VCP INHIBITORS AND THEIR USE FOR TREATMENT - Patent application

Abstract

VCP inhibitors and their use for treatment. The present invention relates to inhibitors of valosin-containing protein (VCP or p97) and their use in the treatment or prevention of diseases such as amyotrophic lateral sclerosis (ALS). In particular, the present invention provides VCP inhibitors for use in methods of treating or preventing ALS, where the subject has been identified as not having a pathogenic genetic mutation in the VCP gene (non-VCP-associated ALS). The present invention also relates to methods of identifying a patient as not having a pathogenic mutation in the VCP gene.

Description

[Technical Field]

[0001] The present invention relates to inhibitors of valosin-containing protein (VCP or p97) and their use in the treatment or prevention of diseases such as amyotrophic lateral sclerosis (ALS). In particular, the present invention provides VCP inhibitors for use in methods of treating or preventing ALS, in which the subject has been identified as not having a pathogenic genetic mutation in the VCP gene (non-VCP-associated ALS). The present invention also relates to methods of identifying patients as not having a pathogenic mutation in the VCP gene. [Background technology]

[0002] Amyotrophic lateral sclerosis (ALS) is a consistently fatal neurological disease characterized by the selective and progressive degeneration of motor neurons. Dysregulated RNA metabolism, particularly the subcellular localization and function of RNA-binding proteins (RBPs), plays a central role in ALS pathogenesis. RBPs orchestrate the RNA life cycle, controlling transcription, splicing, RNA localization, function, and decay.

[0003] Several ALS-causing gene mutations encode RBPs, including transactive response DNA-binding protein 43 (TARDBP, encoding TDP-43), FUS / TLS (fused in sarcoma / translocated in liposarcoma) or FUS, and heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1). Subcellular mislocalization of RBPs is also a pathological hallmark of ALS, with TDP-43 mislocalized from the nucleus to the cytoplasm in 97% of ALS cases [1].

[0004] More recently, widespread mislocalization of SFPQ and FUS across various ALS models and postmortem tissues from sporadic ALS has also been reported [2, 3]. Accumulation of RBPs in the cytoplasm is thought to contribute to the formation of RBP oligomers and fibrillar pathological cytoplasmic inclusions seen in ALS [4, 5]. Because a single RBP can bind to thousands of RNA targets, impairment of even a single RBP can have broad and diverse effects on RNA metabolism [6].

[0005] Valosin-containing protein (VCP or p97) is an abundant AAA+ ATPase (an ATPase associated with diverse cellular activities) with diverse intracellular functions encompassing almost every aspect of cellular physiology. VCP functions include maintaining protein homeostasis, mitochondrial quality control, and apoptosis [7]. The structure of VCP is important for its many functions; it is a hexameric protein, with each subunit containing an N-terminal domain, two ATPase domains (D1 and D2), and a disordered C-terminal domain. Autosomal dominant VCP mutations account for approximately 2% of familial ALS cases [8].

[0006] Although limited, pathogenic variants in VCP have also been identified in sporadic cases of the disease. [9] Due to its role in many cellular pathways, disruption of VCP function can result in several forms of the disease. For example, mutations in VCP have also been identified in other neurodegenerative disorders, including inclusion body myopathy, Paget's disease, and frontotemporal dementia (IBMPFD).

[0007] Pathogenic mutations in VCP are most commonly found in the N-terminal domain, which is involved in binding cofactors and ubiquitinated substrates, but also occur in the D1 and D2 domains

[10] . The majority of VCP mutations have been biochemically shown to be associated with normal or increased ATPase activity in cell models, with the R155C mutation exhibiting more than twice the activity of its wild-type counterpart

[11] . However, it remains controversial whether disease mutants with increased ATPase activity cause disease through a dominant-active or dominant-negative mechanism. Furthermore, this has not yet been systematically addressed in patient-derived motor neurons, which have the advantage of transmitting the mutation at a pathophysiological level.

[0008] Missense mutations in VCP account for 1–2% of familial ALS cases but can also cause an autosomal dominant disorder known as inclusion body myopathy, Paget's disease, and frontotemporal dementia (IBMPFD). ALS-causing VCP mutations recapitulate key features of sporadic ALS, including nuclear-to-cytoplasmic mislocalization of key RBPs, including TDP-43, FUS, and SFPQ [2, 3, 8, 29]. However, the mechanism by which VCP mutations lead to RBP mislocalization in ALS remains elusive.

[0009] It was previously reported that human induced pluripotent stem cells (iPSCs) can be reliably differentiated into highly enriched, functionally validated spinal motor neurons with a time-resolved molecular phenotype of VCP-associated ALS [2, 3, 12, 13]. Herein, we first investigated the nuclear-cytoplasmic distribution of key RBPs compared to control motor neurons using this established human stem cell model of VCP mutation-associated ALS.

[0010] We demonstrate that TDP-43, FUS, and SFPQ exhibit an abnormally reduced nuclear-to-cytoplasmic ratio in VCP mutant motor neurons, which extends to their abnormal presence within neurites. We find that treatment of control motor neurons with targeted VCP D2 ATPase inhibitors does not recapitulate the ALS RBP mislocalization phenotype, arguing against their loss of function in disease. Importantly, we find that in VCP mutant motor neurons, the nuclear-to-cytoplasmic mislocalization of both TDP-43 and FUS, as well as the nuclear-to-neurite mislocalization of TDP-43, FUS, and SFPQ, is reversible by treatment with pharmacological inhibitors of the VCP D2 ATPase domain.

[0011] Taken together, these findings support a model in which VCP mutations cause increased D2 ATPase activity, which in turn leads to mislocalization of TDP-43, FUS, and SFPQ from the nucleus to the cytosol. Our study raises the possibility of utilizing FDA-approved VCP inhibitors that target the D2 ATPase domain in the treatment of VCP-associated ALS. Summary of the Invention

[0012] This Summary introduces concepts that are further described in the Detailed Description. It is not intended to identify essential features of the claimed subject matter, nor should it be used to limit the scope of the claimed subject matter.

[0013] As described in Example 3, the present invention is the first to disclose that pharmacological inhibition of the VCP D2 ATPase domain does not induce an ALS phenotype in healthy human motor neurons (e.g., motor neurons that do not have a pathogenic mutation in the VCP gene). Instead, the present invention is the first to demonstrate that a VCP inhibitor can reverse the mislocalization of an RNA-binding protein in control (non-VCP mutant) motor neurons. Thus, the present application provides the first disclosure that a VCP inhibitor can be used to treat or prevent ALS in subjects identified as not having a pathogenic mutation in the VCP gene (non-VCP-associated ALS).

[0014] A significant result is the altered localization of TDP-43 and FUS, as shown in Example 3. Mislocalization of TDP-43 is a key disease hallmark of ALS. It is mislocalized from the nucleus to the cytoplasm in over 97% of ALS cases. Figure 2 (described in Example 3) demonstrates for the first time that VCP inhibitors can enhance the nuclear localization of TDP-43 in healthy human motor neurons, providing evidence for a therapeutic pathway for non-VCP-related ALS.

[0015] The present invention provides a VCP (valosin-containing protein) inhibitor for use in a method for treating or preventing amyotrophic lateral sclerosis (ALS) in a subject. In some embodiments, the ALS is non-VCP-associated ALS.

[0016] In some embodiments, the subject is not identified as having a pathogenic mutation in the VCP gene. In some embodiments, the subject is identified as not having a pathogenic mutation in the VCP gene. In some embodiments, the ALS is non-VCP-associated ALS.

[0017] In some embodiments, the subject may not have certain pathogenic genetic mutations in the VCP gene. In some embodiments, the subject is identified as not having a pathogenic genetic mutation in the VCP gene at any of positions R155 and R191. In some embodiments, the subject is identified as not having a pathogenic genetic mutation in the VCP gene selected from the list consisting of R155C and R191Q. In some embodiments, the subject is identified as not having a pathogenic genetic mutation in the VCP gene at any of positions R95, I114, I151, R155, G156, M158, R159, R191, N387, N401, R487, D592, R662, and N750. In some embodiments, the subject is identified as not having any pathogenic genetic mutation in the VCP gene selected from the list consisting of: R95C, R95G, I114V, I151V, R155H, R155C, G156C, M158V, R159G, R159C, R159H, R191G, R191Q, N387T, N401S, R487H, D592N, R662C, and N750S.

[0018] In some embodiments, the subject may have one or more pathogenic genetic mutations in the TARDBP gene, hi some embodiments, the subject has been identified as having one or more pathogenic genetic mutations in the TARDBP gene.

[0019] In some embodiments, amyotrophic lateral sclerosis is associated with a decreased nuclear-to-cytoplasmic ratio of one or more of TDP-43, FUS, and / or SFPQ. In some embodiments, a VCP inhibitor ameliorates one or more symptoms associated with a decreased nuclear-to-cytoplasmic ratio of one or more of TDP-43, FUS, and / or SFPQ.

[0020] In some embodiments, amyotrophic lateral sclerosis is associated with a decreased nuclear-to-cytoplasmic ratio of TDP-43. In some embodiments, a VCP inhibitor ameliorates one or more symptoms associated with a decreased nuclear-to-cytoplasmic ratio of TDP-43.

[0021] In some embodiments, amyotrophic lateral sclerosis is associated with a decreased nuclear-to-cytoplasmic ratio of FUS, and in some embodiments, a VCP inhibitor ameliorates one or more symptoms associated with a decreased nuclear-to-cytoplasmic ratio of FUS.

[0022] In some embodiments, amyotrophic lateral sclerosis is associated with a decreased nuclear-to-cytoplasmic ratio of SFPQ. In some embodiments, a VCP inhibitor ameliorates one or more symptoms associated with a decreased nuclear-to-cytoplasmic ratio of SFPQ.

[0023] In some embodiments, treating or preventing ALS includes partially or completely alleviating, ameliorating, eliminating, inhibiting, delaying the onset, reducing the severity and / or incidence of neurological dysfunction in a patient suffering from or susceptible to ALS. In some embodiments, neurological dysfunction includes one or more of the symptoms associated with dysfunction of the central nervous system, such as developmental delay, progressive cognitive impairment, hearing loss, impaired language development, motor skill deficits, hyperactivity, aggression, and / or sleep disorders.

[0024] In some embodiments, treating or preventing ALS with a VCP inhibitor results in an improvement or amelioration of one or more symptoms of neurological dysfunction by more than about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% compared to the symptoms of neurological dysfunction in the absence of the VCP inhibitor. In some embodiments, treating or preventing ALS with a VCP inhibitor results in an improvement or amelioration of one or more symptoms of neurological dysfunction by more than about 50%, about 80%, about 90%, or about 95%.

[0025] In some embodiments, the VCP inhibitor inhibits the D2 ATPase domain of VCP.

[0026] In some embodiments, the VCP inhibitor is selected from the group consisting of ML240 (2-(2-amino-1H-benzimidazol-1-yl)-8-methoxy-N-(phenylmethyl)-4-quinazolinamine), ML241, 2-anilino-4-aryl-1,3-thiazole, 3,4-methylenedioxy-6-nitrostyrene, DBeQ (N2,N4-dibenzylquinazoline-2,4-diamine), CB-5083 (1-[7,8-dihydro-4-[(phenylmethyl)amino]-5H-pyrano[4,3-d]pyrimidin-2-yl]-2-methyl- methyl-1H-indole-4-carboxamide), CB-5339 (1-[4-(benzylamino)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-2-yl]-2-methylindole-4-carboxamide), UPCDC-30245 (1-(3-(5-fluoro-1H-indol-2-yl)phenyl)-N-(2-(4-isopropylpiperazin-1-yl)ethyl)piperidin-4-amine), NMS-873, NMS-859, iyerestatin I, and xanthohumol.

[0027] In some embodiments, the VCP inhibitor is ML240 (2-(2-amino-1H-benzimidazol-1-yl)-8-methoxy-N-(phenylmethyl)-4-quinazolinamine).

[0028] In some embodiments, the VCP inhibitor is CB-5083 (1-[7,8-dihydro-4-[(phenylmethyl)amino]-5H-pyrano[4,3-d]pyrimidin-2-yl]-2-methyl-1H-indole-4-carboxamide) or CB-5339 (1-[4-(benzylamino)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-2-yl]-2-methylindole-4-carboxamide).

[0029] The present invention also provides a method of diagnosing a subject as having or suspected of having non-VCP-associated ALS, comprising determining whether the subject has a pathogenic mutation in the VCP gene and providing a diagnosis of non-VCP-associated ALS based on the absence of a pathogenic mutation in the VCP gene.

[0030] In some embodiments, the method comprises identifying the absence of a pathogenic genetic mutation in the VCP gene at any of positions R95, I114, I151, R155, G156, M158, R159, R191, N387, N401, R487, D592, R662, and N750. In some embodiments, the method comprises identifying the absence of any pathogenic genetic mutation in the VCP gene selected from the list consisting of R95C, R95G, I114V, I151V, R155H, R155C, G156C, M158V, R159G, R159C, R159H, R191G, R191Q, N387T, N401S, R487H, D592N, R662C, and N750S.

[0031] The invention also provides a VCP inhibitor for use in a method of treating or preventing non-VCP-related ALS in a subject, comprising diagnosing a patient as having or suspected of having non-VCP-related ALS using a method according to the invention, and administering a VCP inhibitor to the patient.

[0032] The invention also provides a VCP inhibitor for use in a method of treating or preventing non-VCP-related ALS in a subject, the method comprising administering a VCP inhibitor to the patient, wherein the patient has been determined to have, or is suspected of having, non-VCP-related ALS using a method according to the invention.

[0033] The present invention also provides a pharmaceutical composition comprising a VCP inhibitor, optionally containing one or more excipients, for use in a method for treating or preventing amyotrophic lateral sclerosis (ALS).

[0034] The present invention also provides methods for treating or preventing amyotrophic lateral sclerosis (ALS), comprising administering a VCP inhibitor to a subject in need thereof. In some embodiments, the subject has not been identified as having a pathogenic mutation in the VCP gene. In some embodiments, the subject has been identified as not having a pathogenic mutation in the VCP gene. In some embodiments, the ALS is non-VCP-associated ALS. The VCP inhibitor may be administered in a therapeutically effective amount.

[0035] The present invention also provides kits for diagnosing a subject as having or suspected of having non-VCP-associated ALS, comprising a means for determining whether the subject has a pathogenic mutation in the VCP gene. In some embodiments, the kit further comprises one or more containers containing one or more VCP inhibitors and, optionally, informational material. In some embodiments, the informational material comprises instructions for using the kit in diagnosing and / or treating non-VCP-associated ALS.

[0036] Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0037] [Figure 1]Figure 1 shows mislocalization of TDP-43 and SFPQ in VCP mutant motor neurons. A) Immunolabeling of TDP-43 in control and VCP mutant motor neurons. B) Individual cell analysis of the nuclear:cytoplasmic ratio of TDP-43 confirms that VCP mutant motor neurons exhibit a reduced nuclear:cytoplasmic ratio (N / C). C) Quantification of TDP-43 in motor neuron neurites shows that VCP mutant motor neurons have a reduced nuclear:neurite ratio (Nu / Ne). D) Immunolabeling of SFPQ in control and VCP mutant motor neurons. E) Individual cell analysis of the nuclear:cytoplasmic ratio of SFPQ shows a small but significant reduction in VCP mutant motor neurons. F) Quantification of SFPQ in motor neuron neurites confirms that VCP mutant motor neurons have a reduced nuclear:neurite ratio. G) Immunolabeling of hnRNPA1 in control and VCP mutant motor neurons. H) Individual cell quantification of hnRNPA1 shows no difference in the nuclear:cytoplasmic ratio in control and VCP mutant motor neurons. I) Localization of hnRNPK in control and VCP mutant motor neurons. J) Quantification of hnRNPK shows no difference in the nuclear:cytoplasmic ratio in control and VCP mutant motor neurons. Scale bar = 10 μm. Data are collected from three control cell lines and four VCP mutant lines. For graphs B, E, H, and J, data are shown as violin plots, each data point represents a well from six independent experimental replicates (CTRL n = 34, VCP n = 45), and p values ​​are from an unpaired T-test.The following approximate numbers of cells were analyzed: B) CTRL1:10000, CTRL2:10000, CTRL3:14000, MUT1:13000, MUT2:15000, MUT3:14000, MUT4:11000, E) CTRL1:9000, CTRL2:10000, CTRL3:13000, MUT1:12000, MUT2:14000, MUT3:14000, MUT4:11000. MUT1:12000, MUT2:12000, MUT3:13000, MUT4:9000; H) CTRL1:9000, CTRL2:10000, CTRL3:13000, MUT1:12000, MUT2:12000, MUT3:13000, MUT4:9000; J) CTRL1:9000, CTRL2:9000, CTRL3:12000, MUT1:11000, MUT2:12000, MUT3:12000, MUT4:9000. For graphs C and F, data are collected from three independent experiments of three control and four VCP mutant lines, with over 5000 neurons analyzed for each line. Data are shown as violin plots, data points indicate fields, and p-values ​​are calculated using the Mann-Whitney test. All data are normalized to the mean control value for each experimental replicate. [Figure 2]Pharmacological inhibition of VCP D2 ATPase does not recapitulate the ALS RBP mislocalization phenotype in control motor neurons. A) Control motor neurons treated with 1 μM ML240 (2-(2-amino-1H-benzimidazol-1-yl)-8-methoxy-N-(phenylmethyl)-4-quinazolinamine) and immunolabeled for TDP-43 and βIII-tubulin, followed by DAPI staining. B) Individual cell quantification of TDP-43 showed that control motor neurons treated with ML240 had an increased nuclear:cytoplasmic ratio (N / C). C) There was no difference in the nuclear:neurite ratio (Nu / Ne) of TDP-43 upon ML240 treatment. D) Control motor neurons treated with 1 μM ML240 and immunolabeled for FUS and βIII-tubulin, followed by DAPI staining. E) Treatment of control motor neurons with ML240 showed no difference in the nuclear:cytoplasmic localization of FUS. F) A slight increase in the nuclear:neurite ratio of FUS was observed upon ML240 treatment. There was no difference in the G) nuclear:cytoplasmic ratio or H) nuclear:neurite ratio of SFPQ upon ML240 treatment. In control motor neurons, there was no difference in the nuclear:cytoplasmic ratio of I) hnRNPA1 or J) hnRNPK upon ML240 treatment. Scale bar = 10 μm. Data are shown as violin plots normalized to the control untreated value in each experimental replicate. Data are collected from three control lines across three independent experimental replicates using approximately the following numbers of cells in both untreated and treated conditions: CTRL1:3000, CTRL2:6000, and CTRL3:6000. For graphs B, E, G, I, and J, each data point represents a well (UT n=16, ML240 n=16), and p-values ​​are calculated from an unpaired t-test. For graphs C, F, and H, each data point represents a field, and p-values ​​are calculated from a Mann-Whitney test. [Figure 3]Figure 1 shows that inhibition of the VCP D2-ATPase domain reverses the mislocalization phenotype of TDP-43, FUS, and SFPQ in VCP mutant motor neurons. A) VCP mutant motor neurons treated with 1 μM ML240 and immunolabeled for TDP-43 and βIII-tubulin. B) Cell-by-cell quantification of the nucleus:cytoplasm ratio (N / C) shows that the decreased nucleus:cytoplasm ratio in VCP motor neurons increases above control values ​​upon ML240 treatment. C) Quantification of TDP-43 in neurites shows an increase in the nucleus:neurite ratio (Nu / Ne) upon ML240 treatment. D) Immunolabeling of FUS and βIII-tubulin in VCP mutant motor neurons treated with ML240. E) Quantification of FUS in the nucleus and cytoplasm confirms the increase in the nucleus:cytoplasm ratio upon ML240 treatment in VCP mutant motor neurons. F) Quantification of FUS in neurites shows an increase in the nucleus:neurite ratio upon ML240 treatment relative to control values. G) VCP mutant motor neurons treated with ML240 and immunolabeled for SFPQ and βIII-tubulin. ML240 treatment does not alter the subcellular distribution of SFPQ when examining the nucleus:cytoplasm ratio (H), but increases it when examining the nucleus:neurite ratio (I). J) Quantification of hnRNPA1 shows no change in the nucleus:cytoplasm ratio upon ML240 treatment in VCP mutant motor neurons. K) Quantification of hnRNPK shows no change in the nucleus:cytoplasm ratio upon ML240 treatment in VCP mutant motor neurons. Scale bar = 10 μm. Data were collected from three independent experimental replicates of four VCP ALS mutant strains, analyzing the following approximate numbers of cells: MUT1:7000, MUT2:6000, MUT3:7000, and MUT4:6000. Data are normalized to the untreated control values ​​for each experimental replicate. Data are presented as violin plots, with each data point representing a well (UT n=24, ML240 n=24) in graphs B, E, H, J, and K, and a field in graphs C, F, and I. For graphs B, E, H, and K, p values ​​are calculated from an unpaired t-test. For graphs C, F, I, and J, p values ​​are calculated from a Mann-Whitney test. [Figure 4] 1 is a graphical depiction of the localization of TDP-43, FUS, and SFPQ in control and mutant motor neurons and the effect of VCP D2 ATPase inhibition. [Figure 5] Examples of neuron segmentation images used in image analysis. A) Examples of nuclear and cytoplasmic compartments used in the analysis of nucleus:cytoplasm ratio. Nucleus:cytoplasmic ratios are calculated for each cell. B) Examples of nuclear and neurite compartments used in the analysis of nucleus:neurite ratios. Nucleus:neurite ratios are calculated for each field of view. [Figure 6] (Figure 1) Characterization of motor neurons. Representative images of control and VCP mutant iPSC-derived motor neurons immunolabeled with the motor neuron-specific markers SMI-32 and ChAT and the neuronal marker βIII-tubulin. Scale bar = 20 μm. [Figure 7] Compartmental analysis of TDP-43 and FUS in VCP mutant motor neurons. A) Nuclear compartmental analysis shows that VCP mutant motor neurons have reduced TDP-43 in the nucleus compared to DAPI. B) Neurite compartmental analysis shows that VCP mutant motor neurons have increased TDP-43 in neuronal processes compared to the neuronal marker βIII-tubulin. C) Compartmental analysis shows reduced SFPQ protein in the nucleus of VCP mutant motor neurons. D) Compartmental analysis shows increased SFPQ in the neurites of VCP mutant motor neurons. Data are shown as violin plots normalized to the control untreated value in each experimental replicate. Data are collected from three control strains from six wells across three independent experimental replicates. Data are plotted per field, and p-values ​​are calculated from the Mann-Whitney test. [Figure 8]Western blot analysis shows that TDP-43, FUS, and SFPQ protein levels are unchanged upon inhibition of the VCP D2 ATPase domain. A) Representative immunoblots of SFPQ, FUS, and TDP-43 from control and VCP mutant MN untreated and treated with 1 μM ML240. B) Quantification of SFPQ, FUS, and TDP-43 from three control and three VCP mutant lines, normalized to GAPDH, showed that ML240 treatment did not alter total protein levels. [Figure 9] Figure 1 shows details of the iPSC cell lines used in this study: MUT1 and MUT2 have an R191Q mutation in VCP, MUT3 and MUT4 have an R155C mutation in VCP, and MUT5 and MUT6 have a G298S mutation in TARDBP. [Figure 10] 1 shows an exemplary VCP protein sequence. This figure discloses an exemplary human VCP protein sequence (UniProtKB-P55072). [Figure 11]Compartmental analysis of TDP-43 in VCP mutant and TARDBP mutant motor neurons. A) Individual cell quantification of TDP-43 showed that control motor neurons treated with DBEQ had an increased nuclear to cytoplasmic ratio (N / C). B) Individual cell quantification of TDP-43 showed that control motor neurons treated with CB-5083 had an increased nuclear to cytoplasmic ratio (N / C). C) Individual cell quantification of TDP-43 showed that VCP mutant motor neurons treated with DBEQ had an increased nuclear to cytoplasmic ratio (N / C). D) Individual cell quantification of TDP-43 showed that VCP mutant motor neurons treated with CB5083 had an increased nuclear to cytoplasmic ratio (N / C). E) Individual cell quantification of TDP-43 showed that TARDP mutant motor neurons treated with CB5083 had an increased nuclear to cytoplasmic ratio (N / C). Data are collected from two independent experimental replicates of four CTRL strains (CTRL2, CTRL3, CTRL4, CTRL5), three VCP ALS mutant strains (MUT1, MUT3, MUT4), and two TARDBP ALS mutant strains (MUT5, MUT6). Data are normalized to the raw value for each experimental replicate. Data are plotted as mean ± SD, and p-values ​​shown are calculated from unpaired t-tests. DETAILED DESCRIPTION OF THE INVENTION

[0038] In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions for these and other terms are set forth throughout the specification.

[0039] As used herein, the term "approximately" or "about" as applied to one or more reference values ​​refers to a value similar to the stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% (greater or less) in either direction of the stated reference value, unless otherwise specified or clear from the context (except where such number exceeds 100% of the possible values).

[0040] As used herein, the term "amelioration" refers to the prevention, reduction, or alleviation of a condition, or the improvement of a subject's condition. Amelioration includes, but does not require, complete reversal or complete prevention of a disease state.

[0041] As used herein, the term "comparable" refers to a system, set of conditions, effect, or result that is sufficiently similar to the test system, set of conditions, effect, or result to allow a scientifically reasonable comparison. Those skilled in the art will recognize and understand which systems, sets of conditions, effects, or results are sufficiently similar to be "comparable" to any particular test system, set of conditions, effect, or result described herein.

[0042] As used herein, the term "correlate" has its ordinary meaning of "exhibiting a correlation with." Those skilled in the art will recognize that two traits, items, or values ​​exhibit a correlation with each other if they exhibit a tendency to appear and / or fluctuate together. In some embodiments, a correlation is statistically significant when its p-value is less than 0.05, and in some embodiments, a correlation is statistically significant when its p-value is less than 0.01. In some embodiments, the correlation is assessed by regression analysis. In some embodiments, the correlation is a correlation coefficient.

[0043] As used herein, the terms "improve," "increase," or "reduce," or grammatical equivalents, refer to a value relative to a reference {e.g., baseline) measurement, e.g., a measurement taken under comparable conditions {e.g., in the same individual prior to the initiation of a treatment described herein, or in a control individual (or control individuals) in the absence of treatment).

[0044] As used herein, a "polypeptide" generally refers to a string of at least two amino acids joined to each other by peptide bonds. In some embodiments, a polypeptide can include at least 3-5 amino acids, each of which is joined to another amino acid by at least one peptide bond. Those skilled in the art will recognize that polypeptides can optionally include "unnatural" amino acids or other entities, which can optionally be incorporated into the polypeptide chain.

[0045] As used herein, the term "protein" refers to a polypeptide (i.e., a string of at least two amino acids linked together by a peptide bond). A protein may contain moieties other than amino acids (e.g., it may be a glycoprotein, proteoglycan, etc.) and / or may be otherwise processed or modified. Those of skill in the art will recognize that a "protein" may be an entire polypeptide chain (with or without a signal sequence) produced by a cell, or a characteristic portion thereof. Those of skill in the art will recognize that a protein may also include two or more polypeptide chains, for example, linked by one or more disulfide bonds or associated by other means. Polypeptides can contain L-amino acids, D-amino acids, or both, and can contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, proteins can include natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids.

[0046] A "reference" entity, system, amount, set of conditions, etc. is one to which a test entity, system, amount, set of conditions, etc. is compared as described herein. For example, in some embodiments, a "reference" individual is a control individual who is not afflicted with or predisposed to any form of ALS disease, and in some embodiments, a "reference" individual is a control individual who is afflicted with the same form of ALS disease as the individual to be treated, and optionally is about the same age as the individual to be treated (to ensure that the stage of disease in the treated and control individual(s) is comparable).

[0047] As used herein, the terms "subject," "individual," or "patient" refer to any organism to which embodiments of the present invention can be used or administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals {e.g., mammals, e.g., mice, rats, rabbits, non-human primates, and humans; insects; worms, etc.). In preferred embodiments of the present invention, the subject is a human.

[0048] As used herein, the term "target cell" or "target tissue" refers to any cell, tissue, or organism affected by ALS that is to be treated, or any cell, tissue, or organism in which a protein involved in ALS is expressed. In some embodiments, the target cell, tissue, or organism includes a cell, tissue, or organism in which a detectable or abnormally high amount of FUS or TDP-43 is present {e.g., comparable to that observed in a patient suffering from or susceptible to ALS). In some embodiments, the target cell, tissue, or organism includes a cell, tissue, or organism that exhibits a pathology, symptom, or feature associated with disease.

[0049] As used herein, the phrase "drug" or "therapeutic agent" refers to any agent that has a therapeutic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject. In some embodiments, the therapeutic agent is a VCP inhibitor. In some embodiments, the primary therapeutic agent is a VCP inhibitor that can be used in combination with one or more additional therapeutic agents.

[0050] As used herein, the term "therapeutic regimen" refers to any method that partially or completely alleviates, ameliorates, eliminates, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. This can optionally include the administration of one or more doses spaced by regular or varying time intervals. In some embodiments, a therapeutic regimen is one whose implementation is designed to achieve and / or correlate with a particular effect {e.g., across a relevant population of cells, tissues, or organisms), e.g., reduction or elimination of a harmful condition or disease, such as ALS. In some embodiments, treatment involves the administration of one or more therapeutic agents, either simultaneously, sequentially, or at different times, for the same or different amounts of time. In some embodiments, a "therapeutic regimen" includes genetic methods, e.g., gene therapy, gene disruption, or other methods known to induce or reduce expression (e.g., transcription, processing, and / or translation of a particular gene product, e.g., primary transcript or mRNA).

[0051] As used herein, the term "therapeutically effective amount" refers to an amount of a therapeutic agent that confers a therapeutic effect on a treated subject, at a reasonable benefit / risk ratio applicable to any medical treatment. Such a therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., the subject gives an indication of or feels an effect). In some embodiments, a "therapeutically effective amount" refers to an amount of a therapeutic agent or composition that is effective to treat, ameliorate, or prevent (e.g., delay the onset of) the associated disease or condition, and / or to exhibit a detectable therapeutic or prophylactic effect, e.g., by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and / or reducing the severity or frequency of symptoms of the disease. A therapeutically effective amount is generally administered in a dosing regimen that can include multiple unit doses. For any particular therapeutic agent, the therapeutically effective amount (and / or an appropriate unit dose within an effective dosing regimen) may vary, for example, depending on the route of administration or combination with other therapeutic agents. Alternatively or additionally, the particular therapeutically effective amount (and / or unit dose) for any particular patient may depend on a variety of factors, including the particular form of ALS being treated; the severity of the ALS; the activity of the particular therapeutic agent used; the particular composition used; the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and / or rate of excretion or metabolism of the particular therapeutic agent used; the duration of treatment; and factors such as are known in the medical arts.

[0052] As used herein, the term "treatment" (also "treat" or "treating") refers to any administration of a therapeutic agent according to a treatment regimen that achieves a desired effect in terms of partially or completely alleviating, ameliorating, eliminating, inhibiting, delaying the onset of, reducing the severity of, and / or reducing the incidence of one or more symptoms or features of a particular disease, disorder, and / or condition (e.g., ALS), and in some embodiments, administration of a therapeutic agent according to a treatment regimen is correlated with achieving a desired effect. Such treatment may be of subjects who do not exhibit symptoms of the associated disease, disorder, and / or condition and / or who exhibit only early symptoms of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be of subjects who exhibit one or more established symptoms of the associated disease, disorder, and / or condition. In some embodiments, treatment may be of subjects who have been diagnosed with the associated disease, disorder, and / or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the relevant disease, disorder and / or condition.

[0053] As used herein, the term neuroprotective agent refers to an agent that prevents or slows the progression of neuronal degeneration and / or prevents neuronal cell death.

[0054] VCP inhibitors (VCP inhibitors) VCP inhibitors can bind to VCP. Binding to a VCP polypeptide can be assessed by any technique known to those skilled in the art. Examples of suitable assays include two-hybrid assay systems that measure interactions in vivo, such as affinity chromatography assays involving binding to a polypeptide immobilized on a column, and fluorescence assays in which binding of a drug(s) to a VCP polypeptide is correlated with a change in fluorescence of one or both partners of the binding pair. Assays performed in vivo in cells, such as two-hybrid assays, are preferred. In a preferred aspect of this embodiment, the present invention provides a method for identifying pharmaceutical agents useful in the treatment of ALS, comprising incubating cells with the drug(s) to be tested and selecting agents that improve or reverse one or more functional parameters associated with ALS.

[0055] Examples of agents that can modulate the functional effects of VCP include agents that are inhibitors of VCP and / or VCP adaptor proteins.

[0056] VCP inhibitors include those agents described above as well as those listed in Table 1 below and agents that inhibit and / or disrupt VCP adaptor proteins.

[0057] TIFF2024527566000001.tif211170

[0058] In some embodiments, the VCP inhibitor is selected from the group consisting of ML240 (2-(2-amino-1H-benzimidazol-1-yl)-8-methoxy-N-(phenylmethyl)-4-quinazolinamine), ML241, 2-anilino-4-aryl-1,3-thiazole, 3,4-methylenedioxy-6-nitrostyrene, DBeQ (N2,N4-dibenzylquinazoline-2,4-diamine), NMS-873, NMS-859, iyarestatin I, and xanthohumol.

[0059] In some embodiments, the VCP inhibitor is ML240 (2-(2-amino-1H-benzimidazol-1-yl)-8-methoxy-N-(phenylmethyl)-4-quinazolinamine), ML241, 2-anilino-4-aryl-1,3-thiazole, 3,4-methylenedioxy-6-nitrostyrene, DBeQ (N2,N4-dibenzylquinazoline-2,4-diamine), CB-5083 (1-[7,8-dihydro-4-[(phenylmethyl)amino]-5H-pyrano[4,3-d]pyrimidin-2-yl]-2-methyl- methyl-1H-indole-4-carboxamide), CB-5339 (1-[4-(benzylamino)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-2-yl]-2-methylindole-4-carboxamide), UPCDC-30245 (1-(3-(5-fluoro-1H-indol-2-yl)phenyl)-N-(2-(4-isopropylpiperazin-1-yl)ethyl)piperidin-4-amine), NMS-873, NMS-859, iyerestatin I, and xanthohumol.

[0060] In some embodiments, the VCP inhibitor is selected from the group consisting of ML240, DBeQ, and CB-5083. In some embodiments, the VCP inhibitor is ML240. In some embodiments, the VCP inhibitor is DBeQ. In some embodiments, the VCP inhibitor is CB-5083. In some embodiments, the VCP inhibitor is CB-5083 or CB-5339.

[0061] The VCP inhibitors described herein can be used to treat VCP-related or non-VCP-related ALS. Preferably, the ALS is non-VCP-related ALS, and the VCP inhibitor inhibits the D2 ATPase domain of VCP. Thus, in a preferred embodiment, the present invention provides a VCP inhibitor for use in a method of treating or preventing non-VCP-related ALS in a subject, the VCP inhibitor inhibiting the D2 ATPase domain of VCP. For example, in some embodiments, the ALS is non-VCP-related ALS, and the VCP inhibitor is CB-5083 or CB-5339. In some embodiments, the subject is identified as not having a pathogenic mutation in the VCP gene, and the VCP inhibitor inhibits the D2 ATPase domain of VCP. In some embodiments, the subject is identified as not having a pathogenic mutation in the VCP gene, and the VCP inhibitor is CB-5083 or CB-5339.

[0062] In some embodiments, the subject has or has been identified as having one or more pathogenic genetic mutations in the TARDBP gene, and the VCP inhibitor inhibits the D2 ATPase domain of VCP. In some embodiments, the subject has or has been identified as having one or more pathogenic genetic mutations in the TARDBP gene, and the VCP inhibitor is CB-5083 or CB-5339. In some embodiments, the subject has or has been identified as having a pathogenic genetic mutation in the TARDBP gene at position G298, and the VCP inhibitor inhibits the D2 ATPase domain of VCP. In some embodiments, the mutation at position G298 is a G298S mutation. In some embodiments, the subject has or has been identified as having a pathogenic genetic mutation in the TARDBP gene at position G298, and the VCP inhibitor is CB-5083 or CB-5339. In some embodiments, the mutation at position G298 is a G298S mutation.

[0063] VCP adaptor proteins are known in the art. See, e.g.,

[21] , especially Table 1 therein. Additionally, methods for identifying VCP adaptor proteins are known. For example,

[22] describes a method based on unbiased mass spectrometry that is used to identify the complex between VCP and the UBXD1 cofactor.

[0064] Agents that affect the activity or localization of VCP can be of nearly any general type, including small molecular weight agents, including organic agents, peptides, polypeptides, including antibodies, or proteins, which may be linear, cyclic, polycyclic, or a combination thereof. Generally, as used herein, "peptide," "polypeptide," and "protein" are considered synonymous. Certain VCP inhibitors are described above in Table 1. See also

[23] for examples of other useful VCP inhibitors.

[0065] As used herein, a "VCP inhibitor" is a drug capable of inhibiting the activity of VCP, which is required for normal neuronal function. VCP inhibitors are known in the art and are regularly discovered because VCP is also a target for cancer therapy and other medical fields. Exemplary inhibitors include those listed above, and methods for identifying VCP inhibitors are described in the prior art. In some embodiments, the VCP inhibitors of the present invention can inhibit the activity of VCP by inhibiting the D2 ATPase domain of VCP.

[0066] VCP inhibitors can be referred to as VCP antagonists.

[0067] Pharmaceutical Composition of Drug The drug may be in the form of a pharmaceutical composition. The pharmaceutical composition may comprise the drug (i.e., a VCP inhibitor). The pharmaceutical composition may comprise about 5 nanograms (ng) to about 10 milligrams (mg) of the drug. In some embodiments, the pharmaceutical composition according to the present invention comprises about 25 ng to about 5 mg of the drug. In some embodiments, the pharmaceutical composition contains about 50 ng to about 1 mg of the drug. In some embodiments, the pharmaceutical composition contains about 0.1 micrograms to about 500 micrograms of the drug. In some embodiments, the pharmaceutical composition contains about 1 microgram to about 350 micrograms of the drug. In some embodiments, the pharmaceutical composition contains about 5 micrograms to about 250 micrograms of the drug. In some embodiments, the pharmaceutical composition contains about 10 micrograms to about 200 micrograms of the drug. In some embodiments, the pharmaceutical composition contains about 15 micrograms to about 150 micrograms of the drug. In some embodiments, the pharmaceutical composition contains about 20 micrograms to about 100 micrograms of the drug. In some embodiments, the pharmaceutical composition contains about 25 micrograms to about 75 micrograms of the drug. In some embodiments, the pharmaceutical composition contains about 30 micrograms to about 50 micrograms of the drug. In some embodiments, the pharmaceutical composition contains about 35 micrograms to about 40 micrograms of drug. In some embodiments, the pharmaceutical composition contains about 100 micrograms to about 200 micrograms of drug. In some embodiments, the pharmaceutical composition contains about 10 micrograms to about 100 micrograms of drug. In some embodiments, the pharmaceutical composition contains about 20 micrograms to about 80 micrograms of drug. In some embodiments, the pharmaceutical composition contains about 25 micrograms to about 60 micrograms of drug. In some embodiments, the pharmaceutical composition contains about 30 ng to about 50 micrograms of drug. In some embodiments, the pharmaceutical composition contains about 35 ng to about 45 micrograms of drug. In some embodiments, the pharmaceutical composition contains about 0.1 micrograms to about 500 micrograms of drug.In some embodiments, the pharmaceutical composition contains about 1 microgram to about 350 micrograms of the drug. In some embodiments, the pharmaceutical composition contains about 25 micrograms to about 250 micrograms of the drug. In some embodiments, the pharmaceutical composition contains about 100 micrograms to about 200 micrograms of the drug.

[0068] In other embodiments, the pharmaceutical composition may contain up to 15 ng, 20 ng, 25 ng, 30 ng, 35 ng, 40 ng, 45 ng, 50 ng, 55 ng, 60 ng, 65 ng, 70 ng, 75 ng, 80 ng, 85 ng, 90 ng, 95 ng or 100 ng inclusive of the drug. In some embodiments, the pharmaceutical composition contains 1 microgram, 5 micrograms, 10 micrograms, 15 micrograms, 20 micrograms, 25 micrograms, 30 micrograms, 35 micrograms, 40 micrograms, 45 micrograms, 50 micrograms, 55 micrograms, 60 micrograms, 65 micrograms, 70 micrograms, 75 micrograms, 80 micrograms, 85 micrograms, 90 micrograms, 95 micrograms, 100 micrograms, 105 micrograms, 110 micrograms, 115 micrograms, 120 micrograms, 125 micrograms, 130 micrograms, 135 micrograms, 140 micrograms, 145 micrograms, 150 micrograms, 155 micrograms, 160 micrograms, 165 micrograms, 170 micrograms, 175 micrograms, 180 micrograms, 185 micrograms, 190 micrograms, 195 micrograms, micrograms, 200 micrograms, 205 micrograms, 210 micrograms, 215 micrograms, 220 micrograms, 225 micrograms, 230 micrograms, 235 micrograms, 240 micrograms, 245 micrograms, 250 micrograms, 255 micrograms, 260 micrograms, 265 micrograms, 270 micrograms, 275 micrograms, 280 micrograms, 285 micrograms, 290 micrograms, 295 micrograms, 300 micrograms, 305 micrograms, 310 micrograms, 315 micrograms, 320 micrograms, 325 micrograms, 330 micrograms, 335 micrograms, 340 micrograms, 345 micrograms, 350 micrograms, 355 micrograms, 360 micrograms, 365 micrograms, 370 micrograms, 375 micrograms, 380 micrograms, 385 micrograms, 390 micrograms,395 micrograms, 400 micrograms, 405 micrograms, 410 micrograms, 415 micrograms, 420 micrograms, 425 micrograms, 430 micrograms, 435 micrograms, 440 micrograms, 445 micrograms, 450 micrograms, 455 micrograms, 460 micrograms, 465 micrograms, 470 micrograms, 475 micrograms, 480 micrograms, 485 micrograms, 490 micrograms, 495 micrograms, 500 micrograms, 605 micrograms Ram, 610 micrograms, 615 micrograms, 620 micrograms, 625 micrograms, 630 micrograms, 635 micrograms, 640 micrograms, 645 micrograms, 650 micrograms, 655 micrograms, 660 micrograms, 665 micrograms, 670 micrograms, 675 micrograms, 680 micrograms, 685 micrograms, 690 micrograms, 695 micrograms, 700 micrograms, 705 micrograms, 710 micrograms, 715 micrograms, 720 micrograms micrograms, 725 micrograms, 730 micrograms, 735 micrograms, 740 micrograms, 745 micrograms, 750 micrograms, 755 micrograms, 760 micrograms, 765 micrograms, 770 micrograms, 775 micrograms, 780 micrograms, 785 micrograms, 790 micrograms, 795 micrograms, 800 micrograms, 805 micrograms, 810 micrograms, 815 micrograms, 820 micrograms, 825 micrograms, 830 micrograms, 83 5 micrograms, 840 micrograms, 845 micrograms, 850 micrograms, 855 micrograms, 860 micrograms, 865 micrograms, 870 micrograms, 875 micrograms, 880 micrograms, 885 micrograms, 890 micrograms, 895 micrograms, 900 micrograms, 905 micrograms, 910 micrograms, 915 micrograms, 920 micrograms, 925 micrograms, 930 micrograms, 935 micrograms, 940 micrograms, 945 micrograms,The pharmaceutical composition may contain up to 950 micrograms, 955 micrograms, 960 micrograms, 965 micrograms, 970 micrograms, 975 micrograms, 980 micrograms, 985 micrograms, 990 micrograms, 995 micrograms, or 1000 micrograms of drug, inclusive. In some embodiments, the pharmaceutical composition may contain up to 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, or 10 mg of drug, inclusive.

[0069] In some embodiments, the dosage of an agent of the invention is from about 0.5 mg to about 5000 mg. In some embodiments, the dosage of an agent of the invention used in the compositions described herein is less than about 5000 mg, or less than about 4000 mg, or less than about 3000 mg, or less than about 2000 mg, or less than about 1000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, the dose of a second agent described herein is less than about 1000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

[0070] In one embodiment, the agents of the present invention are administered to a patient in dosages ranging from once to five or more times per day. In another embodiment, the agents of the present invention are administered to a patient in dosages ranging from once daily, every two days, every three days, to once weekly and once every two weeks. It will be readily apparent to those skilled in the art that the frequency of administration of the various combination compositions of the present invention will vary from subject to subject, depending on many factors, including, but not limited to, age, disease or disorder being treated, gender, overall health, and other factors. Therefore, the present invention should not be construed as limited to any particular dosing regimen, and the exact dosage and composition to be administered to any patient will be determined by the attending physician, taking into account all other factors relevant to the patient.

[0071] Pharmaceutical compositions can further contain other agents for formulation purposes according to the mode of administration used.When the pharmaceutical composition is an injectable pharmaceutical composition, it is sterile, pyrogen-free, and particulate-free.An isotonic preparation is preferably used.Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose.In some cases, isotonic solutions such as phosphate buffered saline are appropriate.Stabilizers include gelatin and albumin.

[0072] The medicament may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be a functional molecule, such as a vehicle, adjuvant, carrier, or diluent.

[0073] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal formulations, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration, etc. It is understood that the formulations and compositions contemplated as useful in the present invention are not limited to the specific formulations and compositions described herein.

[0074] For oral administration, tablets, sugar-coated tablets, liquids, drops, suppositories, or capsules, caplets, and gel capsules are particularly suitable. Other formulations suitable for oral administration include, but are not limited to, powdered or granular formulations, aqueous or oily suspensions, aqueous or oily solutions, pastes, gels, toothpastes, mouthwashes, coatings, mouthwashes, or emulsions. Compositions intended for oral use can be prepared according to any method known in the art, and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutical excipients suitable for the manufacture of tablets. Such excipients include, for example, inert diluents such as lactose; granulating and disintegrating agents such as cornstarch; binders such as starch; and lubricants such as magnesium stearate.

[0075] The tablets may be uncoated or may be coated using known methods to achieve delayed disintegration in the subject's digestive tract, thereby resulting in sustained release and absorption of the active ingredient. For example, tablets may be coated using materials such as glyceryl monostearate or glyceryl distearate. The tablets may further contain sweeteners, flavoring agents, coloring agents, preservatives, or any combination thereof to provide a palatable preparation.

[0076] Controlled Release Formulations and Drug Delivery Systems The controlled or sustained release formulation of the pharmaceutical composition of the present invention can be prepared using conventional technology.In some cases, the dosage form used can be provided as a sustained or controlled release of one or more active ingredients therein, for example, by using hydropropylmethylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, or microspheres, or a combination thereof, to provide a desired release profile at various ratios.Suitable controlled release formulations known to those skilled in the art, including those described herein, can be easily selected for use with the pharmaceutical composition of the present invention.Therefore, the present invention encompasses single-unit dosage forms suitable for oral administration, such as tablets, capsules, gel capsules, and caplets, which are suitable for controlled release.

[0077] Most controlled-release pharmaceutical products share a common goal of improving drug therapy over that achieved by their non-controlled counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by curing or controlling a condition in a minimum amount of time using a minimum amount of drug substance.

[0078] Advantages of controlled-release formulations include extended activity of the drug, reduced dosage frequency, and increased patient compliance. Furthermore, controlled-release formulations can be used to affect the time of onset of action or other characteristics, such as blood levels of the drug, and can thereby affect the occurrence of side effects.

[0079] Most controlled-release formulations are designed to initially release an amount of drug that quickly produces the desired therapeutic effect, and then gradually and continuously release another amount of drug to maintain this level of therapeutic effect over an extended period of time. To maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body.

[0080] Controlled-release of an active ingredient can be stimulated by various inducers, for example, pH, temperature, enzymes, water, or other physiological conditions or compounds. The term "controlled-release ingredient" in the context of the present invention is defined herein as a compound(s), including but not limited to, polymers, polymer matrices, gels, permeable membranes, liposomes, or microspheres, or combinations thereof, that facilitates controlled release of an active ingredient.

[0081] In certain embodiments, the formulations of the present invention may be, but are not limited to, short-acting formulations, rapid offset formulations, and controlled release formulations, including sustained release formulations, delayed release formulations, and pulsed release formulations.

[0082] The term sustained release is used in its conventional sense to refer to a drug formulation that releases drug gradually over an extended period of time, resulting in substantially constant blood levels of drug over an extended period of time, which may, but need not, extend to a month or more and should be released longer than the same amount of drug administered in bolus form.

[0083] For sustained release, the compound can be formulated with a suitable polymer or hydrophobic material that provides sustained release to the compound. Thus, the compound for use in the method of the present invention can be administered, for example, in the form of microparticles by injection, or in the form of wafers or disks by implantation. In a preferred embodiment of the present invention, the compound of the present invention is administered to a patient alone or in combination with another pharmaceutical agent using a sustained release formulation.

[0084] The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides an initial release of drug after some delay following drug administration, including delays from about 10 minutes to about 12 hours.

[0085] The term immediate release is used in its conventional sense to refer to a drug formulation that provides release of the drug immediately after drug administration.

[0086] As used herein, short-term refers to any period of time up to about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes (inclusive) after drug administration, and any and all whole or partial increments thereof.

[0087] As used herein, rapid offset refers to any period of time up to about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes inclusive, and any and all whole or partial increments thereof, after drug administration.

[0088] kit The agents described herein can be provided in a kit. In some examples, the kit includes (a) a container containing an agent described herein and, optionally, (b) informational material. The informational material can be explanatory, instructional, marketing, or other material relating to the methods described herein and / or the use of the agent, e.g., for therapeutic benefit.

[0089] The informational material of the kit is not limited in format. In some examples, the informational material can include information regarding the manufacture of the therapeutic agent, the molecular weight, concentration, expiration date, batch or manufacturing site information, etc. In other aspects, the informational material relates to, for example, methods for administering the therapeutic agent in an appropriate amount, mode, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein). The method can be a method for treating a subject with ALS.

[0090] In some cases, the informational material, e.g., instructions, is provided in printed form, e.g., printed text, drawings, and / or photographs, e.g., labels or printed sheets. The informational material can also be provided in other formats, e.g., Braille, computer-readable material, video recordings, or audio recordings. In other examples, the informational material of the kit is contact information, e.g., a physical address, email address, website, or phone number, where a user of the kit can obtain essential information regarding the therapeutic agent therein and / or its use in the methods described herein. The informational material can also be provided in any combination of formats.

[0091] In addition to the therapeutic agent, the kit can include other components, such as a solvent or buffer, a stabilizer, or a preservative. The kit can also include additional agents, such as a second or third agent, e.g., another therapeutic agent. The components can be provided in any form, such as liquid, dried, or lyophilized. The components can be substantially pure (but they can be combined together or delivered separately from each other) and / or sterile. When the components are provided in a liquid solution, the liquid solution can be an aqueous solution, e.g., a sterile aqueous solution. When the components are provided in a dry form, reconstitution is generally by the addition of a suitable solvent. A solvent, e.g., sterile water or a buffer, can optionally be provided in the kit.

[0092] The kit can include one or more containers for the therapeutic agent or other agents. In some cases, the kit includes separate containers, dividers, or compartments for the therapeutic agent and the informational material. For example, the therapeutic agent can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other aspects, the separate elements of the kit are contained in a single, undivided container. For example, the therapeutic agent can be contained in a bottle, vial, or syringe that has attached thereto the informational material in the form of a label. In some cases, the kit can include multiple (e.g., a pack) individual containers, each containing one or more unit dosage forms of the therapeutic agent (e.g., a dosage form described herein). The containers can contain unit doses, e.g., units containing the therapeutic agent. For example, the kit can include multiple syringes, ampoules, foil packets, blister packs, or medical devices, each containing a unit dose, e.g., a unit dose. The containers of the kit can be airtight, waterproof (e.g., impervious to changes in moisture or evaporation), and / or light-tight.

[0093] The kit can optionally include a device suitable for administering the therapeutic agent, e.g., a syringe or other suitable delivery device. The device can be provided pre-filled with, e.g., a unit dose of the therapeutic agent, or it can be empty but suitable for filling.

[0094] Amyotrophic lateral sclerosis (ALS) Amyotrophic lateral sclerosis (ALS) is an adult-onset, fatal neurodegenerative disorder characterized by degeneration of both upper motor neurons in the primary motor cortex and lower motor neurons in the brainstem and spinal cord. ALS symptoms initially include muscle atrophy and weakness, often followed by widespread paralysis of voluntary muscles and eventually the respiratory muscles. Approximately 50% of patients with ALS die within 30 months of symptom onset, often from respiratory failure, although approximately 10% of patients can survive longer than 10 years.

[24]

[0095] Approximately 10%–15% of ALS patients have a familial form of the disease, with at least two first- or second-degree relatives having ALS.

[25] If no family history is confirmed, the diagnosis is considered sporadic (or non-familial). The incidence of sporadic ALS shows little variation in Western countries, ranging from 1 to 2 cases per 100,000 person-years, with an estimated lifetime risk of 1 in 400. ALS is rare before the age of 40 and then increases exponentially with age. The mean age of onset is 58–63 years for sporadic ALS and 40–60 years for familial ALS, with peak incidence occurring between 70 and 79 years. Men are at higher risk for ALS than women, resulting in a male-to-female ratio of 1.2–1.5.

[24]

[0096] In some embodiments, ALS can be familial ALS. In some embodiments, ALS can be sporadic ALS. In some embodiments, familial ALS is defined as a patient with two or more occurrences of the disease in the family history. In some embodiments, sporadic ALS is defined as a patient with no known family history of the disease. In some embodiments, ALS can be associated with one or more pathogenic gene mutations in the VCP protein. In some embodiments, ALS can be familial ALS associated with one or more pathogenic gene mutations in the VCP protein. In some embodiments, ALS can be sporadic ALS associated with one or more pathogenic gene mutations in the VCP protein.

[0097] In some embodiments, the subject has one or more pathogenic genetic mutations in a gene other than the VCP gene. The pathogenic mutations can be known pathogenic mutations. The pathogenic mutations can be mutations that cause ALS. For example, in some embodiments, the subject has one or more pathogenic genetic mutations in the TARDBP gene. Thus, in some embodiments, ALS may be associated with one or more genetic mutations in the TARDBP gene. In some embodiments, the subject has been identified as having one or more pathogenic genetic mutations in the TARDBP gene. Such mutations will be known in the art. In some embodiments, the subject has or has been identified as having a pathogenic genetic mutation in the TARDBP gene at any one or more of positions S292, G294, G295, G298, A315, A382, M337, G348, or S393. In some embodiments, the subject has or is identified as having one or more pathogenic genetic mutations in the TARDBP gene selected from the list consisting of S292N, G294V, G295S, G298S, A315T, A382T, M337V, G348C, and S393L. In some embodiments, the subject has or is identified as having a pathogenic genetic mutation in the TARDBP gene at position G298. In some embodiments, the mutation at position G298 is a G298S mutation.

[0098] Pathogenic gene mutations in VCP A subject with ALS can be identified as having one or more known pathogenic mutations in the VCP gene. Such a subject can be characterized as having VCP-associated ALS. A subject with ALS can be identified as not having one or more known pathogenic mutations in the VCP gene. Such a subject can be characterized as having non-VCP-associated ALS. The present invention provides methods for treating or preventing ALS in a subject not identified as having a pathogenic mutation in the VCP gene. The present invention also provides methods for treating or preventing ALS in a subject not identified as having a pathogenic mutation in the VCP gene. The present invention also provides VCP inhibitors for use in methods for treating or preventing ALS in a subject not identified as having a pathogenic mutation in the VCP gene. The present invention also provides VCP inhibitors for use in methods for treating or preventing ALS in a subject not identified as having a pathogenic mutation in the VCP gene.

[0099] In some embodiments, the subject is identified as not having any of the VCP pathogenic genetic mutations listed in Table 2. In some embodiments, the subject is identified as not having a pathogenic genetic mutation in the VCP gene at any of positions R95, I114, I151, R155, G156, M158, R159, R191, N387, N401, R487, D592, R662, and N750. In some embodiments, the patient is identified as not having a pathogenic genetic mutation in the VCP gene selected from the list consisting of: R95C, R95G, I114V, I151V, R155H, R155C, G156C, M158V, R159G, R159C, R159H, R191G, R191Q, N387T, N401S, R487H, D592N, R662C, and N750S.

[0100] An estimated 5 to 10 percent of ALS cases are familial and are caused by mutations in one of several genes. The pattern of inheritance varies depending on the gene involved. Most cases are inherited in an autosomal dominant pattern, meaning that one copy of the altered gene in each cell is sufficient to cause the disorder. Most affected people have one parent with the condition. Some people who inherit a familial gene mutation known to cause ALS may never develop any of the features of the condition. It is unclear why some people with the mutated gene develop the disease while others do not.

[0101] Although ALS is rare, it is inherited in an autosomal recessive pattern, which means that both copies of the gene in each cell have mutations.The parents of an individual with autosomal recessive condition each carry one copy of the mutated gene, but they typically do not show the signs and symptoms of the condition.Because the parents of affected person are not affected, autosomal recessive ALS is often mistaken for sporadic ALS, even though it is caused by familial gene mutations.

[0102] Very rarely, ALS is inherited in an X-linked dominant pattern. X-linked conditions occur when the gene associated with the condition is located on the X chromosome, one of the two sex chromosomes. In women (who have two X chromosomes), a mutation in one of the two copies of the gene in each cell is sufficient to cause the disorder. In men (who only have one X chromosome), a mutation in only one copy of the gene in each cell causes the disorder. In most cases, men tend to develop the disease early and have a shorter life expectancy than women. A characteristic of X-linked inheritance is that fathers do not transmit X-linked traits to their sons.

[0103] Approximately 90 to 95 percent of ALS cases are sporadic, meaning that they are not inherited.

[0104] In both sporadic and familial ALS, patients may have one or more pathogenic genetic mutations in the VCP gene. Some pathogenic mutations in the VCP gene have been well characterized in the prior art, and those skilled in the art know appropriate gene panels that can be used to identify patients carrying pathogenic mutations. Table 2 shows a non-exhaustive list of some known pathogenic mutations in the VCP gene.

[0105] TIFF2024527566000002.tif206170

[0106] Diagnostic methods The present invention also encompasses a method for diagnosing a subject as having non-VCP-associated ALS, in order to determine whether the subject has any pathogenic mutations in the VCP gene. Accordingly, embodiments of the present invention can include determining whether a subject has any pathogenic mutations in the VCP gene. Several suitable methods for determining the sequence of the VCP gene in a biological sample from a subject will be known to those skilled in the art. In some embodiments, determining whether a subject has any pathogenic mutations in the VCP gene comprises determining the sequence of the VCP gene, wherein the sequence of the VCP gene is determined using any one or more of the following techniques: DNA sequencing, RNA sequencing, microarray analysis, real-time quantitative PCR, Northern blot analysis, in situ hybridization, and / or detection and quantification of binding molecules. In a preferred embodiment, determining whether a subject has any pathogenic mutations in the VCP gene involves the use of DNA sequencing. The decision to provide or recommend treatment to the subject can be made based on the determination of the presence of one or more pathogenic mutations in the VCP gene. The recommendation to provide a treatment may be provided in the form of a report. Accordingly, embodiments of the present invention may include providing a report, wherein the report includes information regarding the presence or absence in the subject of one or more pathogenic mutations in the VCP gene. The report may additionally or alternatively include a recommendation to provide a treatment (e.g., a VCP inhibitor) to the subject, e.g., based on the presence of one or more pathogenic mutations in the VCP gene, or a recommendation not to provide a treatment (e.g., a VCP inhibitor) to the subject, e.g., based on the absence of one or more pathogenic mutations in the VCP gene.

[0107] The step of determining whether a subject has one or more pathogenic mutations can be performed on a sample from the subject. The method can include obtaining the sample from the subject, or the sample can be obtained from the subject at an earlier time point. The sample can include, for example, a plasma or blood sample.

[0108] Treatment of ALS with the Agents of the Present Invention In some embodiments, the agent is administered to the central nervous system of a subject, e.g., a subject suffering from or susceptible to ALS. In some embodiments, the agent is administered to one or more target cells or tissues in the brain, spinal cord, and / or peripheral organs. In some embodiments, the target cells or tissues include cells or tissues that exhibit pathology, symptoms, or characteristics associated with a disease. In some embodiments, the target cells or tissues include cells or tissues that express elevated levels of TDP-43 or FUS / TLS, e.g., cells that express elevated levels of TDP-43 or FUS / TLS in the cytoplasm of the cells. As used herein, a target tissue may be a brain target tissue, a spinal cord target tissue, and / or a peripheral target tissue.

[0109] The compositions described herein can be administered directly into the CNS of a subject suffering from or at risk of developing ALS, thereby achieving therapeutic concentrations in affected cells and tissues of the CNS (e.g., brain). For example, to treat ALS, one or more agents can be administered to target cells or tissues in the brain, spinal cord, and / or peripheral organs. As used herein, the term "treat" or "treatment" refers to the amelioration of one or more symptoms associated with a disease, the prevention or delay of the onset of one or more symptoms of ALS.

[0110] In some embodiments, treatment refers to partial or complete alleviation, amelioration, elimination, inhibition, delay in onset, reduction in severity and / or incidence of neurological dysfunction in a patient suffering from or susceptible to ALS. As used herein, the term "neurological dysfunction" includes a variety of symptoms associated with dysfunction of the central nervous system (e.g., the brain and spinal cord). Symptoms of neurological dysfunction can include, for example, developmental delay, progressive cognitive impairment, hearing loss, impaired language development, deficits in motor skills, hyperactivity, aggression, and / or sleep disorders, among others.

[0111] In some embodiments, treatment refers to a reduction in toxicity of various cells or tissues. In some embodiments, treatment refers to a reduction in FUS- or TDP-43-induced neuronal toxicity in brain target tissues, spinal cord neurons, and / or peripheral target tissues. In certain embodiments, toxicity is reduced by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more compared to a control. In some embodiments, toxicity is reduced by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to a control. In some embodiments, toxicity is measured by tests known to those skilled in the art, including, but not limited to, neuroimaging methods (e.g., CT scan, MRI, functional MRI, etc.).

[0112] In certain embodiments, treatment according to the present disclosure results in a reduction (e.g., about a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 97.5%, 99% or more reduction) of one or more pathological, clinical, or biological markers associated with ALS, or a complete elimination of the presence of, or alternatively, the accumulation of, the marker. For example, in some embodiments, upon administration to a subject, the pharmaceutical compositions described herein exhibit or achieve a reduction in muscle loss, muscle spasms, muscle weakness, spasticity, abnormal tendon reflexes, Babinski's sign, difficulty breathing, facial weakness, slurred speech, loss of sensation, loss of reasoning ability, loss of judgment, and / or loss of imagination.

[0113] In some embodiments, treatment refers to an increase in survival (e.g., survival time). For example, treatment can result in an increase in the patient's life expectancy. In some embodiments, treatment can result in an increase in the patient's life expectancy by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% compared to the life expectancy of one or more control individuals with ALS who are not receiving treatment. In some embodiments, treatment results in an increase in patient life expectancy of more than about 100%, about 105%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 195%, about 200% or more. In some embodiments, treatment results in an increase in patient life expectancy of more than about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years or more compared to the life expectancy of one or more control individuals with ALS who are not receiving treatment. In some embodiments, treatment results in long-term survival of the patient. As used herein, the term "long-term survival" refers to a survival or life expectancy of greater than about 40, 45, 50, 55, 60 years or more.

[0114] As used herein, the terms "improve," "increase," or "reduce" refer to a value compared to a control. In some embodiments, a suitable control is a baseline measurement, e.g., a measurement from the same individual prior to the initiation of a treatment described herein, or a measurement from a control individual (or individuals) in the absence of a treatment described herein. A "control individual" is an individual who has ALS and is approximately the same age and / or sex as the individual being treated (to ensure that the stage of disease in the treated and control individual(s) is comparable).

[0115] In some embodiments, the pathogenic genetic mutation is associated with a loss of VCP-dependent endocytic mechanisms of cytoplasmic proteostasis. In some embodiments, the pathogenic genetic mutation is associated with mislocalization of an RBP (e.g., TDP-43, FUS, and / or SFPQ). In some embodiments, the pathogenic genetic mutation is associated with a reduced nuclear-to-cytoplasmic ratio of one or more of TDP-43, FUS, and / or SFPQ. In some embodiments, the pathogenic genetic mutation is associated with a reduced nuclear-to-cytoplasmic ratio of TDP-43. In some embodiments, the pathogenic genetic mutation is associated with a reduced nuclear-to-cytoplasmic ratio of FUS. In some embodiments, the pathogenic genetic mutation is associated with a reduced nuclear-to-cytoplasmic ratio of SFPQ. In some embodiments, the pathogenic genetic mutation is associated with one or more (e.g., one, two, three, four, five, or more) of the above-mentioned normal functions of VCP. In some embodiments, the pathogenic genetic mutation is associated with one or more point mutations in the VCP protein. In some embodiments, the pathogenic genetic mutation is associated with a mutation at one or more positions selected from the list consisting of R95, I114, I151, R155, G156, M158, R159, R191, N387, N401, R487, D592, R662, and N750. In some embodiments, the pathogenic genetic mutation is associated with one or more mutations selected from the list consisting of R95C, R95G, I114V, I151V, R155H, R155C, G156C, M158V, R159G, R159C, R159H, R191G, R191Q, N387T, N401S, R487H, D592N, R662C, and N750S.

[0116] The term "associated with" is used herein to describe an observed correlation between two items or events. For example, a pathogenic gene mutation in VCP can be considered "associated with" a particular neurological dysfunction or disorder if its presence or level correlates with the presence or level of that dysfunction or disorder.

[0117] The individual to be treated (also referred to as a "patient" or "subject") is an individual (fetus, infant, child, adolescent, or adult) who has ALS or is at risk of developing ALS. In some examples, the subject to be treated is genetically predisposed to developing ALS. For example, the subject to be treated may have a mutation in the VCP gene, SOD1 gene, ALS2 gene, VAPB gene, SETX gene, TDP-43 gene, FUS / TLS gene, and / or OPTN gene. In some embodiments, the patient does not have a genetic predisposition to developing ALS. In some embodiments, the subject to be treated may not have a known pathogenic mutation in the VCP gene, SOD1 gene, ALS2 gene, VAPB gene, SETX gene, TDP-43 gene, FUS / TLS gene, and / or OPTN gene. In preferred embodiments, the subject to be treated may not have a known pathogenic mutation in the VCP gene.

[0118] Combination therapy In some embodiments, an agent described herein (e.g., a VCP inhibitor) is administered to a subject in combination with one or more additional therapies for treating ALS or one or more symptoms of ALS. For example, the agent can be administered in combination with riluzole, baclofen, diazepam, trihexyphenidyl, or amitriptyline.

[0119] In some embodiments, combined administration of a first agent (e.g., a VCP inhibitor) and a second agent results in amelioration of ALS or its symptoms to an extent greater than that produced by either the first agent or the second agent alone, and the difference between the combined effect and the effect of each agent alone can be statistically significant.

[0120] In some embodiments, the co-administration of a first agent and a second agent allows for the administration of the second agent at a reduced dose, at a reduced number of doses, and / or at a reduced dosing frequency compared to the standard approved dosing regimen for the second agent.

[0121] In some embodiments, an immunosuppressant known to one skilled in the art can be administered to a subject in combination with the agents described herein. Exemplary immunosuppressants include, but are not limited to, cyclosporine, FK506, rapamycin, CTLA4-Ig, anti-TNF agents (e.g., etanercept), daclizumab (e.g., Zenapax™), anti-CD2 agents, anti-CD4 agents, and anti-CD40 agents.

[0122] Route of administration The agent or pharmaceutical composition can be administered by a variety of routes, including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal administration, intrapleurally, intravenously, intraarterially, intraperitoneally, subcutaneously, intramuscularly, intranasally, intrathecally, and / or intraarticularly, or a combination thereof. In some embodiments, the agent or pharmaceutical composition is administered orally.

[0123] The present invention is further illustrated in the following examples. It should be understood that these examples, while illustrating embodiments of the present invention, are given for illustrative purposes only. From the above discussion and these examples, those skilled in the art will be able to ascertain the essential features of the present invention and can make various changes and modifications to the present invention to adapt it to various uses and conditions without departing from the spirit and scope thereof. Accordingly, various modifications of the present invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Furthermore, the features of each aspect of the present invention relate to each of the other aspects, mutatis mutandis. For example, embodiments relating to the type of VCP inhibitor, pathogenic mutation, type of ALS, etc., provided in the context of VCP inhibitors for use according to the present invention equally apply to the diagnostic methods, compositions, and kits of the present invention. [Example]

[0124] Example 1 - Materials and Methods Human fibroblasts and iPSCs. Dermal fibroblasts were cultured in OptiMEM + 10% FCS medium. For iPSC generation, episomal plasmids, pCXLE hOct4 shp53, pCXLE hSK, and pCXLE hUL (Addgene)

[26] , were transfected into dermal fibroblasts. The three control lines used were commercially available (Control 2, Control 3, and Control 5) and were purchased from Coriell (catalog number ND41866). * C), were purchased from ThermoFisher Scientific (catalog number A18945) and Cedars-Sinai (CS02iCTR-NTn4). The TARDBP mutant lines (MUT5 and MUT6) used were commercially available and purchased from NINDS (ND50007) and Cedars-Sinai (CS47i). Details of the iPSC lines used in this study can be seen in Figure 9.

[0125] Cell culture and motor neuron differentiation. iPSCs were maintained on Geltrex (Life Technologies) in Essential 8 Medium (Life Technologies) and passaged using EDTA (Life Technologies, 0.5 mM). iPSC cultures were maintained at 37°C and 5% carbon dioxide. iPSCs were differentiated into spinal motor neurons as described in Hall et al., 2017

[12] .

[0126] iPSCs were plated to 100% confluency and then differentiated into neuroepithelia in a medium consisting of DMEM / F12 Glutamax, Neurobasal, L-glutamine, N2 supplement, non-essential amino acids, B27 supplement, β-mercaptoethanol (all from Life Technologies), and insulin (Sigma). Cells were treated sequentially with small molecules: Days 0–7: 1 μM dorsomorphin (Millipore), 2 μM SB431542 (Tocris Bioscience), and 3.3 μM CHIR99021 (Miltenyi Biotec); Days 7–14: 0.5 μM retinoic acid (Sigma) and 1 μM palmorphagen (Sigma); Days 14–18: 0.1 μM palmorphagen. Following 18 days of neural conversion and patterning, cells were terminally differentiated into neural precursors in 0.1 μM Compound E (Enzo Life Sciences).

[0127] The neuroepithelial layer was enzymatically dissociated throughout using dispase (GIBCO, 1 mg / ml). Neural precursors were dissociated with Accutase (Life Technologies) and finally plated onto 96-well plates (Falcon) coated with polyethyleneimine (PEI) (2.2 mg / ml in 0.1 M sodium borate (Sigma)) and Geltrex. Following 6 days of terminal differentiation, cells were fixed in 4% paraformaldehyde for immunolabeling.

[0128] Inhibitor treatment. Motor neuron cultures were treated with 1 μM ML240 (Sigma; SML1071; CAS: 1346527-98-7) for 2 hours, 5 μM DBeQ for 3 hours, or 1 μM CB-5083 for 3 hours.

[0129] Immunofluorescence staining. Cells were fixed in 4% paraformaldehyde in PBS for 15 minutes at room temperature (RT). For permeabilization and blocking of nonspecific antibodies, 0.3% Triton-X containing 5% bovine serum albumin (BSA) (Sigma) in PBS was added for 60 minutes. Primary antibodies were prepared in 5% BSA and then added overnight at 4°C. The primary antibodies used were SMI-32 (BioLegend; 801701; mouse; 1:1000), ChAT (Millipore; AB144P; goat; 1:100), βIII-tubulin (abcam; ab41489; chicken; 1:1000), TDP-43 (ProteinTech; 12892-1-AP; rabbit; 1:400), SFPQ (abcam; ab11825; mouse; 1:400), FUS (Santa Cruz; sc-47711; mouse; 1:200), hnRNPA1 (Cell Signaling; 8443S; rabbit; 1:500), and hnRNPK (Santa Cruz; sc-28380; mouse; 1:500). Species-specific Alexa Fluor-conjugated secondary antibodies (Life Technologies) diluted 1:1000 in 5% BSA were added for 90 min at RT in the dark. Cells were washed once for 10 min in PBS containing DAPI, 4',6-diamidino-2-phenylindole nuclear stain (1:1000).

[0130] Image Acquisition and Analysis. Images were acquired using an Opera Phenix High-Content Screening System (Perkin Elmer). Images were acquired as confocal z-stacks with a 1 μm z-step using a 40× objective. Stacks were processed to obtain maximum intensity projections. A minimum of 12 fields were acquired for each well. To calculate the nuclear:cytoplasmic ratio of RNA-binding proteins (RBPs) in single cells, images were analyzed using a Columbus image analysis system (Perkin Elmer). A DAPI mask defined the nucleus, and a trained machine learning function automatically selected neurons based on nuclear characteristics. The mean nuclear intensity of the RBP of interest was measured for each individual cell. For cytoplasmic measurements, a 1.5 μm cytoplasmic region was defined around the nucleus within the cytoplasmic mask, and the mean intensity was measured. An example of the nuclear and cytoplasmic compartments defined by this analysis can be seen in Figure 5A. The ratio of the mean nuclear:cytoplasmic intensity measurements was calculated for each cell. The mean of each field was calculated and then averaged across wells.

[0131] For the nucleus:neurite ratio, we performed a semi-automated image analysis pipeline combining Ilastik

[27] , Cellprofiler

[28] , and ImageJ. Nuclei were segmented using DAPI-stained images scaled from 0 to 500 in intensity in ImageJ to allow for improved detection of nuclei. A randomly selected subset of images was used in Ilastik to generate a binary nuclei segmentation mask. To define the neurite compartment, a neuron mask was created using βIII-tubulin, as this is a reliable marker of axons and dendrites. To remove nuclei and cytoplasm from the βIII-tubulin mask, nuclei were magnified by 30 pixels and removed, ensuring that only neurites were included in the analysis. An example of a compartment defined by this analysis can be seen in Figure 5B. Intensity measurements of proteins of interest were performed in Cellprofiler using the nucleus and neurite masks. Calculation of intensity values ​​and ratios was performed using custom R scripts.

[0132] When examining the nucleus:cytoplasm or nucleus:neurite ratio, if an increase or decrease in the ratio is detected, it is unclear which subcellular compartment contributes to the change. To address this for the nucleus:neurite ratio, we took advantage of the presence of compartment-specific markers. As described above, we performed the same semi-automated image analysis pipeline for the nucleus:neurite ratio, but in addition to the proteins of interest, we performed intensity measurements for DAPI and βIII-tubulin, which were used to calculate the specific ratios.

[0133] Western blot analysis. Protein levels of TDP-43, FUS, and SFPQ were assessed in whole cells in control and VCP mutant motor neurons. Prior to protein extraction, cells were left untreated or treated with 1 μM ML240 for 2 hours. Cells were lysed, and proteins were extracted by RIPA disruption. Total protein concentration was quantified using a BCA protein assay (Sigma). Equal amounts of protein samples were then loaded onto gels, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Samples were then blocked with PBS, 0.1% Tween, and 5% dry milk powder for 1 hour at room temperature, followed by overnight incubation with primary antibodies at 4°C. The following antibodies were diluted in PBS 5% BSA: TDP-43 (ProteinTech; 12892-1-AP; rabbit; 1:1000), SFPQ (Abcam; 11825; mouse; 1:250), FUS (Santa Cruz; sc-47711; mouse; 1:500), and GAPDH (GeneTex; GT239; mouse; 1:10,000). For detection, membranes were incubated with species-specific near-infrared fluorescent antibodies (IRDye, Licor) for 1 h at RT and imaged using the Odyssey Fc Imaging System (Licor).

[0134] Statistical analysis. There were three control and four VCP mutant iPSC lines, details of which can be seen in Figure 9. The number of cells used in each experiment is stated in the figure legend. At a minimum, for each line, data were collected from 34 fields from 6 wells across three independent experimental replicates. Data were plotted as violin plots, either per field or per well. When data were presented as normalized to the untreated control, each raw value was divided by the mean of the untreated control within each experimental replicate. When comparing two individual groups with Gaussian distributions, an unpaired two-tailed Student's t-test was used. When a Gaussian distribution was not achieved, a Mann-Whitney test was used. Statistical analysis was performed using Prism8. A p-value of 0.05 or less was considered statistically significant ( * p<0.05, ** p<0.01,*** p<0.001).

[0135] Example 2 - TDP-43, SFPQ and FUS are mislocalized to neurites in VCP mutant motor neurons We took advantage of our established robust differentiation of human iPSCs into highly abundant, characterized spinal motor neurons (MNs) that are positive for choline acetyltransferase (ChAT), SMI-32, and βIII-tubulin (TUJ1) (Figure 6).

[0136] Importantly, we have previously functionally validated our enriched MN cultures by i) demonstrating cytosolic calcium responses to physiological calcium stimuli (glutamate and KCl), ii) whole-cell patch clamping, and iii) coculture with iPSC-derived skeletal muscle with multielectrode array (MEA) analysis

[12] and demonstrating neuromuscular junction formation

[13] . Using this model, we have previously reported time-resolved pathogenic phenotypes of VCP-associated ALS, including novel features of ALS, such as reduced nuclear-to-cytoplasmic ratios of SFPQ and FUS in mutant neural precursors [2, 3, 12]. Furthermore, we have confirmed the intracellular TDP-43 and FUS mislocalization phenotype in terminally differentiated motor neurons [12, 29, 30].

[0137] However, our previous studies and those of others have not systematically examined these aforementioned RBPs together, nor have they addressed the specific sites of mislocalization of these RBPs with respect to their presence within neurites. Against this background, we utilized our VCP mutant iPSC-derived motor neurons to comprehensively investigate the subcellular localization of five ALS-associated RBPs. Single-cell analysis of the nuclear-to-cytoplasmic ratios of over 70,000 neurons revealed decreased TDP-43 and SFPQ in VCP mutant human motor neurons (Figure 1A, B, D, and E), which is in line with our recent report of a reduced nuclear-to-cytoplasmic ratio of FUS

[29] .

[0138] Based on further analysis, we detected that TDP-43 and SFPQ also had a reduced nuclear-to-neurite ratio, thereby also abnormally localizing within the neurites of VCP mutant motor neurons (Fig. 1C, F). The presence of compartment-specific markers (nucleus: DAPI, neurite: βIII-tubulin) then allowed us to examine the nuclear and neurite compartments independently, revealing that the reduced nuclear-to-neurite ratios of these RBPs were caused by both their reduction in the nucleus and their increase in the neurite (Fig. 7A-D).

[0139] To rule out the possibility that RBPs are generally mislocalized in the iPSC model, we next examined the subcellular localization of hnRNPA1 and hnRNPK, which have previously been implicated in ALS [31, 32]. However, hnRNPA1 and hnRNPK showed no detectable change in their nuclear-to-cytoplasmic localization in VCP mutant motor neurons, consistent with the selective mislocalization of TDP-43, FUS, and SFPQ in iPSC-derived VCP mutant motor neurons (Figure 1G–J).

[0140] Example 3 - Pharmacological inhibition of the VCP D2 ATPase domain does not induce ALS phenotypes in healthy human motor neurons Whether VCP disease mutations exert dominant-active or dominant-negative effects remains a matter of debate in the field. To gain mechanistic insight into the effects of VCP mutations in human motor neurons, we utilized ML240, a potent and selective inhibitor of the D2 ATPase domain of the VCP protein

[33] .

[0141] Control motor neurons were treated with 1 μM ML240 for 2 hours before fixation and immunocytochemistry. Interestingly, inhibition of D2 ATPase increased the nuclear-to-cytoplasmic ratio of TDP-43 (Figure 2A and B). However, no such increase was observed in the nuclear-to-neurite ratio of TDP-43, possibly suggesting a proximal-to-distal shift in protein distribution in this setting (Figure 2C).

[0142] Interestingly, we found that the nuclear-to-cytoplasmic ratio of FUS was unchanged, but a small but statistically significant increase in the nuclear-to-neurite ratio of FUS was observed (Fig. 2D–F). This suggests that the D2 ATPase domain may have an RBP-specific role in a cellular compartment-specific manner. Further analysis of the aforementioned RBPs; SFPQ, hnRNPA1, and hnRNPK, revealed no change in their nuclear-to-cytoplasmic or nuclear-to-neurite ratios (SFPQ) upon VCP D2 ATPase inhibition (Fig. 2G–I). Collectively, these data argue against loss of function of the VCP D2 ATPase domain as a mechanism for the observed RBP mislocalization phenotype.

[0143] Example 4 - Pharmacological inhibition of the D2 ATPase domain reverses TDP-43 and FUS mislocalization associated with VCP mutations in human motor neurons Noting the clear effect of ML240 on TDP-43 in control motor neurons, we reasoned that its application to VCP mutant motor neurons might ameliorate their RBP mislocalization phenotype. In particular, we hypothesized that the mutations that cause ALS VCP (VCP R155C and VCP R191Q) result in a dominant-active effect of the D2 ATPase domain.

[0144] Application of ML240 in VCP mutant motor neurons indeed robustly reversed the mislocalization of both TDP-43 and FUS when examining both nucleus-to-cytoplasmic and nucleus-to-neurite mislocalization ( Fig. 3, A–F ).

[0145] For SFPQ, ML240 treatment also significantly reversed the mislocalization from the nucleus to the neurites. However, although ML240 treatment increased the nuclear-to-cytoplasmic ratio (mean; UT = 0.90, ML240 = 0.97), this difference did not reach statistical significance for SFPQ (Fig. 3G–I).

[0146] The present data further demonstrate that this reversal is due to relocalization of TDP-43, FUS, and SFPQ from neurites and / or the cytoplasm to the nucleus, as total protein levels were unchanged upon ML240 treatment (Figure 8A, B).

[0147] Notably, hnRNPA1 and hnRNPK showed no change in nuclear-to-cytoplasmic ratio, and ML240 treatment only affected the localization of RBPs that were significantly mislocalized as a result of the VCP mutation (Fig. 3J,K).

[0148] In this study, we used our extensive, functionally validated, iPSC-derived, patient-specific motor neuron model [2, 12, 13] to systematically investigate the effects of ALS-causing VCP mutations on RBP nucleocytoplasmic localization and the ability of inhibitors of the VCP D2 ATPase to suppress these VCP-associated disease phenotypes. Importantly, this model closely resembles the physiology of ALS motor neurons because it conveys pathophysiological levels of mutations and does not rely on artificial overexpression.

[0149] The data presented here are the first to demonstrate that VCP mutations (R155C and R191Q) cause a selective reduction in the nuclear-to-cytoplasmic ratio of TDP-43, FUS, and SFPQ in terminally differentiated motor neurons (where normal nucleocytoplasmic distribution of both hnRNPK and hnRNPA1 is observed). We further show that TDP-43, FUS, and SFPQ are also mislocalized in motor neuron neurites (Figure 1). Recent studies have demonstrated novel roles for these RBPs in axonal mRNA translation and viability, suggesting that impaired axonal RNA processing may contribute to specific pathophysiology in motor neurons [34, 35, 36].

[0150] However, the main finding of our study is that VCP mutation-associated mislocalization of TDP-43, FUS, and (partially) SPFQ is reversible by pharmacological inhibition of the D2 ATPase domain of the VCP protein using the potent and selective inhibitor ML240 [11, 33, 37]. This dominant-active VCP mutation mechanism has also been demonstrated for other phenotypes [38, 39]. However, there is controversy in the field, and some studies have suggested that VCP mutants function as dominant-negatives [40, 41, 42].

[0151] These seemingly contrasting studies can be reconciled by hypothesizing that inhibition of ATP hydrolysis can reverse downstream effects associated with excessive ATP hydrolysis while also enhancing a dominant-negative effect on ATPase activation. Furthermore, because VCP has a wide range of intracellular functions, it can be hypothesized that VCP mutations can produce both dominant-active or dominant-negative effects, depending on cofactor binding and subsequent downstream cellular pathways.

[0152] The molecular mechanisms underlying how VCP interacts with TDP-43, FUS, and SFPQ are unclear and should be explored in further studies. To date, studies have demonstrated direct interactions between VCP and RBPs, including TDP-43

[43] and FUS

[44] , but our understanding of the molecular consequences of these interactions is limited.

[0153] Recent studies in yeast have demonstrated a role for Cdc48 / VCP in the endocytosis-dependent turnover of TDP-43 and FUS

[45] . Together with the present study, this raises the possibility that increased D2 ATPase activity can disrupt the VCP-dependent endocytic machinery of cytoplasmic proteostasis. Understanding the precise consequences of VCP interaction with RBPs will help elucidate the mechanisms underlying its mislocalization in pathological conditions.

[0154] Our finding that VCP disease mutants exhibit increased D2 ATPase activity may have important therapeutic implications: indeed, VCP inhibitors have been found to rescue multiple VCP disease phenotypes in Drosophila models and patient fibroblasts

[39] .

[0155] Notably, the rescue was multifaceted, including improvement of mitochondrial phenotype, p62, and ubiquitin pathology. Our findings suggest that pharmacological inhibitors of VCP D2 ATPase may be effective across the spectrum of multisystem pathologies caused by VCP mutations. This extends beyond ALS and IBMPFD to some cases of VCP-associated Charcot-Marie-Tooth disease and hereditary spastic paraplegia [46, 47]. However, studies have shown that VCP inhibitors can disrupt the maintenance of cellular homeostasis in a dose-dependent manner, so the therapeutic balance should be explored and optimized in future studies. However, since VCP inhibitors are already in phase II clinical trials for cancer treatment, it is important to recognize their therapeutic potential for these devastating and, so far, incurable diseases.

[0156] Example 5 - Additional VCP inhibitors reverse TDP-43 mislocalization in VCP mutant and TARDBP mutant human motor neurons To gain further insight into the role of VCP inhibition in motor neurons, motor neurons were treated with additional VCP inhibitors, DBeQ and CB-5083. DBeQ is a reversible, ATP-competitive VCP inhibitor that targets both the D1 and D2 ATPase domains of VCP. CB-5083 is a potent, reversible, ATP-competitive VCP inhibitor that selectively targets the D2 ATPase domain.

[0157] Control motor neurons were treated with 5 μM DBeQ for 3 hours or 1 μM CB-5083 for 3 hours before fixation and immunocytochemistry. Inhibition of VCP by DBeQ and CB-5083, respectively, increased the nuclear-to-cytoplasmic ratio of TDP-43 (Figure 11A and B), supporting the findings observed upon ML240 treatment (see Example 3).

[0158] ML240 was shown to reliably reverse TDP-43 mislocalization in VCP mutant motor neurons (see Example 4). VCP mutant motor neurons were treated with DBeQ and CB-5083, respectively. These additional VCP inhibitors also reversed TDP-43 mislocalization from the nucleus to the cytoplasm (Figure 11, C and D).

[0159] To investigate whether VCP inhibition reverses the nuclear-to-cytoplasmic mislocalization of TDP-43 in other genetic backgrounds of ALS (i.e., non-VCP mutants), we treated TARDBP mutant (G298S) motor neurons with CB-5083. VCP inhibition by CB-5083 increased the nuclear-to-cytoplasmic ratio of TDP-43 in these cell lines (Figure ​(Figure11E). 11E).

[0160] These data demonstrate that VCP inhibitors other than ML240 are effective in reversing TDP-43 mislocalization in ALS mutant cell lines for both VCP and non-VCP mutants.

[0161] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. Furthermore, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent section headings are used, they should not be construed as necessarily limiting.

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Claims

1. A VCP (valosin-containing protein) inhibitor for treating or preventing amyotrophic lateral sclerosis (ALS) in a subject, wherein the subject does not have a known pathogenic mutation in the VCP gene, SOD1 gene, ALS2 gene, VAPB gene, SETX gene, TDP-43 gene, FUS / TLS gene, and / or OPTN gene.

2. The VCP inhibitor according to claim 1, wherein the subject has not been identified as having a pathogenic mutation in the VCP gene.

3. The VCP inhibitor according to claim 1, wherein the subject has been identified as not having a pathogenic mutation in the VCP gene.

4. The VCP inhibitor according to claim 1, wherein the ALS is non-VCP-related ALS.

5. wherein the subject, in the VCP gene, (i) any one of positions R155 and R191, (ii) selected from the list consisting of R155C and R191Q, (iii) any one of positions R95, I114, I151, R155, G156, M158, R159, R191, N387, N401, R487, D592, R662, and N750, and / or (iv) selected from the list consisting of R95C, R95G, I114V, I151V, R155H, R155C, G156C, M158V, R159G, R159C, R159H, R191G, R191Q, N387T, N401S, R487H, D592N, R662C, and N750S, The VCP inhibitor according to claim 1, wherein the subject has been identified as not having a pathogenic gene mutation.

6. The amyotrophic lateral sclerosis is associated with a reduction in the nuclear-to-cytoplasmic ratio of one or more of TDP-43, FUS, and / or SFPQ. Optionally, the VCP inhibitor improves one or more symptoms associated with a reduction in the nuclear-to-cytoplasmic ratio of one or more of TDP-43, FUS, and / or SFPQ; Further optionally, (i) the amyotrophic lateral sclerosis is associated with a reduction in the nuclear-to-cytoplasmic ratio of TDP-43, and optionally, the VCP inhibitor improves one or more symptoms associated with a reduction in the nuclear-to-cytoplasmic ratio of TDP-43, (ii) the amyotrophic lateral sclerosis is associated with a reduction in the nuclear-to-cytoplasmic ratio of FUS, and optionally, the VCP inhibitor improves one or more symptoms associated with a reduction in the nuclear-to-cytoplasmic ratio of FUS, and / or (iii) the amyotrophic lateral sclerosis is associated with a reduction in the nuclear-to-cytoplasmic ratio of SFPQ, and optionally, the VCP inhibitor improves one or more symptoms associated with the reduction in the nuclear-to-cytoplasmic ratio of SFPQ The VCP inhibitor according to claim 1 **Claim 7** Treating or preventing ALS comprises partial or complete alleviation, improvement, elimination, inhibition, delay in onset, reduction in severity and / or incidence of neurological dysfunction in a patient suffering from or susceptible to ALS, optionally, the neurological dysfunction comprises symptoms associated with a dysfunction of the central nervous system such as one or more of developmental delay, progressive cognitive dysfunction, hearing loss, speech developmental disorder, lack of motor skills, hyperactivity, aggressiveness and / or sleep disorder, and further optionally, by treating or preventing ALS with a VCP inhibitor, a reversal or improvement of one or more neurological dysfunction symptoms by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or more than about 100% compared to the neurological dysfunction symptoms in the absence of the VCP inhibitor is brought about. The VCP inhibitor according to claim 1 **Claim 8** The VCP inhibitor according to claim 1, which inhibits the D2 ATPase domain of VCP **Claim 9** The VCP inhibitor is (i) selected from the group consisting of ML240 (2-(2-amino-1H-benzoimidazol-1-yl)-8-methoxy-N-(phenylmethyl)-4-quinazolinamine), ML241, 2-anilino-4-aryl-1,3-thiazole, 3,4-methylenedioxy-6-nitrostyrene, DBeQ (N2,N4-dibenzylquinazoline-2,4-diamine), CB-5083 (1-[7,8-dihydro-4-[(phenylmethyl)amino]-5H-pyrano[4,3-d]pyrimidin-2-yl]-2-methyl-1H-indole-4-carboxamide), CB-5339 (1-[4-(benzylamino)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-2-yl]-2-methylindole-4-carboxamide), UPCDC-30245 (1-(3-(5-fluoro-1H-indol-2-yl)phenyl)-N-(2-(4-isopropylpiperazin-1-yl)ethyl)piperidin-4-amine), NMS-873, NMS-859, iiyaresatatin I, and xanthohumol; (ii) selected from the group consisting of ML240 (2-(2-amino-1H-benzoimidazol-1-yl)-8-methoxy-N-(phenylmethyl)-4-quinazolinamine), ML241, 2-anilino-4-aryl-1,3-thiazole, 3,4-methylenedioxy-6-nitrostyrene, DBeQ (N2,N4-dibenzylquinazoline-2,4-diamine), NMS-873, NMS-859, iiyaresatatin I, and xanthohumol; (iii) ML240 (2-(2-amino-1H-benzoimidazol-1-yl)-8-methoxy-N-(phenylmethyl)-4-quinazolinamine), or (iv) CB-5083 (1-[7,8-dihydro-4-[(phenylmethyl)amino]-5H-pyrano[4,3-d]pyrimidin-2-yl]-2-methyl-1H-indole-4-carboxamide) or CB-5339 (1-[4-(benzylamino)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-2-yl]-2-methylindole-4-carboxamide); The VCP inhibitor according to claim 1.

10. A method for determining a subject as having or suspected of having non-VCP-related ALS, comprising determining whether the subject has a pathogenic mutation in the VCP gene, wherein the absence of a pathogenic mutation in the VCP gene means that the subject has or is suspected of having non-VCP-related ALS, and optionally, comprising identifying the absence of a pathogenic gene mutation at any of positions R95, I114, I151, R155, G156, M158, R159, R191, N387, N401, R487, D592, R662, and N750 in the VCP gene, or comprising identifying the absence of any pathogenic gene mutation in the VCP gene selected from the list consisting of R95C, R95G, I114V, I151V, R155H, R155C, G156C, M158V, R159G, R159C, R159H, R191G, R191Q, N387T, N401S, R487H, D592N, R662C, and N750S, Method.

11. A VCP inhibitor for treating or preventing non-VCP-related ALS in a subject, comprising determining a patient as having or suspected of having non-VCP-related ALS using the method according to claim 10.

12. A VCP inhibitor for treating or preventing non-VCP-related ALS in a subject determined to have or suspected of having non-VCP-related ALS using the method according to claim 10.

13. A pharmaceutical composition comprising a VCP inhibitor for use in a method of treating or preventing amyotrophic lateral sclerosis (ALS), optionally comprising one or more excipients, wherein the subject does not have a known pathogenic mutation in the VCP gene, SOD1 gene, ALS2 gene, VAPB gene, SETX gene, TDP-43 gene, FUS / TLS gene, and / or OPTN gene. Pharmaceutical composition.

14. A kit for diagnosing a subject as having or suspected of having non-VCP-related ALS, comprising means for determining whether the subject has a pathogenic mutation in the VCP gene, optionally further comprising one or more containers containing one or more VCP inhibitors and optionally information materials, and further optionally, the information materials comprising instructions for use of the kit in the diagnosis and treatment of non-VCP-related ALS. Kit.

15. The VCP inhibitor is the VCP inhibitor according to Claim 8 or 9, the VCP inhibitor according to Claim 11 or 12, the pharmaceutical composition according to Claim 13, or the kit according to Claim 14.

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