WO2023205579A1 - Compositions and methods for disrupting pathological aggregates - Google Patents

Compositions and methods for disrupting pathological aggregates Download PDF

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Publication number
WO2023205579A1
WO2023205579A1 PCT/US2023/065671 US2023065671W WO2023205579A1 WO 2023205579 A1 WO2023205579 A1 WO 2023205579A1 US 2023065671 W US2023065671 W US 2023065671W WO 2023205579 A1 WO2023205579 A1 WO 2023205579A1
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disease
protein aggregate
protein
branched
cell
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PCT/US2023/065671
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French (fr)
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Julia Sabine SCHALETZKY
Michael RAPE
Edward Eric WEHRI
Stephanie Kum-Yin SEE
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The Regents Of The University Of California
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Publication of WO2023205579A1 publication Critical patent/WO2023205579A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/36—Post-translational modifications [PTMs] in chemical analysis of biological material addition of addition of other proteins or peptides, e.g. SUMOylation, ubiquitination
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • a Sequence Listing is provided herewith as a Sequence Listing XML, “BERK- 460WO_SEQ_LLST” created on April 3, 2023 and having a size of 1 1 .8 KB.
  • the contents of the Sequence Listing XML are incorporated by reference herein in their entirety.
  • Ubiquitin is a small protein that has important regulatory roles in a wide variety of cellular pathways. The best known of these is ubiquitin’s role in protein degradation, where covalent attachment of ubiquitin to a target protein enables that target protein to be recognized and destroyed by the 26S proteasome. Ubiquitin contains seven lysine residues (Lys6, Lysl l, Lys27, Lys33, Lys29, Lys48, and Lys63). The molecule produced upon ubiquitination of a ubiquitin protein is termed polyubiquitin, and may comprise two or more ubiquitin moieties.
  • a common feature in many neurodegenerative diseases is accumulation of misfolded proteins, which form characteristic intracellular aggregates that are toxic to neurons. While afflicting millions of people worldwide, a therapeutic strategy to treat neurodegenerative diseases such as Huntington’s Disease and Parkinson’s Disease is lacking.
  • the present disclosure provides methods of disrupting a protein aggregate comprising branched polyubiquitin.
  • the present disclosure provides a method of treating a disease associated with a protein aggregate comprising branched polyubiquitin.
  • FIG. 1A-1B provide amino acid sequences of p97/VCP polypeptides (SEQ ID NOs: 1-2, respectively).
  • FIG. 2A-2C provide amino acid sequences of UFD1 polypeptides (SEQ ID NOs:3-5, respectively).
  • FIG. 3A-3B provide amino acid sequences of NPL4 polypeptides (SEQ ID NOs:6-7, respectively).
  • FIG. 4 provides an amino acid sequence of an NPL4 polypeptide (SEQ ID NO: 8).
  • FIG. 5 provides structures of molecules that increase the affinity of a p97/VCP-adaptor protein complex to polyubiquitin.
  • FIG. 6 provides structures of molecules that increase the affinity of a p97/VCP-adaptor protein complex to polyubiquitin.
  • treatment used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease or symptom in a mammal, and includes: (a) preventing the disease or symptom from occurring in a subject which may or may not be predisposed to acquiring the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease or one or more symptoms associated with the disease, e.g., arresting its development; and/or (c) relieving the disease, i.e., causing regression of the disease.
  • the therapeutic agent may be administered before, during and/or after the onset of disease or injury.
  • the treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment may be performed prior to complete loss of function in the affected tissues.
  • the subject therapy may be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.
  • the terms “individual,” “subject,” “host,” and “patient,” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired. Mammals include, e.g., humans, non-human primates, rodents (e.g., rats; mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, and the like), etc. Unless otherwise indicated, the terms “individual,” “subject,” “host,” and “patient,” refer to a human.
  • the present disclosure provides methods of disrupting a protein aggiegate comprising branched polyubiquitin.
  • the present disclosure provides a method of treating a disease associated with a protein aggregate comprising branched polyubiquitin.
  • the present disclosure provides agents that disrupt a protein aggregate comprising branched polyubiquitin.
  • the agents increase the binding affinity of p97/VCP to such protein aggregates. Binding of p97/VCP to a protein aggregate comprising branched polyubiquitin accelerates unfolding and proteasomal degradation of the protein aggregate.
  • Such protein aggregates can be cytotoxic, e.g., neurotoxic, toxic to pancreatic beta islet cells, toxic to cardiomyocytes, etc. Therefore, an agent that increases the binding affinity of p97/VCP to a protein aggregate comprising branched polyubiquitin is useful for treating a disease associated with a protein aggregate comprising branched polyubiquitin.
  • An agent described herein can increase binding (e.g., increase the binding affinity) of p97/VCP to polyubiquitin by at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 100% (or two-fold), at least 2.5-fold, at least 5- fold, at least 10-fold, or more than 10-fold, compared to the binding affinity of p97/VCP to polyubiquitin in the absence of the agent.
  • Whether an agent can increase binding of p97/VCP to polyubiquitin can be determined using any convenient assay, e.g., a fluorescence polarization assay, e.g., as described below.
  • an agent that increases binding of p97/VCP to polyubiquitin also disrupts a protein aggregate comprising polyubiquitin.
  • an agent increases binding of p97/VCP to polyubiquitin disrupts a protein aggregate comprising polyubiquitin, thereby reducing the level of the protein aggregate in a cell.
  • an agent that increases binding of p97/VCP to polyubiquitin reduces the level (amount) of a protein aggregate comprising polyubiquitin in a cell by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or more than 80%, compared to the level of the protein aggregate in the cell not contacted with the agent.
  • suitable agents are provided in FIG. 5 and FIG. 6.
  • the agent is a compound of FIG. 5, or an analog or derivative thereof.
  • the agent is a compound of FIG. 6, or an analog or derivative thereof.
  • the present disclosure provides a pharmaceutical composition comprising an active agent of the present disclosure.
  • the pharmaceutical composition is suitable for administering to an individual in need thereof.
  • the pharmaceutical composition is suitable for administering to an individual in need thereof, where the individual is a human.
  • a protein aggregate comprising polyubiquitin comprises (e.g., is modified with) a branched polyubiquitin.
  • a protein aggregate comprising polyubiquitin comprises huntingtin (comprising poly(glutamine)) and a branched polyubiquitin; where such protein aggregates are associated with Huntington’s Disease.
  • a protein aggregate comprising polyubiquitin comprises amyloid-P protein and a branched ubiquitin.
  • a protein aggregate comprising polyubiquitin comprises a tau protein and a branched ubiquitin; where such protein aggregates are associated with Alzheimer’s Disease.
  • a protein aggregate comprising polyubiquitin comprises islet amyloid protein and a branched ubiquitin; where such protein aggregates are associated with type 2 diabetes.
  • a protein aggregate comprising polyubiquitin comprises a-synuclein and a branched ubiquitin; where such protein aggregates are associated with Parkinson’s Disease.
  • a protein aggregate comprising polyubiquitin comprises TAR DNA-binding protein-43 (TDP43) and a branched ubiquitin; where such protein aggregates are associated with amyotrophic lateral sclerosis (ALS).
  • a protein aggregate comprising polyubiquitin comprises ataxin-3 (ATX 3) and a branched ubiquitin; where such protein aggregates are associated with spinocerebellar ataxia.
  • a protein aggregate (e.g., a disease-associated protein aggregate) can be modified with any of a variety of forms of branched ubiquitin.
  • Branched ubiquitin includes, e.g., KI 1/K48, K29/K48, and K48/K6 ubiquitin.
  • Branched ubiquitin also includes, e.g., K6/K11, K6/K48, K27/K29, and K29/K33.
  • An active agent of the present disclosure can be formulated with one or more pharmaceutically acceptable excipients.
  • pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein.
  • Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy,” 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds., 7 th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H.
  • the pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents, arc readily available to the public.
  • pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
  • Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (1 1 ) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium
  • a subject pharmaceutical composition can optionally include, without limitation, other pharmaceutically acceptable components, including, without limitation, buffers, preservatives, tonicity adjusters, salts, antioxidants, physiological substances, pharmacological substances, bulking agents, emulsifying agents, wetting agents, sweetening or flavoring agents, and the like.
  • buffers include, without limitation, acetate buffers, citrate buffers, phosphate buffers, neutral buffered saline, phosphate buffered saline and borate buffers. It is understood that acids or bases can be used to adjust the pH of a composition as needed.
  • antioxidants include, without limitation, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole and butylated hydroxytoluene.
  • Useful preservatives include, without limitation, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuric nitrate and a stabilized oxy chloro composition, for example, PURITETM.
  • Tonicity adjustors suitable for inclusion in a subject pharmaceutical composition include, without limitation, salts such as, e.g., sodium chloride, potassium chloride, mannitol or glycerin and other pharmaceutically acceptable tonicity adjustor. It is understood that these and other substances known in the art of pharmacology can be included in a subject pharmaceutical composition.
  • Agents that can increase binding (e.g., increase the binding affinity) of p97/VCP to polyubiquitin can be identified using an assay described herein.
  • a suitable assay is a fluorescence polarization (FP) assay. See, e.g., Moerke (2009) Current Protocols in Chemical Biology 1:1-15; and Lea and Simeonov (2011) Expert Opin. Drug Discov. 6:17.
  • FP fluorescence polarization
  • An FP assay can be carried out as follows.
  • a complex comprising p97/VCP and adaptor proteins UFD1, NPL4, and FAF1 can be combined with fluorescently labeled linear polyubiquitin or branched polyubiquitin, in the presence or absence of a test agent, to form a test sample.
  • the test sample is excited using polarized light; the degree of polarization of the emitted light is determined.
  • a test agent that results in a polarized state of the emitted light is considered a candidate agent for disrupting a protein aggregate comprising polyubiquitin.
  • An example of a suitable FP assay is described in Example 1.
  • Valosin-containing protein is also known as “transitional endoplasmic reticulum ATPase” (TERA), “p97”, “CDC48”, “FTDALS6”, and “IBMPFD.”
  • TERA transitional endoplasmic reticulum ATPase
  • p97/VCP belongs to the AAA+ (ATPases associated with various cellular activities) ATPase family.
  • p97/VCP is highly conserved and, in conjunction with adaptor proteins, couples ATP hydrolysis to segregation of polypeptides from immobile cellular structures such as protein assemblies, membranes, ribosomes, and chromatin; this segregation can result in proteasomal degradation of the segregated polypeptides. See, e.g., Ye et al. (2017) Front. Mol. Biosci.
  • a p97/VCP polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the p97/VCP amino acid sequence depicted in FIG. 1A. In some cases, a p97/VCP polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the p97/VCP amino acid sequence depicted in FIG. IB.
  • UFD1 Ubiquitin recognition factor in endoplasmic reticulum-associated degradation protein 1
  • UFD1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the UFD1 amino acid sequence depicted in FIG. 2 A.
  • a UFD1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the UFD1 amino acid sequence depicted in FIG. 2B.
  • a UFD1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the UFD1 amino acid sequence depicted in FIG. 2C.
  • NPL4 Nuclear protein localization 4 homolog
  • NPLOC4 Nuclear protein localization 4 homolog
  • an NPL4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the NPL4 amino acid sequence depicted in FIG. 3A.
  • an NPL4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the NPL4 amino acid sequence depicted in FIG. 3B.
  • an FAF1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the FAF1 amino acid sequence depicted in FIG. 4.
  • p97/VCP adaptor proteins are known in the art. Such adaptor proteins include, e.g., PLAA/Ufd3, PNGase, HOIP, and Ufd2, which bind to the C-terminal appendage of p97/VCP.
  • Adaptor proteins that bind to the N-terminal domain of p97/VCP include, e.g., UBXN1/SAKS1, UBXN2A/UBXD4, UBXN2B/p37, UBXN2C/p47, UBXN3B/UBXD8/FAF2/ETEA, UBXN4/UBXD2/Erasin, UBXN6/UBXD1, UBXN7/UBXD7, UBXN8/UBXD6, UBXN9/UBXD9/ASPSCR1, UBXN10/UBXD3, UBXN11/UBXD5, OTU1/YOD1, VCP1P/VC1P135, V1MP, gp78/AMFR, SV1P, ZFAND2B/A1RAPL, ANKZF1, Hrdl/SYVVNl, Ataxin3/MJD/SCA3, UBE4B/Ufd2, NUB1/NYREN18
  • the present disclosure provides a method of disrupting a protein aggregate comprising branched polyubiquitin, the method comprising contacting the protein aggregate with an agent that promotes binding of a p97/VCP polypeptide to the protein aggregate, where binding of the p97/VCP polypeptide to the protein aggregate disrupts the protein aggregate.
  • suitable agents are provided in FIG. 5 and FIG. 6.
  • the agent is a compound of FIG. 5, or an analog or derivative thereof.
  • the agent is a compound of FIG. 6, or an analog or derivative thereof.
  • the present disclosure provides a method of decreasing the amount of a pathological protein aggregate comprising branched polyubiquitin in a cell, the method comprising contacting the cell with an agent that promotes binding of a p97/VCP polypeptide to the protein aggregate (e.g., that promotes binding of p97/VCP polypeptide to polyubiquitin present in the protein aggregate), where the binding of the p97/VCP polypeptide to the protein aggregate promotes unfolding and proteasomal degradation of the protein aggregate, thereby reducing the amount of the protein aggregate in the cell.
  • the cell is a neuron.
  • the cell is a pancreatic beta islet cell.
  • the cell is a cardiomyocyte.
  • contacting a cell comprising the protein aggregate reduces the amount of the protein aggregate in the cell by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or more than 80%, compared to the level of the protein aggregate in the cell not contacted with the agent.
  • the present disclosure provides a method of treating a disease associated with a protein aggregate comprising branched polyubiquitin, the method comprising administering to an individual having such a disorder an effective amount of an agent that promotes binding of a p97/VCP polypeptide to the protein aggregate. Binding of the p97/VCP polypeptide to the protein aggregate disrupts the protein aggregate and treats the disease.
  • the disease is a neurodegenerative disease.
  • neurodegenerative disease refers to or describes the physiological condition in nerve-containing animals that is typically characterized by deterioration of nervous tissue or deterioration of communication between cells in nervous tissue.
  • neurodegenerative diseases include, but arc not limited to, Lewy body disease, postpoliomyelitis syndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, amyotrophic lateral sclerosis, Guillain-Barre syndrome, Carcot Marie Tooth syndrome, striatonigral degeneration, and nervous ccll/tissue destruction caused by or associated with tauopathies, prion diseases, bulbar palsy, motor neuron disease, dementia, Canavan disease, Huntington's disease, neuronal ceroidlipofuscinosis, Alexander's disease, Tourette's syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Halervorden-Spatz syndrome, lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome.
  • the neurodegenerative disease is Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis, frontotemporal dementia, Creutzfeldt- Jacob disease, spinocerebellar ataxia, or Lewy body dementia.
  • the disease is type 2 diabetes. In some cases, the disease is amyloid transthyretin cardiomyopathy.
  • an “effective amount of’ an agent that promotes binding of a p97/VCP polypeptide to a protein aggregate modified with a branched ubiquitin is an amount that, when administered in one or more doses to an individual in need thereof, reduces the severity of an adverse symptom associated with the neurodegenerative disease by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or more than 50%, compared to the severity of the symptom in the individual not treated with the agent or compared to the severity of the symptom in the individual before treatment with the agent.
  • an “effective amount of’ an agent that promotes binding of a p97/VCP polypeptide to a protein aggregate modified with a branched ubiquitin is an amount that, when administered in one or more doses to an individual having type 2 diabetes, reduces at least one adverse symptom of type 2 diabetes in the individual by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, compared to the symptoms in the individual before administration of the composition, or in the absence of administration with the composition.
  • Adverse symptoms can include, e.g., a body mass index that is above the normal range; blood insulin levels that are outside of the normal range; and the like.
  • a method of disrupting a protein aggregate modified with branched polyubiquitin comprising contacting the protein aggregate with an agent that promotes binding of a p97/V CP polypeptide to the protein aggregate, wherein catalytic action of the p97/VCP polypeptide on the protein aggregate disrupts the protein aggregate.
  • Aspect 2 The method of aspect 1, wherein the branched polyubiquitin is KI 1/K48, K29/K48, K48/K6, K6/K11, K6/K48, K27/K29, or K29/K33 branched ubiquitin.
  • Aspect 3 The method of aspect 1 or aspect 2, wherein the p97/VCP is present in a complex with one or more adaptor proteins.
  • Aspect 4 The method of aspect 3, wherein the one or more adaptor proteins are selected from NPL4, UFD1 , FAF1 , PLAA/Ufd3, PNGase, HO1P, Ufd2, UBXN1/SAKS1 , UBXN2A/UBXD4, UBXN2B/p37, UBXN2C/p47, UBXN3B/UBXD8/FAF2/ETEA, UBXN4/UBXD2/Erasin, UBXN6/UBXD1, UBXN7/UBXD7, UBXN8/UBXD6, UBXN9/UBXD9/ASPSCR1, UBXN10/UBXD3, UBXN11/UBXD5, OTU1/YOD1, VCPIP/VCIP135, VIMP, gp78/AMFR, SVIP, ZFAND2B/AIRAPL, ANKZF1, Hrdl/SYVVNl
  • Aspect 5 The method of aspect any one of aspects 1-4, wherein the protein aggregate is present in a neuron.
  • Aspect 6 The method of any one of aspects 1-4, wherein the protein aggregate is present in a cardiac cell.
  • Aspect 7 The method of any one of aspects 1-4, wherein the protein aggregate is present in a pancreatic beta islet cell.
  • Aspect 8 The method of any one of aspects 1-7, wherein the agent is: i) a compound of FIG. 5, or an analog or derivative thereof; or ii) a compound of FIG. 6, or an analog or derivative thereof.
  • a method of reducing the amount of protein aggregate modified with branched polyubiquitin in a cell comprising contacting the cell with an agent that promotes binding of a p97/VCP polypeptide to the protein aggregate, wherein catalytic action of the p97/VCP polypeptide on the protein aggregate results in a reduction in the amount of the protein aggregate in the cell.
  • Aspect 10 The method of aspect 9, wherein the cell is a neuron.
  • Aspect 11 The method of aspect 9, wherein the cell is a pancreatic beta islet cell.
  • Aspect 12 The method of aspect 9, wherein the cell is a cardiomyocyte.
  • Aspect 13 The method of any one of aspects 9-12, wherein the branched polyubiquitin is a KI 1/K48 branched polyubiquitin.
  • Aspect 14 The method of any one of aspects 9-13, wherein the agent is: i) a compound of FIG. 5, or an analog or derivative thereof; or ii) a compound of FIG. 6, or an analog or derivative thereof.
  • a method of treating a disease associated with a protein aggregate modified with branched polyubiquitin comprising administering to an individual having such a disorder an effective an agent that promotes binding of a p97/VCP polypeptide to the protein aggregate, wherein catalytic action of the p97/VCP polypeptide on the protein aggregate disrupts the protein aggregate and treats the disease.
  • Aspect 16 The method of aspect 15, wherein the branched polyubiquitin is KI 1/K48, K29/K48, K48/K6, K6/K11, K6/K48. K27/K29, or K29/K33 branched ubiquitin.
  • Aspect 17 The method of aspect 15 or aspect 16, wherein the disease is a ncurodcgcncrativc disease.
  • Aspect 18 The method of aspect 17, wherein the neurodegenerative disease is Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis, frontotemporal dementia, Creutzfeldt- Jacob disease, spinocerebellar ataxia type 3, or Lewy body dementia.
  • the neurodegenerative disease is Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis, frontotemporal dementia, Creutzfeldt- Jacob disease, spinocerebellar ataxia type 3, or Lewy body dementia.
  • Aspect 19 The method of aspect 15, wherein the disease is type 2 diabetes.
  • Aspect 20 The method of aspect 15, wherein the disease is amyloid transthyretin cardiomyopathy.
  • Aspect 21 The method of aspect 17, wherein the protein aggregate comprises a pathological form of poly(glutamine) huntingtin protein, and where the disease is Huntington’s Disease.
  • Aspect 22 The method of aspect 17, wherein the protein aggregate comprises alpha- synuclein, and wherein the disease is Parkinson’s Disease.
  • Aspect 23 The method of aspect 17, wherein the protein aggregate comprises TAR DNA-binding protein-43 (TDP43), and wherein the disease is amyotrophic lateral sclerosis.
  • TDP43 TAR DNA-binding protein-43
  • Aspect 24 The method of aspect 17, wherein the protein aggregate comprises tau protein, and wherein the disease is Alzheimer’ s Disease.
  • Aspect 25 The method of aspect 17, wherein the protein aggregate comprises ataxin-3 (ATXN3), and wherein the disease is spinocerebellar ataxia type 3.
  • Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.
  • Example 1 Example 1
  • a fluorescence polarization (FP) assay used to identify compounds that increase p97 complex binding to linear ubiquitin was set up as follows.
  • p97-UFDl/NPL4/FAFl protein complex was first generated by combining 10 mM His-FLAG-p97, 5 mM FAFl-His, and 1.6 mM His-UFD1/NPL4 in FP buffer containing 20 mM HEPES pH 7.4, 150 mM KC1, ImM MgCF, and 0.2% (v/v) NP-40 (non-ionic detergent).
  • IP Immunoprecipitation
  • Htt-73Q-GFP Hauntingtin protein with 73 Gin repeats fused to green fluorescent protein (GFP)
  • DMSO dimethyl sulfoxide
  • Cells were then isolated from plates, washed once in phosphate-buffered saline (PBS), and then lysed in 1 mL buffer containing 50 mM HEPES pH 7.5, 1.5 mM MgCl 2 , 5 mM KC1, 150 mM NaCl + 0.1% NP-40, protease inhibitor, 10 pM carfilzomib and 5 pL benzonase. The cell extract was cleared at top speed on a table-top centrifuge to remove insoluble debris.
  • PBS phosphate-buffered saline

Abstract

The present disclosure provides methods of disrupting a protein aggregate modified with branched polyubiquitin, contacting the protein aggregate with an agent that promotes binding of a p97/VCP polypeptide to the protein aggregate to disrupt the protein aggregate, and further, treating diseases, such as neurodegenerative diseases, associated with a protein aggregate.

Description

COMPOSITIONS AND METHODS FOR DISRUPTING PATHOLOGICAL AGGREGATES
CROSS -REFERENCE
[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/332,054, filed April 18, 2022, and U.S. Provisional Patent Application No. 63/448,760, filed February 28, 2023, which applications are incorporated herein by reference in their entirety.
INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED
[0002] A Sequence Listing is provided herewith as a Sequence Listing XML, “BERK- 460WO_SEQ_LLST” created on April 3, 2023 and having a size of 1 1 .8 KB. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.
INTRODUCTION
[0003] Ubiquitin is a small protein that has important regulatory roles in a wide variety of cellular pathways. The best known of these is ubiquitin’s role in protein degradation, where covalent attachment of ubiquitin to a target protein enables that target protein to be recognized and destroyed by the 26S proteasome. Ubiquitin contains seven lysine residues (Lys6, Lysl l, Lys27, Lys33, Lys29, Lys48, and Lys63). The molecule produced upon ubiquitination of a ubiquitin protein is termed polyubiquitin, and may comprise two or more ubiquitin moieties.
[0004] A common feature in many neurodegenerative diseases is accumulation of misfolded proteins, which form characteristic intracellular aggregates that are toxic to neurons. While afflicting millions of people worldwide, a therapeutic strategy to treat neurodegenerative diseases such as Huntington’s Disease and Parkinson’s Disease is lacking.
SUMMARY
[0005] The present disclosure provides methods of disrupting a protein aggregate comprising branched polyubiquitin. The present disclosure provides a method of treating a disease associated with a protein aggregate comprising branched polyubiquitin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1A-1B provide amino acid sequences of p97/VCP polypeptides (SEQ ID NOs: 1-2, respectively).
[0007] FIG. 2A-2C provide amino acid sequences of UFD1 polypeptides (SEQ ID NOs:3-5, respectively). [0008] FIG. 3A-3B provide amino acid sequences of NPL4 polypeptides (SEQ ID NOs:6-7, respectively).
[0009] FIG. 4 provides an amino acid sequence of an NPL4 polypeptide (SEQ ID NO: 8).
[0010] FIG. 5 provides structures of molecules that increase the affinity of a p97/VCP-adaptor protein complex to polyubiquitin.
[0011] FIG. 6 provides structures of molecules that increase the affinity of a p97/VCP-adaptor protein complex to polyubiquitin.
DEFINITIONS
[0012] The terms “treatment”, “treating” and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. “Treatment” as used herein covers any treatment of a disease or symptom in a mammal, and includes: (a) preventing the disease or symptom from occurring in a subject which may or may not be predisposed to acquiring the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease or one or more symptoms associated with the disease, e.g., arresting its development; and/or (c) relieving the disease, i.e., causing regression of the disease. The therapeutic agent may be administered before, during and/or after the onset of disease or injury. The treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment may be performed prior to complete loss of function in the affected tissues. The subject therapy may be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.
[0013] The terms “individual,” “subject,” “host,” and “patient,” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired. Mammals include, e.g., humans, non-human primates, rodents (e.g., rats; mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, and the like), etc. Unless otherwise indicated, the terms “individual,” “subject,” “host,” and “patient,” refer to a human.
[0014] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. [0015] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
[0016] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials arc now described. All publications mentioned herein arc incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
[0017] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a p97/VCP polypeptide” includes a plurality of such polypeptides and reference to “the branched polyubiquitin” includes reference to one or more branched polyubiquitins and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
[0018] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. [0019] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
DETAILED DESCRIPTION
[0020] The present disclosure provides methods of disrupting a protein aggiegate comprising branched polyubiquitin. The present disclosure provides a method of treating a disease associated with a protein aggregate comprising branched polyubiquitin.
AGENTS TH T INCREASE BINDING OF P97/VCP TO POLYUBIQUITIN
[0021] The present disclosure provides agents that disrupt a protein aggregate comprising branched polyubiquitin. The agents increase the binding affinity of p97/VCP to such protein aggregates. Binding of p97/VCP to a protein aggregate comprising branched polyubiquitin accelerates unfolding and proteasomal degradation of the protein aggregate. Such protein aggregates can be cytotoxic, e.g., neurotoxic, toxic to pancreatic beta islet cells, toxic to cardiomyocytes, etc. Therefore, an agent that increases the binding affinity of p97/VCP to a protein aggregate comprising branched polyubiquitin is useful for treating a disease associated with a protein aggregate comprising branched polyubiquitin.
[0022] An agent described herein (also referred to as an “active agent”) can increase binding (e.g., increase the binding affinity) of p97/VCP to polyubiquitin by at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 100% (or two-fold), at least 2.5-fold, at least 5- fold, at least 10-fold, or more than 10-fold, compared to the binding affinity of p97/VCP to polyubiquitin in the absence of the agent. Whether an agent can increase binding of p97/VCP to polyubiquitin can be determined using any convenient assay, e.g., a fluorescence polarization assay, e.g., as described below.
[0023] In some cases, an agent that increases binding of p97/VCP to polyubiquitin also disrupts a protein aggregate comprising polyubiquitin. In some cases, an agent increases binding of p97/VCP to polyubiquitin disrupts a protein aggregate comprising polyubiquitin, thereby reducing the level of the protein aggregate in a cell. For example, in some cases, an agent that increases binding of p97/VCP to polyubiquitin reduces the level (amount) of a protein aggregate comprising polyubiquitin in a cell by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or more than 80%, compared to the level of the protein aggregate in the cell not contacted with the agent. [0024] Non-limiting examples of suitable agents are provided in FIG. 5 and FIG. 6. In some cases, the agent is a compound of FIG. 5, or an analog or derivative thereof. In some cases, the agent is a compound of FIG. 6, or an analog or derivative thereof.
[0025] The present disclosure provides a pharmaceutical composition comprising an active agent of the present disclosure. In some cases, the pharmaceutical composition is suitable for administering to an individual in need thereof. In some cases, the pharmaceutical composition is suitable for administering to an individual in need thereof, where the individual is a human.
[0026] In some cases, a protein aggregate comprising polyubiquitin comprises (e.g., is modified with) a branched polyubiquitin. In some cases, a protein aggregate comprising polyubiquitin comprises huntingtin (comprising poly(glutamine)) and a branched polyubiquitin; where such protein aggregates are associated with Huntington’s Disease. In some cases, a protein aggregate comprising polyubiquitin comprises amyloid-P protein and a branched ubiquitin. In some cases, a protein aggregate comprising polyubiquitin comprises a tau protein and a branched ubiquitin; where such protein aggregates are associated with Alzheimer’s Disease. In some cases, a protein aggregate comprising polyubiquitin comprises islet amyloid protein and a branched ubiquitin; where such protein aggregates are associated with type 2 diabetes. In some cases, a protein aggregate comprising polyubiquitin comprises a-synuclein and a branched ubiquitin; where such protein aggregates are associated with Parkinson’s Disease. In some cases, a protein aggregate comprising polyubiquitin comprises TAR DNA-binding protein-43 (TDP43) and a branched ubiquitin; where such protein aggregates are associated with amyotrophic lateral sclerosis (ALS). In some cases, a protein aggregate comprising polyubiquitin comprises ataxin-3 (ATX 3) and a branched ubiquitin; where such protein aggregates are associated with spinocerebellar ataxia.
[0027] A protein aggregate (e.g., a disease-associated protein aggregate) can be modified with any of a variety of forms of branched ubiquitin. Branched ubiquitin includes, e.g., KI 1/K48, K29/K48, and K48/K6 ubiquitin. Branched ubiquitin also includes, e.g., K6/K11, K6/K48, K27/K29, and K29/K33.
[0028] An active agent of the present disclosure can be formulated with one or more pharmaceutically acceptable excipients. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy,” 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds., 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc. [0029] The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, arc readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
[0030] Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (1 1 ) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogcn-frcc water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or poly anhydrides; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.
[0031] A subject pharmaceutical composition can optionally include, without limitation, other pharmaceutically acceptable components, including, without limitation, buffers, preservatives, tonicity adjusters, salts, antioxidants, physiological substances, pharmacological substances, bulking agents, emulsifying agents, wetting agents, sweetening or flavoring agents, and the like. Various buffers and means for adjusting pH can be used to prepare a pharmaceutical composition disclosed in the present specification, provided that the resulting preparation is pharmaceutically acceptable. Such buffers include, without limitation, acetate buffers, citrate buffers, phosphate buffers, neutral buffered saline, phosphate buffered saline and borate buffers. It is understood that acids or bases can be used to adjust the pH of a composition as needed. Pharmaceutically acceptable antioxidants include, without limitation, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole and butylated hydroxytoluene. Useful preservatives include, without limitation, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuric nitrate and a stabilized oxy chloro composition, for example, PURITE™. Tonicity adjustors suitable for inclusion in a subject pharmaceutical composition include, without limitation, salts such as, e.g., sodium chloride, potassium chloride, mannitol or glycerin and other pharmaceutically acceptable tonicity adjustor. It is understood that these and other substances known in the art of pharmacology can be included in a subject pharmaceutical composition. IDENTIFICATION OF ACTIVE AGENTS
[0032] Agents that can increase binding (e.g., increase the binding affinity) of p97/VCP to polyubiquitin can be identified using an assay described herein. A suitable assay is a fluorescence polarization (FP) assay. See, e.g., Moerke (2009) Current Protocols in Chemical Biology 1:1-15; and Lea and Simeonov (2011) Expert Opin. Drug Discov. 6:17.
[0033] An FP assay can be carried out as follows. A complex comprising p97/VCP and adaptor proteins UFD1, NPL4, and FAF1 can be combined with fluorescently labeled linear polyubiquitin or branched polyubiquitin, in the presence or absence of a test agent, to form a test sample. The test sample is excited using polarized light; the degree of polarization of the emitted light is determined. A test agent that results in a polarized state of the emitted light is considered a candidate agent for disrupting a protein aggregate comprising polyubiquitin. An example of a suitable FP assay is described in Example 1.
[0034] Valosin-containing protein (VCP) is also known as “transitional endoplasmic reticulum ATPase” (TERA), “p97”, “CDC48”, “FTDALS6”, and “IBMPFD.” In this disclosure, the protein is referred to as “p97/VCP.” p97/VCP belongs to the AAA+ (ATPases associated with various cellular activities) ATPase family. p97/VCP is highly conserved and, in conjunction with adaptor proteins, couples ATP hydrolysis to segregation of polypeptides from immobile cellular structures such as protein assemblies, membranes, ribosomes, and chromatin; this segregation can result in proteasomal degradation of the segregated polypeptides. See, e.g., Ye et al. (2017) Front. Mol. Biosci.
[0035] In some cases, a p97/VCP polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the p97/VCP amino acid sequence depicted in FIG. 1A. In some cases, a p97/VCP polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the p97/VCP amino acid sequence depicted in FIG. IB.
[0036] The adaptor protein “Ubiquitin recognition factor in endoplasmic reticulum-associated degradation protein 1” (UFD1) is also known as “ubiquitin fusion degradation protein 1.” In some cases, a UFD1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the UFD1 amino acid sequence depicted in FIG. 2 A. In some cases, a UFD1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the UFD1 amino acid sequence depicted in FIG. 2B. In some cases, a UFD1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the UFD1 amino acid sequence depicted in FIG. 2C.
[0037] The adaptor protein “Nuclear protein localization 4 homolog” (NPL4) is also known as “NPLOC4” and “NPLOC4 ubiquitin recognition factor.” In some cases, an NPL4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the NPL4 amino acid sequence depicted in FIG. 3A. In some cases, an NPL4 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the NPL4 amino acid sequence depicted in FIG. 3B.
[0038] The adaptor protein “Fas associated factor 1” (FAF1) is also known as “UBX domaincontaining protein 12” (UBXD12), and “UBX domain-containing protein 3A” (UBXN3 A). In some cases, an FAF1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the FAF1 amino acid sequence depicted in FIG. 4.
[0039] Other p97/VCP adaptor proteins are known in the art. Such adaptor proteins include, e.g., PLAA/Ufd3, PNGase, HOIP, and Ufd2, which bind to the C-terminal appendage of p97/VCP. Adaptor proteins that bind to the N-terminal domain of p97/VCP include, e.g., UBXN1/SAKS1, UBXN2A/UBXD4, UBXN2B/p37, UBXN2C/p47, UBXN3B/UBXD8/FAF2/ETEA, UBXN4/UBXD2/Erasin, UBXN6/UBXD1, UBXN7/UBXD7, UBXN8/UBXD6, UBXN9/UBXD9/ASPSCR1, UBXN10/UBXD3, UBXN11/UBXD5, OTU1/YOD1, VCP1P/VC1P135, V1MP, gp78/AMFR, SV1P, ZFAND2B/A1RAPL, ANKZF1, Hrdl/SYVVNl, Ataxin3/MJD/SCA3, UBE4B/Ufd2, NUB1/NYREN18, RHBDD1/RHBDL4, Derlinl, Derlin2, and SPRTN/DVCl/Clorfl24. See, e.g., Ye et al. (2017) Front. Mol. Biosci. 4:39.
METHODS
[0040] The present disclosure provides a method of disrupting a protein aggregate comprising branched polyubiquitin, the method comprising contacting the protein aggregate with an agent that promotes binding of a p97/VCP polypeptide to the protein aggregate, where binding of the p97/VCP polypeptide to the protein aggregate disrupts the protein aggregate. Non-limiting examples of suitable agents are provided in FIG. 5 and FIG. 6. In some cases, the agent is a compound of FIG. 5, or an analog or derivative thereof. In some cases, the agent is a compound of FIG. 6, or an analog or derivative thereof.
[0041] The present disclosure provides a method of decreasing the amount of a pathological protein aggregate comprising branched polyubiquitin in a cell, the method comprising contacting the cell with an agent that promotes binding of a p97/VCP polypeptide to the protein aggregate (e.g., that promotes binding of p97/VCP polypeptide to polyubiquitin present in the protein aggregate), where the binding of the p97/VCP polypeptide to the protein aggregate promotes unfolding and proteasomal degradation of the protein aggregate, thereby reducing the amount of the protein aggregate in the cell. In some cases, the cell is a neuron. In some cases, the cell is a pancreatic beta islet cell. In some cases, the cell is a cardiomyocyte. In some cases, contacting a cell comprising the protein aggregate reduces the amount of the protein aggregate in the cell by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or more than 80%, compared to the level of the protein aggregate in the cell not contacted with the agent.
[0042] The present disclosure provides a method of treating a disease associated with a protein aggregate comprising branched polyubiquitin, the method comprising administering to an individual having such a disorder an effective amount of an agent that promotes binding of a p97/VCP polypeptide to the protein aggregate. Binding of the p97/VCP polypeptide to the protein aggregate disrupts the protein aggregate and treats the disease.
[0043] In some cases, the disease is a neurodegenerative disease. The term “neurodegenerative disease” refers to or describes the physiological condition in nerve-containing animals that is typically characterized by deterioration of nervous tissue or deterioration of communication between cells in nervous tissue. Examples of neurodegenerative diseases include, but arc not limited to, Lewy body disease, postpoliomyelitis syndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, amyotrophic lateral sclerosis, Guillain-Barre syndrome, Carcot Marie Tooth syndrome, striatonigral degeneration, and nervous ccll/tissue destruction caused by or associated with tauopathies, prion diseases, bulbar palsy, motor neuron disease, dementia, Canavan disease, Huntington's disease, neuronal ceroidlipofuscinosis, Alexander's disease, Tourette's syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Halervorden-Spatz syndrome, lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome. In some cases, the neurodegenerative disease is Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis, frontotemporal dementia, Creutzfeldt- Jacob disease, spinocerebellar ataxia, or Lewy body dementia.
[0044] In some cases, the disease is type 2 diabetes. In some cases, the disease is amyloid transthyretin cardiomyopathy.
[0045] For the treatment of a neurodegenerative disease, in some cases, an “effective amount of’ an agent that promotes binding of a p97/VCP polypeptide to a protein aggregate modified with a branched ubiquitin is an amount that, when administered in one or more doses to an individual in need thereof, reduces the severity of an adverse symptom associated with the neurodegenerative disease by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or more than 50%, compared to the severity of the symptom in the individual not treated with the agent or compared to the severity of the symptom in the individual before treatment with the agent.
[0046] For the treatment of type 2 diabetes, an “effective amount of’ an agent that promotes binding of a p97/VCP polypeptide to a protein aggregate modified with a branched ubiquitin is an amount that, when administered in one or more doses to an individual having type 2 diabetes, reduces at least one adverse symptom of type 2 diabetes in the individual by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, compared to the symptoms in the individual before administration of the composition, or in the absence of administration with the composition. Adverse symptoms can include, e.g., a body mass index that is above the normal range; blood insulin levels that are outside of the normal range; and the like.
Examples of Non-Limiting Aspects of the Disclosure
[0047] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:
[0048] Aspect 1. A method of disrupting a protein aggregate modified with branched polyubiquitin, the method comprising contacting the protein aggregate with an agent that promotes binding of a p97/V CP polypeptide to the protein aggregate, wherein catalytic action of the p97/VCP polypeptide on the protein aggregate disrupts the protein aggregate.
[0049] Aspect 2. The method of aspect 1, wherein the branched polyubiquitin is KI 1/K48, K29/K48, K48/K6, K6/K11, K6/K48, K27/K29, or K29/K33 branched ubiquitin.
[0050] Aspect 3. The method of aspect 1 or aspect 2, wherein the p97/VCP is present in a complex with one or more adaptor proteins.
[0051] Aspect 4. The method of aspect 3, wherein the one or more adaptor proteins are selected from NPL4, UFD1 , FAF1 , PLAA/Ufd3, PNGase, HO1P, Ufd2, UBXN1/SAKS1 , UBXN2A/UBXD4, UBXN2B/p37, UBXN2C/p47, UBXN3B/UBXD8/FAF2/ETEA, UBXN4/UBXD2/Erasin, UBXN6/UBXD1, UBXN7/UBXD7, UBXN8/UBXD6, UBXN9/UBXD9/ASPSCR1, UBXN10/UBXD3, UBXN11/UBXD5, OTU1/YOD1, VCPIP/VCIP135, VIMP, gp78/AMFR, SVIP, ZFAND2B/AIRAPL, ANKZF1, Hrdl/SYVVNl, Ataxin3/MJD/SCA3, UBE4B/Ufd2, NUB1/NYREN18, RHBDD1/RHBDL4, Derlinl, Derlin2, and SPRTN/DVCl/Clorfl24.
[0052] Aspect 5. The method of aspect any one of aspects 1-4, wherein the protein aggregate is present in a neuron.
[0053] Aspect 6. The method of any one of aspects 1-4, wherein the protein aggregate is present in a cardiac cell.
[0054] Aspect 7. The method of any one of aspects 1-4, wherein the protein aggregate is present in a pancreatic beta islet cell.
[0055] Aspect 8. The method of any one of aspects 1-7, wherein the agent is: i) a compound of FIG. 5, or an analog or derivative thereof; or ii) a compound of FIG. 6, or an analog or derivative thereof.
[0056] Aspect 9. A method of reducing the amount of protein aggregate modified with branched polyubiquitin in a cell, the method comprising contacting the cell with an agent that promotes binding of a p97/VCP polypeptide to the protein aggregate, wherein catalytic action of the p97/VCP polypeptide on the protein aggregate results in a reduction in the amount of the protein aggregate in the cell.
[0057] Aspect 10. The method of aspect 9, wherein the cell is a neuron.
[0058] Aspect 11. The method of aspect 9, wherein the cell is a pancreatic beta islet cell.
[0059] Aspect 12. The method of aspect 9, wherein the cell is a cardiomyocyte.
[0060] Aspect 13. The method of any one of aspects 9-12, wherein the branched polyubiquitin is a KI 1/K48 branched polyubiquitin.
[0061] Aspect 14. The method of any one of aspects 9-13, wherein the agent is: i) a compound of FIG. 5, or an analog or derivative thereof; or ii) a compound of FIG. 6, or an analog or derivative thereof.
[0062] Aspect 15. A method of treating a disease associated with a protein aggregate modified with branched polyubiquitin, the method comprising administering to an individual having such a disorder an effective an agent that promotes binding of a p97/VCP polypeptide to the protein aggregate, wherein catalytic action of the p97/VCP polypeptide on the protein aggregate disrupts the protein aggregate and treats the disease.
[0063] Aspect 16. The method of aspect 15, wherein the branched polyubiquitin is KI 1/K48, K29/K48, K48/K6, K6/K11, K6/K48. K27/K29, or K29/K33 branched ubiquitin. [0064] Aspect 17. The method of aspect 15 or aspect 16, wherein the disease is a ncurodcgcncrativc disease.
[0065] Aspect 18. The method of aspect 17, wherein the neurodegenerative disease is Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis, frontotemporal dementia, Creutzfeldt- Jacob disease, spinocerebellar ataxia type 3, or Lewy body dementia.
[0066] Aspect 19. The method of aspect 15, wherein the disease is type 2 diabetes.
[0067] Aspect 20. The method of aspect 15, wherein the disease is amyloid transthyretin cardiomyopathy.
[0068] Aspect 21. The method of aspect 17, wherein the protein aggregate comprises a pathological form of poly(glutamine) huntingtin protein, and where the disease is Huntington’s Disease.
[0069] Aspect 22. The method of aspect 17, wherein the protein aggregate comprises alpha- synuclein, and wherein the disease is Parkinson’s Disease.
[0070] Aspect 23. The method of aspect 17, wherein the protein aggregate comprises TAR DNA-binding protein-43 (TDP43), and wherein the disease is amyotrophic lateral sclerosis.
[0071] Aspect 24. The method of aspect 17, wherein the protein aggregate comprises tau protein, and wherein the disease is Alzheimer’ s Disease.
[0072] Aspect 25. The method of aspect 17, wherein the protein aggregate comprises ataxin-3 (ATXN3), and wherein the disease is spinocerebellar ataxia type 3.
EXAMPLES
[0073] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like. Example 1
ASSAYS
FP assay:
[0074] A fluorescence polarization (FP) assay used to identify compounds that increase p97 complex binding to linear ubiquitin was set up as follows. p97-UFDl/NPL4/FAFl protein complex was first generated by combining 10 mM His-FLAG-p97, 5 mM FAFl-His, and 1.6 mM His-UFD1/NPL4 in FP buffer containing 20 mM HEPES pH 7.4, 150 mM KC1, ImM MgCF, and 0.2% (v/v) NP-40 (non-ionic detergent). An equal volume of p97 complex was then combined with an equal volume of 60 nM Ml -linked linear tetra-Ub conjugated to the dye ATTO-550 also diluted in FP buffer and added to either DMSO or test compounds 30 min before a FP reading was taken on a Perkin Elmer EnVision® Plate Reader. Immunoprecipitation (IP) assay:
[0075] To determine if the p97 complex could bind branched or aggregated Huntingtin protein from cell extracts, an immunoprecipitation experiment was carried out as follows: Cell extracts from Htt-73Q-GFP (Huntingtin protein with 73 Gin repeats fused to green fluorescent protein (GFP)) expressing H4 cells were generated by growing up five 15 cm plates treated with either dimethyl sulfoxide (DMSO) or 10 uM Mgl32 for 4 hours. Cells were then isolated from plates, washed once in phosphate-buffered saline (PBS), and then lysed in 1 mL buffer containing 50 mM HEPES pH 7.5, 1.5 mM MgCl2, 5 mM KC1, 150 mM NaCl + 0.1% NP-40, protease inhibitor, 10 pM carfilzomib and 5 pL benzonase. The cell extract was cleared at top speed on a table-top centrifuge to remove insoluble debris. Cleared cell extract was then transferred to new tubes and incubated with 8 pg of His-FLAG-p97, 8 pg Fafl-His, 16 pg of His-Ufd-1/Npl4, and 10 pL v/v slurry anti-FLAG magnetic beads in the presence and absence of molecular glues for 1 hour at 4C. The beads were then washed with ImL buffer containing 20 mM HEPES pH 7.4, 150 mM KC1, ImM MgCh, and 0.05% (v/v) NP-40 three times and p97 complex bound to beads was eluted in 25 pL of 2x Urea loading buffer. 15 pL of sample was run on a 4-12% Bis-Tris sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to nitrocellulose. Branched ubiquitin (Ub) was detected with anti-Kl 1/K48 branched Ub antibody, Htt-73Q was detected with an anti-GFP antibody and tubulin was detected with an anti-tubulin antibody.
RESULTS
[0076] From a library of > 100,000 compounds, molecules were identified, using the FP assay described above, that increase the affinity of a p97/VCP-adaptor complex to KI l/K48-branched ubiquitin. The molecules are depicted in FIG. 5. [0077] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMS What is claimed is:
1. A method of disrupting a protein aggregate modified with branched polyubiquitin, the method comprising contacting the protein aggregate with an agent that promotes binding of a p97/VCP polypeptide to the protein aggregate, wherein catalytic action of the p97/VCP polypeptide on the protein aggregate disrupts the protein aggregate.
2. The method of claim 1, wherein the branched polyubiquitin is KI 1/K48, K29/K48, K48/K6, K6/K11, K6/K48, K27/K29, or K29/K33 branched ubiquitin.
3. The method of claim 1 or claim 2, wherein the p97/VCP is present in a complex with one or more adaptor proteins.
4. The method of claim 3, wherein the one or more adaptor proteins are selected from NPL4, UFD1, FAF1, PLAA/Ufd3, PNGase, H01P, Ufd2, UBXN1/SAKS1, UBXN2A/UBXD4, UBXN2B/p37, UBXN2C/p47, UBXN3B/UBXD8/FAF2/ETEA, UBXN4/UBXD2/Erasin, UBXN6/UBXD1, UBXN7/UBXD7, UBXN8/UBXD6, UBXN9/UBXD9/ASPSCR1, UBXN10/UBXD3, UBXN11/UBXD5, OTU1/YOD1, VCPIP/VCIP135, VIMP, gp78/AMFR, SVIP, ZFAND2B/AIRAPL, ANKZF1, Hrdl/SYVVNl, Ataxin3/MJD/SCA3, UBE4B/Ufd2, NUB1/NYREN18, RHBDD1/RHBDL4, Derlinl, Derlin2, and SPRTN/DVCl/Clorfl24.
5. The method of claim any one of claims 1 -4, wherein the protein aggregate is present in a neuron.
6. The method of any one of claims 1-4, wherein the protein aggregate is present in a cardiac cell.
7. The method of any one of claims 1-4, wherein the protein aggregate is present in a pancreatic beta islet cell.
8. The method of any one of claims 1-7, wherein the agent is: i) a compound of FIG. 5, or an analog or derivative thereof; or ii) a compound of FIG. 6, or an analog or derivative thereof.
9. A method of reducing the amount of protein aggregate modified with branched polyubiquitin in a cell, the method comprising contacting the cell with an agent that promotes binding of a p97/VCP polypeptide to the protein aggregate, wherein catalytic action of the p97/VCP polypeptide on the protein aggregate results in a reduction in the amount of the protein aggregate in the cell.
10. The method of claim 9, wherein the cell is a neuron.
11. The method of claim 9, wherein the cell is a pancreatic beta islet cell.
12. The method of claim 9, wherein the cell is a cardiomyocyte.
13. The method of any one of claims 9-12, wherein the branched polyubiquitin is a KI 1/K48 branched polyubiquitin.
14. The method of any one of claims 9-13, wherein the agent is: i) a compound of FIG. 5, or an analog or derivative thereof; or ii) a compound of FIG. 6, or an analog or derivative thereof.
15. A method of treating a disease associated with a protein aggregate modified with branched polyubiquitin, the method comprising administering to an individual having such a disorder an effective an agent that promotes binding of a p97/VCP polypeptide to the protein aggregate, wherein catalytic action of the p97/V CP polypeptide on the protein aggiegate disrupts the protein aggregate and treats the disease.
16. The method of claim 15, wherein the branched polyubiquitin is K1 1/K48, K29/K48, K48/K6, K6/K11 , K6/K48, K27/K29, or K29/K33 branched ubiquitin.
17. The method of claim 15 or claim 16, wherein the disease is a neurodegenerative disease.
18. The method of claim 17, wherein the neurodegenerative disease is Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis, frontotemporal dementia, Creutzfeldt -Jacob disease, spinocerebellar ataxia type 3, or Lewy body dementia.
19. The method of claim 15, wherein the disease is type 2 diabetes.
20. The method of claim 15, wherein the disease is amyloid transthyretin cardiomyopathy.
21. The method of claim 17, wherein the protein aggregate comprises a pathological form of poly(glutamine) huntingtin protein, and where the disease is Huntington’ s Disease.
22. The method of claim 17, wherein the protein aggregate comprises alpha-synuclein, and wherein the disease is Parkinson’s Disease.
23. The method of claim 17, wherein the protein aggregate comprises TAR DNA-binding protein-43 (TDP43), and wherein the disease is amyotrophic lateral sclerosis.
24. The method of claim 17, wherein the protein aggregate comprises tau protein, and wherein the disease is Alzheimer’s Disease.
25. The method of claim 17, wherein the protein aggregate comprises ataxin-3 (ATXN3), and wherein the disease is spinocerebellar' ataxia.
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