2-(4-chloro-3-fluorophenoxy)-n-[trans-4-[5-[3-(trifluoromethoxy)-1-azetidinyl]-1,3,4-oxadiazol-2-YL]cyclohexyl]-acetamide for use in the treatment of vanishing white matter, huntington's disease, charcot marie tooth syndrome, amyotrophic lateral sclerosis or for increasing the guanine nucleotide exchange factor activit

Abstract

The invention described herein provides eIF2B agonist (e.g., COMPOUND 1 ) and uses thereof for treatment of diseases, especially neurodegenerative diseases, including vanishing white matter (VWM), Huntington's disease (HD), Charcot Marie Tooth syndrome (CMT), and Amyotropic Lateral Sclerosis (ALS).

Description

[0001] -(4-CHLORO-3-FLUOROPHENOXY)-N-[TRANS-4-[5-[3-(TRIFLUOROMETHOXY)-1-AZETIDINYL]-1 ,3,4-OXADIAZOL-2- YL]CYCLOHEXYL]-ACETAMIDE FOR USE IN THE TREATMENT OF VANISHING WHITE MATTER, HUNTINGTON'S DISEASE, CHARCOT MARIE TOOTH SYNDROME, AMYOTROPHIC LATERAL SCLEROSIS OR FOR INCREASING THE GUANINE NUCLEOTIDE EXCHANGE FACTOR ACTIVIT

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to and the benefit of the filing date of International Application No. PCT / CN2024 / 094951 , filed on May 23, 2024, the entire content of which is incorporated herein by reference.

[0004] JOINT RESEARCH AGREEMENT

[0005] Subject matter disclosed herein was developed, and the claimed invention was made by, or on behalf of, one or more parties to a Joint Research Agreement (JRA), within the meaning of 35 U.S.C. § 100(h) and 37 C.F.R. § 1.9(e), that was in effect on or before the effective filing date of the claimed invention. Said one or more parties to the JRA consist of ReviR Therapeutics, Inc., and XTALPI INC. (including its affiliate Shenzhen Zhongge Biological Technology Co., Ltd.). The claimed invention was made as a result of activities undertaken within the scope of said Joint Research Agreement.

[0006] BACKGROUND OF THE INVENTION

[0007] Loss of protein folding homeostasis characterizes many of the most prevalent neurodegenerative diseases. As a mechanism for coping with folding stress within the endoplasmic reticulum (ER), the unfolded protein response (UPR) comprises a set of signaling mechanisms that initiate relevant gene expression programs to restore protein homeostasis or promote neuronal death when chronic stress or stress overload is present. This function of the UPR has been proposed to play a key role in some neurodegenerative diseases.

[0008] The integrative stress response (ISR) is an evolutionarily conserved intracellular signaling network that helps cells, tissues, and organisms adapt to variable environments and stay healthy. The ISR responds to changes by reprogramming relevant gene expressions to restore homeostasis. In the brain, the formation of long-term memories requires the synthesis of new proteins, so inhibition of the ISR enhances long-term memory formation. Activation of the ISR, on the other hand, prevents this process, and age-associated cognitive deficits are often associated with ISR activation.

[0009] As a central regulator of protein homeostasis, ISR activation occurs in a wide range of brain diseases. This activation process was confirmed by examining eIF2-P and PKR, PERK, and GCN2 phosphorylation in the brain, including from patient samples as well as samples from animal models of neurodegenerative diseases, e.g., Alzheimer’s disease, Parkinson’s disease, Huntington’ s disease, traumatic brain injury, Down syndrome, and neurodegenerative disorders such as Charcot-Marie-Tooth disease. Notably, ISR activation leads to cognitive deficits in mouse models of traumatic brain injury, aging, and neurodegenerative diseases. eIF2B (eukaryotic translation initiation factor 2B) is a key enzyme in the regulation of protein synthesis. It is a type of Guanine Nucleotide Exchange Factor (GEF) specialized for eukaryotic translation initiation factor 2 (eIF2). During mRNA translation / protein synthesis through eIF2, the GDP to GTP exchange on eIF2 is catalized by the guanine nucleotide exchange factor (GEF) eIF2B, which is composed of five subunits (a, |3, y, 8, s). eIF2B subunits (a, 0, y, 5, s) assemble into a decamer composed of two copies of each subunit.

[0010] SUMMARY OF THE INVENTION

[0011] Disclosed herein is a potent eIF2B agonist COMPOUND 1 (also indicated as “CMPD 1” herein in some figures) and uses thereof for treatment of diseases associated with ISR, such as neurodegenerative diseases (e.g., vanishing white matter (VWM), Huntington’s disease (HD), Charcot Marie Tooth syndrome (CMT) (including, for example CMT1 A, CMT1B, CMT2A or CMT2D), and Amyotropic Lateral Sclerosis (ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD), Chronic traumatic encephalopathy (CTE), and Aicardi-Goutieres Syndrome), for inhibition of integrated stress response (ISR), and / or for increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B.

[0012] Both in vitro and in vivo data have demonstrated significantly higher potency of COMPOUND 1 in modulating ISR, and its stronger efficacy in animal disease models, in comparison to several known eIF-2B agonists as therapeutic agents (e.g. , Reference 1, see below). The chemical structure of COMPOUND 1 is shown below:

[0013] Accordingly, in some aspects, disclosed herein is a method of treating vanishing white matter (VWM) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and a compound of the following formula

[0014] - 2 -

[0015] MEI 48415584v.l (COMPOUND 1) or a pharmaceutically acceptable salt thereof

[0016] In some aspects, disclosed herein is a use of a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof for treatment of vanishing white matter (VWM) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and the compound or a pharmaceutically acceptable salt thereof

[0017] In some aspects, disclosed herein is a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof for use in treatment of vanishing white matter (VWM) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and the compound or a pharmaceutically acceptable salt thereof

[0018] In some aspects, disclosed herein is a method of treating Huntington’s disease (HD) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof

[0019] - 3 -

[0020] MEI 48415584v.l

[0021] In some aspects, disclosed herein is a use of a compound of the following formula (COMPOUND 1 ) or a pharmaceutically acceptable salt thereof for treatment of Huntington’ s disease (HD) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and the compound or a pharmaceutically acceptable salt thereof

[0022] In some aspects, disclosed herein is a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof for use in treatment of Huntington’s disease (HD) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and the compound or a pharmaceutically acceptable salt thereof

[0023] In some aspects, disclosed herein is a method of treating Amyotropic Lateral Sclerosis (ALS) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof

[0024] - 4 -

[0025] MEI 48415584v.l In some aspects, disclosed herein is a use of a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof for treatment of Amyotropic Lateral Sclerosis (ALS) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and the compound or a pharmaceutically acceptable salt thereof

[0026] In some aspects, disclosed herein is a compound of the following formula (COMPOUND 1 ) or a pharmaceutically acceptable salt thereof for use in treatment of Amyotropic Lateral Sclerosis (ALS) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and the compound or a pharmaceutically acceptable salt thereof

[0027] In some aspects, disclosed herein is a method of inhibiting integrated stress response (ISR), or of increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the subject a composition comprising a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof, wherein the subject has vanishing white matter (VWM)

[0028] In some aspects, disclosed herein is a use of a composition comprising a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof for inhibition of integrated stress response (ISR), or of increasing the guanine nucleotide

[0029] - 5 -

[0030] MEI 48415584v.l exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the subject the composition comprising a compound of the following formula or a pharmaceutically acceptable salt thereof, wherein the subject has vanishing white matter (VWM),

[0031] In some aspects, disclosed herein is a composition comprising a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof for use in inhibition of integrated stress response (ISR), or of increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the subject the composition comprising a compound of the following formula or a pharmaceutically acceptable salt thereof, wherein the subject has vanishing white matter (VWM)

[0032] In some aspects, disclosed herein is a method of inhibiting integrated stress response (ISR), or of increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the subject a composition comprising a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof, wherein the subject has Huntington’s disease (HD)

[0033] In some aspects, disclosed herein is a use of a composition comprising a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof for inhibition of integrated stress response (ISR), or of increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the

[0034] - 6 -

[0035] MEI 48415584v.l subject the composition comprising a compound of the following formula or a pharmaceutically acceptable salt thereof, wherein the subject has Huntington’s disease (HD)

[0036] In some aspects, disclosed herein is a composition comprising a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof for use in inhibition of integrated stress response (ISR), or of increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the subject the composition comprising a compound of the following formula or a pharmaceutically acceptable salt thereof, wherein the subject has Huntington’s disease (HD)

[0037] In some aspects, disclosed herein is a method of inhibiting integrated stress response (ISR), or of increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the subject a composition comprising a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof, wherein the subject has Charcot Marie Tooth syndrome (CMT)

[0038] In some aspects, disclosed herein is a use of a composition comprising a compound of the following formula or a pharmaceutically acceptable salt thereof for inhibition of integrated stress response (ISR), or of increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the subject the composition comprising a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof, wherein the subject has Charcot Marie Tooth syndrome (CMT)

[0039] MEI 48415584v.l

[0040] In some aspects, disclosed herein is a composition comprising a compound of the following formula or a pharmaceutically acceptable salt thereof for use in inhibition of integrated stress response (ISR), or of increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the subject the composition comprising a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof, wherein the subject has Charcot Marie Tooth syndrome (CMT)

[0041] In some aspects, disclosed herein is a method of inhibiting integrated stress response (ISR), or of increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the subject a composition comprising a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof, wherein the subject has Amyotropic Lateral Sclerosis (ALS)

[0042] In some aspects, disclosed herein is a use of a composition comprising a compound of the following formula or a pharmaceutically acceptable salt thereof for inhibition of integrated stress response (ISR), or of increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the subject the composition comprising a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof, wherein the subject has Amyotropic Lateral Sclerosis (ALS)

[0043] - 8 -

[0044] MEI 48415584v.l

[0045] In some aspects, disclosed herein is a composition comprising a compound of the following formula or a pharmaceutically acceptable salt thereof for use in inhibition of integrated stress response (ISR), or of increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the subject the composition comprising a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof, wherein the subject has Amyotropic Lateral Sclerosis (ALS)

[0046] In some aspects, disclosed herein is a method of treating Charcot Marie Tooth syndrome (CMT) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and a compound of the following formula (COMPOUND 1 ) or a pharmaceutically acceptable salt thereof

[0047] In some aspects, disclosed herein is a use of a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof for treatment of Charcot Marie Tooth syndrome (CMT) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and the compound or a pharmaceutically acceptable salt thereof

[0048] - 9 -

[0049] MEI 48415584v.l

[0050] In some aspects, disclosed herein is a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof for use in treatment of Charcot Marie Tooth syndrome (CMT) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and the compound or a pharmaceutically acceptable salt thereof

[0051] The invention disclosed herein also provides methods, uses, and compositions of COMPOUND 1 for use in treating a number of other diseases as described herein below.

[0052] In some embodiments, the CMT comprises I is CMT1A, CMT1B, CMT1C, CMT1D, IE, CMT1F, CMT1X, CMT2A, CMT2B, CMT2C, CMT2D, CMT2E, CMT2F, CMT2I, CMT2K, CMT2L, CMT2M, CMT2P, CMT2S, CMT2T, CMT4A, CMT4B1, CMT4B2, CMT4B3, CMT4C, CMT4D, CMT4E, CMT4F, CMT4G, CMT4AH, CMT4J, CMTX2, CMTX3, CMTX4, CMTX5, or CMTX6, or a combination thereof.

[0053] In some embodiments, the CMT comprises / is CMT2A. In some embodiments, the CMT comprises I is CMT2D.

[0054] In some embodiments, the compound (e.g., COMPOUND 1) or the pharmaceutical composition there of is administered orally, intravenously, or intragastrically.

[0055] In some embodiments, the compound e.g., COMPOUND 1) or the pharmaceutical composition thereof is administered at a dose of about 0.1 mg / kg to 100 mg / kg e.g., about 0.1 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 12 mg / kg, 14 mg / kg, 16 mg / kg, 18 mg / kg, 20 mg / kg, 22 mg / kg, 24 mg / kg, 26 mg / kg, 28 mg / kg, 30 mg / kg, 40 mg / Kg, 50, mg / Kg, 60 mg / Kg, 70 mg / Kg, 75 mg / Kg, 80 mg / Kg, 90, mg / Kg, or 100 mg / Kg).

[0056] In some embodiments, the compound (e.g., COMPOUND 1) or the pharmaceutical

[0057] - 10 -

[0058] MEI 48415584v.l composition thereof is administered at a dose of about 3 mg / kg.

[0059] In some embodiments, the compound (e.g., COMPOUND 1 ) or the pharmaceutical composition thereof is administered at a dose of about 30 mg / kg.

[0060] In some embodiments, the compound e.g., COMPOUND 1) or the pharmaceutical composition thereof is administered at a dose of about 1 mg to 300 mg per day (e.g. , about Img per day, 5 mg per day, 10 mg per day, 15 mg per day, 20 mg per day, 25 mg per day, 30 mg per day, 35 mg per day, 40 mg per day, 45 mg per day, 50 mg per day, 50 mg per day, 60 mg per day, 70 mg per day, 80 mg per day, 90 mg per day, 100 mg per day, 100 mg per day, 125 mg per day, 150 mg per day, 175 mg per day, 200 mg per day, 225 mg per day, 250 mg per day, 275 mg per day, or 300 mg per day) per patient.

[0061] In some embodiments, administering the compound (e.g., COMPOUND 1) or the pharmaceutical composition thereof results in increased nerve conduction velocity (NCV) (e.g., about 1.2 times, 1.5 times, 1.8 times, 2 times, 2.2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, or 5 times higher than level before treatment).

[0062] In some embodiments, administering the compound (e.g., COMPOUND 1) or the pharmaceutical composition thereof results in improvement of one or more of symptoms selected from the group consisting of muscle weakness, paralysis, muscle atrophy, decreased reflexes, hammertoes, foot drop, trips and falls, repeated ankle sprains, breathing problems, numbness or tingling, chronic pain, and loss or decrease in vision and hearing.

[0063] In some embodiments, administering the compound (e.g., COMPOUND 1) or composition of any preceding aspects inhibits integrated stress response (ISR) in the subject and / or increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B.

[0064] In some embodiments, the compound (e.g., COMPOUND 1) or composition thereof is administered orally and / or intravenously.

[0065] In some embodiments, a level of one or more ISR-associated biomarkers is reduced in the subject after administering the compound. In some embodiments, a level of one or more ISR-associated biomarkers is reduced in the subject after administering the compound.

[0066] It should be understood that any one embodiment of the invention described herein, including those only described in the examples or claims, can be combined with any one or more other embodiments disclosed herein, unless the combination is improper or expressly disclaimed.

[0067] BRIEF DESCRIPTION OF THE DRAWINGS

[0068] - 11 -

[0069] MEI 48415584v.l FIGs. 1A and IB show dose response curves of COMPOUND 1 and reference compound (“Reference 1”) in inhibition of ATF4 expression as assessed by luciferase reporter assay.

[0070] FIGs. 2A-2C show dose response curves of COMPOUND 1 and Reference 1 (FIG. 2B) or Reference 2 (FIG. 2C) in inhibition of ATF4 expression as assessed by Western blot assay.

[0071] FIG. 3 shows a non-limiting illustration of generation of Eif2b5R19IH / RI9IHmice.

[0072] FIG. 4 shows a non-limiting illustration of study design for assessing efficacy of COMPOUND 1 in mouse model of VWM.

[0073] FIGs. 5A-5C show body weight of Eif2b5RI91H / RI9IHmice (also indicated as R191-H0 or HO mice) following treatment of the indicated doses of COMPOUND 1 , Reference 1 , or vehicle.

[0074] FIGs. 6A-6F show results of beam walking test to assess motor function. Time to cross the beam was measured (FIGS. 6A-6C) and the number of foot slips / falls was counted (FIGS. 6D-6F).

[0075] FIG. 7 shows the modulation of the integrated stress response (ISR) by Compound 1 and Reference 1 in the brains of Ez 27>5R191H / R191Hmice after treatment.

[0076] FIG. 8A shows the increased activation of the integrated stress response (ISR) in medium spiny neurons isolated from post-mortem brains of advanced (green) versus pre- symptomatic (grey) Huntington’s disease patients.

[0077] FIG. 8B shows the increased activation of the integrated stress response (ISR) in medium spiny neurons derived from induced pluripotent stem cells (iPSCs) from advanced (green) versus pre-symptomatic (grey) Huntington’s disease patients.

[0078] FIGs. 9A and 9B show the increase in expression of GDF15 and ATF4 (genes that are markers of ISR) in fibroblasts isolated from patients with Charcot-Marie-Tooth type 2A (CMT2A).

[0079] FIGs. 10A and 10B show body weight of mice after treatment.

[0080] FIGs. 11A-11C show grip strength of mice at 4- (FIG. 11 A), 6- (FIG. 1 IB), and 8 (FIG. 11b) weeks post treatment.

[0081] FIGs. 12A-12F show rotarod test of mice at 4- (FIGs. 12A and 12D), 6- (FIGs. 12B and 12E), and 8 weeks (FIGs. 12C and 12F) post treatment.

[0082] FIGs. 13A-13F show beam walk test of mice at 2- (FIG. 13A), 4- (FIG. 13B), 6-

[0083] - 12 -

[0084] MEI 48415584v.l (FIG. 13C), and 8 (FIGs. 13D-13F) weeks post treatment.

[0085] FIGs. 14A- 14D show NCV test of mice at 2- (FIG. 14A), 4- (FIG. 14B), 6- (FIG. 14C), and 8 (FIG. 14D) weeks post treatment.

[0086] FIG. 15 shows PK results of mice treated with compound in chow.

[0087] FIG. 16 shows PK results of mice treated with Compound 1 through oral administration.

[0088] FIG. 17 shows representative transmission electron micrographs of the femoral nerve at 6000x and 15,000x magnification of wild-type and CMT2D Gur.s,P278KY / +mice after treatment.

[0089] FIG. 18 shows the integrated Stress Response (ISR) score in the spinal cord of CMT2D G’( / / '.S7P27SKY / +and wild-type mice

[0090] DETAILED DESCRIPTION OF THE INVENTION

[0091] Integrated Stress Response (ISR) is an evolutionarily conserved signaling pathway that helps tissues adapt to stress and return to homeostasis. eIF2B is the guanine nucleotide exchange factor that activates eIF2 (eukaryotic translation initiation factor 2) and promotes formation of the ternary complex that initiates mRNA translation in the absence of cellular stress. eIF2B’s substrate, eIF2, is composed of three subunits (a, p, y) and binds methionine initiator tRNA and guanosine triphosphate (GTP) to form the ternary complex required to initiate translation on AUG start codons. GTP is hydrolyzed at the ribosome and eIF2-GDP is released, requiring reactivation by eIF2B to enable a new round of protein synthesis involving eIF2.

[0092] Under various stress conditions including ISR, however, eIF2 is phosphorylated by various stress-sensing kinases. ISR leads to the phosphorylation of eIF2, converting eIF2 from a substrate of eIF2B into a competitive inhibitor which reduces translation initiation events and decreases global protein synthesis.

[0093] ISR activation is a hallmark of several neurodegenerative disorders. Chronic ISR activation is observed in numerous neurodegenerative disorders including amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), vanishing white matter disease (VWM), frontotemporal degeneration (FTD), and Charcot-Marie-Tooth syndrome type 2D (CMT2D), and Parkinson’s disease (PD). Thus, ablation / inhibition of ISR by activation of eIF2B can be a promising treatment avenue for these neurodegenerative diseases.

[0094] While not wishing to be bound by any particular theory, it is believed that eIF2B

[0095] - 13 -

[0096] MEI 48415584v.l activators / agonists stabilize the decameric form of eIF2B. Specifically, eIF2B activators I agonists may enhance GEF activity of eIF2B by stabilizing the decameric form of the enzyme and increase thermostability of the eIF2B5 subunit. Thus, eIF2B agonists can make cells insensitive to the effects of eIF2a-P by activating the GEF activity of eIF2B, thus allowing global protein synthesis to proceed with residual unphosphorylated eIF2a. This reverses I alleviates / eliminates the effects of eIF2a phosphorylation (e.g., by ISR), restoring translation in stressed cells, and blocking translation of ISR-activated mRNAs such as ATF4.

[0097] Indeed, IS RIB, a small molecule inhibitor of ISR initially identified in a phenotypic screen, was later determined to bind to the eIF2B decamer, and to enhance the GEF activity of eIF2B by stabilizing the decameric form of the enzyme and increase thermostability of the eIF2B5 subunit), thereby restoring protein translation and UPR transcription to basal levels, leading to reduction of the integrated stress response (ISR).

[0098] Numerous animal experiments have further confirmed that the eIF2B activators have therapeutic efficacy in a number of neurodegenerative disorders.

[0099] For example, eIF2B agonists have been found to increase long-term memory in mouse models. After 3 days of oral administration of the eIF2B activator ABBV-CLS-7262 (see Cho et al., Neurology 100(17supp2), 2023, doi.org / l 0.l212 / WNL.00000000002038l), the brain function of the model animals could be restored to youthful levels.

[0100] In another example, eIF2B agonist Reference 1 (see Craig et al. , J. Med. Chem. 67(7):5758-5782, 2024) has been shown to reduce stress granules in ALS patient-derived iPSC-MNs, suggesting its utility in ALS treatment. Reference 1 also restores body weight and motor function deficits in mice model of Vanishing white matter disease (VWM).

[0101] Further, ISRIB was shown to provide protective effects against the A -mediated AD.

[0102] These results provide evidence that eIF2B agonists may inhibit some neurodegenerative diseases at a later stage and have the potential to treat diseases such as Alzheimer’s disease and Parkinson’s disease.

[0103] Indeed, the eIF2B agonists ABBV-CLS-7262 and DNL-343 are both in Phase 1 clinical trials for ALS. Published Phase 1 data from the DNL-343 trial shows that it was safely tolerated in healthy individuals.

[0104] Accordingly, disclosed herein are potent eIF2B activators / agonists (e.g., COMPOUND 1), composition comprising said eIF2B activators / agonists, and uses thereof for treating or preventing diseases and disorders (e.g., neurodegenerative disease).

[0105] - 14 -

[0106] MEI 48415584v.l Definition

[0107] The term “pharmaceutically acceptable carrier” refers to a carrier that does not cause an allergic reaction or other untoward effect in patients to whom it is administered and are compatible with the other ingredients in the formulation. Pharmaceutically acceptable carriers include, for example, pharmaceutical diluents, excipients or carriers suitably selected with respect to the intended form of administration, and consistent with conventional pharmaceutical practices. For example, solid carriers / diluents include, but are not limited to, a gum, a starch (e.g., corn starch, pregelatinized starch), a sugar (e.g., lactose, mannitol, sucrose, dextrose), a cellulosic material (e.g., microcrystalline cellulose), an acrylate (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the therapeutic agent.

[0108] As used herein, the term “salt” refers to acid or base salts of the compounds used in the methods of the present invention. Illustrative examples of acceptable salts are mineral acid (hydrochloric acid, hydrobromic acid, phosphoric acid, and the like) salts, organic acid (acetic acid, propionic acid, glutamic acid, citric acid and the like) salts, quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts.

[0109] An “effective amount” is an amount sufficient to accomplish a stated purpose (e.g. achieve the effect for which it is administered, treat a disease, reduce enzyme activity, increase enzyme activity, reduce one or more symptoms of a disease or condition). An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.” A “reduction” of a symptom or symptoms (and grammatical equivalents of this phrase) means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s).

[0110] Compositions and Uses

[0111] In some aspects, disclosed herein is a method of treating vanishing white matter (VWM) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof:

[0112] - 15 -

[0113] MEI 48415584v.l

[0114] In some aspects, disclosed herein is a use of a compound of the following formula (COMPOUND 1 ) or a pharmaceutically acceptable salt thereof for treatment of vanishing white matter (VWM) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and the compound or a pharmaceutically acceptable salt thereof

[0115] In some aspects, disclosed herein is a compound of the following formula or a pharmaceutically acceptable salt thereof for use in treatment of vanishing white matter (VWM) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and the compound (COMPOUND 1) or a pharmaceutically acceptable salt thereof

[0116] In some aspects, disclosed herein is a method of treating Huntington’s disease (HD) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof:

[0117] - 16 -

[0118] MEI 48415584v.l

[0119] In some aspects, disclosed herein is a use of a compound of the following formula or a pharmaceutically acceptable salt thereof for treatment of Huntington’ s disease (HD) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and the compound (COMPOUND 1) or a pharmaceutically acceptable salt thereof

[0120] In some aspects, disclosed herein is a compound of the following formula (COMPOUND 1 ) or a pharmaceutically acceptable salt thereof for use in treatment of Huntington’s disease (HD) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and the compound or a pharmaceutically acceptable salt thereof

[0121] In some aspects, disclosed herein is a method of treating Charcot Marie Tooth syndrome (CMT) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof

[0122] - 17 -

[0123] MEI 48415584v.l In some aspects, disclosed herein is a use of a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof for treatment of Charcot Marie Tooth syndrome (CMT) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and the compound or a pharmaceutically acceptable salt thereof

[0124] In some aspects, disclosed herein is a compound of the following formula (COMPOUND 1 ) or a pharmaceutically acceptable salt thereof for use in treatment of Charcot Marie Tooth syndrome (CMT) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and the compound or a pharmaceutically acceptable salt thereof

[0125] In some embodiments, the CMT comprises I is CMT1A, CMT1B, CMT1C, CMT1D, IE, CMT1F, CMT1X, CMT2A, CMT2B, CMT2C, CMT2D, CMT2E, CMT2F, CMT2I, CMT2K, CMT2L, CMT2M, CMT2P, CMT2S, CMT2T, CMT4A, CMT4B1, CMT4B2, CMT4B3, CMT4C, CMT4D, CMT4E, CMT4F, CMT4G, CMT4AH, CMT4J, CMTX2, CMTX3, CMTX4, CMTX5, or CMTX6, or a combination thereof.

[0126] In some embodiments, the CMT comprises / is CMT2A. In some embodiments, the CMT comprises I is CMT2D.

[0127] In some embodiments, the compound (e.g., COMPOUND 1) or the pharmaceutical composition thereof is administered orally, intravenously, or intragastrically.

[0128] In some embodiments, the compound (e.g., COMPOUND 1) or the pharmaceutical composition thereof is administered at a dose of about 0.1 mg / kg to 100 mg / kg e.g., about 0.1 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7

[0129] - 18 -

[0130] MEI 48415584v.l mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 12 mg / kg, 14 mg / kg, 16 mg / kg, 18 mg / kg, 20 mg / kg, 22 mg / kg, 24 mg / kg, 26 mg / kg, 28 mg / kg, 30 mg / kg, 40 mg / kg, 50, mg / kg, 60 mg / Kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 90, mg / kg, or 100 mg / kg, or about 0.1 mg / kg to 1 mg / kg, 0.1 mg / kg to 10 mg / kg, 0.1 mg / kg to 20 mg / kg, 1 mg / kg to 5 mg / kg, 1 mg / kg to 10 mg / kg, 1 mg / kg to 20 mg / kg, 5 mg / kg to 25 mg / kg, 5 mg / kg to 50 mg / kg, 10 mg / kg to 40 mg / kg, 10 mg / kg to 50 mg / kg, 10 mg / kg to 100 mg / kg, 20 mg / kg to 40 mg / kg, 25 mg / kg to 50 mg / kg, 25 mg / kg to 75 mg / kg, 25 mg / kg to 100 mg / kg, or 50 mg / kg to 100 mg / kg). The term “kg” refers to body weight of the subject.

[0131] In some embodiments, the compound (e.g., COMPOUND 1) or the pharmaceutical composition thereof is administered at a dose of about 3 mg / kg.

[0132] In some embodiments, the compound (e.g., COMPOUND 1) or the pharmaceutical composition thereof is administered at a dose of about 30 mg / kg.

[0133] In some embodiments, the compound (e.g. COMPOUND 1) or the pharmaceutical composition thereof is administered at a dose of about 1 mg to 300 mg per day (e.g., about Img per day, 5 mg per day, 10 mg per day, 15 mg per day, 20 mg per day, 25 mg per day, 30 mg per day, 35 mg per day, 40 mg per day, 45 mg per day, 50 mg per day, 50 mg per day, 60 mg per day, 70 mg per day, 80 mg per day, 90 mg per day, 100 mg per day, 100 mg per day, 125 mg per day, 150 mg per day, 175 mg per day, 200 mg per day, 225 mg per day, 250 mg per day, 275 mg per day, or 300 mg per day, or about 1 mg to 10 mg per day, 1 mg to 30 mg per day, 1 mg to 100 mg per day, 5 mg to 25 mg per day, 5 mg to 50 mg per day, 5 mg to 100 mg per day, 5 mg to 150 mg per day, 5 mg to 200 mg per day, 10 mg to 50 mg per day, 10 mg to 100 mg per day, 10 mg to 150 mg per day, 10 mg to 200 mg per day, 50 mg to 100 mg per day, 50 mg to 150 mg per day, 50 mg to 200 mg per day, 50 mg to 250 mg per day, 50 mg to 300 mg per day, 75 mg to 100 mg per day, 75 mg to 125 mg per day, 75 mg to 150 mg per day, 75 mg to 175 mg per day, 75 mg to 200 mg per day, 75 mg to 250 mg per day, 75 mg to 300 mg per day, 100 mg to 150 mg per day, 100 mg to 200 mg per day, 100 mg to 250 mg per day, 100 mg to 300 mg per day, 150 mg to 200 mg per day, 150 mg to 250 mg per day, 150 mg to 300 mg per day, 200 mg to 300 mg per day, 200 mg to 250 mg per day, or 250 mg to 300 mg perdsay) per patient

[0134] In some embodiments, administering the compound (e.g., COMPOUND 1) or the pharmaceutical composition thereof results in increased nerve conduction velocity (NCV) (e.g., about 1.2 times, 1.5 times, 1.8 times, 2 times, 2.2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, or 5 times higher than level before treatment).

[0135] - 19 -

[0136] MEI 48415584v.l In some embodiments, administering the compound (e.g., COMPOUND 1) or the pharmaceutical composition thereof results in improvement of one or more of symptoms of CMT selected from the group consisting of muscle weakness, paralysis, muscle atrophy, decreased reflexes, hammertoes, foot drop, trips and falls, repeated ankle sprains, breathing problems, numbness or tingling, chronic pain, and loss or decrease in vision and hearing.

[0137] In some aspects, disclosed herein is a method of treating Amyotropic Lateral Sclerosis (ALS) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof

[0138] In some aspects, disclosed herein is a use of a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof for treatment of Amyotropic Lateral Sclerosis (ALS) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and the compound or a pharmaceutically acceptable salt thereof

[0139] In some aspects, disclosed herein is a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof for use in treatment of Amyotropic Lateral Sclerosis (ALS) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and the compound or a pharmaceutically acceptable salt thereof

[0140] - 20 -

[0141] MEI 48415584v.l

[0142] Accumulating evidence has shown that eIF2B activators / agonists can enhance the GEF activity of eIF2B and ablate / alleviate the effect of ISR. In some aspects, administering the compound or composition disclosed herein (e.g., COMPOUND 1) inhibits integrated stress response (ISR) in the subject and / or increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B.

[0143] In some embodiments, a level of one or more ISR- associated biomarkers is reduced in the subject after administering the compound (e.g., COMPOUND 1). In some embodiments, the one or more ISR-associated biomarkers are selected from the group consisting of: Activating Transcription Factor 4 (ATF4), ChaC Glutathione Specific Gamma- Glutamylcyclotransferase 1 (CHAC1), and Tribbles Pseudokinase 3 (TRIB3). In some embodiments, the ISR-associated biomarker is ATF4.

[0144] In some embodiments, a level of ATF4 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0145] In some embodiments, a level of CHAC1 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0146] In some embodiments, a level of TRIB3 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0147] Accordingly, in some aspects, disclosed herein is a method of treating vanishing white matter (VWM) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and a compound of the following formula (e.g., COMPOUND 1) or a pharmaceutically acceptable salt thereof

[0148] - 21 -

[0149] MEI 48415584v.l wherein administering the compound inhibits integrated stress response (ISR) in the subject and / or increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B. In some embodiments, a level of one or more ISR-associated biomarkers is reduced in the subject after administering the compound. In some embodiments, the one or more ISR-associated biomarkers are selected from the group consisting of: Activating Transcription Factor 4 (ATF4), ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1), and Tribbles Pseudokinase 3 (TRIB3). In some embodiments, the ISR-associated biomarker is ATF4. In some embodiments, a level of ATF4 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of CHAC1 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of TRIB3 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0150] In some aspects, disclosed herein is a use of a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof for treatment of vanishing white matter (VWM) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and the compound or a pharmaceutically acceptable salt thereof administering the compound inhibits integrated stress response (ISR) in the subject and / or increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B. In some embodiments, a level of one or more ISR-associated biomarkers is reduced in the subject

[0151] - 22 -

[0152] MEI 48415584v.l after administering the compound. In some embodiments, the one or more ISR-associated biomarkers are selected from the group consisting of: Activating Transcription Factor 4 (ATF4), ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1), and Tribbles Pseudokinase 3 (TRIB3). In some embodiments, the ISR-associated biomarker is ATF4. In some embodiments, a level of ATF4 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of CHAC1 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of TRIB3 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0153] In some aspects, disclosed herein is a compound of the following formula (COMPOUND 1 ) or a pharmaceutically acceptable salt thereof for use in treatment of vanishing white matter (VWM) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and the compound or a pharmaceutically acceptable salt thereof wherein administering the compound inhibits integrated stress response (ISR) in the subject and / or increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B. In some embodiments, a level of one or more ISR-associated biomarkers is reduced in the subject after administering the compound. In some embodiments, the one or more ISR-associated biomarkers are selected from the group consisting of: Activating Transcription Factor 4 (ATF4), ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1), and Tribbles Pseudokinase 3 (TRIB3). In some embodiments, the ISR-associated biomarker is ATF4. In some embodiments, a level of ATF4 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of CHAC1 in the subject is reduced by about 20%, about 25%, about

[0154] - 23 -

[0155] MEI 48415584v.l 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of TRIB3 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0156] In some aspects, disclosed herein is a method of treating Huntington’s disease (HD) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof wherein administering the compound inhibits integrated stress response (ISR) in the subject and / or increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B. In some embodiments, a level of one or more ISR-associated biomarkers is reduced in the subject after administering the compound. In some embodiments, the one or more ISR-associated biomarkers are selected from the group consisting of: Activating Transcription Factor 4 (ATF4), ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1), and Tribbles Pseudokinase 3 (TRIB3). In some embodiments, the ISR-associated biomarker is ATF4. In some embodiments, a level of ATF4 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of CHACl in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of TRIB3 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0157] In some aspects, disclosed herein is a use of a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof for treatment of Huntington’s disease (HD) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a

[0158] - 24 -

[0159] MEI 48415584v.l pharmaceutically acceptable carrier or excipient, and the compound or a pharmaceutically acceptable salt thereof wherein administering the compound inhibits integrated stress response (ISR) in the subject and / or increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B. In some embodiments, a level of one or more ISR-associated biomarkers is reduced in the subject after administering the compound. In some embodiments, the one or more ISR-associated biomarkers are selected from the group consisting of: Activating Transcription Factor 4 (ATF4), ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1), and Tribbles Pseudokinase 3 (TRIB3). In some embodiments, the ISR-associated biomarker is ATF4). In some embodiments, a level of ATF4 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of CHAC1 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of TRIB3 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0160] In some aspects, disclosed herein is a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof for use in treatment of Huntington’s disease (HD) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and the compound or a pharmaceutically acceptable salt thereof wherein administering the compound inhibits integrated stress response (ISR) in the subject and / or

[0161] - 25 -

[0162] MEI 48415584v.l increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B. In some embodiments, a level of one or more ISR-associated biomarkers is reduced in the subject after administering the compound. In some embodiments, the one or more ISR-associated biomarkers are selected from the group consisting of: Activating Transcription Factor 4 (ATF4), ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1), and Tribbles Pseudokinase 3 (TRIB3). In some embodiments, the ISR-associated biomarker is ATF4. In some embodiments, a level of ATF4 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of CHAC1 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of TRIB3 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0163] In some aspects, disclosed herein is a method of treating Charcot Marie Tooth syndrome (CMT) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and a compound of the following formula or a pharmaceutically acceptable salt thereof , wherein administering the compound inhibits integrated stress response (ISR) in the subject and / or increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B. In some embodiments, a level of one or more ISR-associated biomarkers is reduced in the subject after administering the compound. In some embodiments, the one or more ISR-associated biomarkers are selected from the group consisting of: Activating Transcription Factor 4 (ATF4), ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1), and Tribbles Pseudokinase 3 (TRIB3). In some embodiments, the ISR-associated biomarker is ATF4. In some embodiments, a level of ATF4 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,

[0164] - 26 -

[0165] MEI 48415584v.l about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of CHAC1 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of TRIB3 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0166] In some aspects, disclosed herein is a use of a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof for treatment of Charcot Marie Tooth syndrome (CMT) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and the compound or a pharmaceutically acceptable salt thereof wherein administering the compound inhibits integrated stress response (ISR) in the subject and / or increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B. In some embodiments, a level of one or more ISR-associated biomarkers is reduced in the subject after administering the compound. In some embodiments, the one or more ISR-associated biomarkers are selected from the group consisting of: Activating Transcription Factor 4 (ATF4), ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1), and Tribbles Pseudokinase 3 (TRIB3). In some embodiments, the ISR-associated biomarker is ATF4. In some embodiments, a level of ATF4 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of CHAC1 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of TRIB3 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0167] - 27 -

[0168] MEI 48415584v.l In some aspects, disclosed herein is a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof for use in treatment of Charcot Marie Tooth syndrome (CMT) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and the compound or a pharmaceutically acceptable salt thereof wherein administering the compound inhibits integrated stress response (ISR) in the subject and / or increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B. In some embodiments, a level of one or more ISR-associated biomarkers is reduced in the subject after administering the compound. In some embodiments, the one or more ISR-associated biomarkers are selected from the group consisting of: Activating Transcription Factor 4 (ATF4), ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1), and Tribbles Pseudokinase 3 (TRIB3). In some embodiments, the ISR-associated biomarker is ATF4. In some embodiments, a level of ATF4 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of CHAC1 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of TRIB3 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0169] In some aspects, disclosed herein is a method of treating Amyotropic Lateral Sclerosis (ALS) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and a compound of the following formula (COMPOUND 1 ) or a pharmaceutically acceptable salt thereof

[0170] - 28 -

[0171] MEI 48415584v.l wherein administering the compound inhibits integrated stress response (ISR) in the subject and / or increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B. In some embodiments, a level of one or more ISR-associated biomarkers is reduced in the subject after administering the compound. In some embodiments, the one or more ISR-associated biomarkers are selected from the group consisting of: Activating Transcription Factor 4 (ATF4), ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1), and Tribbles Pseudokinase 3 (TRIB3). In some embodiments, the ISR-associated biomarker is ATF4. In some embodiments, a level of ATF4 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of CHAC1 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of TRIB3 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0172] In some aspects, disclosed herein is a use of a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof for treatment of Amyotropic Lateral Sclerosis (ALS) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and the compound or a pharmaceutically acceptable salt thereof wherein administering the compound inhibits integrated stress response (ISR) in the subject and / or increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B. In some embodiments, a level of one or more ISR-associated biomarkers is reduced in the subject

[0173] - 29 -

[0174] MEI 48415584v.l after administering the compound. In some embodiments, the one or more ISR-associated biomarkers are selected from the group consisting of: Activating Transcription Factor 4 (ATF4), ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1), and Tribbles Pseudokinase 3 (TRIB3). In some embodiments, the ISR-associated biomarker is ATF4. In some embodiments, a level of ATF4 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of CHAC1 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of TRIB3 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0175] In some aspects, disclosed herein is a compound of the following formula (COMPOUND 1 ) or a pharmaceutically acceptable salt thereof for use in treatment of Amyotropic Lateral Sclerosis (ALS) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and the compound or a pharmaceutically acceptable salt thereof wherein administering the compound inhibits integrated stress response (ISR) in the subject and / or increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B. In some embodiments, a level of one or more ISR-associated biomarkers is reduced in the subject after administering the compound. In some embodiments, the one or more ISR-associated biomarkers are selected from the group consisting of: Activating Transcription Factor 4 (ATF4), ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1), and Tribbles Pseudokinase 3 (TRIB3). In some embodiments, the ISR-associated biomarker is ATF4. In some embodiments, a level of ATF4 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of CHAC1 in the subject is reduced by about 20%, about 25%, about

[0176] - 30 -

[0177] MEI 48415584v.l 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of TRIB3 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0178] In some aspects, disclosed herein is a method of inhibiting integrated stress response (ISR), or of increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the subject a composition comprising a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof, wherein the subject has vanishing white matter (VWM) wherein administering the compound inhibits integrated stress response (ISR) in the subject and / or increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B. In some embodiments, a level of one or more ISR-associated biomarkers is reduced in the subject after administering the compound. In some embodiments, the one or more ISR-associated biomarkers are selected from the group consisting of: Activating Transcription Factor 4 (ATF4), ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1), and Tribbles Pseudokinase 3 (TRIB3). In some embodiments, the ISR-associated biomarker is

[0179] ATF4. In some embodiments, a level of ATF4 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of CHAC1 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of TRIB3 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0180] In some aspects, disclosed herein is a use of a composition comprising a compound of the following formula or a pharmaceutically acceptable salt thereof for inhibition of integrated stress response (ISR), or of increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the subject the

[0181] - 31 -

[0182] MEI 48415584v.l composition comprising a compound of the following formula(COMPOUND 1) or a pharmaceutically acceptable salt thereof, wherein the subject has vanishing white matter (VWM), wherein administering the compound inhibits integrated stress response (ISR) in the subject and / or increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B. In some embodiments, a level of one or more ISR-associated biomarkers is reduced in the subject after administering the compound. In some embodiments, the one or more ISR-associated biomarkers are selected from the group consisting of: Activating Transcription Factor 4 (ATF4), ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1), and Tribbles Pseudokinase 3 (TRIB3). In some embodiments, the ISR-associated biomarker is

[0183] ATF4. In some embodiments, a level of ATF4 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of CHAC1 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of TRIB3 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0184] In some aspects, disclosed herein is a composition comprising a compound of the following formula or a pharmaceutically acceptable salt thereof for use in inhibition of integrated stress response (ISR), or of increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the subject the composition comprising a compound of the following formula(COMPOUND 1) or a pharmaceutically acceptable salt thereof, wherein the subject has vanishing white matter (VWM)

[0185] - 32 -

[0186] MEI 48415584v.l wherein administering the compound inhibits integrated stress response (ISR) in the subject and / or increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B. In some embodiments, a level of one or more ISR-associated biomarkers is reduced in the subject after administering the compound. In some embodiments, the one or more ISR-associated biomarkers are selected from the group consisting of: Activating Transcription Factor 4 (ATF4), ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1), and Tribbles Pseudokinase 3 (TRIB3). In some embodiments, the ISR-associated biomarker is ATF4. In some embodiments, a level of ATF4 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of CHAC1 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of TRIB3 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0187] In some aspects, disclosed herein is a method of inhibiting integrated stress response (ISR), or of increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the subject a composition comprising a compound of the following formula(COMPOUND 1) or a pharmaceutically acceptable salt thereof, wherein the subject has Huntington’s disease (HD) wherein administering the compound inhibits integrated stress response (ISR) in the subject and / or increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B. In some embodiments, a level of one or more ISR-associated biomarkers is reduced in the subject after administering the compound. In some embodiments, the one or more ISR-associated

[0188] - 33 -

[0189] MEI 48415584v.l biomarkers are selected from the group consisting of: Activating Transcription Factor 4 (ATF4), ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1), and Tribbles Pseudokinase 3 (TRIB3). In some embodiments, the ISR-associated biomarker is ATF4. In some embodiments, a level of ATF4 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of CHAC1 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of TRIB3 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0190] In some aspects, disclosed herein is a use of a composition comprising a compound of the following formula or a pharmaceutically acceptable salt thereof for inhibition of integrated stress response (ISR), or of increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the subject the composition comprising a compound of the following formula(COMPOUND 1) or a pharmaceutically acceptable salt thereof, wherein the subject has Huntington’s disease (HD) wherein administering the compound inhibits integrated stress response (ISR) in the subject and / or increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B. In some embodiments, a level of one or more ISR-associated biomarkers is reduced in the subject after administering the compound. In some embodiments, the one or more ISR-associated biomarkers are selected from the group consisting of: Activating Transcription Factor 4 (ATF4), ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1), and Tribbles Pseudokinase 3 (TRIB3). In some embodiments, the ISR-associated biomarker is ATF4. In some embodiments, a level of ATF4 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of CHAC1 in the subject is reduced by about 20%, about 25%, about

[0191] - 34 -

[0192] MEI 48415584v.l 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of TRIB3 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0193] In some aspects, disclosed herein is a composition comprising a compound of the following formula or a pharmaceutically acceptable salt thereof for use in inhibition of integrated stress response (ISR), or of increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the subject the composition comprising a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof, wherein the subject has Huntington’s disease (HD) wherein administering the compound inhibits integrated stress response (ISR) in the subject and / or increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B. In some embodiments, a level of one or more ISR-associated biomarkers is reduced in the subject after administering the compound. In some embodiments, the one or more ISR-associated biomarkers are selected from the group consisting of: Activating Transcription Factor 4 (ATF4), ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1), and Tribbles Pseudokinase 3 (TRIB3). In some embodiments, the ISR-associated biomarker is ATF4. In some embodiments, a level of ATF4 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of CHAC1 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of TRIB3 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0194] In some aspects, disclosed herein is a method of inhibiting integrated stress response (ISR), or of increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B in a

[0195] - 35 -

[0196] MEI 48415584v.l subject, comprising administering to the subject a composition comprising a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof, wherein the subject has Charcot Marie Tooth syndrome (CMT) wherein administering the compound inhibits integrated stress response (ISR) in the subject and / or increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B. In some embodiments, a level of one or more ISR-associated biomarkers is reduced in the subject after administering the compound. In some embodiments, the one or more ISR-associated biomarkers are selected from the group consisting of: Activating Transcription Factor 4 (ATF4), ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1), and Tribbles Pseudokinase 3 (TRIB3). In some embodiments, the ISR-associated biomarker is

[0197] ATF4. In some embodiments, a level of ATF4 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of CHAC1 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of TRIB3 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0198] In some aspects, disclosed herein is a use of a composition comprising a compound of the following formula or a pharmaceutically acceptable salt thereof for inhibition of integrated stress response (ISR), or of increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the subject the composition comprising a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof, wherein the subject has Charcot Marie Tooth syndrome (CMT)

[0199] - 36 -

[0200] MEI 48415584v.l wherein administering the compound inhibits integrated stress response (ISR) in the subject and / or increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B. In some embodiments, a level of one or more ISR-associated biomarkers is reduced in the subject after administering the compound. In some embodiments, the one or more ISR-associated biomarkers are selected from the group consisting of: Activating Transcription Factor 4 (ATF4), ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1), and Tribbles Pseudokinase 3 (TRIB3). In some embodiments, the ISR-associated biomarker is ATF4. In some embodiments, a level of ATF4 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of CHAC1 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of TRIB3 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0201] In some aspects, disclosed herein is a composition comprising a compound of the following formula or a pharmaceutically acceptable salt thereof for use in inhibition of integrated stress response (ISR), or of increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the subject the composition comprising a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof, wherein the subject has Charcot Marie Tooth syndrome (CMT) wherein administering the compound inhibits integrated stress response (ISR) in the subject and / or increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B. In some

[0202] - 37 -

[0203] MEI 48415584v.l embodiments, a level of one or more ISR-associated biomarkers is reduced in the subject after administering the compound. In some embodiments, the one or more ISR-associated biomarkers are selected from the group consisting of: Activating Transcription Factor 4 (ATF4), ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1), and Tribbles Pseudokinase 3 (TRIB3). In some embodiments, the ISR-associated biomarker is ATF4. In some embodiments, a level of ATF4 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of CHAC1 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of TRIB3 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0204] In some aspects, disclosed herein is a method of inhibiting integrated stress response (ISR), or of increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the subject a composition comprising a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof, wherein the subject has Amyotropic Lateral Sclerosis (ALS) wherein administering the compound inhibits integrated stress response (ISR) in the subject and / or increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B. In some embodiments, a level of one or more ISR-associated biomarkers is reduced in the subject after administering the compound. In some embodiments, the one or more ISR-associated biomarkers are selected from the group consisting of: Activating Transcription Factor 4 (ATF4), ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1), and Tribbles Pseudokinase 3 (TRIB3). In some embodiments, the ISR-associated biomarker is ATF4. In some embodiments, a level of ATF4 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some

[0205] - 38 -

[0206] MEI 48415584v.l embodiments, a level of CHAC1 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of TRIB3 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0207] In some aspects, disclosed herein is a use of a composition comprising a compound of the following formula or a pharmaceutically acceptable salt thereof for inhibition of integrated stress response (ISR), or of increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the subject the composition comprising a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof, wherein the subject has Amyotropic Lateral Sclerosis (ALS) wherein administering the compound inhibits integrated stress response (ISR) in the subject and / or increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B. In some embodiments, a level of one or more ISR-associated biomarkers is reduced in the subject after administering the compound. In some embodiments, the one or more ISR-associated biomarkers are selected from the group consisting of: Activating Transcription Factor 4 (ATF4), ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1), and Tribbles Pseudokinase 3 (TRIB3). In some embodiments, the ISR-associated biomarker is ATF4. In some embodiments, a level of ATF4 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of CHAC1 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of TRIB3 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0208] - 39 -

[0209] MEI 48415584v.l In some aspects, disclosed herein is a composition comprising a compound of the following formula or a pharmaceutically acceptable salt thereof for use in inhibition of integrated stress response (ISR), or of increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the subject the composition comprising a compound of the following formula (COMPOUND 1) or a pharmaceutically acceptable salt thereof, wherein the subject has Amyotropic Lateral Sclerosis (ALS) wherein administering the compound inhibits integrated stress response (ISR) in the subject and / or increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B.

[0210] In some embodiments, a level of one or more ISR- associated biomarkers is reduced in the subject after administering the compound. In some embodiments, the one or more ISR- associated biomarkers are selected from the group consisting of: Activating Transcription Factor 4 (ATF4), ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1), and Tribbles Pseudokinase 3 (TRIB3). In some embodiments, the ISR-associated biomarker is ATF4. In some embodiments, a level of ATF4 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of CHAC1 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment. In some embodiments, a level of TRIB3 in the subject is reduced by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more compared to that prior to the treatment.

[0211] In some embodiments, the compound (e.g., COMPOUND 1) or composition thereof is administered orally and / or intravenously.

[0212] Diseases and Treatments

[0213] Amyotrophic lateral sclerosis (ALS) is also known as motor neuron disease (MND), commonly known as amyotrophic lateral sclerosis. It is an irreversible and fatal motor neuron

[0214] - 40 -

[0215] MEI 48415584v.l disease, with the main symptom of progressive muscle weakness and atrophy of the limbs and trunk muscles, and a gradual loss of motor function, as if being “frozen”, so it is commonly known as “ALS.” Most people with ALS die from respiratory failure, usually within three to five years after symptoms first appear. Currently, there is no cure for ALS, and there are no effective treatments to stop or reverse the progression of the disease. The central pathologic finding in ALS is the death of motor neurons in the motor cortex and spinal cord. Degeneration of corticospinal axons leads to thinning and scarring (sclerosis) of the lateral aspect of the spinal cord.

[0216] ALS involves the death of motor neurons in the brain and spine and is related to protein homeostasis as well as cytoskeletal function. eIF2B activators, such as COMPOUND 1 , show efficacy in rescuing motor deficits in mice similar to ALS. Specifically, ISR is activated in ALS patients, and the TDP-43 stress granules have been found to accumulate in motor neurons. eIF2B agonist helps to dissolve the TDP-43 stress granules, thus treating ALS. eIF2B agonist also rescued mice from neurological deficits caused by persistent ISR in the brain / spinal cord. The eIF2B agonist DNL343 penetrated the CNS and showed robust inhibition of the ISR pathway.

[0217] In some embodiments, the compounds (e.g., COMPOUND 1) and / or the pharmaceutical compositions disclosed herein are used to treat or prevent ALS, and / or ameliorate the symptoms thereof, including, for example, trouble walking or doing usual daily activities, tripping and falling, and / or slurred speech or trouble swallowing.

[0218] In some embodiments, the compounds (e.g., COMPOUND 1) and / or the pharmaceutical compositions disclosed herein reduce stress proteins, decrease CNS ISR activity, restore white matter in the spinal cord, improve motor function and balance, and / or dissolve stress granules in ALS patient’s motor neurons.

[0219] Huntington’s disease (HD) is a fatal, incurable genetic disorder that causes nerve cells in the brain to break down. HD is caused by a mutation in the gene for a protein called huntingtin (HTT). An upstream open reading frame (uORF) within the HTT transcript decreased HTT translation from the main ORF. During stress conditions, HTT uORF translation is reduced, leading to increased translation of HTT from the main ORF (similar mechanism as ATF4 translation upregulated by uORF during ISR) with increase in HTT protein levels. A proline stretch after the HTT CAG repeat causes ribosomes to slow down and stall on the HTT mRNA. This activates stress and sequestering eIF5A which in turn leads to further ribosome stalling across the transcriptome.

[0220] - 41 -

[0221] MEI 48415584v.l Mutant huntingtin protein (mHTT) induces ER stress by interference of toxic oligomers with ER-associated degradation (ERAD) components. mHTT induces PERK, ATF6, and IRE1 branch of the unfolded protein response in vitro. Phosphorylation of eIF2a increases due to expression of mHTT.

[0222] Thus, un some embodiments, the compounds (e.g., COMPOUND 1) and / or the pharmaceutical compositions disclosed herein are effective to treat, prevent HD and / or ameliorate the related symptoms, including, for example, movement disorders, trouble with cognitive skills, and / or brain cell damage and loss of brain tissue assessed by CT scan.

[0223] In some embodiments, the compounds (e.g., COMPOUND 1) and / or the pharmaceutical compositions disclosed herein can reduce eIF2a phosphorylation in HD patient.

[0224] Alzheimer’s Disease (AD) is characterized by extracellular neuritic plaques composed of P-amyloid (AP), and intracellular neurofibrillary tangles composed of tau protein. The pathogenesis of this disease is associated with aberrant translational machinery through eIF2a.

[0225] In some embodiments, the compounds (e.g., COMPOUND 1) and / or the pharmaceutical compositions disclosed herein are effective to treat, prevent AD and / or ameliorate the related symptoms, including, for example, memory impairment, trouble with language, confusion with location or passage of time, and / or loss of brain cells over time as determined by brain imaging.

[0226] In some embodiments, the compounds e.g., COMPOUND 1) and / or the pharmaceutical compositions disclosed herein can reduce eIF2a phosphorylation in AD patient.

[0227] In some embodiments, the compounds (e.g., COMPOUND 1) and / or the pharmaceutical compositions disclosed herein can prevent brain damaging, slow down and / or ameliorate brain damaging as assessed by magnetic resonance imaging (MRI), computerized tomography (CT), positron emission tomography (PET).

[0228] In some embodiments, the compounds (e.g., COMPOUND 1) and / or the pharmaceutical compositions disclosed herein can reduce and / or prevent increase of amyloid and / or tau protein in a biological sample (e.g. , cerebrospinal fluid) of AD patient, and / or prevent and / or ameliorate Amyloid P protein (A ) accumulation and accumulations of neurofibrillary tangles (NFTs), as assessed by imaging (MRI), computerized tomography (CT), positron emission tomography (PET), or histological staining.

[0229] - 42 -

[0230] MEI 48415584v.l In some embodiments, the compounds (e.g., COMPOUND 1) and / or the pharmaceutical compositions disclosed herein can improve behavioral phenotypes in AD animal model, including, for example, improvement of platform crossing and prolonged target quadrant occupancy.

[0231] Charcot-Marie-Tooth (CMT) disease is a hereditary and progressive neurological disease affecting both sensory and motor nerves in the arms, hands, legs, and feet. The affected nerves slowly degenerate and lose the ability to communicate with their distant targets, resulting in muscle weakness and atrophy. There are many different types of CMT, which may share some symptoms but vary by pattern of inheritance, age of onset, and whether the axon or myelin sheath is involved.

[0232] The tRNA synthetases (aaRSes) are the largest gene family associated with CMT. These enzymes charge tRNAs with their cognate amino acid so the tRNA can participate in protein synthesis. Data from multiple mouse and cell-based models of aaRS-associated CMT show that the mutant aaRS sequesters tRNAs, causing ribosome stalling, and activation of the integrated stress response (ISR) in motor and sensory neurons through the activating kinase. Mutations in several tRNA synthetase genes have been identified in patients with CMTC, CMT2N and CMTB.

[0233] CMT1

[0234] CMT type 1 is the most common subtype of CMT, accounting for roughly two-thirds of all cases. CMT1 is inherited in an autosomal dominant pattern.

[0235] CMT1A (caused by a duplication in the PMP22 gene on chromosome 17) accounts for around 70% to 80% of CMT1 cases, making it the most common subtype of CMT1. This autosomal dominantly inherited demyelinating form of CMT is caused by a 1.5 Mb duplication on chromosome 17pl 1.2, containing the gene coding for peripheral myelin protein 22 (PMP22) and thus leading to three copies of the PMP22 gene. Patients with CMT1A are usually slow runners in childhood, develop high arches and hammertoes and often require orthotics (braces) for ankle support. Varying degrees of hand weakness occur, often appearing as much as 10 years after foot and leg problems. Problems with balance because of ankle weakness and loss of proprioception (the brain’s ability to know where the limbs are in space) are common.

[0236] CMT1B is the second most common subtype of CMT1. CMT1B is caused by a defect within the MPZ gene, which lies on chromosome 1. The MPZ gene produces myelin protein

[0237] - 43 -

[0238] MEI 48415584v.l zero (MPZ protein), and the disruption of this mutated protein causes deficits within the myelin sheath. About 40 percent of people with CMT1B have an infantile onset with delayed walking (after 15 months), and development of symptoms before 5 years of age. Hip dysplasia and optic nerve atrophy have been observed in this group (though hip dysplasia may happen in up to 20 percent of kids with CMT), and scoliosis in about a third of people. People with early-onset CMT1B tend to have very slow nerve conduction velocities, with speeds <15 m / s in the arms (normal >50 m / s). A minority (7 %) of people with this diseasecausing variant in the MPZ gene have a childhood onset, with symptoms between 6 and 20 years of age. This group tends to have slow nerve conductions, between 15 and 25 m / s. The remainder of people with CMT1B have an adult onset, with symptoms tending to begin after age 40 and a comparatively milder presentation. Their nerve conduction studies tend to be in the intermediate range of between 35 and 45 m / s.

[0239] CMT1C is caused by disease-causing variants in the LITAF gene. CMT1C affects fewer than 1% of people who have the disease. Presentation is similar to CMT1A, with onset between the first and third decades, and weakness in the feet and hands, atrophy, and sensory loss and slow nerve conduction velocities (16-25 m / s).

[0240] CMT1D is caused by mutations in a gene on chromosome 10 called EGR2, which codes for the early growth response protein 2. CMT ID causes less than 1% of cases of CMT. Most people with CMT ID show severe symptoms in the first decade of life, including delayed motor milestones and nerve conduction velocities of 10 m / s or less (sometimes referred to as Dejerine Sottas syndrome). A few cases of CMT1D have milder symptoms that appear later in life. Other symptoms may include cranial nerve dysfunction and respiratory difficulties.

[0241] Point mutations in the PMP22 gene cause CMT IE. People with CMT IE have earlier onset and more severe symptoms than those with CMT1 A. Children often present within the first two years of life with delayed walking. Conduction velocities in patients tend to be markedly reduced, usually under 10 m / s (normal in the arms is >50 m / s).

[0242] CMT IF is an autosomal dominant form of CMT in which the defect is on Chromosome 8 and mutations in gene NEFL that encodes the neurofilament light chain protein.

[0243] CMT2

[0244] CMT2 represents 12% to 36% of all CMT cases. CMT2 is characterized by distal

[0245] - 44 -

[0246] MEI 48415584v.l weakness, atrophy, sensory loss, decreased deep-tendon reflexes, and variable foot deformity. CMT2 can be caused by direct damage to nerve axons themselves. CMT2 is commonly referred to as “axonal” CMT.

[0247] CMT2A is the most common subtype of CMT2 (it accounts for 20% of the cases of axonal CMT) and is caused by defects in the MFN2 gene. The MFN2 gene encodes for mitofusin 2, which is a protein involved in the fusion of cellular mitochondria. MFN2 is a 757-amino acid long, nuclear encoded protein, anchored to the outer mitochondrial membrane by two transmembrane domains (TM1 and TM2).

[0248] CMT2B is a debilitating autosomal dominant hereditary sensory neuropathy. Patients with this disease lose pain sensation and frequently need amputation. Axonal dysfunction and degeneration of peripheral sensory neurons is a major clinical manifestation of CMT2B. CMT2B is characterized by severe ulceration problems and the defect IN in gene RAB7 located on chromosome 3. CMT2B may be caused by missense point mutations (L129F, K157N, N161T / I, V162M) in Rab7 GTPase.

[0249] CMT2C is an autosomal dominant neuropathy characterized by limb, diaphragm, and laryngeal muscle weakness. Two unrelated families with CMT2C showed significant linkage to chromosome 12q24.1 1 . All genes in this region were sequenced and heterozygous missense mutations were identified in the TRPV4 gene at positions C.805OT and c.806G>A, causing the amino acid substitutions R269C and R269H. TRPV4 is a well known member of the TRP superfamily of cation channels. Onset is between infancy and the 6th decade.

[0250] CMT2D is caused by defects in the GARS gene, located on chromosome 7, which codes glycyl-tRNA synthetase. The symptoms of CMT2D vary in patients, ranging from motor symptoms only to both sensory and motor symptoms.

[0251] CMT2E is caused by dominantly inherited mutations in the NEFL gene, located on chromosome 8, which codes for the neurofilament light chain protein. Neurofilaments form the structural framework determining the shape and size of nerve cells. Notably, mutations in NEFL protein also cause CMT IF. Onset is in the first to sixth decade with a gait anomaly and a leg weakness that reaches the arms secondarily. Tendon reflexes are reduced or absent and after years all patients have a pes cavus. Other signs may be present including hearing loss and postural tremor.

[0252] CMT2F is caused by dominant mutations in HSPB1 gene, located on chromosome 7, which codes for heat shock protein beta-1 (HSPB1). This protein helps neurofilaments maintain the diameter of axons. This is essential for the transmission of nerve impulses. A

[0253] - 45 -

[0254] MEI 48415584v.l peripheral sensorimotor neuropathy with symmetric weakness primarily occurs in the lower limbs and reaching the arms only after 5 to 10 years, occasional and predominantly distal sensory loss and reduced tendon reflexes.

[0255] CMT2I is caused by mutations in the myelin protein zero gene (MPZ), located on chromosome 1. CMT2I is characterized by a late onset with with severe sensory loss associated with distal weakness mainly of the legs and absent or reduced deep tendon reflexeS. Mutations in MPZ also cause CMT1B.

[0256] CMT2K is Caused by mutations in the GDAP1 gene (8q 13.3), encoding a protein required for mitochondrial fission. About 25% of people with mutations in the GDAP1 gene have CMT2K. GDAP1 codes for a protein called ganglioside-induced differentiation- associated protein 1 found in mitochondria. Onset occurs with a clinical picture including hypotonia, scoliosis, a hoarse voice, vocal cord paralysis and respiratory insufficiency. Nerve conduction velocities and pathological findings from sural nerve biopsies are indicative of a predominantly axonal neuropathy with some demyelinating features.

[0257] CMT2L is an autosomal dominant neuromuscular disorder characterized by muscle weakness and atrophy and sensory impairment of the distal lower and upper limbs resulting from a length-dependent axonal peripheral neuropathy. It is caused by mutations in the HSPB8 gene that encodes heat shock protein beta- 8.

[0258] CMT subtypes and their respective gene defects

[0259] - 46 -

[0260] MEI 48415584v.l

[0261] Mutations in the glycyl-tRNA synthetase gene (GARS) cause CMT2D. CMT-GARS mutant models activate the ISR, specifically in alpha motor neurons and in a subset of sensory neurons. Gcn2 significantly alleviated neuropathy in CMT-GARS mice.

[0262] CMT1B mice (e.g., PO glycoprotein mutation or MpzR98C / + mice) and CMT1A mice e.g. , C3-PMP22 mice) show enhanced activation of ISR e.g., increased levels of P- eIF2a and / or total eIF2a). P0S63del is not detected in the myelin sheath, but is retained in the ER, where its accumulation triggers a canonical UPR, indicating a toxic gain of function. Inactivation of Gadd34 restores motor function and rescues the neurophysiological and morphological deficits in S63del mice.

[0263] GCN2iB improves body weight and motor performance in CMT-Gars mice. Further, Gadd34 inhibition improves myelination in S63del DRG explant cultures and reduces demyelination in S63del mice.

[0264] The data herein also show increased expression of GDF15 and ATF4 (genes that are markers of ISR) in fibroblasts isolated from patients with CMT2A, indicating that inhibition of ISR can treat CMT2A.

[0265] Thus, in some embodiments, the compounds (e.g. , COMPOUND 1) and / or the pharmaceutical compositions disclosed herein are effective to treat, prevent CMT and / or ameliorate the related symptoms, including, for example, weakness in legs, ankles and feet, high foot arches, curled toes, footdrop, and / or gait.

[0266] - 47 -

[0267] MEI 48415584v.l In some embodiments, the compounds (e.g., COMPOUND 1) and / or the pharmaceutical compositions disclosed herein can reduce eIF2a phosphorylation in CMT patient. In some embodiments, the CMT comprises / is CMT1 A, CMT1B, CMT1C, CMT1D, IE, CMT1F, CMT1X, CMT2A, CMT2B, CMT2C, CMT2D, CMT2E, CMT2F, CMT2I, CMT2K, CMT2L, CMT2M, CMT2P, CMT2S, CMT2T, CMT4A, CMT4B1, CMT4B2, CMT4B3, CMT4C, CMT4D, CMT4E, CMT4F, CMT4G, CMT4AH, CMT4J, CMTX2, CMTX3, CMTX4, CMTX5, or CMTX6, or a combination thereof. In some mebodiments, the compounds (e.g., COMPOUND 1 ) and / or the pharmaceutical compositions thereof disclosed herein can reduce eIF2a phosphorylation in CMT1 A patient. In some mebodiments, the compounds (e.g., COMPOUND 1) and / or the pharmaceutical compositions thereof disclosed herein can reduce eIF2a phosphorylation in CMT2A patient. In some mebodiments, the compounds (e.g., COMPOUND 1) and / or the pharmaceutical compositions thereof disclosed herein can reduce eIF2a phosphorylation in CMT1B patient. In some mebodiments, the compounds (e.g., COMPOUND 1) and / or the pharmaceutical compositions thereof disclosed herein can reduce eIF2a phosphorylation in CMT2D patient.

[0268] The following are non-limiting diseases, e.g., neurodegenerative disease, that may also be treated utilizing the methods, compounds (e.g., COMPOUND 1), and compositions of the present disclosure.

[0269] In some embodiments, the neurodegenerative disease includes but is not limited to: leukodystrophy, white matter lesions, dysmyelinating Disorders or demyelinating disease, intellectual disability syndrome, cognitive dysfunction, glial cell dysfunction or brain injury (e.g., traumatic brain injury or toxin-induced brain injury), Alexander’s disease, Alper’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), Ataxia-telangiectasia, Batten disease (also called Spielmeyer- Vogt- Sjogren-Batten disease), Bovine spongiform encephalopathy (BSE), Canavan disease, Cockayne syndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, dystonia, frontotemporal dementia (FTD), Gerstmann-Straussler- Scheinker syndrome, Huntington’s disease, HIV-associated dementia, Kennedy disease, Krabbe disease, kuru, Lewy body dementia, Machado-Joseph disease (spinocerebellar ataxia type 3), multiple system atrophy, multisystem proteinopathy, narcolepsy, neuroborreliosis, Parkinson’s disease, Pelizaeus-Merzbacher disease, Pick’s disease, primary lateral sclerosis, prion disease, Refsum disease, Sandhoff disease, Schilder’s disease, subacute combined degeneration of spinal cord secondary to pernicious anemia, schizophrenia, spinocerebellar disorders (various types with different characteristics, e.g., spinocerebellar disorder type 2 or

[0270] - 48 -

[0271] MEI 48415584v.l type 8 spinocerebellar disorder), spinal muscular atrophy, Steele-Richardson-Olszewski syndrome, progressive supranuclear palsy, corticobasal degeneration, adrenoleukodystrophy, X-linked adrenoleukodystrophy, cerebral adrenoleukodystrophy, Pelizaeus-Merzbacher disease, Krabbe disease, leukodystrophy due to mutations in the DARS2 gene (sometimes called leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL), DARS2-associated spectrum disorders or Tabes dorsalis.

[0272] In some embodiments, the methods, compounds (e.g., COMPOUND 1), and compositions of the present disclosure can be utilized for treatment of cancer, including but are not limited to: human cancers and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, melanomas, etc., including solid and lymphoid cancers, kidney cancer, breast cancer, lung cancer, bladder cancer, colon cancer, ovarian cancer, prostate cancer, pancreatic cancer, stomach cancer, brain cancer, head and neck cancer, skin cancer, uterine cancer, testicular cancer, glioma, esophageal cancer, liver cancer (including hepatocarcinoma), lymphoma (including B-acute lymphoblastic lymphoma, non- Hodgkin lymphoma) (e.g., Burkitt lymphoma, small cell lymphoma, and large cell lymphoma), Hodgkin lymphoma, leukemia (including AML, ALL, and CML), and / or multiple myeloma. In some other contexts, “cancer” refers to: lung cancer, breast cancer, ovarian cancer, leukemia, lymphoma, melanoma, pancreatic cancer, sarcoma, bladder cancer, bone cancer, brain cancer, cervical cancer, colon cancer, esophageal cancer, stomach cancer, liver cancer, head and neck cancer, kidney cancer, myeloma, thyroid cancer, prostate cancer, metastatic cancer or carcinoma.

[0273] The above-mentioned leukemias include but are not limited to: acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute promyelocytic leukemia, adult T-cell leukemia, nonleukemic leukemia, nonleukocytosis leukemia, basophilic leukemia, blastic leukemia, bovine leukemia, chronic myelogenous leukemia, cutaneous leukemia, stem cell leukemia, eosinophilic leukemia, Gross leukemia, hairy cell leukemia, hemoblastic leukemia, hemoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphoid leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenic leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryoblastic leukemia, small myeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myeloid leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasma cell leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling’s leukemia, stem cell leukemia, sub-leukemic leukemia or

[0274] - 49 -

[0275] MEI 48415584v.l undifferentiated cell leukemia.

[0276] In some embodiments, the methods, compounds (e.g., COMPOUND 1), and compositions of the present disclosure can be utilized for treatment of inflammatory diseases, which include but are not limited to: postoperative cognitive dysfunction, arthritis e.g., rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis), systemic lupus erythematosus (SUE), myasthenia gravis, juvenile onset diabetes, type 1 diabetes, Guillain- Barre syndrome, Hashimoto’s encephalitis, Hashimoto’s thyroiditis, ankylosing spondylitis, psoriasis, Sjogren’s syndrome, vasculitis, glomerulus nephritis, autoimmune thyroiditis, Behcet’s disease, Crohn’s disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, ichthyosis, Graves’ ophthalmopathy, inflammatory bowel disease, Addison’s disease, vitiligo, asthma e.g., allergic asthma), acne vulgaris, celiac disease, chronic prostatitis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplant rejection, interstitial cystitis, atherosclerosis and atopic dermatitis.

[0277] In some embodiments, the methods, compounds (e.g., COMPOUND 1), and compositions of the present disclosure can be utilized for treatment of musculoskeletal diseases, which include but are not limited to: muscular dystrophy (e.g., Duchenne muscular dystrophy), Becker muscular dystrophy, distal muscular dystrophy, congenital muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, type-1 myotonic dystrophy or type-2 myotonic dystrophy, limb-girdle muscular dystrophy, multisystem proteinopathy, Rhizomelic chondrodysplasia punctata, X-linked recessive chondrodysplasia punctata, Conradi-Hunermann syndrome, autosomal dominant punctate chondrodysplasia, stress-induced bone disease (e.g., stress-induced osteoporosis), amyotrophic lateral sclerosis (ALS), primary lateral sclerosis, progressive muscular atrophy, progressive bulbar palsy, pseudobulbar palsy, spinal muscular atrophy, progressive myelobulbar muscular atrophy, spinal spasm, spinal muscular atrophy, myasthenia gravis, neuralgia, fibromyalgia, Machado-Joseph disease, Paget’s disease of bone, fasciculation syndrome, Freidrich’s ataxia, muscle wasting disorders (e.g., sarcopenia, cachexia), inclusion body myopathy, motor neuron disease, or paralysis.

[0278] In some embodiments, the methods, compounds (e.g., COMPOUND 1), and compositions of the present disclosure can be utilized for treatment of metabolic diseases, which include but are not limited to: non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), liver fibrosis, obesity, heart disease, atherosclerosis, arthritis, cystinosis, diabetes (e.g., type 1 diabetes, type 2 diabetes, or gestational diabetes),

[0279] - 50 -

[0280] MEI 48415584v.l phenylketonuria, proliferative retinopathy or Kearns-Sayre disease.

[0281] In some embodiments, the methods, compounds (e.g., COMPOUND 1), and compositions of the present disclosure can be utilized for treatment of mitochondrial diseases, which include but are not limited to: Barth syndrome, chronic progressive external ophthalmoplegia (cPEO), Kerns-Sell syndrome (KSS), Leigh syndrome (e.g., MILS or maternally inherited Leigh syndrome), mitochondrial DNA deletion syndrome (MDDS, such as Alpers syndrome), mitochondrial encephalomyopathy (e.g., mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS)), mitochondrial neurogastrointestinal encephalopathy syndrome (MNGIE), myoclonic epilepsy with ragged red fibers (MERRF), neuropathy, ataxia, neuropathy, ataxia, and retinitis pigmentosa (NARP), Leber’s hereditary optic neuropathy (LHON) and Pearson syndrome.

[0282] In some embodiments, the methods, compounds (e.g., COMPOUND 1), and compositions of the present disclosure can be utilized for treatment of hearing loss diseases, which include but are not limited to: mitochondrial nonsyndromic hearing loss and deafness, hair cell death, age-associated hearing loss, noise-induced hearing loss, hereditary hearing loss, hearing loss due to ototoxic exposure, disease, and trauma. In some embodiments, the mitochondrial non-syndromic hearing loss and deafness is MT-RNR1 -associated hearing loss.

[0283] In some embodiments, the methods, compounds (e.g., COMPOUND 1), and compositions of the present disclosure can be utilized for treatment of eye diseases, which include but are not limited to: cataracts, glaucoma, endoplasmic reticulum (ER) stress, autophagy deficiency, age-associated macular degeneration (AMD), or diabetic retinopathy.

[0284] In some embodiments, the methods, compounds (e.g., COMPOUND 1), and compositions of the present disclosure can be utilized for treatment of kidney diseases, which include but are not limited to: Abderhalden-Kaufmann-Lignac syndrome (nephropathic cystinosis), abdominal compartment syndrome, acetaminophen-induced nephrotoxicity, acute renal failure / acute kidney injury, acute lobar nephropathy, acute phosphate nephropathy, acute tubular necrosis, adenine phosphoribosyltransferase deficiency, adenovirus nephritis, Alagille Syndrome, Alport Syndrome, amyloidosis, ANCA vasculitis-associated with endocarditis and other infections, angiomyolipoma, analgesic nephropathy, anorexia nervosa nephropathy, vasoconstrictor peptide antibodies and focal segmental glomerulosclerosis, antiphospholipid syndrome, glomerulonephritis-associated with anti-TNF-alpha therapy, AP0L1 mutations, apparent mineralocorticoid excess syndrome, aristolochic acid

[0285] - 51 -

[0286] MEI 48415584v.l nephropathy, herbal medicine-associated nephropathy, Balkan endemic nephropathy, urinary tract arteriovenous malformations and fistulas, autosomal dominant hypocalcemia, Bardet- Biedl syndrome, Bartter syndrome, bath salt-induced acute kidney injury, Beer Potomania, beeturia, P-thalassemia nephropathy, bile cast nephropathy, autologous kidney BK polyomavirus nephropathy, bladder rupture, bladder sphincter dyssynergia, bladder tamponade, Border-Crossers’ nephropathy, Bourbon virus acute kidney Injury, burnt sugar cane harvesting and acute renal dysfunction, Byetta kidney failure, Clq nephropathy, C3 glomerulopathy, C3 glomerulopathy with monoclonal gammopathy, C4 glomerulopathy, calcineurin inhibitor nephrotoxicity, Callilepis Laureola poisoning, cannabinoid hyperemesis- induced acute renal failure, cardiorenal syndrome, Carfilzomib-induced renal injury, CFHR5 nephropathy, Charcot-Marie-Tooth Disease with glomerulopathy, herbal medicine-associated nephropathy, cherry concentrate-induced acute kidney injury, cholesterol embolism, Churg- Strauss syndrome, chyluria, ciliary lesion, cocaine-induced nephropathy, cold-induced diuresis, colistin nephrotoxicity, collagen fibrous glomerulopathy, collapsing glomerulopathy collapsing glomerulopathy- associated with CMV, combined antiretroviral (cART)-associated nephropathy, congenital anomalies of the kidney and urinary tract (CAKUT), congenital nephrotic syndrome, congestive renal failure, cone epiphyseal nephrotic syndrome (Mainzer- Saldino syndrome or Saldino-Mainzer disease), contrast agent nephropathy, copper sulfate poisoning, cortical necrosis, Crizotinib-associated acute kidney injury, crystalline cryoglobulinemia, cryoglobulinemia, crystalglobulin-induced nephropathy, crystal-induced acute kidney injury, crystal reserve histiocytosis, acquired cystic kidney disease, cystinuria, Dasatinib-induced nephrotic range proteinuria, dense deposit disease (MPGN type 2), Dent Disease (X-linked recessive nephrolithiasis), DHA crystal nephropathy, dialysis imbalance syndrome, diabetes and diabetic nephropathy, diabetes insipidus, dietary supplements kidney failure, diffuse mesangial sclerosis, diuretic, Burmese Djenkol Bean poisoning (Djenkolism), Down syndrome kidney disease, drug abuse-associated nephropathy, duplicated ureter, EAST syndrome, Ebola nephropathy, ectopic kidney, ectopic ureter, edema, swelling, Erdheim- Chester disease, Fabry’s disease, familial hypocalciuric hypercalcemia, Fanconi syndrome, Fraser syndrome, fibronectin glomerulopathy, fibrillary glomerulonephritis and immune vibrissae glomerulopathy, Fraley syndrome, excessive fluid, hypervolemia, focal segmental glomerulosclerosis, focal sclerosis, focal glomerulosclerosis, Galloway Mowat syndrome, giant cell arteritis involving the kidneys, gestational hypertension, Gitelman syndrome, glomerular disease, glomerular tubular reflux, diabetes, Goodpasture syndrome, Green Smoothie Cleanse nephropathy, HANAC syndrome, Harvoni (Ledipasvir and Sofosbuvir)-

[0287] - 52 -

[0288] MEI 48415584v.l induced kidney injury, hair dye ingestion-induced acute kidney injury, Hantavirus infection podocytopathy, heat stress nephropathy, hematuria (blood in urine), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), hemophagocytic syndrome, hemorrhagic cystitis, hemorrhagic fever with renal syndrome (HFRS, Hantavirus nephropathy, Korean hemorrhagic fever, epidemic hemorrhagic fever, nephropathis epidemica), hemosiderinuria, hemosiderinosis-associated with paroxysmal nocturnal, hemoglobinuria and hemolytic anemia, hepatic glomerulopathy, hepatic veno-occlusive disease, sinusoidal obstruction syndrome, hepatitis C -related kidney disease, hepatocyte nuclear factor IB-associated nephropathy, hepatorenal syndrome, herbal medicine-associated nephropathy, high altitude renal syndrome, hypertensive nephropathy, HIV-associated immune complex kidney disease (HIVICK), HIV-associated nephropathy (HIV AN), HNF1B- associated autosomal dominant tubulointerstitial nephropathy, horseshoe kidney (renal fusion), Hunner’s ulcer, hydroxychloroquine-induced renal phospholipidosis, hyperaldosteronism, hypercalcemia, hypertension, hypermagnesemia, hypernatremia, hyperoxaluria, hyperphosphatemia, hypocalcemia, hypocomplement urticarial vasculitis syndrome, hypokalemia, hypokalemia-associated renal dysfunction, hypokalemic periodic paralysis, hypomagnesemia, hyponatremia, hypophosphatemia, cannabis-associated hypophosphatemia, hypertension, monogenic hypertension, ice tea nephropathy, ifosfamide nephrotoxicity, IgA nephropathy, IgG4 nephropathy, soak-induced diuresis, immune checkpoint therapy-associated interstitial nephritis, infliximab-associated nephrotic interstitial cystitis, bladder pain syndrome (questionnaire), interstitial nephritis, megakaryocytic interstitial nephritis, Ivemark syndrome, JC viral nephropathy, Joubert syndrome, ketamine- associated bladder dysfunction, kidney stones, nephrolithiasis, kombucha tea toxicity, lead nephropathy and lead-associated nephrotoxicity, lecithin cholesterol acyltransferase deficiency (LCAT deficiency), leptospirosis nephropathy, light chain deposition disease, monoclonal immunoglobulinosis, light chain proximal tubulopathy, Liddle syndrome, Lightwood- Albright syndrome, lipoprotein glomerulopathy, lithium nephrotoxicity, LMX1B mutations-induced hereditary FSGS, low back pain with hematuria, lupus, systemic lupus erythematosus, lupus nephropathy, lupus nephritis, lupus nephritis with seropositive antineutrophil cytoplasmic antibodies, lupus podocytopathy, Lyme disease -associated glomerulonephritis, lysinuria protein intolerance, lysozyme nephropathy, malarial, nephropathy, malignant disease-associated nephropathy, malignant hypertension, Malakoplakia, McKittrick- Wheelock syndrome, MDMA (Molly; Ecstacy; 3,4- methylenedioxymethamphetamine) renal failure, urethral stricture, medullary cystic kidney

[0289] - 53 -

[0290] MEI 48415584v.l disease, uromodulin-associate nephropathy, type 1 adolescent hyperuricemic nephropathy, medullary sponge kidney, megaureterosis, meloxamine toxic-associated nephropathy, MELAS syndrome, membranous proliferative glomerulonephritis, membranous nephropathy, membranous glomerulopathy with occult IgGk deposition, Mesoamerican nephropathy, metabolic acidosis, metabolic alkalosis, methotrexate-associated renal failure, microscopic polyangiitis, milk-alkali syndrome, minimal change nephropathy, monoclonal gammopathy with renal significance, dysproteinemia, mouthwash toxicity, MUC1 nephropathy, polycystic dysplastic kidney, multiple myeloma, myeloproliferative neoplastic glomerulopathy, nail- patellar syndrome, NARP syndrome, nephrocalcinosis, nephrogenic systemic fibrosis, nephroptosis (floating kidney, renal ptosis), nephrotic syndrome, neurogenic bladder, 9 / 11 kidney disease, nodular glomerulosclerosis, nongonococcal urethritis, Nutcracker syndrome, nephron paucity and gigantism, orofacial digital syndrome, orotic aciduria, orthostatic hypotension, orthostatic proteinuria, osmotic diuresis, osmotic nephropathy, ovarian hyperstimulation syndrome, oxalate nephropathy, Page kidney, renal papillary necrosis, papillorenal syndrome (renal coloboma syndrome, solitary renal agenesis), PARN mutation- associated nephropathy, Parvovirus B19 nephropathy, peritoneal-renal syndrome, posterior urethral valve, POEMS syndrome, podocyte infolding glomerulopathy, postinfectious glomerulonephritis, poststreptococcal glomerulonephritis, atypical postinfectious glomerulonephritis, postinfectious glomerulonephritis (IgA dominant), mimics IgA nephropathy, polyarteritis nodosa, posterior urethral valve polycystic kidney disease, postobstructive diuresis, pre-renal disease, propofol infusion-associated syndrome, proliferative glomerulonephritis with monoclonal IgG deposition (Nasr disease, propolis (bee resin)- associated kidney failure, proteinuria (protein in urine), pseudohyperaldosteronism, pseudohypobicarbonaemia, pseudohypoparathyroidism, pulmonary-renal syndrome, nephropyelitis (kidney infection), pyonephrosis, pyridium-associated renal failure, radiation nephropathy, ranolazine nephropathy, refeeding syndrome, reflux nephropathy, rapidly progressive glomerulonephritis, renal abscess, perirenal abscess, renal agenesis, acute kidney injury associated with renal arcuate vein, microthrombosis, renal aneurysm, spontaneous renal artery dissection, renal artery stenosis, renal cell carcinoma, renal cyst, renal hypouricemia with exercise-induced acute renal failure, renal infarction, renal osteodystrophy, renal tubular acidosis, renin mutations-associated and autosomal dominant, tubulointerstitial nephropathy, renin- secreting tumor (juxtaglomerloblastoma), reset osmostat, retro- vena cava ureter, retroperitoneal fibrosis, rhabdomyolysis, obesity treatment surgery- associated rhabdomyolysis, rheumatoid arthritis-associated kidney disease, sarcoidosis

[0291] - 54 -

[0292] MEI 48415584v.l nephropathy, salt loss from kidneys and brain, schistosomiasis glomerulopathy, Schimke immunoosseous dysplasia, scleroderma renal crisis, serpentine fibula-polycystic kidney syndrome, Exner Syndrome, sickle cell nephropathy, silica exposure-associated chronic kidney disease, Sri Lankan Farmers’ Kidney Disease, Sjogren’s syndrome nephropathy, synthetic cannabinoids-induced acute kidney injury, post-hematopoietic cell transplantation nephropathy, stem cell transplantation-associated kidney disease, TAFRO syndrome, tea and toast hyponatremia, tenofovir-induced nephrotoxicity, thin basement membrane disease, benign familial hematuria, monoclonal gammopathy-associated ranolazine nephropathy, refeeding syndrome, reflux nephropathy, rapidly progressive glomerulonephritis, kidney abscess perirenal abscess, renal agenesis, renal arcuate vein microthrombosis-associated acute kidney injury, renal aneurysm, spontaneous renal artery dissection, renal artery stenosis, renal cell carcinoma, renal cyst, renal hypouricemia with exercise-induced acute renal failure, renal infarction, renal osteodystrophy, renal tubular acidosis, renin mutation- associated autosomal dominant tubulointerstitial, nephropathy, renin-secreting tumors (juxtaglomerloblastoma), reset osmostat, retrovena cava ureter, retroperitoneal fibrosis, rhabdomyolysis, obesity treatment surgery-associated rhabdomyolysis, rheumatoid arthritis- associated kidney disease, sarcoidosis nephropathy, salt loss from kidney and brain, schistosomiasis glomerulopathy, Schimke immunoosseous dysplasia, scleroderma renal crisis, serpentine fibula-polycystic kidney syndrome, Exner syndrome, sickle cell nephropathy, silica exposure-associated chronic kidney disease, Sri Lankan Farmers’ Kidney Disease, Sjogren’s syndrome nephropathy, synthetic cannabinoids-associated acute kidney injury, post-hematopoietic cell transplantation nephropathy, stem cell, transplantation- associated kidney disease, TAFRO syndrome, tea and toast hyponatremia, tenofovir-induced nephrotoxicity, thin basement membrane disease, benign familial hematuria, thrombotic microangiopathy associated with monoclonal gammopathy, battlefield nephritis, trigoneitis of the bladder, urogenital tuberculosis, tuberous sclerosis, renal tubular agenesis, immune complex tubulointerstitial nephritis due to autoantibodies directed against the proximal tubule brush border, tumor lysis syndrome, uremia, uremic optic neuropathy, cystic ureteritis, ureteral hernia, urethral caruncle, urethral stricture, urinary incontinence, urinary tract infection, urinary tract obstruction, urogenital impotence, uromodulin-related nephropathy, vancomycin-associated cast nephropathy, vasomotor nephropathy, bladder ileus, vesicoureteral reflux, VGEF inhibitory renal thrombotic microangiopathy, volatile anesthetic acute kidney injury, Von HippeLLindau Disease, Waldenstrom’s macroglobulinemic glomerulonephritis, Warfarin-related nephropathy, wasp sting-induced acute kidney injury,

[0293] - 55 -

[0294] MEI 48415584v.l Wegener’s granulomatosis, granulomatosis with poly angiitis, West Nile Virus-associated chronic kidney disease, Wunderlich syndrome, Zellweger Syndrome or cerebral hepatorenal syndrome.

[0295] In some embodiments, the methods, compounds (e.g., COMPOUND 1), and compositions of the present disclosure can be utilized for treatment of skin diseases, which include but are not limited to: acne, alopecia areata, basal cell carcinoma, Bowen’s disease, congenital erythropoietic porphyria, contact dermatitis, Darier’s disease, disseminated superficial actinic porokeratosis, dystrophic epidermolysis bullosa, eczema (atopic eczema), Paget’s disease of the breast, epidermolysis bullosa simplex, erythropoietic protoporphyrin, fungal infection of fingernails, Hailey-Hailey disease, herpes simplex, hidradenitis suppurativa, hirsutism, hyperhidrosis, ichthyosis, impetigo, epileptic scars, keratosis pilaris, lichen planus, lichen sclerosus, melanoma, black skin disease, mucosal pemphigoid, pemphigoid, pemphigus vulgaris, pityriasis licheniformis, pityriasis rubra pilaris, plantar warts, polymorphic light eruption, psoriasis, plaque psoriasis, pyoderma gangrenosum, rosacea, scabies, scleroderma, shingles, squamous cell carcinoma, sweet’s syndrome, measles and angioedema and vitiligo.

[0296] In some embodiments, the methods, compounds e.g., COMPOUND 1), and compositions of the present disclosure can be utilized for treatment of fibrotic diseases, which include but are not limited to: adhesive shoulder capsulitis, arterial stiffness, arthrofibrosis, atrial fibrosis, cardiac fibrosis and sclerosis, congenital liver fibrosis, Crohn’s disease, cystic fibrosis, Dupuytren’s contracture, endomyocardial fibrosis, colloid scar, hepatitis C, hypertrophic cardiomyopathy, hypersensitivity pneumonitis, idiopathic pulmonary fibrosis, idiopathic interstitial pneumonia, interstitial lung disease, scars, mediastinal fibrosis, myelofibrosis, nephrogenic systemic fibrosis, nonalcoholic fatty liver disease, old myocardial infarction, Peyronie’s disease, pneumoconiosis, pneumonia, progressive massive fibrosis, pulmonary fibrosis, radiation-induced lung injury, retroperitoneal fibrosis, scleroderma / systemic sclerosis, silicosis and ventricular remodeling.

[0297] In some embodiments, the methods, compounds (e.g., COMPOUND 1), and compositions of the present disclosure can be utilized for treatment of hemoglobin disorders, which include but are not limited to: dominant beta-thalassemia, acquired (toxic) methemoglobinemia, carboxyhemoglobinemia, congenital Heinz body hemolytic anemia, HbH disease, HbS / p thalassemia, HbE / p thalassemia, HbSC disease, pure alpha-i- thalassemia (phenotype of alphaO-thalassemia), hydrops fetalis with Hb Bart’s, sickle cell anemia, sickle

[0298] - 56 -

[0299] MEI 48415584v.l cell traits, sickle P-thalassemia, a-F-thalassemia, aO-thalassemia, alpha thalassemia associated with myelodysplastic syndromes, a-thalassemia with mental retardation syndrome (ATR), pO-thalassemia, P+-thalassemia, 5-thalassemia, y-thalassemia, P-thalassemia major, P- thalassemia intermedia, 5P-thalassemia and sySP-thalassemia.

[0300] In some embodiments, the methods, compounds (e.g., COMPOUND 1), and compositions of the present disclosure can be utilized for treatment of autoimmune diseases, which include but are not limited to: flaccid inability (achalasia), Addison’s disease, adult Still’ sdisease, agammaglobulinemia, pemphigus, amyloidosis, ankylosing Spondylitis, anti- GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune autonomic dysfunction, autoimmune encephalomyelitis, autoimmunehepatitis, autoimmune inner ear disease (ATED), autoimmune myocarditis, autoimmune ovarian inflammation, autoimmune pancreatitis, autoimmune retinitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, acute motor axonal neuropathy (AMAN), Balo Disease, leukodystrophy, benign mucocutaneous pemphigoid, herpetic pemphigoid, Castleman disease (CD), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic relapsing multifocal osteomyelitis (CRMO), Chagas-Strauss syndrome (CSS) or eosinophilic granulomatosis with polyangiitis (EGPA), epileptic scar-like pemphigoid, Cogan’s syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn’s disease,, herpes-like dermatitis, dermatomyositis, Devic’s disease (neuromyelitis optica spectrum disorder), discoid lupus, Dressier’s syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, primary mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (arteritis nervosa), giant cell myocarditis, glomerulonephritis, Guldpatrick’s syndrome, granulomatous polyangiitis, Graves’ disease, Guillain-Barre syndrome, Hashimoto’s thyroiditis, hemolytic anemia, Henoch- Schonlein purpura (HSP), herpes gestationis or pemphigus gestationis (PG), hidradenitis suppurativa (HS) (acne inversa), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purple (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis juvenile diabetes mellitus (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome (LES), leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus, chronic Lyme disease, Meniere’s disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren’s ulcer, Mucha-Habermann disease (MH), Multifocal motor neuropathy

[0301] - 57 -

[0302] MEI 48415584v.l (MMN or MMNCB), multiple sclerosis, myasthenia gravis, myositis, episodic sleeping sickness, neonatal Lupus, optic neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, pars planitis (peripheral uveitis),, Parsonnage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, type I polyglandular syndrome, type II polyglandular syndrome, type III polyglandular syndrome, wet polymyalgia, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud’s phenomenon, reactive arthritis, reflex sympathetic dystrophy, recurrent polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, schmidt syndrome, scleritis, scleroderma, Sjogren’s syndrome, semen and testicular autoimmunity, Stiff Person Syndrome (SPS), subacute bacterial endocarditis (SBE), Susac’s syndrome, sympathetic ophthalmia (SO), Takayasu’s arteritis, arteritis cerebrovascularis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, leukoepilepsy, Vogt-Koyanagi-Harada Disease and Wegener’s Granulomatosis (or Granulomatosis with Poly angiitis (GPA)).

[0303] In some embodiments, the methods, compounds (e.g., COMPOUND 1), and compositions of the present disclosure can be utilized for treatment of viral infections, which include but are not limited to: influenza, human immunodeficiency virus (HIV), and herpes.

[0304] In some embodiments, the methods, compounds e.g., COMPOUND 1), and compositions of the present disclosure can be utilized for treatment of malaria infections, which include but are not limited to: infections caused by plasmodium vivax, plasmodium ovale, plasmodium malariae and plasmodium falciparum.

[0305] In some embodiments, the methods, compounds e.g., COMPOUND 1), and compositions of the present disclosure can be utilized for treatment of diseases with mutations that lead to induction of the unfolded protein response (UPR), which include but are not limited to: Marinesco-Sjogren syndrome., neuropathic pain, diabetic neuropathic pain, noise-induced hearing loss, non-syndromic sensory nerve hearing loss, age-related hearing loss, Wolfram syndrome, Darier White disease, Usher syndrome, collagen lesions, thin basal

[0306] - 58 -

[0307] MEI 48415584v.l nephropathy, Allport syndrome, metaphyseal chondrodysplasia type Schmid metaphyseal chondrodysplasia and pseudochondrodysplasia.

[0308] The compounds (e.g., COMPOUND 1) and derivatives provided in the present invention may be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) naming systems.

[0309] Additional detailed aspects of the invention are further described in the sections below.

[0310] EXAMPLES

[0311] Example 1. Synthesis of COMPOUND 1

[0312] Step 1: Preparation of intermediate 6b

[0313] At 25°C, Compound 6a (Bide Pharma, BD00841787, 1.50 g, 7.75 mmol) and DMF (30 mL) were added to a 50 mL single-neck bottle, followed by compound li (Bide Pharma, BD00901377, 1.58 g, 7.75 mmol), diisopropylethylamine (5.13 mL, 30.98 mmol) and 2-(7- Azobenzotriazole)-N,N,N’,N’ -tetramethylurea hexafluorophosphate (4.42 g, 11.62 mmol) under stirring conditions. The reaction was completed after 18 hours at 25°C. The mixture was poured into 200 mL of water, extracted with 300 mL of ethyl acetate twice. The obtained organic layer was washed with 100 mL saturated brine, dried with anhydrous sodium sulfate overnight, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether : ethyl acetate = 10:1 ~ 1 :2) to obtain intermediate 6b. LC-MS: m / z : 344.0 (M-i-H)+.

[0314] Step 2: Preparation of intermediate 6c

[0315] At 25°C, to a 50 mL single-neck bottle was added Compound 6b (2.2 g, 6.40 mmol)

[0316] - 59 -

[0317] MEI 48415584v.l and EtOH (20 mL), followed by hydrazine hydrate (20 mL) under stirring conditions. The reaction was completed after 16 hours at 85 °C. The reaction mixture was cooled down to room temperature, and then concentrated under reduced pressure to obtain a crude product. The obtained crude product was suspended in acetonitrile (50 mL), and the suspension was stirred 2 hours at 25 °C, and then filtered to obtain intermediate 6c.]H NMR (400 MHz, DMSO-d6) 58.93 (s, 1H), 7.99 (d, J = 8.1 Hz, 1H), 7.50 (t, J = 8.9 Hz, 1H), 7.07 (dd, J = 11.4, 2.8 Hz, 1H), 6.88-6.77 (m, 1H), 4.49 (s, 2H), 3.56 (tt, J = 8.0, 4.0 Hz, 1H), 3.18 (d, J = 6.9 Hz, 1H), 2.00 (s, 1 H), 1.79-1.62 (m, 4H), 1.46-1.22 (m, 4H). LC-MS: m / z : 344.0 (M+H)+.

[0318] Step 3: Preparation of intermediate 6d

[0319] At 25°C, to a 50 mL single-neck bottle was added intermediate 6c (1g, 2.91 mmol) and CH2CI2 (30 mL), followed by N,N’ -carbonyldiimidazole (0.71 g, 4.37 mmol) under stirring conditions. The reaction was completed after 16 hours at 25°C. The reaction mixture was concentrated under reduced pressure to obtain a crude product. The obtained crude product was purified by column chromatography (CH2CI2 : CH3OH = 10:1 ~ 5:l) to obtain intermediate 6d. LC-MS: m / z : 370.0 (M+H)+.

[0320] Step 4: Preparation of Compound 6

[0321] At 25°C, to a 50 mL single-neck bottle was added intermeidate 6d (100 mg, 0.27 mmol) and DMF (4 mL), followed by diisopropylethylamine (0.18 mL, 1.08 mmol), 1H- benzotriazole-l-yloxytripyrrolidinyl hexafluorophosphate (179.41 mg, 0.41 mmol) and compound 6f (71.50 mg, 0.40 mmol) under stirring conditions. The reaction was completed after 18 hours at 25 °C. The reaction mixture was poured into 20 mL water, extracted with 30 mL ethyl acetate twice. The obtained organic layer was washed with 10 mL brine, dried with sodium sulfate overnight, filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The obtained crude product was purified by HPLC (trifluoroacetic acid / acetonitrile / water system) to obtain Compound 6. *H NMR (400 MHz, DMSO-d6) 58.05 (d, J = 8.0 Hz, 1H), 7.50 (t, J = 8.9 Hz, 1H), 7.07 (dd, J = 11.4, 2.9 Hz, 1H), 6.85 (dd, J = 8.7, 2.9 Hz, 1H), 5.37-5.24 (m, 1H), 4.56- 4.37 (m, 4H), 4.19 (dd, J = 9.7, 4.0 Hz, 2H), 3.66 (t, J = 3.8 Hz, 1H), 2.73 (td, J = 11.9, 10.1, 6.1 Hz, 1H), 2.09-1.97 (m, 2H), 1.90-1.79 (m, 2H), 1.55-1.35 (m, 4H)OLC-MS: m / z :493.1 (M+H)+.

[0322] Example 2. COMPOUND 1 shows superior potency in cellular models

[0323] - 60 -

[0324] MEI 48415584v.l This example demonstrates that the compound of the invention (e.g. , COMPOUND 1) is an activator I agonist for the GEF activity of e-IF2, in that it inhibits ATF4 translation (as measured by a luciferase reporter gene under the control of the ATF4 5’ UTR, in an ATF4 luciferase reporter plasmid) through activation of the eIF2.GTP.tRNAiMetternary complex.

[0325] The ATF4 luciferase reporter plasmid is composed of two parts, the 5’ untranslated region sequence of the ATF4 gene and the luciferase coding sequence. Specifically, the 5’ untranslated region sequence of ATF4 containing two upstream open reading frames (uORFs) (NCBI database number BC022088.2) and the firefly luciferase coding gene are cloned into the pLVX-Puro vector (YouBio, VT1465). The packaging plasmids for lentivirus are psPAX2 (YouBio, VT1444) and pMD2.G (YouBio, VT1443). The above three plasmids are transfected simultaneously with X-tremeGENE 9 DNA transfection reagent in HEK293T / 17 cells to create the HEK293T / 17-ATF4 uORF-Luc-Puro monoclonal cell, and the lentivirus-containing culture medium is collected after 48 hours. Puromycin at 1 pg / mL is used to screen the virus-transduced HEK293T / 17 cells, after which the monoclonal cells are obtained by limited dilution.

[0326] This cell line (HEK293T / 17-ATF4 uORF-Luc-Puro) was utilized to detect the translational regulation of ATF4 and test the activity of eIF2B activator by cold fluorescence. The experimental procedure was as follows: Spread 6000 HEK293T / 17-ATF4uORF-Luc- Puro monoclonal cells in a 384- well plate to allow them to attach to the wall overnight. The test compound was dissolved in DMSO and then added to the cell culture solution together with 50 nM of toxic carotene. Incubate it for 6 hours. Toxic carotene acted to cause cellular stress and upregulate ATF4 protein translation. After 6 hours, cells were lysed using the One- Glo Luciferase Assay Kit (Promega #E6120), followed by reading the cold fluorescence values using the LUM program of the EnVision 2104 plate reader.

[0327] The relative expression of the ATF4 reporter gene (ATF4 reporter expression %) was calculated as follows:

[0328] ATF4 reporter expression % = (ave_sample-ave_vc) / (ave_pc-ave_vc)*100%. ave_vc: average signal value of negative control ave_pc: average signal value of positive control ave_sample: average signal value of the sample

[0329] Fit dose-response curves and calculate EC50 values:

[0330] The method of nonlinear regression log (inhibitor) vs. response — variable slope (four

[0331] - 61 -

[0332] MEI 48415584v.l parameters) of GraphPad 9 software was used to fit the correspondence between the relative expression of ATF4 reporter genes and compound concentrations.

[0333] X-axis: log value of compound concentration; Y-axis: relative expression of ATF4 reporter gene; Top: asymptote valuation on the curve; Bottom: asymptote valuation under the curve; Hillslope: slope of the fitted curve.

[0334] Formula: Y = Bottom 4- (Top - Bottom) / (1 4- 10A((Log EC so - X) x HillSlope)), i.e., Log ECso = X + (1 / HillSlope) x log ((Top - Y) / (Y - Bottom)).

[0335] The data regarding the HEK293T / 17-ATF4 uORF-Luc-Puro monoclonal cell activity showed that COMPOUND 1 has ECso less than 1 nM.

[0336] FIGs. 1A-1B show that COMPOUND 1 effectively inhibited the expression of ATF4 as assessed by luciferase reporter assay, with the ECso value about 2 pM, which was about 10,000 times lower than the ECso value of Reference 1 (about 23 nM).

[0337] Example 3. Additional biological assessments

[0338] Western blot assay

[0339] Consistent with the results of the luciferase assay, COMPOUND 1 effectively inhibited ATF4 translation in a dose-dependent manner as assessed by Western blot analysis (FIGs. 2A-2C). The ECso value of COMPOUND 1 was around 39 pM, which was about 333 times lower than the ECso value of a comparator compound Reference 1 (about 13 nM). These data demonstrate the superior potency of COMPOUND 1 over the known therapeutic agent on the inhibition of ATF4 expression.

[0340] The experimental results show that COMPOUND 1 can enhance / activate the activity of eIF2B, and can reduce the amount of ATF4 expression, which leads to the reduction of fluorescence intensity. This indicates that the present compounds can significantly alleviate the cellular stress caused by toxic carotene, reduce the integration stress response of the cell, and normalize the synthesis of intracellular proteins. Therefore, the present compound is an eIF2B agonist.

[0341] Kinetic Solubility

[0342] Kinetic solubility of COMPOUND 1 was assessed using the following procedure: dissolve the test compound (COMPOUND 1) in DMSO to prepare 10 mM stock solution; prepare a 100 mM K2HPO4 solution by adding 8.71 g K2HPO4 to 500 mL deionized water;

[0343] - 62 -

[0344] MEI 48415584v.l add 2.05 g of potassium dihydrogen phosphate to 150 mL of deionized water to prepare a 100 mM potassium dihydrogen phosphate solution; mix 405 mL of 100 mM K2HPO4 and 95 mL of 100 mM KH2PO4, and adjust the pH value of the mixed solution to 7.4 with 100 mM K2HPO4 / KH2PO4 solution; prepare the buffer solution (simulating intestinal fluid in the fast state, pH = 6.5) by adding 10.41 g of FaSSIF buffer concentrate to 240.3 g deionized water, prepare the buffer (simulating intestinal fluid in the fed state, pH = 5.0) by adding 4.071 g of FeSSIF buffer concentrate to 45.97 g deionized water.

[0345] Using a 96- well plate, 16 pL of 10 mM compound stock solution was added to 784 pL of different buffers (n = 3), and the plate was sealed and shaken at 1000 rpm at 25 °C (PBS) and 37°C (other) for 1.5 h. After incubation, the solution was transferred to a filter plate. AU samples were filtered. About 5 pL of the filtrate was taken, to which 5 pL DMSO and 490 pL acetonitrile aqueous solution (1 :1) containing the internal standard was added and mix well. Then, an acetonitrile aqueous solution (1 :1) containing the internal standard was used to dilute it, according to the properties of the compound and its response in the mass spectrum. The dilution factor was changed according to the solubility value and UPLC- MS / MS signal response.

[0346] The experiment indicates that COMPOUND 1 has good solubility in the different simulated environments described above. Specifically, COMPOUND 1 has a solubility greater than 10 pg / mL in a simulated intestinal fluid (pH = 6.5) environment in a fast state, and a solubility greater than 80 pg / mL in a simulated intestinal fluid (pH = 5.0) environment in a fed state.

[0347] Liver microsomal stability in vitro

[0348] The experiment indicates that COMPOUND 1 has good in vitro liver microsomal stability, based on the livers microsomal stability assay performed according to the following procedure:

[0349] Preheat 100 mM K-Mg-buffer containing 5 mM MgCL. Add 5 pL 10 mM of COMPOUND 1 and reference stock solution to 95 pL acetonitrile (ACN) to prepare a labeling solution. Add 1.5 pL 500 pM labeling solution and 18.75 pL 20 mg / mL liver microsomes to 479.8 pL K-Mg buffer. Prepare the NADPH stock solution (3 mM) by dissolving NADPH into K-Mg buffer. At different time points (0, 5, 15, 30 and 45 minutes), evenly drop 30 pL 1.5 pM labeling solution containing microsomes into the detection plate. Pre-incubate at 37 °C for 5 min. At 0 min, add 200 pL of ACN containing IS (internal

[0350] - 63 -

[0351] MEI 48415584v.l standard, tolbutamid / terfenadine) into the well, and then add 15 pL of NADPH stock solution (6 mM). For other time points, start the reaction by adding 15 pL NADPH stock solution (6 mM) to the wells and start to time. Add 200 pL of ACN containing IS to the corresponding plate wells at 5, 15, 30, and 45 minutes to stop the reaction.

[0352] After quenching, shake the plate at 600 rpm for 10 minutes and then centrifuge at 4000 rpm for 50 minutes. Pipette 80 pL of supernatant from each well and transfer it to a 96- well sample plate containing 160 pL of pure water for UPLC / MS / MS analysis.

[0353] In vitro hepatic microsomal stability of the exemplary compounds.

[0354] Cell membrane permeability

[0355] The experiment indicates that COMPOUND 1 has good cell membrane permeability, based on the assay performed according to the following procedure:

[0356] Dilute COMPOUND 1 from 10 mM stock solution to a concentration of 10 pM in transport buffer (HBSS + BSA) and then apply it to the apical or 5 basolateral sides of the cell monolayer. Measure the permeability of COMPOUND 1 from the A to B direction or B to A direction in duplicate after 120 minutes of incubation at 37°C, 5% CO2 and 95% relative humidity. In addition, determine the exclusion ratio of COMPOUND 1 . Use the LC-MS / MS method to quantitatively analyze the test substance and reference substance based on the analyte / IS peak area ratio.

[0357] Cell membrane permeability of the exemplary compounds.

[0358] P450 enzyme inhibition

[0359] The experiment indicates that COMPOUND 1 has weak inhibition activity on the five

[0360] - 64 -

[0361] MEI 48415584v.l major P450 enzymes (CYP1 A2, CYP2C9, CYP2D6, CYP2C19, CYP3A4) and has a low risk of drug-drug interactions. Specifically, the IC50 values of COMPOUND 1 for these five major P450 enzymes are greater than 10 pM, especially the IC50 values of CYP2D6 and CYP3A4 are both greater than 30 pM. The assay was performed according to the following procedure:

[0362] Prepare phosphate buffer for liver microsomes. Add 169 pL of liver microsomes in phosphate buffer and 1 pL of working solutions of various concentrations of COMPOUND 1 or positive control compound to a 96-well plate. Place the culture plate in a water bath and preheat at 37°C for 15 minutes. After the incubation, add 10 pL of substrate to the culture plate (for CYP3A4-T, add 1 pL of substrate and 9 pL of K-Mg buffer to the culture plate), mix the incubation mixture on a rotary mixer for 10 seconds, and then add 20 pL of 10 mM NADPH solution to start the reaction at a final concentration of 1 mM. Repeat the experiment twice. At predetermined time points listed in Table 4, quench the reaction by adding 400 pL of quenching solution (cold ACN containing 500 nM toluene and 10 nM terfenadine). Centrifuge at 3220 g for 50 min at 4°C. Transfer 100 pL of supernatant to a new plate. Dilute the supernatant with 100 pL pure water. Mix well and analyze the sample using UPLC- MS / MS.

[0363] Configuration of substrate stock solution.

[0364] Mouse pharmacokinetic (PK) evaluation

[0365] The experiment indicates that COMPOUND 1 has excellent pharmacokinetic properties in mice, including but not limited to Cl (clearance rate), T1 / 2 (half-life), Cmax (peak concentration), AUC (drug concentration), area under the curve), F (bioavailability), etc.). The PK evaluation was performed according to the following procedure:

[0366] Dissolve COMPOUND 1 in the solvent to prepare a clear solution or homogeneous suspension. To each group of 3 mice, administer 1 mg / kg (1 mpk) of COMPOUND 1 via tail

[0367] - 65 -

[0368] MEI 48415584v.l vein, or administer 10 mg / kg (10 mpk) orally (PO). Collect blood at 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h and 24 h after intravenous administration, and at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, after oral administration. Collect blood at 6 h, 8 h and 24 h. Centrifuge plasma samples and take the supernatant to prepare samples for quantitative analysis by LC / MS / MS.

[0369] Mouse PK properties of COMPOUND 1

[0370] In comparison, Reference 1 orally administered (PO) to mouse at the same 30 mg / kg dose yielded AUC(o-inf) of 216,018 ng*h / ml, roughly 40% of that of COMPOUND 1. The bioavailability of Reference 1 was also 7-fold less (2% vs. 14%) than that of COMPOUND 1.

[0371] Certain non-limiting protocols useful for the experiments are provided below as illustration only.

[0372] Cell culture and maintenance:

[0373] 1. Culture HEK293T / 17 WT or mutant cells in T75 cell culture flask in DMEM (11965-092, Gibco), 10%FBS, 1%P / S in 37C, 5% CO2incubator.

[0374] 2. Digest cells with 0.25% trypsin EDTA (25200-056, Gibco) when cell density reached 70-80%.

[0375] 3. Use cells less than 20th generations for assay.

[0376] Protocol for Thapsigargin (Tg) titration:

[0377] 1 . Seed 400,000 HEK293T / 17 WT or mutant cells per well in a 12-well plate in 1 mL DMEM Complete Medium plus 10% FBS. Incubate cells at 37°C, 5% CO2overnight.

[0378] 2. Add Tg into the 12-well plate according to the plate map, staring from 100 nM, 2- fold serial dilution, 10 points. Incubate cells at 37°C, 5% CO2 for 2 h.

[0379] 3. Remove supernatant and wash with PBS. Add 200 pL RIPA lysis and extraction buffer containing Protease Inhibitor Cocktail (4693116001, Roche) and Phosphatase Inhibitor

[0380] - 66 -

[0381] MEI 48415584v.l Cocktail (4906845001, Roche), lyse cells on ice for 10 min, then centrifuge at 15,000 rpm at 4 °C for 10 min, collect supernatant as samples.

[0382] 4. Mix 75 pL samples and 25 pL NuPAGE™LDS 4x sample buffer (NP0007, Invitrogen) (add 5% P-sulfhydryl ethanol before use). Heat samples at 100°C for 5 min using Compact Dry Bath (88871003, ThermoFisher).

[0383] 5. Load 15 pL of the above sample per well into 4-12% BIS-Tris gel (WG1402BOX, Invitrogen), perform electrophoresis using lx MES SDS running buffer (Invitrogen, NP0001), at 100 V for 120 min.

[0384] 6. Activate PVDF membrane in methanol for 3 min, then wash with lx NuPAGE Transfer Buffer (NP00061, Invitrogen). Transfer protein to PVDF membrane using constant current at 300 mA for 90 min.

[0385] 7. Block PVDF membrane using Intercept (TBS) Blocking Buffer (927-60001, LL COR) at room temperature for 1 h. Probe the membrane with 1 : 1 ,000 dilution of ATF4 (32364, ThermoFisher) and 1:3,000 dilution of GAPDH (97166S, CST) overnight at 4°C.

[0386] 8. Wash the membrane with lx TBST for 5 min, 3 times. Incubate with 1:10,000 diluted IRDye 680RD Goat Anti-Mouse IgG (926-68070, LI-COR) and 1 :10,000 diluted IRDye 800CW goat anti-rabbit IgG (926-32211, LI-COR) for Ih. Note: protect from light.

[0387] 9. Wash the membrane with lx TBST for 5 min, 3 times. Western blot analysis was performed using LI-COR Odyssey imaging system (LI-COR).

[0388] 10. Data analysis: Fluorescence signal of samples in the membrane was obtained by Image studio. Then calculate the ratio of ATF4 signal to GAPDH signal. The SignalATF4 / SignalGAPDH was analyzed using non-linear regression in GraphPad Prism 9 to obtain the EC80 value of the Tg.

[0389] Protocol for compounds test:

[0390] 1. Seed 400,000 HEK293T / 17 WT or mutant cells per well in a 12-well plate in 1 mL DMEM Complete Medium plus 10% FBS. Incubate cells at 37°C, 5% CO2 overnight.

[0391] 2. Add test compounds into the 12-well plate according to the plate map, staring from 1,000 or 100 nM, 3-fold serial dilution, 9 points, then add the Tg (final cone. 10 nM) into 12- well plate. Incubate cells at 37°C, 5% CO2 for 2 h.

[0392] 3. Remove supernatant and wash with PBS. Add 200 pL RIPA lysis and extraction buffer containing Protease Inhibitor Cocktail (4693116001, Roche) and Phosphatase Inhibitor Cocktail (4906845001, Roche), lyse cells on ice for 10 min, then centrifuge at 15,000 rpm at

[0393] - 67 -

[0394] MEI 48415584v.l 4°C for 10 min, collect supernatant as samples.

[0395] 4. Mix 75 pL samples and 25 L NuPAGE™LDS 4x sample buffer (NP0007, Invitrogen) (add 5% P-sulfhydryl ethanol before use). Heat samples at 100°C for 5 min using Compact Dry Bath (88871003, ThermoFisher).

[0396] 5. Load 15 pL of the above sample per well into 4-12% BIS-Tris gel (WG1402BOX, Invitrogen), perform electrophoresis using lx MES SDS running buffer (Invitrogen, NP0001), at 100 V for 120 min.

[0397] 6. Activate PVDF membrane in methanol for 3 min, then wash with lx NuPAGE Transfer Buffer (NP00061, Invitrogen). Transfer protein to PVDF membrane using constant current at 300 mA for 90 min.

[0398] 7. Block PVDF membrane using Intercept (TBS) Blocking Buffer (927-60001 , LL COR) at room temperature for 1 h. Probe the membrane with 1 : 1000 dilution of ATF4 (32364, ThermoFisher) and 1:3,000 dilution of GAPDH (97166S, CST) overnight at 4°C.

[0399] 8. Wash the membrane with lx TBST for 5 min, 3 times. Incubate with 1 :10,000 diluted IRDye 680RD Goat Anti-Mouse IgG (926-68070, LI-COR) and 1 :10,000 diluted IRDye 800CW goat anti-rabbit IgG (926-32211, LI-COR) for Ih. Note: protect from light.

[0400] 9. Wash the membrane with IX TBST for 5 min, 3 times. Western blot analysis was performed using LI-COR Odyssey imaging system (LI-COR).

[0401] 10. Data analysis: Fluorescence signal of samples in the membrane was obtained by Image studio. Then calculate the ratio of ATF4 signal to GAPDH signal (SignalATF4 1 SignalGAPDH). By comparing the SignalATF4 / SignalGAPDH of control group of 0.1% DMSO and control group of Tg, calculated the relative expression of ATF4. The relative expression of ATF4 was analyzed using non-linear regression in GraphPad Prism 9 to obtain the EC50 value of the test compounds.

[0402] Example 4. COMPOUND 1 is efficacious in mouse model of Vanishing While Matter (VWM)

[0403] Vanishing White Matter (VWM) is a neurological disease caused by mutations in eIF2B that reduce its activity. Introduction of the human mutation (R191H) into mice leads to persistent ISR in the CNS and behavioral changes consistent with the disease phenotype of human VWM.

[0404] - 68 -

[0405] MEI 48415584v.l To assess the effects of COMPOUND 1 on VWM, Eif2b5R191H / R191Hmice with homozygous R191H knock-in (R191H-H0 mice) were generated as shown in FIG. 3. Experiments were performed as shown in FIG. 4. Briefly, R191H-H0 mice of 10 to 12 weeks of age were treated with COMPOUND 1 (indicated as “CMPD 1” in the figure), Reference 1, or vehicle. Behavior tests were performed at 10-12 weeks, 23-25 weeks, 30-32 weeks, and 33-35 weeks of age. All the mice were sacrificed at 34-36 weeks of age and samples were collected for measurement of expression of ISR markers and myelin loss in the brain and spinal cord. Body weight was monitored through the course of the experiment.

[0406] At the initiation of the study (10-12 weeks old), R191H-HO mice had body weight of around 21 g, significantly lower than the body weight of WT control mice (about 25 g) (FIG. 5 A). The body weight of 1 mg / kg COMPOUND 1 -treated and 10 mg / kg COMPOUND 1 - treated R191H-HO mice quickly restored to around 24 g by 13 weeks of age, which was about 1 to 2 weeks post dosing. Weight gain of these groups of mice then slowed down and gradually increased to 25 g by 33-35 weeks of age, at which point WT mice had body weight of about 26 g. These results demonstrate the efficacy of COMPOUND 1 in treatment of VWM.

[0407] Similar degrees of weight gain was observed in 1 mg / kg COMPOUND 1 -treated and 10 mg / kg Reference 1 -treated group (to 25 g by 33-35 weeks of age). Moreover, treatment of 1 mg / kg Reference 1 restored the body weight of R191H-H0 mice to about 22.5 g by 33-35 weeks of age, a degree of restoration that was significantly less than treatment by the same dose of COMPOUND 1 (to 25 g). These results demonstrate the higher potency of COMPOUND 1 over Reference 1 for treating VWM in vivo. Similar results were observed in male and female groups plotted separately (FIGs. 5B and 5C).

[0408] Next, behavioral assessment of motor function was performed. In a beam-crossing test of balance and motor coordination, vehicle-treated R191H-H0 mice showed significant increase in beam-crossing time (FIG. 6A) and exhibited more foot slips and falls from the beam while crossing (FIG. 6D) in comparison to vehicle-treated WT control. By contrast, COMPOUND 1 -treated R191H-H0 mice were indistinguishable from vehicle-treated WT in the results of beam-crossing time and slips and falls (FIGs. 6A and 6D). Similar results were observed in male (FIGs. 6B and 6E) and female groups (FIGs. 6C and 6F) in both assays. Notably, COMPOUND 1 shows efficacy at doses as low as 0.1 mg / kg in restoring motor coordination in VWM mice, demonstrating the high potency of COMPOUND 1 for treatment of VWM.

[0409] - 69 -

[0410] MEI 48415584v.l Example 5. COMPOUND 7 was more efficacious than Reference 1 in mouse model of Vanishing White Matter (VWM)

[0411] This example demonstrates that COMPOUND 1 of the invention was far more potent than Reference 1 in suppresing ISR activity in the mouse model of VWM - Eif2b5RI9IH / R,91Hmice having homozygous R191H knock-in (R191H-HO mice) (see above in FIG. 3).

[0412] Vanishing White Matter is a chronic and progressive neurodegenerative disease that is driven by chronic activation of the integrated stress response (TSR) caused by recessive mutations in eIF2B. To assess the efficacy and potency of COMPOUND 1 to agonize eIF2B and resolve the ISR, the Eif2b5RI9IH / R19mouse model for this disease was created by knocking in the R191H point mutation into both alleles of the Eif2b5 gene locus. Such homozygous Knock-In (KI) “R191H-HO mice” were then treated with increasing doses of COMPOUND 1, or Reference 1, and the efficacy of compound treatment was measured via different modalities.

[0413] For example, to measure ISR activity, R191H-HO mice of 10 to 12 weeks of age were treated with COMPOUND 1 (indicated as “CMPD 1” in the figure), Reference 1 (indicated as “REF 1” in the figure), or vehicle, at the indicated compound concentrations. ISR activity in the mice brians of each experimental group was assessed and normalized.

[0414] To assess ISR activity, a set of genes from the literature were curated, which genes have previously been shown to be activated under ISR activation. The expression of these genes were then measured from bulk RNA-seq data obtained from the brains of vehicle- or compound-treated WT and R191H mice. The log2 fold change of expression for each gene, as compared to its average expression in the WT vehicle-treated samples, was shown in the heatmap in FIG. 7. Genes were depicted in the rows, and the relative expression from each individual mouse was shown in the columns. The overall activity of ISR activation per sample was calculate by simply taking a weighted average of the expression of the genes in the signature, as shown by the boxplots on the right. The weights per gene were assigned by performing an eigen value decomposition of the genes in this signature across the samples, and extracting the contribution of the gene to the first eigen vector.

[0415] It is apparent that in vehicle treated R191H-HO mice, normalized ISR activity was significantly higher than wild-type mice similarly treated by vehicle.

[0416] COMPOUND 1 treatment of the R191H-HO mice significantly reduced normalized ISR activity in a dose responsive manner. Similar dose-responsive results were also seen in

[0417] - 70 -

[0418] MEI 48415584v.l Reference 1 treated R191H-H0 mice. However, COMPOUND 1 at 1 mg / kg (Impk) was significantly more potent that Reference 1 at 1 mpk. Indeed, COMPOUND 1 at 0. 1 mpk was about similarly potent as Reference 1 at 1 mpk, suggesting much higher potency of COMPOUND 1 compared to Reference 1.

[0419] Example 6. COMPOUND 1 is efficacious to treat Huntington’s Disease (HD)

[0420] This example demonstrates that COMPOUND 1 is effective to reduce ISR activity in Huntington’s Disease, thus treating HD.

[0421] FIG. 8A shows increased activation of the integrated stress response (ISR) in medium spiny neurons isolated from post-mortem brains of advanced (right side) versus pre- symptomatic (left side) Huntington’s disease patients.

[0422] Specifically, Fluorescence-Activated Nuclear Sorting (FANS) was used to quantitate cell type-specific gene expression from various cell types of the striatum and cerebellum in HD and control donors from post mortem human brain (Matlik et al., Nat Genet 56, 383-394, 2024, incorporated herein by reference). The ISR pathway activation within each cell type from the individual samples was calculated as described previously.

[0423] It is apparent that ISR Activation Score in advanced patient’s brains were significantly higher than that in the brains of the pre- symptomatic HD patients. Both the striatonigral and striatopallidal medium spiny neurons from HD donors had increased activation of the ISR as compared to the normal donors. Thus, administering COMPOUND 1 to HD patients reduces ISR activity in the brains of the HD patients, thus treating HD.

[0424] FIG. 8B shows increased activation of the integrated stress response (ISR) in medium spiny neurons derived from induced pluripotent stem cells (iPSCs) from advanced (right side) versus pre-symptomatic (left side) Huntington’s disease patients. Specifically, direct neuronal reprogramming was employed to generate medium spiny neurons from fibroblasts isolated from patients with CAG repeat lengths >= 40 (Mi Oh et al., Nat Neurosci 25(11): 1420-1433, 2022). In an attempt to model the effects of disease progression on molecular phenotypes of MSNs, some fibroblasts were collected >= 15 years before the onset of HD symptoms (preHD) and others after (HD). The ISR activation score was calculated from RNA-seq data collected from the iPSC-derived MSN's indicate a slight increase in ISR activation in those MSNs derived from patients after the onset of HD symptoms.

[0425] Again, it is apparent that ISR Activation Score in iPSC derived from advanced HD

[0426] - 71 -

[0427] MEI 48415584v.l patients were significantly higher than that in iPSC derived from pre- symptomatic HD patients. Thus, administering COMPOUND 1 to HD patients reduces ISR activity in the brains of the HD patients, thus treating HD.

[0428] Example 7. COMPOUND 1 is efficacious to treat Char cot-Marie-Tooth type 2A (CMT2A)

[0429] This example demonstrates that COMPOUND 1 is effective to reduce ISR activity in Charcot-Marie-Tooth type 2A (CMT2A), thus treating CMT2A.

[0430] FIGs. 9A and 9B show the increase in expression of GDF15 and ATF4 (genes that are markers of ISR) in fibroblasts isolated from patients with Charcot-Marie-Tooth type 2A (CMT2A). It is apparent that ISR activity, as measured by expression of marker genes GDF15 and ATF4, was significantly higher in CMT2A patient fibroblasts. Thus, administering COMPOUND 1 to CMT2A patients reduces ISR activity in CMT2A patients, thus treating CMT2A.

[0431] Example 8. In vivo evaluation of efficacy of COMPOUND 1 on CMT2D mouse model.

[0432] 8.1 Experimental Design

[0433] Table 1 Group and treatment_efficacy

[0434] Note:

[0435] 1] Dosing start time: 3 weeks old (upon weaning)

[0436] 21 EOL: 5, 7 and 1 1 weeks old

[0437] 3] Daily body weight monitoring (except weekend)

[0438] 4] Behavioral tests: Rotarod and grip strength at 4, 6 and 8 weeks post dosing; Beam walk at

[0439] 2, 4, 6 and 8 weeks post dosing

[0440] 5] Nerve conduction velocity (NCV) test: at 2, 4, 6 and 8 weeks post dosing

[0441] - 72 -

[0442] MEI 48415584v.l 6] Spinal cord (Thoracic and lumbar, RNA later, snap frozen), sciatic nerve (RNA later), femoral nerves (FFPE), Cervical spinal cord anterior horn (RNA later), Dorsal root ganglion (RNA later), TA (RNA later, snap frozen, FFPE), GA (RNA later, snap frozen, FFPE) and CSF collection at EOL

[0443] 7] Toluidine blue staining and motor axon number quantification on femoral nerves

[0444] 8] Plasma from Groups 3 and 4 were collected on Day 4, Day 7, 2 weeks, 4 weeks, 6 weeks and 8 weeks post dosing for Compound 1 PK analysis. Samples were collected between 14:00 and 16:00 at each timepoint.

[0445] Table 2 Group and treatment_PK

[0446] 1] 3 mice from G3 and G4 were switched to normal chow for 3 days at 10 weeks old (7 weeks of dosing) before PO dosing

[0447] 2] Plasma sample collection: 0.5, 1, 2, 4, 8 and 24h post dosing

[0448] 3] Vehicle: 10% (v / v) TPGS in H2O

[0449] 8.2 Materials

[0450] 8.2.1. Animals and Housing Condition

[0451] 8.2.1.1. Animals

[0452] Species: Mus Musculus

[0453] Strain: B6;CAST-GarGNmf249 / RwbJ (Gars™3™*, #033165)

[0454] Age: 3 weeks old

[0455] Sex: mixed

[0456] Body weight: 6-18 g

[0457] Number of animals: 57

[0458] Animal breeding: IVF was performed for colony expansion using 1 male heterozygous Gars™3™ mouse from JAX and 20 4-week-old female C57BL / 6J mice from

[0459] - 73 -

[0460] MEI 48415584v.l Charles River.

[0461] 8.2.1.2. Housing Condition

[0462] The mice were kept in individual ventilation cages at constant temperature and humidity with 3 mice in each cage.

[0463] • Temperature: 20-26 °C.

[0464] • Humidity: 40-70%.

[0465] Cages: Made of polycarbonate. The size is 375 mm x 215 mm x 180 mm. The bedding material is com cob, which is changed twice per week.

[0466] Diet: Animals had free access to irradiation sterilized dry granule food during the entire study period.

[0467] Water: Animals had free access to sterile drinking water.

[0468] Cage identification: number of animals, sex, strain, date received, treatment, study number, group number and the date of the treatment.

[0469] Animal identification: Animals were marked by ear coding.

[0470] 8.3. Experimental Methods and Procedures

[0471] 8.3.1. Grouping

[0472] Fifty seven heterozygous Gar.yP278KY / +mice were divided into 4 groups based on their weights after finishing quarantine, ensuring that the average weights of all 3 groups were as similar as possible.

[0473] 8.3.2. Chow Preparation

[0474] Customized chow containing 2 concentrations of COMPOUND 1 , namely 30 and 300 mg / kg. Typically, COMPOUND 1 was mixed into a measured amount of feed ingredients, followed by routine production and irradiation sterilization.

[0475] 8.3.3 Grip Strength

[0476] 1) Fix the force meter to a firm base (so to be stable during the pull and not move as a result of mouse grasp), at a defined height above the table or surface.

[0477] 2) Attach the net-bar to the force meter in a defined manner.

[0478] - 74 -

[0479] MEI 48415584v.l 3) Turn on the meter. Choose gf (gram force) as unit of values.

[0480] 4) Set to zero.

[0481] 5) Lift the mouse by the tail to the height where the four paws are at all on the bar.

[0482] 6) Move the mouse horizontally towards the bar until it becomes within reach.

[0483] 7) Gently pull the mouse away until its grasp is broken. The pulling should be at a constant speed and sufficiently slow to permit the mouse to build up a resistance against it. The transducer saves the value at this point.

[0484] 8) Record the transducer as the Peak Force of this trial and place mice back to their home cage. Let them rest at least 1 min before the next trial.

[0485] 9) After a rest period, repeat 5-8 to complete a total of 3 trials.

[0486] 10) Corrected grip strength = grip strength X body weight.

[0487] 8.3.4 Rotarod

[0488] Acclimate all mice in the test environment and train the mice on the rotarod at a fixed speed (4 rpm~14 rpm) before the day of the test. Usually it takes about 4 times (each day one time) of training. Rotarod test will be performed the next day after the training. The fall-off latency and speed of each mouse will be recorded at accelerated speed from 4~40 rpm, 90s are set as cut-off value.

[0489] 8.3.5 Beam walk

[0490] Training

[0491] 1) Set up for 0 2.5 cm beam on the 2 poles, and place the mouse in the dark box for 3 minutes.

[0492] 2) Place the mouse on the beam that are 10 cm away from the box opening. If the mouse can cross and back to the box, mark “A / ” in the record. Then let the mouse rest in the box for about 1 min and continue the training for the next beam length. If the mouse falls, mark “x” in the record, and repeat the same procedure to complete up to 6 trials regardless of falling or crossing, then resume the training for the next beam length. 1 min rest in the box for the mouse will be needed between each trial.

[0493] 3) Place the mouse on the beam that are 15cm, 30cm, 45cm, 60cm respectively

[0494] - 75 -

[0495] MEI 48415584v.l away from the box and repeat the same training procedure.

[0496] 4) Clean the beam and box with 75% ethanol spray and tissue papers before the training for the next mouse. Only 0 2.5cm beam is used for training.

[0497] Testing

[0498] 1) Set up for 0 2.5 cm beam on the 2 poles, and place the mouse in the dark box for 3 minutes.

[0499] 2) Place the mouse 60 cm away from the box on the beam. If the mouse crosses successfully, record the time it takes. If the mouse falls, record the time and the actual traveled distance. Let the mouse rest in the box for about Imin, and repeat the same procedure for 4 times regardless of mouse falling or crossing.

[0500] 3) Place the mouse back to its cage, clean the beam and box with 75% ethanol spray and tissue papers and start the same test for the next mouse on the same beam.

[0501] 4) Cross speed = cross distance / cross time.

[0502] 8.3.6 NCV test

[0503] 1) Stimulus Site: Achilles tendon (AT) and sciatic notch (SN).

[0504] 2) Two sterile acupuncture needles (diameter, 0.25mm) are inserted into AT, and then SN. They are stimulated with constant- voltage (20 v) square- wave pulses (duration, 0.1 ms; interval 3s, repeat 10 times), respectively.

[0505] 3) Recording Site: Interosseous muscle of the hindpaw.

[0506] 4) Compound muscle action potentials (M waves) and H reflexes are recorded by another two acupuncture needles (diameter, 0.25mm) inserted into the interosseous muscle of hindpaw. The firings are amplified and monitored using standard electrophysiological techniques and recorded onto a PC using CED Spike 2 software (Cambridge Electronics Design). An average of 10 recordings for each is used for measurements.

[0507] 5) The latencies of the compound muscle action potentials are measured and recorded. The latency from the stimulus artifact to the onset of the negative M-wave deflection or to the positive peak time of M-wave is calculated. Measure and record the distance between SN and AT.

[0508] 6) NCV= (sciatic M wave latency - Achilles tendon M wave latency) / distance between SN and AT stimulation point

[0509] - 76 -

[0510] MEI 48415584v.l 8.3.7 Sample Collection

[0511] Blood Collection and Plasma Preparation:

[0512] 1) Collect 30 pL blood samples from mice through submandibular venipuncture into the 1.5 mL commercial EDTA anticoagulant tube at predetermined time points post the treatment (as shown in Table 1 and 2).

[0513] 2) Centrifuge the anticoagulant blood samples at 2,500 g at 4 °C for 15 min.

[0514] 3) Transfer the plasma samples into pre-labeled tubes. The plasma samples were stored at -80°C before PK.

[0515] CSF Collection:

[0516] 1) Identify the atlanto-occipital membrane at the base of the skull.

[0517] 2) Insert a sterile needle (25-30 gauge) at a 30-degree angle, aiming for the cistema magna.

[0518] 3) Gently aspirate to collect CSF into the syringe, collect 2 pL CSF samples from mice through into the 1.5 mL commercial tube at predetermined time points post the treatment (as shown in Table 1). Store all samples at -80°C before further analysis.

[0519] Tissue Collection:

[0520] 1) Collect Spinal cord (Thoracic and lumbar, RNA later, snap frozen), sciatic nerve (RNA later), femoral nerves (FFPE), Cervical spinal cord anterior horn (RNA later), Dorsal root ganglion (RNA later), TA (RNA later, snap frozen, FFPE), GA (RNA later, snap frozen, FFPE) from each mouse at predetermined time points post the treatment (as shown in Table 1).

[0521] 2) Transfer the samples into pre-labeled tubes, and snap freeze the samples in liquid nitrogen within 2 min after dissection, store the samples at -80°C.

[0522] 3) Transfer the samples into pre-labeled tubes with RNAlater, soak for 4-8h, store the samples at -80°C.

[0523] 4) Transfer the samples into pre-labeled tubes with 10% formalin at room temperature for further FFPE.

[0524] 8.3.8. Pharmacokinetics Assay

[0525] The harvested plasmas were processed for pharmacokinetics analysis by LC / MS / MS

[0526] - 77 -

[0527] MEI 48415584v.l with a concentration range of 20-20000 ng / mL.

[0528] 8.3.9. Statistical Analysis

[0529] Statistical analysis was performed, and experimental data are presented as mean ± standard error of the mean (Mean ± SEM). Statistical analysis was conducted using GraphPad Prism 10 software. Comparisons between two groups were analyzed using a t-test for significance, while comparisons among three or more groups were analyzed using one-way ANOVA (Sfdak's multiple comparison test) for significance, ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001.

[0530] 8.3.10 Electron Microscopy Analysis of frozen tissue

[0531] Femoral nerves were dissected and processed for transmission electron microscopy (TEM) analysis by fixating. The motor and sensory branches of the femoral nerve were plastic-embedded, sectioned at 0.5 pm thickness, and stained with toluidine blue to visualize myelin. Ultrathin sections were mounted on grids for TEM and imaged with a Transmission Electron Microscope. Samples examined for signs of pathology including abnormal, thin, or missing myelination, degenerating and / or regenerating axons, abnormalities in the axoplasm or cell bodies such as the presence of vacuoles, disrupted cytoskeletal components or organelles, with particular attention paid to abnormalities in mitochondria shape or localization. Axon number, nerve area, and diameter were measured and compared between genotypes and treatment.

[0532] 8.4. Results

[0533] Garsl encodes a tRNA synthetase that covalently links amino acids onto their cognate tRNAs. Mutations in GARS are associated with Charcot-Marie-Tooth neuropathies, type 2D (CMT2D). Gur.yP278KY / +heterozygous mice have a severe axonal neuropathy of both sensory and motor axons. Specifically, mice have abnormal neuromuscular junction morphology and impaired transmission, reduced nerve conduction velocities, and a loss of large-diameter peripheral axons. On the C57BL / 6 background, heterozygous mice fail to thrive and are always smaller than their littermates and develop muscle weakness and motor dysfunction like unsteady gait after weaning.

[0534] - 78 -

[0535] MEI 48415584v.l 8.4.1. Body weight

[0536] The body weight as well as body weight change of mice after chow administration were monitored and are shown in FIGs. 10A-10B. Compared with their wild-type (WT) littermates, the heterozygous mice exhibited delayed growth and development, along with significantly lower body weight. COMPOUND 1 in chow at either 30 mg / kg and 300 mg / kg could markedly accelerate the growth rate and improve body weight gain of heterozygous mice, achieving levels comparable to WT after 2-week feeding.

[0537] 8.4.2. Grip strength

[0538] To investigate the effect of COMPOUND 1 on the muscle weakness phenotype of CTM2D mice, four limb muscle strength tests were conducted at 4, 6 and 8 weeks after drug intervention (FIGs. 11A-11C). Compared to WT mice, G / / '.s P27SKY / +heterozygous mice failed to grip the mesh bar of the grip strength meter, resulting in near- zero limb force. In contrast, heterozygous mice treated with COMPOUND 1 either in 30 mg / kg or 300 mg / kg in chow led to dramatic increase in grip strength starting from 4 weeks after treatment (p<0.0001). No significant difference could be observed between these two COMPOUND 1 treated groups.

[0539] 8.4.3. Rotarod

[0540] In order to evaluate whether COMPOUND 1 could improve the motor dysfunction phenotype of CTM2D mice, rotarod tests were performed at 4, 6 and 8 weeks after treatment (FIGs. 12A-12F). Compared to WT mice, Gar5P278KY / +heterozygous mice showed shorter latency to fall and significant decrease in the maximum speed in the rod (p<0.01). After 4 weeks treatment with COMPOUND 1, heterozygous mice exhibited improved performance on the rod with sharply increased latency to fall (p<0.0001) and maximum speed when fell (p<0.05), especially those in the high dose group. However, no significant difference could be observed among vehicle and COMPOUND 1 treated groups at 6- and 8-weeks post dosing.

[0541] 8.4.4. Beam walk

[0542] Beam walk test is usually used to evaluate the motor coordination, balance as well as gait of the mice. WT mice in this study could quickly cross the beam with no foot slips during the test. In contrast, GarsP278KY / +heterozygous mice required significantly longer time

[0543] - 79 -

[0544] MEI 48415584v.l to cross the beam (p<0.01), with a subset failing to complete the full 60-cm crossing, multiple falls were observed during testing (FIGs. 13A-13F). In comparison, heterozygous mice after COMPOUND 1 treatment (30 and 300 mg / kg in chow) showed significantly faster crossing speed when tested at 4 and 6 weeks after administration (p<0.05). Although the COMPOUND 1 treated group did not demonstrate statistically significant differences in speed parameters compared to the vehicle treated group at the 8-week post-treatment assessment (potentially due to sample size limitations), it still exhibited superior performance in maximum traversed distance (p<0.05). No significant difference could be observed between these two COMPOUND 1 treated groups (FIG. 13F) during the whole process.

[0545] 8.4.5. NCV

[0546] Reduced nerve conduction velocity (NCV) is typical hallmark of CMT2D mice, therefore the conduction velocity of sciatic axons was investigated and calculated by measuring the latency of compound motor action potentials recorded in the muscle of the left rear paw. Compared with the WT, Gar5P278KY / +heterozygous mice showed dramatically slower NCV (p<0.05), indicating severe motor axon damage due to disease onset (FIGs. 14A-14D). Starting from 2 weeks post treatment, 300 mg / kg COMPOUND 1 in chow could significantly improve the NCV of Gur5P278KY / +heterozygous mice (p<0.05). Administration of 30 mg / kg COMPOUND 1 in chow also demonstrated beneficial effects on NCV improvement, although statistically significant differences were only observed at the 6-week post-treatment timepoint (potentially again could be attributable to limited sample size).

[0547] 8.4.6. PK

[0548] The concentrations of COMPOUND 1 in plasma samples are shown in FIGs. 16-17. Dose-dependent drug exposure was observed. Moreover, a stable drug concentration could be achieved with chow formulation of drug from 2 to 6 weeks. Since 30 and 300 mg / kg COMPOUND 1 in chow is equivalent to 3 and 30 mg / kg PO dose, the drug PK through single intragastric administration was investigated. COMPOUND 1 displayed time dependent elimination in plasma and peaked at Ih and 2h at the dosage of 3 mg / kg and 30 mg / kg respectively.

[0549] 8.4.7 Electron Microscopic Analysis of Femoral Nerve

[0550] - 80 -

[0551] MEI 48415584v.l Histological analysis of the femoral nerve of mice by transmission electron microscope (TEM) revealed that vehicle treated G« / ;s7P27SKY / +animals had a significant reduction in axon size, and myelination thickness compared to the WT control samples. COMPOUND 1 treated animals demonstrate an increase in axon size and myelination more closely resembling the WT control (FIG. 18).

[0552] 8.4.8 Integrated Stress Response (ISR) Score in Spinal Cord

[0553] The spinal cords of mice from each group was analyzed by RNAseq. The ISR activity score was calculated for each sample by taking a weighted average of the expression of 88 genes previously reported to be direct targets of the ATF4 transcription factor as previously described [Labbe K, et al Nat. Commun. 15, 8301 (2024)]. Gene weights were determined by the loading of each gene on to the first principal component of the 88 gene expression matrix.

[0554] 8.5. Results Summary

[0555] In this study, the efficacy and PK of COMPOUND 1 in the treatment of mice in the chow were investigated.

[0556] For efficacy study, COMPOUND 1 was administered in chow to achieve the goal of maintaining relatively stable and sustained plasma drug concentrations while minimizing stress and injury to animals caused by frequent manual dosing. Both 30 mg / kg and 300 mg / kg COMPOUND 1 in chow resulted in increased body weight gain of Gar.yP278KY / +heterozygous mice, and improved their performance in motor functional behavioral tests, including Beam walk (starting from 2 weeks post dosing) and Rotarod (only at 4 weeks post dosing), as well as muscle weakness reflected by grip strength test (starting from 4 weeks after dosing). Regarding NCV, 300 mg / kg COMPOUND 1 demonstrated more pronounced therapeutic improvements than 30 mg / kg. Furthermore, electron microscopic analysis of the femoral nerve showed increased axon diameter and myelination due to COMPOUND 1 treatment. Finally, COMPOUND 1 treatment led to an amelioration of the ISR score in spinal cord tissue of CMT2D G«r.yP278KY / +mice, suggesting robust target and pathway engagement.

[0557] For PK study, the concentrations of COMPOUND 1 in plasma samples are shown in FIG. 169. The data showed that COMPOUND 1 displayed dose-dependent and stable exposure with chow formulation of drug. Regarding single intragastric dosing of COMPOUND 1 , dose dependent exposure and time dependent elimination in plasma, as well

[0558] - 81 -

[0559] MEI 48415584v.l as peaking at 1 h (3 mg / kg) and 2h (30 mg / kg) after dosing were observed.

[0560] Although the present application has been described with reference to the exemplified embodiments, various improvements can be made thereto and parts thereof can be replaced with equivalents without departing from the scope of the present application. In particular, as long as there is no conflict or contradiction, each of the technical features / embodiments herein can be combined with any other technical features I embodiments in any manner. The present application is not limited to the particular Embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

[0561] - 82 -

[0562] MEI 48415584v.l

Claims

1. WHAT IS CLAIMED IS:

1. A method of treating vanishing white matter (VWM) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and a compound of the following formula or a pharmaceutically acceptable salt thereof2. A method of treating Huntington’s disease (HD) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and a compound of the following formula or a pharmaceutically acceptable salt thereof3. A method of treating Charcot Marie Tooth syndrome (CMT) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and a compound of the following formula or a pharmaceutically acceptable salt thereof4. The method of claim 3, wherein the CMT comprises I is CMT1A, CMT1B, CMT1C, CMT1D, IE, CMT1F, CMT1X, CMT2A, CMT2B, CMT2C, CMT2D, CMT2E,- 83 -MEI 48415584v.lCMT2F, CMT2I, CMT2K, CMT2L, CMT2M, CMT2P, CMT2S, CMT2T, CMT4A, CMT4B1, CMT4B2, CMT4B3, CMT4C, CMT4D, CMT4E, CMT4F, CMT4G, CMT4AH, CMT4J, CMTX2, CMTX3, CMTX4, CMTX5, or CMTX6, or a combination thereof.

5. The method of claim 4, wherein the CMT comprises / is CMT1A.

6. The method of claim 4, wherein the CMT comprises / is CMT2A.

7. The method of claim 4, wherein the CMT comprises / is CMT1B.

8. The method of claim 4, wherein the CMT comprises / is CMT2D.

9. The method of any one of claims 3-8, wherein administering the compound (e.g. , COMPOUND 1) or the pharmaceutical composition thereof results in improvement of one or more of symptoms selected from the group consisting of muscle weakness, paralysis, muscle atrophy, decreased reflexes, hammertoes, foot drop, trips and falls, repeated ankle sprains, breathing problems, numbness or tingling, chronic pain, and loss or decrease in vision and hearing.

10. The method of any one of claims 3-9, wherein administering the compound (e.g., COMPOUND 1) or the pharmaceutical composition thereof results in increased nerve conduction velocity (NCVj e.g. , about 1.2 times, 1.5 times, 1.8 times, 2 times, 2.2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, or 5 times higher than level before treatment).

11. A method of treating Amyotropic Lateral Sclerosis (ALS) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, and a compound of the following formula or a pharmaceutically acceptable salt thereof- 84 -MEI 48415584v.l12. A method of inhibiting integrated stress response (ISR), or of increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the subject a composition comprising a compound of the following formula or a pharmaceutically acceptable salt thereof, wherein the subject has vanishing white matter (VWM)13. A method of inhibiting integrated stress response (ISR) , or of increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the subject a composition comprising a compound of the following formula or a pharmaceutically acceptable salt thereof, wherein the subject has Huntington’ s disease (HD)14. A method of inhibiting integrated stress response (ISR) , or of increasing the guanine nucleotide exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the subject a composition comprising a compound of the following formula or a pharmaceutically acceptable salt thereof, wherein the subject has Charcot Marie Tooth syndrome (CMT)15. The method of claim 14, wherein the CMT comprises / is CMT1A, CMT1B, CMT1C,CMT1D, IE, CMT1F, CMT1X, CMT2A, CMT2B, CMT2C, CMT2D, CMT2E, CMT2F, CMT2I, CMT2K, CMT2L, CMT2M, CMT2P, CMT2S, CMT2T, CMT4A, CMT4B 1, CMT4B2, CMT4B3, CMT4C, CMT4D, CMT4E, CMT4F, CMT4G, CMT4AH, CMT4J, CMTX2, CMTX3, CMTX4, CMTX5, or CMTX6, or a- 85 -MEI 48415584v.lcombination thereof.

16. The method of claim 15, wherein the CMT comprises / is CMT1A.

17. The method of claim 15, wherein the CMT comprises / is CMT2A.

18. The method of claim 15, wherein the CMT comprises / is CMT1B.

19. The method of claim 15, wherein the CMT comprises / is CMT2D.

20. The method of any one of claims 14-19, wherein administering the compound (e.g., COMPOUND 1) or the pharmaceutical composition thereof results in improvement of one or more of symptoms selected from the group consisting of muscle weakness, paralysis, muscle atrophy, decreased reflexes, hammertoes, foot drop, trips and falls, repeated ankle sprains, breathing problems, numbness or tingling, chronic pain, and loss or decrease in vision and hearing.

21. The method of any one of claims 14-20, wherein administering the compound (e.g., COMPOUND 1) or the pharmaceutical composition thereof results in increased nerve conduction velocity (NCV) (e.g. , about 1.2 times, 1.5 times, 1.8 times, 2 times, 2.2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, or 5 times higher than level before treatment).

22. A method of inhibiting integrated stress response (ISR) , or of increasing the guanne nucleotide exchange factor (GEF) activity of eIF-2B in a subject, comprising administering to the subject a composition comprising a compound of the following formula or a pharmaceutically acceptable salt thereof, wherein the subject has Amyotropic Lateral Sclerosis (ALS)23. The method of any one of claims 1-22, wherein administering the compound inhibits integrated stress response (ISR) in the subject and / or increases the guanine nucleotide exchange factor (GEF) activity of eIF-2B.

24. The method of any one of claims 1-23, wherein a level of one or more ISR-associated- 86 -MEI 48415584v.lbiomarkers is reduced in the subject after administering the compound.

25. The method of claim 24, wherein the one or more ISR-associated biomarkers are selected from the group consisting of: Activating Transcription Factor 4 (ATF4), ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1), and Tribbles Pseudokinase 3 (TRIB3).

26. The method of claim 24 or 25, wherein the ISR-associated biomarker is ATF4.

27. The method of any one of claims 1-26, wherein the compound (e.g., COMPOUND 1) or the pharmaceutical composition thereof is administered orally, intravenously, or intragastrically.

28. The method of any one of claims 1-27, wherein the compound (e.g., COMPOUND 1) or the pharmaceutical composition thereof is administered at a dose of about 0.1 mg / kg to 100 mg / kg (e.g., about 0.1 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 12 mg / kg, 14 mg / kg, 16 mg / kg, 18 mg / kg, 20 mg / kg, 22 mg / kg, 24 mg / kg, 26 mg / kg, 28 mg / kg, 30 mg / kg, 40 mg / kg, 50, mg / kg, 60 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 90, mg / kg, or 100 mg / kg) .

29. The method of claim 28, wherein the compound (e.g. , COMPOUND 1) or the pharmaceutical composition thereof is administered at a dose of about 3 mg / kg.

30. The method of claim 28, wherein the compound (e.g., COMPOUND 1) or the pharmaceutical composition thereof is administered at a dose of about 30 mg / kg.

31. The method ofany one of claims 1-27, wherein the compound (e.g., COMPOUND 1) or the pharmaceutical composition thereof at a dose of about 1 mg to 300 mg per day (e.g., about Img per day, 5 mg per day, 10 mg per day, 15 mg per day, 20 mg per day, 25 mg per day, 30 mg per day, 35 mg per day, 40 mg per day, 45 mg per day, 50 mg per day, 50 mg per day, 60 mg per day, 70 mg per day, 80 mg per day, 90 mg per day, 100 mg per day, 100 mg per day, 125 mg per day, 150 mg per day, 175 mg per day, 200 mg per day, 225 mg per day, 250 mg per day, 275 mg per day, or 300 mg per day) per patient for the treatment of VWM, CMT, HD, or ALS.- 87 -MEI 48415584v.l

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