Use of bromodomain inhibitors for the treatment of huntington's disease

Abstract

The present disclosure relates to the treatment of Huntington’s disease using BRD9 inhibitors.

Description

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 007,161, filed April 8, 2020, the entire contents of which are incorporated herein by reference.

[0003] 2. BACKGROUND

[0004] Huntington’s disease (HD) is a progressive, fatal neurodegenerative disease that is inherited in an autosomal dominant manner and caused by a mutation of a polymorphic trinucleotide (CAG) tract expansion in the Huntingtin gene (HTT). The American College of Medical Genetics / American Society of Human Genetics Huntington Disease Genetic Testing Workgroup (Am J Hum Genet. 1998; 62: 1243-7) indicated that 26 or fewer CAG repeat sequences in the HTT gene are considered “normal”; 27-35 CAG repeat sequences are considered variable normal alleles; and 36 or more CAG repeat sequences are considered pathogenic alleles. The HTT gene encodes the HTT protein, and the expanded CAG tract leads to a pathologically increased polyglutamine repeat sequence near the N-terminus of the protein. It is an autosomal dominant disorder, and although individuals carry two copies of the HTT gene, one mutant allele is sufficient to cause HD.

[0005] HD is associated with a triad of motor, behavioral, and cognitive symptoms. Motor impairment is the main feature of the disease, with chorea being the most prominent motor symptom. Although useful for diagnosis, chorea is a poor marker of disease severity. In contrast, disability and disease severity are most associated with negative motor features, such as impaired fine motor skills, bradykinesia, and gross motor coordination skills, including speech difficulties, gait and postural dysfunction (Mahant et al., 2003, Neurology 61(8): 1085-92).

[0006] Numerous medications are prescribed to improve motor and emotional problems associated with HD; however, there is insufficient scientific evidence that the various medications are useful for HD (Mestre et al., 2009, Cochrane Database Syst Rev. (3): CD006455; Mestre et al., 2009, Cochrane Database Syst Rev. (3): CD006456). Thus, there is a significant unmet medical need in developing medications to improve HD symptoms. 3. SUMMARY

[0008] BRD9 is a bromodomain-containing protein that contains a bromodomain at the amino-terminal half of its sequence and has a domain of unknown function (DUF3512) at its carboxy-terminal end. BRD9 is part of the chromatin-remodeling BAF (also known as SWI / SNF) complex (Kadoch et al., 2013, Nat. Genet. 45, 592-601; Middeljans et al., 2012, PLoS. One. 7, e33834). Amplification of the BRD9 locus has been observed in ovarian and breast cancers (see, e.g., Kang et al., 2008, Cancer Genet. Cytogenet. 182: 1-11; Scotto et al., 2008, Mol. Cancer 7:58), and BRD9 inhibitors are being developed as potential cancer therapeutics (see, e.g., Martin et al., 2016, J. Med. Chem. 59(10):4462-4475). The present disclosure is based on the discovery that BRD9 inhibitors are effective in reversing the disease phenotype in human organoid HD models, and thus are useful in treating patients with HD.

[0009] Accordingly, the present disclosure provides methods of treating HD by administering to a subject in need thereof an effective amount of a BRD9 inhibitor. Examples of methods and BRD9 inhibitors used therein are described below in Section 6 and specific embodiments 1 to 47.

[0010] In another aspect, the present disclosure provides BRD9 inhibitors for use in treating HD in a subject in need thereof. Examples of BRD9 inhibitors for use in treating HD in a subject in need thereof are provided below in Section 6 and specific embodiments 48 to 94.

[0011] In yet another aspect, the present disclosure provides the use of a BRD9 inhibitor in the manufacture of a medicament for treating HD. Examples of the use of BRD9 inhibitors in the manufacture of a medicament for treating HD are provided below in Section 6 and specific embodiments 95 and 96. 4. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figures 1A-1B Immunofluorescence analysis of neuruloids on disc-shaped micropatterns is shown. Figure 1A Top view. Bottom view. Stained with DAPI, PAX6 and N-CAD. Figure 1B Left panel: cartoon picture of the ectodermal compartments within a human embryo at the stage of neural development. Right panel: representative of human neuruloids. Neural cells 102, neural crest 104, cranial placode 106 and epidermis 108. The reorganized embryonic parts show the developing central nervous system organized in a neural rosette (PAX6+ cells, Figure 1A), along with neural crest (SOX10+) and placode fates (SIX1+), are covered by a layer of epidermal cells (restricted to TFAP2+). Comparison of in vitro neuroepithelium to in vivo counterpart at around day 21 post-fertilization Figure 1B reveals a high degree of similarity, making them ideal preclinical endpoints for studying human genetic diseases and discovering drugs based on phenotypic reversion.

[0014] Figure 2 shows phenotypic characterization of HD lines. (Left panel) Representative images of PAX6 area for different HD isogenic lines in the neuroepithelium assay. PAX6 staining allows visualization of Pax6 area. (Right panel) Relevant quantification of PAX6 area normalized by colony area. Note that HTT- / - line shows the most significant phenotype. This suggests that poly-Q expansion of HTT protein represents a major loss-of-function, rather than a gain-of-toxic function, as usually hypothesized.

[0015] Figure 3 illustrates the concept of phenotypic reversion of HD neuroepithelium, which will be used as the basis for high-throughput screening campaigns.

[0016] Figure 4 shows the scheme of AI-mediated drug screening analysis. A specific network is used to input all images in the screening experiment. The network is specifically trained to output two quantities: drug toxicity and drug efficacy.

[0017] Figure 5 shows the results of the screening campaign. The effect of 2080 compounds is plotted as a function of efficacy (phenotype rescue) and toxicity. WT control (RUES2) and HD-56CAG (56CAG) controls are plotted as triangles pointing to the right and left, respectively. Upward triangles represent the effect of each compound. Diamonds represent hit compounds, highlighting molecules with high efficacy and low toxicity.

[0018] Figure 6 shows that bromosporin rescues HD neuroepithelium phenotype. (Upper panel) Results of the primary screen. From left to right: examples of WT control and HD control wells, and HD wells treated with 10 mM bromosporin. Each well contains approximately 27 neuroepithelium replicates. Neuroepithelium is stained with DAPI (nuclei), PAX6 (neural marker), and phalloidin (filamentous actin). (Lower panel) Hit validation in small-scale experiments using bromosporin stock. Neuroepithelium is stained with SOX10 (neural crest marker), PAX6 (neural marker), and N-CAD (cell-cell adhesion). 0.5 mM bromosporin rescues HD phenotype.

[0019] Figure 7 Quantification of the potency and toxicity of bromosporin. The efficacy of bromosporin in rescuing the HD-neuraloid phenotype of HD-56CAG was measured (open diamonds and curves). This shows an EC 50 Toxicity of bromosporin as a function of concentration was measured in the WT-20CAG background and in the HD-56CAG background.

[0020] Figure 8 A panel of BRD inhibitors was shown to be active in the neuraloid assay at a single concentration of 10 mM. For each molecule, the point represents its known molecular target, and the rescue efficacy (dotted bars) and the level of toxicity (dashed bars) are shown. Only compounds with rescue activity above the threshold indicated by the dashed line on the right and toxicity below the level of the dashed line on the left were considered as hits. In this experiment, only BI7273 (a BRD9 / 7 inhibitor) was a hit.

[0021] Figures 9A-9B A panel of BRD9 inhibitors was shown to be active in the neuraloid assay in a dose-dependent manner. The potency ( Figure 9A ) and toxicity ( Figure 9B ) of 5 different small molecules inhibiting BRD9 are shown. All compounds were potent (with sub-micromolar EC 50 ) and showed low toxicity (below micromolar range).

[0022] Figure 10 BRD inhibitors showing sub-micromolar potency in rescuing HD neuraloids are illustrated. Bromosporin, BI7273, I-BRD9, dBRD9 and BI9564 all rescued the HD neuraloid to the WT configuration. Bromosporin is a broad-spectrum bromodomain inhibitor with IC50 of 0.41 mM, 0.29 mM, 0.122 mM and 0.017 mM for BRD2, BRD4, BRD9 and CECR2, respectively. BI-7273 is a potent, selective, cell-permeable BRD9 BD inhibitor with IC50 of 19 nM and 117 nM for BRD9 and BRD7 in a AlphaLISA® assay, respectively. I-BRD9 (GSK602) is a potent selective BRD9 inhibitor with a pIC50 of 7.3, while its pIC50 for BRD4 is 5.3. dBRD9 is a potent and selective degradation-PROTAC BRD9. BI-9564 is a selective inhibitor of BRD9 and BRD7 bromodomains with IC50 of 75 nM and 3.4 mM, respectively.

[0023] Figures 11A-11BBromosporaxin is shown to have HTT lowering activity. Total and amplified HTT levels were measured in neuroblast-like cells treated with DMSO control (concentration 0), 5 mM (concentration 1) or 1 mM (concentration 2) bromosporaxin, BI7273, dBRD9 or BI9564. The assay was performed in two genetic backgrounds: 56CAG( Figure 11A ) and 72CAG( Figure 11B ). The dotted line on each graph refers to the control level of HTT in DMSO treated controls. The signal values measured by the MSD assay are specific to the antibody used, and therefore are different in the case of total and amplified HTT, as the two measurements are performed with different antibodies, and therefore absolute levels in the two measurements cannot be compared.

[0024] Figures 12A-12B HD gastruloids are shown to be rescued by BRD inhibitors. Figure 12A Gastruloids were generated by applying CHIR and Activin for two days on pluripotent micropatterned media. In the WT-20CAG configuration, SOX17+ rings form at the periphery of the colonies. The rings are amplified in the HD-56CAG background and significantly occupy the entire colony in the HTT- / - knockout background. Figure 12B Quantification of SOX17+ rings in gastruloids treated with BRD inhibitors. All treatments show a reduction in the area of SOX17+ rings compared to the WT-20CAG background.

[0025] Figures 13A-13C BRD9 knockdown is shown to partially rescue HD-56CAG neuroblasts. Figure 13A Inducible CRISPR interference constructs can reduce BRD9 mRNA levels by 50%. Figure 13B HD-56CAG (left panel) show an enlarged PAX6 area and a lower number of SOX10+ cells compared to WT-20CAG neuroblasts (right panel). BRD9 knockdown partially rescues both features (middle panel). Figure 13C Quantification of the relevant features. N > 40 colonies for each case.

[0026] 5. Definitions

[0027] Administration: The terms “administration,” “application,” or “giving” refer to the introduction of a compound or pharmaceutical composition into a subject, such as by subcutaneous injection, intraperitoneal injection, intramuscular injection, intravenous injection, epidermal or transdermal administration, mucosal administration, oral administration, nasal administration, rectal administration, or vaginal administration. Targeting compounds and pharmaceutical compositions to tissues of the central nervous system may involve delivery to the CSF and brain via intrathecal, intraventricular, or intraparenchymal administration. The carrier formulation may be selected or modified depending on the route of administration. For general reference, see, for example, Remington—The Science and Practice of Pharmacy, 21 st edition. Gennaro et al. editors. Lippincott Williams & Wilkins Philadelphia.

[0028] BRD9 Inhibitor: The term "BRD9 inhibitor" refers to a compound that inhibits the activity of BRD9. In some embodiments, the BRD9 inhibitor is a broad-spectrum bromine-domain inhibitor with activity against one or more bromine-domain proteins, including BRD9. In some embodiments, the BRD9 inhibitor is a selective inhibitor of BRD9. For example, the BRD9 inhibitor may have at least two, at least five, or at least ten times higher anti-BRD9 activity compared to one, two, or three other bromine-domain proteins (e.g., but not limited to BRD2, BRD3, BRD4, or any combination thereof). Because BRD9 and BRD7 are closely related, in some embodiments, the selective inhibitor of BRD9 may inhibit BRD7 to a similar degree as BRD9, provided that it has less inhibitory activity against BRD2, BRD3, and / or BRD4.

[0029] Bromine domain: The term "bromine domain" refers to a protein domain that recognizes acetylated lysine residues (such as those at the N-terminal tails of histones). In some embodiments, the bromine domain (e.g., a protein containing the bromine domain, such as a bromine and extra terminal (BET) protein) comprises approximately 110 amino acids and shares a conserved fold consisting of a left-handed bundle of four α-helices linked by different loop regions that interact with chromatin. In certain embodiments, the bromodomain is ASH1L (GenBank ID: gi|8922081), ATAD2 (GenBank ID: gi|24497618), BAZ2B (GenBank ID: gi|7304923), BRD1 (GenBank ID: gi|11321642), BRD2(1) (GenBank ID: gi|4826806), BRD2(2)(GenBank ID: gi|4826806), BRD3(1)(GenBank ID: gi|11067749), BRD3(2)(GenBankID: gi|11067749), BRD4(1)(GenBank ID: gi|19718731), BRD4(2)(GenBank ID: gi|19718731), BRD9 (GenBank ID: gi|57770383), BRDT(1)(GenBank ID: gi|46399198), BRPF1 (GenBank ID: gi|51173720), CECR2 (GenBank ID: gi|148612882), CREBBP (GenBank ID: gi|4758056), EP300 (GenBank ID: gi|50345997), FALZ (GenBank ID: gi|38788274), GCN5L2 (GenBank ID: gi|10835101), KIAA1240 (GenBank ID: gi|51460532), LOC93349 (GenBank ID: gi|134133279), PB1(1) (GenBank ID: gi|30794372), PB1(2) (GenBank ID: gi|30794372), PB1(3)(GenBank ID: gi|30794372), PB1(5) (GenBank ID: gi|30794372), PB1(6) (GenBank ID: gi|30794372), PCAF (GenBank ID: gi|140805843), PHIP(2) (GenBankID: gi|34996489), SMARCA2 (GenBank ID: gi|48255900), SMARCA4 (GenBank ID: gi|21071056), SP140 (GenBank ID: gi|52487219), TAF1(1) (GenBank ID: gi|20357585), TAF1(2) (GenBank ID: gi|20357585), TAF1L(1) (GenBank ID: gi|24429572), TAF1L(2) (GenBank ID: gi|24429572), TIF1 (GenBank ID: gi 14971415), TRIM28 (GenBank ID: gi|5032179), or WDR9(2) (GenBank ID: gi|16445436).

[0030] Degron: The term "degron" refers to an amino acid sequence that provides a degradation signal that directs the cellular degradation of a polypeptide. The degron can facilitate the degradation of the attached polypeptide by the proteasome or the autophagy-lysosome pathway. See, e.g., Kanemaki et al., 2013, Pflugers Arch. 465(3):419-425 and Erales et al., 2014, Biochim Biophys Acta 1843(1):216-221.

[0031] Dendrimer: As used herein, the term “dendrimer” is intended to include, but is not limited to, a molecular structure having an inner core, an inner layer (or “generation”) of repeating units regularly attached to this initiator core, and an outer surface of terminal groups attached to the outermost generation. Examples of dendrimers include, but are not limited to, poly(amidoamine) (PAMAM), polyesters, polylysine, and poly(propylene imine) (PPI). The PAMAM dendrimers can have carboxyl, amine, and hydroxyl termini, and can be dendrimers of any generation, including but not limited to, a first generation PAMAM dendrimer, a second generation PAMAM dendrimer, a third generation PAMAM dendrimer, a fourth generation PAMAM dendrimer, a fifth generation PAMAM dendrimer, a sixth generation PAMAM dendrimer, a seventh generation PAMAM dendrimer, an eighth generation PAMAM dendrimer, a ninth generation PAMAM dendrimer, or a tenth generation PAMAM dendrimer. Dendrimers suitable for use in the present application include, but are not limited to, polyamide-amine (PAMAM), polypropylamine (POPAM), polyethyleneimine, polylysine, polyesters, iptycenes, aliphatic poly(ether), and / or aromatic polyether dendrimers. Each dendrimer of a dendrimer complex can have similar or different chemical properties as the other dendrimers (e.g., a first dendrimer can include a PAMAM dendrimer, while a second dendrimer can include a POPAM dendrimer). In some embodiments, the first or second dendrimer can further include an additional agent. The multi-armed PEG polymer includes a polyethylene glycol having at least two branched chains with a thiol or thio pyridine terminal group; however, embodiments disclosed herein are not limited thereto, and PEG polymers with other terminal groups such as succinimidyl or maleimide terminals can be used. PEG polymers having a molecular weight of 10 kDa to 80 kDa can be used.

[0032] Inhibition: The term “inhibition,” “inhibiting,” “inhibit,” or “inhibitor” refers to the ability of a compound to decrease, retard, stop, or prevent a particular protein or biological process activity (e.g., activity of a bromodomain and / or bromodomain-containing protein). In some embodiments, the activity in a cell or tissue is decreased and / or the activity is decreased relative to a vehicle.

[0033] Nuruloid: The term “nuruloid” refers to a self-organizing organoid on a micropattern with neural progenitor cells, neural crest, sensory placode, and epidermis. Nuruloids can be generated from embryonic stem cells (e.g., human embryonic stem cells), as described in Harekani et al., 2019, Nature Biotechnology 37:1198-1208.

[0034] Selective inhibition: As used herein, when a compound has the ability to “selectively” or “specifically” reduce BRD9 activity as compared to one or more other bromodomain-containing proteins, the compound can inhibit the activity of BRD9 at least about 2-fold as compared to another bromodomain. In various embodiments, the compound has greater inhibitory activity against BRD9 as compared to another bromodomain other than BRD7, at least about 5-fold, at least about 10-fold, at least about 25-fold, or at least about 50-fold. Inhibition activity can be measured as percent (%) inhibition of activity and / or IC50values in in vitro assays, as described in Section 6. Because BRD9 and BRD7 are closely related, in certain embodiments, a selective inhibitor of BRD9 inhibits BRD7 to a similar extent as BRD9, provided that it has lower inhibitory activity against one or more more distantly related bromodomain-containing proteins (e.g., BRD2, BRD3, and / or BRD4). 50

[0035] ​Subject or patient: The terms “subject” and “patient” refer to a human (i.e., a male or female of any age group, such as a pediatric subject (e.g., an infant, child, or adolescent) or an adult subject (e.g., a young adult, middle-aged adult, or elderly adult)) or a non-human animal (e.g., a mammal such as a non-human primate, a domestic animal such as a cat or dog, or a farm animal such as a cow, horse, or pig). In particular embodiments, the subject has an expanded polyglutamine or polyQ repeat in at least one HTT allele. The expanded polyglutamine repeat can comprise one or two glutamine codons (i.e., CAA and / or CAG) and encodes an HTT protein having 36 or more, preferably 40 or more glutamines. In particular embodiments, the polyglutamine repeat encodes an HTT protein having 42 to 265 glutamines, and in certain particular embodiments 45, 48, 50, 55, 56, 58, 60, 65, 67, 70, 72, 74, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, or 265 glutamines, as well as any derivable range of glutamines having two of the foregoing glutamine repeat numbers as endpoints (e.g., 48 to 180, 50 to 150, or 56 to 130). Because HD is an autosomal dominant genetic disorder, a subject can have an expanded glutamine repeat sequence in only one HTT allele, but a subject having an expanded glutamine repeat sequence in both HTT alleles is within the scope of the present disclosure.

[0036] Treatment: The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease described herein. In some embodiments, treatment can be administered after one or more signs or symptoms of the disease have developed or have been observed. In other embodiments, treatment can be administered in the absence of signs or symptoms of the disease. For example, treatment can be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a genetic history of HD or the presence of an expanded glutamine or CAG repeat in the huntingtin gene).

[0037] 6. Detailed description

[0038] BRD9 inhibitors are known in the art and can be used to treat Huntington’s disease (HD). BRD9 inhibitors can have different properties and origins, including but not limited to nucleic acids, polypeptides, or small molecules.

[0039] In one aspect, the inhibitor is an antisense nucleic acid capable of inhibiting transcription of a BRD9 gene or translation of a BRD9 mRNA. The antisense nucleic acid can comprise all or part of a sequence encoding a bromodomain-containing protein, or a sequence complementary thereto. The antisense sequence can be DNA, RNA (e.g., siRNA), a ribozyme, etc. It can be single-stranded or double-stranded. It can also be an RNA encoded by an antisense gene. When using an antisense nucleic acid comprising a portion of a sequence of a gene or mRNA, it is preferred to use a portion comprising at least 10 consecutive bases from the sequence, more preferably at least 15 consecutive bases from the sequence, to ensure specific hybridization. In the case of an antisense oligonucleotide, it typically comprises fewer than 100 bases, e.g., about 10 to 50 bases or 18 to 30 bases. The antisense oligonucleotide can be modified to increase its stability, its nuclease resistance, its cell penetration, etc. Perfect complementarity between the sequence of the antisense molecule and the sequence of the BRD9 gene or mRNA is not required, but is generally desirable.

[0040] In other embodiments, the BRD9 inhibitor is a polypeptide or peptide. For example, it can be a peptide comprising a region of a bromodomain-containing protein and capable of antagonizing the activity of a bromodomain-containing protein. The peptide advantageously comprises 5 to 50 consecutive amino acids, typically 7 to 40 consecutive amino acids, of the primary sequence of a BRD9 protein. The polypeptide can also be an antibody against a bromodomain-containing protein, or a fragment or derivative of such an antibody, e.g., a Fab fragment or a single-chain antibody (e.g., ScFv). Such antibodies, fragments or derivatives can be produced by conventional techniques.

[0041] In yet other embodiments, however, the BRD9 inhibitor is a small molecule. BRD9 inhibitors include, but are not limited to, I-BRD9, TP-472, BI-7273, BI-9564, dBRD9, GNE-375 and LP-99, a methyquinolone compound, a thienopyridinone, and the BRD9 inhibitors disclosed in Remillard et al., 2017, Angew. Chem. Int. Ed. 56: 1-7 and Theodoulou et al., 2016, J. Med. Chem. 99: 1425-39 (all incorporated herein by reference). The small molecule BRD9 inhibitors can be administered in the form of a free base or a physiologically acceptable salt.

[0042] In one particular embodiment, the BRD9 inhibitor is BI-9564 or a pharmaceutically acceptable salt thereof:

[0043]

[0044] In another particular embodiment, the BRD9 inhibitor is BI-7273 or a pharmaceutically acceptable salt thereof:

[0045]

[0046] In another specific embodiment, the BRD9 inhibitor is LP-99 or a pharmaceutically acceptable salt thereof:

[0047]

[0048] In another specific embodiment, the BRD9 inhibitor is I-BRD9 or a pharmaceutically acceptable salt thereof:

[0049]

[0050] In another specific embodiment, the BRD9 inhibitor is d-BRD9 or a pharmaceutically acceptable salt thereof:

[0051]

[0052] In yet another specific embodiment, the BRD9 inhibitor is TP-472 or a pharmaceutically acceptable salt thereof:

[0053]

[0054] In yet another specific embodiment, the BRD9 inhibitor is GNE-375 or a pharmaceutically acceptable salt thereof:

[0055]

[0056] In a further specific embodiment, the BRD9 inhibitor is a compound of formula (I) or an enantiomer, diastereomer, stereoisomer or a pharmaceutically acceptable salt thereof:

[0057]

[0058] wherein:

[0059] A is phenyl or 5- or 6-membered heteroaryl containing 1 or 2 heteroatoms selected from N and S, wherein the phenyl or heteroaryl is unsubstituted or substituted by 1 to 3 R 3 groups;

[0060] R 1 is H, (Ci-C4)alkyl or (Ci-C4)haloalkyl;

[0061] each R 2 is independently (Ci-C4)alkyl, (Ci-C4)haloalkyl, (Ci-C4)alkoxy, (Ci-C4)haloalkoxy, halogen, OH or NH2;

[0062] each R3 Independently, it is (C1-C4)alkyl, (C1-C4)haloalkyl, (C1-C4)alkoxy, (C1-C4)haloalkoxy, halogen, OH, NH2 or

[0063] X1 is NR 5 Or O;

[0064] Y1 is S(O) a or NR 5 ;

[0065] Each R 4 It is independently (C1-C4)alkyl, (C1-C4)haloalkyl, halogen or -C(O)(C1-C3)alkyl;

[0066] Each R 5 Independently H or (C1-C4) alkyl;

[0067] Each R 6 Independently H or (C1-C4) alkyl;

[0068] a is 0, 1, or 2; and

[0069] n and r are each independently 0, 1, 2 or 3.

[0070] In a further specific embodiment, the BRD9 inhibitor is a compound of formula (II) or its enantiomers, diastereomers, stereoisomers, or pharmaceutically acceptable salts:

[0071]

[0072] in:

[0073] R 1 For (C) 1- C3) Alkyl or cyclopropyl;

[0074] R 2 Halogen, (C 1- C3)alkyl, (C 1- C3) haloalkyl, NH2, NH(C 1- C3) Alkyl or OH;

[0075] X 1 For N or CR 3 And X 2 For N or CR 4 The condition is X 1 and X 2 Cannot be N at the same time;

[0076] R 3 For H or (C1- C3)alkyl;

[0077] R 4 is H or (C 1- C3)alkyl; provided that R 3 and R 4 cannot both be (C 1- C3)alkyl;

[0078] or, R 2 and R 3 together form a phenyl ring or a 5-6 membered heteroaromatic ring, wherein each ring can independently be unsubstituted or substituted with one or more groups that are independently halogen, OH, NH2, NH(C 1- C3)alkyl, or (C 1- C3)alkyl, wherein said (C 1- C3)alkyl can be unsubstituted or substituted with 5-6 membered heteroaryl or phenyl;

[0079] R 5 and R 9 are the same or different and independently H, O(C 1- C3)alkyl, or (C 1- C3)alkyl;

[0080] R 6 and R 8 are the same or different and independently H, OH, halogen, NH2, (C 1- C3)alkyl, O(C 1- C3)alkyl, O(C 1- C3)haloalkyl, (C 1- C3)alkyl-O-(C 1- C3)alkyl, 4-7 membered heterocycloalkyl, (C 1- C3)alkyl-SO2-(C 1- C3)alkyl, (C 1- C3)alkyl-NH2, (C 1- C3)alkyl-N((C 1- C3)alkyl)2, N((C 1- C3)alkyl)2, or NHR 13 ;

[0081] R 13 is, at each occurrence, independently SO2-(C 1- C3)alkyl, or (C 1- C3)alkyl, wherein the (C 1- C3)alkyl is unsubstituted or substituted with 5-6 membered heteroaryl;

[0082] or, R 5 and R 6together form a benzene ring;

[0083] R 7 and R 6 or R 7 and R 8 together form a 5-7 membered heterocycloalkyl, which is unsubstituted or substituted by (C 1- C3)alkyl;

[0084] R 7 is H, NH2, Y-R 12 , (C 1- C3)alkyl or 4-7 membered heterocycloalkyl;

[0085] Y is CR 10 R 11 , SO2 or CO;

[0086] R 10 and R 11 are the same or different and independently H or (C 1- C3)alkyl; or R 10 and R 11 together form a C 3-4 cycloalkyl;

[0087] R 12 is NH2, OH, (C 1- C3)alkyl, N(R 15 , R 16 ), OR 17 , aryl or 5-6 membered heteroaryl, wherein the aryl or heteroaryl is independently unsubstituted or substituted by one or more halogen or 4-7 membered heterocycloalkyl, wherein each heterocycloalkyl is independently unsubstituted or substituted by one or more groups selected from halogen, OH, NH2, (C 1- C3)alkyl, NH(C 1- C3)alkyl, N((C 1- C3)alkyl)2, O(C 1- C3)alkyl and CH2R 14 ;

[0088] R 14 is 5-10 membered monocyclic or bicyclic aryl or heteroaryl, which is unsubstituted or substituted by NH2, OH, halogen, CN, (C 1- C3)alkyl or O(C 1- C3)alkyl;

[0089] R 15 is H or (C 1- C3)alkyl;

[0090] R 16 is (C 1-C3)alkyl, C 2-3 alkyl-N((C 1- C3)alkyl)2, C 2-3 alkyl-NH(C 1- C3)alkyl or 4-7 membered heterocycloalkyl, wherein heterocycloalkyl is unsubstituted or substituted by (C 1- C3)alkyl;

[0091] R 17 is (C 1- C3)alkyl or 4-7 membered heterocycloalkyl, wherein heterocycloalkyl is unsubstituted or substituted by (C 1- C3)alkyl;

[0092] wherein when R 7 is YR 12 , R 6 and R 8 can be the same or different and independently H, OH, halogen, NH2, CN, (C 1- C3)alkyl, (C 1- C3)haloalkyl, O(C 1- C3)alkyl, O(C 1- C3)haloalkyl or (C 1- C3)alkyl-O-(C 1- C3)alkyl; and

[0093] wherein at least one of the substituents R 5 to R 9 is not hydrogen.

[0094] In yet another specific embodiment, the BRD9 inhibitor is a bromosporin or a pharmaceutically acceptable salt thereof:

[0095]

[0096] The present disclosure is not limited to a particular BRD9 antagonist. Suitable BRD9 inhibitors can reduce BRD9 activity by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 98%. In certain embodiments, the BRD9 inhibitor reduces BRD9 activity by at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%. Ranges combining any two of the preceding values (e.g., from at least about 30% to at most about 90% or from at least about 50% to at most 90%) are also within the scope of the present disclosure.

[0097] Methods of determining BRD9 inhibitory activity are known. For example, inhibitory activity can be determined using the TR-FRET method described in Theodoulou et al., 2016, J. Med. Chem. 99: 1425-39. Briefly, compounds can be incubated with Alexa Fluor 647 ligand (GSK2833930A) in Greiner 384-well black low volume microtiter plates and incubated at room temperature for 30 minutes protected from light. Assay reagents can include Eu-W1024 Anti-6xHis antibody. Plates can be read to determine donor and acceptor counts. From this, the ratio of acceptor / donor is calculated (λex = 337 nm, λemdonor = 615 nm, emacceptor = 665 nm) and used for data analysis. In various embodiments, antagonists can reduce BRD9 activity by at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 100%, at least 200%, or even at least 1000% or more compared to the absence of an inhibitor. This assay can be adapted to determine the inhibitory effect of a given BRD9 inhibitor on other bromodomain-containing proteins to assess selectivity for BRD9. A selective BRD9 inhibitor can have at least two-fold, at least five-fold, or at least ten-fold greater percent inhibition (%) for BRD9 relative to one, two, or three other bromodomain-containing proteins, such as, but not limited to, BRD2, BRD3, BRD4, or any combination of the foregoing. In the case of BRD4, which has two binding domains (binding domain 1 or BD1 and binding domain 2, BD2), a single residue mutation in the BD2 acetyl lysine binding pocket (Y390A) can be introduced to reduce the affinity of the fluoro-ligand for the mutant BD2 domain with the goal of determining the binding of the BRD9 inhibitor to the single non-mutated BD1 bromodomain.

[0098] Alternatively, BRD9 inhibitors can be evaluated for inhibitory activity as follows. His / Flag epitope tagged BRD9 134-239Cloned, expressed and purified to homogeneity. BRD9 binding and inhibition can be assessed by monitoring the binding of biotinylated H4-tetraacetyl peptide (New England Peptide, NEP2069-11 / 13) to the target using AlphaLisa technology (Perkin-Elmer). Specifically, in 384-well, ProxiPlate BRD9 (final 50 nM) is bound to peptide (final 3 nM) in 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM TCEP, 0.01% (w / v) BSA and 0.008% (w / v) Brij-35 in the presence of DMSO (final 0.8% DMSO) or serial dilutions of compound in DMSO. After incubation at room temperature for 20 minutes, AlphaLisa streptavidin acceptor beads (Perkin-AL125C) and AlphaLisa nickel donor beads (Perkin AS 10ID) are added at final concentrations of 15 pg / mL, respectively. After equilibration in the dark for 90 minutes, plates are read on an Envision instrument and IC50values are calculated using four-parameter non-linear curve fitting 50 . This assay can be adapted to determine the inhibitory effect of a given BRD9 inhibitor on other bromodomain-containing proteins to assess selectivity for BRD9. A selective BRD9 inhibitor has an IC50value for BRD9 that is at least one-half, at least one-fifth, or at least one-tenth of the IC50value for one, two, or three other bromodomain-containing proteins, such as, but not limited to, BRD2, BRD3, BRD4, or any combination of the foregoing. 50

[0099] ​The ability of BRD9 inhibitors to reverse the HD neurocyte-like phenotype can be tested. Neuroblastocytes are self-organizing cellular components derived from the ectoderm and based on micropatterns that mimic neurocyte formation (Harekami et al., 2019, Nature Biotechnology 37: 1198–1208). Specifically, these neurocytes exhibit a developing central nervous system forming a neural rosette in the center. Comparison of in vitro neurocytes with their in vivo counterparts around day 21 post-fertilization revealed a high degree of similarity, making them ideal preclinical endpoints for studying human genetic diseases and thus substrates for phenotype-reversal-based drug discovery. Neuroblastocytes modified to carry the HTT gene with amplified CAG repeat sequences (e.g., repeats 43, 48, 56, 65, 72, and 150) reflect the diversity of poly-Q lengths observed in patients with HD and are characterized by the HD phenotype, including the amplification of the PAX6 neural rosette (Harekami et al., 2019, Nature Biotechnology 37: 1198–1208). The BRD9 inhibitor can act on neuroid embryos to reverse the amplification of PAX6-expressing neural rosettes induced by CAG amplification. The BRD9 inhibitor used in the methods of this disclosure preferably partially or completely reverses the HD neuroid embryo phenotype and has an EC50 concentration of less than 1 μM. 50 In certain embodiments, the BRD9 inhibitors of this disclosure partially or completely reverse the HD phenotype, having EC50 values ​​of less than 750 nM, less than 500 nM, less than 300 nM, or less than 200 nM. 50 .

[0100] In some implementations, wild-type (WT) neurouloids are used in ECs with reversed HD phenotypes. 50 The effect remained unaffected at that time, indicating a lack of pleiotropic effect, which may reflect toxicity at therapeutic doses. In some respects, the BRD9 inhibitors used in the methods disclosed herein have ECMO effects for reversing the HD neuroblastoma. 50 The EC 50 Compared to EC used to induce pleiotropic effects in WT neurites 50 At least 5 times lower. In a specific implementation, EC is used to reverse the HD neuroblastic phenotype. 50 Compared to EC used to induce pleiotropic effects in WT neurites 50 Low, at least one-tenth, at least one-twentieth, or at least one-fiftieth of its value.

[0101] The BRD9 inhibitor is typically administered in the form of a pharmaceutical composition with one or more adjuvants, excipients, carriers, buffers, diluents, and / or other conventional pharmaceutical auxiliaries. For further details of formulations and administration techniques, see the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).

[0102] The BRD9 inhibitor can be formulated or co-administered with an agent to improve delivery across the blood-brain barrier ("BBB"), as described in Pardridge et al., 2007, Drug. Discov. Today 12(1-2): 54-61.

[0103] In certain embodiments, the BRD9 inhibitor is formulated or co-administered with an exosome or a carbon nanotube.

[0104] In certain embodiments, the BRD9 inhibitor is formulated or co-administered with a brain permeability enhancer (such as cereport, regadenoson, or menthol). Brain permeability enhancers (e.g., cereport) bind to receptors on the surface of endothelial cells and initiate a biochemical cascade that loosens tight junctions.

[0105] The BRD9 inhibitor can also be administered as an amino acid conjugate, such as a lysine conjugate or a phenylalanine conjugate.

[0106] Other drugs that improve delivery across the BBB are transport peptides and proteins that allow delivery of specific molecules across the BBB without damaging the BBB. Peptidomimetic mAbs (e.g., against transferrin receptor) can be used as molecular "Trojan horses" to ferry any attached drugs or genes across the BBB.

[0107] The BRD9 inhibitor can be encapsulated in a nanoparticle, such as a lipid-based nanoparticle, an albumin-based nanoparticle, an apolipoprotein-based nanoparticle, a polymer-based nanoparticle, a dendrimer-based nanoparticle, or an inorganic-based nanoparticle.

[0108] Administration of the BRD9 inhibitor can be accompanied by physical or electrical methods, such as microbubble-enhanced ultrasound or transcranial magnetic stimulation, to improve uptake across the BBB.

[0109] The effective amount of the BRD9 inhibitor to be administered depends on many factors, including but not limited to the type of disease or disorder that causes the intended cerebral ischemic episode, the general health status, body size, age of the patient, and the nature of the treatment, i.e. short-term versus chronic treatment. Typically, the treatment can be administered in a single dose or in multiple doses, i.e. once, twice, three times or more doses per day. The time frame of the administration can range from one week or one month to long-term administration over a long period of time.

[0110] In certain embodiments, the BRD9 inhibitors described herein can be used for the manufacture of a medicament for the treatment of Huntington’s disease.

[0111] 7. Example

[0112] 7.1. Example 1: Micro-pattern based neuroblast-like induction and analysis

[0113] Gene expression within the neuroblast-like was analyzed and compared to equivalent expression in the neural development stage in vivo in the ectodermal compartment.

[0114] 7.1.1. Materials and methods

[0115] Micro-pattern cell culture on-chip and neuroblast-like induction: First, micro-patterned glass coverslips (CYTOO CHIPS Arena A, Arena 500A, Arena EMB A) were coated with 10 pg / ml laminin-521 (BioLamina, LN521-03) diluted in PBS+ / + (Gibco) for 3 hours at 37°C. The micro-patterns were then placed face-up on parafilm in a 10 cm petri dish, and 800 pl of laminin solution was added to the micro-patterns. After 3 hours at 37°C, the coated micro-patterns were transferred to a 35 mm petri dish containing 5 ml PBS+ / +. The laminin was removed with 6 serial dilutions in PBS+ / + (1 :4 dilution) followed by two complete washes in PBS+ / +. The coated micro-patterns were then stored in PBS+ / + at 37°C. Cells were seeded as follows: Cells grown in MEF-CM in the petri dish were washed once with PBS- / - (Gibco) and then treated with accutase (Stem Cell Technologies) for 5 minutes. The cells were then pipetted to ensure a single cell suspension and accutase was added to the petri dish to a final concentration of 20 ng / ml. The cells were then seeded on the micro-patterns at a density of 5 x 104cells / cm2. The cells were then incubated at 37°C for 24 hours. -1 Recombinant laminin-521 (BioLamina, LN521-03) was coated for 3 hours. The micro-patterns were then placed face-up on parafilm in a 10 cm petri dish, and 800 pl of laminin solution was added to the micro-patterns. After 3 hours at 37°C, the coated micro-patterns were transferred to a 35 mm petri dish containing 5 ml PBS+ / +. The laminin was removed with 6 serial dilutions in PBS+ / + (1 :4 dilution) followed by two complete washes in PBS+ / +. The coated micro-patterns were then stored in PBS+ / + at 37°C. Cells were seeded as follows: Cells grown in MEF-CM in the petri dish were washed once with PBS- / - (Gibco) and then treated with accutase (Stem Cell Technologies) for 5 minutes. The cells were then pipetted to ensure a single cell suspension and accutase was added to the petri dish to a final concentration of 20 ng / ml. The cells were then seeded on the micro-patterns at a density of 5 x 104cells / cm2. The cells were then incubated at 37°C for 24 hours. -1 bFGF and ROCK inhibitor Y27632 (10 mM; Abeam ab120129) were diluted 4x in HUESM medium. Cells were further diluted with the same medium, and 5 x 104cells in 3.0 ml medium were seeded on the micro-patterns. The cells were then incubated at 37°C for 24 hours. 5(or as indicated in the figure) cells were plated on micro-patterns in 35-mm tissue culture dishes and then incubated at 37°C. After 3h, the micro-patterns in the culture dishes were washed once with PBS+ / +. For SB+LDN conditions: PBS+ / + was replaced with 3N medium 73 containing 10 mM SB431542 (Stemgent 04-0010-10) and 0.2 mM LDN 193189 (Stemgent 04-0019). On day 3 and day 5, the medium was replaced with fresh medium of the same and incubated at 37°C until day 7. For SB+BMP4 conditions (neural-like), PBS was replaced with HUESM containing 10 mM SB431542 and 0.2 mM LDN 193189. On day 3, the medium was replaced with HUESM containing 10 mM SB431542 and BMP4 (50 ng ml -1 or as indicated for each experiment). On day 5, the medium was replaced with fresh medium of the same and incubated until day 7.

[0116] Immunofluorescence: Micro-pattern coverslips were fixed with 4% paraformaldehyde (Electron Microscopy Sciences 15713) in warm medium for 30 min, washed 3 times with PBS - / -, then blocked and permeabilized with 3% normal donkey serum (Jackson Immunoresearch 017-000-121) and 2% Triton X-100 (Sigma 93443) in PBS - / - for 30 min. Micro-patterns were incubated with primary antibodies for 1.5 h, washed 3 times for 5 min in PBS - / -, incubated with Alexa 488, Alexa 555, Alexa 594 or Alexa 647 (1 : 1,000 dilution, Molecular Probes) and 10 ng ml -1 of DAPI (ThermoFisher Scientific D1306) conjugated secondary antibodies for 30 min, then washed twice with PBS - / -. For double staining using antibodies from the same species, Alexa 488 Fab fragments (Jackson Immunoresearch, 715-547-003) and Fab fragment IgG (Jackson Immunoresearch, 715-007-003) were used. Coverslips were mounted on glass slides using ProLong Gold antifade mountant (Molecular Probes P36934).

[0117] Microscopy: Micro-pattern coverslips were obtained on a Zeiss Inverted LSM 780 laser scanning confocal microscope with x10, x20 or x40 water immersion objectives.

[0118] 7.1.2. Results

[0119] Around day 21 after fertilization, in vitro neural embryos (...) Figure 1A ) and its in vivo counterpart ( Figure 1B The comparison revealed a high degree of similarity, making them ideal preclinical endpoints for studying human genetic diseases and screening drugs based on phenotype reversal.

[0120] 7.2. Example 2: Characterization of neuroblastoids derived from HD cell lines

[0121] Libraries of eight HD RUES2 and other gene cell lines were induced to form neurocytes, and the expression of many molecular markers was screened.

[0122] 7.2.1. Materials and Methods

[0123] Cell culture: All hESC lines were grown in HUESM medium regulated with mouse embryonic fibroblasts and supplemented with 20 ng / ml -1 βFGF (MEF-CM)27. Mycoplasma spp. were detected in cells every 2 months. Cells were grown on tissue culture dishes coated with Geltrex (Life Technologies) solution, and the expression of various markers was analyzed.

[0124] 7.2.2. Results

[0125] Neuroblastoids exhibit a distinctive feature: PAX6 region / rosette extension. In neuroblastoids, CAG extension is associated with an increase in PAX6+ area. Figure 2 The discovery of this CAG amplification-specific phenotype opens up the possibility of using this phenotypic feature for high-throughput screening activities to discover small molecules that can reverse the HD phenotype back to WT.

[0126] 7.3. Example 3: AI screening for HD phenotypic reversal

[0127] AI screening was performed on molecules that could reverse the HD neuroblastic phenotype to the wild type (see [link]). Figure 3 In such screening, molecules that can act at the colony level to reverse the harmful effects caused by CAG amplification while leaving WT colonies unaffected are specific to the disease allele and are therefore considered promising therapeutic candidates for in vivo validation in HD animal models.

[0128] The simplest feature that can be used to characterize the neural-like phenotype is the expansion of the PAX6+ domain. This represents a useful specific feature, but does not encompass the full range of phenotypic variation that can result from a large-scale screen. Ideally, an analytical tool is needed that 1) strongly discriminates between WT and HD neural-like embryos to minimize the number of false positives / negatives; 2) quantitatively measures the degree of phenotypic reversion by plotting the proportion between WT and HD phenotypes; 3) measures the cytotoxicity and off-target effects of the test small molecules.

[0129] In the past few years, machine learning tools based on deep neural networks have been used to perform similar face recognition tasks in security videos or search engines. These tools can also be used for drug discovery. Specifically, the screening campaign returns a large number of images of neural-like embryos: WT controls, HD controls, and HD treated with drugs. All of these images can be used as input to a deep neural network that is specifically trained to return two predicted quantities of clinical drug success: drug toxicity and drug efficacy Figure 4 ).

[0130] 7.3.1. Materials and methods

[0131] Micro-patterned cell culture and imaging on 96-well plates: The micro-patterned cell culture and neural-like embryo induction protocol on chips was used with the micro-patterned 96-well plates (CYTOOPLATES Arena A, Arena 700). The neural-like embryo induction was modified so that all steps requiring media change, cell dispensing, and immunofluorescence washes and incubations were performed using the EL406 wash / dispensing robot. For cell seeding, a cell suspension with a volume of 200 pl was used with a density of 0.18 M cells / ml. Plate imaging was performed by a 4X lens using an InCell Analyzer 2000 high-content imager.

[0132] Multiple libraries consisting of 2080 compounds were screened in 96-well plates with micro-patterned glass bottoms. In these plates, there are approximately 27 individual neural-like embryos per well. During the screening campaign, two plates of WT controls and two plates of untreated HD controls were kept to have enough control data to train the deep neural network for later analysis. Compounds were applied to the test plates on day 0 of differentiation and reapplied during the media change steps on days 3 and 5. Each compound was applied at a unique concentration of 10 uM in a unique well. On day 7, the plates were fixed and stained for DAPI, PAX6, and Phalloidin. After staining, the plates were imaged and individual neural-like embryos in each well were segmented and labeled before being fed into the specific deep neural network.

[0133] Image analysis: Images obtained from micro-pattern or plating experiments were stitched and background corrected. A foreground mask was created by thresholding the DAPI channel and computing an alpha shape related to the colony size to detect colonies. From the corrected and stitched images, each detected colony was extracted.

[0134] Deep learning for quantification of phenotype rescue: The multi-channel WT and disease organoid images were split into training (70%) and validation (30%) images. A neural network (NN) was then trained on the training dataset. The NN was coded using a machine learning framework, e.g. Pytorch. For 2D images, this framework provides a convolutional neural network pre-trained on the ImageNet database. Residual networks (ResNets) are a subclass of convolutional networks that are particularly effective at image classification. Pre-trained ResNets of different depths (with 18, 34, 50, 101 or 152 layers) are available in all major machine learning frameworks. ResNet50 was chosen. It consists of blocks of convolutional, BatchNorm and linear rectification (ReLU) layers. The final average pooling and dense connection layers were removed and replaced by custom layers. First, the pre-trained network classifies the images into more classes than WT and disease. In addition, the last layer of the NN is more specific to the dataset than the initial layers. Therefore, the last average pooling and fully connected layers were removed and replaced by an untrained adaptive average pooling, adaptive max pooling, BatchNorm layer, Dropout layer and fully connected layer, followed by a final softmax operation. Here, a fully connected layer of 512 units was used, followed by a final fully connected layer consisting of only two units, one for WT and one for disease. The final softmax converts the activations of these units into probabilities that sum to 1.

[0135] Training was performed by showing images to the network, comparing the output probabilities of WT or disease to the true values, and changing the network weights so that the next time an image is shown, the network will give a prediction closer to the true value. This fitting process is performed using the backpropagation algorithm, which is implemented in all major neural network frameworks. Images are shown to the network multiple times (each run is called an “epoch”). Images are “augmented”, i.e. a set of image transformations is applied to the images that do not change the content of the image significantly, but enlarge the pool of images the network can learn from. These data augmentation operations include rotation, cropping, scaling the image from 90% to 110%, and changing the contrast of the image. Multiple training runs were performed using different hyperparameters (number of layers, momentum and learning rate, dropout percentage, number of epochs) to find the best set of these parameters.

[0136] The screened images are then analyzed using the network trained on the training images. First, the accuracy of the network is verified by using the unprocessed control organoids in the screen. Then, the images of the organoids treated with the drug compounds are analyzed by the network, which assigns a score between 0 and 1 to each image, where 1 represents WT and 0 represents disease. In the screen analysis, wells with less than 10 complete organoids are excluded from the analysis, assuming that in those cases the compounds have a toxic effect. For the other cases, the network measures the ability of each well’s compound to reverse the phenotype according to the tools described in this section, and this score is averaged across wells.

[0137] Deep learning for quantification of cytotoxicity: An autoencoder-based approach is used to assess toxicity. These unsupervised neural networks encode and compress data into a low-dimensional latent representation. The advantage of the unsupervised nature of this machine learning approach is that the autoencoder learns the representation of the data without any additional information about the data (e.g., whether it comes from a wild-type or disease cell line), thus it is unbiased when estimating the toxicity of a compound. Representing the data as a vector also has the advantage that the wild-type and disease phenotype differences can be eliminated from the vector space, as this difference is not relevant for determining toxicity. Toxicity is determined by first eliminating the difference between wild-type and disease from the latent space. Then, the distance to the mean vector of wild-type and disease phenotypes is calculated and compared to the standard deviation of wild-type and disease phenotypes. This distance is defined as toxicity.

[0138] 7.3.2. Results

[0139] The analysis results are shown in Figure 5 . Most of the compounds belong to the lower left quadrant, whose molecules do not substantially affect the HD neuroepithelium. Many molecules have a toxic profile (toxicity score > 3), and a few hits fall in the hit space defined by the characterization of the AI analysis tool, which is defined as a high phenotype rescue score (> 0.95) and a low toxicity (< 3). In particular, bromosporin was found to be a particularly effective low-toxicity molecule (phenotype rescue = 0.98, toxicity score = 1.93).

[0140] Figure 6 (top panel) shows examples of images taken in the primary screen using WT control wells, HD control wells, and HD wells treated with 10 mM bromosporin. This qualitatively shows the enlarged PAX6 area in the HD background and the reduction to WT levels with bromosporin, and the reversal of the HD phenotype to the WT configuration when the HD neuroepithelium is contacted with bromosporin, which matches the quantitative results obtained by the AI algorithm, as shown in Figure 5

[0141] ​Bromosporangin was described in the literature as a broad-spectrum inhibitor of BRDs with IC50values of 0.41 μΜ, 0.29 μΜ, 0.122 μΜ and 0.017 μΜ for BRD2, BRD4, BRD9 and CECR2, respectively. To confirm bromosporangin as a modulator of the HD phenotype in vitro, bromosporangin was reconstituted from fresh stock powder to verify its properties in small scale experiments. 50 Figure 6 (Next figure) shows the qualitative results, which clearly show the rescue of the HD gene-induced disarray by the application of 0.5 μΜ bromosporangin.

[0142] 7.4. Example 4: Quantification of bromosporangin potency and toxicity

[0143] To quantitatively measure the potency of bromosporangin to reverse the HD neuroblast phenotype, a dose response experiment was performed.

[0144] 7.4.1. Materials and methods

[0145] HD neuroblasts were contacted in triplicates with 10 different concentrations of bromosporangin. The degree of phenotype reversal for each bromosporangin concentration was quantified using an Al algorithm.

[0146] 7.4.2. Results

[0147] Bromosporangin was fully potent at around 300 nM with an EC 50 of 120 nM Figure 7 Toxicity profiles of bromosporangin were assessed on WT and HD-56CAG neuroblasts. Toxicity reactions started to appear at concentrations higher than 1 μΜ. Overall, this indicates that bromosporangin is potent in the complex in vitro model of HD and that there is a concentration window, between 0.3 μΜ and 1 μΜ, where bromosporangin does not affect WT neuroblasts but can rescue HD neuroblasts. This highlights the specific window of the disease allele in this assay system. Moreover, in this concentration range, bromosporangin shows low toxicity.

[0148] 7.5. Example 5: Assay for BRD inhibitors for HD neuroblast phenotype reversal

[0149] An assay was performed to find out whether other BRD inhibitors have HD neuroblast phenotype reversal activity.

[0150] 7.5.1. Materials and methods

[0151] A selection of BRD inhibitors was tested using 96-well plates. The potency and toxicity of each compound were determined.

[0152] 7.5.2. Results

[0153] ​A selection of 19 BRD inhibitors were tested at a single concentration of 10 mM using a 96-well plate. Efficacy and toxicity were plotted alongside each molecule’s target in Figure 8 50

[0154] The fact that BI7273 scored positively also reinforces the finding that BRD inhibitors are effective in the HD in vitro model. Moreover, since BRD9 is the only commonality between the molecular targets of Bromosporin and BI7273, the possibility that BRD9 is in fact the relevant target that can be inhibited to rescue the HD phenotype in this assay system is raised.

[0155] While other BRD9 inhibitors (such as BI9564) did not score positively in the experiment using the panel of BRD inhibitors Figure 8 ), these molecules can potentially hinder the activity of the highly potent molecules when tested at a single and rather high concentration of 10 mM, which can start to appear toxic and off-target effects at high concentrations.

[0156] In line with this hypothesis, concentration-dependent responses of other BRD9-centric inhibitors were tested at lower concentrations. These included Bromosporin (targets: BRD2, BRD4, BRD9, and CECR2) and BI7273 (targets: BRD9 and BRD7) as positive controls, but also BI9564 (targets: BRD9 and BRD7), dBRD9 (selective BRD9 protac), and I-BRD9 (BRD9 and BRD4). The results are shown in Figure 9. Strikingly, all of these molecules were effective in rescuing the HD neuroblasts with sub-micromolar activity (Figure 9a). Moreover, BI7273, BI9564, d-BRD9, and I-BRD9 all had lower toxicity profiles than Bromosporin (Figure 9b). This can suggest that the broad activity of Bromosporin becomes detrimental at high concentrations compared to the more BRD9-centric compounds. Figure 10 A brief explanation of the successful BRD9 inhibiting compounds and their activity is shown.

[0157] 7.6. Example 6: Mechanism of action of BRD inhibitors

[0158] ​​To decipher the mechanism of action of BRD inhibitors, the HTT lowering capacity of BRD inhibitors was tested, in part based on the fact that lowering HTT strategies have shown efficacy in HD mouse models and are the basis of the latest clinical development for HD. Thus, lowering HTT is the only known mechanism of action that is hoped to treat HD.

[0159] Therefore, we repeated our protocol for the generation of HD-like neuroembryoids in two genetic backgrounds (56CAG and 72CAG) and treated them with the 5 BRD inhibitors disclosed in Figure 9 at two concentrations (1 and 5 mM). At the end of the differentiation protocol, neuroembryoid lysates were frozen and then analyzed for their HTT content. Amplified HTT levels and total HTT levels (amplified + regular) were quantified by ELISA-based Mesoscale Discovery (MSD) electrochemiluminescence assay.

[0160] 7.6.1. Materials and methods

[0161] HD-like neuroembryoids were generated in two genetic backgrounds (56CAG and 72CAG); and treated with the 5 BRD inhibitors disclosed in Figure 9 at two concentrations (1 and 5 mM). At the end of the differentiation protocol, neuroembryoid lysates were analyzed for their HTT content. Amplified HTT levels and total HTT levels (amplified + regular) were quantified by ELISA-based Mesoscale Discovery (MSD) electrochemiluminescence assay. Figure 10

[0162] 7.6.2. Results

[0163] Results are shown in Figure 11. Among the 4 drugs tested (bromosporangin, BI7273, BI9564 and dBRD9), only bromosporangin has the capacity to lower HTT levels. Specifically, bromosporangin lowered both amplified and total HTT levels in both tested concentrations and in the two genetic backgrounds of 56CAG and 72CAG. The lowering of HTT levels was significant, with more than 75% reduction in amplified HTT levels in all backgrounds at 5 mM and more than 50% reduction at the lower concentration of 1 mM. Interestingly, no HTT lowering capacity was measured with BI7273, BI9564 and dBRD9. This suggests that these three molecules rescue HD-like neuroembryoids through a mechanism of action different from the lowering of HTT levels.

[0164] 7.7. Example 7: Rescue of HD phenotype in human-like gastruloids mediated by BRD inhibitors

[0165] ​BRD inhibitors were tested to determine if they were able to rescue another HD phenotype in human primitivum. When human embryonic stem cell colonies were stimulated with CHIR and Activin for 48 hours, a peripheral induced primitive streak-like population SOX17+ was induced (see Figure 12a). Furthermore, in HD primitivum made from HD-56CAG cells, the SOX17+ domain in the periphery was greatly expanded. This effect was even more pronounced when using the null HTT- / - cell line, which supports the finding that the HD mutation is in fact a loss-of-function mutation in the developmental context, as it phenocopies the loss of protein.

[0166] 7.7.1. Materials and methods

[0167] Primitivum induction: Protocol using coated micropatterned chips. 8x10 5 cells were plated on each coverslip in a defined volume of MEF-CM supplemented with 20 ng / ml bFGF (R&D Systems), 10 mM ROCK inhibitor (Y-27632, Abeam), 1X Pen-Strep (Thermo Fisher Scientific), 100 pg / ml Normocin in PBS+ / + and left for 10 minutes to ensure even distribution across the pattern. The ROCK inhibitor was removed from the medium 3 hours after plating and the next day cells were induced with 50 ng / ml BMP4 (R&D Systems), 2 mM IWP2 (Stemgent), 6 mM CHIR99021 (EMD Millipore), 100 ng / ml Activin (ACTIVIN) (R&D Systems) and small molecule treatment. Samples were fixed after 48 hours and analysed by immunofluorescence.

[0168] 7.7.2. Results

[0169] When 1 mM of Bromosporangin, BI7273 or dBRD9 were applied to HD primitivum, in each case the SOX17+ region in the periphery was significantly reduced, reverting to the WT configuration (see Figure 12b). This clearly demonstrates that BRD inhibitors retain their ability to perform phenotypic rescue in a second HD phenotype that is orthogonal to the primitivum.

[0170] 7.8. Example 8: Reducing BRD9 levels using an inducible CRISPR / Cas system to rescue HD phenotype

[0171] 7.8.1. Materials and methods

[0172] An inducible BRD9 knockdown line was generated in the HG-56CAG background that efficiently reduces BRD9 transcript. Using a Tet-based CRISPR / Cas9 system induced by doxycycline, BRD9 transcript was reduced upon addition of doxycycline (DOX). Optimization of three individual gRNAs led to a construct that was able to reduce BRD9 mRNA levels by 50% within 2 days of DOX application Figure 13A HD phenotypes were analyzed and compared in the inducible knockdown line and WT-20CAG class neural tubes used as control.

[0173] 7.8.2. Results

[0174] Analysis of HD-56CAG and WT-20CAG class neural tubes showed that compared to control, the disintegration of the central PAX6+ domain became expanded and the peripheral SOX10+ cells were reduced in HD-56CAG Figure 13B and 13C Upon application of DOX and reduction of BRD9 levels in the 56CAG background, a significant rescue of both HD related parameters to their WT values was observed Figure 13B and 13C These data confirm that BRD9 is a target of interest for HD and that its inhibition leads to a reversal of the HD phenotype.

[0175] 8. Specific embodiments

[0176] While various specific embodiments have been illustrated and described, it will be understood that various changes can be made without departing from the spirit and scope of the disclosure. The disclosure is exemplified by the numbered embodiments listed below.

[0177] 1. A method of treating a subject having Huntington’s Disease (HD), the method comprising administering to the subject a therapeutically effective amount of a bromodomain 9 (BRD9) inhibitor.

[0178] 2. The method of embodiment 1, wherein the BRD9 inhibitor is not conjugated to a degron.

[0179] 3. The method of embodiment 1, wherein administration of the BRD9 inhibitor does not induce proteasome-mediated BRD9 degradation in vivo.

[0180] 4. The method of any one of embodiments 1 to 3, wherein the BRD9 inhibitor is a selective BRD9 inhibitor.

[0181] 5. The method of embodiment 4, wherein the BRD9 inhibitor has at least 2-fold or at least 5-fold greater inhibition of BRD9 compared to BRD2.

[0182] 6. The method of either embodiment 4 or embodiment 5, wherein the BRD9 inhibitor is at least 2-fold or at least 5-fold more potent against BRD9 compared to BRD3.

[0183] 7. The method of any one of embodiments 4-6, wherein the BRD9 inhibitor is at least 2-fold or at least 5-fold more potent against BRD9 compared to BRD4.

[0184] 8. The method of any one of embodiments 1-7, wherein the BRD9 inhibitor is a pyridinone compound.

[0185] 9. The method of any one of embodiments 1-7, wherein the BRD9 inhibitor is BI-9564 or a pharmaceutically acceptable salt thereof:

[0186]

[0187] 10. The method of any one of embodiments 1-7, wherein the BRD9 inhibitor is BI-7273 or a pharmaceutically acceptable salt thereof:

[0188]

[0189] 11. The method of any one of embodiments 1-7, wherein the BRD9 inhibitor is a methylquinolinone compound.

[0190] 12. The method of any one of embodiments 1-7, wherein the BRD9 inhibitor is LP-99 or a pharmaceutically acceptable salt thereof:

[0191]

[0192] 13. The method of any one of embodiments 1-7, wherein the BRD9 inhibitor is a thienopyridinone compound.

[0193] 14. The method of any one of embodiments 1-7, wherein the BRD9 inhibitor is I-BRD9 or a pharmaceutically acceptable salt thereof:

[0194]

[0195] 15. The method of any one of embodiments 1-7, wherein the BRD9 inhibitor is d-BRD9 or a pharmaceutically acceptable salt thereof:

[0196]

[0197] 16. The method of any one of embodiments 1 to 7, wherein the BRD9 inhibitor is TP-472 or a pharmaceutically acceptable salt thereof:

[0198]

[0199] 17. The method of any one of embodiments 1 to 7, wherein the BRD9 inhibitor is GNE-375 or a pharmaceutically acceptable salt thereof:

[0200]

[0201] 18. The method of any one of embodiments 1 to 7, wherein the BRD9 inhibitor is a compound of Formula (I) or an enantiomer, diastereomer, stereoisomer, or a pharmaceutically acceptable salt thereof:

[0202]

[0203] wherein:

[0204] A is phenyl or 5- or 6-membered heteroaryl containing 1 or 2 heteroatoms selected from N and S, wherein the phenyl or heteroaryl is unsubstituted or substituted with 1 to 3 R 3 groups;

[0205] R 1 is H, (C1-C4)alkyl, or (C1-C4)haloalkyl;

[0206] each R 2 is independently (C1-C4)alkyl, (C1-C4)haloalkyl, (C1-C4)alkoxy, (C1-C4)haloalkoxy, halogen, OH, or NH2;

[0207] each R 3 is independently (C1-C4)alkyl, (C1-C4)haloalkyl, (C1-C4)alkoxy, (C1-C4)haloalkoxy, halogen, OH, NH2, or

[0208] X1is NR 5 or O;

[0209] Y1is S(O) a or NR 5 ;

[0210] each R 4 is independently (C1-C4)alkyl, (C1-C4)haloalkyl, halogen, or -C(O)(C1-C3)alkyl;

[0211] each R 5independently H or (C1-C4)alkyl;

[0212] each R 6 independently H or (C1-C4)alkyl;

[0213] a is 0, 1, or 2; and

[0214] n and r are each independently 0, 1, 2, or 3.

[0215] 19. The method of any one of embodiments 1 to 7, wherein the BRD9 inhibitor is a compound of Formula (II), or an enantiomer, diastereomer, stereoisomer, or pharmaceutically acceptable salt thereof:

[0216]

[0217] wherein:

[0218] R 1 is (C 1- C3)alkyl or cyclopropyl;

[0219] R 2 is halogen, (C 1- C3)alkyl, (C 1- C3)haloalkyl, NH2, NH(C 1- C3)alkyl, or OH;

[0220] X 1 is N or CR 3 , and X 2 is N or CR 4 ; provided that X 1 and X 2 cannot both be N;

[0221] R 3 is H or (C 1- C3)alkyl;

[0222] R 4 is H or (C 1- C3)alkyl; provided that R 3 and R 4 cannot both be (C 1- C3)alkyl;

[0223] or, R 2 and R 3 together form a phenyl ring or a 5-6 membered heteroaromatic ring, wherein each ring can be independently unsubstituted or substituted with one or more groups that are independently halogen, OH, NH2, NH(C 1- C3)alkyl, or (C 1- C3)alkyl, wherein the (C 1-C3) alkyl groups may be unsubstituted or substituted with 5-6 membered heteroaryl groups or phenyl groups;

[0224] R 5 and R 9 Same or different, and independently H, O(C) 1- C3)alkyl or (C 1- C3)alkyl;

[0225] R 6 and R 8 Same or different, and independently H, OH, halogen, NH2, (C 1- C3)alkyl, O(C) 1- C3)alkyl, O(C) 1- C3) haloalkyl, (C 1- C3)alkyl-O-(C 1- C3)alkyl, 4-7 membered heterocyclic alkyl, (C 1- C3)alkyl-SO2-(C 1- C3)alkyl, (C 1- C3)alkyl-NH2, (C 1- C3)alkyl-N((C 1- C3)alkyl)2、N((C 1- C3)alkyl)2 or NHR 13 ;

[0226] R 13 Each time it exists, it is independently SO2-(C 1- C3)alkyl or (C 1- C3)alkyl, wherein the (C) 1- C3) alkyl groups are either unsubstituted or substituted with 5 to 6-membered heteroaryl groups;

[0227] Or, R 5 and R 6 Together they form a benzene ring;

[0228] Or, R 7 and R 6 or R 7 and R 8 Together they form 5-7 membered heterocyclic alkyl groups, which are unsubstituted or (C) 1- C3) Alkyl substitution;

[0229] R 7 For H, NH2, YR 12 (C) 1- C3) alkyl or 4-7 membered heterocyclic alkyl;

[0230] Y is CR 10 R 11 SO2 or CO;

[0231] R 10 and R 11 are the same or different and independently H or (C 1- C3)alkyl; or R 10 and R 11 together form a C 3-4 cycloalkyl;

[0232] R 12 is NH2, OH, (C 1- C3)alkyl, N(R 15 ,R 16 ), OR 17 , aryl, or 5-6 membered heteroaryl, wherein the aryl or heteroaryl is independently unsubstituted or substituted with one or more halogen or 4-7 membered heterocycloalkyl, wherein each heterocycloalkyl is independently unsubstituted or substituted with one or more groups selected from halogen, OH, NH2, (C 1- C3)alkyl, NH(C 1- C3)alkyl, N((C 1- C3)alkyl)2, O(C 1- C3)alkyl, and CH2R 14 ;

[0233] R 14 is 5-10 membered monocyclic or bicyclic aryl or heteroaryl, which is unsubstituted or substituted with NH2, OH, halogen, CN, (C 1- C3)alkyl, or O(C 1- C3)alkyl;

[0234] R 15 is H or (C 1- C3)alkyl;

[0235] R 16 is (C 1- C3)alkyl, C 2-3 alkyl-N((C 1- C3)alkyl)2, C 2-3 alkyl-NH(C 1- C3)alkyl, or 4-7 membered heterocycloalkyl, wherein heterocycloalkyl is unsubstituted or substituted with (C 1- C3)alkyl;

[0236] R 17 is (C 1- C3)alkyl or 4-7 membered heterocycloalkyl, wherein heterocycloalkyl is unsubstituted or substituted with (C 1- C3)alkyl;

[0237] wherein when R 7 is YR 12 , R 6and R 8 may be the same or different and are independently H, OH, halogen, NH2, CN, (C 1- C3)alkyl, (C 1- C3)haloalkyl, O(C 1- C3)alkyl, O(C 1- C3)haloalkyl, or (C 1- C3)alkyl-O-(C 1- C3)alkyl; and

[0238] wherein the substituents R 5 to R 9 are not hydrogen.

[0239] 20. The method of any one of embodiments 1 to 3, wherein the BRD9 inhibitor is a non-selective BRD9 inhibitor.

[0240] 21. The method of embodiment 20, wherein the BRD9 inhibitor is a bromosporin:

[0241] or a pharmaceutically acceptable salt thereof.

[0242] 22. The method of any one of embodiments 1 to 21, wherein the BRD9 inhibitor is administered as an amino acid conjugate.

[0243] 23. The method of embodiment 22, wherein the amino acid conjugate is a lysine conjugate.

[0244] 24. The method of embodiment 22, wherein the amino acid conjugate is a phenylalanine conjugate.

[0245] 25. The method of any one of embodiments 1 to 24, wherein the BRD9 inhibitor is administered to the subject with a carrier.

[0246] 26. The method of embodiment 25, wherein the carrier comprises a nanoparticle, an exosome, or a carbon nanotube.

[0247] 27. The method of embodiment 26, wherein the carrier comprises a nanoparticle.

[0248] 28. The method of embodiment 27, wherein the nanoparticle comprises a lipid-based nanoparticle.

[0249] 29. The method of embodiment 27, wherein the nanoparticle comprises a human serum albumin-based nanoparticle.

[0250] 30. The method of embodiment 27, wherein the nanoparticle comprises an apolipoprotein-based nanoparticle.

[0251] 31. The method of embodiment 27, wherein the nanoparticle comprises a polymer-based nanoparticle.

[0252] 32. The method of embodiment 27, wherein the nanoparticle comprises a dendrimer-based nanoparticle.

[0253] 33. The method of embodiment 27, wherein the nanoparticle comprises an inorganic-based nanoparticle.

[0254] 34. The method of embodiment 26, wherein the carrier comprises an exosome.

[0255] 35. The method of embodiment 27, wherein the carrier comprises a carbon nanotube.

[0256] 36. The method of any one of embodiments 1-35, further comprising administering a brain permeability enhancer adjunctively to the subject.

[0257] 37. The method of embodiment 36, wherein the BRD9 inhibitor and brain permeability enhancer are co-administered in a single pharmaceutical composition.

[0258] 38. The method of embodiment 36 or embodiment 37, wherein the brain permeability enhancer comprises cereport, regadenoson, or borneol.

[0259] 39. The method of embodiment 38, wherein the brain permeability enhancer comprises cereport.

[0260] 40. The method of embodiment 38, wherein the brain permeability enhancer comprises regadenoson.

[0261] 41. The method of embodiment 38, wherein the brain permeability enhancer comprises borneol.

[0262] 42. The method of any one of embodiments 1-41, further comprising subjecting the subject to microbubble-enhanced ultrasound.

[0263] 43. The method of any one of embodiments 1-42, wherein the BRD9 inhibitor is administered intranasally to the subject.

[0264] 44. The method of any one of embodiments 1-42, wherein the BRD9 inhibitor is administered intravenously to the subject.

[0265] 45. The method of any one of embodiments 1 to 42, wherein the BRD9 inhibitor is administered to the subject by intra-arterial injection.

[0266] 46. The method of any one of embodiments 1 to 42, wherein the BRD9 inhibitor is administered to the CSF of the subject.

[0267] 47. The method of embodiment 46, wherein the BRD9 inhibitor is administered intrathecally, intracerebroventricularly, or intraparenchymally.

[0268] 48. A BRD9 inhibitor for use in treating Huntington’s Disease (HD) in a subject in need thereof.

[0269] 49. The BRD9 inhibitor of embodiment 48, wherein the BRD9 inhibitor is not conjugated to a degron.

[0270] 50. The BRD9 inhibitor of embodiment 48, wherein administration of the BRD9 inhibitor does not induce proteasome-mediated BRD9 degradation in vivo.

[0271] 51. The BRD9 inhibitor of any one of embodiments 48 to 50, wherein the BRD9 inhibitor is a selective BRD9 inhibitor.

[0272] 52. The BRD9 inhibitor of embodiment 51, wherein the BRD9 inhibitor is at least 2-fold or at least 5-fold more potent against BRD9 compared to BRD2.

[0273] 53. The BRD9 inhibitor of embodiment 51 or embodiment 52, wherein the BRD9 inhibitor is at least 2-fold or at least 5-fold more potent against BRD9 compared to BRD3.

[0274] 54. The BRD9 inhibitor of any one of embodiments 51 to 53, wherein the BRD9 inhibitor is at least 2-fold or at least 5-fold more potent against BRD9 compared to BRD4.

[0275] 55. The BRD9 inhibitor of any one of embodiments 48 to 54, wherein the BRD9 inhibitor is a pyridinone compound.

[0276] 56. The BRD9 inhibitor of any one of embodiments 48 to 54, wherein the BRD9 inhibitor is BI-9564 or a pharmaceutically acceptable salt thereof:

[0277]

[0278] 57. The BRD9 inhibitor of any one of embodiments 48-54, wherein the BRD9 inhibitor is BI- 7273 or a pharmaceutically acceptable salt thereof:

[0279]

[0280] 58. The BRD9 inhibitor of any one of embodiments 48-54, wherein the BRD9 inhibitor is a methylquinolinone compound.

[0281] 59. The BRD9 inhibitor of any one of embodiments 48-54, wherein the BRD9 inhibitor is LP-99 or a pharmaceutically acceptable salt thereof:

[0282]

[0283] 60. The BRD9 inhibitor of any one of embodiments 48-54, wherein the BRD9 inhibitor is a thienopyridinone compound.

[0284] 61. The BRD9 inhibitor of any one of embodiments 48-54, wherein the BRD9 inhibitor is I- BRD9 or a pharmaceutically acceptable salt thereof:

[0285]

[0286] 62. The BRD9 inhibitor of any one of embodiments 48-54, wherein the BRD9 inhibitor is d- BRD9 or a pharmaceutically acceptable salt thereof:

[0287]

[0288] 63. The BRD9 inhibitor of any one of embodiments 48-54, wherein the BRD9 inhibitor is TP- 472 or a pharmaceutically acceptable salt thereof:

[0289]

[0290] 64. The BRD9 inhibitor of any one of embodiments 48-54, wherein the BRD9 inhibitor is GNE- 375 or a pharmaceutically acceptable salt thereof:

[0291]

[0292] 65. The BRD9 inhibitor of any one of embodiments 48-54, wherein the BRD9 inhibitor is a compound of Formula (I) or an enantiomer, diastereomer, stereoisomer, or a pharmaceutically acceptable salt thereof:

[0293]

[0294] wherein:

[0295] A is phenyl or 5- or 6-membered heteroaryl containing 1 or 2 heteroatoms selected from N and S, wherein the phenyl or heteroaryl is unsubstituted or substituted with 1 to 3 R 3 groups;

[0296] R 1 is H, (Ci-C4)alkyl or (Ci-C4)haloalkyl;

[0297] each R 2 is independently (Ci-C4)alkyl, (Ci-C4)haloalkyl, (Ci-C4)alkoxy, (Ci-C4)haloalkoxy, halogen, OH or NH2;

[0298] each R 3 is independently (Ci-C4)alkyl, (Ci-C4)haloalkyl, (Ci-C4)alkoxy, (Ci-C4)haloalkoxy, halogen, OH, NH2or

[0299] X1is NR 5 or O;

[0300] Y1is S(O) a or NR 5 ;

[0301] each R 4 is independently (Ci-C4)alkyl, (Ci-C4)haloalkyl, halogen or -C(O)(Ci-C3)alkyl;

[0302] each R 5 is independently H or (Ci-C4)alkyl;

[0303] each R 6 is independently H or (Ci-C4)alkyl;

[0304] a is 0, 1 or 2; and

[0305] n and r are each independently 0, 1, 2 or 3.

[0306] 66. The BRD9 inhibitor according to any one of embodiments 48 to 54, wherein the BRD9 inhibitor is a compound of Formula (II) or an enantiomer, diastereomer, stereoisomer, or a pharmaceutically acceptable salt thereof:

[0307]

[0308] wherein:

[0309] R 1 is (C 1- C3)alkyl or cyclopropyl;

[0310] R 2 is halogen, (C 1- C3)alkyl, (C 1- C3)haloalkyl, NH2, NH(C 1- C3)alkyl or OH;

[0311] X 1 is N or CR 3 , and X 2 is N or CR 4 ; provided that X 1 and X 2 cannot be N at the same time;

[0312] R 3 is H or (C 1- C3)alkyl;

[0313] R 4 is H or (C 1- C3)alkyl; provided that R 3 and R 4 cannot be (C 1- C3)alkyl at the same time;

[0314] or, R 2 and R 3 together form a phenyl ring or a 5-6 membered heteroaromatic ring, wherein each ring can independently be unsubstituted or substituted with one or more groups that are independently halogen, OH, NH2, NH(C 1- C3)alkyl or (C 1- C3)alkyl, wherein the (C 1- C3)alkyl can be unsubstituted or substituted with 5-6 membered heteroaryl or phenyl;

[0315] R 5 and R 9 are the same or different and independently H, O(C 1- C3)alkyl or (C 1- C3)alkyl;

[0316] R 6 and R 8 are the same or different and independently H, OH, halogen, NH2, (C 1- C3)alkyl, O(C 1- C3)alkyl, O(C 1- C3)haloalkyl, (C 1- C3)alkyl-O-(C 1- C3)alkyl, 4-7 membered heterocycloalkyl, (C1- C3)alkyl-SO2-(C 1- C3)alkyl, (C 1- C3)alkyl-NH2, (C 1- C3)alkyl-N((C 1- C3)alkyl)2, N((C 1- C3)alkyl)2, or NHR 13 ;

[0317] R 13 is independently at each occurrence SO2-(C 1- C3)alkyl or (C 1- C3)alkyl, wherein the (C 1- C3)alkyl is unsubstituted or substituted with 5- to 6-membered heteroaryl;

[0318] or, R 5 and R 6 together form a phenyl ring;

[0319] or, R 7 and R 6 or R 7 and R 8 together form a 5- to 7-membered heterocycloalkyl, which is unsubstituted or substituted with (C 1- C3)alkyl;

[0320] R 7 is H, NH2, Y-R 12 , (C 1- C3)alkyl, or 4- to 7-membered heterocycloalkyl;

[0321] Y is CR 10 R 11 , SO2, or CO;

[0322] R 10 and R 11 are the same or different and independently H or (C 1- C3)alkyl; or R 10 and R 11 together form a C 3-4 cycloalkyl;

[0323] R 12 is NH2, OH, (C1-C3)alkyl, N(R 15 ,R 16 ), OR 17, aryl or 5-6 membered heteroaryl, wherein the aryl or heteroaryl is independently unsubstituted or substituted with one or more halogen or 4-7 membered heterocycloalkyl, wherein each heterocycloalkyl is independently unsubstituted or substituted with one or more groups selected from halogen, OH, NH2, (Ci-C3)alkyl, NH(Ci-C3)alkyl, N((Ci-C3)alkyl)2, O(Ci-C3)alkyl, and CH2R 1- ; 1- ; 14 ;

[0324] R 14 is 5-10 membered monocyclic or bicyclic aryl or heteroaryl, unsubstituted or substituted with NH2, OH, halogen, CN, (Ci-C3)alkyl, or O(Ci-C3)alkyl; 1- ; 1- ;

[0325] R 15 is H or (Ci-C3)alkyl; 1- ;

[0326] R 16 is (Ci-C3)alkyl, C 1- alkyl-N((Ci-C3)alkyl)2, C 2-3 alkyl-NH(Ci-C3)alkyl, or 4-7 membered heterocycloalkyl, wherein heterocycloalkyl is unsubstituted or substituted with (Ci-C3)alkyl; 1- ; 2-3 ; 1- ; 1- ;

[0327] R 17 is (Ci-C3)alkyl or 4-7 membered heterocycloalkyl, wherein heterocycloalkyl is unsubstituted or substituted with (Ci-C3)alkyl; 1- ; 1- ;

[0328] wherein when R 7 is YR 12 , R 6 and R 8 can be the same or different and are independently H, OH, halogen, NH2, CN, (Ci-C3)alkyl, (Ci-C3)haloalkyl, O(Ci-C3)alkyl, O(Ci-C3)haloalkyl, or (Ci-C3)alkyl-O-(Ci-C3)alkyl; and 1- ; 1- ; 1- ; 1- ; 1- ; 1- ;

[0329] wherein at least one of the substituents R 5 through R 9 is not hydrogen.

[0330] 67. The BRD9 inhibitor of any one of embodiments 48-54, wherein the BRD9 inhibitor is a non-selective BRD9 inhibitor.

[0331] 68. The BRD9 inhibitor of embodiment 67, wherein the BRD9 inhibitor is a bromosporin or a pharmaceutically acceptable salt thereof:

[0332]

[0333] 69. The BRD9 inhibitor of any one of embodiments 48-68, wherein the BRD9 inhibitor is administered as an amino acid conjugate.

[0334] 70. The BRD9 inhibitor of embodiment 69, wherein the amino acid conjugate is a lysine conjugate.

[0335] 71. The BRD9 inhibitor of embodiment 69, wherein the amino acid conjugate is a phenylalanine conjugate.

[0336] 72. The BRD9 inhibitor of any one of embodiments 48-71, wherein the BRD9 inhibitor is administered to a subject with a carrier.

[0337] 73. The BRD9 inhibitor of embodiment 72, wherein the carrier comprises a nanoparticle, an exosome, or a carbon nanotube.

[0338] 74. The BRD9 inhibitor of embodiment 73, wherein the carrier comprises a nanoparticle.

[0339] 75. The BRD9 inhibitor of embodiment 74, wherein the nanoparticle comprises a lipid-based nanoparticle.

[0340] 76. The BRD9 inhibitor of embodiment 74, wherein the nanoparticle comprises a human serum albumin-based nanoparticle.

[0341] 77. The BRD9 inhibitor of embodiment 74, wherein the nanoparticle comprises an apolipoprotein-based nanoparticle.

[0342] 78. The BRD9 inhibitor of embodiment 74, wherein the nanoparticle comprises a polymer-based nanoparticle.

[0343] 79. The BRD9 inhibitor of embodiment 74, wherein the nanoparticle comprises a dendrimer-based nanoparticle.

[0344] 80. The BRD9 inhibitor of embodiment 74, wherein the nanoparticle comprises an inorganic-based nanoparticle.

[0345] 81. The BRD9 inhibitor of embodiment 73, wherein the carrier comprises an exosome.

[0346] 82. The BRD9 inhibitor of embodiment 73, wherein the carrier comprises a carbon nanotube.

[0347] 83. The BRD9 inhibitor of any one of embodiments 48-82, wherein the treatment of the subject further comprises adjunctive administration of a brain permeability enhancer to the subject.

[0348] 84. The BRD9 inhibitor of embodiment 83, wherein the BRD9 inhibitor and brain permeability enhancer are co-administered in a single pharmaceutical composition.

[0349] 85. The BRD9 inhibitor of embodiment 83 or embodiment 84, wherein the brain permeability enhancer comprises cereport, regadenoson, or borneol.

[0350] 86. The BRD9 inhibitor of embodiment 85, wherein the brain permeability enhancer comprises cereport.

[0351] 87. The BRD9 inhibitor of embodiment 85, wherein the brain permeability enhancer comprises regadenoson.

[0352] 88. The BRD9 inhibitor of embodiment 85, wherein the brain permeability enhancer comprises borneol.

[0353] 89. The BRD9 inhibitor of any one of embodiments 48-88, wherein the treatment of the subject further comprises subjecting the subject to microbubble-enhanced ultrasound.

[0354] 90. The BRD9 inhibitor of any one of embodiments 48-89, wherein the BRD9 inhibitor is administered intranasally to the subject.

[0355] 91. The BRD9 inhibitor of any one of embodiments 48-89, wherein the BRD9 inhibitor is administered intravenously to the subject.

[0356] 92. The BRD9 inhibitor of any one of embodiments 48-89, wherein the BRD9 inhibitor is administered to the subject by intra-arterial injection.

[0357] 93. The BRD9 inhibitor according to any one of embodiments 48-89, wherein the BRD9 inhibitor is administered to the CSF of the subject.

[0358] 94. The BRD9 inhibitor according to embodiment 93, wherein the BRD9 inhibitor is administered intrathecally, intracerebroventricularly, or intraparenchymally.

[0359] 95. Use of a BRD9 inhibitor in the manufacture of a medicament for the treatment of Huntington’s disease.

[0360] 96. The use according to embodiment 95, wherein the BRD9 inhibitor is a BRD9 inhibitor according to any one of embodiments 49-94.

[0361] 9. Citation of Reference

[0362] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes. In the event that there is an inconsistency between the teachings of one or more of the references incorporated herein and the present disclosure, the teachings of the present specification are intended to prevail.

Claims

1. Use of a bromodomain 9 (BRD9) inhibitor in the manufacture of a medicament for treating a subject having Huntington’s Disease (HD), wherein the BRD9 inhibitor is BI-9564 or a pharmaceutically acceptable salt thereof: 。 2. Use of a bromodomain 9 (BRD9) inhibitor in the manufacture of a medicament for treating a subject having Huntington’s Disease (HD), wherein the BRD9 inhibitor is BI-7273 or a pharmaceutically acceptable salt thereof: 。 3. Use of a bromodomain 9 (BRD9) inhibitor in the manufacture of a medicament for treating a subject having Huntington’s Disease (HD), wherein the BRD9 inhibitor is I-BRD9 or a pharmaceutically acceptable salt thereof: 。 4. Use of a bromodomain 9 (BRD9) inhibitor in the manufacture of a medicament for treating a subject having Huntington’s Disease (HD), wherein the BRD9 inhibitor is d-BRD9 or a pharmaceutically acceptable salt thereof: 。 5. Use of a bromodomain 9 (BRD9) inhibitor in the manufacture of a medicament for treating a subject having Huntington’s Disease (HD), wherein the BRD9 inhibitor is bromosporin or a pharmaceutically acceptable salt thereof: 。 6. The use of any one of claims 1-5, wherein the BRD9 inhibitor is used in the form of an amino acid conjugate.

7. The use of claim 6, wherein the amino acid conjugate is a lysine conjugate.

8. The use of claim 6, wherein the amino acid conjugate is a phenylalanine conjugate.

9. The use of any one of claims 1-8, wherein the BRD9 inhibitor is used with a carrier.

10. The use of claim 9, wherein the carrier comprises a nanoparticle, an exosome, or a carbon nanotube.

11. The use of claim 10, wherein the carrier comprises a nanoparticle.

12. The use of claim 11, wherein the nanoparticle comprises a lipid-based nanoparticle.

13. The use of claim 11, wherein the nanoparticle comprises a human serum albumin-based nanoparticle.

14. The use of claim 11, wherein the nanoparticle comprises an apolipoprotein-based nanoparticle.

15. The use of claim 11, wherein the nanoparticle comprises a polymer-based nanoparticle.

16. The use of claim 11, wherein the nanoparticle comprises a dendrimer-based nanoparticle.

17. The use of claim 11, wherein the nanoparticle comprises an inorganic-based nanoparticle.

18. The use of claim 10, wherein the carrier comprises an exosome.

19. The use of claim 10, wherein the carrier comprises a carbon nanotube.

20. The use of any one of claims 1-19, further comprising adjunctive use of a brain permeability enhancer.

21. The use of claim 20, wherein the BRD9 inhibitor and brain permeability enhancer are co-administered in a single pharmaceutical composition.

22. The use according to claim 20 or claim 21, wherein the brain permeability enhancer comprises CeReport, Reganoxan, or Borneol.

23. The use according to claim 22, wherein the brain permeability enhancer comprises CeReport.

24. The use according to claim 22, wherein the brain permeability enhancer comprises reganosin.

25. The use according to claim 22, wherein the brain permeability enhancer comprises borneol.

26. The use according to any one of claims 1 to 25, further comprising performing microbubble-enhanced ultrasound on the subject.

27. The use according to any one of claims 1 to 26, wherein the BRD9 inhibitor is administered intranasally to the subject.

28. The use according to any one of claims 1 to 26, wherein the BRD9 inhibitor is administered intravenously to the subject.

29. The use according to any one of claims 1 to 26, wherein the BRD9 inhibitor is administered to the subject by intra-arterial injection.

30. The use according to any one of claims 1 to 26, wherein the BRD9 inhibitor is administered to the subject's CSF.

31. The use according to claim 30, wherein the BRD9 inhibitor is administered intrathecally, intraventricularly, or intraparenchymalally.

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