Lysosome-associated membrane protein-targeting compounds and their use

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 of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 149,730, filed on 16 February 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] The present invention relates to the field of preventing and treating certain diseases or disorders associated with lysosomal accumulation, polyglucosane accumulation or abnormal glycogen accumulation, abnormal protein accumulation, and autophagy misregulation-related disorders, as well as to the field of screening for agents that 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 disorders (LSDs) represent a diverse group of dozens of rare genetic disorders characterized by the accumulation of undigested or partially digested macromolecules, ultimately leading to cellular dysfunction and clinical abnormalities. LSDs result from gene mutations in one or more lysosomal enzymes, leading to the accumulation of enzyme substrates within lysosomes. This can result in organ enlargement, connective tissue and ocular pathologies, and central nervous system dysfunction.

[0004] Neurological disorders and neurodegenerative processes are associated with lysosomal dysfunction and are the main features of most LSDs. Neuropathology can occur in multiple brain regions (e.g., thalamus, cortex, hippocampus, and cerebellum) and involves unique temporal and spatial changes, often accompanied by initial region-specific neurodegeneration and inflammation. For example, Purkinje neurons degenerate in many of these disorders, leading to cerebellar ataxia.

[0005] Glycogen is a branched polysaccharide with a molecular weight of 9 to 10 million daltons. An average glycogen molecule contains approximately 55,000 glucose residues linked by α-1,4 glycosidic bonds (92%) and α-1,6 glycosidic bonds (8%). Glycogen synthesis is catalyzed by two enzymes: (i) glycogen synthase, which "links" glucose units to form a linear chain, and (ii) glycogen branching enzyme (GBE), which links new, short branches of glucose units into a linear chain via α-1,6 glycosidic bonds. Glycogen is primarily stored in the liver and muscles, where it becomes an energy store that can be rapidly mobilized. The most common disorder of glycogen metabolism is seen in diabetes, where abnormal amounts of insulin or abnormal insulin responses lead to the accumulation or depletion of hepatic 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 axonal leukodystrophy between the ages of 45 and 50. 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 complex disease. APBD is caused by a deficiency in glycogen branching enzyme (GBE), which results in insoluble glycogen (polyglucosan, PG) due to insufficient branching. This PG precipitates, aggregates, and accumulates to form PG bodies (PB). Because PB aggregates when out of solution, it cannot be digested by glycogen phosphorylase. The aggregated bodies lead to liver failure and death in childhood (Anderson's disease; GSD type IV). The milder the GBE mutation (e.g., p.Y329S in APBD), the smaller the PB becomes, which simply accumulates on the sides of cells without interfering with hepatocytes and most other cell types. However, in neurons and astrocytes, over time, PB can block narrow regions of the axon and process, leading to APBD.

[0007] Effective treatments for APBD have not been found at present, and despite the urgent need, APBD is a larger group of GSD. GSD is a diverse group consisting of 15 incurable diseases, with a total frequency of 1 in 20,000 to 43,000 people. All GSDs, from pediatric liver disorders such as GSD1 to juvenile myoclonic epilepsy such as Lafora disease (LD) and adult progressive neurodegenerative disorders such as APBD, are currently incurable. Therapies, drugs, and improved correlated diagnostic methods for lysosomal storage diseases and glycogenoses are still needed.

Summary of the Invention

[0008] In one aspect of the present invention, a pharmaceutical composition for use in preventing or treating a disease or disorder selected from lysosomal storage-related diseases and autophagy dysregulation-related diseases, comprising a compound and its pharmaceutically acceptable salt, isomer or tautomer, wherein the compound has the formula I:

[0009]

Chemical formula

[0010]

Chemical formula

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

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

[0013] In some embodiments, the compound is

[0014] [Chemical formula] selected from or both.

[0015] In some embodiments, the lysosomal storage-related diseases are selected from the group consisting of Gaucher disease, Fabry disease, Tay-Sachs disease, mucopolysaccharidosis (MPS) disease, aspartylglucosaminuria, GM1-gangliosidosis, Krabbe disease (globoid cell leukodystrophy or galactosylceramide lipidoses), metachromatic, leukodystrophy, Sandhoff disease, mucolipidosis type II (I-cell disease), mucolipidosis type IIIA (pseudo-Hurler polydystrophy), Niemann-Pick disease type C2 and type C1, Danon disease, free sialic acid storage disorder, mucolipidosis type IV, and multiple sulfatase deficiency (MSD), metabolic disorders, obesity, type II diabetes, and insulin resistance.

[0016] In some embodiments, autophagy misregistration-related disorders are characterized by decreased or misregulated autophagy activity. In some embodiments, autophagy misregistration-related disorders characterized by decreased or misregulated autophagy activity are selected from the group consisting of Alzheimer's disease and cancers associated with decreased autophagy activity.

[0017] Another aspect of the present invention provides a method for treating or preventing the development of a disease or disorder selected from lysosomal storage-related diseases and autophagy misregulation-related diseases in a subject requiring such treatment, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention to the subject.

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

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

[0020] In some embodiments, the drug is intended for use in the prevention or treatment of diseases or disorders related to lysosome storage, polyglucosane accumulation, or abnormal glycogen storage. In some embodiments, the drug is intended for use in the prevention or treatment of autophagy misregulation-related diseases.

[0021] In some embodiments, the disease or disorder is selected from the group consisting of glycogen storage disease (GSD), adult polyglucosane body disease (APBD), and Lafora disease, Gaucher disease, Fabry disease, Tay-Sachs disease, mucopolysaccharidosis (MPS) disease, aspartylglucosamiuria, GML-gangliosidosis, Krabbe disease (globoid cell leukodystrophy or galactosylceramidridosis), metachromatic leukodystrophy, Sandhoff disease, mucolibidosis type II (I-Cell disease), mucolibidosis type IIIA (pseudo-Hurler polydystrophy), Niemann-Pick disease types C2 and Cl, Danon disease, free sialic acid storage disorder, mucolibidosis type IV, and multiple sulfatase deficiency (MSD), metabolic disorders, obesity, type II diabetes mellitus, and insulin resistance.

[0022] In some embodiments, the drug is

[0023] [ka] It is selected from the group consisting of the following.

[0024] In another aspect of the present invention, a pharmaceutical composition comprising the agent of the present invention and a pharmaceutically acceptable carrier is provided.

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

[0026] In some embodiments, the pharmaceutical composition contains a drug in a concentration of 100 nM to 5 mM.

[0027] Another aspect of the present invention provides a method for treating or preventing the development of diseases or disorders related to lysosomal accumulation, polyglucosane accumulation, or abnormal glycogen accumulation, and autophagy misregulation-related diseases in a subject requiring such treatment, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention to the subject.

[0028] Another aspect of the present invention provides a method for determining the suitability of a compound for preventing or treating diseases or disorders related to lysosome accumulation, polyglucosane accumulation or abnormal glycogen accumulation, and autophagy misregulation-related disorders, comprising contacting the compound with a pocket domain in the N-terminal domain of lysosome-associated membrane protein 1 (LAMP-1; SEQ ID NO: 1), wherein binding of the compound to the pocket indicates that the compound is effective in treating the diseases or disorders.

[0029] In some embodiments, the provided drug includes a conjugation comprising either SEQ ID NO: 2 (FSVNYD) or SEQ ID NO: 3 (NVTV).

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

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

[0032] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the invention relates. Similar or equivalent methods and materials may be used in the practice or testing of embodiments of the invention, but exemplary methods and / or materials are described below. In case of any conflict, the definitions included in this specification shall prevail. Furthermore, materials, methods, and examples are illustrative and not necessarily intended to be limiting.

[0033] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given below. However, 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, so it should be understood that the detailed description and specific examples, while illustrating preferred embodiments of the invention, are given only as examples. [Brief explanation of the drawing]

[0034] [Figure 1A] Figures 1A to 1M show the chemical structure of compound 1 (1A), a graph of Kaplan-Meier survival curves (log-rank test p-value < 0.000692) based on 17 animals (n=17) treated twice a week with 250 mg / kg of compound 1 (144DG11) compared with 9 animals (n=9) treated with a 5% DMSO vehicle (1B), a graph of body weight curves (in g) (1C), a graph of average movement time in open field (1D), and graphs of stretch reflexes as a function of time (degree of hind limb extension after grasping the animal by the tail) as a function of time after wild-type mice (n=8) were treated with the vehicle and after Gbeys / ys mice were treated with the vehicle (n=8) or compound 1 (n=9) (1E-1F), and a photograph of a movement heatmap showing quantification of open field performance experiments (1G) (upper panel, n=9, 9-month-old females). The lower panel shows a single-animal visual tracking example, where wt represents an untreated wild-type animal as a control, tg represents a Gbeys / ys (transgenic) mouse treated with compound 1, and tg represents an APBD mouse treated with vehicle. The graph (1H) shows the gait analysis (stride length) of n=9, 9-month-old female mice from each arm (showing mean (+ / -sd) stride length), the graph (1I) shows the mean duration of the motor curve in open field, the graph (in g) shows the body weight curve (1J), and the graph (1K) shows the stretch reflex curve as a function of time after Gbeys / ys mice shown at 6 months of age (at onset) were treated with vehicle or compound 1. The image also includes a photograph of a 7-month-old Gbeys / ys mouse treated with vehicle (1L) or compound 1 (1M) 3 months prior to the photograph. [Figure 1B]Figures 1A to 1M show the chemical structure of compound 1 (1A), a graph of Kaplan-Meier survival curves (log-rank test p-value < 0.000692) based on 17 animals (n=17) treated twice a week with 250 mg / kg of compound 1 (144DG11) compared with 9 animals (n=9) treated with a 5% DMSO vehicle (1B), a graph of body weight curves (in g) (1C), a graph of average movement time in open field (1D), and graphs of stretch reflexes as a function of time (degree of hind limb extension after grasping the animal by the tail) as a function of time after wild-type mice (n=8) were treated with the vehicle and after Gbeys / ys mice were treated with the vehicle (n=8) or compound 1 (n=9) (1E-1F), and a photograph of a movement heatmap showing quantification of open field performance experiments (1G) (upper panel, n=9, 9-month-old females). The lower panel shows a single-animal visual tracking example, where wt represents an untreated wild-type animal as a control, tg represents a Gbeys / ys (transgenic) mouse treated with compound 1, and tg represents an APBD mouse treated with vehicle. The graph (1H) shows the gait analysis (stride length) of n=9, 9-month-old female mice from each arm (showing mean (+ / -sd) stride length), the graph (1I) shows the mean duration of the motor curve in open field, the graph (in g) shows the body weight curve (1J), and the graph (1K) shows the stretch reflex curve as a function of time after Gbeys / ys mice shown at 6 months of age (at onset) were treated with vehicle or compound 1. The image also includes a photograph of a 7-month-old Gbeys / ys mouse treated with vehicle (1L) or compound 1 (1M) 3 months prior to the photograph. [Figure 1C]Figures 1A to 1M show the chemical structure of compound 1 (1A), a graph of Kaplan-Meier survival curves (log-rank test p-value < 0.000692) based on 17 animals (n=17) treated twice a week with 250 mg / kg of compound 1 (144DG11) compared with 9 animals (n=9) treated with a 5% DMSO vehicle (1B), a graph of body weight curves (in g) (1C), a graph of average movement time in open field (1D), and graphs of stretch reflexes as a function of time (degree of hind limb extension after grasping the animal by the tail) as a function of time after wild-type mice (n=8) were treated with the vehicle and after Gbeys / ys mice were treated with the vehicle (n=8) or compound 1 (n=9) (1E-1F), and a photograph of a movement heatmap showing quantification of open field performance experiments (1G) (upper panel, n=9, 9-month-old females). The lower panel shows a single-animal visual tracking example, where wt represents an untreated wild-type animal as a control, tg represents a Gbeys / ys (transgenic) mouse treated with compound 1, and tg represents an APBD mouse treated with vehicle. The graph (1H) shows the gait analysis (stride length) of n=9, 9-month-old female mice from each arm (showing mean (+ / -sd) stride length), the graph (1I) shows the mean duration of the motor curve in open field, the graph (in g) shows the body weight curve (1J), and the graph (1K) shows the stretch reflex curve as a function of time after Gbeys / ys mice shown at 6 months of age (at onset) were treated with vehicle or compound 1. The image also includes a photograph of a 7-month-old Gbeys / ys mouse treated with vehicle (1L) or compound 1 (1M) 3 months prior to the photograph. [Figure 1D]Figures 1A to 1M show the chemical structure of compound 1 (1A), a graph of Kaplan-Meier survival curves (log-rank test p-value < 0.000692) based on 17 animals (n=17) treated twice a week with 250 mg / kg of compound 1 (144DG11) compared with 9 animals (n=9) treated with a 5% DMSO vehicle (1B), a graph of body weight curves (in g) (1C), a graph of average movement time in open field (1D), and graphs of stretch reflexes as a function of time (degree of hind limb extension after grasping the animal by the tail) as a function of time after wild-type mice (n=8) were treated with the vehicle and after Gbeys / ys mice were treated with the vehicle (n=8) or compound 1 (n=9) (1E-1F), and a photograph of a movement heatmap showing quantification of open field performance experiments (1G) (upper panel, n=9, 9-month-old females). The lower panel shows a single-animal visual tracking example, where wt represents an untreated wild-type animal as a control, tg represents a Gbeys / ys (transgenic) mouse treated with compound 1, and tg represents an APBD mouse treated with vehicle. The graph (1H) shows the gait analysis (stride length) of n=9, 9-month-old female mice from each arm (showing mean (+ / -sd) stride length), the graph (1I) shows the mean duration of the motor curve in open field, the graph (in g) shows the body weight curve (1J), and the graph (1K) shows the stretch reflex curve as a function of time after Gbeys / ys mice shown at 6 months of age (at onset) were treated with vehicle or compound 1. The image also includes a photograph of a 7-month-old Gbeys / ys mouse treated with vehicle (1L) or compound 1 (1M) 3 months prior to the photograph. [Figure 1E]Figures 1A to 1M show the chemical structure of compound 1 (1A), a graph of Kaplan-Meier survival curves (log-rank test p-value < 0.000692) based on 17 animals (n=17) treated twice a week with 250 mg / kg of compound 1 (144DG11) compared with 9 animals (n=9) treated with a 5% DMSO vehicle (1B), a graph of body weight curves (in g) (1C), a graph of average movement time in open field (1D), and graphs of stretch reflexes as a function of time (degree of hind limb extension after grasping the animal by the tail) as a function of time after wild-type mice (n=8) were treated with the vehicle and after Gbeys / ys mice were treated with the vehicle (n=8) or compound 1 (n=9) (1E-1F), and a photograph of a movement heatmap showing quantification of open field performance experiments (1G) (upper panel, n=9, 9-month-old females). The lower panel shows a single-animal visual tracking example, where wt represents an untreated wild-type animal as a control, tg represents a Gbeys / ys (transgenic) mouse treated with compound 1, and tg represents an APBD mouse treated with vehicle. The graph (1H) shows the gait analysis (stride length) of n=9, 9-month-old female mice from each arm (showing mean (+ / -sd) stride length), the graph (1I) shows the mean duration of the motor curve in open field, the graph (in g) shows the body weight curve (1J), and the graph (1K) shows the stretch reflex curve as a function of time after Gbeys / ys mice shown at 6 months of age (at onset) were treated with vehicle or compound 1. The image also includes a photograph of a 7-month-old Gbeys / ys mouse treated with vehicle (1L) or compound 1 (1M) 3 months prior to the photograph. [Figure 1F]Figures 1A to 1M show the chemical structure of compound 1 (1A), a graph of Kaplan-Meier survival curves (log-rank test p-value < 0.000692) based on 17 animals (n=17) treated twice a week with 250 mg / kg of compound 1 (144DG11) compared with 9 animals (n=9) treated with a 5% DMSO vehicle (1B), a graph of body weight curves (in g) (1C), a graph of average movement time in open field (1D), and graphs of stretch reflexes as a function of time (degree of hind limb extension after grasping the animal by the tail) as a function of time after wild-type mice (n=8) were treated with the vehicle and after Gbeys / ys mice were treated with the vehicle (n=8) or compound 1 (n=9) (1E-1F), and a photograph of a movement heatmap showing quantification of open field performance experiments (1G) (upper panel, n=9, 9-month-old females). The lower panel shows a single-animal visual tracking example, where wt represents an untreated wild-type animal as a control, tg represents a Gbeys / ys (transgenic) mouse treated with compound 1, and tg represents an APBD mouse treated with vehicle. The graph (1H) shows the gait analysis (stride length) of n=9, 9-month-old female mice from each arm (showing mean (+ / -sd) stride length), the graph (1I) shows the mean duration of the motor curve in open field, the graph (in g) shows the body weight curve (1J), and the graph (1K) shows the stretch reflex curve as a function of time after Gbeys / ys mice shown at 6 months of age (at onset) were treated with vehicle or compound 1. The image also includes a photograph of a 7-month-old Gbeys / ys mouse treated with vehicle (1L) or compound 1 (1M) 3 months prior to the photograph. [Figure 1G]Figures 1A to 1M show the chemical structure of compound 1 (1A), a graph of Kaplan-Meier survival curves (log-rank test p-value < 0.000692) based on 17 animals (n=17) treated twice a week with 250 mg / kg of compound 1 (144DG11) compared with 9 animals (n=9) treated with a 5% DMSO vehicle (1B), a graph of body weight curves (in g) (1C), a graph of average movement time in open field (1D), and graphs of stretch reflexes as a function of time (degree of hind limb extension after grasping the animal by the tail) as a function of time after wild-type mice (n=8) were treated with the vehicle and after Gbeys / ys mice were treated with the vehicle (n=8) or compound 1 (n=9) (1E-1F), and a photograph of a movement heatmap showing quantification of open field performance experiments (1G) (upper panel, n=9, 9-month-old females). The lower panel shows a single-animal visual tracking example, where wt represents an untreated wild-type animal as a control, tg represents a Gbeys / ys (transgenic) mouse treated with compound 1, and tg represents an APBD mouse treated with vehicle. The graph (1H) shows the gait analysis (stride length) of n=9, 9-month-old female mice from each arm (showing mean (+ / -sd) stride length), the graph (1I) shows the mean duration of the motor curve in open field, the graph (in g) shows the body weight curve (1J), and the graph (1K) shows the stretch reflex curve as a function of time after Gbeys / ys mice shown at 6 months of age (at onset) were treated with vehicle or compound 1. The image also includes a photograph of a 7-month-old Gbeys / ys mouse treated with vehicle (1L) or compound 1 (1M) 3 months prior to the photograph. [Figure 1H]Figures 1A to 1M show the chemical structure of compound 1 (1A), a graph of Kaplan-Meier survival curves (log-rank test p-value < 0.000692) based on 17 animals (n=17) treated twice a week with 250 mg / kg of compound 1 (144DG11) compared with 9 animals (n=9) treated with a 5% DMSO vehicle (1B), a graph of body weight curves (in g) (1C), a graph of average movement time in open field (1D), and graphs of stretch reflexes as a function of time (degree of hind limb extension after grasping the animal by the tail) as a function of time after wild-type mice (n=8) were treated with the vehicle and after Gbeys / ys mice were treated with the vehicle (n=8) or compound 1 (n=9) (1E-1F), and a photograph of a movement heatmap showing quantification of open field performance experiments (1G) (upper panel, n=9, 9-month-old females). The lower panel shows a single-animal visual tracking example, where wt represents an untreated wild-type animal as a control, tg represents a Gbeys / ys (transgenic) mouse treated with compound 1, and tg represents an APBD mouse treated with vehicle. The graph (1H) shows the gait analysis (stride length) of n=9, 9-month-old female mice from each arm (showing mean (+ / -sd) stride length), the graph (1I) shows the mean duration of the motor curve in open field, the graph (in g) shows the body weight curve (1J), and the graph (1K) shows the stretch reflex curve as a function of time after Gbeys / ys mice shown at 6 months of age (at onset) were treated with vehicle or compound 1. The image also includes a photograph of a 7-month-old Gbeys / ys mouse treated with vehicle (1L) or compound 1 (1M) 3 months prior to the photograph. [Figure 1I]Figures 1A to 1M show the chemical structure of compound 1 (1A), a graph of Kaplan-Meier survival curves (log-rank test p-value < 0.000692) based on 17 animals (n=17) treated twice a week with 250 mg / kg of compound 1 (144DG11) compared with 9 animals (n=9) treated with a 5% DMSO vehicle (1B), a graph of body weight curves (in g) (1C), a graph of average movement time in open field (1D), and graphs of stretch reflexes as a function of time (degree of hind limb extension after grasping the animal by the tail) as a function of time after wild-type mice (n=8) were treated with the vehicle and after Gbeys / ys mice were treated with the vehicle (n=8) or compound 1 (n=9) (1E-1F), and a photograph of a movement heatmap showing quantification of open field performance experiments (1G) (upper panel, n=9, 9-month-old females). The lower panel shows a single-animal visual tracking example, where wt represents an untreated wild-type animal as a control, tg represents a Gbeys / ys (transgenic) mouse treated with compound 1, and tg represents an APBD mouse treated with vehicle. The graph (1H) shows the gait analysis (stride length) of n=9, 9-month-old female mice from each arm (showing mean (+ / -sd) stride length), the graph (1I) shows the mean duration of the motor curve in open field, the graph (in g) shows the body weight curve (1J), and the graph (1K) shows the stretch reflex curve as a function of time after Gbeys / ys mice shown at 6 months of age (at onset) were treated with vehicle or compound 1. The image also includes a photograph of a 7-month-old Gbeys / ys mouse treated with vehicle (1L) or compound 1 (1M) 3 months prior to the photograph. [Figure 1J]Figures 1A to 1M show the chemical structure of compound 1 (1A), a graph of Kaplan-Meier survival curves (log-rank test p-value < 0.000692) based on 17 animals (n=17) treated twice a week with 250 mg / kg of compound 1 (144DG11) compared with 9 animals (n=9) treated with a 5% DMSO vehicle (1B), a graph of body weight curves (in g) (1C), a graph of average movement time in open field (1D), and graphs of stretch reflexes as a function of time (degree of hind limb extension after grasping the animal by the tail) as a function of time after wild-type mice (n=8) were treated with the vehicle and after Gbeys / ys mice were treated with the vehicle (n=8) or compound 1 (n=9) (1E-1F), and a photograph of a movement heatmap showing quantification of open field performance experiments (1G) (upper panel, n=9, 9-month-old females). The lower panel shows a single-animal visual tracking example, where wt represents an untreated wild-type animal as a control, tg represents a Gbeys / ys (transgenic) mouse treated with compound 1, and tg represents an APBD mouse treated with vehicle. The graph (1H) shows the gait analysis (stride length) of n=9, 9-month-old female mice from each arm (showing mean (+ / -sd) stride length), the graph (1I) shows the mean duration of the motor curve in open field, the graph (in g) shows the body weight curve (1J), and the graph (1K) shows the stretch reflex curve as a function of time after Gbeys / ys mice shown at 6 months of age (at onset) were treated with vehicle or compound 1. The image also includes a photograph of a 7-month-old Gbeys / ys mouse treated with vehicle (1L) or compound 1 (1M) 3 months prior to the photograph. [Figure 1K]Figures 1A to 1M show the chemical structure of compound 1 (1A), a graph of Kaplan-Meier survival curves (log-rank test p-value < 0.000692) based on 17 animals (n=17) treated twice a week with 250 mg / kg of compound 1 (144DG11) compared with 9 animals (n=9) treated with a 5% DMSO vehicle (1B), a graph of body weight curves (in g) (1C), a graph of average movement time in open field (1D), and graphs of stretch reflexes as a function of time (degree of hind limb extension after grasping the animal by the tail) as a function of time after wild-type mice (n=8) were treated with the vehicle and after Gbeys / ys mice were treated with the vehicle (n=8) or compound 1 (n=9) (1E-1F), and a photograph of a movement heatmap showing quantification of open field performance experiments (1G) (upper panel, n=9, 9-month-old females). The lower panel shows a single-animal visual tracking example, where wt represents an untreated wild-type animal as a control, tg represents a Gbeys / ys (transgenic) mouse treated with compound 1, and tg represents an APBD mouse treated with vehicle. The graph (1H) shows the gait analysis (stride length) of n=9, 9-month-old female mice from each arm (showing mean (+ / -sd) stride length), the graph (1I) shows the mean duration of the motor curve in open field, the graph (in g) shows the body weight curve (1J), and the graph (1K) shows the stretch reflex curve as a function of time after Gbeys / ys mice shown at 6 months of age (at onset) were treated with vehicle or compound 1. The image also includes a photograph of a 7-month-old Gbeys / ys mouse treated with vehicle (1L) or compound 1 (1M) 3 months prior to the photograph. [Figure 1L]Figures 1A to 1M show the chemical structure of compound 1 (1A), a graph of Kaplan-Meier survival curves (log-rank test p-value < 0.000692) based on 17 animals (n=17) treated twice a week with 250 mg / kg of compound 1 (144DG11) compared with 9 animals (n=9) treated with a 5% DMSO vehicle (1B), a graph of body weight curves (in g) (1C), a graph of average movement time in open field (1D), and graphs of stretch reflexes as a function of time (degree of hind limb extension after grasping the animal by the tail) as a function of time after wild-type mice (n=8) were treated with the vehicle and after Gbeys / ys mice were treated with the vehicle (n=8) or compound 1 (n=9) (1E-1F), and a photograph of a movement heatmap showing quantification of open field performance experiments (1G) (upper panel, n=9, 9-month-old females). The lower panel shows a single-animal visual tracking example, where wt represents an untreated wild-type animal as a control, tg represents a Gbeys / ys (transgenic) mouse treated with compound 1, and tg represents an APBD mouse treated with vehicle. The graph (1H) shows the gait analysis (stride length) of n=9, 9-month-old female mice from each arm (showing mean (+ / -sd) stride length), the graph (1I) shows the mean duration of the motor curve in open field, the graph (in g) shows the body weight curve (1J), and the graph (1K) shows the stretch reflex curve as a function of time after Gbeys / ys mice shown at 6 months of age (at onset) were treated with vehicle or compound 1. The image also includes a photograph of a 7-month-old Gbeys / ys mouse treated with vehicle (1L) or compound 1 (1M) 3 months prior to the photograph. [Figure 1M]Figures 1A to 1M show the chemical structure of compound 1 (1A), a graph of Kaplan-Meier survival curves (log-rank test p-value < 0.000692) based on 17 animals (n=17) treated twice a week with 250 mg / kg of compound 1 (144DG11) compared with 9 animals (n=9) treated with a 5% DMSO vehicle (1B), a graph of body weight curves (in g) (1C), a graph of average movement time in open field (1D), and graphs of stretch reflexes as a function of time (degree of hind limb extension after grasping the animal by the tail) as a function of time after wild-type mice (n=8) were treated with the vehicle and after Gbeys / ys mice were treated with the vehicle (n=8) or compound 1 (n=9) (1E-1F), and a photograph of a movement heatmap showing quantification of open field performance experiments (1G) (upper panel, n=9, 9-month-old females). The lower panel shows a single-animal visual tracking example, where wt represents an untreated wild-type animal as a control, tg represents a Gbeys / ys (transgenic) mouse treated with compound 1, and tg represents an APBD mouse treated with vehicle. The graph (1H) shows the gait analysis (stride length) of n=9, 9-month-old female mice from each arm (showing mean (+ / -sd) stride length), the graph (1I) shows the mean duration of the motor curve in open field, the graph (in g) shows the body weight curve (1J), and the graph (1K) shows the stretch reflex curve as a function of time after Gbeys / ys mice shown at 6 months of age (at onset) were treated with vehicle or compound 1. The image also includes a photograph of a 7-month-old Gbeys / ys mouse treated with vehicle (1L) or compound 1 (1M) 3 months prior to the photograph. [Figure 2A-1]Figures 2A-2C contain images and graphs of the histopathological effects and pharmacokinetics of compound 1. The right panel shows images of the indicated tissues stained for PG (arrow) with PAS from mice treated with diastase and then sacrificed. The left panel shows bar graphs quantifying PAS staining (2A), bar graphs quantifying total glycogen in each tissue (2B), and graphs of the pharmacokinetics of compound 1 (2C), based on the analysis of four sections obtained from the tissues of n=2 wild-type mice, n=7 Gbeys / ys vehicle-treated mice, and n=9 compound 1-treated mice. Nine-month-old Gbeys / ys mice were injected with 150 μL of compound 1 at a dose of 250 mg / kg via SC injection. Mice were sacrificed 30, 60, 90, and 210 minutes after injection, and the indicated tissues were removed, with 200 μL of serum collected. The graphs show the levels of compound 1 in different tissues as measured by LC-MS / MS. The mean and SEM results obtained from n=3 mice at each time point are shown. The repeated measures two-way ANOVA test shows that the pharmacokinetic profile of each tissue is significantly different from that of all other tissues (p<0.05). * indicates a significant difference determined by Student's t-test (p<0.05). [Figure 2A-2]Figures 2A-2C contain images and graphs of the histopathological effects and pharmacokinetics of compound 1. The right panel shows images of the indicated tissues stained for PG (arrow) with PAS from mice treated with diastase and then sacrificed. The left panel shows bar graphs quantifying PAS staining (2A), bar graphs quantifying total glycogen in each tissue (2B), and graphs of the pharmacokinetics of compound 1 (2C), based on the analysis of four sections obtained from the tissues of n=2 wild-type mice, n=7 Gbeys / ys vehicle-treated mice, and n=9 compound 1-treated mice. Nine-month-old Gbeys / ys mice were injected with 150 μL of compound 1 at a dose of 250 mg / kg via SC injection. Mice were sacrificed 30, 60, 90, and 210 minutes after injection, and the indicated tissues were removed, with 200 μL of serum collected. The graphs show the levels of compound 1 in different tissues as measured by LC-MS / MS. The mean and SEM results obtained from n=3 mice at each time point are shown. The repeated measures two-way ANOVA test shows that the pharmacokinetic profile of each tissue is significantly different from that of all other tissues (p<0.05). * indicates a significant difference determined by Student's t-test (p<0.05). [Figure 2B]Figures 2A-2C contain images and graphs of the histopathological effects and pharmacokinetics of compound 1. The right panel shows images of the indicated tissues stained for PG (arrow) with PAS from mice treated with diastase and then sacrificed. The left panel shows bar graphs quantifying PAS staining (2A), bar graphs quantifying total glycogen in each tissue (2B), and graphs of the pharmacokinetics of compound 1 (2C), based on the analysis of four sections obtained from the tissues of n=2 wild-type mice, n=7 Gbeys / ys vehicle-treated mice, and n=9 compound 1-treated mice. Nine-month-old Gbeys / ys mice were injected with 150 μL of compound 1 at a dose of 250 mg / kg via SC injection. Mice were sacrificed 30, 60, 90, and 210 minutes after injection, and the indicated tissues were removed, with 200 μL of serum collected. The graphs show the levels of compound 1 in different tissues as measured by LC-MS / MS. The mean and SEM results obtained from n=3 mice at each time point are shown. The repeated measures two-way ANOVA test shows that the pharmacokinetic profile of each tissue is significantly different from that of all other tissues (p<0.05). * indicates a significant difference determined by Student's t-test (p<0.05). [Figure 2C]Figures 2A-2C contain images and graphs of the histopathological effects and pharmacokinetics of compound 1. The right panel shows images of the indicated tissues stained for PG (arrow) with PAS from mice treated with diastase and then sacrificed. The left panel shows bar graphs quantifying PAS staining (2A), bar graphs quantifying total glycogen in each tissue (2B), and graphs of the pharmacokinetics of compound 1 (2C), based on the analysis of four sections obtained from the tissues of n=2 wild-type mice, n=7 Gbeys / ys vehicle-treated mice, and n=9 compound 1-treated mice. Nine-month-old Gbeys / ys mice were injected with 150 μL of compound 1 at a dose of 250 mg / kg via SC injection. Mice were sacrificed 30, 60, 90, and 210 minutes after injection, and the indicated tissues were removed, with 200 μL of serum collected. The graphs show the levels of compound 1 in different tissues as measured by LC-MS / MS. The mean and SEM results obtained from n=3 mice at each time point are shown. The repeated measures two-way ANOVA test shows that the pharmacokinetic profile of each tissue is significantly different from that of all other tissues (p<0.05). * indicates a significant difference determined by Student's t-test (p<0.05). [Figure 3A]Figures 3A–3I include graphs of the effect of compound 1 on in vivo metabolism, with mice monitored over 24 hours using the Promethion high-resolution behavioral phenotyping system (Sable Instruments, Inc.). Effective mass was calculated as a power of 0.75. Data are mean ± SEM obtained 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 administered from 4 months of age. Untreated GBEys / ys mice showed lower (in light) respiratory quotient (3A), total energy expenditure (TEE) (3B), and fatty acid oxidation (3C) compared to wild-type controls. Carbohydrate oxidation and walking activity, which were not significantly affected by disease status, increased with compound 1 to levels even exceeding 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). Hematologic metabolism panel based on n=5, 9.5-month-old mice treated as shown (3I). In Gbeys / ys cells, compound 1 increased blood glucose and decreased blood triglycerides (p<0.05, Student's t-test). *p<0.05v wt. controls, #p<0.05v GBEys / ys vehicle-treated mice. [Figure 3B]Figures 3A–3I include graphs of the effect of compound 1 on in vivo metabolism, with mice monitored over 24 hours using the Promethion high-resolution behavioral phenotyping system (Sable Instruments, Inc.). Effective mass was calculated as a power of 0.75. Data are mean ± SEM obtained 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 administered from 4 months of age. Untreated GBEys / ys mice showed lower (in light) respiratory quotient (3A), total energy expenditure (TEE) (3B), and fatty acid oxidation (3C) compared to wild-type controls. Carbohydrate oxidation and walking activity, which were not significantly affected by disease status, increased with compound 1 to levels even exceeding 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). Hematologic metabolism panel based on n=5, 9.5-month-old mice treated as shown (3I). In Gbeys / ys cells, compound 1 increased blood glucose and decreased blood triglycerides (p<0.05, Student's t-test). *p<0.05v wt. controls, #p<0.05v GBEys / ys vehicle-treated mice. [Figure 3C]Figures 3A–3I include graphs of the effect of compound 1 on in vivo metabolism, with mice monitored over 24 hours using the Promethion high-resolution behavioral phenotyping system (Sable Instruments, Inc.). Effective mass was calculated as a power of 0.75. Data are mean ± SEM obtained 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 administered from 4 months of age. Untreated GBEys / ys mice showed lower (in light) respiratory quotient (3A), total energy expenditure (TEE) (3B), and fatty acid oxidation (3C) compared to wild-type controls. Carbohydrate oxidation and walking activity, which were not significantly affected by disease status, increased with compound 1 to levels even exceeding 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). Hematologic metabolism panel based on n=5, 9.5-month-old mice treated as shown (3I). In Gbeys / ys cells, compound 1 increased blood glucose and decreased blood triglycerides (p<0.05, Student's t-test). *p<0.05v wt. controls, #p<0.05v GBEys / ys vehicle-treated mice. [Figure 3D]Figures 3A–3I include graphs of the effect of compound 1 on in vivo metabolism, with mice monitored over 24 hours using the Promethion high-resolution behavioral phenotyping system (Sable Instruments, Inc.). Effective mass was calculated as a power of 0.75. Data are mean ± SEM obtained 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 administered from 4 months of age. Untreated GBEys / ys mice showed lower (in light) respiratory quotient (3A), total energy expenditure (TEE) (3B), and fatty acid oxidation (3C) compared to wild-type controls. Carbohydrate oxidation and walking activity, which were not significantly affected by disease status, increased with compound 1 to levels even exceeding 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). Hematologic metabolism panel based on n=5, 9.5-month-old mice treated as shown (3I). In Gbeys / ys cells, compound 1 increased blood glucose and decreased blood triglycerides (p<0.05, Student's t-test). *p<0.05v wt. controls, #p<0.05v GBEys / ys vehicle-treated mice. [Figure 3E]Figures 3A–3I include graphs of the effect of compound 1 on in vivo metabolism, with mice monitored over 24 hours using the Promethion high-resolution behavioral phenotyping system (Sable Instruments, Inc.). Effective mass was calculated as a power of 0.75. Data are mean ± SEM obtained 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 administered from 4 months of age. Untreated GBEys / ys mice showed lower (in light) respiratory quotient (3A), total energy expenditure (TEE) (3B), and fatty acid oxidation (3C) compared to wild-type controls. Carbohydrate oxidation and walking activity, which were not significantly affected by disease status, increased with compound 1 to levels even exceeding 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). Hematologic metabolism panel based on n=5, 9.5-month-old mice treated as shown (3I). In Gbeys / ys cells, compound 1 increased blood glucose and decreased blood triglycerides (p<0.05, Student's t-test). *p<0.05v wt. controls, #p<0.05v GBEys / ys vehicle-treated mice. [Figure 3F]Figures 3A–3I include graphs of the effect of compound 1 on in vivo metabolism, with mice monitored over 24 hours using the Promethion high-resolution behavioral phenotyping system (Sable Instruments, Inc.). Effective mass was calculated as a power of 0.75. Data are mean ± SEM obtained 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 administered from 4 months of age. Untreated GBEys / ys mice showed lower (in light) respiratory quotient (3A), total energy expenditure (TEE) (3B), and fatty acid oxidation (3C) compared to wild-type controls. Carbohydrate oxidation and walking activity, which were not significantly affected by disease status, increased with compound 1 to levels even exceeding 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). Hematologic metabolism panel based on n=5, 9.5-month-old mice treated as shown (3I). In Gbeys / ys cells, compound 1 increased blood glucose and decreased blood triglycerides (p<0.05, Student's t-test). *p<0.05v wt. controls, #p<0.05v GBEys / ys vehicle-treated mice. [Figure 3G]Figures 3A–3I include graphs of the effect of compound 1 on in vivo metabolism, with mice monitored over 24 hours using the Promethion high-resolution behavioral phenotyping system (Sable Instruments, Inc.). Effective mass was calculated as a power of 0.75. Data are mean ± SEM obtained 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 administered from 4 months of age. Untreated GBEys / ys mice showed lower (in light) respiratory quotient (3A), total energy expenditure (TEE) (3B), and fatty acid oxidation (3C) compared to wild-type controls. Carbohydrate oxidation and walking activity, which were not significantly affected by disease status, increased with compound 1 to levels even exceeding 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). Hematologic metabolism panel based on n=5, 9.5-month-old mice treated as shown (3I). In Gbeys / ys cells, compound 1 increased blood glucose and decreased blood triglycerides (p<0.05, Student's t-test). *p<0.05v wt. controls, #p<0.05v GBEys / ys vehicle-treated mice. [Figure 3H]Figures 3A–3I include graphs of the effect of compound 1 on in vivo metabolism, with mice monitored over 24 hours using the Promethion high-resolution behavioral phenotyping system (Sable Instruments, Inc.). Effective mass was calculated as a power of 0.75. Data are mean ± SEM obtained 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 administered from 4 months of age. Untreated GBEys / ys mice showed lower (in light) respiratory quotient (3A), total energy expenditure (TEE) (3B), and fatty acid oxidation (3C) compared to wild-type controls. Carbohydrate oxidation and walking activity, which were not significantly affected by disease status, increased with compound 1 to levels even exceeding 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). Hematologic metabolism panel based on n=5, 9.5-month-old mice treated as shown (3I). In Gbeys / ys cells, compound 1 increased blood glucose and decreased blood triglycerides (p<0.05, Student's t-test). *p<0.05v wt. controls, #p<0.05v GBEys / ys vehicle-treated mice. [Figure 3I]Figures 3A–3I include graphs of the effect of compound 1 on in vivo metabolism, with mice monitored over 24 hours using the Promethion high-resolution behavioral phenotyping system (Sable Instruments, Inc.). Effective mass was calculated as a power of 0.75. Data are mean ± SEM obtained 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 administered from 4 months of age. Untreated GBEys / ys mice showed lower (in light) respiratory quotient (3A), total energy expenditure (TEE) (3B), and fatty acid oxidation (3C) compared to wild-type controls. Carbohydrate oxidation and walking activity, which were not significantly affected by disease status, increased with compound 1 to levels even exceeding 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). Hematologic metabolism panel based on n=5, 9.5-month-old mice treated as shown (3I). In Gbeys / ys cells, compound 1 increased blood glucose and decreased blood triglycerides (p<0.05, Student's t-test). *p<0.05v wt. controls, #p<0.05v GBEys / ys vehicle-treated mice. [Figure 4A]Figures 4A–4D include bar graphs of PAS staining for total glycogen in cutaneous fibroblasts from different APBD patients (4A), images of PAS staining for total glycogen in APBD87 fibroblasts (left) that were glucose-starved for 48 hours, or APBD87 fibroblasts (right) that were glucose-starved and then supplemented for the last 24 hours to induce glycogen loading (4B) (image acquisition was performed using a Nikon Eclipse Ti2 microscope with a 40x PlanFluor objective lens and CY3 filter), image-based multiparametric phenotype graphs of APBD fibroblasts under 48 hours of glucose starvation, or under starvation and glucose supplementation 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 hours (chronic) or 20 minutes (acute) in the assay. Measurements were normalized to the number of cells measured by crystal violet staining. Mean and standard deviation values ​​are shown based on n=6 replicates. [Figure 4B]Figures 4A–4D include bar graphs of PAS staining for total glycogen in cutaneous fibroblasts from different APBD patients (4A), images of PAS staining for total glycogen in APBD87 fibroblasts (left) that were glucose-starved for 48 hours, or APBD87 fibroblasts (right) that were glucose-starved and then supplemented for the last 24 hours to induce glycogen loading (4B) (image acquisition was performed using a Nikon Eclipse Ti2 microscope with a 40x PlanFluor objective lens and CY3 filter), image-based multiparametric phenotype graphs of APBD fibroblasts under 48 hours of glucose starvation, or under starvation and glucose supplementation 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 hours (chronic) or 20 minutes (acute) in the assay. Measurements were normalized to the number of cells measured by crystal violet staining. Mean and standard deviation values ​​are shown based on n=6 replicates. [Figure 4C]Figures 4A–4D include bar graphs of PAS staining for total glycogen in cutaneous fibroblasts from different APBD patients (4A), images of PAS staining for total glycogen in APBD87 fibroblasts (left) that were glucose-starved for 48 hours, or APBD87 fibroblasts (right) that were glucose-starved and then supplemented for the last 24 hours to induce glycogen loading (4B) (image acquisition was performed using a Nikon Eclipse Ti2 microscope with a 40x PlanFluor objective lens and CY3 filter), image-based multiparametric phenotype graphs of APBD fibroblasts under 48 hours of glucose starvation, or under starvation and glucose supplementation 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 hours (chronic) or 20 minutes (acute) in the assay. Measurements were normalized to the number of cells measured by crystal violet staining. Mean and standard deviation values ​​are shown based on n=6 replicates. [Figure 4D]Figures 4A–4D include bar graphs of PAS staining for total glycogen in cutaneous fibroblasts from different APBD patients (4A), images of PAS staining for total glycogen in APBD87 fibroblasts (left) that were glucose-starved for 48 hours, or APBD87 fibroblasts (right) that were glucose-starved and then supplemented for the last 24 hours to induce glycogen loading (4B) (image acquisition was performed using a Nikon Eclipse Ti2 microscope with a 40x PlanFluor objective lens and CY3 filter), image-based multiparametric phenotype graphs of APBD fibroblasts under 48 hours of glucose starvation, or under starvation and glucose supplementation 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 hours (chronic) or 20 minutes (acute) in the assay. Measurements were normalized to the number of cells measured by crystal violet staining. Mean and standard deviation values ​​are shown based on n=6 replicates. [Figure 5A] Figures 5A–5E include experimental images (5A) showing that heteroassemblies are formed around compound 1 and not around endogenous molecules, as indicated by the liquid crystals formed in experiments 1–3; images of the target string network in the interactome of compound 1 (5B); cell thermal shift assay (CETSA) of different targets of the heteroassembly of compound 1 (5C); surface plasmon resonance sensorgram of compound 1 binding to LAMP1 (5D) (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 by LAMP1 grids predicted by SiteMap, fPocket, and FtSite (5E). [Figure 5B]Figures 5A–5E include experimental images (5A) showing that heteroassemblies are formed around compound 1 and not around endogenous molecules, as indicated by the liquid crystals formed in experiments 1–3; images of the target string network in the interactome of compound 1 (5B); cell thermal shift assay (CETSA) of different targets of the heteroassembly of compound 1 (5C); surface plasmon resonance sensorgram of compound 1 binding to LAMP1 (5D) (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 by LAMP1 grids predicted by SiteMap, fPocket, and FtSite (5E). [Figure 5C] Figures 5A–5E include experimental images (5A) showing that heteroassemblies are formed around compound 1 and not around endogenous molecules, as indicated by the liquid crystals formed in experiments 1–3; images of the target string network in the interactome of compound 1 (5B); cell thermal shift assay (CETSA) of different targets of the heteroassembly of compound 1 (5C); surface plasmon resonance sensorgram of compound 1 binding to LAMP1 (5D) (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 by LAMP1 grids predicted by SiteMap, fPocket, and FtSite (5E). [Figure 5D]Figures 5A–5E include experimental images (5A) showing that heteroassemblies are formed around compound 1 and not around endogenous molecules, as indicated by the liquid crystals formed in experiments 1–3; images of the target string network in the interactome of compound 1 (5B); cell thermal shift assay (CETSA) of different targets of the heteroassembly of compound 1 (5C); surface plasmon resonance sensorgram of compound 1 binding to LAMP1 (5D) (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 by LAMP1 grids predicted by SiteMap, fPocket, and FtSite (5E). [Figure 5E] Figures 5A–5E include experimental images (5A) showing that heteroassemblies are formed around compound 1 and not around endogenous molecules, as indicated by the liquid crystals formed in experiments 1–3; images of the target string network in the interactome of compound 1 (5B); cell thermal shift assay (CETSA) of different targets of the heteroassembly of compound 1 (5C); surface plasmon resonance sensorgram of compound 1 binding to LAMP1 (5D) (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 by LAMP1 grids predicted by SiteMap, fPocket, and FtSite (5E). [Figure 6A]Figures 6A–6E show bar graphs of autophagy flux determined by the degree of lysosomal inhibitor-dependent increase in the ratio of lipidized LC3 to non-lipidized LC3 (LC3II / LC3I) (6A), representative TEM images of liver tissue from 9.5-month-old Gbeys / ys mice treated with compound 11 or a 5% DMSO vehicle (6B) (G: glycogen (α-particles) and polyglucosane (structure with variable electron density), L: lysosomes, M: mitochondria, right panel: lysosomal glycogen staining quantified by the ImageJ "Count Particles" tool), and LAMP1 knockdown and control APBD primary dermal fibroblasts treated with compound 1 and the lysosomal inhibitor (LI). The images include micrographs of treated or untreated cells, quantifications of the three experiments and results of Student's t-tests (** for p<0.1, ** for p<0.05, *** for p<0.01) (6C), graphs of lysosomal pH changes measured in APBD primary fibroblasts transduced with lentivirus encoding GFP or GFP-shLAMP1 and treated or untreated with compound 1 for 24 hours, and confocal micrographs of cells treated with Lysosensor and stained for PAS (6D), and bar graphs of ATP production rate assays in LAMP1-KD cells and GFP (control) cells treated with compound 1 for 24 hours (chronic) or assay (acute) (6E). [Figure 6B]Figures 6A–6E show bar graphs of autophagy flux determined by the degree of lysosomal inhibitor-dependent increase in the ratio of lipidized LC3 to non-lipidized LC3 (LC3II / LC3I) (6A), representative TEM images of liver tissue from 9.5-month-old Gbeys / ys mice treated with compound 11 or a 5% DMSO vehicle (6B) (G: glycogen (α-particles) and polyglucosane (structure with variable electron density), L: lysosomes, M: mitochondria, right panel: lysosomal glycogen staining quantified by the ImageJ "Count Particles" tool), and LAMP1 knockdown and control APBD primary dermal fibroblasts treated with compound 1 and the lysosomal inhibitor (LI). The images include micrographs of treated or untreated cells, quantifications of the three experiments and results of Student's t-tests (** for p<0.1, ** for p<0.05, *** for p<0.01) (6C), graphs of lysosomal pH changes measured in APBD primary fibroblasts transduced with lentivirus encoding GFP or GFP-shLAMP1 and treated or untreated with compound 1 for 24 hours, and confocal micrographs of cells treated with Lysosensor and stained for PAS (6D), and bar graphs of ATP production rate assays in LAMP1-KD cells and GFP (control) cells treated with compound 1 for 24 hours (chronic) or assay (acute) (6E). [Figure 6C]Figures 6A–6E show bar graphs of autophagy flux determined by the degree of lysosomal inhibitor-dependent increase in the ratio of lipidized LC3 to non-lipidized LC3 (LC3II / LC3I) (6A), representative TEM images of liver tissue from 9.5-month-old Gbeys / ys mice treated with compound 11 or a 5% DMSO vehicle (6B) (G: glycogen (α-particles) and polyglucosane (structure with variable electron density), L: lysosomes, M: mitochondria, right panel: lysosomal glycogen staining quantified by the ImageJ "Count Particles" tool), and LAMP1 knockdown and control APBD primary dermal fibroblasts treated with compound 1 and the lysosomal inhibitor (LI). The images include micrographs of treated or untreated cells, quantifications of the three experiments and results of Student's t-tests (** for p<0.1, ** for p<0.05, *** for p<0.01) (6C), graphs of lysosomal pH changes measured in APBD primary fibroblasts transduced with lentivirus encoding GFP or GFP-shLAMP1 and treated or untreated with compound 1 for 24 hours, and confocal micrographs of cells treated with Lysosensor and stained for PAS (6D), and bar graphs of ATP production rate assays in LAMP1-KD cells and GFP (control) cells treated with compound 1 for 24 hours (chronic) or assay (acute) (6E). [Figure 6D-1]Figures 6A–6E show bar graphs of autophagy flux determined by the degree of lysosomal inhibitor-dependent increase in the ratio of lipidized LC3 to non-lipidized LC3 (LC3II / LC3I) (6A), representative TEM images of liver tissue from 9.5-month-old Gbeys / ys mice treated with compound 11 or a 5% DMSO vehicle (6B) (G: glycogen (α-particles) and polyglucosane (structure with variable electron density), L: lysosomes, M: mitochondria, right panel: lysosomal glycogen staining quantified by the ImageJ "Count Particles" tool), and LAMP1 knockdown and control APBD primary dermal fibroblasts treated with compound 1 and the lysosomal inhibitor (LI). The images include micrographs of treated or untreated cells, quantifications of the three experiments and results of Student's t-tests (** for p<0.1, ** for p<0.05, *** for p<0.01) (6C), graphs of lysosomal pH changes measured in APBD primary fibroblasts transduced with lentivirus encoding GFP or GFP-shLAMP1 and treated or untreated with compound 1 for 24 hours, and confocal micrographs of cells treated with Lysosensor and stained for PAS (6D), and bar graphs of ATP production rate assays in LAMP1-KD cells and GFP (control) cells treated with compound 1 for 24 hours (chronic) or assay (acute) (6E). [Figure 6D-2]Figures 6A–6E show bar graphs of autophagy flux determined by the degree of lysosomal inhibitor-dependent increase in the ratio of lipidized LC3 to non-lipidized LC3 (LC3II / LC3I) (6A), representative TEM images of liver tissue from 9.5-month-old Gbeys / ys mice treated with compound 11 or a 5% DMSO vehicle (6B) (G: glycogen (α-particles) and polyglucosane (structure with variable electron density), L: lysosomes, M: mitochondria, right panel: lysosomal glycogen staining quantified by the ImageJ "Count Particles" tool), and LAMP1 knockdown and control APBD primary dermal fibroblasts treated with compound 1 and the lysosomal inhibitor (LI). The images include micrographs of treated or untreated cells, quantifications of the three experiments and results of Student's t-tests (** for p<0.1, ** for p<0.05, *** for p<0.01) (6C), graphs of lysosomal pH changes measured in APBD primary fibroblasts transduced with lentivirus encoding GFP or GFP-shLAMP1 and treated or untreated with compound 1 for 24 hours, and confocal micrographs of cells treated with Lysosensor and stained for PAS (6D), and bar graphs of ATP production rate assays in LAMP1-KD cells and GFP (control) cells treated with compound 1 for 24 hours (chronic) or assay (acute) (6E). [Figure 6E]Figures 6A–6E show bar graphs of autophagy flux determined by the degree of lysosomal inhibitor-dependent increase in the ratio of lipidized LC3 to non-lipidized LC3 (LC3II / LC3I) (6A), representative TEM images of liver tissue from 9.5-month-old Gbeys / ys mice treated with compound 11 or a 5% DMSO vehicle (6B) (G: glycogen (α-particles) and polyglucosane (structure with variable electron density), L: lysosomes, M: mitochondria, right panel: lysosomal glycogen staining quantified by the ImageJ "Count Particles" tool), and LAMP1 knockdown and control APBD primary dermal fibroblasts treated with compound 1 and the lysosomal inhibitor (LI). The images include micrographs of treated or untreated cells, quantifications of the three experiments and results of Student's t-tests (** for p<0.1, ** for p<0.05, *** for p<0.01) (6C), graphs of lysosomal pH changes measured in APBD primary fibroblasts transduced with lentivirus encoding GFP or GFP-shLAMP1 and treated or untreated with compound 1 for 24 hours, and confocal micrographs of cells treated with Lysosensor and stained for PAS (6D), and bar graphs of ATP production rate assays in LAMP1-KD cells and GFP (control) cells treated with compound 1 for 24 hours (chronic) or assay (acute) (6E). [Figure 7A]Figures 7A to 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 different variables (cell characteristics) shown on the x-axis (random forest analysis demonstrated that APBD and HC cell populations are separated with a 93% confidence level) (7A), n=5 HC and n=5 7B) A graph of multiparametric cellular phenotypic characterization of APBD patient dermal fibroblasts (the degree of deviation of the shown cellular features from HC, ordered by the amount of deviation (-log(p-value)). Features whose values ​​are above the dashed line (box) indicate a deviation from HC with a p-value < 0.01. Various comparisons analyzed (compound A = compound 1) are shown) a bar graph of lysosomal parameters affected by compound 1 in APBD and HC cells, analyzed by IBP (7C) a volcano plot of proteins affected by APBD and compound 1 under starvation and glycogen loading conditions (7D) a 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) a gene ontology of proteins upregulated by compound 1 (left) and downregulated by compound 1 (right) (7F). [Figure 7B]Figures 7A to 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 different variables (cell characteristics) shown on the x-axis (random forest analysis demonstrated that APBD and HC cell populations are separated with a 93% confidence level) (7A), n=5 HC and n=5 7B) A graph of multiparametric cellular phenotypic characterization of APBD patient dermal fibroblasts (the degree of deviation of the shown cellular features from HC, ordered by the amount of deviation (-log(p-value)). Features whose values ​​are above the dashed line (box) indicate a deviation from HC with a p-value < 0.01. Various comparisons analyzed (compound A = compound 1) are shown) a bar graph of lysosomal parameters affected by compound 1 in APBD and HC cells, analyzed by IBP (7C) a volcano plot of proteins affected by APBD and compound 1 under starvation and glycogen loading conditions (7D) a 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) a gene ontology of proteins upregulated by compound 1 (left) and downregulated by compound 1 (right) (7F). [Figure 7C]Figures 7A to 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 different variables (cell characteristics) shown on the x-axis (random forest analysis demonstrated that APBD and HC cell populations are separated with a 93% confidence level) (7A), n=5 HC and n=5 7B) A graph of multiparametric cellular phenotypic characterization of APBD patient dermal fibroblasts (the degree of deviation of the shown cellular features from HC, ordered by the amount of deviation (-log(p-value)). Features whose values ​​are above the dashed line (box) indicate a deviation from HC with a p-value < 0.01. Various comparisons analyzed (compound A = compound 1) are shown) a bar graph of lysosomal parameters affected by compound 1 in APBD and HC cells, analyzed by IBP (7C) a volcano plot of proteins affected by APBD and compound 1 under starvation and glycogen loading conditions (7D) a 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) a gene ontology of proteins upregulated by compound 1 (left) and downregulated by compound 1 (right) (7F). [Figure 7D]Figures 7A to 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 different variables (cell characteristics) shown on the x-axis (random forest analysis demonstrated that APBD and HC cell populations are separated with a 93% confidence level) (7A), n=5 HC and n=5 7B) A graph of multiparametric cellular phenotypic characterization of APBD patient dermal fibroblasts (the degree of deviation of the shown cellular features from HC, ordered by the amount of deviation (-log(p-value)). Features whose values ​​are above the dashed line (box) indicate a deviation from HC with a p-value < 0.01. Various comparisons analyzed (compound A = compound 1) are shown) a bar graph of lysosomal parameters affected by compound 1 in APBD and HC cells, analyzed by IBP (7C) a volcano plot of proteins affected by APBD and compound 1 under starvation and glycogen loading conditions (7D) a 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) a gene ontology of proteins upregulated by compound 1 (left) and downregulated by compound 1 (right) (7F). [Figure 7E]Figures 7A to 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 different variables (cell characteristics) shown on the x-axis (random forest analysis demonstrated that APBD and HC cell populations are separated with a 93% confidence level) (7A), n=5 HC and n=5 7B) A graph of multiparametric cellular phenotypic characterization of APBD patient dermal fibroblasts (the degree of deviation of the shown cellular features from HC, ordered by the amount of deviation (-log(p-value)). Features whose values ​​are above the dashed line (box) indicate a deviation from HC with a p-value < 0.01. Various comparisons analyzed (compound A = compound 1) are shown) a bar graph of lysosomal parameters affected by compound 1 in APBD and HC cells, analyzed by IBP (7C) a volcano plot of proteins affected by APBD and compound 1 under starvation and glycogen loading conditions (7D) a 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) a gene ontology of proteins upregulated by compound 1 (left) and downregulated by compound 1 (right) (7F). [Figure 7F]Figures 7A to 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 different variables (cell characteristics) shown on the x-axis (random forest analysis demonstrated that APBD and HC cell populations are separated with a 93% confidence level) (7A), n=5 HC and n=5 7B) A graph of multiparametric cellular phenotypic characterization of APBD patient dermal fibroblasts (the degree of deviation of the shown cellular features from HC, ordered by the amount of deviation (-log(p-value)). Features whose values ​​are above the dashed line (box) indicate a deviation from HC with a p-value < 0.01. Various comparisons analyzed (compound A = compound 1) are shown) a bar graph of lysosomal parameters affected by compound 1 in APBD and HC cells, analyzed by IBP (7C) a volcano plot of proteins affected by APBD and compound 1 under starvation and glycogen loading conditions (7D) a 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) a gene ontology of proteins upregulated by compound 1 (left) and downregulated by compound 1 (right) (7F). [Figure 8] Figure 8 shows in silico ADMET (absorption, distribution, metabolism, and excretion toxicity)-compliant polyglucosane reduction compounds and analyses using three different ADMET algorithms. [Figure 9] Figure 9 includes images of the results for an ADMET-incompatible compound (88095528 in Figure 8) that causes wounds in Gbeys / ys mice. [Figure 10] Figure 10 includes a graph of body weight of wild-type C57Bl6J mice treated with compound 1 for 3 months. Mice were injected twice weekly with either 150 μL of compound 1 in 5% DMSO at a dose of 250 mg / kg, or an equal volume of 5% DMSO(V, vehicle) control. Injections were administered intravenously for the first month and subcutaneously for the following two months. [Figure 11]Figure 11 shows 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 and 12B include micrographs of the glycosylation status of LAMP1 and RNase B tested by a 15% SDS-PAGE mobility shift gel stained with QC colloidal Coomersie stain (#1610803, Bio-Rad) after short-term (24-hour) or long-term (72-hour) dialysis (12A), and a sensorgram showing no interaction between the deglycosylated LAMP1-Nter protein (degLAMP1-Nt) and compound 11 (12B). [Figure 12B] Figures 12A and 12B include micrographs of the glycosylation status of LAMP1 and RNase B tested by a 15% SDS-PAGE mobility shift gel stained with QC colloidal Coomersie stain (#1610803, Bio-Rad) after short-term (24-hour) or long-term (72-hour) dialysis (12A), and a sensorgram showing no interaction between the deglycosylated LAMP1-Nter protein (degLAMP1-Nt) and compound 11 (12B). [Figure 13A] Figures 13A and 13B include the predicted binding site of compound 1 in the N-terminal domain of LAMP1, an image of the LAMP1 N-terminus:LAMP1 N-terminal protein:protein docking calculation (13A), and a schematic diagram of the lysosomal membrane (LM), LAMP1, LAMP2, and compound 1, a potential inhibitor (13B). [Figure 13B] Figures 13A and 13B include the predicted binding site of compound 1 in the N-terminal domain of LAMP1, an image of the LAMP1 N-terminus:LAMP1 N-terminal protein:protein docking calculation (13A), and a schematic diagram of the lysosomal membrane (LM), LAMP1, LAMP2, and compound 1, a potential inhibitor (13B). [Figure 14A]Figures 14A–14B include images of heteroaggregates (circles) obtained by NPOT® on fibroblasts (14A) or HC fibroblasts (14B) from APBD patients in the presence of 10⁻⁶M compound 1 and OKMW-XXC (negative control). Each experiment was performed in triplicate. A technical negative control was obtained without the addition of any compound. Each photograph represents a well in a 96-well plate. [Figure 14B] Figures 14A–14B include images of heteroaggregates (circles) obtained by NPOT® on fibroblasts (14A) or HC fibroblasts (14B) from APBD patients in the presence of 10⁻⁶M compound 1 and OKMW-XXC (negative control). Each experiment was performed in triplicate. A technical negative control was obtained without the addition of any compound. Each photograph represents a well in a 96-well plate. [Figure 15] Figure 15 includes micrographs and vertical bar graphs showing autophagy flux in serum-starved and serum-treated (or untreated, shown as Comparative Example A) dermal fibroblasts from PD patients. [Figure 16] Figure 16 includes fluorescence micrographs and vertical bar graphs showing that treatment of primary PD fibroblasts with 144DG11 (50 μM, 24 hours) significantly reduced PAS staining (magenta), indicating glycogen depletion. Yellow: calcein used for cell segmentation, blue: DAPI nuclear staining. The central panel shows quantification of segmented autophagy flux in serum-starved and treated (or not treated, shown as Comparative Example A) dermal fibroblasts from PD patients. [Figure 17]Figure 17 includes vertical bar graphs showing glycolysis (1) and mitochondrial (2) ATP production as measured by Agilent's Seahorse instrument and ATP rate assay kit. Fibroblasts from HC and PD patients were subjected to 48 hours of serum / glucose starvation, followed by 24 hours of supplementation with complete medium either without (untreated) or with (chronic) 50 μM 144DG11. Acutely, 50 μM 144DG11 was added to a 20-minute assay 24 hours after serum / glucose supplementation. Measurements were normalized to the number of cells measured by crystal violet staining. Mean and SD values ​​are shown based on n=6 replicates. In acutely 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 a vertical bar graph showing a blood metabolism panel based on 6-month wild-type or Agl- / - mice (n=5–6) treated for 3 months as shown. Serum triglycerides were reduced by 144DG11, suggesting correction of hyperlipidemia. *p<0.049, ***p<0.004. [Figure 19A] Figures 19A and 19B include fluorescence micrographs showing microglia cells isolated from the brains of AD-modeled 5XFAD mice using CD11b magnetic beads. The microglia were incubated for 24 hours with or without 50 μM 144DG11, fixed, and stained with PAS for the autophagocytic substrates LC3 (19A) and p62 (19B), and for glycogen (all as shown). Decreased levels of both LC3 and p62 indicate that autophagocytosis of these substrates was induced. [Figure 19B]Figures 19A and 19B include fluorescence micrographs showing microglia cells isolated from the brains of AD-modeled 5XFAD mice using CD11b magnetic beads. The microglia were incubated for 24 hours with or without 50 μM 144DG11, fixed, and stained with PAS for the autophagocytic substrates LC3 (19A) and p62 (19B), and for glycogen (all as shown). Decreased levels of both LC3 and p62 indicate that autophagocytosis of these substrates was induced. [Figure 20A] Figures 20A and 20B include fluorescence micrographs showing primary non-small cell lung cancer. Cells were processed and stained for autophagy substrates LC3 (20A) and p62 (20B), as shown in 19A and 19B. [Figure 20B] Figures 20A and 20B include fluorescence micrographs showing primary non-small cell lung cancer. Cells were processed and stained for autophagy substrates LC3 (20A) and p62 (20B), as shown in 19A and 19B. [Figure 21] Figure 21 includes vertical bar graphs showing that cutaneous fibroblasts derived from Gsd1a patients were treated with a solvent or 50 μM compound A for 24 hours, analyzed for NAD+ / NADH ratio using the Promega kit (left panel), and analyzed for Sirt1 expression (center panel) and p62 expression (right panel) by Western immunoblotting. [Modes for carrying out the invention]

[0035] The present invention relates to a pharmaceutical composition for use in the prevention or treatment of diseases or disorders related to lysosome storage.

[0036] The present invention further relates to pharmaceutical compositions for use in the prevention or treatment of diseases or disorders related to polyglucosane 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 autophagy misregulation-related diseases. The present invention further relates to a pharmaceutical composition for use in the prevention or treatment of diseases or disorders related to decreased autophagy.

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

[0040] The present invention also relates to a method for treating or preventing the development of diseases or disorders associated with lysosomal accumulation, polyglucosane accumulation, or abnormal glycogen accumulation in subjects requiring such treatment.

[0041] According to several embodiments, the present invention relates to a compound, a pharmaceutically acceptable salt thereof, an isomer or tautomer thereof, for use in the prevention or treatment of a disease or disorder selected from lysosomal accumulation-related diseases and autophagy misregulation-related diseases, wherein the compound is of formula I:

[0042] [ka] (In the formula,

[0043] [ka] represents a single or double bond, n and m each independently represent integers in the range of 1 to 3, and R and R 1 Each of these independently represents hydrogen, or is absent, R 3 , R 4 , R 5 , R 6 , R 7 and R 8Each of these is independently selected from the group consisting of alkyl, cycloalkyl, alkoxy, hydroxy, thiohydroxy, thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halo, trihalomethyl, cyano, amide, carboxy, sulfonyl, sulfoxy, sulfinyl, and sulfonamide, representing hydrogen, or substituted or unsubstituted. The present invention provides compounds represented by [formula], their pharmaceutically acceptable salts, isomers, or tautomers.

[0044] In some embodiments, R or R 1 One of these represents hydrogen. In some embodiments, R is hydrogen, and R 1 It does not exist. In some embodiments, R 1 R is hydrogen, and R does not exist.

[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] It is either selected from or both.

[0049] According to several embodiments, the present invention provides a pharmaceutical composition for use in the prevention or treatment of a disease or disorder selected from lysosome accumulation-related diseases and autophagy misregulation-related diseases, comprising a compound, a pharmaceutically acceptable salt thereof, an isomer or tautomer thereof, wherein the compound is represented by Formula I as described herein.

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

[0051] In some embodiments, lysosome-related diseases or disorders refer to diseases or disorders related to the inability of lysosomal enzymes to degrade accumulated substrates, lysosome swelling, lysosome rupture, impaired lysosomal signaling, or any combination thereof.

[0052] In some embodiments, the pharmaceutical composition is intended for use in the prevention or treatment of diseases or disorders associated with the inability of lysosomal enzymes to degrade accumulated substrates. In some embodiments, the pharmaceutical composition is intended for use in the prevention or treatment of diseases or disorders associated with swollen lysosomes. In some embodiments, the pharmaceutical composition is intended for use in the prevention or treatment of diseases or disorders associated with lysosomal rupture, which causes the leakage of toxic contents into the cytosol.

[0053] In some embodiments, diseases or disorders associated with lysosome accumulation are selected from the group consisting of Gaucher disease, Fabry disease, Tay-Sachs disease, mucopolysaccharidosis (MPS) disease, aspartylglucosamiuria, GML-gangliosidosis, Krabbe disease (globoid cell leukodystrophy or galactosylceramidripodosis), metachromatic leukodystrophy, Sandhoff disease, mucolibidosis type II (I-Cell disease), mucolibidosis type IIIA (pseudo-Hurler polydystrophy), Niemann-Pick disease types C2 and Cl, Danon disease, free sialic acid storage disorder, mucolibidosis type IV, and multiple sulfatase deficiency (MSD), as well as metabolic disorders.

[0054] The terms “lysosome accumulation,” “lysosomal storage disorder,” and “lysosomal storage disorder” (LSD) are used interchangeably herein to refer to a group of genetic disorders characterized by lysosomal dysfunction and neurodegeneration. These disorders typically result from a single gene deficiency, i.e., a deficiency in a specific enzyme normally required for the degradation of glycosaminoglycans (GAGs), which prevents cells from excreting carbohydrate residues, resulting in their accumulation in the cell’s lysosomes. This accumulation disrupts the normal function of the cell and leads to the clinical symptoms of LSD. Non-exclusive examples of diseases or disorders associated with lysosome accumulation include sphingolipidosis, ceramidase (e.g., Faber disease, Krabbe disease), galactosialidosis, gangliosidosis including alpha-galactosidase (e.g., Fabry disease (α-galactosidase A), Schindler disease (α-galactosidase B)), beta-galactosidase (e.g., GM1 gangliosidosis, GM2 gangliosidosis, Sandhoff disease, Tay-Sachs disease), glucocerebroucidosis (e.g., Gaucher disease (types I, II, III), sphingomyelinase (e.g., lysosomal acid lipase deficiency, Niemann-Pick disease), sulfatidosis (e.g., metachromatic leukodystrophy, multiple sulfatase deficiency), mucopolysaccharidosis (e.g., type I (MPS I (Harler syndrome, MPS IS, Chaille syndrome, MPS I HS)), Hurler-Scheyet syndrome, type II (Hunter syndrome), type III (Sanfilippo syndrome), type IV (Morcchio syndrome), type VI (Malotorami syndrome), type VII (Sly syndrome), type IX (hyaluronidase deficiency), mucolipidosis (e.g., type I (sialidosis), type II (I-Cell disease), type III (pseudo-Hurler polydystrophy / phosphotransferase deficiency), type IV (mucolipidine 1 deficiency)), lipidosis (e.g., Niemann-Pick disease), neuronal ceroid lipofuscinosis (e.g., type 1 Santavuori-Hartia disease / infant NCL (CLN2 / LINCL TPP1)), type 2 Jansky-Birschoski disease / infant NCL (CLN2 / LINCLExamples include TPP1), Batten-Spielmeyer-Voigt disease type 3 / juvenile NCL (CLN3), Kuffs disease type 4 / adult NCL (CLN4), Finnish variant type 5 / late infancy (CLN5), late infancy variant type 6 (CLN6), CLN7 type 7, Northern mannose disease type 8 (CLN8), Turkish late infancy disease type 8 (CLN8), German / Serbian late infancy disease type 9, Congenital cathepsin D deficiency type 10 (CTSD), Wolmann disease, oligosaccharides (e.g., alpha-epilepsy, beta-mannosidosis, aspartylglucosamiuria, fucose disease), lysosomal transport disorders (e.g., cystinosis, pycnodystostosis, Salla disease / sialic acid storage, infant free sialic acid storage), Pompe disease type II, Danon disease type Iib), and cholesterol ester storage disorders.

[0055] In some embodiments, the use of the compound represented by formula I, its pharmaceutically acceptable salts, isomers, or tautomers in the prevention or treatment of diseases or disorders associated with lysosome accumulation does not include or exclude glycogen storage disease (GSD) or related conditions. In some embodiments, the use of the compound represented by formula I, its pharmaceutically acceptable salts, isomers, or tautomers in the prevention or treatment of diseases or disorders associated with lysosome storage does not include or exclude GSD type IV, GSD type VII, APDB, or any combination thereof.

[0056] In some embodiments, the use of compounds represented by formula I, their pharmaceutically acceptable salts, isomers, or tautomers in the prevention or treatment of diseases or disorders associated with lysosome accumulation does not include or excludes glycogen storage disease (GSD)-related neurodegenerative diseases.

[0057] According to some embodiments, the present invention provides a method for treating or preventing the development of a disease or disorder related to lysosome accumulation in a subject requiring such treatment, the method comprising administering a therapeutically effective amount of the above-described pharmaceutical composition to the subject.

[0058] In some embodiments, the therapeutically effective dose is an amount effective in slowing, stopping, or reversing the progression of protein accumulation / aggregation associated with lysosomal storage disorders or impairments. In some embodiments, the therapeutically effective dose is an amount effective in slowing, stopping, or reversing the progression of polyglucosane accumulation or abnormal glycogen accumulation. In some embodiments, the therapeutically effective dose is an amount effective in increasing autophagynolytic activity.

[0059] In some embodiments, the therapeutically effective dose is an amount effective in improving one or more symptoms of pathology associated with lysosomal storage disease and / or reducing neurodegeneration and / or neuroinflammation associated with lysosomal storage disease.

[0060] In another aspect, the present invention provides a method for treating or preventing the development of diseases or disorders associated with reduced or misregulated autophagy activity.

[0061] In some embodiments, autophagy misregulation-related disorders are disorders caused by misfolded protein aggregates. In another embodiment of this aspect, disorders caused by misfolded protein aggregates are 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 misregistration-related disorders” also includes, but is not limited to, any disease or disorder, such as cancer, cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders, in which induction of autophagy contributes to the delay of onset, slowing of progression, cessation, or reversal of one or more symptoms associated with that disease or disorder.

[0063] The term “autophagy misregistration-related disorders” 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 misregistration-related disorders” also includes mental illness or disorder, for example, any mental illness or disorder in which the induction of autophagy contributes to the delay of onset, slowing of progression, cessation, or reversal of one or more symptoms associated with the mental illness or disorder. In one embodiment, the mental illness or disorder is selected from schizophrenia and bipolar disorder.

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

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

[0066] The term "autophagy" refers to a catabolic process involving 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 may include (i) the formation of a membrane around a target region of the cell, separating its contents from the rest of the cytoplasm, and (ii) the fusion of the resulting vesicles with lysosomes and the subsequent degradation of the vesicle contents.

[0067] In some embodiments, methods are provided for reducing neurodegeneration, reducing neuroinflammation, slowing progression, or reducing memory impairment, reducing abnormal lysosome size, reactivating autophagy flux, or any combination thereof, which include administering a therapeutically effective amount of the pharmaceutical composition described herein to a subject.

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

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

[0070] In some embodiments, subjects are identified as having lysosomal storage disorder by the presence of genetic markers for lysosomal storage disorder.

[0071] In some embodiments, administration is within one month, two months, three months, six months, one year, or three years of birth (including any value in between). Each possibility represents a separate embodiment of the present invention.

[0072] In some embodiments, the compounds and pharmaceutical compositions described herein can inhibit and / or modulate the aggregation of one or more proteins, and / or promote the deaggregation of protein fibrils or other protein aggregates, or both. In some embodiments, the compounds and pharmaceutical compositions described herein can inhibit and / or modulate the aggregation of one or more amyloid-forming proteins (e.g., one or more of α-synuclein, Ab, tau, etc.), and / or promote the deaggregation of amyloid protein fibrils or other amyloid protein aggregates, or both.

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

[0074] As used herein, LAMP1 relates to lysosome-associated membrane glycoprotein 1 having UniProt accession number P11279. In some embodiments, LAMP1 is sequence number 4 (MAAPGSARRPLLLLLLLLLLGLMHCASAAMFMVKNGNGTACIMANFSAAFSVNYDTKSGPKNMTFDLPSDATVVLNRSSCGKENTSDPSLVIAFGRGHTLTLNFTRNATRYSVQLMSFVYNLSDTHLFPNASSKEIKTVESITDIRADIDKKYRCVSGTQVHMNNVTVTLHDATIQAYLSNSSFSRGETRCEQDRPSPTTAPP It has the amino acid sequence shown in APPSPSPSPVPKSPSVDKYNVSGTNGTCLLASMGLQLNLTYERKDNTTVTRLLNINPNKTSASGSCGAHLVTLELHSEGTTVLLFQFGMNASSSRFFLQGIQLNTILPDARDPAFKAANGSLRALQATVGNSYKCNAEEHVRVTKAFSVNIFKVWVQAFKVEGGQFGSVEECLLDENSMLIPIAVGGALAGLVLIVLIAYLVGRKRSHAGYQTI).

[0075] In some embodiments, the drug binds to at least one region of LAMP1 selected from either SEQ ID NO: 2 (FSVNYD) or SEQ ID NO: 3 (NVTV) or its homolog.

[0076] In some embodiments, the drug binds to amino acid residues selected from the 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 drug binds to combinations of amino acid residues selected from the 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., substitution) that allows the drug 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., interfering with or inhibiting LAMP1:LAMP1 interactions or inhibiting LAMP1-to-LAMP1 interactions).

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

[0079] Non-exclusive examples of identifying pockets include the following algorithms used by SiteMap, FtSite, or fPocket. In some embodiments, pockets are identified using the SiteMap, FtSite, or fPocket program.

[0080] As used herein, the term “pocket” refers to a cavity, indentation, or depression on the surface of a protein molecule created as a result of the folding of a peptide chain into a three-dimensional structure that makes the protein functional. Pockets can be readily identified by examining the protein structure and / or by using commercially available modeling software.

[0081] As used herein, the term “drug” refers to any small organic molecule that can enter and / or bind to the protein pocket described herein.

[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 naturally occurring biological macromolecules (e.g., proteins, nucleic acids, etc.). In some embodiments, the organic molecules have sizes up to 5,000 Da, up to 2,000 Da, or up to 1,000 Da (including any value in between). Each possibility represents a distinct embodiment of the present invention.

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

[0084] In some embodiments, the drug

[0085] [ka] It is selected from the group consisting of the following.

[0086] In some embodiments, the bond is a specific bond.

[0087] The terms “specific binding” or “preferential binding” refer to binding that occurs between two pairs of species (e.g., enzyme / substrate, receptor / agonist, antibody / antigen, and lectin / carbohydrate) and can be mediated by covalent and / or non-covalent interactions. When the interaction between 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 interactions exist that produce a binding complex between them. Specifically, specific binding is characterized by one member of the pair preferentially binding to a particular species compared to the binding of that member of the pair to other species within the compound family to which that species belongs. Thus, for example, a drug may exhibit an affinity at least twice, preferably at least ten times, at least 100 times, at least 1,000 times, or at least 10,000 times (including any value in between) greater affinity to a particular pocket on a LAMP-1 molecule (i.e., a pocket as defined herein) than to an affinity to a different pocket on the same or related protein. Each possibility represents a distinct embodiment of the present invention.

[0088] In some embodiments, the drug inhibits LAMP1:LAMP1 interaction. In some embodiments, the drug inhibits LAMP1-to-LAMP1 interaction.

[0089] In some embodiments, the drug is intended for use in the prevention or treatment of diseases or disorders selected from among those related to lysosome accumulation, polyglucosane accumulation or abnormal glycogen accumulation and abnormal protein accumulation, and autophagy misregulation-related diseases.

[0090] In some embodiments, the drug is intended for use in the prevention or treatment of diseases or disorders associated with the inability of lysosomal enzymes to degrade accumulated substrates. In some embodiments, the drug is intended for use in the prevention or treatment of diseases or disorders associated with swollen lysosomes. In some embodiments, the drug is intended for use in the prevention or treatment of diseases or disorders associated with lysosomal rupture, which leads to the leakage of toxic contents into the cytosol.

[0091] In some embodiments, the disease or disorder is selected from the group consisting of glycogen storage disease (GSD), adult polyglucosane body disease (APBD), and Lafora disease, Gaucher disease, Fabry disease, Tay-Sachs disease, mucopolysaccharidosis (MPS) disease, aspartylglucosamiuria, GML-gangliosidosis, Krabbe disease (globoid cell leukodystrophy or galactosylceramidridosis), metachromatic leukodystrophy, Sandhoff disease, mucolibidosis type II (I-Cell disease), mucolibidosis type IIIA (pseudo-Hurler polydystrophy), Niemann-Pick disease types C2 and Cl, Danon disease, free sialic acid storage disorder, mucolibidosis type IV, and multiple sulfatase deficiency (MSD), metabolic disorders, obesity, and insulin resistance.

[0092] In some embodiments, the disease or disorder is glycogen storage disease (GSD). In some embodiments, GSD is associated with glycogen branching enzyme deficiency. In some embodiments, GSD is selected from types I-XV. In some embodiments, GSD is type GSD0. In some embodiments, GSD is type GSD1. In some embodiments, GSD is type GSD2. In some embodiments, GSD is type GSD3. In some embodiments, GSD is type GSD4. In some embodiments, GSD is type GSD5. In some embodiments, GSD is type GSD6. In some embodiments, GSD is type GSD7. In some embodiments, GSD is type GSD9. In some embodiments, GSD is type GSD10. In some embodiments, GSD is type GSD11. In some embodiments, GSD is type GSD12. In some embodiments, GSD is type GSD13. In some embodiments, GSD is GSD14 type (also classified as congenital glycosylation disorder type 1 (CDG1T)). In some embodiments, GSD is GSD15 type.

[0093] In some embodiments, the medical condition is one or more of the following: adult polyglucosane body disorder (APBD), Andersen disease, Forbes disease, and Danon disease.

[0094] In some embodiments, the medical condition associated with GSD, or "glycogen branching enzyme deficiency," means a disease or disorder characterized by the deposition, accumulation, or aggregation of polyglucosane bodies in muscles, nerves, 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 systems of the subject.

[0095] Embodiments of the present invention include various methods for customizing the treatment, prevention, or reduction of incidence or severity of GSD and other disorders associated with the accumulation of polyglucosanes.

[0096] In some embodiments, this drug is used to treat neurodegenerative diseases. In some embodiments, this drug is used to treat inflammatory diseases. In some embodiments, this drug is used to treat GSD-related cancers.

[0097] In some embodiments, cancer is cancer associated with reduced autophagy activity. In some embodiments, cancer includes or is lung cancer. In some embodiments, lung cancer is or includes non-small cell lung cancer (NSCLC).

[0098] In some embodiments, the drug is characterized by activity that reduces polyglucosane (PB) cell content. In some embodiments, "reducing PB cell content" means shaping the size of PBs (e.g., shrinking them). In some embodiments, "reducing PB cell content" means degrading PBs (e.g., by regulating glycogen branching enzyme, GBE).

[0099] In some embodiments, the drug can modulate (e.g., inhibit, or in some embodiments, increase) the activity of at least one enzyme.

[0100] In some embodiments, the drug can inhibit one or more enzymes. Non-limiting examples of such enzymes include glycosyltransferases, such as glycogen synthase (GS) and protein phosphatase-1 (PP1).

[0101] In some embodiments, autophagy misregistration-related disorders are disorders caused by misfolded protein aggregates. In another embodiment of this aspect, disorders caused by misfolded protein aggregates are 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. The term “autophagy misregistration-related disorders” 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 misregistration-related disorders” also includes any mental disorder or disorder in which the induction of autophagy contributes to the delay of the onset, slowing of the progression, cessation, or reversal of one or more symptoms associated with the mental disorder or disorder. In one embodiment, the mental disorder or disorder is selected from schizophrenia and bipolar disorder.

[0102] As used herein in relation to enzymes, the term "inhibitory" or any grammatical derivative thereof means that the activity of the enzyme can be prevented, blocked, weakened, or reduced.

[0103] In some embodiments, “reducing activity” means that the activity is 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 value and range in between) compared to an equivalent situation in which the presence of the compound of the Disclosure or a composition of the substance containing it is absent.

[0104] The agents of this disclosure can be designed and used alone, in combination with them, or in combination with any other therapeutic agent to exert dual and possibly synergistic activity when used in combination with them or with any other therapeutic agent.

[0105] According to some embodiments, the present invention provides pharmaceutical compositions comprising the agents described herein.

[0106] In some embodiments, the pharmaceutical composition has a pH in solution 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 in between). Each possibility represents a separate embodiment of the present invention.

[0107] In some embodiments, the drug 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 in between). Each possibility represents a distinct embodiment of the present invention.

[0108] In some embodiments, the drug exhibits specific binding to LAMP-1 in solution at lysosomal pH values ​​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 in between). Each possibility represents a distinct embodiment of the present invention.

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

[0110] According to several embodiments, the present invention provides a method for treating or preventing the development of a disease or disorder related to lysosomal accumulation, polyglucosane accumulation, or abnormal glycogen accumulation in a subject requiring such treatment, the method comprising administering a therapeutically effective amount of the above-mentioned pharmaceutical composition to the subject.

[0111] In some embodiments, diseases or disorders associated with lysosome accumulation are selected from the group consisting of Gaucher disease, Fabry disease, Tay-Sachs disease, mucopolysaccharidosis (MPS) disease, aspartylglucosamiuria, GML-gangliosidosis, Krabbe disease (globoid cell leukodystrophy or galactosylceramidridosis), metachromatic leukodystrophy, Sandhoff disease, mucolibidosis type II (I-Cell disease), mucolibidosis type IIIA (pseudohaller-polydystrophy), Niemann-Pick disease types C2 and Cl, Danon disease, free sialic acid storage disorder, mucolibidosis 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 disease (GSD) due to morphologies of GSD (including, but not limited to, GSD-IV, GSD-VI, GSD-IX, and GSD-XI) and AMP-activated protein kinase γ subunit 2 deficiency. In some embodiments, the compounds of the present disclosure may reduce pathogenic PB accumulation in PB associated with GSD, GSD type IV (APBD and Andersen disease), GSD type VII (Tarui disease), and Lafora 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, orthologues, variants (e.g., allelic variants), and modified forms (e.g., including one or more naturally occurring or engineered mutations). In one embodiment, 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 comprising a domain found in the membrane of an endosome / lysosomal compartment or lysosomal-associated organelle, and further comprising a luminal domain.

[0114] Pharmaceutical compositions comprising the compounds and agents disclosed herein According to one embodiment of the present invention, a pharmaceutical composition is provided comprising one or more compounds and / or agents described herein and a pharmaceutically acceptable carrier.

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

[0116] As used herein, the term "therapeutic dose" refers to the amount of compound administered that can alleviate, to some extent, one or more symptoms of the condition being treated.

[0117] The term “subject” (which should be read to include “individual,” “animal,” “patient,” or “mammal,” where the context allows) defines any subject to which treatment is applied, in particular mammalian subjects. In some embodiments, the subject is human.

[0118] The compounds described herein may be administered in their entirety, or as any of their pharmaceutically acceptable salts, enantiomers, tautomers, diastereomers, protonated or unprotonated forms, solvates, hydrates, or prodrugs, or otherwise utilized.

[0119] The term "pharmaceutically acceptable salt" refers to the charged species of the parent compound and its counterion, which are typically used to modify the solubility of the parent compound and / or reduce any significant irritation to the organism by the parent compound without rendering the compound inactive and beneficial to 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 by conventional methods. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but in other respects, these salts are equivalent to the parent form of the compound in this invention.

[0120] The phrase "pharmaceutically acceptable salt" means that salts of the active compound prepared with relatively non-toxic acids or bases, depending on the specific substituents present in the compounds described herein.

[0121] Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monocarbonate, phosphoric acid, monohydrogen-phosphoric acid, dihydrogen-phosphoric acid, sulfuric acid, monohydrogen-sulfuric acid, hydroiodic acid, or phosphorous acid, as well as salts 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, and methanesulfonic acid. Salts of amino acids such as alginates, and salts of organic acids such as glucuronic acid or galacturonic acid are also included (see, for example, Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain compounds of the present invention contain both a basic functional group and an acidic functional group that enable the compounds described herein to be converted to either a base addition salt or an acid addition salt.

[0122] In some embodiments, the neutral form of the compounds described herein is regenerated by contacting the salt with a base or acid and isolating the parent compound in the 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 in other respects, these salts are equivalent to the parent form of the compound in the present invention.

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

[0124] In some embodiments, the compounds described herein have an asymmetric carbon atom (optical center) or a double bond, and racemates, diastereomers, tautomers, geometric isomers, and individual isomers are included within the scope of the present invention.

[0125] As used herein and in the art, the term “enantiomer” refers to a stereoisomer of a compound that can be superimposed on its counterpart only by complete inversion / reflection (mirror image) of each other. Enantiomers are said to be “palmar-like” because they refer to each other like a right hand and a left hand. Enantiomers have identical chemical and physical properties except when they exist in a palmar-like environment (e.g., all living systems).

[0126] In some embodiments, the compounds described herein may exist in a non-solvated form and in a solvated form, such as a hydrated form. Generally, the solvated form is equivalent to the non-solvated form and is included within the scope of the invention. Certain compounds of the invention may exist in multiple crystalline or amorphous forms. Generally, all physical forms are equivalent for the uses intended by the invention and are intended to be within the scope of the invention.

[0127] The term "solvate" refers to a variable stoichiometric complex (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 and acetic acid.

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

[0129] In some embodiments, “pharmaceutical composition” refers to preparations of one or more compounds described herein (as active ingredients), or physiologically acceptable salts or prodrugs thereof, with, but not limited to, physiologically appropriate carriers, excipients, lubricants, buffers, antimicrobial agents, fillers (e.g., mannitol), antioxidants (e.g., ascorbic acid or sodium bisulfite), anti-inflammatory agents, antiviral agents, chemotherapeutic agents, antihistamines, and other chemical components.

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

[0131] The terms "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" may be used interchangeably and refer to carriers or diluents that do not cause significant irritation to the organism and do not inhibit the biological activity and properties of the administered compound.

[0132] In this specification, the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of a drug. Non-limiting examples of excipients include calcium carbonate, calcium phosphate, various sugars and various types of starches, cellulose derivatives, gelatin, vegetable oils and polyethylene glycol.

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

[0134] Accordingly, in some embodiments, pharmaceutical compositions for use according to the present invention may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and adjuvants that facilitate the processing of the compound into pharmaceutically usable preparations. The appropriate formulation depends on the selected route of administration. The dosage may vary depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dosage may 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 composition may be formulated for administration by one or more routes, depending on whether topical or systemic treatment or administration is selected and the area to be treated. As further described throughout this specification, administration may be orally, dental, by inhalation, or parenterally, for example, by intravenous infusion or intraperitoneal, subcutaneous, intramuscular or intravenous injection, or topically (e.g., into the eye, vagina, rectum, or nasal cavity).

[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, etc., may be necessary or desirable.

[0137] Compositions for oral administration may include powders or granules, suspensions, dental compositions, solutions in water or a non-aqueous medium, pouches, pills, caplets, capsules, or tablets. Thickeners, diluents, flavoring agents, dispersing agents, emulsifiers, or binders may be desirable.

[0138] Examples of parenteral administration formulations include, but are not limited to, sterile solutions that may also contain buffers, diluents, and other suitable additives. Sustained-release compositions are intended for therapeutic use.

[0139] The amount of the composition administered will naturally depend on the patient being treated, the severity of their suffering, the method of administration, and the judgment of the prescribing physician.

[0140] This pharmaceutical composition may further contain, but are not limited to, antimicrobial agents, antioxidants, buffers, fillers, surfactants, anti-inflammatory agents, antiviral agents, chemotherapeutic agents, and antihistamines.

[0141] The compositions of the present invention may, if necessary, be supplied 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. The pack may include, for example, 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 relating to a type of container prescribed by a government agency that regulates the manufacture, use, or sale of a drug, which reflects the agency's approval of the composition or the form of administration in humans or veterinary medicine. Such notice may be, for example, a label approved by the U.S. Food and Drug Administration for a prescription drug, or a package insert for an approved product.

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

[0143] Screening method According to one aspect of several embodiments of the present invention, a method is provided for determining the suitability of a compound for preventing or treating diseases or disorders related to lysosome accumulation, polyglucosane accumulation or abnormal glycogen accumulation, and diseases or disorders related to abnormal protein accumulation, as well as autophagy misregulation-related diseases, the method comprising contacting the compound with a pocket domain in the N-terminal domain of lysosome-associated membrane protein 1 (LAMP-1; SEQ ID NO: 1), wherein binding of the compound to the pocket indicates that the compound is effective in treating the diseases or disorders.

[0144] In some embodiments, the provided drug includes a conjugation comprising either SEQ ID NO: 2 (FSVNYD) or SEQ ID NO: 3 (NVTV).

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

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

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

[0148] In some embodiments, the method includes detecting the reduction of PB exerted by one or more selected compounds (e.g., small molecules).

[0149] Thanks to its ability to enable computer-based screening of libraries of compounds inherently possessing any of a variety of chemical, biological, and / or physical characteristics, this method is understood to allow for the identification of compounds that can exhibit optimal in vivo pharmacokinetics, optimally low immunogenicity, and optimal efficacy for all prior art compounds that can reduce PB cell content by, for example, modifying impaired enzyme activity associated with glycogen storage disease (e.g., glycogen synthase or glycogen branching enzyme).

[0150] In some embodiments, the method includes biochemically determining the ability of a compound to reduce PB cell content.

[0151] In some embodiments, biochemical identification involves subjecting cells to periodate Schiff (PAS) staining to provide PAS-stained cells. In some embodiments, the method further includes washing the sample to remove unreacted Schiff reagent, and then detecting a signal (e.g., photofluorescence) derived from the PAS-stained sample at a specified wavelength.

[0152] Further embodiments of the method of this disclosure are shown in the following Examples section.

[0153] definition As used herein, the term “alkyl” refers to aliphatic hydrocarbons including linear and branched groups. Preferably, alkyl groups have 21 to 100 carbon atoms, more preferably 21 to 50 carbon atoms. Whenever a numerical range is given, for example, when “21 to 100” is mentioned herein, it means that the group (in this case, alkyl group) may contain 100 or fewer carbon atoms, such as 21 carbon atoms, 22 carbon atoms, 23 carbon atoms, and so on. In the context of the present invention, a “long-chain alkyl” is an alkyl having at least 20 carbon atoms in its back chain (the longest path of consecutive covalent atoms). Thus, a short-chain alkyl has 20 or fewer back chain carbons. Alkyls may be substituted or unsubstituted, as defined herein.

[0154] As used herein, the term "alkyl" also includes saturated and unsaturated hydrocarbons, and therefore the term further includes alkenyls and alkynyls.

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

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

[0157] The term "cycloalkyl" refers to a group of all-carbon monocyclic or fused ring (i.e., a ring sharing adjacent pairs of carbon atoms) that does not have a fully conjugated π-electron system of one or more rings. Cycloalkyl groups may be substituted or unsubstituted, as shown herein.

[0158] The term "aryl" refers to a monocyclic or polycyclic (i.e., a ring sharing adjacent carbon atom pairs) all-carbon group having a fully conjugated π-electron system. The aryl group may be substituted or unsubstituted, as shown herein.

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

[0160] The term "aryloxy" represents the -O-aryl as defined herein.

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

[0162] The terms "halogen," "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 halogenated compounds.

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

[0165] The term "hydroxyl" or "hydroxy" represents the -OH group.

[0166] The terms "thiohydroxy" or "thiol" represent the -SH group.

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

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

[0169] The term "amine" represents the -NR'R'' group, where R' and R'' are as described herein.

[0170] The term "heteroaryl" refers to a monocyclic or fused ring (i.e., a ring sharing adjacent pairs of atoms) that has one or more atoms in the ring, such as nitrogen, oxygen, and sulfur, and also has a fully conjugated π-electron system. Non-exclusive 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 containing one or more atoms such as nitrogen, oxygen, and sulfur in the ring. The ring may also contain one or more double bonds. However, the ring does not have a fully conjugated π-electron system. Typical examples include piperidine, piperazine, tetrahydrofuran, tetrahydropyran, and morpholino.

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

[0173] The term "carbonyl" refers to the -C(=O)-R' group (wherein R' is as previously defined herein).

[0174] The above terms also include their thio derivatives (thiocarboxy and thiocarbonyl).

[0175] The term "thiocarbonyl" represents a -C(=S)-R' group (wherein R' is as previously defined herein).

[0176] The "thiocarboxyl" group represents a -C(=S)-OR' group (wherein R' is as defined herein).

[0177] The "sulfinyl" group represents the -S(=O)-R' group (wherein R' is as defined herein).

[0178] The "sulfonyl" or "sulfonate" group represents a -S(=O)2-R' group (wherein Rx is as defined herein).

[0179] The "carbamyl" or "carbamate" group represents the -OC(=O)-NR'R'' group (wherein R' is as defined herein, and R'' is as defined for R').

[0180] The "nitro" group refers to the -NO2 group.

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

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

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

[0184] The terms "phosphonyl" or "phosphonate" represent two -OP(=O)(OR') groups, where R' is as previously defined herein.

[0185] The term "phosphenyl" represents the -PR'R'' group, where R' and R'' are as previously defined herein.

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

[0187] The term "heteroaryl" refers to a monocyclic or fused ring (i.e., a ring sharing adjacent pairs of atoms) that has one or more atoms in the ring, such as nitrogen, oxygen, and sulfur, and also has a fully conjugated π-electron system. Non-limiting examples of heteroaryl groups include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, and purine. Heteroaryl groups may be substituted with one or more substituents or may be unsubstituted, as described herein. Typical examples include thiadiazole, pyridine, pyrrole, oxazole, indole, and purine.

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

[0189] The term "haloalkyl" refers to the previously defined alkyl group that is further substituted by one or more halogenated compounds.

[0190] general As used herein, the term “approximately” refers to ±10%.

[0191] The terms "comprises," "comprising," "includes," "including," and "have," as well as their variations, all mean "to include, but not limited to."

[0192] The phrase "~consists of" means "to include and be limited to ~".

[0193] The term "essentially derived from" means that the composition, method, or structure may include additional components, steps, and / or parts, provided that the additional components, steps, and / or parts do not substantially alter the basic and novel properties of the claimed composition, method, or structure.

[0194] The term “exemplary” is used herein to mean “serving as an example, case, or illustration.” Any embodiment described as “exemplary” should not necessarily be construed as being preferable or advantageous to other embodiments, and / or preclude the incorporation of features from other embodiments.

[0195] The phrase “optionally” is used herein to mean “provided in some embodiments but not in other embodiments.” Any particular embodiment of the present invention may include several “optionally” features, insofar as such features do not conflict.

[0196] As used herein, the singular forms "a," "an," and "the" include multiple references unless the context clearly indicates otherwise. For example, the terms "compound" or "at least one compound" may include multiple compounds, including mixtures thereof.

[0197] Throughout this application, various embodiments of the present invention may be presented in range form. It should be understood that the range form is merely for convenience and brevity and should not be interpreted as an inflexible limitation on the scope of the invention. Therefore, the range description should be considered to specifically disclose all possible subranges and the individual numbers within those ranges. For example, a range description such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and the individual numbers within those ranges such as 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0198] Where a numerical range is indicated herein, it is always intended to include any cited digit (fraction or integer) within that range. The expressions “ranging / ranges” between a first number and a second number, and “ranging / ranges from” the first number to the second number, are used interchangeably herein and mean including the first and second numbers, as well as all fractions and integers between them.

[0199] As used herein, the term “method” means a style, means, technique and procedure for accomplishing a given task, and includes, but is not limited to, styles, means, techniques and procedures known to practitioners in the fields of chemistry, pharmacy, biology, biochemistry and medicine, or readily developed from styles, means, techniques and procedures known to them.

[0200] As used herein, the term “to treat” includes inhibiting, substantially inhibiting, delaying or reversing the progression of a condition, substantially improving the clinical or aesthetic symptoms of a condition, or substantially preventing the appearance of the clinical or aesthetic symptoms of a condition.

[0201] For clarity, certain features of the present invention described in relation to separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the present invention, described in the context of a single embodiment for brevity, may be provided separately, in any suitable subcombination, or appropriately in other described embodiments of the present invention. Certain features described in relation to various embodiments should not be considered essential features of those embodiments unless the embodiments would not function without those elements.

[0202] Various embodiments and aspects of the present invention, as described above and claimed in the following claims, are experimentally supported in the following examples. [Examples]

[0203] The following examples, in conjunction with the above description, illustrate some non-limiting embodiments of the present invention.

[0204] Materials and methods research design The presented experiment combines 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, the inventors used Compound 1 in Gbe ys / ysThe ability to correct disease phenotypes in female mice was tested. Initially, two groups (5% DMSO vehicle and Compound 1) consisting of n=7–9 animals each were used. Based on the obtained mean and SD, it was retrospectively demonstrated that these numbers provide sufficient power, as 80% power had already been achieved with n=5 animals / group. Additional open field, walking, and stretch reflex tests (Figures 1E–1H) also included a C57BL / 6 wild-type control group of n=9 animals. Animals were excluded from the experiment if their body weight decreased by less than 10% during sequential weight measurements or if their body weight decreased by less than 20% from the start. Sample size decreased slightly over time due to death. Compound 1 was injected twice weekly at a dose of 250 mg / kg in 150 μL of 5% DMSO. The vehicle control was 5% DMSO. The injections were administered intravenously (IV) for the first month, and then subcutaneously (SC) due to limited space for injection and scarring of the animals' tails. The inventors initiated injections at 4 months of age, two months before disease onset, or at 6 months of age (onset) for comparison, anticipating a favorable preventative effect. Treatment continued until 10 months of age. The effects of compound 1 on various motor parameters were tested approximately every two weeks. At the end of these experiments, some mice were sacrificed by cervical dislocation, with n=2 wild-type and n=7 Gbe mice. ys / ys Tissues were collected from vehicle-treated mice and n=9 compound 1-treated mice, sectioned, fixed, and stained with PAS for diastase-resistant prostaglandins (Figures 2A-2C). Tissue glycogen was biochemically measured as described. In addition, the pharmacokinetic profile of compound 1 was measured by LC-MS / MS of serum and tissue obtained from n=3 mice / time point. Experimenters were blinded to treatment assignments.

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

[0206] Histological PG and glycogen measurement To characterize the histopathological effects of compound 1, we used wt and Gbe treated with compound 1 and vehicle. ys / ys Brain, heart, muscle, nerve bundles (peripheral nerves), and liver tissue were dissected from the animals. The tissues were extracted, fixed, embedded in paraffin, and sectioned. After deparaffinization, the sections were treated with 0.5% diastase for 5 minutes to digest non-polyglucosane glycogen, leaving polyglucosane. Next, the sections were washed, stained with PAS for polyglucosane, counterstained with hematoxylin, and analyzed by light microscopy, all as described above. For biochemical glycogen measurement, 100 mg of each tissue was subjected to alkaline hydrolysis and boiling, followed by ethanol precipitation of glycogen. The glycogen was then enzymatically digested into glucose using 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 special microscopic-grade 96-well plates (Grenier Bio-One, Germany). Following different treatments, a mixture of Thermo Scientific cell fluorescent dyes in PBS was added to each well in a 5% CO2 incubator at 37°C for 30 minutes. This mix (Figures 4C and 7B) contained DAPI (1 μg / ml, nuclear (DNA) staining), MitoTracker Green (500 nM, voltage-independent mitochondrial staining), TMRE (500 μM, voltage-gated mitochondrial staining), and Cell Mask Deep Red (0.5 μg / ml, cytosolic staining). In Figure 7C, only lysosomes were stained with LysoTracker Deep Red (75 nM). Next, the cells were fixed with 4% paraformaldehyde (PFA), washed with PBS, and the plates were transferred to an InCell2200 instrument (GE Healthcare, UK) to acquire images at 40x magnification. The generated output was based on comparative fluorescence intensity. Target segmentation was performed using multi-target analysis on a GE analytical workstation to identify nuclear and cell boundaries. All assay parameters (including acquisition exposure time, objective lens, and analytical 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, as well as tissue samples from the brain, kidney, hind limb quadriceps, heart, liver, and spleen, were collected, homogenized, and extracted with acetonitrile according to established guidelines. Calibration curves were prepared using compound 1 at concentrations of 0, 1, 10, 100, and 1,000 ng / ml in a 1 mg / ml solution of 4-tert-butyl-2-(4H-1,2,4-triazole-4-yl)phenol (ChemBridge) as an internal standard (IS). Tissue samples were then dissolved in a 1 mg / ml IS solution and spiked with compound 1 from 0 to 1,000 ng / ml to create a standard curve, from which the tissue levels of compound 1 were determined. Samples were analyzed by LC-MS / MS Sciex Triple Quad™ 5500 mass spectrometer.

[0209] ethics The in vivo experiment was approved by the IACUC at Hebrew University.

[0210] statistical analysis In Figures 1A–1M, the significance of the overall trend was tested using a two-way ANOVA with repeated measures. This test examines how the response is influenced by two factors: compound 1v control and duration of administration, which are given repeatedly (and therefore measured repeatedly). Compound 1 and the vehicle were compared using Bonferroni's test, which is very robust because it corrects for multiple comparisons (the threshold for determining significance at each time point decreases inversely with the number of comparisons). As a result, most differences at specific time points became insignificant with increasing numbers of comparisons, and the inventors sometimes chose to also show data from multiple t-tests that did not correct for multiple comparisons. In Figures 4D and 6E, the inventors used a one-way ANOVA with Sidak's post-hoc correction for multiple comparisons. Another statistical test used was Student's t-test.

[0211] Target identification using nematic protein organization (NPOT) Human healthy fibroblasts and fibroblasts derived from two APBD patients were subjected to NPOT®. All analyses were performed blindly by Inoviem Scientific. Protein homogenates from these fibroblast dry pellets were prepared by three cycles of rapid freezing (liquid nitrogen) and slow thawing (on ice), and mixed at maximum vortex speed for 30 seconds. Sample protein concentrations were 50–66 mg / ml when measured by BCA. NPOT® is a proprietary technology provided by Inoviem Scientific (Food Scientific) specializing in the isolation and identification of specific polymer scaffolds directly from human tissue under basic or pathological conditions. This technology is based on Kirkwood-Buff molecular crowding and aggregation theory. It enables the formation and label-free identification of polymer complexes involved in physiological or pathological processes. A unique strength of Inoviem Scientific is its ability to directly analyze drug-protein and protein-protein interactions in human tissues from complex mixtures, without disrupting the natural molecular conformation and consequently maintaining the original physiological or pathological state.

[0212] Under laminar flow and sterilization conditions, 10 -6 Compound 1 of M and negative controls from HTS screening were separately mixed with protein homogenates (containing soluble and membrane proteins) and subjected to NPOT® isolation. Macromolecular assemblies associated with ligands were separated using a differential microdialysis system, and the macromolecules (protein groups) moved through the liquid phase based on their physicochemical properties. The moving macromolecules gradually grew from nematic crystals to macromolecular heteroassemblies thanks to molecular interactions between the test drug and its target. The heteroassemblies were left overnight, isolated in 96-well plates, and identified by LC-MS / MS.

[0213] Figures 14A-14B show heteroassemblies formed in APBD patients and HC fibroblasts in the presence of compound 1 and a negative control. Each compound, upon contact with the indicated protein homogenate, produced distinctive heteroassemblies with a common reticular morphology. These experiments were performed in triplicate for each compound. For each of these biological replicas, the heteroassemblies were isolated and their protein content was analyzed by LC-MS / MS. The negative control was obtained using protein homogenates under NPOT® conditions without the addition of the compound and showed no aggregation. This further supports the idea that heteroassembly formation is initiated by the compound, rather than by endogenous small molecules, through their interaction with the primary target.

[0214] Under a Zeiss SteREO Discovery V8 microscope, each formed heteroassembly was isolated by microdissection, 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 and the relative amount of protein to be used for subsequent digestion steps and injection into the LC-MS / MS instrument for proteomics analysis.

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

[0216] For data analysis and targeted deconvolution, Inoviem Scientific (Texas Scientific) developed a proprietary database and software that enables 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 includes all NPOT® datasets obtained from various tissues, organs, or cell lines, various species, and unrelated compounds. The InoPerA® software can calculate the occurrence of a given gene in the entire database or in specific datasets that meet defined criteria such as species or organ. Inoviem removed contaminants observed in NPOT® performed on human tissues and cells, corresponding to 613 NPOT®-bound LC-MS / MS analyses. As a result, this tool can rapidly highlight rare proteins that create new therapeutic targets within the dataset (Figure 5B).

[0217] We also used DAVID, another bioinformatics resource, to detect tissue-specific expression, gene ontology, and function-related gene enrichment. We investigated network enrichment within the dataset using STRING analysis (string-db.org). STRING is one of ELIXIR's core data resources (like Ensembl or UniProt) and contains known predicted protein-protein interactions. Inoviem uses stringent parameters and retains only known interactions ("experimentally determined" and "curated database" interaction sources). This allowed us to decipher protein-protein associations within complex datasets and further enabled DAVID pathway analysis. Additionally, we used Reactome (reactome.org), a free, open-source, curated, and peer-reviewed pathway database. This database provides intuitive bioinformatics tools for visualizing, interpreting, and analyzing pathway knowledge to support findings obtained elsewhere.

[0218] In the bioinformatics pipeline, the first step of filtering involved removing "false positives" from mass spectrometry, i.e., proteins found in only one replica and containing only one specific peptide. The dataset was then compared in a 2x2 matrix (144DG11 and its respective negative control) in human dermal fibroblast tissue. The next step in protein list analysis was the identification of nonspecific proteins, i.e., proteins repeatedly found in all NPOT® experiments (InoPERA®). Contaminants (or "frequent hits") observed in human dermal fibroblasts were removed. This cleared the protein list of the interactome, representing 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 function-related gene groups. The primary canonical, disease, and functional pathways underlying the interactome of compound 1 were the lysosomal membrane (References: GO:0005765 and KEGG pathway hsa04142). In parallel, STRING analysis (string-db.org) was used to visualize prominent nodes and enhanced networks. For this initial ranking of specific proteins in the compound interactome, the inventors did not use the signal intensity of peptides sequenced by MS because 1) the inherent characteristics of the technique (contrary to, for example, classical immunoprecipitation protocols) could not be based on protein quantification, and 2) the inventors did not use LC-MS / MS quantification protocols (which imply higher costs and longer analysis times). This unbiased analysis allowed Innoviem to classify potentially relevant proteins and classify them according to their involvement in specific pathways or in relation to specific diseases. Following this bioinformatics selection, eight proteins belonging to the autophagosome-autolysosome pathway were discovered (Figure 5B).The discovery of this clear and enhanced network indicates the overall success of the NPOT (registered trademark) experiment.

[0219] Computer 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: interdomain linker, (4) residues S217-D378: C-terminal domain, and (5) residues E379-I417: transmembrane segment. The inventors considered that 1. the signal sequence and transmembrane segment are irrelevant to small molecule binding, and 2. the interdomain linker is unstructured and highly glycosylated (7 out of 20 residues), and therefore too complex to model, so they analyzed only the N-terminal and C-terminal domains. The inventors did not consider glycosylation at the N-terminus and C-terminus. The C and N-terminal domains were modeled based on the known crystal structure of the 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 5 gv 0s themselves) were prepared at pH 5 using the "Protein Preparation Wizard" implemented in Schrodinger 2020-2. Possible binding sites were identified using three different computational tools: SiteMap, FtSite, and fPocket. In total, 130 arbitrary sites were identified in the 3D structures of 11 LAMP1s. Docking calculations were performed for each of the proposed binding sites: 418 molecules were selected as decoys from a large and diverse database of approximately 30 million molecules, according to the compound 1 applicability domain (Lipinski rule property). This 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 its 233 decoys (prepared at pH 5) were performed for all estimated binding sites (130 sites in total) in all models. These calculations were performed using the Glide algorithm, as implemented in Schrodinger 2020-2.According to the docking result analysis, among 130 sites, compound 1 was ranked in the top 10% at 3 grids from SiteMap, 3 grids from FtSite, and 12 grids from fPocket at 18 sites. Eight grids were in the C-terminal domain and the other ten GRIDS were in the N-terminal domain.

[0220] The inventors 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, the inventors understood that site 1 of SiteMap, site 3 of fPocket, and site 2 of FtSite pointed to the same pocket (residues F50-D55, N62, L67, F118, Y120-L122, T125, L127-S133, N164-V166).

[0221] The inventors investigated the differences among the three binding modes (Figure 5E): two of the three binding modes (SiteMap and fPocket) were identical, and the part of compound 1 in the third (FtSite) seemed to be rotated relative to the other two.

[0222] To predict the probability of obtaining the unique binding observed for compound 1 purely by chance, the inventors repeated the analysis previously performed for compound 1 for all 233 decoys. Only in 14 of the 234 molecules (233 decoys + compound 1) did the inventors observe the same results for compound 1, i.e., the molecule whose binding to the pocket was predicted by three different tools (Table 1). This indicates a relatively small probability (14 / 234, approximately 6%). Furthermore, the pocket identified for compound 1 (Figure 5E) is the most common (5 out of 14 matches, 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 anticipated medicinal chemistry of compound 1. The table shows when molecules enter the same pocket according to three different tools (for predicting binding sites). The three digits following "Site" in the first column indicate the site ranking by SiteMap (1st), FtSite (2nd), and fPocket (3rd).

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

[0224] The inventors repeated the analysis with a less restrictive definition of the binding site and obtained similar results, namely, that 45 out of 234 molecules (approximately 19%) successfully docked to at least one of the predicted pockets. However, in 14 of the 45 molecules, the inventors observed binding to multiple sites, indicating non-discrimination. Therefore, overall, 31 out of 234 molecules (approximately 12.7%) successfully docked to one of the predicted sites. In summary, the inventors computationally identified possible binding sites for compound 1 in the N-terminal domain of LAMP1 and predicted with high confidence that these results were specific to compound 1, given the low probability of obtaining the same results for decoy molecules.

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

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

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

[0228] MS Data Analysis: Mass spectral data were processed using the MaxQuant computing 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. This search included cysteine ​​carbamide methylation as a fixed modification and N-terminal acetylation and methionine oxidation as variable modifications, allowing for up to two miscleavages. The run-to-run matching option was used. Peptides of at least 7 amino acid length were considered, and the required FDR was set to 1% at both the peptide and protein levels. Relative protein quantification in MaxQuant was performed using the label-free quantification (LFQ) algorithm. Statistical analysis (n=4-7) was performed using the Perseus statistical package. Only proteins with at least three valid LFQ values ​​in at least one sample group were accepted for statistical analysis using t-tests (p<0.05).

[0229] Example 1 Compound 1 is Gbe ys / ys Improving survival and motor function in mice The inventors used compound 1 (Figure 1A) in the APBD mouse model Gbe ys / ysThe compound was tested for its ability to correct the deficient motor phenotype and short lifespan in the subject. Compound 1 is one of the 19PG-reducing HTS hits previously discovered by the inventors. It was selected by in silico ADMET (absorption, distribution, metabolism, elimination, and toxicity) tests performed to predict which of these hits are safe and pharmacokinetically and pharmacodynamically preferable, and therefore worth further investigation (Figure 8, compound "A"). In fact, compounds with low ADMET scores, such as "B" (Figure 8), were ineffective and caused adverse effects such as wounds (Figure 9). Furthermore, safety evaluations in wild-type mice confirmed that compound 1 administered at 250 mg / kg in 5% DMSO (the highest possible dose due to solubility and DMSO toxicity issues) for 3 months did not affect the animals' weight gain 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 hour and 24 hours of treatment, mice were again tested for abnormal spontaneous behaviors (e.g., immobility, excessive running, stereotypic movements, and postural abnormalities) (Irwin test). Compound 1 did not cause any adverse effects in these Irwin tests (Table 2).

[0230] [Table 2]

[0231] Importantly, as shown in Figure 1B, treatment with compound 1 significantly improved animal survival compared to vehicle-treated animals (log-rank test p-value < 0.000692). The extension of lifespan likely reflects improvements in several parameters related to the animals' growth capacity. The most notable parameter in this regard is animal body weight. Compound 1 actually mitigated the time-dependent loss of animal body weight caused by the disease (Figure 1C). The inventors also tested the effects of compound 1 on various motor 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 manifested as increased movement and an increased tendency to move towards the center, which may also be related to improved stress and anxiety (Figure 1E). Gbe in open-field performance ys / ys The progressive deterioration in mice is associated with gait disturbances. Therefore, we tested the effect of compound 1 on gait in 9-month-old mice with severely affected gait. At that age, compound 1 did indeed improve gait or increase stride length (Figure 1F). This data also indicates that the most significant improvement among all motor parameters tested was on the overall stretch reflex (Figure 1G). Overall stretch reflexes throughout the entire study period were significantly improved by compound 1 (Figure 1G, p<0.05) at nine specific time points (asterisks in Figure 1G). This effect is particularly significant because its patient correlation is with pyramidal limb paresis or upper motor neuron sign, one of the major neurological deficits in APBD patients. Importantly, open-field performance (Figure 1E), gait (Figure 1F), and stretch reflex (Figure 1H) were significantly improved by compound 1, but they did not recover to wild-type levels, indicating that while compound 1's performance is effective, there is still some room for future improvement.

[0232] To investigate the effects of compound 1 on motor parameters, the inventors initiated injections of compound 1 at 4 months of age, two months before the onset of the disease, assuming a favorable preventive effect. Such an effect is expected in neurodegenerative disorders such as APBD, where already dead neurons cannot be affected by post-onset treatment. This assumption was validated for all parameters improved by compound 1—open field (Figure 1I), body weight (Figure 1J), and total stretch reflex (Figure 1K). When administered after the onset of the disease at 6 months of age, no improvement was observed. Notably, the stretch reflex, the parameter most affected by compound 1, was also the only parameter improved by the compound from the disease progression stage at 9 months of age (Figure 1K). The overall beneficial effect of compound 1 is best understood by animal photographs showing reduced kyphosis and a neater coat in the treated animals (Figures 1L-1M).

[0233] Example 2 Compound 1 reduces the histopathological accumulation of polyglucosane and glycogen according to its in vivo distribution. Since compound 1 significantly improved motor and survival parameters, the inventors initiated an investigation into its histopathological effects. This information is important to determine whether the expected mode of action of compound 1 (reduction of polyglucosane levels in fibroblasts), as observed ex vivo, also occurs in vivo, and if so, in which tissues. Brain, heart, muscle, nerve bundles (peripheral nerves), and liver tissue obtained from animals treated with compound 1 and vehicle were collected after sacrifice at 9.5 months of age. The same tissues obtained from wild-type mice were used as a control. Following diastase treatment to digest non-polyglucosane glycogen while preserving polyglucosane, sections were stained for polyglucosane with periodate Schiff (PAS) reagent, counterstained with hematoxylin, and analyzed under a light microscope. The results (Figure 2A) show a significant decrease in polyglucosane levels in the brain, liver, heart, and peripheral nerves, with no apparent effect on muscle polyglucosane. Biochemically determined total glycogen levels were also affected accordingly (Figure 2B). These results likely explain the improvements observed in motor parameters and animal reproduction (Figures 1A–1M).

[0234] Pharmacokinetic analysis is useful in describing the effects of compound 1 in situ, regardless of its intrinsic ability to modify polyglucosanes in isolated cells. This is because the timing, distribution, and stability of arrival 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 250 mg / kg of compound 1 by subcutaneous injection to Gbe, as performed in efficacy experiments. ys / ysMice were treated. The mice were then sacrificed at 0, 30, 60, 90, and 210 minutes after administration. 100 μL of serum, as well as tissue from the brain, kidney, hindlimb skeletal muscle, heart, liver, and spleen, were collected, homogenized, extracted, and analyzed for compound 1 levels by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The results are shown in Figure 2C. The different effects of compound 1 on glycogen and polyglucosane content in different tissues are consistent with their different distributions and residence times in each tissue. The greatest degree of polyglucosane / glycogen reduction was observed in the liver, which was consistent with the longest residence time / persistence of compound 1 observed in that organ (estimated half-life exceeding 3 hours). The heart and brain showed intermediate levels of compound 1. However, these levels persisted up to 60 minutes after injection, which may explain the compound 1-mediated reduction of polyglucosane and the glycogen content observed in these tissues. On the other hand, the muscles showed only a very small accumulation of compound 1, which is consistent with the insufficient effect of this compound on muscle glycogen and polyglucosane content. Based on the sampling time used, C max The time to the highest C was 30 minutes for all observed tissues, and all of these tissues showed similar absorption rates. max This was observed in the liver and kidneys, consistent with their well-established and rapid perfusion. As expected, the lowest C max This was observed in the quadriceps skeletal muscle, which is known to be an organ with insufficient perfusion.

[0235] Example 3 Compound 1 enhances carbohydrate metabolism and improves the metabolic panel in vivo. The effects of Compound 1 on various metabolic parameters were investigated in vivo using metabolic cages. Nutritional 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, and 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 in wild-type (wt) animals. The parallel increases in total energy consumption (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 ys / ys a therapeutic advantage since Gbe mice store glycogen as insoluble and pathogenic polysaccharides. The stimulation of locomotor activity (Figure 3E) and the stimulation of food and water intake (Figures 3F - 3H) are consistent with this observation of the stimulation of carbohydrate catabolism in animals affected by Compound 1. Furthermore, taken together, the increased nutrient burning and food intake indicate that the metabolic efficiency in animals affected by Compound 1 can be improved.

[0236] The inventors further tested whether Compound 1 can correct the hypoglycemia and hyperlipidemia observed in Gbe ys / ys mice. Such effects are expected from agents that can induce the catabolism of liver glycogen and subsequently increase blood glucose. The results of blood biochemical tests in 9.5-month-old Gbe ys / ys mice showed that treatment with Compound 1 corrected the characteristic hypoglycemia and hyperlipidemia in mice to control levels (Figure 3I). The functions of muscle (creatine kinase) and liver (alanine transaminase) were not affected by this treatment (Figure 3I).

[0237] Example 4 Compound 1 enhances catabolism in glycogen-overloaded APBD patient cells The RQ shift for carbohydrate catabolism observed in vivo prompted us to investigate whether carbohydrate catabolism is also upregulated intracellularly. To that end, given the significant variability in glycogen levels among fibroblasts derived from different APBD patients (Figure 4A), we first aimed to induce a physiological glycogen overload or glycogen-loaded state equivalent to that found in tissues. We determined that glycogen loading was generated by 48 hours of glucose starvation followed by 24 hours of sugar supplementation, which presumably induces accelerated glucose uptake, subsequently inducing glycogen synthesis. This starvation / supplementation condition did indeed increase intracellular glycogen levels, as shown by PAS staining (Figure 4B). Furthermore, phenotypic analysis based on multiparametric high-content imaging revealed that under glycogen-loaded conditions, cell area, nuclear intensity, and, importantly, mitochondrial mass features (see box in Figure 4C) deviated from healthy controls (HC) compared to cells subjected to glucose starvation alone. Therefore, we selected this glycogen loading condition and analyzed cellular-level catabolism 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 cutaneous fibroblasts from both HC and APBD patients. Acute assay supplementation with 144DG11 was more effective than 48-hour pretreatment with the compound in increasing the glycolytic contribution to ATP production. 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. The inventors investigated the mechanism of action of 144DG11. To this end, they first identified its molecular targets. Nematic protein retrieval (NPOT, Inoviem, Ltd.) was applied to homogenates of APBD patient fibroblasts. NPOT analysis revealed a protein heteroassembly that spontaneously generated 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 autophagocytic proteins or lysosomal proteins (Figure 5B). Furthermore, the inventors tested the specific interactions of 144DG11 with six of the eight targets discovered by NPOT using a cell thermal shift assay. These results (Figure 5C) suggest that LAMP1, rather than other protein targets, directly interacts with 144DG11. This finding relates to a novel pathogenicity hypothesis linking cellular glycogen overload to glycogen transport to lysosomes via the starch-binding domain containing protein 1. To verify the interaction of 144DG11 with LAMP1, the inventors used surface plasmon resonance (SPR) technology. SPR data (Figure 5D) show specific and dose-dependent binding of 144DG11 to the luminal portion of LAMP1 only at lysosomal pH 4.5–5, and not at cytoplasmic pH 7, with some binding initiating at an intermediate pH of 6. In summary, these results constitute strong and satisfactory 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 of this binding D It is relatively high (6.3mM), which is probably due to a slow K onThis is explained by (Figure 5D, association rate at pH 4.5). The inventors hypothesized that this slow association rate could be explained by the inhibition of 144DG11 diffusion by bulky oligosaccharides at the glycosylation site. Therefore, the inventors repeated SPR experiments using chemically deglycosylated luminal LAMP1 domains. However, deglycosylated LAMP1 did not bind to 144DG11, which is likely due to a significant structural change induced by deglycosylation (Figures 12A-12B), and therefore the inventors were unable to test whether oligosaccharide steric hindrance affects the binding kinetics of 144DG11 to LAMP1. The inventors further investigated the binding of 144DG11 to LAMP1 by structural computer docking. In searching for the predicted binding site of compound 1 in LAMP1, we analyzed the N-terminal and C-terminal subdomains of the luminal domain (residues A29-R195 and S217-D378, respectively) that have similar topologies. These domains were modeled and computer-docked to compound 1 against a decoy at intralysomal pH 5 based on the known crystal structure of the 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 extremely 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 the autophagy flux in primary APBD fibroblasts. This is demonstrated by increased sensitivity to lysosomal inhibitors in the presence of compound 1. As seen in Figure 6A, the lysosomal inhibitor increased the LC3ii / LC3i ratio (autophagy arrest) in cells treated with compound 1 compared to untreated cells. The increase in autophagy flux by compound 1 is also shown by a decrease in the level of the autophagy substrate p62 (Figure 6A). Furthermore, APBD modeling Gbe ys / ys Transmission electron microscopy analysis of mouse liver sections demonstrates a decrease in lysosomal glycogen after treatment with compound 1 (Figure 6B).

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

[0241] To test the effects of LAMP1-KD and compound 1 on nutrient utilization, the inventors again used ATP rate assays in LAMP1-KD and control APBD fibroblasts acutely or chronically treated with compound 1 (Figures 4A-4D). These results (Figure 6E) show that starvation was more limited in LAMP1-KD (LAMP1-KD-S UT vs. LAMP1-KD+S UT, p<0.0001) than in GFP-transduced controls (control UT-S vs. control UT+S, p<0.36) (resulting in a decrease in overall ATP production). In LAMP1-KD cells, starvation also increased the relative contribution of respiration to ATP production (78% in LAMP1-KD-S UT vs. 48% in LAMP1-KD+S UT (orange bar)). These observations are consistent with the higher ATP production efficiency of respiration compared to glycolysis, as suggested by their higher overall ATP production rates under basal conditions, and possibly the higher ATP demand of LAMP-KD compared to control cells (see: LAMP1-KD+SUT vs. control+SUT, p<0.01). The effect of compound 1 on LAMP1-KD cells and control cells followed its selective increase in catabolic (ATP-producing) autophagy flux in LAMP1-KD cells compared to control cells (Figure 6C). Under non-starvation conditions, supplementation with compound 1 significantly increased total ATP production and respiratory ATP production in LAMP1-KD cells (see LAMP1-KD+S UT and LAMP1-KD+S chronic (p<0.03 for total, p<0.0008 for respiratory) and LAMP1-D+S acute (p<0.01 for overall and respiratory)), but had little effect on ATP production in control cells, and rather sharply decreased it (see control UT+S and control+S chronic (p<0.1) and control UT+S acute (p<0.0008 for decrease)). Under starvation conditions, control cells only increased respiratory ATP production in response to the transient effect of acutely supplemented compound 1 (see control UT-S and control-S acute, p<0.004). No significant effect of long-term supplementation with compound 1 was observed in control cells (see reference). Control UT-S vs. Control-S (chronic), p<0.3).In contrast, starved LAMP1-KD cells increased both respiratory ATP and glycolytic ATP in response to acute supplementation of compound 1, which likely reflects the short-term conversion of glucose from glycogenolysis to glycolysis (see LAMP1-KD-S UT and 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 increased in KD cells (see LAMP1-KD-S UT and LAMP-KD-S chronic (p<0.15 for glycoATP, p<0.0002 for mitoATP)).

[0242] Example 7 Compound 1 restores abnormal mitochondrial and lysosome characteristics at the cellular level. Since the inventors have shown that the mode of action of compound 1 is involved in lysosomal catabolism that increases ATP production, the inventors decided to investigate whether the cellular characteristics regulated by compound 1 are related to its catabolic effect. As a first step, the inventors needed a holistic and feature-specific classification method that would allow for the quantification of differences between APBD cells and HC cells, and thus the estimation of the restorative effect of compound 1 on APBD cells. Using an InCell2200 high-content image analyzer, the inventors performed a complete multiparametric analysis of APBD and age- and sex-matched HC dermal fibroblasts. This image-based phenotypic determination (IBP) campaign included 45 independent cellular parameters encompassing a broad cytomorphological spectrum. By analyzing dermal fibroblasts derived from 17 APBD patients and 5 HC individuals, the inventors demonstrated that dermal fibroblasts derived from APBD patients are phenotypically distinguishable from HC dermal fibroblasts (Figure 7A). Once IBP was established as a useful and highly sensitive classification tool, the inventors tested the effect of compound 1 on IBP signatures. The analysis (Figure 7B, upper panel), limited to four color channels and thus excluding lysosomal markers, and analyzed separately (Figure 7C), shows that compound 1 primarily affected nuclear and mitochondrial membrane potential (TMRE) parameters, which were among the features most influenced by the disease phenotype. As expected, this effect was more pronounced (higher -logP values) when APBD fibroblasts treated with compound 1 were compared to untreated HC fibroblasts (a comparison more relevant to the clinical context). As demonstrated for other features (Figures 4D and 6C-6E), treatment with compound 1 alone may have similar effects on both affected and healthy cells, thereby potentially bringing the two phenotypes closer together in treated cells and partially masking the effect of this compound on APBD versus HC.The lower panel of Figure 7B clearly demonstrates that for most features, compound 1 induced the same trend (increase or decrease) in both affected and healthy cells (note that APBD / compound 1 (dot bar) should be compared to APBD (blank bar), and HC / compound 1 (black bar) should be compared to the horizontal line). Compound 1 also reduced lysosome size in APBD cells (Figure 7C), which may be related to improved autophagy flux (Figure 6) and lysosomal function, as observed in healthy cells compared to lysosomal-damaged cells. Furthermore, compound 1 also hyperpolarized mitochondrial membrane potential (MMP) and depolarized it in response to the disease state in APBD (Figure 7B), which likely follows increased mitochondrial nutrient supply due to enhanced autophagy catabolism.

[0243] To validate the image-based analysis of disease states and cellular characteristics regulated by compound 1, the inventors analyzed the effects of disease and treatment on protein expression. As shown in Figure 7D, under 48 hours of starvation, 12.2% of the 2,898 proteins analyzed in APBD patients were upregulated and 6.8% were downregulated compared to HC cells. As an important control, GBE was indeed downregulated in APBD cells (Figure 7D). When glucose supplementation was performed after starvation (glycogen loading, Figures 4A-4D), only 6% of proteins were upregulated and 5% were downregulated, which likely suggests that a more specific subset of proteins is needed to manage excessive glycogen loading. For example, autophagocytic proteins (Fyco1, Rab12, Rab7A, PIP4K2B, SQSTM1, and SNAP29) were only upregulated in APBD cells after glycogen loading. Next, the inventors investigated the proteomics effects of compound 1 in starvation (48-hour starvation) and glycogen overload (48-hour starvation / 24-hour glucose) APBD cells, modifying only 1.7% and 1.3% of all proteins, respectively. The apparent corrective effect of compound 1 could be revealed by proteins downregulated or upregulated by the APBD disease state, which were conversely upregulated or downregulated by compound 1 (Figure 7E). The discovered proteins (49 upregulated, 39 downregulated, Figure 7E) were analyzed using the DAVID functional annotation tool according to the cellular component category containing the largest number of proteins. Proteins upregulated by compound 1 belonged to eight key gene ontology (GO) terms, which included lysosomes, secretory pathways, and oxidatively phosphorylated 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 lysosomal glycosylation enzymes idulonidase and phosphomannomutase 2 under glycogen loading, while under starvation, they were the nucleic acid-binding proteins GRSF1 and HNRPCL1, which are clearly not directly related to glycogen and lysosome catabolism. The lipid-producing protein HSD17B12 was decreased by APBD and induced by compound 1 under both conditions. The proteins downregulated by compound 1 belonged to four GO terms, including secretory pathways and macromolecular complexes (Figure 7F, right panel). The proteins increased by APBD and, in contrast, decreased by compound 1 belonged to lysosomal sorting (VPS16) and carbohydrate biosynthesis (NANS) in starvated cells, and to transcription (RUBL1), signaling (STAM2), and pH regulation (SLC9A1) in glycogen-overloaded cells. Interestingly, Na + / H + Pharmacological inhibition of the antiporter SLC9A1 induces autophagy flux in cardiomyocytes, as does its downregulation in APBD fibroblasts by compound 1 (Figure 6). The protein downregulated by compound 1 under both starvation and glycogen-loaded conditions is the retrograde transport regulator VPS51, which is also involved in lysosome sorting. In summary, the APBD corrective effect of compound 1 is at least partially related to lysosomal functions whose regulation by this compound has been well established by the inventors (Figures 5-6).

[0245] This experiment demonstrates that hit compound 1, discovered by HTS, can treat APBD in in vivo and ex vivo models. After treatment with compound 1, the inventors observed improvements in motor, survival, and histological parameters (Figures 1-2). Since APBD is caused by indigestible carbohydrates, these improvements suggest that compound 1 affects carbohydrate metabolism, which in turn prompted the inventors to conduct in vivo metabolic experiments (Figures 3A-3I). This was the first in vivo metabolic experiment in a GSD animal model. Since APBD mice store glycogen as insoluble polyglucosanes, the inventors used a metabolic cage to test whether compound 1 could affect the ability of these animals to use alternative nutrition (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 increased total energy expenditure, walking activity, food intake, and water intake induced by compound 1 (all consistent with catabolic stimulation). ys / ys Since mice and APBD patients store glycogen as insoluble and pathogenic polyglucosanes, its catabolism constitutes a therapeutic benefit. 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 pre-formed PGs or glycogen. PG formation depends on the balance between GYS activity and GBE activity - the higher the GYS / GBE activity ratio, the more elongated, less branched soluble glycogen is formed, which preferentially forms PGs compared to shorter chains. On the other hand, the degradation of existing PGs and glycogen (PG precursors) by compound 1 is a more direct target and is expected to be more effective than the de novo inhibition of PG formation by the GYS inhibitor guaiacol, which preemptively avoids the formation of harmful PGs. In fact, studies in LD model mice have shown that conditional GYS knockdown after disease onset cannot remove pre-formed harmful Lafora PGs.

[0246] A key challenge in drug discovery is determining the relevant targets and mechanisms of action of drug candidates. To this end, we hereby applied Inoviem's ​​NPOT® protein target identification approach. Recognized as a primary tool for identifying protein targets of small molecules, and having identified several therapeutically relevant targets, this technique identifies compound-target interactions within the cell's natural physiological environment. This means that what is identified is not the target itself, as in other techniques, but a primary target with its signaling pathway or functional quaternary network. Determining the cellular pathway regulated by the test compound, as done for compound 1, is crucial for presumptive formulation of other drugs into the same pathway, which can eventually significantly improve therapeutic efficacy in clinical settings. Furthermore, NPOT® can also confirm the specificity of target binding by filtering out miscellaneous binders and eliminating binding to negative controls (in this case, negative compounds in HTS) and endogenous ligands (Figure 5A). Nevertheless, according to these criteria, the binding of compound 1 to LAMP1 and, through it, to its functional quaternary network was specific (Figure 5B), and in SPR validation it showed dose response and lysosomal pH dependence (Figure 5D), but its apparent LAMP1 binding K D The iodine content is relatively high (6.3 mM), which at first glance could be an obstacle to its clinical application. This problem can be addressed as follows: 1. Pharmacologically relevant findings include that compound 1 specifically interacted with the lysosomal-autophagosome interactome (Figure 5B) and that this interaction was not toxic (Figures 8-11, Table 2).

[0247] This finding suggests low affinity (high K D) Eliminate nonspecific interactions with putative off-targets, which is a major concern with ligands. Conventional approaches to improving the affinity of low-affinity drug candidates are based on medicinal chemistry. In GSD, such approaches have been used to increase the affinity of GYS inhibitors. However, in contrast to GYS, whose reduction is relatively tolerable, LAMP proteins belong to the housekeeping autolysosome mechanism (Figure 5B), and their inhibition can impair perinatal survival, for example, LAMP1-KD without compensatory elevation of LAMP2. Therefore, high-affinity LAMP1 inhibitors may be as toxic to APBD fibroblasts as LAMP1-KD (Figure 6), and the low affinity of compound 1, a LAMP1 inhibitor discovered by the inventors, may actually constitute a clinical benefit by mitigating the suppression of a function essential for cell proliferation (household function). Furthermore, computer analysis has shown that the compound 1 binding pocket of LAMP1 is highly draggable (Figure 5E), meaning that medicinal chemistry analysis is expected to discover various substitutes for compound 1 that could improve its efficacy.

[0248] The discovery of LAMP1 (Figure 5B), which contains a heterogeneous assembly as a functional network target rather than a single protein, opens up the possibility of autophagy-based therapies and truly expands the landscape of therapeutic targets. The autolysosome network was discovered not only in Figure 5B but also by our multi-feature imaging analysis, along with bioenergy parameters that may be modified by changes in nutrient availability associated with autophagy (Figures 7B and 7C). Further support for the relevance of this pathway as a target of compound 1 is provided by proteomics data (Figures 7D-7F) and the actual enhancement of autophagy flux by compound 1 in cells (Figure 6).

[0249] Mechanistically, LAMP1 is a type I lysosomal membrane protein that, along with LAMP2, plays a central role in lysosomal integrity and function. Consequently, LAMP1 is also important for lysosomal involvement in the autophagocytic process, although LAMP2 is more important. Therefore, LAMP1 knockdown is often associated with a decrease in autophagocytosis. However, consistent with these results, other studies have shown that LAMP1-KD actually increased autophagocytic function, and this has also been shown for another transmembrane lysosomal protein, TMEM192. Since autophagocytic flux is not always defined by sensitivity to lysosomal inhibitors, these apparent discrepancies likely depend on cell type, assay conditions, and even the definition of autophagocytosis. To predict the molecular mechanism of action of compound 1 on LAMP1, we used computational chemistry. The 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 this compound inhibits LAMP1-LAMP1 interactions. Experimental data show that cleavage of the N-terminal domain of LAMP1 impairs LAMP1 / LAMP1 and LAMP1 / LAMP2 assemblies, while cleavage of the more mobile N-terminal domain of LAMP2 has the opposite effect (Figure 13B). Therefore, we can hypothesize that the LAMP1 N-terminal domain promotes LAMP1 / LAMP1 and LAMP1 / LAMP2 interactions, and that inhibition of LAMP1 / LAMP1 or LAMP1 / LAMP2 interactions at the N-terminal domain by compound 1 reduces the LAMP1-effective lysosomal membrane density. Thus, we can hypothesize that treatment with compound 1 is equivalent to LAMP1-KD, which may explain its enhancement of the LAMP1-KD effect. The slight increase in LAMP1 levels induced by compound 1 (1.2-fold) likely reflects binding-mediated stabilization (Figure 5C) and does not significantly counteract the compound 1-mediated decrease in membrane density. The inventors hypothesize that a decrease in compound 1-mediated LAMP1 membrane density increases glycophagy due to a demonstrated increase in LAMP2 in the lysosomal membrane during LAMP1-KD.LAMP2 has been observed to enhance autophagosome-lysosome fusion (and consequently, autophagocytic flux) through interaction with the autophagosome peripheral protein GORASP2. Alternatively, lysosomal membrane spacing by LAMP1-KD / compound 1 may enable glycogen transfer into lysosomes (and resulting degradation) by the STBD1 protein. Importantly, lysosomal glycogen degradation occurs in parallel with cytoplasmic degradation, and in particular, in the GSDIV mouse model, which also models APBD in mice, overexpression of the lysosomal glycogenase α-glucosidase-corrected pathological condition occurs.

[0250] In summary, this study demonstrates that compound 1 is a novel catabolic compound capable of breaking down PG and excess glycogen by activating the autophagy pathway. This study lays the foundation for the clinical use of compound 1 in the treatment of APBD patients for whom there are currently no treatment options. Furthermore, it positions compound 1 as a lead compound for treating other GSDs through a safe reduction of glycogen surcharge.

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

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

[0253] These results (Figure 17) indicate that 144DG11 increased the relative contribution of overall ATP production and glycolytic ATP production, at the expense of mitochondrial (OxPhos) ATP production. This phenomenon was selectively observed in PD cells, rather than in healthy control (HC) primary cutaneous fibroblasts. Assay supplementation with 144DG11 was more effective in increasing the glycolytic contribution to ATP production than 24-hour pretreatment with the compound, and its effect on ATP production was not significant, likely due to cellular adaptation. These results suggest that glucose derived from 144DG11-mediated enhanced autophagy catabolism is available for ATP production. These observations are consistent with those made in APBD fibroblasts, and therefore indicate 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 agent. We anticipate that 144DG11 will be more effective in obesity induced by a Western-style diet (high-fat / high-carbohydrate). Observations in GSD4 (Kakhlon et al., (2021)) and GSD3 (Figure 18) that 144DG11 can reduce plasma triglyceride levels strongly suggest that 144DG11 can be developed as an effective anti-obesity therapy.

[0255] As indicated by the 144DG11-mediated reduction of total LC3 and p62, this compound induced autophagy in brain microglia derived from Alzheimer's disease (AD) model mice (Figure 19). This observation is significant as it demonstrates the therapeutic potential of 144DG11 for treating AD. Microglia are the most pro-inflammatory tissue in the brain and are currently at the center of innovative therapeutic research for AD. Furthermore, since neuroinflammation is now accepted as a major virulence factor in AD, and activation of microglial autophagy and mitophagy is a major therapeutic agent (see, e.g., 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). The induction of autophagy has demonstrated therapeutic value in NSCLC (see, e.g., Wang et al., (2021)). Notably, 144DG11 did not reduce glycogen levels in either microglial or NSCLC cells, suggesting that glycogen is not degraded in these cells by the autolysosomal pathway modifiable by 144DG11. This lack of effect on glycogen also suggests that glycogen accumulation may not be pathogenic in these cells. However, the autophagocytic clearance of harmful inclusions by 144DG11 is likely beneficial in many different disease states, as demonstrated herein.

[0257] NAD+ and NADH are crucial 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 signifies an acceleration of electron flow in the mitochondrial electron transport chain (note that this is not mitochondrial ATP production) and an acceleration of glycolysis for better management of metabolic demands. Furthermore, Sirt1 induction, which is often associated with an increase in the NAD+ / NADH ratio, is a well-established and innovative anti-aging, calorie restriction mimic and anti-cancer therapy (see, e.g., Hyun et al., (2020)). Therefore, the results in Gsd1a cells showing an increased NAD+ / NADH ratio and Sirt1 induction (Figure 21) indicate that 144DG11 is a promising therapy for numerous different metabolic disorders, age-related complications, and cancers. In addition, 144DG11 downregulated p62 and showed increased autophagy flux in Gsd1a cells, as demonstrated in GSD4 and PD cells.

[0258] While the present invention has been described in relation to its particular embodiments, it is evident that many alternatives, modifications, and variations are apparent to those skilled in the art. Therefore, the appended claims are intended to encompass the technical concept and broader scope of all such alternatives, modifications, and variations.

[0259] All publications, patents, and patent applications referenced herein are incorporated herein by reference in the same manner as any individual publication, patent, or patent application is specifically and individually indicated to be incorporated herein by reference in whole. Furthermore, any citation or specification of any reference in this application should not be construed as an acknowledgment that such reference is available as prior art of the present invention. Section headings, insofar as they are used, should not necessarily be construed as restrictive.

Claims

1. A pharmaceutical composition for use in the prevention or treatment of lysosome storage-related diseases selected from the group consisting of mucopolysaccharidosis (MPS), Gaucher disease, Fabry disease, Tay-Sachs disease, aspartylglucosamiuria, GM1-gangliosidosis, Krabbe disease, metachromatic leukodystrophy, Sandhoff disease, mucolipidosis, Niemann-Pick disease, free sialic acid storage disorder, multiple sulfatase deficiency (MSD), sphingolipidosis, Faber disease, GM2-gangliosidosis, lysosomal acid lipase deficiency, neuronal ceroid lipofuscinosis, Wolmann disease, cholesterol ester storage disorder, galactosialidosis, alpha-mannosidosis, beta-mannosidosis, fucose disease, Salla disease, Schindler disease, cystinosis, and pycnodysosstosis, comprising a compound or a pharmaceutically acceptable salt thereof, wherein the compound is: 【Chemistry 1】 A pharmaceutical composition that is either or both.

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

3. A pharmaceutical composition for use in the treatment of Alzheimer's disease, comprising a compound or a pharmaceutically acceptable salt thereof, wherein the compound is: 【Chemistry 2】 A pharmaceutical composition that is either or both.

4. A pharmaceutical composition for use in the treatment of non-small cell lung cancer (NSCLC), comprising a compound or a pharmaceutically acceptable salt thereof, wherein the compound is: 【Transformation 3】 A pharmaceutical composition that is either or both.

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