administered together with an adjuvant. It can be done. The term "adjuvant" lacks significant activity when administered alone, but can be a compound that can enhance the activity of another therapeutic agent. In some embodiments, the adjuvant is selected from the group consisting of buffers, antibacterial preservatives, surfactants, antioxidants, tonic regulators, preservatives, thickeners, and viscosity increasing agents. The pharmaceutical compositions of the present invention can be in various forms. These include, for example, liquid solutions (such as injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories, including liquid, semi-solid, and solid dosage forms. The preferred form depends on the intended mode of administration and therapeutic use. In one embodiment, the preferred mode of administration is oral delivery. For administering the compounds of the present invention, various solid oral dosage forms can be used, including solid forms such as tablets, gel caps, capsules, caplets, granules, lozenges, and bulk powders. For administering the compounds of the present invention, various liquid oral dosage forms can also be used, including aqueous and non-aqueous solutions, emulsions, suspensions, syrups, and elixirs. Such dosage forms can also contain suitable inert diluents known in the art, such as water, and suitable excipients known in the art, such as preservatives, wetting agents, sweeteners, flavoring agents, and agents for emulsifying and / or suspending the compounds of the present invention. The compounds of the present invention may be injected intravenously, for example, in the form of an isotonic sterile solution.

[0042] The pharmaceutical compositions of the present invention can be in various forms. These include, for example, liquid solutions (such as injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories, including liquid, semi-solid, and solid dosage forms. The preferred form depends on the intended mode of administration and therapeutic use. In one embodiment, the preferred mode of administration is oral delivery. For administering the compounds of the present invention, various solid oral dosage forms can be used, including solid forms such as tablets, gel caps, capsules, caplets, granules, lozenges, and bulk powders. For administering the compounds of the present invention, various liquid oral dosage forms can also be used, including aqueous and non-aqueous solutions, emulsions, suspensions, syrups, and elixirs. Such dosage forms can also contain suitable inert diluents known in the art, such as water, and suitable excipients known in the art, such as preservatives, wetting agents, sweeteners, flavoring agents, and agents for emulsifying and / or suspending the compounds of the present invention. The compounds of the present invention may be injected intravenously, for example, in the form of an isotonic sterile solution. For administering the compounds of the present invention, various solid oral dosage forms can be used, including solid forms such as tablets, gel caps, capsules, caplets, granules, lozenges, and bulk powders. For administering the compounds of the present invention, various liquid oral dosage forms can also be used, including aqueous and non-aqueous solutions, emulsions, suspensions, syrups, and elixirs. Such dosage forms can also contain suitable inert diluents known in the art, such as water, and suitable excipients known in the art, such as preservatives, wetting agents, sweeteners, flavoring agents, and agents for emulsifying and / or suspending the compounds of the present invention. The compounds of the present invention may be injected intravenously, for example, in the form of an isotonic sterile solution.

[0043] For administering the compounds of the present invention, various solid oral dosage forms can be used, including solid forms such as tablets, gel caps, capsules, caplets, granules, lozenges, and bulk powders. For administering the compounds of the present invention, various liquid oral dosage forms can also be used, including aqueous and non-aqueous solutions, emulsions, suspensions, syrups, and elixirs. Such dosage forms can also contain suitable inert diluents known in the art, such as water, and suitable excipients known in the art, such as preservatives, wetting agents, sweeteners, flavoring agents, and agents for emulsifying and / or suspending the compounds of the present invention. The compounds of the present invention may be injected intravenously, for example, in the form of an isotonic sterile solution. For administering the compounds of the present invention, various solid oral dosage forms can be used, including solid forms such as tablets, gel caps, capsules, caplets, granules, lozenges, and bulk powders.

[0044] For administering the compounds of the present invention, various liquid oral dosage forms can also be used, including aqueous and non-aqueous solutions, emulsions, suspensions, syrups, and elixirs. Such dosage forms can also contain suitable inert diluents known in the art, such as water, and suitable excipients known in the art, such as preservatives, wetting agents, sweeteners, flavoring agents, and agents for emulsifying and / or suspending the compounds of the present invention. The compounds of the present invention may be injected intravenously, for example, in the form of an isotonic sterile solution. For administering the compounds of the present invention, various liquid oral dosage forms can also be used, including aqueous and non-aqueous solutions, emulsions, suspensions, syrups, and elixirs. Such dosage forms can also contain suitable inert diluents known in the art, such as water, and suitable excipients known in the art, such as preservatives, wetting agents, sweeteners, flavoring agents, and agents for emulsifying and / or suspending the compounds of the present invention. The compounds of the present invention may be injected intravenously, for example, in the form of an isotonic sterile solution. For administering the compounds of the present invention, various liquid oral dosage forms can also be used, including aqueous and non-aqueous solutions, emulsions, suspensions, syrups, and elixirs. Such dosage forms can also contain suitable inert diluents known in the art, such as water, and suitable excipients known in the art, such as preservatives, wetting agents, sweeteners, flavoring agents, and agents for emulsifying and / or suspending the compounds of the present invention. The compounds of the present invention may be injected intravenously, for example, in the form of an isotonic sterile solution. For administering the compounds of the present invention, various liquid oral dosage forms can also be used, including aqueous and non-aqueous solutions, emulsions, suspensions, syrups, and elixirs. Such dosage forms can also contain suitable inert diluents known in the art, such as water, and suitable excipients known in the art, such as preservatives, wetting agents, sweeteners, flavoring agents, and agents for emulsifying and / or suspending the compounds of the present invention. The compounds of the present invention may be injected intravenously, for example, in the form of an isotonic sterile solution. For administering the compounds of the present invention, various liquid oral dosage forms can also be used, including aqueous and non-aqueous solutions, emulsions, suspensions, syrups, and elixirs. Such dosage forms can also contain suitable inert diluents known in the art, such as water, and suitable excipients known in the art, such as preservatives, wetting agents, sweeteners, flavoring agents, and agents for emulsifying and / or suspending the compounds of the present invention. The compounds of the present invention may be injected intravenously, for example, in the form of an isotonic sterile solution. For administering the compounds of the present invention, various liquid oral dosage forms can also be used, including aqueous and non-aqueous solutions, emulsions, suspensions, syrups, and elixirs. Such dosage forms can also contain suitable inert diluents known in the art, such as water, and suitable excipients known in the art, such as preservatives, wetting agents, sweeteners, flavoring agents, and agents for emulsifying and / or suspending the compounds of the present invention. The compounds of the present invention may be injected intravenously, for example, in the form of an isotonic sterile solution.

[0045] Therapeutic compositions generally must be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high drug concentration. Sterile injectable solutions can be prepared by incorporating the required amount of the active compound (i.e., the agent) into a suitable solvent containing, as required, one or a combination of the ingredients enumerated above, followed by filter sterilization.

[0046] Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile freeze-dried powders for the preparation of sterile injectable solutions, the preferred method of preparation is vacuum drying and spray drying which yield a powder of the active ingredient plus any additional desired ingredients obtained from a previously sterile-filtered solution. The fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition agents that delay absorption, such as monostearate and gelatin.

[0047] Supplementary active compounds can also be incorporated into the compositions. In certain embodiments, type I PRMT inhibitors are co-formulated and / or co-administered with one or more additional therapeutic agents useful for improving the pharmacokinetics of the agent and / or treating a degenerative disease.

[0048] [III. Methods] Provided herein are type I PRMTs by various methods known in the art. ​​​​​​​​​​​​​A clinical method for treating neurodegenerative diseases, including administration of an inhibitor. The dosing regimen can be adjusted to provide the most suitable desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the urgency of the treatment situation. In one embodiment, an initial bolus dose and subsequent lower maintenance doses are administered. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate the composition in unit dosage form.

[0049] As used herein, the term "unit dosage form" refers to physically discrete units suitable as a single dosage for a mammalian subject to be treated; each unit contains a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in association with the necessary pharmaceutical carrier. The form of the unit dosage form of the present invention is determined by and directly depends on (a) the unique properties of the active compound and the specific therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding the above-mentioned active compound with respect to the treatment of sensitivity in an individual.

[0050] It should be noted that the dosage value can vary depending on the type and severity of the condition to be alleviated. Furthermore, for a particular subject, a particular dosing regimen should be adjusted over time according to the individual needs and expert judgment of the person administering or supervising the administration of the composition, and it should be further understood that the dosage ranges described herein are merely exemplary and are not intended to limit the scope or practice of the claimed composition.

[0051] A suitable treatment for treating neurodegenerative diseases (e.g., ALS or FTD) in human patients The protocol includes, for example, administering to the patient an effective amount of a type I PRMT inhibitor that results in a reduction, improvement, remission, alleviation, delay, and / or mitigation of one or more of the signs, symptoms, or causes of the disease (e.g., a reduction in muscle weakness), or other desirable changes in the biological system Alternatively, the type I PRMT inhibitor is administered at a flat dose of about 0.01, 0.03, 0.1, 0.3, 1, or 3.0 μM, preferably about 1.0 μM or 3.0 μM

[0052] In other embodiments, the dose of the type I PRMT inhibitor is calculated per body weight, e.g., in mg / kg body weight. In another embodiment, the dose of the type I PRMT inhibitor is a flat fixed dose. In another embodiment, the dose of the type I PRMT inhibitor changes over time For example, the type I PRMT inhibitor may initially be administered at a high dose and then decreased over time In another embodiment, the type I PRMT inhibitor antibody is initially administered at a low dose

[0053] and increased over time In another embodiment, the amount of the type I PRMT inhibitor administered is constant for each dose In another embodiment, the amount of the inhibitor administered varies with each dose. For example the maintenance (or subsequent) dose of the inhibitor can be higher than, the same as, or lower than the loading dose initially administered

[0054] The following examples are merely illustrative, and many variations and equivalents will be apparent to those skilled in the art upon reading this disclosure ​​​It should not be construed as limiting the scope of the present disclosure so as to become apparent.

[0055] All references, GenBank entries, patents and the contents of published patent applications cited throughout this application are hereby expressly incorporated by reference into this specification.

[0056] The present invention will be further illustrated by the following examples which should not be construed as further limitations. All figures and all references, patents, as well as the contents of published patent applications cited throughout this application are hereby expressly incorporated by reference into this specification.

Example

[0057] [Materials and Methods] In the examples described below, the following materials and methods were used.

[0058] WST-1 assay: The WST-1 assay is a colorimetric assay for measuring cell metabolism. Cells with good metabolism contain mitochondrial dehydrogenase enzymes that remove hydrogen atoms from specific molecules, resulting in the release of energy that cells can use to carry out important reactions. In the WST-1 assay, a water-soluble tetrazolium substrate (known as WST-1 ) is applied, which readily penetrates cells and their mitochondria upon application. If the cells are viable, their mitochondrial dehydrogenase enzymes catalyze a reaction to convert this WST-1 substrate into a colored product called formazan. The production of this colored formazan product by live cells can be quantified using a spectrophotometer (plate reader). The higher the absorbance reading, the higher the metabolic activity / cell viability rate. ​​

[0059] LDH assay: The LDH assay is a colorimetric assay that measures cell toxicity and death. When cells are exposed to cytotoxic compounds, they can undergo a type of cell death called necrosis , which initially results in cell swelling and loss of cell membrane integrity. As cells lose membrane integrity , an enzyme found in live cells called lactate dehydrogenase (LDH) is released into the solution surrounding the dying cells. Programmed cell death, apoptosis, as well as other forms of cell death also ultimately lead to the destruction of the cell membrane and a massive release of LDH into the extracellular space. Therefore, LDH is an excellent marker for not only necrosis but also all examples of cell death. In the LDH assay, a sample of the solution surrounding the cells is separated and transferred to a plate containing an LDH reaction mixture. This reaction mixture contains a tetrazolium salt intermediate and other reaction components, which, when exposed to LDH, convert the tetrazolium salt into a colored formazan product. This formazan product can be quantified by spectrophotometry. The higher the measured absorbance value, the more cell death there is.

[0060] Caspase-3 / CPP32 assay: The caspase-3 assay is a fluorescence measurement assay that measures apoptotic cell death. Apoptosis is a form of "programmed" cell death in which a specific family of proteins within the cell triggers a series of chemical reactions that cause the cell to self-destruct. One family of proteins involved in this process is the caspase protein family. Of the caspase proteins, caspase-2, 8, 9, and 10 are known as "initiators" and trigger the death cascade ​ become ligases, and caspases - 3, 6, and 7 are known as "executioners" and actually kill cells. In the caspase - 3 assay, cells treated with the test compound are lysed, and a substrate known as DEVD - AFC is added to the lysate. When active caspase - 3 enzyme is present in the lysate, it cleaves AFC from the DEVD - AFC substrate, resulting in a fluorescent signal that can be quantified using a fluorescence microtiter plate reader. Thus, the apoptotic activity of cells treated with the test compound can be quantified. The higher the fluorescence measurement value, the more apoptotic cell death there is. -3 enzyme cleaves AFC from the DEVD - AFC substrate, resulting in a fluorescent signal that can be quantified using a fluorescence microtiter plate reader. Thus, the apoptotic activity of cells treated with the test compound can be quantified. The higher the fluorescence measurement value, the more apoptotic cell death there is.

[0061] BrdU ELISA: BrdU ELISA is an ELISA for detecting cell proliferation activity. BrdU is a pyrimidine analogue and, when inside cells, can be incorporated into newly synthesized DNA strands in place of thymidine. Incorporation of BrdU into cellular DNA, as indicated by incorporation of thymidine into DNA, indicates DNA synthesis activity, which is required for cell proliferation. In BrdU ELISA, cells are treated with the test compound and BrdU reagent and incubated for 24 hours before the test. During the test, an anti - BrdU antibody detects the incorporation of BrdU into cellular DNA that occurred over 24 hours as a surrogate for cell proliferation activity. This anti - BrdU antibody is then labeled with a secondary antibody tagged with a chromogenic substrate that can be quantified spectrophotometrically. The higher the absorbance reading, the higher the cell proliferation activity.

[0062] PRMT inhibitors: Type I and Type II PRMT inhibitors are the examples listed in Table 1 ​​​​​​​​​​​​​​was tested. IC50 for inhibition of dimethylation activity, and GR 15 or PR 15 EC50 for inactivation of toxicity induced by challenge, and also refer to Figure 33 summarizing the chemical structure of each compound tested.

[0063] [Table 1]

[0064] Synthetic dipeptide repeat protein (DRP): DRP toxicity was induced by administering to cells the 15-mer dipeptide repeat sequences of GR, PR, GP or PA.

[0065] [Table 2]

[0066] NSC-34 cell culture: NSC-34 cells (Cedarlane Laboratories, Burlington, Ontario, California) were cultured in high-glucose Dulbecco's modified Eagle's medium (Millipore-Sigma, Burlington, Massachusetts, USA) supplemented with 10% US-origin fetal bovine serum (Thermo-Fisher Scientific, Cambridge, Massachusetts, USA), 1% 200 mM L-glutamine solution (Thermo-Fisher Scientific, Cambridge, Massachusetts, USA), and 1% 10,000 U / mL penicillin-streptomycin solution (Thermo-Fisher Scientific, Cambridge, Massachusetts, USA). It was cultured in the resulting complete medium. Before preparing the NSC-34 complete medium, L-glutamine and penicillin-streptomycin solution were aliquoted and stored at -20 °C, and DMEM / high glucose was stored at 4 °C. At each passage, the cells were washed once with Dulbecco's phosphate-buffered saline with calcium and magnesium (Thermo-Fisher Scientific, Cambridge, Massachusetts, USA), and treated with 0.25% trypsin-EDTA solution (Thermo-Fisher Scientific , Cambridge, Massachusetts, USA) at 37 °C and 5% CO2 for 5 minutes to dissociate them. The prepared complete medium, DPBS, and trypsin were always heated in a 37 °C water bath before use and stored at 4 °C between uses. Cell counting for plating was performed using a Hausser Scientific cell counting chamber (Thermo-Fisher Scientific, Cambridge, Massachusetts, USA).

[0067] Preparation of exogenous dipeptide repeat protein solution: Synthetic proteins GR 15 and PR 15 (GenicBio Limited, Kowloon, Hong Kong, China), and ADMe-GR 15 (Eurogent ec, Leige, Belgium) were purchased as lyophilized powders and stored in a desiccator at - 20 °C before reconstitution. The proteins were reconstituted with sterile DMSO (Millipore-Sigma, Burlington, Massachusetts, USA) to a stock concentration of 10 mM and stored at 4 °C.

[0068] Preparation of PRMT inhibitor solution: Small molecule PRMT inhibitors MS023 and GSK591 (T ocris Bioscience, Bristol, UK), MS049 (an inactive analog of MS023) and also EPZ020411 (Cayman Chemical, Ann Arbor, Michigan, USA), GSK33 68715 (Medchem Express, Monmouth Junction, New Jersey, USA), as well as the negative control MS094 (Millipore-Sigma, Billerica, Massachusetts, USA) were purchased and stored at -20 °C before and after reconstitution. After reconstitution with the solvents specified in Table _, the stock was aliquoted into 15 - 20 μL and immediately stored at -20 °C.

[0069]

Table 3

[0070] Plating of NSC-34 and administration of DRP and PRMT inhibitors: NSC-34 cells were plated at a density of 3.77×10 cells per well in clear, flat-bottomed, full-volume 96-well tissue culture-treated plates (Thermo-Fisher Scientific, Cambridge, Massachusetts, USA). One row at the top and bottom of the plate and two tiers on both sides of the plate were left cell-free, and only medium was included to minimize evaporation of the experimental well volume. When adding DRP / PRMT inhibitors, the desired doses of DRP for challenge and inhibitors for treatment were achieved by diluting aliquots of each stock in warm medium. In experiments using both PRMT inhibitors and DRP, the PRMT inhibitor was always added to the well first, followed by DRP. The vehicle control was DRP treatment, 4 ​​​​​​​​​​Wells treated with DMSO at a concentration equivalent to that of those treated with the drug treatment or both were included in the same manner. The control for inhibitor toxicity was included in the same manner as wells treated with the desired dose of the drug in the experiment, but did not contain DRP. The control for DRP toxicity was included in the same manner as wells treated only with the dose of DRP used in the challenge. When administered, the plates were incubated at 37 °C and 5% CO2 for 24 hours before performing the endpoint of the WST-1 or LDH assay. The additional controls required for each endpoint are specified in the "WST-1 Assay" and "LDH Assay" sections of these methods. WST-1 Assay: Using the steps described in the "Plating of NSC-34 and Administration of DRP and PRMT Inhibitors" section of these methods, the cells were plated and prepared. The other controls for this experiment included wells containing only cells in the medium and wells containing only the medium. At the time of testing, the medium was removed from the wells and replaced with warm, sterile-filtered DPBS containing calcium and magnesium, and a solution of 4.5 g / L D-glucose (Millipore-Sigma, Burlington, Massachusetts, USA). To the wells containing 200 μL of the DPBS-glucose solution, 20 μL / well of the WST-1 reagent (Millipore-Sigma, Burlington, California) was added, and then the plate was incubated at 37 °C and 5% CO2 for 1 hour. After incubation, the plate was read on a SpectraMax M3 microplate reader (Molecular Devices, Sunnyvale, California). The control for inhibitor toxicity was included in the same manner as wells treated with the desired dose of the drug in the experiment, but did not contain DRP. The control for DRP toxicity was included in the same manner as wells treated only with the dose of DRP used in the challenge. When administered, the plates were incubated at 37 °C and 5% CO2 for 24 hours before performing the endpoint of the WST-1 or LDH assay. The additional controls required for each endpoint are specified in the "WST-1 Assay" and "LDH Assay" sections of these methods.

[0071] WST-1 Assay: Using the steps described in the "Plating of NSC-34 and Administration of DRP and PRMT Inhibitors" section of these methods, the cells were plated and prepared. The other controls for this experiment included wells containing only cells in the medium and wells containing only the medium. At the time of testing, the medium was removed from the wells and replaced with warm, sterile-filtered DPBS containing calcium and magnesium, and a solution of 4.5 g / L D-glucose (Millipore-Sigma, Burlington, Massachusetts, USA). To the wells containing 200 μL of the DPBS-glucose solution, 20 μL / well of the WST-1 reagent (Millipore-Sigma, Burlington, California) was added, and then the plate was incubated at 37 °C and 5% CO2 for 1 hour. After incubation, the plate was read on a SpectraMax M3 microplate reader (Molecular Devices, Sunnyvale, California). To the wells containing 200 μL of the DPBS-glucose solution, 20 μL / well of the WST-1 reagent (Millipore-Sigma, Burlington, California) was added, and then the plate was incubated at 37 °C and 5% CO2 for 1 hour. After incubation, the plate was read on a SpectraMax M3 microplate reader (Molecular Devices, Sunnyvale, California). ​​​​​Read at 450 nm in San Jose, CA, USA. Experiments included three replicates per condition. Each experiment was repeated twice (for a total of three runs).

[0072] LDH assay: Cells were plated and prepared using the steps described in the “Plating of NSC-34 and Administration of DRP and PR MT Inhibitors” section of these methods. Other controls for this experiment included multiple sets of wells with only cells in the medium (one triplicate designated “untreated,” one triplicate designated “lysed” positive control), and wells with only medium. Additional controls added only to the transfer plate at the time of testing were 5 μL of LDH only. The test was performed using the colorimetric LDH-cytotoxicity assay kit II (Abcam, Cambridge, Massachusetts, USA) as per the manufacturer's instructions. The final read at 450 nm was performed on a SpectraMax M3 microplate reader (Molecular Devices, San Jose, California, USA). Data analysis included calculation of the LDH release rate using the following formula: USA). The final read at 450 nm was performed on a SpectraMax M3 microplate reader (Molecular Devices, San Jose, California, USA). Data analysis included (Molecular Devices, San Jose, CA, USA). Data analysis included calculation of the LDH release rate using the following formula: Calculation of the LDH release rate using the following formula was included:

Equation

[0073] Experiments included three replicates per condition. Each experiment was repeated twice (for a total of three runs).

[0074] (Example 1. In NSC-34 motor neuron-like cells measured by WST-1 assay, to abrogate the metabolic abnormalities induced by 3 μM GR by MS023 (type I 15 ​​Comparison of PRMT inhibitors) and GSK591 (type II PRMT, especially PRMT5 inhibitor) ) Cells were plated in the medium of two 96-well plates at a density of 3.7×10 4 cells per well and incubated overnight at 37°C and 5% CO2. The next day, just before adding the test compounds, the existing medium was removed and replaced with alternating volumes of medium corresponding to the treatments each well would receive (to ensure that the final volume in all wells was 200 μL after treatment). A 10 mM stock of MS023 was thawed to room temperature and diluted with warm medium to final concentrations of 60, 30, 10, 3, and 1 μM in the plate. According to the same protocol, a 10 mM stock of GSK591 was thawed to room temperature and diluted with warm medium to final concentrations of 200, 100, 33, 10, and 3 μ M in the plate. In addition, a 10 mM stock of GR was equilibrated to room temperature and diluted with warm medium to a final concentration of 3 μM in the wells. The following conditions were plated in triplicate. To prevent evaporation of the volume within the experimental wells during incubation, the samples were surrounded by outer boundary wells of the plate filled with sterile phosphate-buffered saline: · Untreated (cells only in medium) · DMSO control (cells treated with only the amount of DMSO to which the GR-treated cells were exposed) 15 · GR only (cells treated with 3 μM GR only)

[0075] · 3 μM GR and MS023: cells treated with any one of the doses of 60 μM, 30 μM, 10 μM, 3 μM or 1 μM · Untreated (cells only in medium) · DMSO control (cells treated with only the amount of DMSO to which the GR-treated cells were exposed) · GR only (cells treated with 3 μM GR only) 15 · GR only (cells treated with 3 μM GR only) 15 and MS023: cells treated with any one of the doses of 60 μM, 30 μM, 10 μM, 3 μM or 1 μM · Among MS023: any one of the doses of 60 μM, 30 μM, 10 μM, 3 μM, 1 μM cells treated with only one of them · 3 μM GR 15 and GSK591: cells treated with any one of the doses of 200 μM, 100 μM, 33 μM, 10 μM, 3 μM · Among GSK591: any one of the doses of 200 μM, 100 μM, 33 μM, 10 μM, 3 μM cells treated with only one of them

[0076] The plates were incubated at 37 °C and 5% CO2 for 24 hours. Immediately before the test, the medium was removed and replaced with 200 μL of PBS -glucose solution (4.5 g / L, sterilized) that had been warmed from 4 °C to 10 minutes in a 37 °C water bath before use. An aliquot of the WST-1 reagent coat was thawed from -20 °C and equilibrated at room temperature before use. 20 μL of the WST-1 reagent was added to each well containing 200 μL of PBS-glucose. The plates were incubated with WST -1 at 37 °C and 5% CO2 for 1.25 hours, and absorbance readings (450 nm) were obtained every 15 minutes using a Molecular Devices plate reader (SpectraMax M3). The data was exported from the SoftMax Pro7.0 software of the plate reader to an Excel file. As shown in Figures 1A to 1D, the type I PRMT inhibitor MS023 abolished GR-induced metabolic abnormalities in NSC-34 motor neuron-like cells at a dose of 3 μM. PRMT T5 (type II PRMT) inhibitor GSK591 did not abolish GR-induced metabolic abnormalities at any of the test doses.

[0077]

[0078] ​​​​(Example 2. MS023 (type I PRMT inhibitor) nullifies the metabolic abnormalities induced by 3 μM GR or 3 μM PR 15 in NSC-34 motor neuron-like cells.) 15 by )([End of sentence]) Cells were plated and incubated overnight as in Example 1. The next day, just prior to adding the test compound, the existing medium was removed and replaced with alternating amounts of medium as in Example 1. A 10 mM stock of MS023 was thawed to room temperature and diluted as in Example 1. In addition, 10 mM stocks of GR and PR 15 were equilibrated to room temperature 15 and diluted in warm medium to a final concentration of 3 μM in the wells. The following conditions were plated in triplicate. To prevent evaporation of the volume within the experimental wells during incubation, samples were surrounded by outer boundary wells filled with sterile phosphate-buffered saline: · Untreated (cells in medium only)

[0079] · DMSO control (cells treated with only the amount of DMSO to which GR- and PR-treated cells were exposed) · GR only (cells treated with 3 μM GR only) · Either 60 μM, 30 μM, 10 μM, 3 μM · or 1 μM of MS023 in combination with 3 μM GR · PR only (cells treated with 3 μM PR only) 15 · Either 60 μM, 30 μM, 10 μM, 3 μM · or 1 μM of MS023 in combination with 3 μM PR 15 · · · 15 · · 15 · · ·Among MS023: any one of the doses of 60 μM, 30 μM, 10 μM, 3 μM, 1 μM Cells treated with only one of them

[0080] The plates were incubated at 37 °C and 5% CO2 for 24 hours. Immediately before the test, the medium was Removed and replaced with 200 μL of PBS-glucose solution (4.5 g / L, sterilized) that had been warmed from 4 °C to 10 minutes in a 37 °C water bath before use. An aliquot of the WST-1 reagent Coat was thawed at -20 °C and equilibrated at room temperature before use. 20 μL of WST-1 reagent was added to each well containing 200 μL of PBS-glucose Solution. The plates were incubated with WST-1 For 1 hour at 37 °C and 5% CO2, and absorbance readings (450 nm) were taken every 15 minutes using a Molecular Devices plate reader (SpectraMax M3). The data was exported from the SoftMaxPro 7 Software of the plate reader to an Excel file. As shown in Figures 2A to 2D, the 3 μM and 1 μM doses of MS023 (type I PRMT Inhibitor) completely abolished the metabolic abnormalities of NS

[0081] C-34 induced by either 3 μM dose of GR Or PR 15 Or PR 15 These doses also completely abolished the metabolic Abnormalities in the above experiments (see Example 1).

[0082] (Example 3. Effect of low doses (0.01 - 3 μM) of MS023 (type I PRMT inhibitor) on the abolition of metabolic abnormalities induced by 3 μM dose of GR 15 Or PR 15 Measured by the WST-1 assay (range) ​​As in Example 1, cells were plated in the medium. The day after, immediately before adding the test compound, the existing medium was removed and replaced with alternating amounts of medium as in Example 1. A 10 mM stock of MS0 23 was thawed to room temperature and diluted with warm medium to give final 15 concentrations in the plate of 3, 1, 0.3, 0.1, 0.03 and 0.01 μM. A 10 mM stock of GR and a 10 mM stock of PR 15 were equilibrated to room temperature and diluted with warm medium to give a final concentration in the wells of 3 μM.

[0083] The following conditions were plated in triplicate. To prevent evaporation of the volume in the experimental wells during incubation, samples were surrounded by outer border wells of the plate filled with sterile phosphate buffered saline: · Untreated (cells in medium only) · DMSO control (cells treated with only the amount of DMSO to which GR- and PR-treated cells were exposed) · GR only (cells treated with 3 μM GR 15 only) · 3 μM GR 15 and MS023: cells treated with any one of the doses 3 μM, 1 μM, 0.3 μM, 0.1 μ M, 0.03 μM and 0.01 μM · PR only (cells treated with 3 μM PR 15 only) · 3 μM PR 15 and MS023: cells treated with any one of the doses 3 μM, 1 μM, 0.3 μM, 0.1 μ M, 0.03 μM and 0.01 μM · MS023: cells treated with only any one of the doses 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM and 0.01 μM

[0084] The plates were incubated at 37 °C in 5% CO2 for 24 h. Immediately before the test, the medium was removed and replaced with 200 μL of PBS-[[]] glucose solution (4.5 g / L, sterilized) that had been warmed from 4 °C for 10 min in a 37 °C water bath before use. An ali quot of the WST-1 reagent was thawed from -20 °C and equilibrated at room temperature before use. 20 μL of the WST-1 reagent was added to each well containing 200 μL of PBS-glu cose. The plates were incubated with WST -1 at 37 °C in 5% CO2 for 1 h, and absorbance readings (450 nm) were taken every 15 min with a Molecular Devices plate reader (SpectraMax M3). The data were exported from the SoftMaxPro 7.0 software of the plate reader to an Excel file.

[0085] As shown in FIGS. 3A to 3D, MS023 abrogates the metabolic abnormalities induced by 3 μM of GR 15 and PR 15 in a dose-dependent manner. The dose-dependent metabolic abrogation profiles differ slightly by the dipeptide repeat protein: a significant partial abrogation of metabolic activity in GR-treated cells is seen at a low MS023 dose of about 0.1 μM, while in PR-treated cells, it was seen at a low MS023 dose of about 0.3 μM. In other words , MS023 more potently abrogated GR-induced metabolic abnormalities in this assay.

[0086] (Example 4. Inactivation of cytotoxicity generated by MS023 (1 - 60 μM) as well as GR 15 and PR 15 (300 nM) measured by the LDH assay) ​As in Example 1, cells were plated in the medium. The day after, immediately before adding the test compound, the existing medium was removed and replaced with alternating amounts of medium as in Example 1. MS0 23 of the 10 mM stock was thawed to room temperature and diluted as in Example 1. GR 15 of 10 mM stock and PR 15 of the 10 mM stock were equilibrated to room temperature and diluted with warm medium to a final concentration of 300 nM in the wells.

[0087] The following conditions were plated in triplicate. To prevent evaporation of the volume within the experimental wells during incubation, the samples were surrounded by outer border wells filled with sterile phosphate-buffered saline: · Background (medium only) · Low control / "0% toxicity" (cells in medium only) · DMSO control (cells treated with only the amount of DMSO to which GR- and PR-treated cells were exposed) · GR only (cells treated with 300 nM GR only) · GR only (cells treated with 300 nM GR only) 15 · GR only (cells treated with 300 nM GR only) 15 and MS023 at any one of the doses: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM · PR only (cells treated with 300 nM PR 15 only) · PR only (cells treated with 300 nM PR 15 only) and MS023 at any one of the doses: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM · MS023 at any one of the doses: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM · High control / "100% toxicity" (cells lysed with lysis buffer) · Positive control (5 μL LDH solution)

[0088] The plate was incubated at 37 °C and 5% CO2 for 24 hours. The cells were tested and the data were analyzed by LDH assay.

[0089] As shown in FIGS. 4A to 4D, when NSC-3 15 4 cells treated with 300 nM dose of GR were given, the two highest doses of MS023 (60, 30 μM) significantly amplified cytotoxicity in the LDH a ssay. This phenotype was not seen in NSC-34 cells treated with 300 nM PR 15 and at all doses of MS023, at least partially abrogated PR-induced cytotoxicity. These results suggest a difference in the mechanism between the two arginine-rich D RPs. This phenomenon was only observed at 300 nM and not at the 3 μM dose of each ta n protein (data shown in Experiment 5).

[0090] (Example 5. Inactivation of cytotoxicity generated by MS023 (1 - 60 μM) and GR 15 and PR 15 (3 μM) measured by the LDH assay) The cells were plated in the medium of two 96-well plates at a density of 3.7×10 4 cells per well and incubated overnight at 37 °C and 5% CO2. The next day, just before adding the test compounds, the existing medium was removed and replaced with alternating amounts of medium as in Example 1 . The 10 mM stock of MS023 was thawed to room temperature and diluted as in Example 1. The 10 mM stock of GR 15 and the 10 mM stock of PR 15 were equilibrated to room temperature and warm culture ​​It was diluted in the local area to make the final concentration in the well 3 μM.

[0091] The following conditions were plated in triplicate. To prevent evaporation of the volume within the experimental wells during incubation, the samples were surrounded by outer boundary wells filled with sterile phosphate-buffered saline: · Background (medium only) · Low control / "0% toxicity" (cells in medium only) · DMSO control (cells treated with only the amount of DMSO to which GR- and PR-treated cells were exposed) · GR only (cells treated with 3 μM GR only) 15 · 3 μM GR 15 and MS023: cells treated with any one of the doses of 60 μM, 30 μM, 10 μM, 3 μM, 1 μM · PR only (cells treated with 3 μM PR only) 15 · 300 nM PR 15 and MS023: cells treated with any one of the doses of 60 μM, 30 μM, 10 μM, 3 μM, 1 μM · MS023: cells treated with only any one of the doses of 60 μM, 30 μM, 10 μM, 3 μM, 1 μM · High control / "100% toxicity" (cells lysed with lysis buffer) · Positive control (5 μL LDH solution)

[0092] The plates were incubated at 37 °C and 5% CO2 for 24 hours. The cells were tested and the data were analyzed by LDH assay.

[0093] As shown in FIGS. 5A-5D, MS023 induced GR at all doses tested. ​​​​​​​​​Partially inactivated toxicity. MS023 induced PR at doses of 10, 30, and 60 μM Partially inactivated toxicity and completely inactivated PR-induced toxicity at doses of 1 and 3 μM. G Differences in the inactivation ability of MS023 in GR-treated cells and PR-treated cells (as seen in Example 4) Suggest further different mechanisms in GR and PR of arginine-rich DRP .

[0094] (Example 6. Inactivation of cytotoxicity generated by MS023 (0.01 - 3 μM) and GR and PR 15 measured by the LDH assay 15 (3 μM)) Cells were plated in the medium of two 96-well plates at a density of 3.7×10 4 cells per well and incubated overnight at 37 °C and 5% CO2. The next day, just before adding the test compounds, the existing medium was removed and replaced with alternating amounts of medium as in Example 1 . A 10 mM stock of MS023 was thawed to room temperature and diluted with warm medium to a final concentration of 0.01, 0.03, 0.1, 0.3, 1, and 3 μM in the plate . A 10 mM stock of GR1 5 and a 10 mM stock of PR were equilibrated to room temperature and diluted with warm medium 15 to a final concentration of 3 μM in the well .

[0095] The following conditions were plated in triplicate. To prevent evaporation of the volume within the experimental wells during incubation, the samples were surrounded by outer boundary wells of the plate filled with sterile phosphate-buffered saline : · Background (medium only) · Low control / "0% toxicity" (cells in medium only) · High control / "100% toxicity" (cells + GR1 · DMSO control (cells treated with only the amount of DMSO to which GR- and PR-treated cells were exposed) · GR only (cells treated with 3 μM GR only) 15 · 3 μM GR 15 and MS023: cells treated with any one of the doses of 3 μM, 1 μM, 0.3 μM, 0.1 μ M, 0.03 μM, 0.01 μM · PR only (cells treated with 3 μM PR only) 15 · 3 μM PR 15 and MS023: cells treated with any one of the doses of 3 μM, 1 μM, 0.3 μM, 0.1 μ M, 0.03 μM, 0.01 μM · MS023: cells treated with only any one of the doses of 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0. 01 μM · High control / "100% toxicity" (cells lysed with lysis buffer) · Positive control (5 μL LDH solution)

[0096] The plates were incubated at 37 °C and 5% CO2 for 24 hours. The cells were tested and the data were analyzed using the procedure described in the instructions of the LDH assay kit.

[0097] As shown in Figures 6A - 6D, MS023 potently and dose-dependently abrogated both GR - and PR-induced toxicity. MS023 partially abrogated GR-induced toxicity at doses of 0.01, 0.03, 0.1, and 0.3 μM and completely abrogated GR-induced toxicity at doses of 0.3, 1, and 3 μM. MS023 partially abrogated PR-induced toxicity at doses of 0.01, 0.03, and 0.1 μM and completely abrogated PR-induced toxicity at doses of 0.03, 0.1, 0.3, 1, and 3 μM. ​​​The PR-induced toxicity was completely abolished at a dose of μM. Consistent with the previous example, MS023 more potently abolished the PR-induced toxicity.

[0098] (Example 7. MS023 (1 - 60 μM) and the abrogation of apoptosis activity induced by GR 15 and PR 15 (300 nM)) ) Cells were plated in the medium of two 96-well plates at a density of 3.7×10 4 cells per well and incubated overnight at 37 °C, 5% CO2. The next day, just prior to adding the test compounds, the existing medium was removed and replaced with alternating amounts of medium as in Example 1. A 10 mM stock of MS023 was thawed to room temperature and diluted as in Example 1. A 10 mM stock of GR 15 and a 10 mM stock of PR 15 were equilibrated to room temperature and diluted in warm medium to a final concentration of 300 nM in the wells.

[0099] The following conditions were plated in triplicate. To prevent evaporation of the volume within the experimental wells during incubation, the samples were surrounded by outer boundary wells of the plate filled with sterile phosphate-buffered saline: · Background (medium only) · Untreated / “cells only” (cells in medium) · DMSO control (cells treated with only the amount of DMSO to which the GR- and PR-treated cells were exposed) · GR only (cells treated with 300 nM GR only) · 3 μM GR 15 and of MS023: 60 μM, 30 μM, 10 μM, 3 μM 15 Cells treated with any one of the doses of 1 μM ·PR only (300 nM PR 15 cells treated only with) ·300 nM PR 15 and MS023 among: 60 μM, 30 μM, 10 μM, 3 cells treated with any one of the doses of μM, 1 μM ·Among MS023: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM, any cells treated with only one of) ·Positive control (cells treated with 5 or 16 μM of PAC-1 caspase-3 activ ator)

[0100] The plates were incubated at 37 °C and 5% CO2 for 24 hours. The cells were tested and the data were analyzed using the procedure described in the caspase-3 assay kit instructions.

[0101] As shown in Figures 7A - 7D, MS023 dose-dependently abrogated GR- and PR-induced apoptotic activity. For the apoptotic activity induced by 300 nM GR, MS023 at doses of 60, 30, and 10 μM partially abrogated it, and MS023 at doses of 3 and 1 μM completely abrogated it. All doses of MS023 completely abrogated the PR-induced apoptotic activity.

[0102] (Example 8. MS023 (0.01 - 3 μM) and the abrogation of apoptotic activity induced by GR and PR 15 measured in the caspase-3 assay 15 (3 μM)) Cells were seeded into the medium of two 96-well plates at a density of 3.7×10 4 cells per well The plates were plated and incubated overnight at 37°C, 5% CO2. The next day, test compounds were Just before addition, the existing medium was removed and replaced with staggered amounts of medium as in Example 1. Thaw a 10 mM stock of MS023 to room temperature and dilute it in warm medium to plate. The final concentrations in GR were 3, 1, 0.3, 0.1, 0.03 and 0.01 μM. 15 10 mM stock of and PR 15 Equilibrate 10 mM stock of 100 mM acetaminophen to room temperature and add warm medium Dilutions were made to give a final concentration in the well of 3 μM.

[0103] The following conditions were plated in triplicate: To prevent evaporation of the volume within the tube, the samples were placed in a tube filled with sterile phosphate buffered saline. Rate outer boundary well enclosed: Background (medium only) Untreated / "cells only" (cells in medium) DMSO control (GR- and PR-treated cells were treated with DMSO alone at the dose (treated cells) GR only (3μM GR 15 (cells treated only with 3μM GR 15 and of MS023: 3 μM, 1 μM, 0.3 μM, 0.1 μ Cells treated with either 0.01 μM, 0.03 μM, or 0.01 μM PR only (3μM PR 15 (cells treated only with 3μM PR 15 and of MS023: 3 μM, 1 μM, 0.3 μM, 0.1 μ Cells treated with either 0.01 μM, 0.03 μM, or 0.01 μM Of MS023: 3μM, 1μM, 0.3μM, 0.1μM, 0.03μM, 0. Cells treated with only any one of the doses of 01 μM · Positive control (cells treated with 5 or 16 μM of PAC-1 caspase-3 activ ator)

[0104] The plates were incubated at 37 °C and 5% CO2 for 24 hours. The cells were tested and the data were analyzed using the procedure described in the caspase-3 assay kit instructions. As shown in Figures 8A to 8D, MS023 dose-dependently abrogated GR- and PR-induced

[0105] apoptotic activity. In both GR- and PR-treated cells, all doses of MS023 partially abrogated GR- and PR-induced apoptotic activity, and a dose of 3 μM of MS023 completely abrogated GR- and PR-induced apoptotic activity.

[0106] (Example 9. Inactivation of growth inhibition induced by MS023 (1 - 60 μM) and GR and PR 15 measured by BrdU ELISA 15 (300 nM)) Cells were plated in the medium of two 96-well plates at a density of 3.7×10 4 cells per well and incubated overnight at 37 °C and 5% CO2. The next day, just before adding the test compounds, the existing medium was removed and replaced with alternating amounts of medium as in Example 1. A 10 mM stock of MS023 was thawed to room temperature and diluted as in Example 1. Stocks of 10 mM GR and 10 mM PR 15 were equilibrated to room temperature and diluted in warm medium to a final concentration of 300 nM in the wells. 15

[0107] The following conditions were plated in triplicate: To prevent evaporation of the volume within the tube, the samples were placed in a tube filled with sterile phosphate buffered saline. Rate outer boundary well enclosed: Medium only Background (no BrdU reagent, just cells in medium) Untreated / (BrdU reagent included, cells only in medium) DMSO control (GR- and PR-treated cells were treated with DMSO alone at the dose (treated cells) and BrdU reagent GR only (300nM GR 15 and BrdU reagent-treated cells) 300nM GR 15 , BrdU reagent and MS023: 60 μM, 30 μM Cells treated with either 10 μM, 3 μM, or 1 μM doses of PR only (300nM PR 15 and BrdU reagent-treated cells) 300nM PR 15 , BrdU reagent and MS023: 60 μM, 30 μM Cells treated with either 10 μM, 3 μM, or 1 μM doses of BrdU reagent and MS023: 60μM, 30μM, 10μM, 3μM, 1 Cells treated with either one of the μM doses

[0108] The plates were incubated at 37°C, 5% CO2 for 24 hours. Cells were tested for Brd Data were analyzed using the procedures described in the U ELISA kit instructions.

[0109] As shown in Figures 9A-9D, MS023 inhibited IL-18 at all doses tested. Abolishes GR- and PR-induced growth inhibition without inducing excessive proliferation At all doses of MS023, it partially abrogated GR-induced growth inhibition, and at a dose of 3 μM of MS023, it completely rescued the GR-induced growth inhibition. All doses of MS023 completely abrogated PR-induced growth inhibition.

[0110] (Example 10. Chinese Hamster Ovary (CHO) and Mouse Neuroblastoma-Spinal Cord Hybrid (NSC-34) Cells, and Arginine-Rich: GR 15 , PR 15 and Non-Arginine-Rich: GP 15 , PA 15 , DRP (30 and 3 μM Doses) for Comparison of DRP-Induced Metabolic Abnormal Phenotypes in Neuronal and Non-Neuronal Cell Types Measured by the WST-1 Assay) Cells were plated at a density of 200 μL of medium per well in two 96-well plates. In Plate 1, CHO cells were plated at a density of 1 × 10 4 cells / well, and in Plate 2 NSC-34 cells were plated at a density of 2.5 × 10 4 cells / well and incubated overnight at 37 °C , 5% CO2. In previous assays, CHO cells had been demonstrated to grow 2.5-fold faster than NSC-34 cells, so the two cell types were plated at different densities. The next day, stocks of GR 15 , PR 15 , GP 15 , and PA 15 at 10 mM were equilibrated at room temperature and diluted in warm medium to concentrations of 60 0 μM (30 μM on the plate) and 60 μM (3 μM on the plate) for each protein.

[0111] The following conditions were plated in triplicate. To prevent evaporation of the volume within the experimental well during incubation, the samples were surrounded by outer border wells filled with sterile phosphate buffered saline: Untreated (cells only in medium) · DMSO control (cells treated with only the amount of DMSO to which the DRP-treated cells were exposed) i. DMSO control 1: 0.3% DMSO corresponding to a DRP dose of 30 μM ii. DMSO control 2: 0.03% DMSO corresponding to a DRP dose of 3 μM O · Cells treated with 30 or 3 μM GR 15 · Cells treated with 30 or 3 μM PR 15 · Cells treated with 30 or 3 μM PA 15 · Cells treated with 30 or 3 μM GP 15

[0112] The plates were incubated at 37 °C, 5% CO2 for 48 hours. Immediately before the test, the medium was removed and replaced with 200 μL of PBS-glucose solution (4.5 g / L, sterilized) that had been warmed from 4 °C to 10 minutes in a 37 °C water bath before use. The aliquot of WST-1 reagent was thawed from -20 °C and equilibrated at room temperature before use. 20 μL of WST-1 reagent was added to each well containing 200 μL of PBS-glucose. The plates were incubated with WST-1 at 37 °C, 5% CO2 for 1 hour, and absorbance readings (450 nm) were obtained every 15 minutes using a Molecular Devices plate reader (SpectraMax M3). The data was analyzed using SoftMaxPro of the plate reader. -1 at 37 °C, 5% CO2 for 1 hour, and absorbance readings were taken every 15 minutes using a Molecular Devices plate reader (SpectraMax M3). The data was analyzed using SoftMaxPro of the plate reader. ​​​​​​​​​​​​​Exported from 7.0 software to an Excel file.

[0113] As shown in FIGS. 10A - 10D, the DRP challenge resulted in a metabolic dysfunction in both non - neuronal CHO and motoneuron - like NSC - 34 cells. However, a significant decrease in metabolic activity was observed in NSC - 34 compared to CHO. These observations were only seen in cells treated with arginine - rich DRP GR and PR 15 and suggested that motoneuron - like NSC - 34 is not only more sensitive to the DRP challenge but is particularly 15 sensitive to an arginine - rich DRP challenge.

[0114] (Example 11. Dose - response pattern of DRP - induced cytotoxicity in NSC - 34 cells determined by LDH assay) Cells were plated in the medium of two 96 - well plates at a density of 5×10 4 cells per well and incubated overnight at 37 °C, 5% CO2. The next day, stocks of GR 15 , PR1 , GP 15 , and PA 15 at 10 mM were equilibrated at room temperature and diluted in warm medium to give DRP concentrations of: 30, 10, 3, 1, 0.3, 0.1 μM (10 - fold the concentrations achieved in the plate). 10 μL of each concentration was added to 100 μL of the base medium in the plate to give final DRP concentrations of 3, 1, 0.3, 0.1, 0.03, and 0.01 μM.

[0115] The following conditions were plated in duplicate. During incubation, the experimental wells ​​​​​​To prevent evaporation of the volume within the well, the sample was surrounded by the outer boundary wells of the plate filled with sterile phosphate buffered saline: : · Background (media only) · Low control / "0% toxicity" (cells only in media) · DMSO control (cells treated with only the amount of DMSO to which DRP - treated cells were exposed at each concentration) i. The DMSO controls tested were as follows and corresponded to the starting DRP doses ( from a maximum of 3 μM to a minimum of 0.01 μM) and were diluted in media. 1. 0.03% DMSO 2. 0.01% DMSO 3. 0.003% DMSO 4. 0.001% DMSO 5. 0.0003% DMSO 6. 0.0001% DMSO · Cells treated with any one of the doses 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM of GR 15 · Cells treated with any one of the doses 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM of PR 15 · Cells treated with any one of the doses 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM of GP 15 · Cells treated with any one of the doses 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM of PA 15 · High control / "100% toxicity" (cells lysed with lysis buffer) · Positive control (5 μL LDH solution)

[0116] The plates were incubated at 37 °C, 5% CO₂ for 24 hours. The cells were tested for LDH​​​​​ Data were analyzed using the procedures described in the assay kit instructions.

[0117] As shown in Figure 11, arginine-rich DRP (GR 15 , PR 15 ) treatment resulted in significant dose-dependent toxicity to NSC-34 cells when incubated for 24 hours, while non-arginine-rich DRP (GP , PA 15 , 15 ) treatment did not. Approximately 10 % toxicity of GR 15 and PR 15 was achieved using a low concentration of 300 nM. GR 15 and PR 15 showed nearly identical dose-response profiles, and the EC50 values of GR 15 and P R 15 were 50 ± 26 nM and 56 ± 20 nM, respectively.

[0118] (Example 12. Dose-response pattern of DRP-induced apoptotic activity in NSC-34 cells determined by caspase-3 assay) Cells were plated in the medium of two 96-well plates at a density of 5 × 10 cells per well and incubated overnight at 37 °C and 5% CO2. The next day, 10 mM stocks of GR 4 , PR1 , GP 15 , and PA 15 , 15 and PA were equilibrated at room temperature and diluted with warm medium to make the DRP concentrations: 30, 10, 3, 1, 0.3, 0.1 μM (10 times the concentrations achieved in the plate). 10 μL of each concentration was added to 100 μL of base medium in the plate to make the final DRP concentrations 3, 1, 0.3, 0.1, 0.03, and 0.01 μM.

[0119] The following conditions were plated in triplicate. To prevent evaporation of the volume within the experimental well during incubation, the samples were surrounded by outer boundary wells filled with sterile phosphate buffered saline: · Background (media only) · Untreated / "cells only" (cells in media) · DMSO control (cells treated with only the amount of DMSO to which the DRP-treated cells were exposed at each concentration) i. The DMSO controls tested were as follows and corresponded to the starting DRP doses ( from a maximum of 3 μM to a minimum of 0.01 μM) and were diluted in media. 1. 0.03% DMSO 2. 0.01% DMSO 3. 0.003% DMSO 4. 0.001% DMSO 5. 0.0003% DMSO 6. 0.0001% DMSO · Cells treated with any one of the doses 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM of GR 15 · Cells treated with any one of the doses 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM of PR 15 · Cells treated with any one of the doses 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM of GP 15 · Cells treated with any one of the doses 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM of PA 15 · Positive control (cells treated with 5 or 16 μM of PAC-1 caspase-3 activator) ​​​​​​​​

[0120] The plate was incubated at 37 °C in 5% CO2 for 24 hours. The cells were tested and data were analyzed using the procedure described in the caspase-3 assay kit instructions.

[0121] As shown in Figure 12, all DRPs induced apoptotic activity in NSC-34 cells in a dose-dependent manner. The arginine-rich DRPs GR and PR 15 and 15 clustered together and produced the most significant amount of apoptotic activity. The non-arginine-rich GR and PR 15 also clustered together and had lower levels of apoptotic activity compared to the control 15 treated with DMSO.

[0122] (Dose-response pattern of DRP-induced growth inhibition in NSC-34 cells determined by Example 13. BrdU ELISA) Cells were plated in the medium of two 96-well plates at a density of 5 × 10 4 cells per well and incubated overnight at 37 °C in 5% CO2. The next day, the 10 mM stocks of GR and PR1 15 5, GP 5, and PA 15 5 were equilibrated at room temperature and diluted with warm medium 15 to give DRP concentrations of: 30, 10, 3, 1, 0.3, 0.1 μM (10-fold the concentrations achieved in the plates). 10 μL of each concentration was added to 100 μL of base medium in the plate to give final DRP concentrations of 3, 1, 0.3, 0.1, 0.03, and 0.01 μM.

[0123] The following conditions were plated in triplicate. To prevent evaporation of the volume within the experimental well during incubation, the samples were surrounded by outer boundary wells filled with sterile phosphate-buffered saline: · Medium only · Background (cells in medium only, without BrdU reagent) · Untreated / (cells in medium only, with BrdU reagent) · DMSO control (cells treated with DMSO in the amount that BrdU reagent and DRP-treated cells were exposed to at each concentration) i. The DMSO controls tested were as follows and corresponded to the starting DRP doses (from a maximum of 3 μM to a minimum of 0.01 μM) and were diluted in medium. 1. 0.03% DMSO 2. 0.01% DMSO 3. 0.003% DMSO 4. 0.001% DMSO 5. 0.0003% DMSO 6. 0.0001% DMSO · Cells treated with BrdU reagent and any one of the doses of 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM of GR 15 · Cells treated with BrdU reagent and any one of the doses of 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM of PR 15 · Cells treated with BrdU reagent and any one of the doses of 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM of GP 15 · Cells treated with BrdU reagent and any one of the doses of 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM of PA 15

[0124] ​​​​​​​​​​​​The plate was incubated at 37 °C and 5% CO2 for 24 hours. The cells were tested and data were analyzed using the procedure described in the caspase-3 assay kit's instructions.

[0125] As shown in Figure 13, non-arginine-rich DRP GR 15 and PR 15 did not significantly affect the proliferation activity compared to the control treated with DMSO. Arginine-rich DRP GR 15 and PR 15 both significantly inhibited the proliferation activity in a dose-dependent manner, and PR 15 was the most potent inhibitor.

[0126] (Example 14. MS023 reduces the asymmetric arginine methylation of NSC-34 cells) On day 0, NSC-34 cells were plated in 48 wells of a 96-well plate, and CHO cells were plated in the other 48 wells of the same 96-well plate. The NSC- 34 cells were plated in DMEM / high glucose medium. The CHO cells were plated in Ham's F-12K (Kaighn's) medium. Both cell lines were incubated overnight to reach a culture density of approximately 50%.

[0127] On day 1, 24 wells of NSC-34 cells and 24 wells of CHO cells were treated with MS023 at concentrations of 1 μM, 3 μM, 6 μM, 10 μM, 30 μM, and 60 μM. The cells were then incubated overnight.

[0128] On day 2, 18 wells of untreated NSC-34 cells and 18 wells of untreated CHO cells was treated with MS023 at the same concentration as on the first day. As a result, each cell line without receiving MS023 had 6 wells remaining. After adding MS023, the cells were incubated overnight in an incubator.

[0129] On the third day, all the medium in the 96-well plates was removed, and the cells were fixed with 3.7% paraformaldehyde (PFA). Then, the cells were washed and permeabilized with 1×PBS / 0.1% Trit onX-100.

[0130] After removing the PBS / Triton wash solution, Odyssey blocking buffer ( LI-COR, 927-40100) was applied to all wells, and the plates were gently shaken at room temperature for 90 minutes. After 90 minutes, the blocking buffer was removed, and the cells were treated with a 1:50 0 dilution of the anti-asymmetric dimethylarginine motif primary antibody (Cell Signaling Technology , #13522). Incubation with the above primary antibody was continued overnight at 4 degrees Celsius without shaking.

[0131] On the fourth day, the primary antibody solution was removed from the plates, and the cells were washed with 1×PBS / 0.1% Tween 20. After washing, the cells were subjected to a secondary antibody mixture containing a 1:1000 dilution of IRDye 800 CW anti-rabbit antibody (LI-C OR, 926-32211) and a 1:500 dilution of Cell Tag 700 S tain (LI-COR, 926). The secondary antibody mixture was gently shaken and left on the cells for 60 minutes while protecting from light . After 60 minutes, the secondary antibody mixture was removed, and the cells were washed again with 1×PBS / 0.1% Tween20

[0132] ​Washed with [solution]. Then, after removing all the washing buffer, the plate was gently tapped to dry and any excess solution was removed. and the plate was read using the LI-COR Odyssey Classic Imaging System, which provides fluorescence-based results. In this experiment, fluorescence occurred as a result of asymmetric arginine methylation of the cells. MS023 was applied to the cells for 24 hours or 48 hours in anticipation of reducing asymmetric arginine methylation of the cells. The number of cells per well was quantified using Cell Tag 700 Stain, which fluorescently labels the total protein level within the cells. Regarding this effect, the fluorescence from asymmetric arginine methylation (800) per well was divided by the fluorescence from the total protein level (700) per well to generate an 800 / 700 result. As shown in Figure 14, all conditions were significantly reduced in their 8 00 / 700 readout information when compared to untreated cells. With 24 hours of treatment, MS02 3 reduced asymmetric arginine methylation of NSC-34 cells by approximately 13% at 1 μM and approximately 45% at 60 μM.

[0133] With 48 hours of treatment, MS023 reduced asymmetric arginine methylation of NSC-34 cells by approximately 33% at 1 μM and approximately 75% at 60 μM. 00 / 700 readout information when compared to untreated cells. With 24 hours of treatment, MS02 3 reduced asymmetric arginine methylation of NSC-34 cells by approximately 13% at 1 μM and approximately 45% at 60 μM. With 48 hours of treatment, MS023 reduced asymmetric arginine methylation of NSC-34 cells by approximately 33% at 1 μM and approximately 75% at 60 μM. symmetric arginine methylation of NSC-34 cells by approximately 33% at 1 μM and approximately 75% at 60 μM.

[0134] (Example 15. In Vitro Arginine Methylation of Synthetic GR 15 ) To evaluate the interaction between PRMT1 and GR 15 an in vitro methylation assay system was developed. This system consists of a recombinant PRMT1 enzyme, S-adenosyl-L-methionine as the methyl donor S-(5‘-Adenosyl)-L-methionine iodide (SAM), and potential substrates of PRMT1 activity consists of a mixture of potential substrates. In this experiment, 11 tubes were prepared, and each tube contained various amounts of the following components: · Recombinant PRMT1 protein (Active Motif, Cat. 31411) · S-(5‘-Adenosyl)-L-methionine iodide (SAM) (Sigma-Aldrich Cat. A4377) · GR 15 · PR 15 · SOD1 from human erythrocytes (S9636-1KU)

[0135] The above components were mixed in 0.5 ml flat cap tubes (Thermo Fisher Scientific, AB0350) according to Table 4 (all values are in microliters (μl)).

[0136]

Table 4

[0137] After all the tubes were prepared, each tube was tapped gently to ensure thorough mixing, and the tubes were placed in an incubator at 37 °C for 2 hours. After 2 hours, 10 μl of NuPage L DS sample buffer (Thermo Fisher Scientific, NP00 07) was added to stop the reaction mixture. The tubes were tapped gently to ensure thorough mixing, and boiled in a water bath at 95 °C for 5 minutes. Then, the samples were processed for SDS-PAGE on a 4-12% Bis-Tris gel (Thermo Fisher Scientific, NP0322BOX). ​

[0138] Next, the gel was transferred using an iBlot device and an iBlot 2 nitrocellulose mini-stack (The rmo Fisher Scientific, IB23002). After transfer, the membrane was placed in Superblock blocking buffer (Thermo Fis her Scientific, 37515) and blocked overnight at 4°C. The next day, the blocking buffer was removed, and a primary antibody solution containing anti-Histone4 H4R3me2a (Active Moti f, catalog 39006) at a ratio of 1:500 and anti-C9ORF72 / C9RAN T (poly-GR) antibody (Millipore, mABN778) at a ratio of 1:1000 in Superblock / 0.2% Tween20 was applied to the membrane and gently shaken at room temperature for 60 minutes. The primary antibody solution was removed, and the membrane was washed with 1×P BS / 0.1% Tween20. A secondary antibody solution containing IRDye 800 CW anti-rabbit antibody diluted 1:10,000 and IRDye 680 RD anti-rat antibody diluted 1: 10,000 (LI-COR, 925-6 8076) in Superblock / 0.2% Twee n20 was applied to the membrane and gently shaken at room temperature for 60 minutes. The secondary antibody was removed, and the membrane was washed with 1×PBS / 0.1% Tween20. Following the washing step the membrane was read using a LI-COR Odyssey Classic ImagingSyst em. As shown in FIGS. 15A and 15B, GR is asymmetrically methylated by PRMT1 and detected by the anti-H4R3me2a histone 4 antibody. This antibody was used to

[0139] the g 15 asymmetrically methylated by PRMT1 and detected by the anti-H4R3me2a histone 4 antibody. This antibody was used to methylated by PRMT1 and detected by the anti-H4R3me2a histone 4 antibody. This antibody was used to Generated against the region of histone 4 protein containing lysine-arginine pairs. P When either RMT1 or the methyl donor SAM is removed from the reaction mixture, GR1 Asymmetric methylation of 5 does not proceed (lanes 2 and 9 in Figure 15A). Figure 15B shows that GR 15 is present in all wells, thus indicating that its methylation is dependent on the interaction with PRMT1 .

[0140] (Example 16. MS049 (0.2 - 100 μM) and cytotoxicity abrogation generated by GR and PR 15 measured in the LDH assay) 15 (3 μM)) Cells were plated in the medium of two 96-well plates at a density of 3.7×10 4 cells per well and incubated overnight at 37 °C and 5% CO2. The next day, just before adding the test compounds, the existing medium was removed and replaced with alternating amounts of medium as in Example 1 . A 10 mM stock of MS049 was thawed to room temperature and diluted with warm medium to give final concentrations in the plate of 0.2, 1, 2, 10, 20 and 100 μM. A 10 mM stock of GR and a 10 mM stock of PR were equilibrated to room temperature and diluted with warm medium to give final concentrations in the wells of 3 μM. 15 10m M stock of GR 15 and 10 mM stock of PR were equilibrated to room temperature and diluted with warm medium to give final concentrations in the wells of 3 μM.

[0141] The following conditions were plated in triplicate. To prevent evaporation of the volume within the experimental wells during incubation, samples were surrounded by outer boundary wells filled with sterile phosphate-buffered saline: · Background (medium only) · Low control / "0% toxicity" (cells in medium only) · Background (medium only) · Low control / "0% toxicity" (cells in medium only) · DMSO control (cells treated with only the amount of DMSO to which GR- and PR-treated cells were exposed) · GR only (cells treated with 3 μM GR only) · GR only (cells treated with 3 μM GR only) 15 · GR only (cells treated with 3 μM GR only) · 3 μM GR and MS049, at any one of the doses: 100 μM, 20 μM, 10 μM, 2 μM, 1 μM, 0.2 μM 15 · 3 μM GR and MS049, at any one of the doses: 100 μM, 20 μM, 10 μM, 2 μM, 1 μM, 0.2 μM · 3 μM GR and MS049, at any one of the doses: 100 μM, 20 μM, 10 μM, 2 μM, 1 μM, 0.2 μM · PR only (cells treated with 3 μM PR only) 15 · PR only (cells treated with 3 μM PR only) · 3 μM PR and MS049, at any one of the doses: 100 μM, 20 μM, 10 μM, 2 μM, 1 μM, 0.2 μM 15 · 3 μM PR and MS049, at any one of the doses: 100 μM, 20 μM, 10 μM, 2 μM, 1 μM, 0.2 μM · 3 μM PR and MS049, at any one of the doses: 100 μM, 20 μM, 10 μM, 2 μM, 1 μM, 0.2 μM · MS049 only, at any one of the doses: 100 μM, 20 μM, 10 μM, 2 μM, 1 μM, 0.2 μM · MS049 only, at any one of the doses: 100 μM, 20 μM, 10 μM, 2 μM, 1 μM, 0.2 μM · High control / “100% toxicity” (cells lysed in lysis buffer) · Positive control (5 μL LDH solution)

[0142] The plates were incubated at 37 °C and 5% CO2 for 24 hours. The cells were assayed and the data were analyzed using the procedure described in the LDH assay kit instructions. The plates were incubated at 37 °C and 5% CO2 for 24 hours. The cells were assayed and the data were analyzed using the procedure described in the LDH assay kit instructions.

[0143] As shown in Figures 16A–16D, MS049 abrogated the induced toxicity of both GR and PR in a dose-dependent manner. MS049 partially abrogated GR- and PR-induced toxicity at doses of 0.2, 1, 2, 20, and 100 μM and completely abrogated GR- and PR-induced toxicity at doses of 2 and 10 μM. MS049 caused significant toxicity at doses of 100 and 20 μM independent of GR and PR treatment, but not at the other doses tested. As shown in Figures 16A–16D, MS049 abrogated the induced toxicity of both GR and PR in a dose-dependent manner. MS049 partially abrogated GR- and PR-induced toxicity at doses of 0.2, 1, 2, 20, and 100 μM and completely abrogated GR- and PR-induced toxicity at doses of 2 and 10 μM. MS049 caused significant toxicity at doses of 100 and 20 μM independent of GR and PR treatment, but not at the other doses tested. As shown in Figures 16A–16D, MS049 abrogated the induced toxicity of both GR and PR in a dose-dependent manner. MS049 partially abrogated GR- and PR-induced toxicity at doses of 0.2, 1, 2, 20, and 100 μM and completely abrogated GR- and PR-induced toxicity at doses of 2 and 10 μM. MS049 caused significant toxicity at doses of 100 and 20 μM independent of GR and PR treatment, but not at the other doses tested. As shown in Figures 16A–16D, MS049 abrogated the induced toxicity of both GR and PR in a dose-dependent manner. MS049 partially abrogated GR- and PR-induced toxicity at doses of 0.2, 1, 2, 20, and 100 μM and completely abrogated GR- and PR-induced toxicity at doses of 2 and 10 μM. MS049 caused significant toxicity at doses of 100 and 20 μM independent of GR and PR treatment, but not at the other doses tested. As shown in Figures 16A–16D, MS049 abrogated the induced toxicity of both GR and PR in a dose-dependent manner. MS049 partially abrogated GR- and PR-induced toxicity at doses of 0.2, 1, 2, 20, and 100 μM and completely abrogated GR- and PR-induced toxicity at doses of 2 and 10 μM. MS049 caused significant toxicity at doses of 100 and 20 μM independent of GR and PR treatment, but not at the other doses tested. As shown in Figures 16A–16D, MS049 abrogated the induced toxicity of both GR and PR in a dose-dependent manner. MS049 partially abrogated GR- and PR-induced toxicity at doses of 0.2, 1, 2, 20, and 100 μM and completely abrogated GR- and PR-induced toxicity at doses of 2 and 10 μM. MS049 caused significant toxicity at doses of 100 and 20 μM independent of GR and PR treatment, but not at the other doses tested.

[0144] (Example 17. MS049 reduces asymmetric arginine methylation in NSC-34 cells ) On day 0, NSC-34 cells were plated in 48 wells of a 96-well plate, and CHO cells were plated in the other 48 wells of the same 96-well plate. The NSC- 34 cells were plated in DMEM / high glucose medium. The CHO cells were plated in Ham's F-12K (Kaighn's) medium. Both cell lines were incubated overnight to reach a culture density of approximately 50%.

[0145] On day 1, 24 wells of NSC-34 cells and 24 wells of CHO cells were treated with MS049 at concentrations of 0.2 μ M, 1 μM, 2 μM, 10 μM, 20 μM, and 100 μM. The cells were then incubated overnight.

[0146] On day 2, 18 wells of untreated NSC-34 cells and 18 wells of untreated CHO cells were treated with MS049 at the same concentrations as on day 1. This left six wells each of the cell lines that had received MS049 and those that had not. After adding MS049, the cells were incubated overnight.

[0147] On day 3, all the medium in the 96-well plate was removed, and the cells were fixed with 3.7% paraformaldehyde (PFA). The cells were then washed and permeabilized with 1× PBS / 0.1% Trit on X-100.

[0148] After removing the PBS / Triton wash solution, Odyssey blocking buffer ( LI-COR, 927-40100) was applied to all wells, and the plate was incubated at room temperature for 90 ​It was gently shaken for 90 minutes. After 90 minutes, the blocking buffer was removed, and the cells were diluted 1:50 with an anti-asymmetric dimethylarginine motif primary antibody (Cell Signaling Technology , #13522) at a dilution of 0. The incubation with the above primary antibody was continued overnight at 4°C without shaking.

[0149] On the 4th day, the primary antibody solution was removed from the plate, and the cells were washed with 1×PBS / 0.1% Tween 20. After washing, the cells were subjected to a secondary antibody mixture containing a 1:1000 dilution of IRDye 800 CW anti-rabbit antibody (LI-C OR, 926-32211) and a 1:500 dilution of Cell Tag 700 S tain (LI-COR, 926). The secondary antibody mixture was gently shaken and left on the cells for 60 minutes while protecting from light . After 60 minutes, the secondary antibody mixture was removed, and the cells were washed again with 1×PBS / 0.1% Tween20

[0150] . Then, after removing all the washing buffer, the plate was gently tapped dry to remove the excess solution. Then, the plate was read using a LI-COR Od yssey Classic ImagingSystem that provides fluorescence-based results. In this experiment, fluorescence was generated as a result of asymmetric arginine methylation of the cells. MS049 was expected to reduce the asymmetric arginine methylation of the cells, so MS049 was applied to the cells for 24 hours or 48 hours. The number of cells per well was counted using Cell Tag 700Stain that fluorescently labels the total protein level in the cells. Regarding this effect, the fluorescence from the asymmetric arginine methylation (800) per well was compared with that per well from the total protein level (700). from the total protein level (700). Dividing by the fluorescence from the total protein level (700) of Rino to generate the result of 800 / 700 was done.

[0151] As shown in Figure 17, all conditions were significantly decreased in their 8 00 / 700 readings when compared to untreated cells. With 24 hours of treatment, MS049 reduced the asymmetric arginine methylation of NSC-34 cells by approximately 17% at 1 μM and by approximately 39% at 100 μM.

[0152] (Example 18. Inactivation of metabolic abnormalities generated by MS023 (0.2 - 60 μM) and negative control MS0 94 (0.2 - 60 μM) and GR 15 and P R 15 (3 μM)) Cells were plated in the medium of a 96-well plate at a density of 3.7×10 4 cells per well and incubated overnight at 37 °C and 5% CO2. The next day, just before adding the test compounds, the existing medium was removed and replaced with alternating amounts of medium as in Example 1. M S023 and 10 mM stocks of MS094 were thawed to room temperature and diluted with warm medium to achieve final concentrations in the plate of 0.2, 1, 3, 10, 30, and 60 μM. GR 10 mM stocks of and 10 mM stocks of PR 15 were equilibrated to room temperature and diluted with warm medium to 15 achieve a final concentration in the well of 3 μM.

[0153] The following conditions were plated in triplicate. To prevent evaporation of the volume within the experimental wells during incubation, the samples were placed in plates filled with sterile phosphate-buffered saline to prevent evaporation of the volume within the experimental wells during incubation, the samples were placed in plates filled with sterile phosphate-buffered saline ​Surrounded by a boundary well outside the rate: · Background (medium only) · Untreated (cells in medium only) · DMSO control (DMSO equivalent to wells treated with MS094, MS09 4 and, GR 15 or PR 15 or DMSO equivalent to wells treated with GR 15 or PR 15 Cells treated with either DMSO equivalent to wells treated with GR ) · GR only (cells treated only with 3 μM GR 15 ) · 3 μM GR 15 and MS023 at one of the doses: 60 μM, 30 μM, 10 μM, 3 μM 、1 μM, 0.2 μM · 3 μM GR 15 and negative control MS094 at one of the doses: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.2 μM · PR only (cells treated only with 3 μM PR 15 ) · 3 μM PR 15 and MS023 at one of the doses: 60 μM, 30 μM, 10 μM, 3 μM 、1 μM, 0.2 μM · 3 μM PR 15 and negative control MS094 at one of the doses: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.2 μM · MS023 at one of the doses: 60 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.2 μM treated with only one of the doses · Negative control MS094 at one of the doses: 60 μM, 30 μM, 10 μM, 3 μM Cells treated with only one of the doses of 1 μM or 0.2 μM

[0154] The plates were incubated at 37 °C and 5% CO2 for 24 hours. Immediately before the test, the medium was removed and replaced with 200 μL of PBS - glucose solution (4.5 g / L, sterilized) that had been warmed from 4 °C in a 37 °C water bath. An aliquot of the WST - 1 reagent was thawed from - 20 °C and equilibrated at room temperature before use. 20 μL of the WST - 1 reagent was added to each well containing 200 μL of PBS - glucose The plates were incubated with WST - 1 at 37 °C and 5% CO2 for 1 hour, and one absorbance reading (450 nm) was obtained using a Molecular Devices SpectraMax M3 plate reader. The data was exported from the SoftMaxPro7.0 software of the plate reader to an Excel file. As shown in FIGS. 18A - 18F, MS023 abolished both GR - and PR - induced metabolic abnormalities in a dose - dependent manner. The 3 μM and 1 μM doses of MS023 completely abolished the metabolic abnormalities induced by a 3 μM G

[0155] R or PR challenge. None of the doses of the negative control MS094 abolished the metabolic abnormalities induced by a GR 15 or PR 15 challenge. The two highest doses of the negative control MS094 tested, 30 and 60 μM, caused metabolic abnormalities compared to the untreated control, which may be due to DMSO - related toxicity, and the DMSO 15 or PR 15 challenge. did not abolish the metabolic abnormalities induced by the challenge. The two highest doses of the negative control MS094 tested, 30 and 60 μM, caused metabolic abnormalities compared to the untreated control, which may be due to DMSO - related toxicity, and the DMSO ​The associated toxicity is also seen in the DMSO O controls corresponding to these doses (0.30% and 0.60% DMSO).

[0156] (Example 19. MS023 (0.2 - 60 μM) and negative control MS0 94 (0.2 - 60 μM), and the neutralization of cytotoxicity generated by GR 15 and PR1 (5 (3 μM))) Cells were plated in the medium of a 96 - well plate at a density of 3.7×10 4 cells per well and incubated overnight at 37 °C, 5% CO2. The next day, just prior to adding the test compounds, the existing medium was removed and replaced with alternating amounts of medium as in Example 1. M S023 and MS094 10 mM stocks were thawed to room temperature and diluted with warm medium to give final concentrations in the plate of 0.2, 1, 3, 10, 30 and 60 μM. GR 10 mM stocks and PR 10 mM stocks were equilibrated to room temperature and diluted with warm medium 15 to give a final concentration in the wells of 3 μM. 15

[0157] The following conditions were plated in triplicate. To prevent evaporation of the volume within the experimental wells during incubation, the samples were surrounded by border wells of the plate filled with sterile phosphate - buffered saline: · Background (medium only) · Untreated (cells in medium only) · DMSO control (equivalent DMSO to wells treated with MS094, MS09 4 and, GR 15 or PR 15 equivalent DMSO to wells treated with, or GR 15or PR 15 Cells treated with either DMSO equivalent to the wells treated with 15 ) ·GR only (3 μM GR 15 Cells treated only with 15 ) ·3 μM GR 15 And MS023: Cells treated with any one of the doses of 60 μM, 30 μM, 10 μM, 3 μM , 1 μM, 0.2 μM ·3 μM GR 15 And negative control MS094: Cells treated with any one of the doses of 60 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.2 μM ·PR only (3 μM PR 15 Cells treated only with 15 ) ·3 μM PR 15 And MS023: Cells treated with any one of the doses of 60 μM, 30 μM, 10 μM, 3 μM , 1 μM, 0.2 μM ·3 μM PR 15 And negative control MS094: Cells treated with any one of the doses of 60 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.2 μM ·MS023: Cells treated with only any one of the doses of 60 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.2 μM ·Negative control MS094: Cells treated with only any one of the doses of 60 μM, 30 μM, 10 μM, 3 μM , 1 μM, 0.2 μM ·High control / "100% toxicity" (Cells lysed with lysis buffer) ·Positive control (5 μL LDH solution)

[0158] The plates were incubated at 37 °C, 5% CO2 for 24 hours. The cells were tested and the data were analyzed using the procedure described in the LDH assay kit instructions. ​

[0159] As shown in FIGS. 19A to 19F, MS023 dose-dependently abrogated both GR and PR induced cytotoxicity (measured as % LDH release). MS023 at 3 μM and 1 μ M doses completely abrogated the cytotoxicity induced by 3 μM GR 15 or PR 15 challenge. None of the doses of the negative control MS094 abrogated the cytotoxicity induced by GR1 5 or PR challenge. The highest dose of MS02 15 3, 60 μM, produced significant cytotoxicity, but this was not one of the doses shown to completely abrogate the cytotoxicity induced by GR or PR 15 challenge. The three highest doses of the negative control MS094 tested, 10, 30, and 60 μM, showed cytotoxicity compared to the untreated control, but at the two highest doses this was likely due to DMSO-related toxicity and was also seen in the DMSO controls corresponding to these doses ( 15 0.30% and 0.60% DMSO). (Example 20. MS049 (0.2 - 100 μM) and abrogation of cytotoxicity produced by GR and PR (3 μM) as measured by the LDH assay) (3 μM) as measured by the LDH assay)

[0160] (Example 20. MS049 (0.2 - 100 μM) and abrogation of cytotoxicity produced by GR and PR 15 and PR 15 (3 μM) as measured by the LDH assay) Cells were plated in the medium of a 96-well plate at a density of 3.7×10 4 cells per well and incubated overnight at 37 °C, 5% CO2. The next day, just prior to adding the test compounds, the existing medium was removed and replaced with alternating amounts of medium as in Example 1. M cells per well and incubated overnight at 37 °C, 5% CO2. The next day, just prior to adding the test compounds, the existing medium was removed and replaced with alternating amounts of medium as in Example 1. M cells per well and incubated overnight at 37 °C, 5% CO2. The next day, just prior to adding the test compounds, the existing medium was removed and replaced with alternating amounts of medium as in Example 1. M Thaw the 10 mM stock of S049 to room temperature and dilute it in warm medium to a final concentration in the plate of 0.2, 1, 2, 10, 20, and 100 μM. The 10 mM stock of GR and the 10 mM stock of PR 15 were equilibrated to room temperature and diluted in warm medium to a final concentration in the well of 3 μM. 15 The following conditions were plated in triplicate. To prevent evaporation of the volume in the experimental well during incubation, the samples were surrounded by border wells outside the plate filled with sterile phosphate-buffered saline:

[0161] · Background (medium only) · Untreated (cells in medium only) · DMSO control (cells treated with only the amount of DMSO to which the GR- and PR-treated cells were exposed) · GR only (cells treated with 3 μM GR only) · GR at 3 μM 15 and MS049 at one of the doses: 100 μM, 20 μM, 10 μM, 2 μ M, 1 μM, 0.2 μM 15 · PR only (cells treated with 3 μM PR 15 only) · PR at 3 μM 15 and MS049 at one of the doses: 100 μM, 20 μM, 10 μM, 2 μ M, 1 μM, 0.2 μM · MS049 at one of the doses: 100 μM, 20 μM, 10 μM, 2 μM, 1 μM, 0.2 μM only · High control / "100% toxicity" (cells lysed in lysis buffer) · Positive control (5 μL LDH solution)

[0162] ​​​​​​The cells were tested and the data were analyzed using the procedure described in the LDH assay kit instructions. The

[0163] As shown in FIGS. 20A - 20D, MS049 abrogated the cytotoxicity induced by both GR 15 and P R 15 in a dose - dependent manner. MS049 partially abrogated the cytotoxicity induced by GR and PR 15 at doses of 1 and 20 μ 15 M, and completely abrogated the cytotoxicity induced by GR and PR 15 at doses of 2 15 and 10 μM. MS049 caused significant cytotoxicity independently of GR and PR challenges at doses of 100 and 20 μM, but not at the other doses tested. .

[0164] (Example 21. Inactivation of metabolic abnormalities generated by EPZ020411 (0.2 - 20 μM) and GR and PR 15 measured by the WST - 1 assay 15 (3 μM)) ) Cells were plated in the medium of a 96 - well plate at a density of 3.7×10 4 cells per well and incubated overnight at 37 °C and 5% CO2. The next day, just before adding the test compounds, the existing medium was removed and replaced with alternating amounts of medium as in Example 1. The 10 mM stock of EPZ020411 was thawed to room temperature and diluted with warm medium to give final concentrations in the plate of 0.2, 1, 2, 10, and 20 μM. The 10 mM stock of GR and the 10 mM stock of PR were equilibrated to room temperature and diluted with warm medium to give final concentrations in the wells of 0.2, 1, 2, 10, and 20 μM. The 10 mM stock of GR 15 and the 10 mM stock of PR were equilibrated to room temperature and diluted with warm medium to give 15 final concentrations in the wells of The final concentration in the well was made 3 μM.

[0165] The following conditions were plated in triplicate. During incubation, the experimental well To prevent evaporation of the volume within the well, the sample was placed in a plate surrounded by outer boundary wells filled with sterile phosphate-buffered saline: : · Background (medium only) · Untreated (cells in medium only) · DMSO control (DMSO equivalent to that in wells treated with EPZ020411, EPZ020411, and GR 15 or PR 15 and DMSO equivalent to that in wells treated with DM SO, or GR 15 or PR 15 or DMSO equivalent to that in wells treated with any of the above and treated cells) · GR only (cells treated only with 3 μM GR 15 and treated cells) · 3 μM GR 15 and EPZ020411 at any one of the doses: 20 μM, 10 μM, 2 μM, 1 μM, 0.2 μM and treated cells · PR only (cells treated only with 3 μM PR 15 and treated cells) · 3 μM PR 15 and EPZ020411 at any one of the doses: 20 μM, 10 μM, 2 μM, 1 μM, 0.2 μM and treated cells · EPZ020411 at any one of the doses: 20 μM, 10 μM, 2 μM, 1 μM, 0.2 μM and treated cells with only one of the doses

[0166] The plates were incubated at 37 °C and 5% CO2 for 24 hours. Immediately before the test, the medium was removed and 200 μL of PBS-glucose pre-warmed from 4 °C in a 37 °C water bath was added. It was replaced with a solution (4.5 g / L, sterilized). An aliquot of the WST-1 reagent was thawed from -20 °C and equilibrated at room temperature before use. To each well containing 200 μL of PBS-glucose, 20 μL of the WST-1 reagent was added. The plate was incubated with WST-1 at 37 °C, 5% CO2 for 1 hour, and one absorbance reading (450 nm) was obtained using a Molecular Devices SpectraMax M3 plate reader. The data was exported from the SoftMaxPro 7.0 software of the plate reader to an Excel file.

[0167] As shown in Figures 21A - 21D, EPZ020411 abrogated the metabolic abnormalities induced by both GR 15 and PR. 15 EPZ020411 partially abrogated GR-induced metabolic abnormalities at doses of 2, 10, and 20 μM and showed maximal abrogation at 20 μ M. Additionally, EPZ020411 completely abrogated PR-induced metabolic abnormalities at doses of 10 and 20 μM. EPZ020411 caused significant metabolic abnormalities independent of GR and 15 PR challenges at doses of 10 and 20 μM, but not at other doses tested. This indicates that it is unlikely to be due to DMSO-induced metabolic abnormalities, as the corresponding DMSO controls (0.10%, 0.20 % DMSO) did not induce metabolic abnormalities. 15 PR challenges at doses of 10 and 20 μM, but not at other doses tested. This indicates that it is unlikely to be due to DMSO-induced metabolic abnormalities, as the corresponding DMSO controls (0.10%, 0.20 % DMSO) did not induce metabolic abnormalities. This is unlikely to be due to DMSO-induced metabolic abnormalities as the corresponding DMSO controls (0.10%, 0.20 % DMSO) did not induce metabolic abnormalities.

[0168] (Example 22. Inactivation of cytotoxicity generated by EPZ020411 (0.2 - 20 μM) and by GR and PR 15 measured by the LDH assay 15 (3 μM)) Cells were plated in the medium of a 96-well plate at a density of 3.7×10 4 cells per well and incubated overnight at 37°C and 5% CO2. The next day, just before adding the test compounds, the existing medium was removed and replaced with alternating amounts of medium as in Example 1. An 10 mM stock of EPZ020411 was thawed to room temperature and diluted with warm medium to give final concentrations in the plate of 0.2, 1, 2, 10 and 20 μM. A 10 mM stock of GR and a 10 mM stock of PR were equilibrated to room temperature and diluted with warm medium to give a final concentration in the well of 3 μM. 15 stock of GR and 15 stock of PR were equilibrated to room temperature and diluted with warm medium to give a final concentration in the well of 3 μM.

[0169] The following conditions were plated in triplicate. To prevent evaporation of the volume in the experimental wells during incubation, the samples were surrounded by border wells outside the plate filled with sterile phosphate-buffered saline: · Background (medium only) · Untreated (cells in medium only) · DMSO control (either DMSO equivalent to that in the wells treated with EPZ020411, EPZ020411 and, GR or PR treated wells, or DMSO equivalent to that in the wells treated with GR 15 or PR 15 treated wells, or DMSO equivalent to that in the wells treated with GR or PR 15 treated wells, or DMSO equivalent to that in the wells treated with PR 15 treated wells) · GR only (cells treated with 3 μM GR only) 15 · GR only (cells treated with 3 μM GR only) 15 · GR and EPZ020411 at any one of the doses: 20 μM, 10 μM, 2 μM, 1 μM, 0.2 μM · PR only (3 μM PR15 cells treated only with) ·3 μM PR 15 and EPZ020411: at any one of the doses of 20 μM, 10 μM, 2 μM, cells treated with any one of the doses of 1 μM, 0.2 μM · EPZ020411: at any one of the doses of 20 μM, 10 μM, 2 μM, 1 μM, 0.2 μM cells treated with only any one of the doses · high control / "100% toxicity" (cells dissolved in lysis buffer) · positive control (5 μL LDH solution)

[0170] The plates were incubated at 37 °C and 5% CO2 for 24 hours. The cells were tested and the data were analyzed using the procedure described in the LDH assay kit instructions.

[0171] As shown in Figures 22A to 22D, EPZ020411 nullified the cytotoxicity induced by both GR 15 and PR 15 . EPZ020411 partially nullified the cytotoxicity induced by GR 15 and PR 15 at all doses except 0.2 μM, and showed the maximum effect at a dose of 20 μM. EPZ020411 caused significant cytotoxicity independent of GR and PR challenges at doses of 1 0 and 20 μM, but not at the other doses tested. This indicates that only the DMSO control corresponding to the highest dose of EPZ0204 11 (0.20% DMSO) caused slight cytotoxicity, so it cannot be entirely attributed to the cytotoxicity induced by DMSO .

[0172] ​​Example 23. GSK3368715 (0.1-10 μM) and WST-1 uptake GR measured in sey 15 and PR 15 (3 μM) ) Cells were plated in medium in a 96-well plate at 3.7 x 10 cells per well. 4 Plate at cell density The plates were then incubated overnight at 37°C, 5% CO2. The next day, test compounds were added. Immediately prior to incubation, the existing medium was removed and replaced with alternating amounts of medium as in Example 1. Thaw a 10 mM stock of SK3368715 to room temperature and dilute in warm medium to prepare The final concentrations in the GR were 0.1, 0.3, 1, 3 and 10 μM. 15 10 mM Stock and PR 15 Equilibrate a 10 mM stock of to room temperature and dilute in warm medium. The final concentration in the well was 3 μM.

[0173] The following conditions were plated in triplicate: To prevent evaporation of the volume within the tube, the samples were placed in a tube filled with sterile phosphate buffered saline. Rate outer boundary well enclosed: Background (medium only) Untreated (cells in medium only) DMSO control (DMSO equivalent to GSK3368715-treated wells) , GSK3368715 and GR 15 Or PR 15 Equivalent to wells treated with DMSO, or GR 15 Or PR 15 The wells treated with DMSO were equivalent to those treated with DMSO. (cells treated with GR only (3μM GR 15 (cells treated only with · 3 μM GR 15 and GSK3368715, at any one of the doses: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM, were treated with any one of the doses · PR only (cells treated only with 3 μM PR 15 only) · 3 μM PR 15 and GSK3368715, at any one of the doses: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM, were treated with any one of the doses · GSK3368715, at any one of the doses: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM were treated with only any one of the doses

[0174] The plates were incubated at 37 °C, 5% CO2 for 24 hours. Immediately before the test, the medium was removed and replaced with 200 μL of PBS - glucose solution (4.5 g / L, sterilized) that had been warmed from 4 °C in a 37 °C water bath. An aliquot of WST - 1 reagent was thawed from - 20 °C and equilibrated at room temperature before use. 20 μL of WST - 1 reagent was added to each well containing 200 μL of PBS - glucose. The plates were incubated with WST - 1 at 37 °C, 5% CO2 for 1 hour, and one absorbance reading (450 nm) was obtained using a Molecular Devices SpectraMax M3 plate reader. The data was exported from the SoftMaxPro7.0 software of the plate reader to an Excel file. As shown in Figures 23A - 23F, GSK3368715 abolished the metabolic abnormalities induced by both GR1 5 and PR dose - dependently.

[0175] 5 and PR 15 GSK3368715 abolished the metabolic abnormalities induced by both GR1 5 completely abolished the metabolism abnormalities induced by GR and PR at doses of 1 and 3 μM when compared with the corresponding DMSO-treated controls (0.06%, 0.034% DMSO). The highest dose of GSK3368715 tested, 10 μM, induced low levels of metabolism abnormalities independent of the GR and PR challenge, but this is likely due to solvent effects as the corresponding 0.10% DMSO control showed similar levels of metabolism abnormalities. 15 and 15 PR 15 and 15 PR

[0176] (Example 24. MS023 (0.1 - 10 μM) and GSK3368715 (0.1 - 10 μM) and the abolition of metabolism abnormalities generated by GR 15 (3 μM) as measured by the WST-1 assay) Cells were plated in the medium of 96-well plates at a density of 3.7×10 4 cells per well and incubated overnight at 37 °C, 5% CO2. The next day, just before adding the test compounds, the existing medium was removed and replaced with alternating amounts of medium as in Example 1. The 10 mM stocks of MS023 and GSK3368715 were thawed to room temperature and diluted in warm medium to give final concentrations in the plate of 0.1, 0.3, 1, 3 and 10 μM. The 10 mM stock of GR was equilibrated to room temperature and diluted in warm medium to give a final concentration in the well of 3 μM. 15

[0177] The following conditions were plated in triplicate. To prevent evaporation of the volume within the experimental wells during incubation, the samples were Enclosed by the boundary well outside the rate: · Background (medium only) · Untreated (cells in medium only) · DMSO control (DMSO equivalent to wells treated with GSK3368715 、GSK3368715, and GR 15 or PR 15 and equivalent to wells treated with DMSO, or GR 15 or PR 15 and equivalent to DMSO in any of the wells treated with either, and cells treated) · GR only (cells treated only with 3 μM GR 15 only) · 3 μM GR 15 and MS023 among: 10 μM, 3 μM, 1 μM, 0.3 μM 、0.1 μM, and cells treated with any one of the doses · 3 μM GR 15 and GSK3368715 among: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM, and cells treated with any one of the doses · MS023 among: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM, and any one of the doses and cells treated only · GSK3368715 among: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM and cells treated with any one of the doses only

[0178] The plates were incubated at 37 °C and 5% CO2 for 24 hours. Immediately before the test, the medium was removed and replaced with 200 μL of PBS - glucose solution (4.5 g / L, sterilized) that had been warmed from 4 °C in a 37 °C water bath. An aliquot of the WST - 1 reagent was thawed from - 20 °C and equilibrated at room temperature before use. Wells containing 200 μL of PBS - glucose For each well, 20 μL of WST-1 reagent was added. The plate was incubated with WST-1 at 37 °C and 5% CO2 for 1 hour, and one absorbance reading (450 nm) was obtained using a Molecular Devices SpectraMax M3 plate reader. The data was exported from the plate reader's SoftMaxPro 7.0 software to an Excel file.

[0179] As shown in FIGS. 24A to 24D, GSK3368715 dose-dependently inactivated the metabolic abnormalities induced by both GR 15 and PR 15 . Both MS023 and GSK3368715 completely inactivated the metabolic abnormalities induced by GR and PR 15 at doses of 1 and 3 μM. The highest tested doses of GSK3368715, 10 and 3 μM, 15 induced metabolic abnormalities independent of the GR -PR 15 challenge. None of the tested doses of MS023 induced metabolic 15 abnormalities independent of the GR -PR 15 challenge. 15 (Example 25. Inactivation of cytotoxicity produced by GSK3368715 (0.1 - 10 μM) and GR

[0180] and PR (3 μM) measured in the LDH assay) 15 Cells were plated in the medium of a 96-well plate at a density of 3.7 × 10 15 cells per well and incubated overnight at 37°C and 5% CO2. The next day, the test compound was added. 4 ​​​Immediately before, the existing medium was removed and replaced with alternating amounts of medium as in Example 1. G The 10 mM stock of SK3368715 was thawed to room temperature and diluted with warm medium to final concentrations of 0.1, 0.3, 1, 3 and 10 μM in the wells. GR 15 10 mM of stock and PR 15 10 mM stock of was equilibrated to room temperature and diluted with warm medium to a final concentration of 3 μM in the wells.

[0181] The following conditions were plated in triplicate. To prevent evaporation of the volume in the experimental wells during incubation, the samples were surrounded by outer boundary wells filled with sterile phosphate buffered saline: · Background (medium only) · Untreated (cells in medium only) · DMSO control (DMSO equivalent to wells treated with GSK3368715, GSK3368715 and, GR or PR 15 or cells treated with DMSO equivalent to wells treated with PR or, GR 15 or PR 15 or any of the DMSO equivalents to wells treated with PR treated cells) 15 · GR only (cells treated only with 3 μM GR 15 15 15 15 · 3 μM GR 15 and GSK3368715 at any one of the doses: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM treated cells · PR only (cells treated only with 3 μM PR 15 15 15 and GSK3368715 at any one of the doses: 10 μM, 3 μM, 1 μM, ·Among GSK3368715: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM Cells treated with only any one of the following doses ·High control / "100% toxicity" (cells dissolved in lysis buffer) ·Positive control (5 μL LDH solution)

[0182] The plates were incubated at 37 °C and 5% CO2 for 24 hours. The cells were tested, and the data were analyzed using the procedure described in the LDH assay kit instructions.

[0183] As shown in Figures 25A to 25D, GSK3368715 abolished the cytotoxicity induced by both GR1 5 and PR 15 GSK336871 5 abolished the cytotoxicity induced by GR 15 and PR 15 at a dose of 1 μM completely. In addition, at a dose of 3 μM, PR 15 induced cytotoxicity was almost completely abolished. All doses of GSK 3368715 induced low-level cytotoxicity independent of GR 15 and PR 15 challenge, but this may be due to the solvent effect because all corresponding DMSO controls showed the same dose-dependent levels of cytotoxicity.

[0184] (Example 26. Inactivation of metabolic abnormalities generated by symmetric PRMT inhibitor GSK591 (3 - 200 μM) and asymmetric PRMT inhibitor MS023 (1 - 60 μM) and PR measured by WST-1 assay 15 (generated by 3 μM)) Cells were seeded into the medium of 96-well plates at 3.7×10 4 ​Plated at the density of cells and incubated overnight at 37°C and 5% CO2. The next day, just before adding the test compound, the existing medium was removed and replaced with alternating amounts of medium as in Example 1. The 10 mM stocks of GSK591 and MS023 were thawed to room temperature and diluted with warm medium to give final concentrations in the plate of 3, 10, 33, 100 and 200 μM (GSK591) and 1, 3, 10, 30 and 60 μM (MS023). The 10 mM stock of PR was equilibrated to room temperature and diluted with warm medium to give a final concentration in the well of 3 μM. Plated at the density of cells and incubated overnight at 37°C and 5% CO2. The next day, just before adding the test compound, the existing medium was removed and replaced with alternating amounts of medium as in Example 1. The 10 mM stocks of GSK591 and MS023 were thawed to room temperature and diluted with warm medium to give final concentrations in the plate of 3, 10, 33, 100 and 200 μM (GSK591) and 1, 3, 10, 30 and 60 μM (MS023). The 10 mM stock of PR was equilibrated to room temperature and diluted with warm medium to give a final concentration in the well of 3 μM. Plated at the density of cells and incubated overnight at 37°C and 5% CO2. The next day, just before adding the test compound, the existing medium was removed and replaced with alternating amounts of medium as in Example 1. The 10 mM stocks of GSK591 and MS023 were thawed to room temperature and diluted with warm medium to give final concentrations in the plate of 3, 10, 33, 100 and 200 μM (GSK591) and 1, 3, 10, 30 and 60 μM (MS023). The 10 mM stock of PR was equilibrated to room temperature and diluted with warm medium to give a final concentration in the well of 3 μM. Plated at the density of cells and incubated overnight at 37°C and 5% CO2. The next day, just before adding the test compound, the existing medium was removed and replaced with alternating amounts of medium as in Example 1. The 10 mM stocks of GSK591 and MS023 were thawed to room temperature and diluted with warm medium to give final concentrations in the plate of 3, 10, 33, 100 and 200 μM (GSK591) and 1, 3, 10, 30 and 60 μM (MS023). The 10 mM stock of PR was equilibrated to room temperature and diluted with warm medium to give a final concentration in the well of 3 μM. Plated at the density of cells and incubated overnight at 37°C and 5% CO2. The next day, just before adding the test compound, the existing medium was removed and replaced with alternating amounts of medium as in Example 1. The 10 mM stocks of GSK591 and MS023 were thawed to room temperature and diluted with warm medium to give final concentrations in the plate of 3, 10, 33, 100 and 200 μM (GSK591) and 1, 3, 10, 30 and 60 μM (MS023). The 10 mM stock of PR was equilibrated to room temperature and diluted with warm medium to give a final concentration in the well of 3 μM. Plated at the density of cells and incubated overnight at 37°C and 5% CO2. The next day, just before adding the test compound, the existing medium was removed and replaced with alternating amounts of medium as in Example 1. The 10 mM stocks of GSK591 and MS023 were thawed to room temperature and diluted with warm medium to give final concentrations in the plate of 3, 10, 33, 100 and 200 μM (GSK591) and 1, 3, 10, 30 and 60 μM (MS023). The 10 mM stock of PR was equilibrated to room temperature and diluted with warm medium to give a final concentration in the well of 3 μM. 15 Plated at the density of cells and incubated overnight at 37°C and 5% CO2. The next day, just before adding the test compound, the existing medium was removed and replaced with alternating amounts of medium as in Example 1. The 10 mM stocks of GSK591 and MS023 were thawed to room temperature and diluted with warm medium to give final concentrations in the plate of 3, 10, 33, 100 and 200 μM (GSK591) and 1, 3, 10, 30 and 60 μM (MS023). The 10 mM stock of PR was equilibrated to room temperature and diluted with warm medium to give a final concentration in the well of 3 μM. Plated at the density of cells and incubated overnight at 37°C and 5% CO2. The next day, just before adding the test compound, the existing medium was removed and replaced with alternating amounts of medium as in Example 1. The 10 mM stocks of GSK591 and MS023 were thawed to room temperature and diluted with warm medium to give final concentrations in the plate of 3, 10, 33, 100 and 200 μM (GSK591) and 1, 3, 10, 30 and 60 μM (MS023). The 10 mM stock of PR was equilibrated to room temperature and diluted with warm medium to give a final concentration in the well of 3 μM.

[0185] Plated the following conditions in triplicate. To prevent evaporation of the volume within the experimental wells during incubation, the samples were surrounded by outer boundary wells of the plate filled with sterile phosphate-buffered saline: Plated the following conditions in triplicate. To prevent evaporation of the volume within the experimental wells during incubation, the samples were surrounded by outer boundary wells of the plate filled with sterile phosphate-buffered saline: Plated the following conditions in triplicate. To prevent evaporation of the volume within the experimental wells during incubation, the samples were surrounded by outer boundary wells of the plate filled with sterile phosphate-buffered saline: · Background (medium only) · Untreated (cells only in medium) · DMSO control (cells treated with DMSO equivalent to wells treated with GR 15 or PR 15 · PR only (cells treated with 3 μM GR only) · 3 μM PR 15 and MS023 of which: cells treated with any one of the doses of 60 μM, 30 μM, 10 μM, 3 μM · 3 μM PR 15 and GSK591 of which: cells treated with any one of the doses of 200 μM, 100 μM, 33 μM, 10 μM, 3 μM · 3 μM PR 15 and GSK591 of which: cells treated with any one of the doses of 200 μM, 100 μM, 33 μM, 10 μM, 3 μM · MS023 of which: cells treated with only any one of the doses of 60 μM, 30 μM, 10 μM, 3 μM, 1 μM only) ·Among GSK591: cells treated with only any one of the dosages of 200 μM, 100 μM, 33 μM, 10 μM, 3 μM The plate was incubated at 37 °C and 5% CO2 for 24 hours. Immediately before the test, the medium was

[0186] removed and replaced with 200 μL of PBS - glucose solution (4.5 g / L, sterilized) that had been warmed from 4 °C in a 37 °C water bath An aliquot of the WST - 1 reagent was thawed from - 20 °C and equilibrated at room temperature before use. 20 μL of the WST - 1 reagent was added to each well containing 200 μL of PBS - glucose The plate was incubated with WST - 1 at 37 °C and 5% CO2 for 1 hour, and one absorbance reading (450 nm) was obtained using a Molecular Devices Spe ctraMax M3 plate reader The data was exported from the SoftMaxPro7.0 software of the plate reader to an Excel file

[0187] As shown in Figures 26A - 26D, MS023 abolished both GR - and PR - induced metabolic abnormalities in a dose - dependent manner. The 3 μM and 1 μM dosages of MS023 completely abolished the metabolic abnormalities induced by 3 μM P R challenge. None of the dosages of GSK591 abolished the metabolic abnormalities induced by the PR 15 challenge. The three highest dosages of GSK591 tested, 33, 100, and 200 μM, caused metabolic abnormalities independently of the PR challenge, compared to untreated controls. None of the dosages of MS023 tested caused metabolic abnormalities independently of the PR 15 challenge The three highest dosages of GSK591 tested, 33, 100, and 200 μM, caused metabolic abnormalities independently of the PR challenge, compared to untreated controls. None of the dosages of MS023 tested caused metabolic abnormalities independently of the PR 15 challenge None of the dosages of MS023 tested caused metabolic abnormalities independently of the PR 15 challenge​​ done.

[0188] (Example 27. Dose - response patterns of metabolic abnormalities generated by GR 15 and asymmetric dimethyl ation (ADMe)-GR 15 )(as measured by the WST - 1 assay)) Cells were plated in the medium of a 96 - well plate at a density of 3.7×10 4 cells per well and incubated overnight at 37°C and 5% CO2. The next day, a 10 mM stock of GR and AD 15 Me - GR was equilibrated to room temperature and diluted with warm medium to a final concentration in the well 15 of 10 nM to 3 μM. The following conditions were plated in triplicate. To prevent evaporation of the volume within the experimental wells during incubation, samples were surrounded by border wells outside the plate filled with sterile phosphate - buffered saline:

[0189] · Background (medium only) · Untreated (cells in medium only) · DMSO control (cells treated with DMSO equivalent to wells treated with GR or ADMe - GR · GR of which: cells treated with any one of the doses of 10 nM, 30 nM, 100 nM, 300 nM, 1 μM, 3 μM 15 · ADMe - GR 15 of which: cells treated with any one of the doses of 10 nM, 30 nM, 100 nM, 300 nM, 1 μM · ADMe - GR of which: cells treated with any one of the doses of 10 nM, 30 nM, 100 nM, 300 nM, 1 μM, 3 μM · GR 15 of which: cells treated with any one of the doses of 10 nM, 30 nM, 100 nM, 300 nM, 1 μM, 3 μM · ADMe - GR of which: cells treated with any one of the doses of 10 nM, 30 nM, 100 nM, 300 nM, 1 μM 15 · ADMe - GR of which: cells treated with any one of the doses of 10 nM, 30 nM, 100 nM, 300 nM, 1 μM · ADMe - GR of which: cells treated with any one of the doses of 10 nM, 30 nM, 100 nM, 300 nM, 1 μM, 3 μM

[0190] The plate was incubated at 37°C and 5% CO2 for 24 hours. Immediately before the test, the medium was Removed and replaced with 200 μL of PBS-glucose solution (4.5 g / L, sterilized) that had been warmed from 4 °C in a 37 °C water bath. An aliquot of WST-1 reagent was thawed from -20 °C and equilibrated at room temperature before use. To each well containing 200 μL of PBS-glucose, 20 μL of WST-1 reagent was added. The plates were incubated with WST-1 at 37 °C, 5% CO2 for 1 hour and one absorbance reading (450 nm) was obtained using a Molecular Devices SpectraMax M3 plate reader. The data was exported from the plate reader's SoftMaxPro 7.0 software to an Excel file.

[0191] As shown in FIGS. 27A-27E, GR 15 and ADMe-GR 15 challenges induced dose-dependent metabolic abnormalities in NSC-34 after 24 hours, and ADMe-GR 15 consistently produced more metabolic abnormalities than non-methylated GR 15 .

[0192] (Example 28. Dose-response pattern of cytotoxicity produced by GR 15 and asymmetric dimethylation (ADMe)-GR 15 ) Cells were plated in the medium of a 96-well plate at a density of 3.7×10 4 cells per well and incubated overnight at 37 °C, 5% CO2. The next day, 10 mM stocks of GR and AD 15 Me-GR 15 were equilibrated to room temperature and diluted in warm medium to a final concentration of 10 nM - 3 μM in the wells. ​​​

[0193] The following conditions were plated in triplicate. To prevent evaporation of the volume within the experimental well during incubation, the samples were surrounded by outer boundary wells filled with sterile phosphate-buffered saline: · Background (medium only) · Untreated (cells in medium only) · DMSO control (equivalent to wells treated with GR 15 or ADMe-GR 15 and treated with DMSO) · GR 15 at one of the doses: 10 nM, 30 nM, 100 nM, 300 nM, 1 μM, 3 μM · ADMe-GR 15 at one of the doses: 10 nM, 30 nM, 100 nM, 300 nM, 1 μM 15 · High control / "100% toxicity" (cells lysed in lysis buffer) · Positive control (5 μL LDH solution)

[0194]

[0195] The plates were incubated at 37 °C and 5% CO2 for 24 hours. The cells were tested and the data were analyzed using the procedure described in the instructions for the LDH assay kit.

[0196] As shown in Figures 28A - 28E, the GR 15 and ADMe-GR 15 challenges induced dose-dependent cytotoxicity in NSC-34 after 24 hours, and ADMe-GR 15 produced consistently more cytotoxicity than non-methylated GR 15

[0196] ​(Example 29. Inhibition of metabolic abnormalities induced by MS023 (0.1 - 10 μM) and GR measured by the WST-1 assay and ADMe-GR 15 (3 μM) 15 (as determined by the WST-1 assay). Cells were plated in the medium of a 96-well plate at a density of 3.7×10 4 cells per well and incubated overnight at 37°C in 5% CO2. The next day, just prior to adding the test compounds, the existing medium was removed and replaced with alternating amounts of medium as in Example 1. The 10 mM stock of MS023 was thawed to room temperature and diluted in warm medium to give final concentrations in the plate of 0.1, 0.3, 1, 3 and 10 μM. The 10 mM stocks of GR and ADMe-GR were equilibrated to room temperature and diluted in warm medium to give a final concentration in the wells of 3 μM. The following conditions were plated in triplicate. To prevent evaporation of the volume within the experimental wells during incubation, the samples were surrounded by outer border wells filled with sterile phosphate-buffered saline: 15 · Background (medium only) · Untreated (cells in medium only) 15 · DMSO control (cells treated with DMSO equivalent to wells treated with GR or ADMe-GR).

[0197] · GR only (cells treated with 3 μM GR only) · 3 μM GR and MS023 at one of the doses: 10 μM, 3 μM, 1 μM, 0.3 μM or 0.1 μM · Background (medium only) · Untreated (cells in medium only) · DMSO control (cells treated with DMSO equivalent to wells treated with GR 15 or ADMe-GR). 15 · GR only (cells treated with 3 μM GR only) · 3 μM GR and MS023 at one of the doses: 10 μM, 3 μM, 1 μM, 0.3 μM or 0.1 μM 15 · Background (medium only) · Untreated (cells in medium only) 15 · DMSO control (cells treated with DMSO equivalent to wells treated with GR or ADMe-GR). · ADMe-GR only (cells treated with 3 μM ADMe-GR 15 only) · 3 μM ADMe-GR 15 and MS023 at one of the doses: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM · MS023 at one of the doses: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM only)

[0198] The plates were incubated at 37 °C, 5% CO2 for 24 hours. Immediately before the test, the medium was removed and replaced with 200 μL of PBS-glucose solution (4.5 g / L, sterilized) that had been warmed from 4 °C in a 37 °C water bath. An aliquot of WST-1 reagent was thawed from −20 °C and equilibrated at room temperature before use. 20 μL of WST-1 reagent was added to each well containing 200 μL of PBS-glucose. The plates were incubated with WST-1 at 37 °C, 5% CO2 for 1 hour, and one absorbance reading (450 nm) was obtained using a Molecular Devices SpectraMax M3 plate reader. The data was exported from the SoftMaxPro 7.0 software of the plate reader to an Excel file. Figure 29A–Figure 29D, Figure 30A–Figure 30D, and Figure 31A–Figure 31D represent repeats of the above experiments performed separately on three different days. As shown in these figures,

[0199] MS023 consistently and dose-dependently abrogated the metabolic abnormalities induced by GR challenge, while the metabolic abnormalities induced by ADMe-GR challenge were not abrogated. 15 15 ​​​​

[0200] (Example 30. Inactivation of cytotoxicity generated by MS023 (0.1 - 10 μM) and GR measured by LDH assay and ADMe - GR 15 15 15 (at 3 μM)) Cells were plated into the medium of 96 - well plates at a density of 3.7×10 4 cells per well and incubated overnight at 37 °C and 5% CO2. The next day, just prior to adding the test compounds, the existing medium was removed and replaced with alternating amounts of medium as in Example 1. A 10 mM stock of MS023 was thawed to room temperature and diluted in warm medium to a final concentration in the plate of 0.1, 0.3, 1, 3, and 10 μM. A 10 mM stock of GR and a 10 mM stock of ADMe - GR 15 were equilibrated to room temperature and diluted in warm medium to a final concentration in the well of 3 μM. 15 15 The following conditions were plated in triplicate. To prevent evaporation of the volume within the experimental wells during incubation, the samples were surrounded by outer boundary wells of the plate filled with sterile phosphate - buffered saline:

[0201] · Background (medium only) · Untreated (cells in medium only) · DMSO control (cells treated with DMSO equivalent to wells treated with GR or PR 15 15 15 · GR only (cells treated with 3 μM 15 GR only) · 3 μM 15 GR and one of the doses of MS023: 10 μM, 3 μM, 1 μM, 0.3 μM · Only ADMe-GR (cells treated only with 3 μM ADMe-GR 15 ). · 3 μM ADMe-GR 15 and MS023, of which: at any one of the doses 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM (cells treated with any one of these doses). · Only MS023, of which: at any one of the doses 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM (cells treated with only any one of these). · High control / "100% toxicity" (cells dissolved in lysis buffer). · Positive control (5 μL LDH solution).

[0202] The plates were incubated at 37 °C and 5% CO2 for 24 hours. The cells were tested and the data were analyzed using the procedure described in the LDH assay kit instructions.

[0203] As shown in FIGS. 32A to 32D, MS023 consistently and dose-dependently abolished the cytotoxicity induced by GR 15 challenge, but did not abolish the cytotoxicity induced by ADMe-GR challenge. 15 challenge .

[0204] (Example 31. Mechanism of toxicity) As shown in the above examples, the toxicity associated with GR 15 and PR 15 is related to their ability to be asymmetrically dimethylated after 24 hours of incubation ( line 1 of FIG. 33). When an I-type PRMT inhibitor (such as MS023) is added, the exact mechanism that occurs remains unknown, but the toxicity is abolished (second line of FIG. 33). When cells are challenged with asymmetrically dimethylated GR 15 , the toxic effect still exists (third row of FIG. 33). However, ADMe-GR 15 When MS023 was added during the challenge no detoxification was observed. Therefore, GR 15 was already dimethylated so PRMT inhibition had no effect on the observed effects (fourth row of FIG. 34 ). In summary, the results suggest that 15 asymmetric dimethylation of GR is the driving mechanism of toxicity .

[0205] The methods, compositions, and embodiments of the present disclosure are not intended to be exhaustive or to limit the present disclosure to the exact forms described herein. Rather, the compositions and examples are selected so that those skilled in the art can recognize and understand the true value of the principles and practices of the present disclosure. However, it should be understood that many variations and modifications can be made without departing from the spirit and scope of the present disclosure. As long as it is not technically impossible, any feature or element described in connection with one embodiment can be used interchangeably with, or additionally combined with, any other feature or element of any other embodiment and all such permutations are included in the present disclosure. All publications, patents, and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this technology pertains. All publications, patents, and patent applications are hereby incorporated by reference into this specification as if each individual publication or patent application were specifically and individually recited herein. patent application were specifically and individually recited herein. patent application were specifically and individually recited herein. patent application were specifically and individually recited herein. patent application were specifically and individually recited herein.

[0206] All publications, patents, and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this technology pertains. All publications, patents, and patent applications are hereby incorporated by reference into this specification as if each individual publication or patent application were specifically and individually recited herein. patent application were specifically and individually recited herein.

Claims

1. A method for reducing the cytotoxicity caused by a dipeptide repeat protein (DRP), comprising the step of contacting a cell with an effective amount of a type I protein arginine methyltransferase (type I PRMT) inhibitor.

2. The method according to claim 1, wherein the DRP is generated by the expansion of a hexanucleotide (GGGGCC) repeat of open reading frame 72 (C9ORF72) on chromosome 9.

3. The method according to claim 1 or 2, wherein the DRP contains arginine (R).

4. The method according to claim 3, wherein the DRP is asymmetrically dimethylated.

5. The method according to any one of claims 1 to 4, wherein the DRP contains at least one polyglycine-arginine peptide (GR) and / or at least one polyproline-arginine peptide (PR).

6. The method according to any one of claims 1 to 5, wherein the cell is a nerve cell.

7. The method according to claim 6, wherein the nerve cell is a sensory neuron, a motor neuron or an interneuron.

8. The method according to any one of claims 1 to 7, wherein the type I PRMT inhibitor is selected from the group consisting of a PRMT1 inhibitor, a PRMT3 inhibitor, a PRMT4 inhibitor, a PRMT6 inhibitor, and a PRMT8 inhibitor.

9. The method according to claim 8, wherein the type I PRMT inhibitor is a PRMT1 inhibitor.

10. The method according to claim 9, wherein the type I PRMT1 inhibitor is MS023, MS049, GSK715, or EPZ020411.

11. The method according to claim 8, wherein the type I PRMT inhibitor is a PRMT3 inhibitor.

12. The method according to claim 11, wherein the type I PRMT3 inhibitor is MS023 or MS049.

13. The method according to claim 8, wherein the type I PRMT inhibitor is a PRMT4 inhibitor.

14. The method according to claim 13, wherein the type I PRMT4 inhibitor is MS023, MS049 or TP064.

15. The method according to claim 8, wherein the type I PRMT inhibitor is a PRMT6 inhibitor.

16. The method according to claim 15, wherein the type I PRMT6 inhibitor is MS023, MS049, EPZ020411 or TP064.

17. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ The method according to claim 8, wherein the type I PRMT inhibitor is a PRMT8 inhibitor.

18. The method according to claim 17, wherein the type I PRMT8 inhibitor is MS023, MS049, EPZ020411 or T P064.

19. The cytotoxicity caused by the DRP is measured by a decrease in cell membrane leakage, an increase in cell metabolic function, or a decrease in cell apoptosis, according to any one of claims 1 to 18. The method according to any one of claims 1 to 18, wherein the cytotoxicity caused by the DRP is measured by a decrease in cell membrane leakage, an increase in cell metabolic function, or a decrease in cell apoptosis. The method according to any one of claims 1 to 18, wherein the cytotoxicity caused by the DRP is measured by a decrease in cell membrane leakage, an increase in cell metabolic function, or a decrease in cell apoptosis.

20. The method according to any one of claims 1 to 18, wherein the cytotoxicity caused by the DRP is measured by one or more assays of WST-1, LDH, caspase 3, or BrdU. The method according to any one of claims 1 to 18, wherein the cytotoxicity caused by the DRP is measured by one or more assays of WST-1, LDH, caspase 3, or BrdU. The method according to any one of claims 1 to 18, wherein the cytotoxicity caused by the DRP is measured by one or more assays of WST-1, LDH, caspase 3, or BrdU.

21. A method for reducing the cytotoxicity caused by GR and / or PR DRP, comprising: contacting the cells with an effective amount of a type I PRMT inhibitor, wherein the inhibitor is selected from the group consisting of 【Chemical 1】 the above compound or a pharmaceutically acceptable salt or derivative thereof, 【Chemical Formula 2】 the above compound or a pharmaceutically acceptable salt or derivative thereof, [Chemical Formula 3] the above compound or a pharmaceutically acceptable salt or derivative thereof, 【Chemical Formula 4】 the above compound or a pharmaceutically acceptable salt or derivative thereof, and 【Chemical Formula 5】 the above compound or a pharmaceutically acceptable salt or derivative thereof, the method comprising the step of contacting.

22. The method according to claim 20, wherein the DRP is asymmetrically dimethylated.

23. A method for treating a neurodegenerative disease associated with the expression of DRP in a subject, comprising administering an effective amount of a type I PRMT inhibitor. A method for treating a neurodegenerative disease associated with the expression of DRP in a subject, comprising administering an effective amount of a type I PRMT inhibitor.

24. The method according to claim 23, wherein the disease is amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD). The method according to claim 23, wherein the disease is amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD).

25. The method according to claim 24, wherein the disease is ALS.

26. The method according to claim 24, wherein the disease is FTD.

27. The method according to any one of claims 23 to 26, wherein the DRP is generated by expansion of a hexanucleotide (GGGGCC) repeat of C9ORF72. The method according to any one of claims 23 to 26, wherein the DRP is generated by expansion of a hexanucleotide (GGGGCC) repeat of C9ORF72.

28. The method according to any one of claims 23 to 27, wherein the DRP contains arginine (R). 。

29. The method according to claim 28, wherein the DRP is asymmetrically dimethylated.

30. The method according to claim 28, wherein the DRP contains GR and / or PR.

31. The type I PRMT inhibitor is selected from the group consisting of a PRMT1 inhibitor, a PRMT3 inhibitor, a PRMT4 inhibitor , a PRMT6 inhibitor, and a PRMT8 inhibitor, according to any one of claims 23 to 29.

32. The method according to claim 31, wherein the type I PRMT inhibitor is a PRMT1 inhibitor.

33. The method according to claim 32, wherein the type I PRMT1 inhibitor is MS023, MS049, GSK715, or EPZ 020411.

34. The method according to claim 31, wherein the type I PRMT inhibitor is a PRMT3 inhibitor.

35. The method according to claim 34, wherein the type I PRMT3 inhibitor is MS023, GSK715 or MS049.

29.

36. The method according to claim 31, wherein the type I PRMT inhibitor is a PRMT4 inhibitor.

37. The method according to claim 36, wherein the type I PRMT4 inhibitor is MS023, MS049, GSK715, or TP0 64.

38. The method according to claim 31, wherein the type I PRMT inhibitor is a PRMT6 inhibitor.

39. The method according to claim 38, wherein the type I PRMT6 inhibitor is MS023, MS049, EPZ020411, GSK 715, or TP064.

40. The method according to claim 31, wherein the type I PRMT inhibitor is a PRMT8 inhibitor.

41. The method according to claim 40, wherein the type I PRMT8 inhibitor is MS023, MS049, EPZ020411, GSK 715, or TP064.

42. The cytotoxicity caused by DRP is measured by a decrease in cell membrane leakage, an increase in cell metabolic function , or a decrease in cell apoptosis, according to any one of claims 23 to 41.

29.

43. The cytotoxicity caused by DRP is measured by one or more assays of WST-1, LDH, caspase 3, or BrdU, according to any one of claims 23 to 41.

29.

44. The method according to any one of claims 23 to 43, further comprising the step of administering a second therapeutic agent.

29.

45. The method according to claim 44, wherein the second therapeutic agent is riluzole and / or edaravone.

29.

46. The method according to claim 27, wherein the second therapeutic agent is an antibody or its antigen-binding site. 。

47. The method according to claim 46, wherein the antibody blocks the interaction between human CD40 and human CD40L. The method according to claim 46, wherein the antibody blocks the interaction between human CD40 and human CD40L. **Claim 48** A method for treating C9ORF72-linked ALS in a subject, comprising: administering an effective amount of a type I PRMT inhibitor, wherein the inhibitor is 【Chemical Formula 6】 the above compound or a pharmaceutically acceptable salt or derivative thereof, 【Chemical Formula 7】 the above compound or a pharmaceutically acceptable salt or derivative thereof, [Chemical Formula 8] the above compound or a pharmaceutically acceptable salt or derivative thereof, and 【Chemical Formula 9】 selected from the group consisting of the above compound or a pharmaceutically acceptable salt or derivative thereof, the method comprising the step of administering. **Claim 49** A method for treating C9ORF72-linked FTD in a subject, comprising: administering an effective amount of a type I PRMT inhibitor, wherein the inhibitor is 【Chemical 10】 the above compound or a pharmaceutically acceptable salt or derivative thereof, 【Chemical 11】 the above compound or a pharmaceutically acceptable salt or derivative thereof, 【Chemical 12】 the above compound or a pharmaceutically acceptable salt or derivative thereof, and 【Chemical 13】 selected from the group consisting of the above compound or a pharmaceutically acceptable salt or derivative thereof, the method comprising the step of administering. **Claim 50** The method according to claim 48 or 49, wherein the disease is associated with the expression of DRP. **Claim 51** The method according to claim 50, wherein the DRP is generated by the expansion of the hexanucleotide (GGGGCC) repeat of C9ORF72. The method according to claim 50, wherein the DRP is generated by the expansion of the hexanucleotide (GGGGCC) repeat of C9ORF72. **Claim 52** The method according to claim 50, wherein the DRP contains arginine (R). **Claim 53** The method according to claim 50, wherein the DRP is asymmetrically dimethylated.

Images