Lysosome-associated membrane protein targeting compounds and uses thereof

Abstract

Disclosed herein is a polypeptide comprising a pharmaceutical composition for use in the prevention or treatment of lysosomal storage-related diseases or disorders and autophagy misregulation-related diseases.Further provided is an agent that binds to the N-terminal domain region of lysosomal-associated membrane protein 1 (LAMP-1), as well as a method for treating or preventing the onset of lysosomal storage, polyglucosan storage or abnormal glycogen storage and autophagy misregulation-related diseases or disorders in a subject in need thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 149,730, filed February 16, 2021, the entire contents of which are incorporated herein by reference in their entirety.

[0002] The present invention is in the field of preventing and treating certain diseases or disorders associated with lysosomal storage, polyglucosan storage or abnormal glycogen storage, abnormal protein storage, and autophagy misregulation related diseases, and screening for drugs to prevent and treat these diseases. [Background technology]

[0003] Lysosomes are intracellular organelles involved in the physiological turnover of cellular components. They contain catabolic enzymes that require a low pH environment to function optimally. Lysosomal storage diseases (LSDs) represent a heterogeneous group of several dozen rare genetic disorders characterized by the accumulation of undigested or partially digested macromolecules, ultimately resulting in cellular dysfunction and clinical abnormalities. LSDs result from genetic mutations in one or more lysosomal enzymes, leading to the accumulation of enzyme substrates within the lysosomes. Organ enlargement, connective tissue and eye pathology, and central nervous system dysfunction can result.

[0004] Neurological disorders and neurodegenerative processes are associated with lysosomal dysfunction and are the main feature in most LSDs. Neuropathology can occur in multiple brain regions (e.g., thalamus, cortex, hippocampus, and cerebellum) with specific temporal and spatial changes, which are often accompanied by early region-specific neurodegeneration and inflammation. As an example, Purkinje neurons degenerate in many of these diseases, resulting in cerebellar ataxia.

[0005] Glycogen is a branched polysaccharide with a molecular weight of 9 to 10 million daltons. An average glycogen molecule contains about 55,000 glucose residues linked by α-1,4 glycosidic bonds (92%) and α-1,6 glycosidic bonds (8%). The synthesis of glycogen is catalyzed by two enzymes: (i) glycogen synthase, which "links" glucose together to form linear chains, and (ii) glycogen branching enzyme (GBE), which attaches new short branches of glucose units to linear chains with α-1,6 glycosidic bonds. Glycogen is stored primarily in the liver and muscle, where it provides an energy reserve that can be rapidly mobilized. The most common disorder of glycogen metabolism is seen as diabetes, where abnormal amounts of insulin or abnormal insulin responses lead to accumulation or depletion of liver glycogen. Although glycogen synthesis and degradation have been studied for decades, their regulation is not fully understood.

[0006] Adult polyglucosan body disease (APBD) is a glycogen storage disease (GSD) that manifests as a debilitating and fatal progressive axonopathic leukodystrophy from age 45-50 years. APBD is further characterized by peripheral neuropathy, autonomic dysfunction, urinary incontinence, and rarely dementia, all of which are important diagnostic criteria for this easily misdiagnosed and highly heterogeneous disease. APBD is caused by a deficiency of glycogen branching enzyme (GBE), resulting in poorly branched and therefore insoluble glycogen (polyglucosan, PG), which precipitates, aggregates, and accumulates into PG bodies (PB). Because they aggregate out of solution, PB cannot be digested by glycogen phosphorylase. The aggregated aggregates lead to liver failure and death in childhood (Anderson's disease; GSD type IV). Milder GBE mutations (e.g., p.Y329S in APBD) result in smaller PBs that simply accumulate on the sides of cells without interfering with hepatocytes and most other cell types, but in neurons and astrocytes, over time, PBs occlude narrow areas of axons and processes, resulting in APBD.

[0007] APBDs are a larger group of GSDs, although effective treatments for APBDs are currently elusive and urgently needed. GSDs are a diverse group of 15 incurable disorders with a combined incidence of 1 in 20,000 to 43,000. All GSDs are currently incurable, ranging from pediatric liver disorders such as GSD1, to adolescent myoclonic epilepsy such as Lafora disease (LD), and adult progressive neurodegenerative disorders such as APBD. There remains a need for therapies, drugs, and improved correlative diagnostics for lysosomal storage and glycogen storage diseases. Summary of the Invention

[0008] In one aspect of the invention, there is provided a pharmaceutical composition for use in the prevention or treatment of a disease or disorder selected from a lysosomal storage associated disease and an autophagy misregulation associated disease, comprising a compound and a pharma- ceutically acceptable salt, isomer or tautomer thereof, wherein the compound is represented by formula I:

[0009] [ka] (In the formula,

[0010] [ka] represents a single bond or a double bond, n and m each independently represent an integer ranging from 1 to 3; R and R 1 each independently represents hydrogen or is absent; R 3 , R 4 , R 5 , R 6 , R 7 and R 8each independently represents hydrogen or is selected from the group consisting of substituted or unsubstituted alkyl, cycloalkyl, alkoxy, hydroxy, thiohydroxy, thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halo, trihalomethyl, cyano, amido, carboxy, sulfonyl, sulfoxy, sulfinyl, and sulfonamido. A pharmaceutical composition is provided comprising the formula:

[0011] In some embodiments, n and m are 1.

[0012] In some embodiments, R 2 , R 7 and R 8 represents methyl.

[0013] In some embodiments, the compound is

[0014] [ka] or both.

[0015] In some embodiments, the lysosomal storage associated disease is selected from the group consisting of Gaucher disease, Fabry disease, Tay-Sachs disease, mucopolysaccharidoses (MPS) diseases, aspartylglucosaminuria, GM1-gangliosidosis, Krabbe disease (globoid cell leukodystrophy or galactosylceramide lipidosis), metachromatic leukodystrophy, Sandhoff disease, mucolipidosis type II (I-cell disease), mucolipidosis type IIIA (pseudo-Hurler polydystrophy), Niemann-Pick disease types C2 and Cl, Danon disease, free sialic acid storage disorders, mucolipidosis type IV, and multiple sulfatase deficiency (MSD), metabolic disorders, obesity, type II diabetes, and insulin resistance.

[0016] In some embodiments, the autophagy misregulation associated disease is characterized by decreased or misregulated autophagic activity. In some embodiments, the autophagy misregulation associated disease characterized by decreased or misregulated autophagic activity is selected from the group consisting of Alzheimer's disease and cancers associated with decreased autophagic activity.

[0017] In another aspect of the present invention, there is provided a method for treating or preventing the onset of a disease or disorder selected from a lysosomal storage associated disease and an autophagy misregulation associated disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present invention.

[0018] In another aspect of the present invention, an agent is provided that binds to an N-terminal domain region of lysosome-associated membrane protein 1 (LAMP-1; SEQ ID NO: 1; FSVNYDTKSGPKNMTFDLPSDATVVLNRSSCGKENTSDPSLVIAFGRGHTLTLNFTRNATRYSV), wherein the region comprises any one of SEQ ID NO: 2 (FSVNYD) and SEQ ID NO: 3 (NVTV).

[0019] In some embodiments, the agent inhibits the LAMP1:LAMP1 interaction.

[0020] In some embodiments, the medicament is for use in the prevention or treatment of a disease or disorder associated with lysosomal storage, polyglucosan accumulation, or abnormal glycogen accumulation, hi some embodiments, the medicament is for use in the prevention or treatment of a disease associated with autophagy misregulation.

[0021] In some embodiments, the disease or disorder is selected from the group consisting of glycogen storage diseases (GSDs), adult polyglucosan body disease (APBDs), and Lafora disease, Gaucher disease, Fabry disease, Tay-Sachs disease, mucopolysaccharidoses (MPS) diseases, aspartylglucosaminuria, GM1-gangliosidosis, Krabbe disease (globoid cell leukodystrophy or galactosylceramide lipidosis), metachromatic leukodystrophy, Sandhoff disease, mucolibridosis type II (I-cell disease), mucolibridosis type IIIA (pseudo-Hurler polydystrophy), Niemann-Pick disease types C2 and Cl, Danon disease, free sialic acid storage disorders, mucolibridosis type IV, and multiple sulfatase deficiency (MSD), metabolic disorders, obesity, type II diabetes, and insulin resistance.

[0022] In some embodiments, the agent is

[0023] [ka] is selected from the group consisting of:

[0024] In another aspect of the invention, there is provided a pharmaceutical composition comprising an agent of the invention and a pharma- ceutically acceptable carrier.

[0025] In some embodiments, the pharmaceutical composition has a pH of 4 to 6.5 in solution.

[0026] In some embodiments, the pharmaceutical composition comprises between 100 nM and 5 mM of an agent.

[0027] In another aspect of the present invention, there is provided a method for treating or preventing the onset of a disease or disorder associated with lysosomal storage, polyglucosan storage or abnormal glycogen storage and autophagic misregulation associated diseases in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present invention.

[0028] In another aspect of the present invention, there is provided a method for determining the suitability of a compound for preventing or treating a disease or disorder associated with lysosomal storage, polyglucosan storage or abnormal glycogen storage, and autophagic misregulation associated diseases, comprising contacting the compound with a pocket domain within the N-terminal domain of lysosomal associated membrane protein 1 (LAMP-1; SEQ ID NO:1), wherein binding of the compound to the pocket indicates that the compound is effective for treating the disease or disorder.

[0029] In some embodiments, an agent is provided wherein the linkage comprises any one of SEQ ID NO:2 (FSVNYD); and SEQ ID NO:3 (NVTV).

[0030] In some embodiments, the binding is determined by inhibition of LAMP1:LAMP1 interaction.

[0031] In some embodiments, the binding is determined by inhibition of LAMP1-LAMP1 interactions.

[0032] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the present patent specification, including definitions, will control. Additionally, the materials, methods, and examples are merely illustrative and are not intended to be necessarily limiting.

[0033] Further embodiments and their full scope of applicability will become apparent from the detailed description given hereinafter. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0034] [Figure 1A] 1A-1M show the chemical structure of Compound 1 (1A); a graph of the Kaplan-Meier survival curve based on 17 animals (n=17) treated twice weekly with 250 mg / kg Compound 1 (144DG11) compared to 9 animals (n=9) treated with 5% DMSO vehicle (log-rank test p-value <0.000692) (1B); a graph of the body weight curve (in g) (1C); a graph of the average movement time in the open field (1D); and graphs of the stretch reflex (extent of hind limbs opening after grasping the animal by the tail) as a function of time for wild-type mice (n=8) after treatment with vehicle and for Gbeys / ys mice after treatment with vehicle (n=8) or Compound 1 (n=9) (1E-1F); and a photograph of the locomotor heatmap showing the quantification of the open field performance experiment (1G) (top panel, n=9, 9-month-old females). (1H) of gait analysis (stride length) of 9 month old female mice (mean (+ / - sd) stride length shown), a graph of mean duration of movement curve in open field (1I), a graph of body weight curve (in g) (1J), and a graph showing stretch reflex curves as a function of time after treatment with vehicle or Compound 1 for Gbeys / ys mice shown at 6 months of age (at onset) (1K), and photographs of 7 month old Gbeys / ys mice treated with vehicle (1L) or Compound 1 (1M) 3 months prior to photography. [Figure 1B]1A-1M show the chemical structure of Compound 1 (1A); a graph of the Kaplan-Meier survival curve based on 17 animals (n=17) treated twice weekly with 250 mg / kg Compound 1 (144DG11) compared to 9 animals (n=9) treated with 5% DMSO vehicle (log-rank test p-value <0.000692) (1B); a graph of the body weight curve (in g) (1C); a graph of the average movement time in the open field (1D); and graphs of the stretch reflex (extent of hind limbs opening after grasping the animal by the tail) as a function of time for wild-type mice (n=8) after treatment with vehicle and for Gbeys / ys mice after treatment with vehicle (n=8) or Compound 1 (n=9) (1E-1F); and a photograph of the locomotor heatmap showing the quantification of the open field performance experiment (1G) (top panel, n=9, 9-month-old females). (1H) of gait analysis (stride length) of 9 month old female mice (mean (+ / - sd) stride length shown), a graph of mean duration of movement curve in open field (1I), a graph of body weight curve (in g) (1J), and a graph showing stretch reflex curves as a function of time after treatment with vehicle or Compound 1 for Gbeys / ys mice shown at 6 months of age (at onset) (1K), and photographs of 7 month old Gbeys / ys mice treated with vehicle (1L) or Compound 1 (1M) 3 months prior to photography. [Figure 1C]1A-1M show the chemical structure of Compound 1 (1A); a graph of the Kaplan-Meier survival curve based on 17 animals (n=17) treated twice weekly with 250 mg / kg Compound 1 (144DG11) compared to 9 animals (n=9) treated with 5% DMSO vehicle (log-rank test p-value <0.000692) (1B); a graph of the body weight curve (in g) (1C); a graph of the average movement time in the open field (1D); and graphs of the stretch reflex (extent of hind limbs opening after grasping the animal by the tail) as a function of time for wild-type mice (n=8) after treatment with vehicle and for Gbeys / ys mice after treatment with vehicle (n=8) or Compound 1 (n=9) (1E-1F); and a photograph of the locomotor heatmap showing the quantification of the open field performance experiment (1G) (top panel, n=9, 9-month-old females). (1H) of gait analysis (stride length) of 9 month old female mice (mean (+ / - sd) stride length shown), a graph of mean duration of movement curve in open field (1I), a graph of body weight curve (in g) (1J), and a graph showing stretch reflex curves as a function of time after treatment with vehicle or Compound 1 for Gbeys / ys mice shown at 6 months of age (at onset) (1K), and photographs of 7 month old Gbeys / ys mice treated with vehicle (1L) or Compound 1 (1M) 3 months prior to photography. [Figure 1D]1A-1M show the chemical structure of Compound 1 (1A); a graph of the Kaplan-Meier survival curve based on 17 animals (n=17) treated twice weekly with 250 mg / kg Compound 1 (144DG11) compared to 9 animals (n=9) treated with 5% DMSO vehicle (log-rank test p-value <0.000692) (1B); a graph of the body weight curve (in g) (1C); a graph of the average movement time in the open field (1D); and graphs of the stretch reflex (extent of hind limbs opening after grasping the animal by the tail) as a function of time for wild-type mice (n=8) after treatment with vehicle and for Gbeys / ys mice after treatment with vehicle (n=8) or Compound 1 (n=9) (1E-1F); and a photograph of the locomotor heatmap showing the quantification of the open field performance experiment (1G) (top panel, n=9, 9-month-old females). (1H) of gait analysis (stride length) of 9 month old female mice (mean (+ / - sd) stride length shown), a graph of mean duration of movement curve in open field (1I), a graph of body weight curve (in g) (1J), and a graph showing stretch reflex curves as a function of time after treatment with vehicle or Compound 1 for Gbeys / ys mice shown at 6 months of age (at onset) (1K), and photographs of 7 month old Gbeys / ys mice treated with vehicle (1L) or Compound 1 (1M) 3 months prior to photography. [Figure 1E]1A-1M show the chemical structure of Compound 1 (1A); a graph of the Kaplan-Meier survival curve based on 17 animals (n=17) treated twice weekly with 250 mg / kg Compound 1 (144DG11) compared to 9 animals (n=9) treated with 5% DMSO vehicle (log-rank test p-value <0.000692) (1B); a graph of the body weight curve (in g) (1C); a graph of the average movement time in the open field (1D); and graphs of the stretch reflex (extent of hind limbs opening after grasping the animal by the tail) as a function of time for wild-type mice (n=8) after treatment with vehicle and for Gbeys / ys mice after treatment with vehicle (n=8) or Compound 1 (n=9) (1E-1F); and a photograph of the locomotor heatmap showing the quantification of the open field performance experiment (1G) (top panel, n=9, 9-month-old females). (1H) of gait analysis (stride length) of 9 month old female mice (mean (+ / - sd) stride length shown), a graph of mean duration of movement curve in open field (1I), a graph of body weight curve (in g) (1J), and a graph showing stretch reflex curves as a function of time after treatment with vehicle or Compound 1 for Gbeys / ys mice shown at 6 months of age (at onset) (1K), and photographs of 7 month old Gbeys / ys mice treated with vehicle (1L) or Compound 1 (1M) 3 months prior to photography. [Figure 1F]1A-1M show the chemical structure of Compound 1 (1A); a graph of the Kaplan-Meier survival curve based on 17 animals (n=17) treated twice weekly with 250 mg / kg Compound 1 (144DG11) compared to 9 animals (n=9) treated with 5% DMSO vehicle (log-rank test p-value <0.000692) (1B); a graph of the body weight curve (in g) (1C); a graph of the average movement time in the open field (1D); and graphs of the stretch reflex (extent of hind limbs opening after grasping the animal by the tail) as a function of time for wild-type mice (n=8) after treatment with vehicle and for Gbeys / ys mice after treatment with vehicle (n=8) or Compound 1 (n=9) (1E-1F); and a photograph of the locomotor heatmap showing the quantification of the open field performance experiment (1G) (top panel, n=9, 9-month-old females). (1H) of gait analysis (stride length) of 9 month old female mice (mean (+ / - sd) stride length shown), a graph of mean duration of movement curve in open field (1I), a graph of body weight curve (in g) (1J), and a graph showing stretch reflex curves as a function of time after treatment with vehicle or Compound 1 for Gbeys / ys mice shown at 6 months of age (at onset) (1K), and photographs of 7 month old Gbeys / ys mice treated with vehicle (1L) or Compound 1 (1M) 3 months prior to photography. [Figure 1G]1A-1M show the chemical structure of Compound 1 (1A); a graph of the Kaplan-Meier survival curve based on 17 animals (n=17) treated twice weekly with 250 mg / kg Compound 1 (144DG11) compared to 9 animals (n=9) treated with 5% DMSO vehicle (log-rank test p-value <0.000692) (1B); a graph of the body weight curve (in g) (1C); a graph of the average movement time in the open field (1D); and graphs of the stretch reflex (extent of hind limbs opening after grasping the animal by the tail) as a function of time for wild-type mice (n=8) after treatment with vehicle and for Gbeys / ys mice after treatment with vehicle (n=8) or Compound 1 (n=9) (1E-1F); and a photograph of the locomotor heatmap showing the quantification of the open field performance experiment (1G) (top panel, n=9, 9-month-old females). (1H) of gait analysis (stride length) of 9 month old female mice (mean (+ / - sd) stride length shown), a graph of mean duration of movement curve in open field (1I), a graph of body weight curve (in g) (1J), and a graph showing stretch reflex curves as a function of time after treatment with vehicle or Compound 1 for Gbeys / ys mice shown at 6 months of age (at onset) (1K), and photographs of 7 month old Gbeys / ys mice treated with vehicle (1L) or Compound 1 (1M) 3 months prior to photography. [Figure 1H]1A-1M show the chemical structure of Compound 1 (1A); a graph of the Kaplan-Meier survival curve based on 17 animals (n=17) treated twice weekly with 250 mg / kg Compound 1 (144DG11) compared to 9 animals (n=9) treated with 5% DMSO vehicle (log-rank test p-value <0.000692) (1B); a graph of the body weight curve (in g) (1C); a graph of the average movement time in the open field (1D); and graphs of the stretch reflex (extent of hind limbs opening after grasping the animal by the tail) as a function of time for wild-type mice (n=8) after treatment with vehicle and for Gbeys / ys mice after treatment with vehicle (n=8) or Compound 1 (n=9) (1E-1F); and a photograph of the locomotor heatmap showing the quantification of the open field performance experiment (1G) (top panel, n=9, 9-month-old females). (1H) of gait analysis (stride length) of 9 month old female mice (mean (+ / - sd) stride length shown), a graph of mean duration of movement curve in open field (1I), a graph of body weight curve (in g) (1J), and a graph showing stretch reflex curves as a function of time after treatment with vehicle or Compound 1 for Gbeys / ys mice shown at 6 months of age (at onset) (1K), and photographs of 7 month old Gbeys / ys mice treated with vehicle (1L) or Compound 1 (1M) 3 months prior to photography. [Figure 1I]1A-1M show the chemical structure of Compound 1 (1A); a graph of the Kaplan-Meier survival curve based on 17 animals (n=17) treated twice weekly with 250 mg / kg Compound 1 (144DG11) compared to 9 animals (n=9) treated with 5% DMSO vehicle (log-rank test p-value <0.000692) (1B); a graph of the body weight curve (in g) (1C); a graph of the average movement time in the open field (1D); and graphs of the stretch reflex (extent of hind limbs opening after grasping the animal by the tail) as a function of time for wild-type mice (n=8) after treatment with vehicle and for Gbeys / ys mice after treatment with vehicle (n=8) or Compound 1 (n=9) (1E-1F); and a photograph of the locomotor heatmap showing the quantification of the open field performance experiment (1G) (top panel, n=9, 9-month-old females). (1H) of gait analysis (stride length) of 9 month old female mice (mean (+ / - sd) stride length shown), a graph of mean duration of movement curve in open field (1I), a graph of body weight curve (in g) (1J), and a graph showing stretch reflex curves as a function of time after treatment with vehicle or Compound 1 for Gbeys / ys mice shown at 6 months of age (at onset) (1K), and photographs of 7 month old Gbeys / ys mice treated with vehicle (1L) or Compound 1 (1M) 3 months prior to photography. [Figure 1J]1A-1M show the chemical structure of Compound 1 (1A); a graph of the Kaplan-Meier survival curve based on 17 animals (n=17) treated twice weekly with 250 mg / kg Compound 1 (144DG11) compared to 9 animals (n=9) treated with 5% DMSO vehicle (log-rank test p-value <0.000692) (1B); a graph of the body weight curve (in g) (1C); a graph of the average movement time in the open field (1D); and graphs of the stretch reflex (extent of hind limbs opening after grasping the animal by the tail) as a function of time for wild-type mice (n=8) after treatment with vehicle and for Gbeys / ys mice after treatment with vehicle (n=8) or Compound 1 (n=9) (1E-1F); and a photograph of the locomotor heatmap showing the quantification of the open field performance experiment (1G) (top panel, n=9, 9-month-old females). (1H) of gait analysis (stride length) of 9 month old female mice (mean (+ / - sd) stride length shown), a graph of mean duration of movement curve in open field (1I), a graph of body weight curve (in g) (1J), and a graph showing stretch reflex curves as a function of time after treatment with vehicle or Compound 1 for Gbeys / ys mice shown at 6 months of age (at onset) (1K), and photographs of 7 month old Gbeys / ys mice treated with vehicle (1L) or Compound 1 (1M) 3 months prior to photography. [Figure 1K]1A-1M show the chemical structure of Compound 1 (1A); a graph of the Kaplan-Meier survival curve based on 17 animals (n=17) treated twice weekly with 250 mg / kg Compound 1 (144DG11) compared to 9 animals (n=9) treated with 5% DMSO vehicle (log-rank test p-value <0.000692) (1B); a graph of the body weight curve (in g) (1C); a graph of the average movement time in the open field (1D); and graphs of the stretch reflex (extent of hind limbs opening after grasping the animal by the tail) as a function of time for wild-type mice (n=8) after treatment with vehicle and for Gbeys / ys mice after treatment with vehicle (n=8) or Compound 1 (n=9) (1E-1F); and a photograph of the locomotor heatmap showing the quantification of the open field performance experiment (1G) (top panel, n=9, 9-month-old females). (1H) of gait analysis (stride length) of 9 month old female mice (mean (+ / - sd) stride length shown), a graph of mean duration of movement curve in open field (1I), a graph of body weight curve (in g) (1J), and a graph showing stretch reflex curves as a function of time after treatment with vehicle or Compound 1 for Gbeys / ys mice shown at 6 months of age (at onset) (1K), and photographs of 7 month old Gbeys / ys mice treated with vehicle (1L) or Compound 1 (1M) 3 months prior to photography. [Figure 1L]1A-1M show the chemical structure of Compound 1 (1A); a graph of the Kaplan-Meier survival curve based on 17 animals (n=17) treated twice weekly with 250 mg / kg Compound 1 (144DG11) compared to 9 animals (n=9) treated with 5% DMSO vehicle (log-rank test p-value <0.000692) (1B); a graph of the body weight curve (in g) (1C); a graph of the average movement time in the open field (1D); and graphs of the stretch reflex (extent of hind limbs opening after grasping the animal by the tail) as a function of time for wild-type mice (n=8) after treatment with vehicle and for Gbeys / ys mice after treatment with vehicle (n=8) or Compound 1 (n=9) (1E-1F); and a photograph of the locomotor heatmap showing the quantification of the open field performance experiment (1G) (top panel, n=9, 9-month-old females). (1H) of gait analysis (stride length) of 9 month old female mice (mean (+ / - sd) stride length shown), a graph of mean duration of movement curve in open field (1I), a graph of body weight curve (in g) (1J), and a graph showing stretch reflex curves as a function of time after treatment with vehicle or Compound 1 for Gbeys / ys mice shown at 6 months of age (at onset) (1K), and photographs of 7 month old Gbeys / ys mice treated with vehicle (1L) or Compound 1 (1M) 3 months prior to photography. [Figure 1M]1A-1M show the chemical structure of Compound 1 (1A); a graph of the Kaplan-Meier survival curve based on 17 animals (n=17) treated twice weekly with 250 mg / kg Compound 1 (144DG11) compared to 9 animals (n=9) treated with 5% DMSO vehicle (log-rank test p-value <0.000692) (1B); a graph of the body weight curve (in g) (1C); a graph of the average movement time in the open field (1D); and graphs of the stretch reflex (extent of hind limbs opening after grasping the animal by the tail) as a function of time for wild-type mice (n=8) after treatment with vehicle and for Gbeys / ys mice after treatment with vehicle (n=8) or Compound 1 (n=9) (1E-1F); and a photograph of the locomotor heatmap showing the quantification of the open field performance experiment (1G) (top panel, n=9, 9-month-old females). (1H) of gait analysis (stride length) of 9 month old female mice (mean (+ / - sd) stride length shown), a graph of mean duration of movement curve in open field (1I), a graph of body weight curve (in g) (1J), and a graph showing stretch reflex curves as a function of time after treatment with vehicle or Compound 1 for Gbeys / ys mice shown at 6 months of age (at onset) (1K), and photographs of 7 month old Gbeys / ys mice treated with vehicle (1L) or Compound 1 (1M) 3 months prior to photography. [Figure 2A-1]Figures 2A-C contain images and graphs of the histopathological effects of Compound 1 and its pharmacokinetics, where the right panel shows images of the indicated tissues stained for PG (arrows) with PAS from mice sacrificed after treatment with diastase, and the left panel shows a bar graph quantifying PAS staining (2A), a bar graph quantifying total glycogen in each tissue (2B), and a graph of the pharmacokinetics of Compound 1 (2C) based on the analysis of four sections from each tissue from n=2 wild-type mice, n=7 Gbeys / ys vehicle-treated mice, and n=9 Compound 1-treated mice. 9-month-old Gbeys / ys mice were injected SC with 150 μL of Compound 1 at 250 mg / kg. Mice were sacrificed 30, 60, 90, and 210 min after injection, the indicated tissues were removed, and 200 μL of serum was collected. The graph shows Compound 1 levels in different tissues as measured by LC-MS / MS. The mean and SEM of results from n=3 mice at each time point are shown. Repeated measures two-way ANOVA test indicates that the pharmacokinetic profile of each tissue is significantly different from that of all other tissues (p<0.05). * indicates significant difference (p<0.05) as determined by Student's t-test. [Figure 2A-2]Figures 2A-C contain images and graphs of the histopathological effects of Compound 1 and its pharmacokinetics, where the right panel shows images of the indicated tissues stained for PG (arrows) with PAS from mice sacrificed after treatment with diastase, and the left panel shows a bar graph quantifying PAS staining (2A), a bar graph quantifying total glycogen in each tissue (2B), and a graph of the pharmacokinetics of Compound 1 (2C) based on the analysis of four sections from each tissue from n=2 wild-type mice, n=7 Gbeys / ys vehicle-treated mice, and n=9 Compound 1-treated mice. 9-month-old Gbeys / ys mice were injected SC with 150 μL of Compound 1 at 250 mg / kg. Mice were sacrificed 30, 60, 90, and 210 min after injection, the indicated tissues were removed, and 200 μL of serum was collected. The graph shows Compound 1 levels in different tissues as measured by LC-MS / MS. The mean and SEM of results from n=3 mice at each time point are shown. Repeated measures two-way ANOVA test indicates that the pharmacokinetic profile of each tissue is significantly different from that of all other tissues (p<0.05). * indicates significant difference (p<0.05) as determined by Student's t-test. [Figure 2B]Figures 2A-C contain images and graphs of the histopathological effects of Compound 1 and its pharmacokinetics, where the right panel shows images of the indicated tissues stained for PG (arrows) with PAS from mice sacrificed after treatment with diastase, and the left panel shows a bar graph quantifying PAS staining (2A), a bar graph quantifying total glycogen in each tissue (2B), and a graph of the pharmacokinetics of Compound 1 (2C) based on the analysis of four sections from each tissue from n=2 wild-type mice, n=7 Gbeys / ys vehicle-treated mice, and n=9 Compound 1-treated mice. 9-month-old Gbeys / ys mice were injected SC with 150 μL of Compound 1 at 250 mg / kg. Mice were sacrificed 30, 60, 90, and 210 min after injection, the indicated tissues were removed, and 200 μL of serum was collected. The graph shows Compound 1 levels in different tissues as measured by LC-MS / MS. The mean and SEM of results from n=3 mice at each time point are shown. Repeated measures two-way ANOVA test indicates that the pharmacokinetic profile of each tissue is significantly different from that of all other tissues (p<0.05). * indicates significant difference (p<0.05) as determined by Student's t-test. [Figure 2C]Figures 2A-C contain images and graphs of the histopathological effects of Compound 1 and its pharmacokinetics, where the right panel shows images of the indicated tissues stained for PG (arrows) with PAS from mice sacrificed after treatment with diastase, and the left panel shows a bar graph quantifying PAS staining (2A), a bar graph quantifying total glycogen in each tissue (2B), and a graph of the pharmacokinetics of Compound 1 (2C) based on the analysis of four sections from each tissue from n=2 wild-type mice, n=7 Gbeys / ys vehicle-treated mice, and n=9 Compound 1-treated mice. 9-month-old Gbeys / ys mice were injected SC with 150 μL of Compound 1 at 250 mg / kg. Mice were sacrificed 30, 60, 90, and 210 min after injection, the indicated tissues were removed, and 200 μL of serum was collected. The graph shows Compound 1 levels in different tissues as measured by LC-MS / MS. The mean and SEM of results from n=3 mice at each time point are shown. Repeated measures two-way ANOVA test indicates that the pharmacokinetic profile of each tissue is significantly different from that of all other tissues (p<0.05). * indicates significant difference (p<0.05) as determined by Student's t-test. [Figure 3A]Figures 3A-I include graphs of the effect of Compound 1 on in vivo metabolism, where mice were monitored over 24 hours by a Promethion high-resolution behavioral phenotyping system (Sable Instruments, Inc.). Effective masses were calculated to the power of 0.75. Data are mean ± SEM from n=11 9-month-old mice in the wt control vehicle group, n=6 9-month-old mice in the GBEys / ys vehicle group, and n=7 9-month-old mice in the Gbeys / ys144DG11 (Compound 1) treatment group. All injections were from 4 months of age. Untreated GBEys / ys mice show lower (light) respiratory quotient (3A), total energy expenditure (TEE) (3B), and fat oxidation (3C) compared to wild-type controls. Carbohydrate oxidation and locomotor activity, which are not significantly affected by disease state, were increased by Compound 1 even above wt control levels (3D-3E). Compound 1 also reversed the decrease in food and water intake observed in Gbeys / ys mice compared to wt controls (3F-3H). Blood metabolic panel (3I) based on n=5, 9.5 month old mice treated as indicated. Compound 1 increased blood glucose and decreased blood triglycerides in Gbeys / ys cells (p<0.05, Student's t-test). *p<0.05v wt. control, #p<0.05v GBEys / ys vehicle treated mice. [Figure 3B]Figures 3A-I include graphs of the effect of Compound 1 on in vivo metabolism, where mice were monitored over 24 hours by a Promethion high-resolution behavioral phenotyping system (Sable Instruments, Inc.). Effective masses were calculated to the power of 0.75. Data are mean ± SEM from n=11 9-month-old mice in the wt control vehicle group, n=6 9-month-old mice in the GBEys / ys vehicle group, and n=7 9-month-old mice in the Gbeys / ys144DG11 (Compound 1) treatment group. All injections were from 4 months of age. Untreated GBEys / ys mice show lower (light) respiratory quotient (3A), total energy expenditure (TEE) (3B), and fat oxidation (3C) compared to wild-type controls. Carbohydrate oxidation and locomotor activity, which are not significantly affected by disease state, were increased by Compound 1 even above wt control levels (3D-3E). Compound 1 also reversed the decrease in food and water intake observed in Gbeys / ys mice compared to wt controls (3F-3H). Blood metabolic panel (3I) based on n=5, 9.5 month old mice treated as indicated. Compound 1 increased blood glucose and decreased blood triglycerides in Gbeys / ys cells (p<0.05, Student's t-test). *p<0.05v wt. control, #p<0.05v GBEys / ys vehicle treated mice. [Figure 3C]Figures 3A-I include graphs of the effect of Compound 1 on in vivo metabolism, where mice were monitored over 24 hours by a Promethion high-resolution behavioral phenotyping system (Sable Instruments, Inc.). Effective masses were calculated to the power of 0.75. Data are mean ± SEM from n=11 9-month-old mice in the wt control vehicle group, n=6 9-month-old mice in the GBEys / ys vehicle group, and n=7 9-month-old mice in the Gbeys / ys144DG11 (Compound 1) treatment group. All injections were from 4 months of age. Untreated GBEys / ys mice show lower (light) respiratory quotient (3A), total energy expenditure (TEE) (3B), and fat oxidation (3C) compared to wild-type controls. Carbohydrate oxidation and locomotor activity, which are not significantly affected by disease state, were increased by Compound 1 even above wt control levels (3D-3E). Compound 1 also reversed the decrease in food and water intake observed in Gbeys / ys mice compared to wt controls (3F-3H). Blood metabolic panel (3I) based on n=5, 9.5 month old mice treated as indicated. Compound 1 increased blood glucose and decreased blood triglycerides in Gbeys / ys cells (p<0.05, Student's t-test). *p<0.05v wt. control, #p<0.05v GBEys / ys vehicle treated mice. [Figure 3D]Figures 3A-I include graphs of the effect of Compound 1 on in vivo metabolism, where mice were monitored over 24 hours by a Promethion high-resolution behavioral phenotyping system (Sable Instruments, Inc.). Effective masses were calculated to the power of 0.75. Data are mean ± SEM from n=11 9-month-old mice in the wt control vehicle group, n=6 9-month-old mice in the GBEys / ys vehicle group, and n=7 9-month-old mice in the Gbeys / ys144DG11 (Compound 1) treatment group. All injections were from 4 months of age. Untreated GBEys / ys mice show lower (light) respiratory quotient (3A), total energy expenditure (TEE) (3B), and fat oxidation (3C) compared to wild-type controls. Carbohydrate oxidation and locomotor activity, which are not significantly affected by disease state, were increased by Compound 1 even above wt control levels (3D-3E). Compound 1 also reversed the decrease in food and water intake observed in Gbeys / ys mice compared to wt controls (3F-3H). Blood metabolic panel (3I) based on n=5, 9.5 month old mice treated as indicated. Compound 1 increased blood glucose and decreased blood triglycerides in Gbeys / ys cells (p<0.05, Student's t-test). *p<0.05v wt. control, #p<0.05v GBEys / ys vehicle treated mice. [Figure 3E]Figures 3A-I include graphs of the effect of Compound 1 on in vivo metabolism, where mice were monitored over 24 hours by a Promethion high-resolution behavioral phenotyping system (Sable Instruments, Inc.). Effective masses were calculated to the power of 0.75. Data are mean ± SEM from n=11 9-month-old mice in the wt control vehicle group, n=6 9-month-old mice in the GBEys / ys vehicle group, and n=7 9-month-old mice in the Gbeys / ys144DG11 (Compound 1) treatment group. All injections were from 4 months of age. Untreated GBEys / ys mice show lower (light) respiratory quotient (3A), total energy expenditure (TEE) (3B), and fat oxidation (3C) compared to wild-type controls. Carbohydrate oxidation and locomotor activity, which are not significantly affected by disease state, were increased by Compound 1 even above wt control levels (3D-3E). Compound 1 also reversed the decrease in food and water intake observed in Gbeys / ys mice compared to wt controls (3F-3H). Blood metabolic panel (3I) based on n=5, 9.5 month old mice treated as indicated. Compound 1 increased blood glucose and decreased blood triglycerides in Gbeys / ys cells (p<0.05, Student's t-test). *p<0.05v wt. control, #p<0.05v GBEys / ys vehicle treated mice. [Figure 3F]Figures 3A-I include graphs of the effect of Compound 1 on in vivo metabolism, where mice were monitored over 24 hours by a Promethion high-resolution behavioral phenotyping system (Sable Instruments, Inc.). Effective masses were calculated to the power of 0.75. Data are mean ± SEM from n=11 9-month-old mice in the wt control vehicle group, n=6 9-month-old mice in the GBEys / ys vehicle group, and n=7 9-month-old mice in the Gbeys / ys144DG11 (Compound 1) treatment group. All injections were from 4 months of age. Untreated GBEys / ys mice show lower (light) respiratory quotient (3A), total energy expenditure (TEE) (3B), and fat oxidation (3C) compared to wild-type controls. Carbohydrate oxidation and locomotor activity, which are not significantly affected by disease state, were increased by Compound 1 even above wt control levels (3D-3E). Compound 1 also reversed the decrease in food and water intake observed in Gbeys / ys mice compared to wt controls (3F-3H). Blood metabolic panel (3I) based on n=5, 9.5 month old mice treated as indicated. Compound 1 increased blood glucose and decreased blood triglycerides in Gbeys / ys cells (p<0.05, Student's t-test). *p<0.05v wt. control, #p<0.05v GBEys / ys vehicle treated mice. [Figure 3G]Figures 3A-I include graphs of the effect of Compound 1 on in vivo metabolism, where mice were monitored over 24 hours by a Promethion high-resolution behavioral phenotyping system (Sable Instruments, Inc.). Effective masses were calculated to the power of 0.75. Data are mean ± SEM from n=11 9-month-old mice in the wt control vehicle group, n=6 9-month-old mice in the GBEys / ys vehicle group, and n=7 9-month-old mice in the Gbeys / ys144DG11 (Compound 1) treatment group. All injections were from 4 months of age. Untreated GBEys / ys mice show lower (light) respiratory quotient (3A), total energy expenditure (TEE) (3B), and fat oxidation (3C) compared to wild-type controls. Carbohydrate oxidation and locomotor activity, which are not significantly affected by disease state, were increased by Compound 1 even above wt control levels (3D-3E). Compound 1 also reversed the decrease in food and water intake observed in Gbeys / ys mice compared to wt controls (3F-3H). Blood metabolic panel (3I) based on n=5, 9.5 month old mice treated as indicated. Compound 1 increased blood glucose and decreased blood triglycerides in Gbeys / ys cells (p<0.05, Student's t-test). *p<0.05v wt. control, #p<0.05v GBEys / ys vehicle treated mice. [Figure 3H]Figures 3A-I include graphs of the effect of Compound 1 on in vivo metabolism, where mice were monitored over 24 hours by a Promethion high-resolution behavioral phenotyping system (Sable Instruments, Inc.). Effective masses were calculated to the power of 0.75. Data are mean ± SEM from n=11 9-month-old mice in the wt control vehicle group, n=6 9-month-old mice in the GBEys / ys vehicle group, and n=7 9-month-old mice in the Gbeys / ys144DG11 (Compound 1) treatment group. All injections were from 4 months of age. Untreated GBEys / ys mice show lower (light) respiratory quotient (3A), total energy expenditure (TEE) (3B), and fat oxidation (3C) compared to wild-type controls. Carbohydrate oxidation and locomotor activity, which are not significantly affected by disease state, were increased by Compound 1 even above wt control levels (3D-3E). Compound 1 also reversed the decrease in food and water intake observed in Gbeys / ys mice compared to wt controls (3F-3H). Blood metabolic panel (3I) based on n=5, 9.5 month old mice treated as indicated. Compound 1 increased blood glucose and decreased blood triglycerides in Gbeys / ys cells (p<0.05, Student's t-test). *p<0.05v wt. control, #p<0.05v GBEys / ys vehicle treated mice. [Figure 3I]Figures 3A-I include graphs of the effect of Compound 1 on in vivo metabolism, where mice were monitored over 24 hours by a Promethion high-resolution behavioral phenotyping system (Sable Instruments, Inc.). Effective masses were calculated to the power of 0.75. Data are mean ± SEM from n=11 9-month-old mice in the wt control vehicle group, n=6 9-month-old mice in the GBEys / ys vehicle group, and n=7 9-month-old mice in the Gbeys / ys144DG11 (Compound 1) treatment group. All injections were from 4 months of age. Untreated GBEys / ys mice show lower (light) respiratory quotient (3A), total energy expenditure (TEE) (3B), and fat oxidation (3C) compared to wild-type controls. Carbohydrate oxidation and locomotor activity, which are not significantly affected by disease state, were increased by Compound 1 even above wt control levels (3D-3E). Compound 1 also reversed the decrease in food and water intake observed in Gbeys / ys mice compared to wt controls (3F-3H). Blood metabolic panel (3I) based on n=5, 9.5 month old mice treated as indicated. Compound 1 increased blood glucose and decreased blood triglycerides in Gbeys / ys cells (p<0.05, Student's t-test). *p<0.05v wt. control, #p<0.05v GBEys / ys vehicle treated mice. [Figure 4A]4A-4D include bar graphs of PAS staining for total glycogen in skin fibroblasts from different APBD patients (4A), images of PAS staining for total glycogen in APBD87 fibroblasts that were glucose starved for 48 hours (left) or glucose starved and then refilled for the final 24 hours to induce glycogen loading (right) (4B) (image acquisition was performed with a Nikon Eclipse Ti2 microscope using a 40x PlanFluor objective and a CY3 filter), image-based multiparametric phenotype graphs of APBD fibroblasts under 48 hours of glucose starvation or starvation and glucose refilling as in 4B (4C) (significance level p=0.01), and bar graphs of glycolysis and mitochondrial ATP production measured by Agilent's Seahorse instrument and ATP rate assay kit (4D). Fibroblasts from healthy controls (HC) and APBD patients were either untreated or treated with 10 μM Compound 1 for 48 h (chronic) or 20 min (acute) in the assay. Measurements were normalized to cell number measured by crystal violet staining. Mean and SD values ​​based on n=6 replicates are shown. [Figure 4B]4A-4D include bar graphs of PAS staining for total glycogen in skin fibroblasts from different APBD patients (4A), images of PAS staining for total glycogen in APBD87 fibroblasts that were glucose starved for 48 hours (left) or glucose starved and then refilled for the final 24 hours to induce glycogen loading (right) (4B) (image acquisition was performed with a Nikon Eclipse Ti2 microscope using a 40x PlanFluor objective and a CY3 filter), image-based multiparametric phenotype graphs of APBD fibroblasts under 48 hours of glucose starvation or starvation and glucose refilling as in 4B (4C) (significance level p=0.01), and bar graphs of glycolysis and mitochondrial ATP production measured by Agilent's Seahorse instrument and ATP rate assay kit (4D). Fibroblasts from healthy controls (HC) and APBD patients were either untreated or treated with 10 μM Compound 1 for 48 h (chronic) or 20 min (acute) in the assay. Measurements were normalized to cell number measured by crystal violet staining. Mean and SD values ​​based on n=6 replicates are shown. [Figure 4C]4A-4D include bar graphs of PAS staining for total glycogen in skin fibroblasts from different APBD patients (4A), images of PAS staining for total glycogen in APBD87 fibroblasts that were glucose starved for 48 hours (left) or glucose starved and then refilled for the final 24 hours to induce glycogen loading (right) (4B) (image acquisition was performed with a Nikon Eclipse Ti2 microscope using a 40x PlanFluor objective and a CY3 filter), image-based multiparametric phenotype graphs of APBD fibroblasts under 48 hours of glucose starvation or starvation and glucose refilling as in 4B (4C) (significance level p=0.01), and bar graphs of glycolysis and mitochondrial ATP production measured by Agilent's Seahorse instrument and ATP rate assay kit (4D). Fibroblasts from healthy controls (HC) and APBD patients were either untreated or treated with 10 μM Compound 1 for 48 h (chronic) or 20 min (acute) in the assay. Measurements were normalized to cell number measured by crystal violet staining. Mean and SD values ​​based on n=6 replicates are shown. [Figure 4D]4A-4D include bar graphs of PAS staining for total glycogen in skin fibroblasts from different APBD patients (4A), images of PAS staining for total glycogen in APBD87 fibroblasts that were glucose starved for 48 hours (left) or glucose starved and then refilled for the final 24 hours to induce glycogen loading (right) (4B) (image acquisition was performed with a Nikon Eclipse Ti2 microscope using a 40x PlanFluor objective and a CY3 filter), image-based multiparametric phenotype graphs of APBD fibroblasts under 48 hours of glucose starvation or starvation and glucose refilling as in 4B (4C) (significance level p=0.01), and bar graphs of glycolysis and mitochondrial ATP production measured by Agilent's Seahorse instrument and ATP rate assay kit (4D). Fibroblasts from healthy controls (HC) and APBD patients were either untreated or treated with 10 μM Compound 1 for 48 h (chronic) or 20 min (acute) in the assay. Measurements were normalized to cell number measured by crystal violet staining. Mean and SD values ​​based on n=6 replicates are shown. [Figure 5A] 5A-5E include images of experiments showing the formation of heteroassemblies around compound 1 and not around endogenous molecules, as indicated by the liquid crystals formed in experiments 1-3 (5A); images of the STRING network of targets in the interactome of compound 1 (5B); cellular thermal shift assays (CETSA) of different targets of compound 1 heteroassembly (5C); surface plasmon resonance sensorgrams of compound 1 binding to LAMP1 (5D) (wherein sensorgram experiments consisting of association and dissociation at the indicated concentration range and pH values ​​were performed. The results show that dose-responsive association of LAMP1 to compound 1 begins at pH 6, is partial at pH 5, and is clearly demonstrated at lysosomal pH 4.5-5); and images of three binding models of compound 1 with LAMP1 grids predicted by SiteMap, fPocket, and FtSite (5E). [Figure 5B]5A-5E include images of experiments showing the formation of heteroassemblies around compound 1 and not around endogenous molecules, as indicated by the liquid crystals formed in experiments 1-3 (5A); images of the STRING network of targets in the interactome of compound 1 (5B); cellular thermal shift assays (CETSA) of different targets of compound 1 heteroassembly (5C); surface plasmon resonance sensorgrams of compound 1 binding to LAMP1 (5D) (wherein sensorgram experiments consisting of association and dissociation at the indicated concentration range and pH values ​​were performed. The results show that dose-responsive association of LAMP1 to compound 1 begins at pH 6, is partial at pH 5, and is clearly demonstrated at lysosomal pH 4.5-5); and images of three binding models of compound 1 with LAMP1 grids predicted by SiteMap, fPocket, and FtSite (5E). [Figure 5C] 5A-5E include images of experiments showing the formation of heteroassemblies around compound 1 and not around endogenous molecules, as indicated by the liquid crystals formed in experiments 1-3 (5A); images of the STRING network of targets in the interactome of compound 1 (5B); cellular thermal shift assays (CETSA) of different targets of compound 1 heteroassembly (5C); surface plasmon resonance sensorgrams of compound 1 binding to LAMP1 (5D) (wherein sensorgram experiments consisting of association and dissociation at the indicated concentration range and pH values ​​were performed. The results show that dose-responsive association of LAMP1 to compound 1 begins at pH 6, is partial at pH 5, and is clearly demonstrated at lysosomal pH 4.5-5); and images of three binding models of compound 1 with LAMP1 grids predicted by SiteMap, fPocket, and FtSite (5E). [Figure 5D]5A-5E include images of experiments showing the formation of heteroassemblies around compound 1 and not around endogenous molecules, as indicated by the liquid crystals formed in experiments 1-3 (5A); images of the STRING network of targets in the interactome of compound 1 (5B); cellular thermal shift assays (CETSA) of different targets of compound 1 heteroassembly (5C); surface plasmon resonance sensorgrams of compound 1 binding to LAMP1 (5D) (wherein sensorgram experiments consisting of association and dissociation at the indicated concentration range and pH values ​​were performed. The results show that dose-responsive association of LAMP1 to compound 1 begins at pH 6, is partial at pH 5, and is clearly demonstrated at lysosomal pH 4.5-5); and images of three binding models of compound 1 with LAMP1 grids predicted by SiteMap, fPocket, and FtSite (5E). [Figure 5E] 5A-5E include images of experiments showing the formation of heteroassemblies around compound 1 and not around endogenous molecules, as indicated by the liquid crystals formed in experiments 1-3 (5A); images of the STRING network of targets in the interactome of compound 1 (5B); cellular thermal shift assays (CETSA) of different targets of compound 1 heteroassembly (5C); surface plasmon resonance sensorgrams of compound 1 binding to LAMP1 (5D) (wherein sensorgram experiments consisting of association and dissociation at the indicated concentration range and pH values ​​were performed. The results show that dose-responsive association of LAMP1 to compound 1 begins at pH 6, is partial at pH 5, and is clearly demonstrated at lysosomal pH 4.5-5); and images of three binding models of compound 1 with LAMP1 grids predicted by SiteMap, fPocket, and FtSite (5E). [Figure 6A]6A-E. Bar graphs of autophagic flux as determined by the extent of lysosomal inhibitor-dependent increase in the ratio of lipidated to non-lipidated LC3 (LC3II / LC3I) (6A); representative TEM images of liver tissue from 9.5-month-old Gbeys / ys mice treated with Compound 11 or 5% DMSO vehicle (6B). (G: glycogen (α particles) and polyglucosan (structures with variable electron density), L: lysosomes, M: mitochondria; right panel: lysosomal glycogen staining was quantified by the ImageJ "count particles" tool.) LAMP1 knockdown and control APBD primary dermal fibroblasts treated with Compound 1 and lysosomal inhibitor (LI). 6C), a graph of lysosomal pH changes measured in APBD primary fibroblasts transduced with lentivirus encoding GFP or GFP-shLAMP1 and treated or not with Compound 1 for 24 hours, and confocal microscopy images of cells treated with Lysosensor and stained for PAS (6D), and a bar graph of ATP production rate assay in LAMP1-KD cells and GFP (control) cells treated with Compound 1 for 24 hours (chronic) or assayed (acute) (6E). [Figure 6B]6A-E. Bar graphs of autophagic flux as determined by the extent of lysosomal inhibitor-dependent increase in the ratio of lipidated to non-lipidated LC3 (LC3II / LC3I) (6A); representative TEM images of liver tissue from 9.5-month-old Gbeys / ys mice treated with Compound 11 or 5% DMSO vehicle (6B). (G: glycogen (α particles) and polyglucosan (structures with variable electron density), L: lysosomes, M: mitochondria; right panel: lysosomal glycogen staining was quantified by the ImageJ "count particles" tool.) LAMP1 knockdown and control APBD primary dermal fibroblasts treated with Compound 1 and lysosomal inhibitor (LI). 6C), a graph of lysosomal pH changes measured in APBD primary fibroblasts transduced with lentivirus encoding GFP or GFP-shLAMP1 and treated or not with Compound 1 for 24 hours, and confocal microscopy images of cells treated with Lysosensor and stained for PAS (6D), and a bar graph of ATP production rate assay in LAMP1-KD cells and GFP (control) cells treated with Compound 1 for 24 hours (chronic) or assayed (acute) (6E). [Figure 6C]6A-E. Bar graphs of autophagic flux as determined by the extent of lysosomal inhibitor-dependent increase in the ratio of lipidated to non-lipidated LC3 (LC3II / LC3I) (6A); representative TEM images of liver tissue from 9.5-month-old Gbeys / ys mice treated with Compound 11 or 5% DMSO vehicle (6B). (G: glycogen (α particles) and polyglucosan (structures with variable electron density), L: lysosomes, M: mitochondria; right panel: lysosomal glycogen staining was quantified by the ImageJ "count particles" tool.) LAMP1 knockdown and control APBD primary dermal fibroblasts treated with Compound 1 and lysosomal inhibitor (LI). 6C), a graph of lysosomal pH changes measured in APBD primary fibroblasts transduced with lentivirus encoding GFP or GFP-shLAMP1 and treated or not with Compound 1 for 24 hours, and confocal microscopy images of cells treated with Lysosensor and stained for PAS (6D), and a bar graph of ATP production rate assay in LAMP1-KD cells and GFP (control) cells treated with Compound 1 for 24 hours (chronic) or assayed (acute) (6E). [Figure 6D-1]6A-E. Bar graphs of autophagic flux as determined by the extent of lysosomal inhibitor-dependent increase in the ratio of lipidated to non-lipidated LC3 (LC3II / LC3I) (6A); representative TEM images of liver tissue from 9.5-month-old Gbeys / ys mice treated with Compound 11 or 5% DMSO vehicle (6B). (G: glycogen (α particles) and polyglucosan (structures with variable electron density), L: lysosomes, M: mitochondria; right panel: lysosomal glycogen staining was quantified by the ImageJ "count particles" tool.) LAMP1 knockdown and control APBD primary dermal fibroblasts treated with Compound 1 and lysosomal inhibitor (LI). 6C), a graph of lysosomal pH changes measured in APBD primary fibroblasts transduced with lentivirus encoding GFP or GFP-shLAMP1 and treated or not with Compound 1 for 24 hours, and confocal microscopy images of cells treated with Lysosensor and stained for PAS (6D), and a bar graph of ATP production rate assay in LAMP1-KD cells and GFP (control) cells treated with Compound 1 for 24 hours (chronic) or assayed (acute) (6E). [Figure 6D-2]6A-E. Bar graphs of autophagic flux as determined by the extent of lysosomal inhibitor-dependent increase in the ratio of lipidated to non-lipidated LC3 (LC3II / LC3I) (6A); representative TEM images of liver tissue from 9.5-month-old Gbeys / ys mice treated with Compound 11 or 5% DMSO vehicle (6B). (G: glycogen (α particles) and polyglucosan (structures with variable electron density), L: lysosomes, M: mitochondria; right panel: lysosomal glycogen staining was quantified by the ImageJ "count particles" tool.) LAMP1 knockdown and control APBD primary dermal fibroblasts treated with Compound 1 and lysosomal inhibitor (LI). 6C), a graph of lysosomal pH changes measured in APBD primary fibroblasts transduced with lentivirus encoding GFP or GFP-shLAMP1 and treated or not with Compound 1 for 24 hours, and confocal microscopy images of cells treated with Lysosensor and stained for PAS (6D), and a bar graph of ATP production rate assay in LAMP1-KD cells and GFP (control) cells treated with Compound 1 for 24 hours (chronic) or assayed (acute) (6E). [Figure 6E]6A-E. Bar graphs of autophagic flux as determined by the extent of lysosomal inhibitor-dependent increase in the ratio of lipidated to non-lipidated LC3 (LC3II / LC3I) (6A); representative TEM images of liver tissue from 9.5-month-old Gbeys / ys mice treated with Compound 11 or 5% DMSO vehicle (6B). (G: glycogen (α particles) and polyglucosan (structures with variable electron density), L: lysosomes, M: mitochondria; right panel: lysosomal glycogen staining was quantified by the ImageJ "count particles" tool.) LAMP1 knockdown and control APBD primary dermal fibroblasts treated with Compound 1 and lysosomal inhibitor (LI). 6C), a graph of lysosomal pH changes measured in APBD primary fibroblasts transduced with lentivirus encoding GFP or GFP-shLAMP1 and treated or not with Compound 1 for 24 hours, and confocal microscopy images of cells treated with Lysosensor and stained for PAS (6D), and a bar graph of ATP production rate assay in LAMP1-KD cells and GFP (control) cells treated with Compound 1 for 24 hours (chronic) or assayed (acute) (6E). [Figure 7A]Figures 7A-7F show graphs of IBP parameters in HC and APBD fibroblasts, as well as variable importance plots as the output of random forest classification performed on the different variables (cell features) shown on the x-axis (random forest analysis demonstrated that APBD and HC cell populations are separated at a 93% confidence level) (7A), n=5 HC and n=5 Graph of multiparametric cell phenotype characterization of APBD patient skin fibroblasts (degree of deviation from HC of the indicated cell features ordered by the amount of deviation (-log(P value)). Features with values ​​above the dashed line (box) indicate deviation from HC with a p value < 0.01. The various comparisons analyzed (compound A = compound 1) are shown) (7B); bar graph of lysosomal parameters affected by compound 1 in APBD and HC cells analyzed by IBP (7C); volcano plot of proteins affected by APBD and compound 1 under starvation and glycogen loading conditions (7D); Venn diagram of proteins downregulated by APBD and upregulated by compound 1 and vice versa under starvation (48) and glycogen loading (48+24) conditions (7E); and gene ontology of proteins upregulated (left) and downregulated (right) by compound 1 (7F). [Figure 7B]Figures 7A-7F show graphs of IBP parameters in HC and APBD fibroblasts, as well as variable importance plots as the output of random forest classification performed on the different variables (cell features) shown on the x-axis (random forest analysis demonstrated that APBD and HC cell populations are separated at a 93% confidence level) (7A), n=5 HC and n=5 Graph of multiparametric cell phenotype characterization of APBD patient skin fibroblasts (degree of deviation from HC of the indicated cell features ordered by the amount of deviation (-log(P value)). Features with values ​​above the dashed line (box) indicate deviation from HC with a p value < 0.01. The various comparisons analyzed (compound A = compound 1) are shown) (7B); bar graph of lysosomal parameters affected by compound 1 in APBD and HC cells analyzed by IBP (7C); volcano plot of proteins affected by APBD and compound 1 under starvation and glycogen loading conditions (7D); Venn diagram of proteins downregulated by APBD and upregulated by compound 1 and vice versa under starvation (48) and glycogen loading (48+24) conditions (7E); and gene ontology of proteins upregulated (left) and downregulated (right) by compound 1 (7F). [Figure 7C]Figures 7A-7F show graphs of IBP parameters in HC and APBD fibroblasts, as well as variable importance plots as the output of random forest classification performed on the different variables (cell features) shown on the x-axis (random forest analysis demonstrated that APBD and HC cell populations are separated at a 93% confidence level) (7A), n=5 HC and n=5 Graph of multiparametric cell phenotype characterization of APBD patient skin fibroblasts (degree of deviation from HC of the indicated cell features ordered by the amount of deviation (-log(P value)). Features with values ​​above the dashed line (box) indicate deviation from HC with a p value < 0.01. The various comparisons analyzed (compound A = compound 1) are shown) (7B); bar graph of lysosomal parameters affected by compound 1 in APBD and HC cells analyzed by IBP (7C); volcano plot of proteins affected by APBD and compound 1 under starvation and glycogen loading conditions (7D); Venn diagram of proteins downregulated by APBD and upregulated by compound 1 and vice versa under starvation (48) and glycogen loading (48+24) conditions (7E); and gene ontology of proteins upregulated (left) and downregulated (right) by compound 1 (7F). [Figure 7D]Figures 7A-7F show graphs of IBP parameters in HC and APBD fibroblasts, as well as variable importance plots as the output of random forest classification performed on the different variables (cell features) shown on the x-axis (random forest analysis demonstrated that APBD and HC cell populations are separated at a 93% confidence level) (7A), n=5 HC and n=5 Graph of multiparametric cell phenotype characterization of APBD patient skin fibroblasts (degree of deviation from HC of the indicated cell features ordered by the amount of deviation (-log(P value)). Features with values ​​above the dashed line (box) indicate deviation from HC with a p value < 0.01. The various comparisons analyzed (compound A = compound 1) are shown) (7B); bar graph of lysosomal parameters affected by compound 1 in APBD and HC cells analyzed by IBP (7C); volcano plot of proteins affected by APBD and compound 1 under starvation and glycogen loading conditions (7D); Venn diagram of proteins downregulated by APBD and upregulated by compound 1 and vice versa under starvation (48) and glycogen loading (48+24) conditions (7E); and gene ontology of proteins upregulated (left) and downregulated (right) by compound 1 (7F). [Figure 7E]Figures 7A-7F show graphs of IBP parameters in HC and APBD fibroblasts, as well as variable importance plots as the output of random forest classification performed on the different variables (cell features) shown on the x-axis (random forest analysis demonstrated that APBD and HC cell populations are separated at a 93% confidence level) (7A), n=5 HC and n=5 Graph of multiparametric cell phenotype characterization of APBD patient skin fibroblasts (degree of deviation from HC of the indicated cell features ordered by the amount of deviation (-log(P value)). Features with values ​​above the dashed line (box) indicate deviation from HC with a p value < 0.01. The various comparisons analyzed (compound A = compound 1) are shown) (7B); bar graph of lysosomal parameters affected by compound 1 in APBD and HC cells analyzed by IBP (7C); volcano plot of proteins affected by APBD and compound 1 under starvation and glycogen loading conditions (7D); Venn diagram of proteins downregulated by APBD and upregulated by compound 1 and vice versa under starvation (48) and glycogen loading (48+24) conditions (7E); and gene ontology of proteins upregulated (left) and downregulated (right) by compound 1 (7F). [Figure 7F]Figures 7A-7F show graphs of IBP parameters in HC and APBD fibroblasts, as well as variable importance plots as the output of random forest classification performed on the different variables (cell features) shown on the x-axis (random forest analysis demonstrated that APBD and HC cell populations are separated at a 93% confidence level) (7A), n=5 HC and n=5 Graph of multiparametric cell phenotype characterization of APBD patient skin fibroblasts (degree of deviation from HC of the indicated cell features ordered by the amount of deviation (-log(P value)). Features with values ​​above the dashed line (box) indicate deviation from HC with a p value < 0.01. The various comparisons analyzed (compound A = compound 1) are shown) (7B); bar graph of lysosomal parameters affected by compound 1 in APBD and HC cells analyzed by IBP (7C); volcano plot of proteins affected by APBD and compound 1 under starvation and glycogen loading conditions (7D); Venn diagram of proteins downregulated by APBD and upregulated by compound 1 and vice versa under starvation (48) and glycogen loading (48+24) conditions (7E); and gene ontology of proteins upregulated (left) and downregulated (right) by compound 1 (7F). [Figure 8] FIG. 8 includes in silico ADMET (absorption, distribution, metabolism, and excretion toxicity) compatible polyglucosan lowering compounds and analysis of three different ADMET algorithms. [Figure 9] FIG. 9 contains images of the results of an ADMET incompatible compound (88095528 in FIG. 8) causing wounds in Gbeys / ys mice. [Figure 10] 10 includes a graph of the body weight of wild-type C57B16J mice treated for three months with Compound 1. Mice were injected twice weekly with 150 μL of Compound 1 in 5% DMSO at 250 mg / kg or an equal volume of 5% DMSO (V, vehicle) control. Injections were intravenous for the first month and subcutaneous for the next two months. [Figure 11]Figure 11 includes images of tissue sections of the brain, liver, skeletal muscle, and heart of wild-type C57Bl6J mice treated with Compound 1 for 3 months. Sections were stained with H&E staining to visualize lesions. No lesions were observed in any of the treatments. Scale bars, 500 μm (brain), 100 μm (liver), 200 μm (muscle), 100 μm (heart). [Figure 12A] Figures 12A-B contain micrographs of the glycosylation status of LAMP1 and RNase B examined by 15% SDS-PAGE mobility shift gel stained with QC colloidal Coomassie stain (#1610803, Bio-Rad) after short-term (24 h) or long-term (72 h) dialysis (12A), and a sensorgram showing no interaction between deglycosylated LAMP1-Nter protein (degLAMP1-Nt) and compound 11 (12B). [Figure 12B] Figures 12A-B contain micrographs of the glycosylation status of LAMP1 and RNase B examined by 15% SDS-PAGE mobility shift gel stained with QC colloidal Coomassie stain (#1610803, Bio-Rad) after short-term (24 h) or long-term (72 h) dialysis (12A), and a sensorgram showing no interaction between deglycosylated LAMP1-Nter protein (degLAMP1-Nt) and compound 11 (12B). [Figure 13A] 13A-B include an image of the predicted binding site of compound 1 in the N-terminal domain of LAMP1 and the LAMP1 N-terminus:LAMP1 N-terminus protein:protein docking calculation (13A), as well as a schematic diagram of the lysosomal membrane (LM), LAMP1, LAMP2, and the potential inhibitor compound 1 (13B). [Figure 13B] 13A-B include an image of the predicted binding site of compound 1 in the N-terminal domain of LAMP1 and the LAMP1 N-terminus:LAMP1 N-terminus protein:protein docking calculation (13A), as well as a schematic diagram of the lysosomal membrane (LM), LAMP1, LAMP2, and the potential inhibitor compound 1 (13B). [Figure 14A]Figures 14A-14B contain images of heteroaggregates (circles) obtained by NPOT® on fibroblasts of APBD patients (14A) or HC fibroblasts (14B) in the presence of 10-6 M of compound 1 and OKMW-XXC (negative control). Each experiment was performed in triplicate. A technical negative control was obtained without adding any compound. Each picture represents a well of a 96-well plate. [Figure 14B] Figures 14A-14B contain images of heteroaggregates (circles) obtained by NPOT® on fibroblasts of APBD patients (14A) or HC fibroblasts (14B) in the presence of 10-6 M of compound 1 and OKMW-XXC (negative control). Each experiment was performed in triplicate. A technical negative control was obtained without adding any compound. Each picture represents a well of a 96-well plate. [Figure 15] FIG. 15 includes photomicrographs and vertical bar graphs showing autophagic flux in skin fibroblasts from PD patients that were serum-starved and treated with 50 μM 144DG11 (or not treated, shown as Comparative Example A). [Figure 16] Figure 16 includes fluorescent micrographs and vertical bar graphs showing that treatment of PD primary fibroblasts with 144DG11 (50 μM, 24 h) significantly reduced PAS staining (magenta), indicative of glycogen depletion. Yellow, calcein used for cell segmentation; blue, DAPI nuclear stain. The center panel shows quantification of segmented autophagic flux in dermal fibroblasts from a PD patient that were serum-starved and treated (or not, shown as Comparative Example A) with 50 μM 144DG11. [Figure 17]FIG. 17 includes vertical bar graphs showing glycolytic (1) and mitochondrial (2) ATP production measured by Agilent's Seahorse instrument and ATP rate assay kit. HC and PD patient fibroblasts were serum / glucose starved for 48 h and then supplemented with complete medium without (untreated) or with (chronic) 50 μM 144DG11 for 24 h. Acute, 50 μM 144DG11 was added to the assay for 20 min 24 h after serum / glucose supplementation. Measurements were normalized to cell number measured by crystal violet staining. Mean and SD values ​​based on n=6 replicates are shown. In acute 144DG11-treated PD fibroblasts, glycolysis and total ATP production were increased compared to untreated PD cells (p<0.002, one-way ANOVA with Sidak post-hoc correction for multiple comparisons). [Figure 18] Figure 18 includes vertical bar graphs showing blood metabolic panels based on n=5-6 6-month-old wild-type or Agl- / - mice treated for 3 months as indicated. Blood triglycerides were reduced by 144DG11, suggesting correction of hyperlipidemia. *p<0.049, ***p<0.004. [Figure 19A] Figures 19A-B contain fluorescent micrographs showing microglial cells isolated from the brains of AD modeling 5XFAD mice by CD11b magnetic beads, where microglia were incubated with (treated) or without (untreated) 50 μM 144DG11 for 24 hours, fixed, and stained for the autophagic substrates LC3 (19A) and p62 (19B) and for glycogen by PAS (all as indicated). The reduction in the levels of both LC3 and p62 indicates that autophagy was induced to degrade these substrates. [Figure 19B]Figures 19A-B contain fluorescent micrographs showing microglial cells isolated from the brains of AD modeling 5XFAD mice by CD11b magnetic beads, where microglia were incubated with (treated) or without (untreated) 50 μM 144DG11 for 24 hours, fixed, and stained for the autophagic substrates LC3 (19A) and p62 (19B) and for glycogen by PAS (all as indicated). The reduction in the levels of both LC3 and p62 indicates that autophagy was induced to degrade these substrates. [Figure 20A] Figures 20A-20B include fluorescent micrographs showing primary non-small cell lung cancer cells treated and stained for the autophagic substrates LC3 (20A) and p62 (20B) as in 19A-19B. [Figure 20B] Figures 20A-20B include fluorescent micrographs showing primary non-small cell lung cancer cells treated and stained for the autophagic substrates LC3 (20A) and p62 (20B) as in 19A-19B. [Figure 21] FIG. 21 includes vertical bar graphs showing that skin fibroblasts derived from a Gsd1a patient were treated with vehicle or 50 μM compound A for 24 hours and analyzed for NAD+ / NADH ratio by Promega kit (left panel), Sirt1 expression (middle panel) and p62 expression (right panel) by Western immunoblotting. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] The present invention relates to a pharmaceutical composition for use in the prevention or treatment of a disease or disorder associated with lysosomal storage.

[0036] The present invention further relates to a pharmaceutical composition for use in the prevention or treatment of a disease or disorder associated with polyglucosan accumulation or abnormal glycogen accumulation.

[0037] The present invention further relates to pharmaceutical compositions for use in the prevention or treatment of diseases or disorders associated with abnormal protein accumulation.

[0038] The present invention further relates to a pharmaceutical composition for use in the prevention or treatment of a disease associated with autophagy misregulation.The present invention further relates to a pharmaceutical composition for use in the prevention or treatment of a disease or disorder associated with decreased autophagy.

[0039] The present invention also relates to agents that bind to the N-terminal domain region of lysosome-associated membrane protein 1 (LAMP-1).

[0040] The present invention also relates to methods of treating or preventing the onset of a disease or disorder associated with lysosomal storage, polyglucosan storage or abnormal glycogen storage in a subject in need thereof.

[0041] According to some embodiments, the present invention provides a compound, or a pharma- ceutically acceptable salt, isomer, or tautomer thereof, for use in the prophylaxis or treatment of a disease or disorder selected from a lysosomal storage-associated disease and an autophagy misregulation-associated disease, the compound being represented by formula I:

[0042] [ka] (In the formula,

[0043] [ka] represents a single bond or a double bond, n and m each independently represent an integer ranging from 1 to 3, R and R 1 each independently represents hydrogen or is absent; R 3 , R 4 , R 5 , R 6 , R 7 and R 8each independently represents hydrogen or is selected from the group consisting of substituted or unsubstituted alkyl, cycloalkyl, alkoxy, hydroxy, thiohydroxy, thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halo, trihalomethyl, cyano, amido, carboxy, sulfonyl, sulfoxy, sulfinyl, and sulfonamido. The present invention provides a compound represented by the formula:

[0044] In some embodiments, R or R 1 In some embodiments, R is hydrogen and R 1 is absent. In some embodiments, R 1 is hydrogen and R is absent.

[0045] In some embodiments, n and m are 1.

[0046] In some embodiments, R 2 , R 7 and R 8 represents methyl.

[0047] In some embodiments, the compound is

[0048] [ka] or both.

[0049] According to some embodiments, the present invention provides a pharmaceutical composition for use in the prevention or treatment of a disease or disorder selected from a lysosomal storage-associated disease and an autophagy misregulation-associated disease, comprising a compound, a pharma- ceutical acceptable salt, isomer or tautomer thereof, wherein the compound is represented by formula I as described herein above.

[0050] According to some embodiments, the present invention provides a pharmaceutical composition for use in the prevention or treatment of a disease selected from lysosomal storage associated disorders, obesity, type II diabetes and insulin resistance, comprising a compound, a pharma- ceutically acceptable salt, isomer or tautomer thereof, wherein the compound is represented by formula I as described herein above.

[0051] In some embodiments, a lysosome-associated disease or disorder refers to a disease or disorder associated with the inability of lysosomal enzymes to degrade accumulated substrates, lysosomal swelling, lysosomal rupture, impaired lysosomal signaling, or any combination thereof.

[0052] In some embodiments, the pharmaceutical composition is for use in the prevention or treatment of a disease or disorder associated with the inability of lysosomal enzymes to degrade accumulated substrates. In some embodiments, the pharmaceutical composition is for use in the prevention or treatment of a disease or disorder associated with distended lysosomes. In some embodiments, the pharmaceutical composition is for use in the prevention or treatment of a disease or disorder associated with lysosomal rupture causing the release of toxic contents into the cytosol.

[0053] In some embodiments, the disease or disorder associated with lysosomal storage is selected from the group consisting of Gaucher disease, Fabry disease, Tay-Sachs disease, mucopolysaccharidoses (MPS) diseases, aspartylglucosaminuria, GM1-gangliosidosis, Krabbe disease (globoid cell leukodystrophy or galactosylceramide lipidosis), metachromatic leukodystrophy, Sandhoff disease, mucolipidosis type II (I-cell disease), mucolipidosis type IIIA (pseudo-Hurler polydystrophy), Niemann-Pick disease types C2 and Cl, Danon disease, free sialic acid storage disorders, mucolipidosis type IV, and multiple sulfatase deficiency (MSD), and metabolic disorders.

[0054] The terms "lysosomal storage", "lysosomal storage disease" and "lysosomal storage disorders" (LSDs) are used interchangeably herein to refer to a group of inherited diseases characterized by lysosomal dysfunction and neurodegeneration. These disorders typically result from a single gene defect, i.e., a deficiency in a specific enzyme normally required for the breakdown of glycosaminoglycans (GAGs), which causes cells to be unable to excrete carbohydrate residues, resulting in their accumulation in the lysosomes of the cells. This accumulation disrupts the normal function of the cells and causes the clinical symptoms of LSDs. Non-limiting examples of diseases or disorders associated with lysosomal storage include sphingolipidoses, ceramidases (e.g., Farber disease, Krabbe disease), galactosialidosis, gangliosidoses including alpha-galactosidases (e.g., Fabry disease (α-galactosidase A), Schindler disease (α-galactosidase B)), beta-galactosidases (e.g., GM1 gangliosidosis, GM2 gangliosidosis, Sandhoff disease, Tay-Sachs disease), glucocerebrosidosis (e.g., Gaucher disease (types I, II, III), sphingomyelinases (e.g., lysosomal acid lipase deficiency, Niemann-Pick disease), sulfatidoses (e.g., metachromatic leukodystrophy, multiple sulfatase deficiency), mucopolysaccharidoses (e.g., type I (MPS I (Hurler syndrome, MPS IS Scheie syndrome, MPS I HS Hurler-Scheie syndrome), type II (Hunter syndrome), type III (Sanfilippo syndrome), type IV (Morquio syndrome), type VI (Marotele-Lamy syndrome), type VII (Sly syndrome), type IX (hyaluronidase deficiency)), mucolipidoses (e.g., type I (sialidosis), type II (I-cell disease), type III (pseudo-Hurler polydystrophy / phosphotransferase deficiency), type IV (mucolipidin 1 deficiency)), lipidoses (e.g., Niemann-Pick disease), neuronal ceroid lipofuscinoses (e.g., type 1 Santavuori-Hartia disease / infantile NCL (CLN2 / LINCL TPP1)), type 2 Jansky-Bielschowski disease / infantile NCL (CLN2 / LINCLTPP1), Batten-Spielmeyer-Vogt disease type 3 / juvenile NCL (CLN3), Kuchs disease type 4 / adult NCL (CLN4), Finnish variant type 5 / late infantile (CLN5), late infantile variant type 6 (CLN6), CLN7 type 7, Northern mannosidosis type 8 (CLN8), Turkish late infantile type 8 (CLN8), German / Serbian late infantile type 9, congenital cathepsin D deficiency type 10 (CTSD), Wolman disease, oligosaccharide diseases (e.g., alpha-mannosidosis, beta-mannosidosis, aspartylglucosaminuria, fucososis), lysosomal transport diseases (e.g., cystinosis, pycnodysostosis, Salla disease / sialic acid storage disease, infantile free sialic acid storage disease), Pompe disease type II, Danon disease type Iib), cholesterol ester storage disease, etc.

[0055] In some embodiments, the use of a compound represented by Formula I, a pharma- ceutically acceptable salt, isomer, or tautomer thereof, in the prophylaxis or treatment of a disease or disorder associated with lysosomal storage does not include or exclude glycogen storage disease (GSD) or conditions associated therewith. In some embodiments, the use of a compound represented by Formula I, a pharma- ceutically acceptable salt, isomer, or tautomer thereof, in the prophylaxis or treatment of a disease or disorder associated with lysosomal storage does not include or exclude GSD IV, GSD VII, APDB, or any combination thereof.

[0056] In some embodiments, the use of a compound represented by Formula I, a pharma- ceutically acceptable salt, isomer, or tautomer thereof in the prophylaxis or treatment of a disease or disorder associated with lysosomal storage does not include or exclude glycogen storage disease (GSD)-associated neurodegenerative diseases.

[0057] According to some embodiments, the present invention provides a method for treating or preventing the onset of a disease or disorder associated with lysosomal storage in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition as described above.

[0058] In some embodiments, a therapeutically effective amount is an amount effective to slow, stop, or reverse the progression of protein accumulation / aggregation associated with a lysosomal storage disease or disorder. In some embodiments, a therapeutically effective amount is an amount effective to slow, stop, or reverse the progression of polyglucosan accumulation or abnormal glycogen accumulation. In some embodiments, a therapeutically effective amount is an amount effective to increase autophagic activity.

[0059] In some embodiments, a therapeutically effective amount is an amount effective to ameliorate one or more symptoms of pathology associated with a lysosomal storage disease and / or reduce neurodegeneration and / or neuroinflammation associated with a lysosomal storage disease.

[0060] In another aspect, the present invention provides a method for treating or preventing the onset of a disease or disorder associated with reduced or misregulated autophagic activity.

[0061] In some embodiments, the autophagy misregulation-related disease is a disease caused by misfolded protein aggregates. In another embodiment of this aspect, the disease caused by misfolded protein aggregates is selected from the group including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar degeneration, oculopharyngeal muscular dystrophy, prion disease, fatal familial insomnia, alpha-1 antitrypsin deficiency, dentatorubral-pallidoluysian atrophy, frontotemporal dementia, progressive supranuclear palsy, X-linked spinal muscular atrophy, and neuronal intranuclear hyaline inclusion disease.

[0062] The term "autophagy misregulation associated disease" also includes any disease or disorder, such as, but not limited to, cancer, cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders, in which induction of autophagy contributes to delaying the onset, slowing the progression, halting, or reversing one or more symptoms associated with the disease or disorder.

[0063] The term "autophagy misregulation associated disease" also includes cancer, for example, any cancer in which induction of autophagy inhibits cell growth and division, reduces mutagenesis, removes mitochondria and other organelles damaged by reactive oxygen species, or kills developing tumor cells. The term "autophagy misregulation associated disease" also includes psychiatric diseases or disorders, for example, any psychiatric disease or disorder in which induction of autophagy contributes to delaying the onset, slowing down, stopping, or reversing one or more symptoms associated with the psychiatric disease or disorder. In one embodiment, the psychiatric disease or disorder is selected from schizophrenia and bipolar disorder.

[0064] In one aspect, the present invention discloses a method of inducing autophagy in a cell, comprising contacting the cell with a pharmaceutical composition of the present invention in an amount effective to induce autophagy in the cell.

[0065] In one embodiment, the cells are present in a subject. In another embodiment, the cells are present in an in vitro cell culture. Non-limiting examples of cells are neuronal cells, glial cells, such as astrocytes, oligodendrocytes, ependymal cells, Schwann cells, lymphatic cells, epithelial cells, endothelial cells, lymphocytes, cancer cells, and hematopoietic cells.

[0066] The term "autophagy" refers to a catabolic process that involves the degradation of components of the cell itself, such as long-lived proteins, protein aggregates, organelles, cell membranes, organelle membranes, and other cellular components. The mechanism of autophagy can include (i) the formation of a membrane around a target area of ​​the cell, separating the contents from the rest of the cytoplasm, and (ii) the fusion of the resulting vesicles with lysosomes and subsequent degradation of the vesicle contents.

[0067] In some embodiments, there is provided a method of reducing neurodegeneration, reducing neuroinflammation, slowing progression, or reducing memory impairment, reducing abnormal lysosomal size, reactivating autophagic flux, or any combination thereof, comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition as described herein above.

[0068] In some embodiments, the method comprises reactivating autophagic flux in a subject suffering from a disease or disorder in which autophagy is disrupted. In some embodiments, the method comprises reactivating autophagic flux in a subject suffering from LDS as disclosed herein. In some embodiments, the method comprises reactivating autophagic flux in a subject suffering from Pompe disease. In some embodiments, the cancer is a cancer associated with decreased autophagic activity.

[0069] In some embodiments, methods are provided for improving one or more symptoms selected from the group consisting of leukodystrophy, scoliosis, hepatosplenomegaly, psychomotor regression, and ichthyosis, and / or delaying the onset of, slowing the progression of, halting, or reversing one or more of these symptoms.

[0070] In some embodiments, a subject is identified as having a lysosomal storage disease by the presence of a genetic marker for a lysosomal storage disease.

[0071] In some embodiments, administration is within 1 month of birth, within 2 months of birth, within 3 months of birth, within 6 months of birth, within 1 year of birth, or within 3 years of birth (including any value in between), with each possibility representing a separate embodiment of the present invention.

[0072] In some embodiments, the compounds and pharmaceutical compositions described herein above are capable of inhibiting and / or modulating the aggregation of one or more proteins and / or promoting the disaggregation of protein fibrils or other protein aggregates, or both. In some embodiments, the compounds and pharmaceutical compositions described herein above are capable of inhibiting and / or modulating the aggregation of one or more amyloid-forming proteins (e.g., one or more of α-synuclein, Ab, tau, etc.) and / or promoting the disaggregation of amyloid protein fibrils or other amyloid protein aggregates, or both.

[0073] Lysosomal membrane protein 1 (LAMP1) targeting agents According to some embodiments, the present invention provides agents that bind to the N-terminal domain region of lysosome-associated membrane protein 1 (LAMP-1; SEQ ID NO: 1; FSVNYDTKSGPKNMTFDLPSDATVVLNRSSCGKENTSDPSLVIAFGRGHTLTLNFTRNATRYSV).

[0074] As used herein, LAMP1 refers to lysosome-associated membrane glycoprotein 1, which has UniProt accession number P11279. In some embodiments, LAMP1 is selected from the group consisting of SEQ ID NO: 4 (MAAPGSARRPLLLLLLLLLLGLMHCASAAMFMVKNGNGTACIMANFSAAFSVNYDTKSGPKNMTFDLPSDATVVLNRSSCGKENTSDPSLVIAFGRGHTLTLNFTRNATRYSVQLMSFVYNLSDTHLFPNASSKEIKTVESITDIRADIDKKYRCVSGTQVHMNNVTVTLHDATIQAYLSNSSFSRGETRCEQDRPSPTTAPP APPSPSPSPVPKSPSVDKYNVSGTNGTCLLASMGLQLNLTYERKDNTTVTRLLNINPNKTSASGSCGAHLVTLELHSEGTTVLLFQFGMNASSSRFFLQGIQLNTILPDARDPAFKAANGSLRALQATVGNSYKCNAEEHVRVTKAFSVNIFKVWVQAFKVEGGQFGSVEECLLDENSMLIPIAVGGALAGLVLIVLIAYLVGRKRSHAGYQTI).

[0075] In some embodiments, the agent binds to at least one region of LAMP1 selected from any one of SEQ ID NO:2 (FSVNYD) and SEQ ID NO:3 (NVTV) or homologs thereof.

[0076] In some embodiments, the agent binds to an amino acid residue selected from residues F50-D55, N62, L67, F118, Y120-L122, T125, L127-S133, and N164-V166 of LAMP-1 (i.e., SEQ ID NO: 4). In some embodiments, the agent binds to a combination of amino acid residues selected from residues F50-D55, N62, L67, F118, Y120-L122, T125, L127-S133, and N164-V166 of LAMP-1 (i.e., SEQ ID NO: 4).

[0077] As used herein, homologs of SEQ ID NO:2 (FSVNYD) and SEQ ID NO:3 (NVTV) refer to at least one mutation (e.g., a substitution) that enables the agent to still bind to the pocket region of the N-terminal domain of LAMP-1 (SEQ ID NO:1) and produce a desired biological or pharmaceutical effect (e.g., disrupting or inhibiting LAMP1:LAMP1 interaction or inhibiting LAMP1-LAMP1 interaction).

[0078] In some embodiments, the N-terminal domain region of LAMP-1 is a pocket.

[0079] Non-limiting examples for identifying pockets include the following algorithms utilized by SiteMap, FtSite, or fPocket: In some embodiments, pockets are identified using the SiteMap, FtSite, or fPocket programs.

[0080] As used herein, the term "pocket" refers to a cavity, depression, or indentation in the surface of a protein molecule that is created as a result of the folding of peptide chains into a three-dimensional structure that renders the protein functional. Pockets can be readily recognized by inspection of the protein structure and / or by using commercially available modeling software.

[0081] As used herein, the term "agent" refers to any small organic molecule capable of entering and / or binding to a protein pocket as described herein above.

[0082] As used herein, the term "small organic molecule" refers to a molecule of a size comparable to organic molecules commonly used in pharmaceuticals. This term excludes natural biological macromolecules (e.g., proteins, nucleic acids, etc.). In some embodiments, the organic molecule has a size of up to 5,000 Da, up to 2,000 Da, or up to 1,000 Da (including any value therebetween). Each possibility represents a separate embodiment of the present invention.

[0083] In some embodiments, the agent is not a compound represented by formula I.

[0084] In some embodiments, the agent is:

[0085] [ka] is selected from the group consisting of:

[0086] In some embodiments, the binding is specific binding.

[0087] The term "specific binding" or "preferential binding" refers to binding that may be mediated by covalent and / or non-covalent interactions occurring between two pairs of species (e.g., enzyme / substrate, receptor / agonist, antibody / antigen, and lectin / carbohydrate). Where the interaction of the two species typically produces a non-covalent complex, the resulting binding is typically the result of electrostatic and / or hydrogen bonds and / or lipophilic interactions. Thus, "specific binding" occurs between pairs of species where there is an interaction between them that produces a binding complex. Specifically, specific binding is characterized by the preferential binding of one member of the pair to a particular species compared to the binding of that member of the pair to other species in the family of compounds to which that species belongs. Thus, for example, an agent may exhibit an affinity for a particular pocket on a LAMP-1 molecule (i.e., a pocket as defined herein) that is at least 2-fold, preferably at least 10-fold, at least 100-fold, at least 1,000-fold, or at least 10,000-fold (including any value in between) greater than its affinity for a different pocket on the same or related protein. Each possibility represents a separate embodiment of the present invention.

[0088] In some embodiments, the agent inhibits LAMP1:LAMP1 interactions. In some embodiments, the agent inhibits LAMP1-LAMP1 interactions.

[0089] In some embodiments, the agent is for use in the prevention or treatment of a disease or disorder selected from a disease or disorder associated with lysosomal storage, a disease or disorder associated with polyglucosan storage or abnormal glycogen storage and abnormal protein storage, and a disease associated with autophagy misregulation.

[0090] In some embodiments, the agent is for use in the prevention or treatment of a disease or disorder associated with the inability of lysosomal enzymes to degrade accumulated substrates. In some embodiments, the agent is for use in the prevention or treatment of a disease or disorder associated with distended lysosomes. In some embodiments, the agent is for use in the prevention or treatment of a disease or disorder associated with lysosomal rupture causing the release of toxic contents into the cytosol.

[0091] In some embodiments, the disease or disorder is selected from the group consisting of glycogen storage diseases (GSDs), adult polyglucosan body disease (APBDs), and Lafora disease, Gaucher disease, Fabry disease, Tay-Sachs disease, mucopolysaccharidoses (MPS) diseases, aspartylglucosaminuria, GM1-gangliosidosis, Krabbe disease (globoid cell leukodystrophy or galactosylceramide lipidosis), metachromatic leukodystrophy, Sandhoff disease, mucolibridosis type II (I-cell disease), mucolibridosis type IIIA (pseudo-Hurler polydystrophy), Niemann-Pick disease types C2 and Cl, Danon disease, free sialic acid storage disorders, mucolibridosis type IV, and multiple sulfatase deficiency (MSD), metabolic disorders, obesity, and insulin resistance.

[0092] In some, the disease or disorder is a glycogen storage disease (GSD). In some embodiments, the GSD is associated with glycogen branching enzyme deficiency. In some embodiments, the GSD is selected from GSD types I-XV. In some embodiments, the GSD is GSD type 0. In some embodiments, the GSD is GSD type 1. In some embodiments, the GSD is GSD type 2. In some embodiments, the GSD is GSD type 3. In some embodiments, the GSD is GSD type 4. In some embodiments, the GSD is GSD type 5. In some embodiments, the GSD is GSD type 6. In some embodiments, the GSD is GSD type 7. In some embodiments, the GSD is GSD type 9. In some embodiments, the GSD is GSD type 10. In some embodiments, the GSD is GSD type 11. In some embodiments, the GSD is GSD type 12. In some embodiments, the GSD is GSD type 13. In some embodiments, the GSD is GSD14 (also classified as congenital glycosylation disorder type 1 (CDG1T)). In some embodiments, the GSD is GSD15.

[0093] In some embodiments, the medical condition is one or more of, but not limited to, adult polyglucosan body disorder (APBD), Andersen's disease, Forbes' disease, and Danon's disease.

[0094] In some embodiments, a medical condition associated with GSD or "glycogen branching enzyme deficiency" is meant to refer to a disease or disorder characterized by the deposition, accumulation or aggregation of polyglucosan bodies in muscle, nerve and / or various other tissues of the body. In some embodiments, the medical condition is characterized by dysfunction of the central and / or peripheral nervous system of the subject.

[0095] Embodiments of the invention encompass a variety of methods for customizing the treatment, prevention, or reduction in incidence or severity of GSD and other disorders associated with the accumulation of polyglucosan bodies.

[0096] In some embodiments, the agent is used to treat a neurodegenerative disease. In some embodiments, the agent is used to treat an inflammatory disease. In some embodiments, the agent is used to treat a GSD-related cancer.

[0097] In some embodiments, the cancer is a cancer associated with decreased autophagic activity. In some embodiments, the cancer comprises or is lung cancer. In some embodiments, the lung cancer is or comprises non-small cell lung cancer (NSCLC).

[0098] In some embodiments, the agent is characterized by an activity of decreasing polyglucosan body (PB) cell content. In some embodiments, "decreasing PB cell content" is meant to refer to shaping (e.g., reducing) the size of PB. In some embodiments, "decreasing PB cell content" is meant to refer to degrading PB (e.g., by modulating glycogen branching enzyme, GBE).

[0099] In some embodiments, the agent is capable of modulating (eg, inhibiting, or, in some embodiments, increasing) the activity of at least one enzyme.

[0100] In some embodiments, the agent is capable of inhibiting one or more enzymes, non-limiting examples of which are glycosyltransferases, such as glycogen synthase (GS) and protein phosphatase-1 (PP1).

[0101] In some embodiments, the autophagy misregulation-related disease is a disease caused by misfolded protein aggregates. In another embodiment of this aspect, the disease caused by misfolded protein aggregates is selected from the group including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar degeneration, oculopharyngeal muscular dystrophy, prion disease, fatal familial insomnia, alpha-1 antitrypsin deficiency, dentatorubral-pallidoluysian atrophy, frontotemporal dementia, progressive supranuclear palsy, X-linked spinal and bulbar muscular atrophy, and neuronal intranuclear hyaline inclusion disease. The term "autophagy misregulation-related disease" also includes cancer, for example, any cancer in which the induction of autophagy inhibits cell growth and division, reduces mutagenesis, removes mitochondria and other organelles damaged by reactive oxygen species, or kills developing tumor cells. The term "autophagy misregulation associated disease" also includes any psychiatric disease or disorder, for example, in which induction of autophagy contributes to delaying the onset, slowing the progression, stopping, or reversing one or more symptoms associated with the psychiatric disease or disorder. In one embodiment, the psychiatric disease or disorder is selected from schizophrenia and bipolar disorder.

[0102] The term "inhibitory" or any grammatical derivative thereof as used herein in reference to an enzyme refers to being capable of preventing, blocking, attenuating, or reducing the activity of the enzyme.

[0103] In some embodiments, "reducing activity" is meant to refer to activity being reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% (including any values ​​and ranges therebetween) compared to an equivalent situation lacking the presence of a compound of the present disclosure or a composition of matter containing same.

[0104] The agents of the present disclosure can be designed and utilized alone or in combination with, or in combination with, any other therapeutically active agent, to exert dual and possibly synergistic activity when combined with, or in combination with, any other therapeutically active agent.

[0105] According to some embodiments, the present invention provides a pharmaceutical composition comprising an agent as described herein above.

[0106] In some embodiments, the pharmaceutical composition, in solution, has a pH of 4 to 6.5, 4.5 to 6.5, 4 to 6, 4 to 5.5, 4 to 5, 4.5 to 6, 4.5 to 5.5, or 4.5 to 5 (including any ranges therebetween), with each possibility representing a separate embodiment of the present invention.

[0107] In some embodiments, the agent exhibits specific binding to LAMP-1 in solution at pH 4-6.5, 4.5-6.5, 4-6, 4-5.5, 4-5, 4.5-6, 4.5-5.5, or 4.5-5 (including any range therebetween), with each possibility representing a separate embodiment of the present invention.

[0108] In some embodiments, the agents exhibit specific binding to LAMP-1 in solution at a lysosomal pH of 4-6.5, 4.5-6.5, 4-6, 4-5.5, 4-5, 4.5-6, 4.5-5.5, or 4.5-5 (including any range therebetween), with each possibility representing a separate embodiment of the present invention.

[0109] In some embodiments, the pharmaceutical composition comprises 100 nM-5 mM, 150 nM-5 mM, 200 nM-5 mM, 500 nM-5 mM, 700 nM-5 mM, 900 nM-5 mM, 1 mM-5 mM, 2 mM-5 mM, 100 nM-3 mM, 150 nM-3 mM, 200 nM-3 mM, 500 nM-3 mM, 700 nM-3 mM, 900 nM-3 mM, 1 mM-3 mM, 2 mM-3 mM, 100 nM-1 mM, 150 nM-1 mM, 200 nM-1 mM, 500 nM-1 mM, or 700 nM-1 mM (including any range therebetween). Each possibility represents a separate embodiment of the present invention.

[0110] According to some embodiments, the present invention provides a method for treating or preventing the onset of a disease or disorder associated with lysosomal storage, polyglucosan storage or abnormal glycogen storage in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition as described above.

[0111] In some embodiments, the disease or disorder associated with lysosomal storage is selected from the group consisting of Gaucher disease, Fabry disease, Tay-Sachs disease, mucopolysaccharidoses (MPS) diseases, aspartylglucosaminuria, GM1-gangliosidosis, Krabbe disease (globoid cell leukodystrophy or galactosylceramide lipidosis), metachromatic leukodystrophy, Sandhoff disease, mucolipidosis type II (I-cell disease), mucolipidosis type IIIA (pseudo-Hurler polydystrophy), Niemann-Pick disease types C2 and Cl, Danon disease, free sialic acid storage disorders, mucolipidosis type IV, and multiple sulfatase deficiency (MSD), metabolic disorders, obesity, and insulin resistance.

[0112] In some embodiments, the present invention provides methods for treating or preventing the development of cardiac glycogen storage diseases due to forms of GSD (including, but not limited to, GSD-IV, -VI, IX, XI) and AMP-activated protein kinase gamma subunit 2 deficiency. In some embodiments, compounds of the present disclosure may reduce pathogenic PB accumulation in PB involved in GSD, GSD type IV (APBD and Andersen's disease), GSD type VII (Tarui's disease), and Lafora's disease (LD).

[0113] As used herein, "lysosomal membrane protein" refers to LAMP-1, LAMP-2, CD63 / LAMP-3, DC-LAMP, or any lysosomal associated membrane protein, or homologs, orthologs, variants (e.g., allelic variants), and modified forms (e.g., containing one or more naturally occurring or engineered mutations). In one aspect, a LAMP polypeptide is a mammalian lysosomal associated membrane protein, e.g., a human or mouse lysosomal associated membrane protein. More generally, "lysosomal membrane protein" refers to any protein that contains a domain found in the membrane of an endosomal / lysosomal compartment or lysosomal-associated organelle and further contains a luminal domain.

[0114] Pharmaceutical compositions containing compounds and agents of the present disclosure According to one aspect of the present embodiments, there is provided a pharmaceutical composition comprising one or more compounds and / or agents described herein and a pharma- ceutically acceptable carrier.

[0115] According to one aspect of the present embodiments, there is provided a pharmaceutical composition comprising a therapeutically effective amount of one or more compounds and / or agents described herein.

[0116] As used herein, the phrase "therapeutically effective amount" refers to that amount of the compound being administered which is capable of relieving to some extent one or more of the symptoms of the condition being treated.

[0117] The term "subject" (which should be read to include "individual," "animal," "patient," or "mammal," where the context permits) defines any subject, particularly a mammalian subject, to which the treatment is indicated. In some embodiments, the subject is a human.

[0118] The compounds described herein above may be administered or otherwise utilized per se, or as either a pharma- ceutically acceptable salt, enantiomer, tautomer, diastereomer, protonated or unprotonated form, solvate, hydrate, or prodrug thereof.

[0119] The phrase "pharmaceutical acceptable salt" refers to a charged species of the parent compound and its counterion, which is typically used to modify the solubility properties of the parent compound and / or reduce any significant irritation to an organism by the parent compound without destroying the biological activity and properties of the administered compound. The neutral form of the compound can be regenerated by contacting the salt with a base or acid and isolating the parent compound in conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise these salts are equivalent to the parent form of the compound in the present invention.

[0120] The phrase "pharmaceutically acceptable salts" is intended to encompass salts of active compounds prepared with relatively non-toxic acids or bases, depending on the particular substituents found on the compounds described herein.

[0121] Examples of pharma- ceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphate, dihydrogenphosphate, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid or phosphorous acid, and those derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, methanesulfonic acid, etc. Also included are salts of amino acids such as alginates, and salts of organic acids such as glucuronic acid or galacturonic acid (see, for example, Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds described herein to be converted into either base or acid addition salts.

[0122] In some embodiments, the neutral forms of the compounds described herein are regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent forms of the compounds differ from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise are equivalent to the parent forms of the compounds herein.

[0123] The term "prodrug" refers to an agent that is converted into an active compound (active parent drug) in vivo. Prodrugs are typically useful for facilitating administration of the parent drug. Prodrugs may also have improved solubility compared to the parent drug in pharmaceutical compositions. Prodrugs are also often used to achieve sustained release of the active compound in vivo.

[0124] In some embodiments, the compounds described herein possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, tautomers, geometric isomers and individual isomers are encompassed within the scope of the invention.

[0125] As used herein and in the art, the term "enantiomer" refers to a stereoisomer of a compound that is superimposable on its counterpart only by complete inversion / reflection (mirror image) of one another. Enantiomers are said to have "handedness" because they refer to one another like right and left hands. Enantiomers have identical chemical and physical properties except when they are present in an environment that itself has handedness (e.g., all living systems).

[0126] In some embodiments, the compounds described herein can exist in unsolvated and solvated forms, such as hydrated forms. In general, solvated forms are equivalent to unsolvated forms and are included within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

[0127] The term "solvate" refers to a complex of variable stoichiometry (e.g., di-, tri-, tetra-, penta-, hexa-, etc.) formed by a solute (conjugate as described herein) and a solvent, where the solvent does not interfere with the biological activity of the solute. Suitable solvents include, for example, ethanol, acetic acid, etc.

[0128] The term "hydrate" refers to a solvate as defined herein above, wherein the solvent is water.

[0129] In some embodiments, a "pharmaceutical composition" refers to a preparation of one or more compounds described herein (as active ingredients), or physiologically acceptable salts or prodrugs thereof, with chemical components such as, but not limited to, physiologically suitable carriers, excipients, lubricants, buffers, antibacterial agents, bulking agents (e.g., mannitol), antioxidants (e.g., ascorbic acid or sodium bisulfite), anti-inflammatory agents, antiviral agents, chemotherapeutic agents, antihistamines, and the like.

[0130] In some embodiments, the purpose of a pharmaceutical composition is to facilitate administration of a compound to a subject. The term "active ingredient" refers to a compound capable of accounting for a biological effect.

[0131] The terms "physiologically acceptable carrier" and "pharmaceutical acceptable carrier" may be used interchangeably and refer to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the compound being administered.

[0132] As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a drug. Non-limiting examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0133] Techniques for drug formulation and administration can be found in the latest edition of "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, incorporated herein by reference.

[0134] Therefore, in some embodiments, pharmaceutical compositions for use according to the present invention may be formulated in a conventional manner using one or more pharma- ceutically acceptable carriers, including excipients and adjuvants that facilitate the processing of the compound into pharma- ceutically usable preparations. Appropriate formulations depend on the route of administration selected. Dosages may vary according to the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dosage can be selected by individual physicians in consideration of the patient's condition (see, for example, Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch.1 p.1).

[0135] In some embodiments, the pharmaceutical compositions can be formulated for administration by any one or more routes, depending on whether local or systemic treatment or administration is selected and on the area to be treated. As further described throughout the specification, administration can be orally, dentally, by inhalation, or parenterally, for example, by intravenous infusion or intraperitoneal, subcutaneous, intramuscular or intravenous injection, or topically (ophthalmically, vaginally, rectally, intranasally, etc.).

[0136] Formulations for topical and / or dental administration may include, but are not limited to, lotions, ointments, gels, creams, suppositories, drops, liquids, sprays and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0137] Compositions for oral administration may include powders or granules, suspensions, dental compositions, or solutions in water or non-aqueous media, sachets, pills, caplets, capsules, or tablets.Thickeners, diluents, flavorings, dispersing aids, emulsifiers, or binders may be desirable.

[0138] Formulations for parenteral administration may include, but are not limited to, sterile solutions which may also contain buffers, diluents and other suitable additives. Sustained release compositions are contemplated for treatment.

[0139] The amount of a composition administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0140] The pharmaceutical compositions may further comprise additional pharma- ceutically active or inactive agents, such as, but not limited to, antibacterial agents, antioxidants, buffers, bulking agents, surfactants, anti-inflammatory agents, antiviral agents, chemotherapeutic agents, and antihistamines.

[0141] The compositions of the present invention may be provided in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient, if desired. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also correspond to a notice associated with the container in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, which notice reflects the agency's approval of the composition or form of administration in human or veterinary medicine. Such notice may, for example, be the label approved by the U.S. Food and Drug Administration for prescription drugs, or an approved product insert.

[0142] It will be understood that these embodiments are susceptible to various modifications and alternatives well known to those skilled in the art.

[0143] Screening Methods According to one aspect of some embodiments of the present invention, there is provided a method for determining the suitability of a compound for preventing or treating a disease or disorder associated with lysosomal storage, a disease or disorder associated with polyglucosan accumulation or abnormal glycogen accumulation, and a disease or disorder associated with abnormal protein accumulation, and autophagic misregulation related diseases, comprising contacting the compound with a pocket domain within the N-terminal domain of lysosomal associated membrane protein 1 (LAMP-1; SEQ ID NO:1), wherein binding of the compound to the pocket indicates that the compound is effective for treating the disease or disorder.

[0144] In some embodiments, an agent is provided wherein the linkage comprises any one of SEQ ID NO:2 (FSVNYD); and SEQ ID NO:3 (NVTV).

[0145] In some embodiments, the binding is determined by inhibition of LAMP1:LAMP1 interaction.

[0146] In some embodiments, the binding is determined by inhibition of LAMP1-LAMP1 interactions.

[0147] In some embodiments, the method includes a step of computational screening of a library of compounds.

[0148] In some embodiments, the methods involve detecting a reduction in PB exerted by one or more selected compounds (eg, small molecules).

[0149] It will be appreciated that by virtue of allowing for computational screening of libraries of compounds essentially having any of a variety of chemical, biological and / or physical characteristics, the present method allows for the identification of compounds that can exhibit optimal in vivo pharmacokinetics, optimally low immunogenicity, and optimal efficacy relative to all prior art compounds capable of reducing PB cell content, for example by correcting impaired enzymatic activity associated with glycogen storage disease (e.g., glycogen synthase or glycogen branching enzyme).

[0150] In some embodiments, the methods involve biochemically qualifying the ability of the compound to reduce PB cell content.

[0151] In some embodiments, biochemically qualifying comprises subjecting the cells to Periodic Acid Schiff (PAS) staining to provide PAS-stained cells. In some embodiments, the method further comprises detecting a signal (e.g., photofluorescence) from the PAS-stained sample at a defined wavelength after washing the sample to remove unreacted Schiff's reagent.

[0152] Further embodiments of the disclosed methods are presented in the Examples section below.

[0153] definition As used herein, the term "alkyl" refers to an aliphatic hydrocarbon, including straight-chain and branched-chain groups. Preferably, an alkyl group has 21 to 100 carbon atoms, more preferably 21 to 50 carbon atoms. Whenever a numerical range is stated herein, for example, "21 to 100," it means that the group (in this case, the alkyl group) may contain up to 100 carbon atoms, such as 21 carbon atoms, 22 carbon atoms, 23 carbon atoms, etc. In the context of the present invention, a "long-chain alkyl" is an alkyl having at least 20 carbon atoms in its backbone (the longest path of consecutive covalently bonded atoms). Thus, a short-chain alkyl has 20 or fewer backbone carbons. An alkyl may be substituted or unsubstituted as defined herein.

[0154] The term "alkyl," as used herein, also encompasses saturated or unsaturated hydrocarbons, and thus, this term further encompasses alkenyls and alkynyls.

[0155] The term "alkenyl" refers to an unsaturated alkyl, as defined herein, having at least two carbon atoms and at least one carbon-carbon double bond. The alkenyl may be unsubstituted or substituted with one or more substituents, as described herein above.

[0156] The term "alkynyl," as defined herein, is an unsaturated alkyl having at least two carbon atoms and at least one carbon-carbon triple bond. Alkynyl can be unsubstituted or substituted with one or more substituents, as described herein above.

[0157] The term "cycloalkyl" refers to an all-carbon monocyclic or fused ring group (i.e., rings which share adjacent pairs of carbon atoms) in which one or more rings do not have a completely conjugated pi-electron system. Cycloalkyl groups can be substituted or unsubstituted as described herein.

[0158] The term "aryl" refers to an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) group having a completely conjugated pi-electron system. Aryl groups can be substituted or unsubstituted as described herein.

[0159] The term "alkoxy" refers to both an --O-alkyl and an --O-cycloalkyl group, as defined herein.

[0160] The term "aryloxy" refers to -O-aryl as defined herein.

[0161] Each of the alkyl, cycloalkyl, and aryl groups in the general formulas herein can be substituted by one or more substituents, where each substituent can be, independently, for example, halide, alkyl, alkoxy, cycloalkyl, alkoxy, nitro, amine, hydroxyl, thiol, thioalkoxy, thiohydroxy, carboxy, amido, aryl, and aryloxy, depending on the substituent and its position in the molecule. Additional substituents are also contemplated.

[0162] The terms "halide," "halogen," or "halo" refer to fluorine, chlorine, bromine, or iodine.

[0163] The term "haloalkyl" refers to an alkyl group, as defined herein, which is further substituted with one or more halide groups.

[0164] The term "haloalkoxy" refers to an alkoxy group, as defined herein, further substituted with one or more halide groups.

[0165] The terms "hydroxyl" or "hydroxy" refer to an --OH group.

[0166] The term "thiohydroxy" or "thiol" refers to a -SH group.

[0167] The term "thioalkoxy" refers to both an --S-alkyl group, and an --S-cycloalkyl group, as defined herein.

[0168] The term "thioaryloxy" refers to both an --S-aryl and an --S-heteroaryl group, as defined herein.

[0169] The term "amine" refers to the group --NR'R'' where R' and R'' are as defined herein.

[0170] The term "heteroaryl" refers to monocyclic or fused ring groups (i.e., rings which share adjacent pairs of atoms) having one or more atoms in the ring, such as, for example, nitrogen, oxygen, and sulfur, and which, in addition, have a completely conjugated pi-electron system. Non-limiting examples of heteroaryl groups include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, and purine.

[0171] The term "heteroalicyclic" or "heterocyclyl" refers to a monocyclic or fused ring group having one or more atoms in the ring, such as nitrogen, oxygen, and sulfur. The ring may also have one or more double bonds, provided that the ring does not have a completely conjugated pi-electron system. Representative examples are piperidine, piperazine, tetrahydrofuran, tetrahydropyran, morpholino, and the like.

[0172] The term "carboxy" or "carboxylate" refers to the group -C(=O)-OR', where R' is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl (bonded through a ring carbon) or heteroalicyclic (bonded through a ring carbon) as defined herein.

[0173] The term "carbonyl" refers to a -C(=O)-R' group, where R' is as defined herein above.

[0174] The term also includes its thio derivatives: thiocarboxy and thiocarbonyl.

[0175] The term "thiocarbonyl" refers to a -C(=S)-R' group, where R' is as defined herein above.

[0176] A "thiocarboxy" group refers to a -C(=S)-OR' group, where R' is as defined herein.

[0177] A "sulfinyl" group refers to a -S(=O)-R' group, where R' is as defined herein.

[0178] A "sulfonyl" or "sulfonate" group refers to a -S(=O)2-R' group, where Rx is as defined herein.

[0179] A "carbamyl" or "carbamate" group refers to an -OC(=O)-NR'R'' group, where R' is as defined herein and R'' is as defined for R'.

[0180] A "nitro" group refers to a -NO2 group.

[0181] A "cyano" or "nitrile" group refers to a -C≡N group.

[0182] As used herein, the term "azido" refers to the group --N3.

[0183] The term "sulfonamide" refers to a -S(=O)2-NR'R'' group, with R' and R'' as defined herein.

[0184] The term "phosphonyl" or "phosphonate" refers to a -OP(=O)(OR')2 group, where R' is as defined herein above.

[0185] The term "phosphinyl" refers to the group -PR'R'' where R' and R'' are as defined herein above.

[0186] The term "alkaryl" refers to an alkyl, as defined herein, substituted with an aryl, as described herein. An exemplary alkaryl is benzyl.

[0187] The term "heteroaryl" refers to a monocyclic or fused ring (i.e., rings sharing adjacent pairs of atoms) group having one or more atoms in the ring, such as, for example, nitrogen, oxygen, and sulfur, and in addition, having a completely conjugated pi-electron system. Non-limiting examples of heteroaryl groups include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, and purine. Heteroaryl groups can be substituted or unsubstituted by one or more substituents as described herein above. Representative examples are thiadiazole, pyridine, pyrrole, oxazole, indole, purine, and the like.

[0188] As used herein, the terms "halo" and "halide" (which are referred to interchangeably herein) refer to an atom of a halogen, which is fluorine, chlorine, bromine, or iodine, which are also referred to herein as fluoride, chloride, bromide, and iodide.

[0189] The term "haloalkyl" refers to an alkyl group, as defined above, which is further substituted with one or more halide groups.

[0190] General As used herein, the term "about" refers to ±10%.

[0191] The terms "comprises," "comprising," "includes," "including," "having" and variations thereof mean "including but not limited to."

[0192] The term "consisting of" means "including and limited to."

[0193] The term "consisting essentially of" means that a composition, method, or structure may include additional components, steps, and / or moieties, but only if the additional components, steps, and / or moieties do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0194] The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any embodiment described as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments and / or does not exclude the incorporation of features from other embodiments.

[0195] The word "optionally" is used herein to mean "present in some embodiments and not present in other embodiments." Any particular embodiment of the invention may include multiple "optional" features, provided such features are not inconsistent.

[0196] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include a plurality of compounds, including mixtures thereof.

[0197] Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0198] Whenever a numerical range is given herein, it is meant to include any recited numbers (fractional or integer) within the given range. The terms "ranging" between a first denoted number and a second denoted number and "ranging" from a first denoted number to a second denoted number are used interchangeably herein and are meant to include the first and second denoted numbers and all fractional and integer numbers therebetween.

[0199] As used herein, the term "method" refers to manner, means, techniques, and procedures for accomplishing a given task, and includes, but is not limited to, manner, means, techniques, and procedures known to or readily developed from known manner, means, techniques, and procedures by practitioners in the chemical, pharmaceutical, biological, biochemical, and medical fields.

[0200] As used herein, the term "treating" includes arresting, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating a clinical or cosmetic symptom of a condition, or substantially preventing the appearance of a clinical or cosmetic symptom of a condition.

[0201] It will be understood that certain features of the invention that are described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention are described in the context of a single embodiment for brevity, but may also be provided separately or in any suitable subcombination, as appropriate, in other described embodiments of the invention. Certain features described in the context of various embodiments are not to be construed as essential features of those embodiments, unless the embodiment cannot function without those elements.

[0202] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below are found to be experimentally supported in the following examples. EXAMPLES

[0203] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

[0204] Materials and Methods research design The presented experiments combine in vivo, ex vivo, and in vitro experiments on the therapeutic potential of Compound 1, a newly discovered compound, for treating APBD. In the in vivo section, we demonstrate that Compound 1 is an effective treatment for Gbe ys / ysIts ability to correct disease phenotypes in female mice was tested. Two groups (5% DMSO vehicle and compound 1) of n=7-9 animals each were used initially. Based on the obtained means and SD, these numbers were retrospectively demonstrated to provide sufficient power, since 80% power was already achieved with n=5 animals / group. Additional open field, ambulatory, and stretch reflex tests (Figure 1E-H) also included a C57BL / 6 wild-type control group of n=9 animals. Animals were removed from the experiment if their body weight was reduced by less than 10% between successive weight measurements or if their body weight was reduced by less than 20% from the start. The sample size decreased slightly over time due to death. Compound 1 was injected twice weekly at 250 mg / kg in 150 μL of 5% DMSO. Vehicle control was 5% DMSO. Injections were intravenous (IV) for the first month, then subcutaneous (SC) due to lack of space to inject and scarring of the animal's tail. We started injections at 4 months of age, 2 months before disease onset, assuming a favorable preventive effect, or at 6 months of age, when disease onset occurred, for comparison. Treatment continued until 10 months of age. The effect of Compound 1 on various locomotor parameters was tested approximately every 2 weeks. At the end of these experiments, some mice were sacrificed by cervical dislocation, and n=2 wild type, n=7 Gbe ys / ys Tissues from vehicle-treated and n=9 compound 1-treated mice were harvested, sectioned, fixed, and stained for diastase-resistant PG with PAS (Figures 2A-C). Tissue glycogen was measured biochemically as described. In addition, the pharmacokinetic profile of compound 1 was measured by LC-MS / MS of serum and tissues obtained from n=3 mice / time point. Experimenters were blinded to treatment allocation.

[0205] Since skin fibroblasts and liver from APBD patients had the highest PG levels, Gbe ys / ys Ex vivo experiments were performed on liver sections from mice. In vitro experiments were performed on cell lysates.

[0206] Histological PG and glycogen measurements To characterize the histopathological effects of Compound 1, we performed histopathological analysis of Gbec2 mice treated with wt, Compound 1, and vehicle. ys / ys Brain, heart, muscle, nerve bundles (peripheral nerves), and liver tissues were dissected from the animals. Tissues were extracted, fixed, embedded in paraffin, and sectioned. After deparaffinization, sections were treated with 0.5% diastase for 5 min to digest non-polyglucosan glycogen and leave polyglucosan. Sections were then washed, stained with PAS for polyglucosan, counterstained with hematoxylin, and analyzed by light microscopy, all as previously described. For biochemical glycogen measurements, 100 mg of each tissue was subjected to alkaline hydrolysis and boiling, followed by ethanol precipitation of glycogen. Glycogen was then enzymatically digested to glucose by amyloglucosidase (Sigma). After digestion, total glycogen was determined based on glucose content using the Sigma GAGO20 kit.

[0207] Imaging and Image-Based Phenotyping APBD dermal fibroblasts were seeded at 1,000 cells / well and cultured in specialized microscopy-grade 96-well plates (Grenier Bio-One, Germany). Following the different treatments, a mixture of Thermo Scientific cell fluorescent dyes in PBS was added to each well for 30 min at 37 °C in a 5% CO2 incubator. This mix (Figure 4C and Figure 7B) contained DAPI (1 μg / ml, nuclear (DNA) stain), MitoTracker Green (500 nM, voltage-independent mitochondrial stain), TMRE (500 μM, voltage-dependent mitochondrial stain), and Cell Mask Deep Red (0.5 μg / ml, cytosol stain). In Figure 7C, only lysosomes were stained with LysoTracker Deep Red (75 nM). Cells were then fixed with 4% paraformaldehyde (PFA), washed with PBS, and plates were transferred to an InCell2200 instrument (GE Healthcare, UK) for image acquisition at 40x magnification. The output generated was based on comparative fluorescence intensity. Object segmentation was performed using multi-target analysis on a GE analysis workstation to identify nuclei and cell boundaries. All assay parameters (including acquisition exposure time, objective lens, and analysis parameters) were kept constant for all assay replicates. For PAS staining of glycogen (Figures 4 and 6C), fixed cells were washed with PBS, permeabilized with 0.1% Triton X-100, washed again, stained, and then imaged.

[0208] Pharmacokinetics For pharmacokinetic analysis, 100 μL of serum and brain, kidney, hind leg quadriceps, heart, liver, and spleen tissues were collected, homogenized, and extracted with acetonitrile according to established guidelines. Compound 1 at 0, 1, 10, 100, and 1,000 ng / ml in a 1 mg / ml solution of 4-tert-butyl-2-(4H-1,2,4-triazol-4-yl)phenol (ChemBridge) was used as an internal standard (IS) to generate a calibration curve. Tissue samples were then dissolved in 1 mg / ml IS solution and spiked with 0 to 1,000 ng / ml of compound 1 to generate a standard curve from which the tissue levels of compound 1 were determined. Samples were analyzed by LC-MS / MS Sciex Triple Quad TM 5500 mass spectrometer.

[0209] ethics In vivo experiments were approved by the Hebrew University IACUC.

[0210] statistical analysis In Figure 1A-1M, the significance of the overall trends was tested by two-way ANOVA with repeated measures. This test examines how the response is affected by two factors: Compound 1 vs. control, which is given repeatedly (and therefore measured repeatedly), and duration of administration. A Bonferroni test was used to compare Compound 1 with vehicle in a manner that is very robust because it corrects for multiple comparisons (because the threshold for determining significance at each time point decreases inversely proportional to the number of comparisons). As a result, most differences at a particular time point become insignificant with increasing number of comparisons, and we sometimes chose to also present data from multiple t-tests that do not correct for multiple comparisons. In Figure 4D and Figure 6E, we used one-way ANOVA with Sidak's post-hoc correction for multiple comparisons. The other statistical test used was the Student's t-test.

[0211] Target Identification by Nematic Protein Organizing Technique (NPOT) NPOT® was applied to human healthy fibroblasts and fibroblasts from two APBD patients. All analyses were performed in a blinded manner by Inoviem Scientific. Protein homogenates from dried pellets of these fibroblasts were prepared by three cycles of fast freezing (liquid nitrogen) and slow thawing (on ice) and mixed for 30 seconds at maximum vortex speed. Sample protein concentrations were 50-66 mg / ml as measured by the BCA method. NPOT® is a proprietary technology offered by Inoviem Scientific (Food Scientific) dedicated to the isolation and identification of specific macromolecular scaffolds performed directly from human tissues in basic conditions or pathological situations. The technology is based on the Kirkwood-Buff molecular crowding and aggregation theory. It allows the formation and label-free identification of macromolecular complexes involved in physiological or pathological processes. A unique strength of Inoviem Scientific is the ability to analyze drug-protein and protein-protein interactions directly in human tissues from complex mixtures without disrupting the native molecular conformation, thereby preserving the original physiological or pathological state.

[0212] Under laminar flow and sterile conditions, -6 Compound 1 of M and negative controls from HTS screening were mixed with protein homogenates (containing soluble and membrane proteins) separately and subjected to NPOT® isolation. The macromolecular assemblies associated with the ligands were separated using a differential microdialysis system, where the macromolecules (protein groups) migrate in the liquid phase based on their physicochemical properties. The migrating macromolecules gradually grow from nematic crystals to macromolecular heteroaggregates thanks to the molecular interactions between the test drug and its target. The heteroaggregates were left overnight and isolated in a 96-well plate, and then identified by LC-MS / MS.

[0213] Heteroaggregates formed in the presence of compound 1 and negative controls in APBD patient and HC fibroblasts are shown in Figures 14A-B. Each compound contacted with the indicated protein homogenates produced well-characterized heteroaggregates with a common reticular morphology. These experiments were performed in triplicate for each compound. For each of these biological replicates, heteroaggregates were isolated and their protein content was analyzed by LC-MS / MS. Negative controls were obtained with protein homogenates in NPOT® conditions without the addition of compound and do not show any aggregation. This further confirmed that the formation of heteroaggregates is initiated by the compounds and not by endogenous small molecules through their interaction with the primary target.

[0214] Each heteroaggregate formed was isolated by microdissection under a Zeiss microscope SteREO Discovery V8, washed in acetone, and then solubilized in standard HBSS solution. The solubilized proteins were filtered through a 4-15% mini-PROTEAN gel. After electrophoresis, the gel was stained with colloidal blue solution to visually estimate the number of proteins present in the gel, as well as the relative amount of proteins used for the subsequent digestion steps and injection into the LC-MS / MS instrument for proteomic analysis.

[0215] Proteomics was outsourced to the "Laboratoire de Spectrometrie de Masse Bio-Organique" (LSMBO) from UMR 7178. Heteroassemblies were directly solubilized in 10 μL of 2D buffer (7 M urea, 2 M thiourea, 4% CHAPS, 20 mM DTT, 1 mM PMSF). Proteins were precipitated in acetate buffer and centrifuged at 7,500 g for 20 min. The pellet was then digested with Trypsin Gold (Promega) for 1 h at 37 °C. Trypsin Gold was resuspended in 50 mM acetic acid at 1 μg / μL and then diluted to 20 μg / mL in 40 mM NH4HCO3. Samples were dried at room temperature in a Speed ​​Vac®. Peptides were purified and concentrated by using ZipTip® pipette tips (Millipore Corporation) before proceeding to mass spectrometry by a 1-hour nano-LC-MS / MS analysis protocol on an ESI-QUAD-TOF instrument. Proteins were identified using Mascot software (rank=1, score=25, minimum length=6 amino acids, FDR=1%). For peptide mapping, the following databases were used:-HumaniRTUN_DCpUN_JUS bank (for human samples).

[0216] For data analysis and target deconvolution, Inoviem Scientific (Texas Scientific) developed a proprietary database and software that allows for accurate and robust analysis of proteins present in NPOT® datasets, simplifying protein ranking while removing protein contaminants. The Inoviem Protein Ranking and Analysis (InoPerA®) database contains all NPOT® datasets obtained with different tissues, organs or cell lines, different species, and unrelated compounds. The InoPerA® software can then calculate the occurrence of a given gene in the entire database or in a specific dataset that matches defined criteria such as species, organ, etc. Inoviem removed contaminants observed in NPOT® performed on human tissues and cells, corresponding to 613 NPOT® coupled LC-MS / MS analyses. As a result, the tool can quickly highlight rare proteins that create new therapeutic targets in the dataset (Figure 5B).

[0217] Another bioinformatics resource, DAVID, was also used to detect tissue-specific expression, gene ontology, and functionally related gene group enrichment. STRING analysis (string-db.org) was used to explore network enrichment in the dataset. STRING is one of the core data resources of ELIXIR (as are Ensembl or UniProt) and contains known and predicted protein-protein interactions. Inoviem uses stringent parameters and only retains known interactions ("experimentally determined" and "curated database" interaction sources). This allows deciphering protein-protein associations in complex datasets, and further achieves DAVID pathway analysis. In addition, Reactome (reactome.org), a free, open-source, curated, and peer-reviewed pathway database, was used. This database provides intuitive bioinformatics tools for visualization, interpretation, and analysis of pathway knowledge to support findings obtained elsewhere.

[0218] In the bioinformatics pipeline, the first step of filtering consisted of removing mass spectrometry "false positives", i.e. proteins found in one replicate and with only one specific peptide. The datasets were then compared in a 2x2 matrix (144DG11 and its respective negative control) in human dermal fibroblast tissue. The next step of protein list analysis was the identification of non-specific proteins, i.e. proteins found repeatedly in all NPOT® experiments (InoPERA®). Contaminants (or "frequent hits") observed in human dermal fibroblasts were removed. The cleared protein list of the interactome thus represents potential specific targets of compound 1. Using this pipeline, 28 proteins were found to specifically interact with compound 1. The specific protein list of the compound 1 interactome was then independently analyzed by DAVID to detect tissue-specific expression, gene ontology, and enrichment of functionally related gene groups. The main canonical and disease and functional pathways underlying the interactome of compound 1 were lysosomal membranes (references: GO:0005765 and KEGG pathway hsa04142). In parallel, STRING analysis (string-db.org) was used to visualize prominent nodes and enriched networks. For this first ranking of specific proteins of the compound interactome, we did not use the signal intensity of peptides sequenced by MS, because 1) the inherent properties of that technique cannot be based on protein quantification (as opposed to, for example, classical immunoprecipitation protocols) and 2) we do not use LC-MS / MS quantification protocols (implying higher costs and longer analysis times). This unbiased analysis allowed Inoviem to classify potential related proteins and classify them according to their involvement in specific pathways or in relation to a specific disease. After this bioinformatic selection, eight proteins were found that belong to the autophagosome-autolysosome pathway (Figure 5B).The discovery of this clear and strengthened network demonstrates the overall success of the NPOT® experiment.

[0219] Computational docking analysis LAMP1 is divided into five domains: (1) residues M1-A28: signal sequence; (2) residues A29-R195: N-terminal domain; (3) residues P196-S216: linker between domains; (4) residues S217-D378: C-terminal domain; and (5) residues E379-I417: transmembrane segment. We analyzed only the N- and C-terminal domains because: 1. the signal sequence and the transmembrane segment are considered irrelevant for small molecule binding; and 2. the linker between domains is unstructured and highly glycosylated (7 out of 20 residues) and therefore too complex to model. We did not consider glycosylation at the N- and C-termini. The C- and N-terminal domains were modeled based on the known crystal structure of mouse LAMP1 C-terminal domain (PDB ID 5gv0), which is structurally very similar to the N-terminal domain. For homology modeling, the MODELLER software tool was used to generate five arbitrary models for each domain. The resulting 10 models (as well as the five gv 0 themselves) were prepared at pH 5 by the "Protein Preparation Wizard" implemented in Schrodinger 2020-2. Possible binding sites were identified by three different computational tools: SiteMap, FtSite and fPocket. Overall, 130 arbitrary sites were identified in the 3D structures of the 11 LAMP1s. Docking calculations were performed for each of the putative binding sites: from a large and diverse database of about 30 million molecules, 418 were selected as decoys according to the compound 1 applicability domain (Lipinski rule characteristics). The decoy library was narrowed down to 233 based on chemical similarity (Tanimoto coefficient >= 0.7). Docking calculations for compound 1 in a set of molecules consisting of compound 1 and these 233 decoys (prepared at pH 5) were performed for all putative binding sites in all models (130 sites in total). These calculations were performed using the Glide algorithm as implemented in Schrodinger 2020-2.According to the docking result analysis, in 18 of 130 sites, compound 1 was ranked in the top 10% in 3 grids from SiteMap, 3 grids from FtSite, and 12 grids from fPocket, 8 grids were in the C-terminal domain, and 10 were in the N-terminal domain.

[0220] We noticed that according to one of the models of the N-terminal domain (model number 4), compound 1 was ranked in the top 10% for 6 of these 18 sites. Analyzing these results, we realized that site 1 of SiteMap, site 3 of fPocket, and site 2 of FtSite point to the same pocket (residues F50-D55, N62, L67, F118, Y120-L122, T125, L127-S133, N164-V166).

[0221] We investigated the differences between the three binding modes (Figure 5E): two of the three binding modes (SiteMap and fPocket) were identical, and the portion of compound 1 in the third (FtSite) appeared to be rotated relative to the other two.

[0222] To estimate the probability of obtaining a unique binding as observed for compound 1 by pure chance, we repeated the analysis shown above for compound 1 for all 233 decoys. Only in 14 of the 234 molecules (233 decoys + compound 1) did we observe the same result as compound 1, i.e., a molecule whose binding to the pocket was predicted by the three different tools (Table 1). This indicates that the probability is relatively small (14 / 234, about 6%). Moreover, the pocket identified for compound 1 (Figure 5E) is the most common (5 matches out of 14, Table 1). This indicates that this pocket is druggable and can bind to a relatively large number of compounds, which is advantageous for improving the envisaged medicinal chemistry of compound 1. The table shows the cases where molecules fall into the same pocket according to the three different tools (for predicting binding sites). The three digits following "Site" in the first column indicate the site ranking according to SiteMap (1st), FtSite (2nd), and fPocket (3rd).

[0223] [Table 1] * The molecule binds to two different binding sites

[0224] We repeated the analysis with a less restrictive definition of the binding site and obtained similar results, i.e., 45 of 234 molecules (about 19%) were successfully docked into at least one of the predicted pockets. However, in 14 of 45 molecules, we observed binding to multiple sites, indicating promiscuity. Thus, overall, 31 of 234 molecules were successfully docked into one of the predicted sites (about 12.7%). In summary, we computationally identified a possible binding site for compound 1 in the N-terminal domain of LAMP1 and predicted with high confidence that this result was specific for compound 1, given the low probability of obtaining the same result for a decoy molecule.

[0225] Transmission electron microscope (TEM) Liver tissue was minced and fixed in a solution containing 2% paraformaldehyde, 2.5% glutaraldehyde (EM grade) in 0.1 M sodium cacodylate buffer (pH 7.3) for 2 h at room temperature followed by 24 h at 4°C. The tissue was then washed four times with sodium cacodylate and post-fixed with 1% osmium tetroxide and 1.5% potassium ferricyanide in sodium cacodylate for 1 h. The samples were then washed four times with the same buffer and dehydrated in a graded series of ethanol solutions (30, 50, 70, 80, 90, 95%) for 10 min each, then 100% ethanol three times for 20 min each. The samples were subsequently treated with two changes of propylene oxide. The samples were then infiltrated with a series of epoxy resins (25, 50, 75, 100%, respectively, for 24 h each) and polymerized in an oven at 60°C for 48 h. Blocks were sectioned using an ultramicrotome (Ultracut E, Riechert-Jung) and 80 nm sections were stained with uranyl acetate and lead citrate. Sections were viewed using a Jeol JEM 1400 Plus transmission electron microscope and images were captured using a Gatan Orius CCD camera.

[0226] Proteomics (Figure 7) Library preparation for MS analysis Cell lysates in RIPA buffer containing protease inhibitors were cleared by centrifugation and 40 μg of protein was used for protein precipitation by the chloroform / methanol method. Precipitated proteins were solubilized in 100 μl of 8 M urea, 10 mM DTT, 25 mM Tris-HCl (pH 8.0) and incubated at 22 °C for 30 min. Iodoacetamide (55 mM) was added and the sample was incubated (22 °C, in the dark) for 30 min, followed by the addition of DTT (10 mM). 50 μl of sample was transferred to a new tube and diluted by adding 7 volumes of 25 mM Tris-HCl (pH 8.0) and sequencing grade modified trypsin (Promega Corp., Madison, WI) was added (0.35 μg / sample), followed by overnight incubation at 37 °C with gentle agitation. Samples were acidified by the addition of 0.2% formic acid and desalted on a C18 homemade stage tip. Peptide concentrations were determined by absorbance at 280 nM and 0.75 μg of peptide was injected into the mass spectrometer.

[0227] NanoLC-MS / MS Analysis MS analysis was performed using a Q Exactive-HF mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) online coupled to a nanoflow UHPLC instrument, Ultimate 3000 Dionex (Thermo Fisher Scientific, Waltham, MA, USA). Peptides dissolved in 0.1% formic acid were separated on a 25 cm long C18 column (75 μm ID, 2 μm, 100 Å, Thermo PepMapRSLC) without a trap column, using an acetonitrile gradient at a flow rate of 0.3 μl / min for 120 min. Instrument settings were as previously described. A survey scan (300-1,650 m / z, target charge value 3E6, maximum ion injection time 20 ms) was acquired, followed by higher energy collisional dissociation (HCD)-based fragmentation (normalized collision energy 27). A resolution of 60,000 was used for survey scans to fragment up to 15 most abundant precursor ions dynamically selected in the "peptide preferred" profile (isolation window 1.6 m / z). MS / MS scans were acquired at a resolution of 15,000 (target value 1E5 charge, maximum ion injection time 25 ms). Dynamic exclusion was 20 s. Data were acquired using Xcalibur software (Thermo Scientific). To avoid carryover, the column was washed with 80% acetonitrile, 0.1% formic acid for 25 min between samples.

[0228] MS data analysis Mass spectral data were processed using the MaxQuant computational platform, version 1.6.14.0. Peak lists were searched against the Uniprot human FASTA sequence database from May 19, 2020, containing 49,974 entries. The search included cysteine ​​carbamidomethylation as fixed modification and N-terminal acetylation and oxidation of methionine as variable modifications, allowing up to two miscleavages. The run-to-run match option was used. Peptides of at least 7 amino acids in length were considered, and the required FDR was set to 1% at the peptide and protein level. Relative protein quantification in MaxQuant was performed using the label-free quantification (LFQ) algorithm. Statistical analysis (n = 4–7) was performed with the Perseus statistical package. Only proteins for which at least three valid LFQ values ​​were obtained in at least one sample group were accepted for statistical analysis by t-test (p < 0.05).

[0229] Example 1 Compound 1 is Gbe ys / ys Improves survival and motor deficits in mice We administered compound 1 (Figure 1A) to the APBD mouse model Gbe ys / ysCompound 1 was tested for its ability to correct the defective locomotor phenotype and short life span in mice. Compound 1 is one of 19 PG-reducing HTS hits previously discovered by the inventors. It was selected by in silico ADMET (absorption, distribution, metabolism, excretion, and toxicity) studies performed on these hits to predict which of them are safe, pharmacokinetically and pharmacodynamically favorable, and therefore worthy of further pursuit (Figure 8, compound "A"). Indeed, compounds with low ADMET scores such as "B" (Figure 8) were not effective and caused adverse effects such as wounds (Figure 9). Furthermore, safety evaluation in wild-type mice confirmed that compound 1 administered at 250 mg / kg (the highest dose possible due to solubility and DMSO toxicity issues) in 5% DMSO for 3 months did not affect the weight gain of the animals over time (Figure 10). The compound also did not cause any histopathological damage or lesions in the brain, liver, skeletal muscle, and heart after 3 months of exposure (Figure 11). After 1 and 24 hours of treatment, mice were also tested for abnormal spontaneous behavior (e.g., immobility, excessive running, stereotypy, and abnormal posture) (Irwin test). Compound 1 did not cause any side effects in these Irwin tests (Table 2).

[0230] [Table 2]

[0231] Importantly, as Figure 1B shows, treatment with Compound 1 significantly improved animal survival compared to vehicle-treated animals (log-rank test p-value < 0.000692). The extension of life span probably reflects an improvement in several parameters related to the growth capacity of the animals. The most prominent parameter in that respect is the weight of the animals. Compound 1 actually mitigated the loss of animal weight over time caused by the disease (Figure 1C). We also tested the effect of Compound 1 on various locomotor parameters every two weeks. Compound 1 improved open field performance (Figure 1D) from a relatively advanced stage of disease progression (8 months, 134 days after injection (Figure 1D)). These improvements were manifested as increased locomotion and an increased tendency to move towards the center (Figure 1E), possibly also related to improved stress and anxiety. Gbe in open field performance ys / ys Progressive deterioration of mice is associated with gait disorders in mice. Therefore, we tested the effect of Compound 1 on gait in 9-month-old mice, whose gait is severely affected. At that age, Compound 1 actually improved gait or increased stride length (Fig. 1F). The data also show that, of all the locomotor parameters tested, the most significant improvement effect was on the overall stretch reflex (Fig. 1G). The overall stretch reflex throughout the entire test period was significantly improved by Compound 1 (Fig. 1G, p<0.05) at nine specific time points (asterisk in Fig. 1G). This effect is particularly important because its patient correlate is pyramidal tetraparesis or upper motor neuron symptoms, which is one of the major neurological deficits in APBD patients. Importantly, open field performance (Figure 1E), ambulation (Figure 1F) and stretch reflex (Figure 1H) were significantly improved by Compound 1, but they were not restored to wild-type levels, indicating that Compound 1 performance, although effective, still leaves some room for future improvement.

[0232] To investigate the effect of Compound 1 on motor parameters, we started injections of Compound 1 at 4 months of age, 2 months before disease onset, assuming a favorable preventive effect. Such an effect is expected in neurodegenerative disorders such as APBD, where already dead neurons cannot be affected by treatment after onset. This assumption was verified for all parameters improved by Compound 1 - open field (Figure 1I), body weight (Figure 1J) and total stretch reflex (Figure 1K). The improving effect did not occur when administered after disease onset at 6 months of age. Of note, the stretch reflex, the parameter most affected by Compound 1, was also the only parameter improved by the compound from the advanced stage of the disease at 9 months of age (Figure 1K). The overall beneficial effect of Compound 1 can be best appreciated by animal photographs showing that treated animals had reduced kyphosis and a more tidy coat (Figure 1L-Figure 1M).

[0233] Example 2 Compound 1 reduces histopathological accumulation of polyglucosan and glycogen according to its biodistribution Since compound 1 significantly improved locomotor and survival parameters, we set out to investigate its histopathological effects. This information is important to determine whether the expected mode of action of compound 1 discovered ex vivo (reduction of polyglucosan levels in fibroblasts) also occurs in vivo, and if so, in which tissues. Brain, heart, muscle, nerve bundles (peripheral nerves), and liver tissues obtained from animals treated with compound 1 and vehicle were harvested after sacrificing the animals at 9.5 months of age. The same tissues obtained from wild-type mice were used as controls. Following diastase treatment to digest non-polyglucosan glycogen leaving polyglucosan behind, sections were stained for polyglucosan with periodic acid Schiff (PAS) reagent, counterstained with hematoxylin, and analyzed by light microscopy. The results (Figure 2A) show a significant reduction in polyglucosan levels in brain, liver, heart, and peripheral nerves, with no apparent effect on muscle polyglucosan. Biochemically determined total glycogen levels were correspondingly affected (Figure 2B). These results likely explain the observed improvements in locomotor parameters and reproduction of the animals (Figures 1A-1M).

[0234] Pharmacokinetic analysis helps to explain the effects of Compound 1 in situ, despite its inherent ability to modify polyglucosan in isolated cells. The reason is that the timing of arrival, distribution and stability in tissues are important determinants of the in situ activity of any pharmacological agent. To determine the distribution and kinetic parameters of Compound 1 in different tissues, we administered Compound 1 at 250 mg / kg by subcutaneous injection to Gbexcultarin, as was done in the efficacy experiments. ys / ysMice were treated. Mice were then sacrificed at 0, 30, 60, 90, and 210 minutes after administration, and 100 μL of serum and brain, kidney, hindlimb skeletal muscle, heart, liver, and spleen tissues were collected, homogenized, extracted, and their compound 1 levels were analyzed by liquid chromatography tandem mass spectrometry (LC-MS / MS). The results are shown in FIG. 2C. The different effects of compound 1 on glycogen and polyglucosan content in different tissues are consistent with its different distribution and residence time in each tissue. The greatest degree of polyglucosan / glycogen reduction was observed in the liver, consistent with the longest residence time / persistence of compound 1 observed in that organ (estimated half-life of over 3 hours). Heart and brain show intermediate levels of compound 1. However, these levels persisted until 60 minutes after injection, which may explain the compound 1-mediated reduction in polyglucosan and glycogen content observed in these tissues. On the other hand, muscle showed only slight accumulation of Compound 1, consistent with the compound's poor effect on muscle glycogen and polyglucosan content. Based on the sampling times used, max The time to absorption was 30 minutes for all tissues observed, indicating similar absorption rates in all these tissues. max was observed in the liver and kidney, consistent with their well-established rapid perfusion. As expected, the lowest C max was observed in the quadriceps skeletal muscle, a known poorly perfused organ.

[0235] Example 3 Compound 1 enhances carbohydrate metabolism and improves metabolic panel in vivo The effect of Compound 1 on various metabolic parameters was investigated in vivo using metabolic cages. Nutrient preference at the whole animal level is determined by the respiratory quotient (RQ, the ratio of CO2 produced to O2 consumed). A lower RQ indicates more fat burning, whereas a higher RQ indicates more carbohydrate burning. As these results (Figure 3A) show, Compound 1 increased the RQ to a level even higher than that of wild-type (wt) animals. The parallel increases in total energy expenditure (Figure 3B) and carbohydrate burning at the expense of fat burning (Figures 3C and 3D) induced by Compound 1 suggest that Compound 1 stimulates glycogen mobilization, which is associated with increased Gbe ys / ys This is of therapeutic benefit since mice store glycogen as insoluble and pathogenic polyglucosan. The stimulation of locomotor activity (Figure 3E) as well as food and water intake (Figures 3F-H) is consistent with this observation of stimulation of carbohydrate catabolism in animals affected by Compound 1. Moreover, taken together, the increased nutrient burn and food intake may improve metabolic efficiency in animals affected by Compound 1.

[0236] The present inventors further found that compound 1 is ys / ys We tested whether the hypoglycemia and hyperlipidemia observed in mice could be corrected. Such an effect would be expected from a drug capable of inducing hepatic glycogen catabolism and subsequently increasing blood glucose. ys / ys Blood biochemistry test results of mice showed that treatment with compound 1 corrected the characteristic hypoglycemia and hyperlipidemia of mice to control levels (Figure 3I). Muscle (creatine kinase) and liver (alanine transferase) functions were not affected by this treatment (Figure 3I).

[0237] Example 4 Compound 1 enhances catabolism in glycogen-overloaded APBD patient cells The RQ shift on carbohydrate catabolism observed in vivo prompted us to investigate whether carbohydrate catabolism is also upregulated in cells. To that end, we first aimed to induce a physiological glycogen overload or glycogen loading state equivalent to that found in tissues, especially since glycogen levels are highly variable among fibroblasts derived from different APBD patients (Figure 4A). We demonstrated that a glycogen load was generated by 48 h of glucose starvation followed by 24 h of sugar repletion, which likely induces accelerated glucose uptake and subsequently glycogen synthesis. This condition of starvation / repletion indeed increased intracellular glycogen levels, as shown by PAS staining (Figure 4B). Moreover, phenotypic analysis based on multiparametric high content imaging revealed that under glycogen loading conditions, cell area, nuclear intensity, and importantly, mitochondrial mass features (see box in Figure 4C) deviated from healthy controls (HC) more than in glucose-starved only cells. Therefore, we chose this glycogen loading condition to analyze catabolism at the cellular level using an ATP rate assay (Agilent's Seahorse ATP rate assay). These results (Figure 4D) show that at the cellular level, 144DG11 A increased not only overall ATP production, but also the relative contribution of glycolytic ATP production at the expense of mitochondrial (OxPhos) ATP production. This phenomenon was observed in both HC and APBD patient skin fibroblasts. Acute assay supplementation with 144DG11 was more effective in increasing glycolytic contribution to ATP production than 48 h pretreatment with the compound. These results suggest that glucose derived from 144DG11-mediated enhanced carbohydrate catabolism is available for ATP production.

[0238] Example 5 Compound 1 binds to the lysosomal membrane protein LAMP1 We investigated the mechanism of action of 144DG11. To that end, we first decided to identify its molecular targets. We applied the nematic protein assembly technique (NPOT, Inoviem, Ltd.) to homogenates of APBD patient fibroblasts. The NPOT analysis found uniquely generated protein heteroassemblies near 144DG11 only when added to the cell homogenate (Figure 5A). The next step in this analysis identified the interactome of protein targets that interact with 144DG11 in APBD patient fibroblasts. Interestingly, as revealed by Inoviem's ​​gene ontology analysis based on several bioinformatics tools, the proteins in the heteroassemblies that interact with 144DG11 in APBD patient fibroblasts are autophagic or lysosomal proteins (Figure 5B). Furthermore, we tested the specific interaction of 144DG11 with six of the eight targets discovered by NPOT by cellular thermal shift assays. These results (Figure 5C) suggest that LAMP1, but not other protein targets, directly interacts with 144DG11. This finding concerns a novel pathogenicity hypothesis linking cellular glycogen excess with glycogen transport to lysosomes via starch-binding domain containing protein 1. To validate the interaction of 144DG11 with LAMP1, we used surface plasmon resonance (SPR) technology. The SPR data (Figure 5D) show specific and dose-dependent binding of 144DG11 to the luminal part of LAMP1 only at lysosomal pH 4.5-5, but not at cytoplasmic pH 7, with some binding starting at an intermediate pH of 6. Taken together, these results constitute strong and satisfying evidence that the specific target of 144DG11 is the type 1 lysosomal protein LAMP1, which is widely used as a lysosomal marker and a known regulator of lysosomal function. However, the apparent K D is relatively high (6.3 mM), which is probably due to the slow K onThe slow association rate is explained by the inhibition of diffusion of 144DG11 by bulky oligosaccharides at the glycosylation site (Figure 5D, rate of association at pH 4.5). We hypothesized that this slow association rate could be explained by inhibition of diffusion of 144DG11 by bulky oligosaccharides at the glycosylation site. Therefore, we repeated the SPR experiment with chemically deglycosylated luminal LAMP1 domain. However, deglycosylated LAMP1 did not bind to 144DG11, likely due to the significant structural changes induced by deglycosylation (Figure 12A-B), and therefore we could not test whether oligosaccharide steric hindrance affects the binding kinetics of 144DG11 to LAMP1. We further investigated 144DG11 binding to LAMP1 by structure-based computer docking. In searching for a putative binding site of compound 1 in LAMP1, we analyzed the N-terminal and C-terminal subdomains of its luminal domain (residues A29-R195 and S217-D378, respectively), which have similar topologies. These domains were modeled and computationally docked to compound 1 against a decoy at intralysosomal pH 5 based on the known crystal structure of mouse LAMP1 C-terminal domain (PDB ID 5gv0). Figure 5E shows the compound 1 LAMP1 binding pocket (residues F50-D55, N62, L67, F118, Y120-L122, T125, L127-S133, N164-V166) predicted by three different algorithms: SiteMap, FtSite, and fPocket. It is very rare for the same binding site to be predicted by three different programs, and therefore this strongly suggests that compound 1 binds to a specific site at the N-terminus of LAMP-1. As seen in Figure 5E, the Asn-linked oligosaccharides face away from the predicted compound 1 binding site and are therefore not expected to directly interfere with its binding, however, they may still affect the diffusion of compound 1.

[0239] Example 6 Compound 1 enhances LAMP1 knockdown-induced autolysosomal degradation and glycogen catabolism Compound 1 increased autophagic flux in APBD primary fibroblasts. This is demonstrated by the increased sensitivity to lysosomal inhibitors in the presence of compound 1. As seen in Figure 6A, lysosomal inhibitors increase the LC3ii / LC3i ratio in cells treated with compound 1 more than in untreated cells (autophagosis stopped). The increase in autophagic flux by compound 1 is also shown by decreasing the levels of the autophagic substrate p62 (Figure 6A). Furthermore, the APBD modeled Gbe ys / ys Transmission electron microscopy analysis of mouse liver sections demonstrates a reduction in lysosomal glycogen following treatment with Compound 1 (FIG. 6B).

[0240] To investigate the functional significance of the interaction between compound 1 and LAMP1, we knocked down the latter using a lentiviral vector carrying a GFP-tagged shRNA against LAMP1. Since LAMP1 knockdown (KD) becomes cytotoxic after 24 hours of expression (or 96 hours after lentiviral infection), the LAMP1-KD experiments in Figures 6C-6D were performed under 24-hour serum starvation conditions without glucose supplementation (Figures 4A-4D) to induce autophagy and maintain cell viability. We expected that LAMP1-KD would neutralize the effect of compound 1, which is purportedly mediated by its interaction with LAMP1. Surprisingly, however, supplementation of LAMP1-knockdown cells with compound 1 enhanced the knockdown effect, i.e., the autophagic flux enhanced by LAMP1-KD was further enhanced by the LAMP1-interacting compound 1 (Figure 6C). The observation that compound 1 enhances the LAMP1-KD effect suggests that the interaction of compound 1 with LAMP1 is inhibitory, similar to many other small molecule-protein interactions. To further test whether LAMP1-KD and compound 1 enhance autophagic flux by improving lysosomal function, we quantified lysosomal acidification using the pH ratiometric dye Lysosensor™, which quantifies pH based on yellow / blue emission ratio. These results show that both LAMP1-KD and compound 1 treatment (in GFP and LAMP1-KD APBD cells) resulted in lysosomal acidification, but LAMP1-KD resulted in more lysosomal acidification. We show overall cellular acidification as an increase in 375 nM excitation yellow / blue emission by flow cytometry (Figure 6D, top panel), and show that this acidification is associated with brighter yellow fluorescence in lysosomes by confocal microscopy (Figure 6D, middle panel). Importantly, LAMP knockdown reduced cellular glycogen levels as shown by PAS staining, and this effect was slightly enhanced by compound 1 in APBD fibroblasts transduced with both GFP control and shLAMP1-GFP lentiviruses (Figure 6D, lower panel).

[0241] To test the effect of LAMP1-KD and Compound 1 on nutrient utilization, we again used ATP rate assays in LAMP1-KD and control APBD fibroblasts treated acutely or chronically with Compound 1 (Figure 4A-D). These results (Figure 6E) show that starvation was more limiting (lower overall ATP production) in LAMP1-KD (LAMP1-KD-S UT v LAMP1-KD+S UT, p<0.0001) than in GFP-transduced controls (Control UT-S v Control UT+S, p<0.36). In LAMP1-KD cells, starvation also increased the relative contribution of respiration to ATP production (78% in LAMP1-KD-S UT v 48% in LAMP 1-KD+S UT (orange bars)). These observations are consistent with the higher ATP production efficiency of respiration compared to glycolysis, as suggested by their higher overall ATP production rate in basal conditions, and possibly the higher ATP demand of LAMP-KD compared to control cells (cf. LAMP1-KD+SUT vs. Control+SUT, p<0.01). The effect of Compound 1 on LAMP1-KD and control cells was in accordance with its selective increase in catabolic (ATP-generating) autophagic flux in LAMP1-KD cells compared to control cells (Figure 6C). In non-starved conditions, supplementation with Compound 1 significantly increased total and respiratory ATP production in LAMP1-KD cells (cf. LAMP1-KD+S UT vs. LAMP1-KD+S Chronic (p<0.03 for total, p<0.0008 for respiratory) and LAMP1-D+S Acute (p<0.01 for total and respiratory)), but only slightly affected ATP production in control cells, rather sharply decreasing it (cf. Control UT+S vs. Control+S Chronic (p<0.1) and Control UT+S Acute (p<0.0008 for decrease)). Under starved conditions, control cells only increased respiratory ATP production in response to the transient effect of acutely supplemented Compound 1 (cf. Control UT-S vs. Control-S Acute, p<0.004). No significant effect of long-term supplementation with Compound 1 was observed in control cells cf. Control UT-S vs. Control-S chronic, p<0.3).In contrast, starved LAMP1-KD cells increased both respiratory and glycolytic ATP in response to acute supplementation of compound 1, likely reflecting short-term diversion of glucose derived from glycogenolysis to glycolysis (cf. LAMP1-KD-S UT vs. LAMP1-KD-S acute (p<0.0003 for glycoATP, p<0.003 for mitoATP). In response to chronically administered compound 1, only respiratory ATP production was increased in KD cells (cf. LAMP1-KD-S UT vs. LAMP-LAMP1-KD-S chronic (p<0.15 for glycoATP, p<0.0002 for mitoATP).

[0242] Example 7 Compound 1 restores abnormal mitochondrial and lysosomal features at the cellular level Since we have shown that the mode of action of compound 1 involves lysosomal catabolism that increases ATP production, we decided to investigate whether the cellular features modulated by compound 1 are related to its catabolic effect. As a first step, we needed a holistic and feature-specific classification method that would allow us to quantify the differences between APBD and HC cells and thus estimate the restorative effect of compound 1 on APBD cells. Using an InCell2200 high content image analyzer, we performed a complete multiparametric analysis of APBD and age- and sex-matched HC dermal fibroblasts. This image-based phenotyping (IBP) campaign included 45 independent cellular parameters encompassing a broad cellular morphological spectrum. Analyzing dermal fibroblasts from 17 APBD patients and 5 HCs, we demonstrated that dermal fibroblasts from APBD patients are phenotypically distinguishable from HC dermal fibroblasts (Figure 7A). Once IBP was established as an informative and sensitive classification tool, we tested the effect of compound 1 on the IBP signature. The analysis (Figure 7B, upper panel), limited to four color channels and thus excluding lysosomal markers, analyzed separately (Figure 7C), shows that compound 1 mainly affected nuclear and mitochondrial membrane potential (TMRE) parameters, which were among the features most affected by the disease phenotype. As expected, this effect was more pronounced (higher -logP values) when compound 1-treated APBD fibroblasts were compared to untreated HC fibroblasts, a comparison more relevant for the clinical situation. As demonstrated for other features (Figure 4D and Figure 6C-E), treatment with compound 1 alone may have a similar effect on both diseased and healthy cells, thus bringing the two phenotypes closer to each other in treated cells and partially masking the effect of this compound on APBD vs. HC.The bottom panel of FIG. 7B indeed reveals that for most features, compound 1 caused the same trends (increase or decrease) in both diseased and healthy cells (note that APBD / compound 1 (stippled bar) should be compared with APBD (blank bar) and HC / compound 1 (black bar) should be compared with the horizontal line). Compound 1 also reduced lysosomal size in APBD cells (FIG. 7C), which may be related to the improvement of autophagic flux (FIG. 6) and lysosomal function as observed in healthy cells compared with lysosomal disordered cells. Furthermore, compound 1 also hyperpolarized mitochondrial membrane potential (MMP) and depolarized by the disease state in APBD (FIG. 7B), which is probably in accordance with the increased nutrient supply of mitochondria due to enhanced autophagic catabolism.

[0243] To validate the image-based analysis of disease states and cellular features regulated by compound 1, we analyzed the effect of disease and treatment on protein expression. As shown in Figure 7D, of the 2,898 proteins analyzed, 12.2% were upregulated and 6.8% were downregulated in APBD patients under 48 h starvation compared to HC cells. As an important control, GBE was indeed downregulated in APBD cells (Figure 7D). When starvation was followed by glucose supplementation (glycogen loading, Figures 4A-4D), only 6% of proteins were upregulated and 5% were downregulated, perhaps suggesting that a more specific subset of proteins is required to manage the excessive glycogen load. For example, autophagy proteins (Fyco1, Rab12, Rab7A, PIP4K2B, SQSTM1, and SNAP29) were only upregulated in APBD cells after glycogen loading. We then investigated the proteomic effects of compound 1 in starved (48 h starvation) and glycogen-overloaded (48 h starvation / 24 h glucose) APBD cells, which only modified 1.7% and 1.3% of all proteins, respectively. The apparent corrective effect of compound 1 could be revealed by proteins that were down- or up-regulated by the APBD disease state, which were conversely up- or down-regulated by compound 1 (Figure 7E). The discovered proteins (49 up-regulated, 39 down-regulated, Figure 7E) were analyzed by the DAVID functional annotation tool according to the cellular component category that contained the largest number of proteins. Proteins up-regulated by compound 1 belonged to eight significant gene ontology (GO) terms, which included lysosomal, secretory pathway and oxidative phosphorylation proteins (Figure 7F, left panel) according to the cellular features regulated by the compound (Figure 7B).

[0244] Interestingly, the proteins downregulated by APBD and upregulated ("corrected") by compound 1 were the lysosomal glycosylation enzymes iduronidase and phosphomannomutase 2 under glycogen loading, whereas under starvation they were the nucleic acid binding proteins GRSF1 and HNRPCL1, which are apparently not directly related to glycogen and lysosomal catabolism. The lipogenic protein HSD17B12 was decreased by APBD and induced by compound 1 under both conditions. Proteins downregulated by compound 1 belonged to four GO terms, including secretory pathway and macromolecular complexes (Figure 7F, right panel). Proteins increased by APBD and contrastingly decreased by compound 1 belonged to lysosomal sorting (VPS16) and carbohydrate biosynthesis (NANS) in starved cells, and to transcription (RUBL1), signal transduction (STAM2) and pH regulation (SLC9A1) in glycogen-overloaded cells. Interestingly, Na + / H + Pharmacological inhibition of the antiporter SLC9A1 induces autophagic flux in cardiomyocytes, similar to its downregulation in APBD fibroblasts by compound 1 (Figure 6). A protein downregulated by compound 1 in both starvation and glycogen-loaded conditions is the retrograde transport regulator VPS51, which is also involved in lysosomal sorting. In summary, the APBD-correcting effect of compound 1 is at least partially related to lysosomal function, whose regulation by this compound has been well established by the inventors (Figures 5-6).

[0245] This experiment shows that the hit compound 1 discovered by HTS can treat APBD in in vivo and ex vivo models. After treatment with compound 1, we observed improvements in locomotion, survival, and histological parameters (Figures 1-2). Because APBD is caused by indigestible carbohydrates, these improvements suggested that compound 1 affects carbohydrate metabolism, which in turn prompted us to perform in vivo metabolic experiments (Figures 3A-3I). This is the first in vivo metabolic experiment in a GSD animal model. Because APBD mice store glycogen as insoluble polyglucosan, we used metabolic cages to test whether compound 1 could affect the ability of these animals to use alternative nutrients (fat) instead of mobilizing glycogen. However, the increase in RQ induced by compound 1 suggested that the treated animals actually increased carbohydrate burning instead of using fat, or that compound 1 increased carbohydrate catabolism. This conclusion was supported by the increases in total energy expenditure, ambulatory activity, food intake, and water intake induced by Compound 1, all consistent with a catabolic stimulus. ys / ys Mice and APBD patients store glycogen as insoluble and pathogenic polyglucosan, so its catabolism constitutes a therapeutic advantage. Glycogen catabolism is also a preferred therapeutic method for the following reasons. Theoretically, therapeutic approaches to APBD should target either PG formation or the degradation of preformed PG or glycogen. PG formation depends on the balance between GYS and GBE activity - the higher the GYS / GBE activity ratio, the more elongated and less branched soluble glycogen is formed, which preferentially forms PG compared to shorter chains. On the other hand, the degradation of existing PG and glycogen (PG precursor) by compound 1 is a more direct target and is expected to be more effective than the inhibition of de novo PG formation by the GYS inhibitor guaiacol, which preemptively avoids harmful PG being made. Indeed, in studies in LD model mice, it was shown that conditional GYS knockdown after disease onset fails to eliminate existing harmful Lafora PG bodies.

[0246] A key challenge in drug discovery is the determination of the relevant targets and mechanisms of action of drug candidates. Towards that end, we have applied here Inoviem's ​​NPOT® protein target identification approach. Recognized as a major tool for identifying protein targets of small molecules and having identified several therapeutically relevant targets, this technique identifies compound-target interactions within the natural physiological environment of cells. This means that what is identified is not the target itself as in other techniques, but the primary target with its signaling pathway or functional quaternary network. Determination of the cellular pathway regulated by a test compound, as is done for compound 1, is important for putatively formulating other drugs into the same pathway, which could significantly improve therapeutic efficacy in time in the clinic. Moreover, NPOT® can also confirm the specificity of target binding by filtering out miscellaneous binders and excluding binding to negative controls (in this case, negative compounds in HTS) and endogenous ligands (Figure 5A). Nevertheless, by these criteria, the binding of compound 1 to LAMP1 and, through it, to its functional quaternary network was specific (Figure 5B) and showed a dose-response and lysosomal pH-dependence in SPR validation (Figure 5D), whereas its apparent LAMP1-binding K D is relatively high (6.3 mM), which at first glance could be an obstacle to its clinical application. This issue can be addressed as follows: 1. A pharmacologically relevant finding is that compound 1 specifically interacted with the lysosome-autophagosome interactome (Figure 5B), and that it was not toxic (Figures 8-11, Table 2).

[0247] This finding supports the finding that low affinity (high K D) to exclude non-specific interactions with putative off-targets, which is a major concern for ligands. Conventional approaches to improve the affinity of low-affinity drug candidates are based on medicinal chemistry. In GSD, such approaches were used to increase the affinity of GYS inhibitors. However, in contrast to GYS, whose reduction is relatively tolerable, LAMP proteins belong to the housekeeping autolysosomal machinery (Figure 5B), and their inhibition may impair perinatal survival, similar to, for example, LAMP1-KD without compensatory elevation of LAMP2. Thus, high-affinity LAMP1 inhibitors may be as toxic as LAMP1-KD for APBD fibroblasts (Figure 6), and the low affinity of the LAMP1 inhibitor we discovered, compound 1, may actually constitute a clinical benefit by relieving the repression of a household function essential for cell proliferation. Furthermore, computational analysis indicates that the compound 1 binding pocket of LAMP1 is highly druggable (Figure 5E), meaning that medicinal chemistry analysis is expected to discover various alternatives to compound 1 that could improve its efficacy.

[0248] The discovery of LAMP1 (Figure 5B), containing hetero-assemblies as functional network targets rather than single proteins, opens up therapies based on autophagy regulation and indeed expands the landscape of therapeutic targets. The autolysosomal network was discovered not only in Figure 5B but also by our multi-feature imaging analysis, together with bioenergetic parameters that may be modified by autophagy-related changes in nutrient availability (Figures 7B and 7C). Further support for the relevance of this pathway as a target of Compound 1 comes from the proteomic data (Figures 7D-7F) and the actual enhancement of autophagic flux by Compound 1 in cells (Figure 6).

[0249] Mechanistically, LAMP1 is a type I lysosomal membrane protein that, together with LAMP2, plays a central role in lysosomal integrity and function. As a result, LAMP1 is also important for lysosomal involvement in the autophagic process, but LAMP2 is more important. Thus, LAMP1 knockdown is often associated with reduced autophagy. However, consistent with the present results, other studies have shown that LAMP1-KD actually increased autophagic function, which was also shown for another transmembrane lysosomal protein, TMEM192. These apparent discrepancies likely depend on the cell type, assay conditions, and even the definition of autophagic flux, since autophagic flux is not always defined by its sensitivity to lysosomal inhibitors. To predict the molecular mechanism of action of compound 1 on LAMP1, we used computational chemistry. Computational results predict that the binding site of compound 1 is located at the LAMP1:LAMP1 interaction interface (Figure 13A), located in the N-terminal domain, suggesting that the compound inhibits LAMP1-LAMP1 interactions. According to experimental data, truncation of the N-terminal domain of LAMP1 impairs LAMP1 / LAMP1 and LAMP1 / LAMP2 assembly, while truncation of the more mobile N-terminal domain of LAMP2 has the opposite effect (Figure 13B). Therefore, we can assume that the LAMP1 N-terminal domain promotes LAMP1 / LAMP1 and LAMP1 / LAMP2 interactions, and inhibition of LAMP1 / LAMP1 or LAMP1 / LAMP2 interactions at the N-terminal domain by compound 1 reduces LAMP1 effective lysosomal membrane density. Therefore, we can assume that compound 1 treatment is equivalent to LAMP1-KD, which may explain its enhancement of LAMP1-KD effect. The slight increase in the level of LAMP1 induced by compound 1 (1.2-fold) probably reflects binding-mediated stabilization (Figure 5C), and probably does not significantly counteract compound 1-mediated reduction in membrane density. We hypothesize that compound 1-mediated decrease in LAMP1 membrane density increases glycophagy due to the demonstrated increase in LAMP2 in lysosomal membranes upon LAMP1-KD.LAMP2 was observed to enhance autophagosome-lysosome fusion (and thus autophagic flux) through interaction with the autophagosomal peripheral protein GORASP2. Alternatively, lysosomal membrane spacing by LAMP1-KD / compound 1 may allow glycogen import (and consequent degradation) into lysosomes by STBD1 protein. Importantly, lysosomal glycogen degradation occurs in parallel with its cytoplasmic degradation, especially in the GSDIV mouse model, which also models APBD in mice, where overexpression of the lysosomal glycogen degrading enzyme α-glucosidase corrects pathology.

[0250] Taken together, this study demonstrates that compound 1 is a novel catabolic compound that can degrade PG and excess stored glycogen by activating the autophagy pathway. This study forms the basis for the clinical use of compound 1 in the treatment of APBD patients who currently have no therapeutic options. Furthermore, it positions compound 1 as a lead compound for treating other GSDs via a safe reduction in glycogen surcharge.

[0251] Example 8 Therapeutic properties of 144DG11 compound 144DG11 can activate autophagy in the lysosomal storage disease (LSD) Pompe disease (PD) where autophagy is disturbed (Figure 15). The data show that in fibroblasts derived from PD patients, the ratio of lipidated autophagy marker LC3 (LC3II) to non-lipidated LC3 (LC3I) LC3 was increased by the autolysosomal inhibitor vinblastine. This ratio serves as the most accepted marker for autophagy and autophagic flux. Sensitivity to vinblastine (i.e., increased LC3II / LC3I ratio indicating accumulation of non-degraded autophagic substrates) was increased by treating serum-starved PD patient-derived fibroblasts with 50 μM 144DG11 for 24 hours. These observations indicate that 144DG11 can activate autophagy in typical LSD PD where autophagy is disturbed, as in GSD APBD. This strongly suggests that 144DG11 also has therapeutic potential for treating LSDs in which disturbance of autophagy is a major pathogenic factor.

[0252] 144DG11 (24 h, 50 μM) can reduce glycogen in PD patient-derived fibroblasts, as also demonstrated in APBD patient-derived fibroblasts (FIG. 16).

[0253] These results (Figure 17) show that 144DG11 increased the relative contribution of overall ATP production as well as glycolytic ATP production at the expense of mitochondrial (OxPhos) ATP production. This phenomenon was selectively observed in PD but not in healthy control (HC) primary skin fibroblasts. Assay supplementation with 144DG11 was more effective in increasing glycolytic contribution to ATP production than 24 h pretreatment with the compound, whose effect on ATP production was not significant, likely due to cellular adaptation. These results suggest that glucose derived from 144DG11-mediated enhanced autophagic catabolism is available for ATP production. These observations are consistent with those made in APBD fibroblasts, thus indicating that 144DG11 is a versatile catabolic enhancer with broad therapeutic potential in common storage disorders.

[0254] The results in Figure 17 suggest that glucose derived from enhanced carbohydrate catabolism is available for ATP production, supporting the future development of 144DG11 as an effective anti-obesity drug. We expect that 144DG11 will be more effective in obesity induced by a Western diet (high fat / high carbohydrate). The observation in GSD4 (Kakhlon et al., (2021)) and GSD3 (Figure 18) that 144DG11 can reduce plasma triglyceride levels strongly suggests that 144DG11 can be developed into an effective anti-obesity therapy.

[0255] As shown by 144DG11-mediated reduction in total LC3 and p62, the compound induced autophagy in brain microglia derived from Alzheimer's disease (AD) model mice (Figure 19). This observation is important because it demonstrates the therapeutic potential of 144DG11 for treating AD. Microglia are the most pro-inflammatory tissue in the brain and are therefore currently at the center of innovative therapeutic research for AD. Furthermore, because neuroinflammation is now accepted as a major pathogenic factor in AD and activation of microglial autophagy and mitophagy is a major therapeutic approach (see, for example, Eshraghi et al., (2021)), 144DG11 is a promising candidate for AD treatment.

[0256] 144DG11 also induced autophagy in primary human non-small cell lung cancer (NSCLC) cells (Figure 20). Induction of autophagy has demonstrated therapeutic value in NSCLC (see, for example, Wang et al., (2021)). Of note, 144DG11 did not reduce glycogen levels in either microglia or NSCLC cells, suggesting that glycogen is not degraded by the autolysosomal pathway that can be modified by 144DG11 in these cells. This lack of effect on glycogen also suggests that glycogen accumulation may not be pathogenic in these cells. However, autophagic clearance of harmful inclusions by 144DG11 is likely beneficial in many different disease states as demonstrated herein.

[0257] NAD+ and NADH are important precursors for the electron transport chain, TCA cycle, glycolysis, amino acid synthesis, fatty acid synthesis, and nucleotide synthesis. The NAD+ / NADH ratio reports the overall degree of catabolism and the balance between glycolysis and OxPhos. An increase in the NAD+ / NADH ratio implies accelerated electron flow in the mitochondrial electron transport chain (note that this is not mitochondrial ATP production) and accelerated glycolysis to better manage metabolic demands. Furthermore, Sirt1 induction, which is often associated with an increase in the NAD+ / NADH ratio, is a well-documented innovative anti-aging, calorie restriction mimicking, and anti-cancer treatment (see, for example, Hyun et al., (2020)). Thus, the results in Gsd1a cells showing the induction of the NAD+ / NADH ratio as well as Sirt1 (Figure 21) indicate that 144DG11 is a promising therapy for a number of different metabolic disorders, age-related complications, and cancer. In addition, 144DG11 downregulated p62 and increased autophagic flux in Gsd1a cells, as demonstrated in GSD4 and PD cells.

[0258] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0259] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. Furthermore, citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. To the extent section headings are used, they should not be construed as necessarily limiting.

Claims

1. A pharmaceutical composition for use in the prevention or treatment of a lysosomal storage-associated disease selected from the group consisting of mucopolysaccharidoses (MPS), Gaucher disease, Fabry disease, Tay-Sachs disease, aspartylglucosaminuria, GM1-gangliosidosis, Krabbe disease, metachromatic leukodystrophy, Sandhoff disease, mucolibidoses, Niemann-Pick disease, free sialic acid storage disorders, multiple sulfatase deficiency (MSD), sphingolipidosis, Farber disease, GM2 gangliosidosis, lysosomal acid lipase deficiency, neuronal ceroid lipofuscinosis, Wolman disease, cholesterol ester storage disease, galactosialidosis, alpha-mannosidosis, beta-mannosidosis, fucososis, Salla disease, Schindler disease, cystinosis, and pycnodysostosis, comprising a compound and a pharma- ceutical acceptable salt, isomer, or tautomer thereof, wherein the compound is represented by formula I: 【Chemistry 1】 (In the formula, 【Chemistry 2】 represents a single bond or a double bond, n and m each independently represent an integer ranging from 1 to 3; R and R １ each independently represents hydrogen or is absent; R ３ , R ４ , R ５ , R ６ , R ７ and R ８ are each independently selected from the group consisting of hydrogen or substituted or unsubstituted alkyl, cycloalkyl, alkoxy, hydroxy, thiohydroxy, thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halo, trihalomethyl, cyano, amido, carboxy, sulfonyl, sulfoxy, sulfinyl, and sulfonamido. A pharmaceutical composition represented by the formula:

2. The pharmaceutical composition according to claim 1, wherein the MPS is selected from the group consisting of MPS I-H type (Hurler syndrome), MPS I-S type (Scheie syndrome), MPS I-HS type (Hurler-Scheie syndrome), MPS II type (Hunter syndrome), MPS III type (Sanfilippo syndrome), MPS IV type (Morquio syndrome), MPS VI type (Maroteaux-Lamy syndrome), MPS VII type (Sly syndrome), and MPS IX type (hyaluronidase deficiency).

3. A pharmaceutical composition for use in the treatment of a psychiatric disorder associated with autophagy dysregulation, comprising a compound and a pharma- ceutically acceptable salt, isomer, or tautomer thereof, wherein said compound has formula I: 【Chemistry 3】 (In the formula, 【Chemistry 4】 represents a single bond or a double bond, n and m each independently represent an integer ranging from 1 to 3; R and R 1 each independently represent hydrogen or are absent; R 3 , R 4 , R 5 , R 6 , R 7 and R 8 each independently represent hydrogen or are selected from the group consisting of substituted or unsubstituted alkyl, cycloalkyl, alkoxy, hydroxy, thiohydroxy, thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halo, trihalomethyl, cyano, amido, carboxy, sulfonyl, sulfoxy, sulfinyl, and sulfonamido. A pharmaceutical composition represented by the formula:

4. The pharmaceutical composition described in claim 3, wherein the psychiatric disorder is selected from schizophrenia and bipolar disorder.

5. A pharmaceutical composition for use in the treatment of lung cancer, comprising a compound and a pharma- ceutically acceptable salt, isomer, or tautomer thereof, wherein the compound has formula I: 【Chemistry 5】 (In the formula, 【Chemistry 6】 represents a single bond or a double bond, n and m each independently represent an integer ranging from 1 to 3; R and R 1 each independently represent hydrogen or are absent; R 3 , R 4 , R 5 , R 6 , R 7 and R 8 each independently represent hydrogen or are selected from the group consisting of substituted or unsubstituted alkyl, cycloalkyl, alkoxy, hydroxy, thiohydroxy, thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halo, trihalomethyl, cyano, amido, carboxy, sulfonyl, sulfoxy, sulfinyl, and sulfonamido. A pharmaceutical composition represented by the formula:

6. The pharmaceutical composition described in claim 5, wherein the lung cancer is non-small cell lung cancer (NSCLC).

7. A pharmaceutical composition for use in treating an ocular disorder associated with autophagy dysregulation, comprising a compound and a pharma- ceutically acceptable salt, isomer, or tautomer thereof, wherein the compound has formula I: 【Chemistry 7】 (In the formula, 【Chemistry 8】 represents a single bond or a double bond, n and m each independently represent an integer ranging from 1 to 3; R and R 1 each independently represent hydrogen or are absent; R 3 , R 4 , R 5 , R 6 , R 7 and R 8 each independently represent hydrogen or are selected from the group consisting of substituted or unsubstituted alkyl, cycloalkyl, alkoxy, hydroxy, thiohydroxy, thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halo, trihalomethyl, cyano, amido, carboxy, sulfonyl, sulfoxy, sulfinyl, and sulfonamido. A pharmaceutical composition represented by the formula:

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein n and m are 1.

9. R ２ , R ７ and R ８ The pharmaceutical composition according to any one of claims 1 to 8, wherein represents methyl.

10. The compound is 【Chemistry 9】 The pharmaceutical composition according to any one of claims 1 to 9, wherein the compound is selected from the group consisting of:

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