WO2020182143A1 - 一种筛选用于治疗或预防polyQ相关的神经退行性疾病的化合物的方法 - Google Patents

一种筛选用于治疗或预防polyQ相关的神经退行性疾病的化合物的方法 Download PDF

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WO2020182143A1
WO2020182143A1 PCT/CN2020/078775 CN2020078775W WO2020182143A1 WO 2020182143 A1 WO2020182143 A1 WO 2020182143A1 CN 2020078775 W CN2020078775 W CN 2020078775W WO 2020182143 A1 WO2020182143 A1 WO 2020182143A1
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polyq
protein
compound
fragment containing
length
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PCT/CN2020/078775
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French (fr)
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鲁伯埙
费义艳
丁滪
党永军
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复旦大学
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Priority claimed from CN201910180717.1A external-priority patent/CN111679082A/zh
Priority claimed from CN201911000415.8A external-priority patent/CN112763718A/zh
Application filed by 复旦大学 filed Critical 复旦大学
Priority to CN202080020716.1A priority Critical patent/CN113711045A/zh
Priority to JP2021555238A priority patent/JP2022525325A/ja
Priority to EP20769239.3A priority patent/EP3940384A4/en
Priority to US17/437,968 priority patent/US20220170930A1/en
Publication of WO2020182143A1 publication Critical patent/WO2020182143A1/zh

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    • G—PHYSICS
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    • 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
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
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    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • 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

  • the present invention relates to the field of biomedicine, and in particular to a method for screening or identifying compounds for the treatment or prevention of polyQ-related neurodegenerative diseases, as well as the compounds obtained by the method and uses.
  • Neurodegenerative disorders refer to diseases caused by the abnormal death of central neurons causing nervous system dysfunction. So far, there is no fundamental treatment that can slow down its development. Take Huntington’s disease (HD), the most common among them, as an example. This is a single-gene inherited disease.
  • HD Huntington
  • the mutation in the CAG repeat region of the exon1 exon of the HTT gene contained in the patient’s chromosome 4 results in a synthetic variant protein (mHTT).
  • the glutamine repeat region (polyQ) is amplified.
  • mHTT is susceptible to shearing, aggregation and toxicity, which eventually leads to specific neuron dysfunction and death.
  • mHTT wild-type HTT protein
  • the present invention provides a method for screening or identifying compounds for the treatment or prevention of polyQ-related neurodegenerative diseases, including
  • the present invention provides the use of a compound obtained by the method of the present invention or a pharmaceutical composition thereof in the preparation of a medicament for the treatment or prevention of polyQ-related neurodegenerative diseases, or in the preparation of a drug for detecting those who are considered suffering Use in diagnostic reagents or kits for subjects who are or are susceptible to polyQ-related neurodegenerative diseases.
  • the present invention provides a compound or a medicament thereof that positively regulates the binding between (1) polyQ abnormally amplified protein or a fragment containing polyQ portion and (2) LC3B protein or a homologue or a fragment thereof
  • the composition is used in the preparation of a medicine for treating or preventing polyQ-related neurodegenerative diseases.
  • Figure 1 Detection of the affinity activity of the compound in a system where both full-length HTT and LC3B are present.
  • FIG. 1 Effects of compounds on HTT levels of cortical neurons in Hdh Q140/Q7 mice.
  • FIG. 3 Effects of Compounds 1 and 2 on HTT levels of cortical neurons in Hdh Q7/Q7 mice.
  • FIG. 4 HTT levels in cortical neurons of Hdh Q140/Q7 mice after treatment with various antibodies.
  • Figure 5 Detection of N-terminal fragments of mHTT in Hdh Q140/Q7 mouse cortical neurons after treatment with antibodies MW1 and 3B5H10.
  • Figure 6 Test results of cortical neuron cell viability in Hdh Q140/Q7 mice after compound treatment.
  • Figure 7 The effect of the compound on the level of mHTT in primary fibroblasts of HD patients at a concentration of 100 nM.
  • Figure 8 The effect of the compound on the mHTT level of immortalized fibroblasts in HD patients.
  • Figure 9 Compound 4 reduces mHTT levels in fibroblasts of immortalized HD patients
  • Figure 10 Effects of compounds on mHTT levels of induced stem cells (iPSC) differentiated neurons in HD patients.
  • iPSC induced stem cells
  • Figure 11 Effects of compounds on apoptosis of neurons differentiated from induced stem cells (iPSC) in HD patients.
  • the ordinate spans 50 ⁇ M.
  • Figure 12 Effects of compounds on neuronal apoptosis induced by stem cell (iPSC) differentiation in HD patients.
  • I, II, III and IV represent compound 1, compound 2, compound 3, and compound 4 respectively.
  • Figure 13 Effects of compounds on mHTT levels in Huntington's disease fruit flies.
  • Figure 14 The effect of compounds on the survival rate of Huntington's disease fruit flies. Wherein I, II, III and IV represent compound 1, compound 2, compound 3, and compound 4 respectively.
  • Figure 15 Effects of compounds on the crawling ability of Huntington's disease fruit flies. Wherein I, II, III and IV represent compound 1, compound 2, compound 3, and compound 4 respectively.
  • Figure 16 The effect of intracerebroventricular injection of compounds on the levels of mHTT and wtHTT in the cortex of Huntington's disease mice.
  • Figure 17 The effect of intraperitoneal injection of compounds on the levels of mHTT and wtHTT in the cortex and striatum of Huntington's disease mice.
  • Figure 18 Detection of mHTT aggregates in the cortex of Huntington's disease mice after intraperitoneal injection of the compound.
  • Figure 19a and Figure 19b Effects of intraperitoneal injection of compounds on behavioral deficits in Huntington's disease mice.
  • Figure 20-1, Figure 20-2, Figure 20-3 and Figure 20-4 The compound does not affect the autophagy function of cultured Hdh Q140/Q7 mouse cortical neurons and the level of control protein.
  • Figure 21 Intraperitoneal injection of compound does not affect the level of SQSTM1/p62 in mouse cortex.
  • Figure 22 Compounds selectively enhance the binding between mHTT and LC3B.
  • Figure 23 Co-localization of mHTT and LC3B in Hela cells under the influence of compounds.
  • White arrows indicate representative colocalization points. Part of the image is zoomed in to show the colocalization points. The ordinate spans 10 ⁇ M.
  • Figure 24 Co-localization of mHTT and LC3B in STHdh Q111/Q111 cells under the influence of compounds.
  • Figure 25 Effect of compounds on the level of proteins containing glutamine repeat regions in HEK293T cells.
  • Figure 26 Effect of compounds on ATXN3 protein levels in fibroblasts of patients with spinocerebellar ataxia type 3.
  • Figure 27 The effect of compounds on the protein levels of mutant ATXN1 in HEK293T cells.
  • one (species) or more (species) can mean, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 (species) Or more (species).
  • any numerical value and any included range falling within the range are specifically disclosed.
  • each value range of the value disclosed herein should be understood to mean each value and range covered in a wider range.
  • the expression "ATXN1 with polyQ length ⁇ 40" may cover the case where polyQ length ⁇ 41, and may cover the case where polyQ length is 92, for example.
  • "ATXN2 with polyQ length ⁇ 33" can cover the case of polyQ length ⁇ 34.
  • "ATXN3 with polyQ length ⁇ 41” can cover the case where polyQ length ⁇ 62, and can cover the case where polyQ length is 74 or 68, for example.
  • ATXN3 with polyQ length ⁇ 41 can cover the case where the polyQ length is 27 or 28.
  • ATXN7 with polyQ length ⁇ 19 can cover the case of polyQ length ⁇ 38.
  • TCP with polyQ length ⁇ 44 can cover the case of polyQ length ⁇ 45.
  • ATN1 with polyQ length ⁇ 39 can cover the case of polyQ length ⁇ 49.
  • HTT with polyQ length ⁇ 36 can cover the case where the polyQ length is 47, 49, 55, 68, 72, 73, 111, 128, or 140.
  • “HTT with polyQ length ⁇ 36” can cover the case where the polyQ length is 7, 16, 19, 23, or 25.
  • AR with polyQ length ⁇ 37 can cover the case of polyQ length ⁇ 38.
  • neurodegenerative disease refers to a disease caused by the loss or pathology of neurons and/or their myelin sheaths. Characteristic pathological structures, such as insoluble aggregates of protein, can be observed in the brain neurons of patients with neurodegenerative diseases. Insoluble aggregates may produce cytotoxicity, which can lead to neuron loss and disease.
  • polyQ or "polyglutamine” refers to the glutamine repeat region in a protein. Glutamine is encoded by cytosine-adenine-guanine (CAG) in the gene. The length of the glutamine repeat region is related to the number of CAG repeats in gene exons. Therefore, the increase in the number of CAG repeats in gene exons will This results in the amplification of the synthesized protein glutamine repeat region. It is known that abnormally amplified polyQ protein is associated with some neurodegenerative diseases.
  • the number of CAG repeats in an exon can be expressed in the form of "Q+number" in a gene name, for example, Q25 or Q72, which respectively represent 25 repeats or 72 repeats of CAG in an exon.
  • the length of the glutamine repeat region can be expressed in the form of "Q+number” as above, for example, Q27 or Q73, which means that the length of the glutamine repeat region is 27 Q (glutamine) or 73 Q respectively.
  • the CAG repeats or glutamine repeats indicated in the form of "Q+number” herein are all continuous repeats. Unless otherwise specified, the length of polyQ herein refers to the length of the continuously repeated glutamine region.
  • polyQ-related neurodegenerative diseases refers to neurodegenerative diseases associated with abnormal expansion of polyQ, or neurodegenerative diseases that respond to protein levels containing amplified polyQ. It is a group of clinical and genetic heterogeneity Of neurodegenerative diseases.
  • Normal polyQ refers to a protein in a normal physiological state that has a length of less than a specific number of polyQ.
  • abnormal amplification of polyQ means that the polyQ length of the protein is greater than the normal length. For diseases or pathological conditions, polyQ length will be longer.
  • normal polyQ protein includes polyQ of normal length (ie its polyQ portion).
  • polyQ abnormally amplified protein includes polyQ of abnormal length (ie its polyQ portion).
  • a polyQ portion having a length of ⁇ 40, ⁇ 33, ⁇ 41, ⁇ 19, ⁇ 46, ⁇ 44, ⁇ 39, ⁇ 36, or ⁇ 37.
  • the length of a fragment of a normal or abnormal polyQ protein can be 1% or higher, 2% or higher, 3% or higher, 4% or higher, 5% or higher, 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 50% or higher, 60% Or higher, 70% or higher, 80% or higher, 90% or higher, or 95% or higher, but containing a complete polyQ portion of normal or abnormal length.
  • polyQ when used in the methods of the present invention, particularly high-throughput methods, polyQ can be used to amplify fragments of proteins abnormally. It contains the polyQ part of the protein, and the length can be only 1% or higher, 2% or higher, 3% or higher, 4% or higher of the corresponding protein.
  • the "polyQ abnormally amplified protein" or a fragment thereof used may comprise the amino acid sequence of SEQ ID No: 1, 2 or 3.
  • the "normal polyQ protein” or a fragment thereof used may comprise the amino acid sequence of SEQ ID No: 4 or 5.
  • the "normal polyQ protein” or a fragment thereof used may include the amino acid sequence of SEQ ID No: 7.
  • the "normal polyQ protein” or a fragment thereof used may comprise the amino acid sequence of SEQ ID No: 6.
  • Normal polyQ protein or “polyQ abnormally amplified protein” may be a naturally occurring protein that contains a polyQ portion of normal or abnormal length, for example, it may be from humans (for example, ATXN1, ATXN2, ATXN3, ATXN7, ATXN12, TBP, ATN1 , HTT or AR) or from other animals, such as from mice (such as mouse HTT protein, whose polyQ length can be, for example, 140 or 111), or from insects, fish, rodents, cloven ungulates, and primates.
  • humans for example, ATXN1, ATXN2, ATXN3, ATXN7, ATXN12, TBP, ATN1 , HTT or AR
  • mice such as mouse HTT protein, whose polyQ length can be, for example, 140 or 111
  • insects, fish, rodents, cloven ungulates, and primates for example, insects, fish, rodents, cloven ungulates, and primates.
  • Normal polyQ protein or “polyQ abnormally amplified protein” can also be artificially engineered or modified proteins, but still have their corresponding polyQ parts. Such transformation or modification can be for processing, purification, characterization, tracing and other purposes, such as adding MBP tags or His tags or fusing with green fluorescent protein, or introducing various amino acid substitutions, additions or deletions. Such techniques are well known to those skilled in the art.
  • a protein contains the corresponding normal or abnormal polyQ part, and has 15% or higher, 20% or higher, 25% or higher, 30% or higher with the corresponding normal or abnormal polyQ amplified protein.
  • Such a protein can be cut or spliced from the full length or fragments of the corresponding normal or abnormal polyQ amplified protein, and optionally includes modification or modification, or it can be designed de novo.
  • a protein containing a longer glutamine repeat region refers to a longer glutamine repeat region in a specific protein.
  • longer glutamine repeat region can refer to "abnormal amplification of polyQ".
  • protein containing longer glutamine repeat region can be referred to as described herein.
  • the "polyQ abnormally amplified protein” has the same meaning. However, in other cases, the polyQ length of the "longer glutamine repeat region” can be satisfied at the same time: (1) For some specific diseases, such polyQ is larger than the normal length, which is abnormal amplification; (2) For other specific diseases, such polyQ lengths are normal lengths and are not abnormal amplifications.
  • Such a "protein containing a longer glutamine repeat region” can also be understood as a “polyQ abnormally amplified protein", which falls within the scope of the present invention and can be used by the skilled person according to actual needs.
  • polyQ-related neurodegenerative diseases include but are not limited to spinocerebellar ataxia (SCA) type 1 (polyQ length ⁇ 41), type 2 (polyQ length ⁇ 34), and type 3 (polyQ length ⁇ 62 ), type 7 (polyQ length ⁇ 38), type 12 (polyQ length ⁇ 46), type 17 (polyQ length ⁇ 45); and dentatorubral atrophy (dentatorubral)-substantia nigra-red nucleus-globus pallidus -Pallidoluysian atrophy, DRPLA, polyQ length ⁇ 49), Huntington's Disease (Huntington's Disease, HD, polyQ length ⁇ 36) and spinal-bulbar muscular atrophy (SBMA, polyQ length ⁇ 38).
  • SCA spinocerebellar ataxia
  • type 1 polyQ length ⁇ 41
  • type 2 polyQ length ⁇ 34
  • type 3 polyQ length ⁇ 62
  • type 7 polyQ length ⁇ 38
  • spinocerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease, MJD) is the most common autosomal dominant spinocerebellar ataxia and common polyQ-related disease second only to HD.
  • ATXN3 also known as MJD1 gene
  • ATXN3 also known as MJD1 gene
  • normal polyQ proteins described herein include, but are not limited to, ATXN1 with polyQ length ⁇ 40, ATXN2 with polyQ length ⁇ 33, ATXN3 with polyQ length ⁇ 41, ATXN7 with polyQ length ⁇ 19, ATXN12 with polyQ length ⁇ 46, polyQ length TBP ⁇ 44, ATN1 with polyQ length ⁇ 39, HTT with polyQ length ⁇ 36, and AR with polyQ length ⁇ 37.
  • examples of the abnormally amplified polyQ proteins described herein include, but are not limited to, ATXN1 with a polyQ length ⁇ 40, ATXN with a polyQ length ⁇ 33, ATXN3 with a polyQ length ⁇ 41, ATXN7 with a polyQ length ⁇ 19, and polyQ length ATXN12 with ⁇ 46, TBP with polyQ length ⁇ 44, ATN1 with polyQ length ⁇ 39, HTT with polyQ length ⁇ 36, and AR with polyQ length ⁇ 37.
  • MAP1LC3B Human microtubule-associated protein 1 light chain 3 ⁇
  • LC3B microtubule-associated protein 1 light chain 3
  • Atg8 protein family which is located on the membranes of pro-autophagosomes and autophagosomes, and can Controlling the formation of autophagosomes is a key protein in the process of autophagy.
  • LC3B protein that can be used includes, for example, LC3B-I and LC3B-II, but it is not limited thereto.
  • LC3B protein for example, see Uniprot Accession: Q9GZQ8.
  • homologues of the LC3B protein can be used, as long as it can be used in the method of the present invention, for example, interacting with a polyQ abnormally amplified protein or a fragment containing a polyQ portion.
  • the homologs of the LC3B protein used herein can be derived from eukaryotes, such as yeast or other non-human animals, such as insects (such as Drosophila), fish, rodents, artiodactyls, primates, etc. Homologs of the LC3B protein can also be derived from other proteins with similar structures and functions, such as LC3A, LC3C, GABARAP and GABARAPL1, but are not limited thereto.
  • LC3A Uniprot Accession: Q9H492-1 and Q9H492-2
  • LC3C Uniprot Accession: Q9BXW4
  • GABARAP Uniprot Accession: O95166
  • GABARAPL1 Uniprot Accession: Q9H0R8-1 and Q9H0R8-2
  • LC3B protein or fragments of homologs thereof can also be used, as long as it can be used in the method of the present invention, for example, interacting with polyQ abnormally amplified protein or fragments containing polyQ portions.
  • Such fragments may have 25% or higher, 30% or higher, 35% or higher, 40% or higher, 50% or higher, 60% or higher with LC3B protein or its homologue. , 70% or higher, 80% or higher, 90% or higher, 95% or higher, or 100% identity.
  • a protein having the same or homology with the LC3B protein or a homologue thereof or a fragment thereof can be used to achieve the purpose of the present invention, as long as it can be used in the method of the present invention.
  • such interactions with polyQ abnormally amplified proteins or fragments containing polyQ portions may be 25% or higher, 30% or higher, 35% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 90% or higher, 95% or higher, or 100%.
  • the LC3B protein or homologues or fragments thereof have 25% or higher, 30% or higher, 35% or higher, 40% or higher with the amino acid sequence of SEQ ID NO: 8 High, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 90% or higher, 95% or higher, or 100% sequence identity.
  • SEQ ID NO: 8 High, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 90% or higher, 95% or higher, or 100% sequence identity.
  • LC3B protein or its homologue or its fragments and/or the mature form of the polyQ abnormally amplified protein or its fragment containing the polyQ portion can be used.
  • the C-terminal amino acid 121-125 of the LC3B protein such as SEQ ID NO: 8 can be excised to obtain its mature form.
  • LC3B protein or its homologues or their fragments and/or polyQ abnormally amplified protein or its polyQ-containing fragments can be modified or transformed according to actual needs. invention. Such a solution is also included in the scope of the present invention.
  • modification or transformation may include, but is not limited to, adding tags (such as GST or HIS) or labeling, or substituting, deleting, adding or replacing partial amino acids, for example.
  • tags such as GST or HIS
  • a small amount of modification or modification can be made to the end of the sequence of SEQ ID NO: 8, for example, the M at the N-terminus of SEQ ID NO: 8 can be replaced with GG, and then the modified or unmodified can be optionally changed.
  • modified sequence adds a tag such as GST to be used in the present invention.
  • modified or modified sequence may have 25% or higher, 30% or higher, 35% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher Schemes corresponding to sequences with higher, 80% or higher, 90% or higher, 95% or higher or 100% sequence identity are also within the scope of the present invention.
  • affinity screening is a process of detecting the affinity binding between a sample and a target.
  • the detection methods used for affinity screening can be, for example, absorbance, radiation (such as proximity scintillation analysis), fluorescence (such as fluorescence resonance energy transfer, fluorescence polarization detection, especially time-dependent fluorescence technology), chemiluminescence (such as amplification Chemiluminescence affinity homogeneous detection, ALPHAScreen), surface plasmon resonance (SPR, for example, using GE's Biacore series), isothermal titration calorimetry (ITC), micro thermophoresis (MST), or oblique incident light reflection Difference method.
  • radiation such as proximity scintillation analysis
  • fluorescence such as fluorescence resonance energy transfer, fluorescence polarization detection, especially time-dependent fluorescence technology
  • chemiluminescence such as amplification
  • SPR surface plasmon resonance
  • ITC isothermal titration calorimetry
  • MST micro
  • sequence identity between two amino acid sequences means the percentage of amino acids that are identical between the sequences.
  • Sequence homology means the percentage of amino acids that are identical or represent conservative amino acid substitutions.
  • sequence comparison usually a sequence is used as a reference sequence to compare the test sequence with.
  • sequence comparison algorithm input the test and reference sequences into the computer, if necessary, specify the sub-sequence coordinates, and specify the sequence algorithm program parameters. Based on the specified program parameters, the sequence comparison algorithm calculates the percent sequence identity of the test sequence relative to the reference sequence. Examples of algorithms suitable for determining sequence identity and sequence similarity percentage include but are not limited to BLAST and BLAST 2.0 algorithms. Software to perform BLAST analysis is available from the National Center for Biotechnology Information (NCBI).
  • target sample includes various sample types that are obtained from a subject and can be used for diagnostic analysis.
  • the target sample herein can be, for example, any cell sample, biological fluid (including blood, serum, spinal fluid, etc.) or any biopsy sample obtained from the tissue of the subject.
  • the concept of the present invention is based at least in part on the discovery of the following new mechanism: a compound that can bind to the key protein LC3B in the autophagy process and the abnormally amplified polyQ protein can promote the degradation of abnormally amplified polyQ protein through autophagy, So as to achieve the purpose of reducing the level of abnormal polyQ amplified protein and treating or preventing corresponding diseases.
  • the present invention provides a method for screening or identifying compounds for the treatment or prevention of polyQ-related neurodegenerative diseases, including
  • the system includes, but is not limited to, a solution system, a subcellular system, a cell (cell culture) system, a tissue system, an organ system, or an animal system.
  • candidate compounds can be provided in the form of a compound library.
  • libraries can be commercially available or synthesized by design.
  • Candidate compounds can include peptides, peptidomimetics, and small organic molecules. For example, it is selected from a compound designed and synthesized, a compound with a determined structure in a database (such as Pubmed), or a compound synthesized de novo. In a specific embodiment, the candidate compound is selected from small organic molecules.
  • small organic molecule or “low molecular weight compound” refers to a molecule that is comparable in size to organic molecules commonly used in drugs.
  • the size of the small organic molecule is about 100 to about 2000 Da, preferably about 200 to about 1000 Da, such as about 200 to about 900 Da, about 200 to about 800 Da, about 200 to about 700 Da, and about 200 Da. About 600 Da, about 200 to about 500 Da.
  • the polyQ abnormally amplified protein or its fragment containing polyQ part used in the method of the present invention is determined by the targeted polyQ-related neurodegenerative diseases.
  • the abnormally amplified polyQ protein comprises the length of polyQ ⁇ 40, ⁇ 33, ⁇ 41, ⁇ 19, ⁇ 46, ⁇ 44, ⁇ 39, ⁇ 36 or ⁇ 37 polyQ part.
  • the polyQ abnormally amplified protein comprises ATXN1 with a polyQ length ⁇ 40 or a fragment containing a polyQ portion.
  • the polyQ abnormally amplified protein comprises ATXN2 with a polyQ length ⁇ 33 or a fragment containing a polyQ portion.
  • the polyQ abnormally amplified protein comprises ATXN3 with a polyQ length ⁇ 41 or a fragment containing a polyQ portion. In another preferred embodiment, the polyQ abnormally amplified protein comprises ATXN7 with a polyQ length ⁇ 19 or a fragment containing a polyQ portion. In a preferred embodiment, the polyQ abnormally amplified protein comprises ATXN12 with a polyQ length ⁇ 46 or a fragment containing a polyQ portion. In another preferred embodiment, the polyQ abnormally amplified protein comprises TBP with a polyQ length ⁇ 44 or a fragment containing a polyQ portion.
  • the polyQ abnormally amplified protein comprises ATN1 with a polyQ length ⁇ 39 or a fragment containing a polyQ portion. In a preferred embodiment, the polyQ abnormally amplified protein comprises HTT with a polyQ length ⁇ 36 or a fragment containing a polyQ portion. In another preferred embodiment, the polyQ abnormally amplified protein comprises an AR with a polyQ length ⁇ 37 or a fragment containing a polyQ portion.
  • the "positive regulation" of binding refers to the determination under the same conditions. Compared with the absence of the compound, when the compound is present, the binding strength between the proteins or fragments involved in the binding becomes greater, or the binding amount becomes greater .
  • the positive modulation of the binding by a compound can be measured at the molecular level, for example, the increase in binding affinity between proteins or fragments thereof in the presence of the compound can be measured. Positive regulation can also be verified by separately measuring the binding strength (such as affinity activity) of the compound to the protein or fragments involved in the binding. If the compound has affinity for the protein or fragments involved in the binding, but does not have or has a lower affinity for the corresponding reference, it can be determined as a positive regulation.
  • step (II) is implemented in the following manner: respectively in the presence/absence of the candidate compound, the characterization of (1) polyQ abnormally amplified protein or a fragment containing polyQ portion and (2) LC3B protein Or its homologues or their fragments, and compare the parameters obtained in the same test in the presence/absence of candidate compounds, and select positive regulation (1) polyQ abnormally amplified protein or It is a compound that combines a fragment of the polyQ part with (2) LC3B protein or its homologue or fragment thereof.
  • the aforementioned “parameters” can be various, but in particular can be, for example, absorbance value, radioactive signal and/or distribution in the sample, fluorescence signal intensity and/or distribution in the sample , Heat change, reflected light intensity, reflected light phase change, etc.
  • step (II) includes : Determine the binding affinity between (1) and (2) in the presence/absence of the candidate compound, and/or, in the presence/absence of the candidate compound, determine ( 1) The binding amount with (2); compare the measured values obtained in the presence/absence of the candidate compound, and if there is a positive change in the binding between the above-mentioned proteins or fragments when the candidate compound is present, select the candidate Compound.
  • the method for determining the binding between proteins or fragments in step (II) is selected from the group consisting of pull-down assay, Split-TEV, co-immunoprecipitation (co-IP), affinity Chromatography (AP), complex co-purification (Copurification), enzyme-linked immunosorbent assay (ELISA), fluorescent molecular sieve (Fluorescence-SEC), yeast two-hybrid (Y2H), HIP-HOP (haploinsufficiency profiling and Homozygous deletion profiling method, time-resolved fluorescence resonance energy transfer (TR-FRET), chemiluminescence method, surface plasmon resonance (surface plasmon resonance, SPR), isothermal titration calorimetry, microthermophoresis and any combination thereof.
  • pull-down assay split-TEV
  • co-IP co-immunoprecipitation
  • AP affinity Chromatography
  • Copurification complex co-purification
  • ELISA enzyme-linked immunosorbent
  • the method for determining the binding is selected from the group consisting of an in vitro pull-down test, co-immunoprecipitation, enzyme-linked immunosorbent assay, fluorescent molecular sieve, time-resolved fluorescence resonance energy transfer, and any combination thereof.
  • step (I) before step (I), the step of pre-screening the candidate compound for affinity activity is further included; including:
  • step (A) and step (B) can be performed independently in any order.
  • step (A) and step (B) can be carried out in different systems at the same time. You can perform step (A) first, then step (B), and vice versa.
  • a compound capable of affinity binding to (1) polyQ abnormally amplified protein or its fragment containing polyQ part and (2) LC3B protein or its homologue or their fragments is selected.
  • step (A) and step (B) may be performed independently or not, or may be performed independently at least once (for example, once, twice, three times or more independently). Such methods can be independently qualitative or quantitative as required.
  • step (A) may include: determining the equilibrium dissociation constant (K d ) of the affinity reaction between the candidate compound and (1) polyQ abnormally amplified protein or a fragment containing polyQ part, and selecting the affinity A compound with a reaction equilibrium dissociation constant of 100 ⁇ M or less is preferably 10 ⁇ M or less, particularly preferably 1 ⁇ M or less, for example, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, etc.
  • step (B) may include: determining the affinity reaction equilibrium dissociation constant of the candidate compound with (2) LC3B protein or its homologues or fragments thereof, and selecting the affinity reaction equilibrium dissociation constant
  • the compound is 100 ⁇ M or less, preferably 10 ⁇ M or less, particularly preferably 1 ⁇ M or less, for example, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, etc.
  • step (A) and step (B) are carried out in the same system at the same time, that is, the candidate compound is simultaneously performed with (1) polyQ abnormally amplified protein or a fragment containing polyQ part and (2) LC3B protein Contact with the homologues or fragments thereof to detect the affinity binding of the candidate compound to the protein or fragments thereof.
  • the method used in step (A) or step (B) to determine affinity binding is selected from the group consisting of proximity scintillation analysis, fluorescence resonance energy transfer, fluorescence polarization detection, fluorescent molecular sieves, microthermophoresis, chemiluminescence, Surface plasmon resonance, isothermal titration calorimetry, oblique incident light reflection difference method and any combination thereof.
  • the method for determining affinity binding in step (A) or step (B) is selected from the group consisting of fluorescence resonance energy transfer, fluorescence polarization detection, oblique incident light reflectance method, and any combination thereof.
  • the method for determining the affinity binding in step (A) or step (B) is the oblique incident light reflectance method.
  • the step of pre-screening candidate compounds for affinity activity is performed by high-throughput screening using the oblique incident light reflectance method; including:
  • the target protein is (1) polyQ abnormally amplified protein or a fragment containing polyQ part or (2) LC3B protein or its homologue or their fragment;
  • step (A) or step (B) to determine the equilibrium dissociation constant is selected from surface plasmon resonance, isothermal titration calorimetry and microthermophoresis.
  • the method of the present invention may further include step (D): determining the binding of the candidate compound to the normal polyQ protein or a fragment containing a polyQ portion. This step may be performed after step (II) or after step (C), for example.
  • the binding is affinity binding.
  • a compound is selected that does not bind affinity to the normal polyQ protein or a fragment containing a polyQ moiety.
  • Step (D) can be carried out or not carried out as needed, and can also be carried out at least once (for example, once, twice, three times or more).
  • the normal polyQ polypeptide used each time may independently be a normal polyQ protein or a fragment containing a polyQ portion.
  • the normal polyQ protein used in step (D) is the normal polyQ protein corresponding to the abnormally amplified polyQ protein used in step (I).
  • the polyQ abnormally amplified protein used in step (I) comprises polyQ abnormally amplified HTT or a fragment containing a polyQ portion.
  • the normal polyQ protein used therein includes normal HTT or a part containing polyQ.
  • the abnormally amplified polyQ protein used in step (I) is an abnormally amplified polyQ HTT or a fragment containing a polyQ portion, for example, an HTT with a polyQ length ⁇ 36 or a polyQ portion containing HTT.
  • the normal polyQ protein used is HTT with normal polyQ length or a fragment containing polyQ part, for example, HTT with polyQ length ⁇ 36 or a fragment containing polyQ part, such as SEQ The amino acid sequence of ID NO: 4 (HTT-Q23) or the amino acid sequence of SEQ ID NO: 5 (HTTexon1-Q25).
  • the polyQ abnormally amplified protein used in step (I) comprises polyQ abnormally amplified ATXN3 or a fragment containing a polyQ portion.
  • the normal polyQ protein used therein includes normal ATXN3 or a part containing polyQ.
  • the abnormally amplified polyQ protein used in step (I) is abnormally amplified polyQ ATXN3 or a fragment containing a polyQ portion, for example, ATXN3 with a length of polyQ ⁇ 41 or a polyQ portion containing ATXN3 Fragments, such as the amino acid sequence of SEQ ID NO: 2 (ATXN3-Q74).
  • the normal polyQ protein used is ATXN3 with normal polyQ length or a fragment containing polyQ part, for example, ATXN3 with polyQ length ⁇ 41 or a fragment containing polyQ part, such as SEQ ID NO: 7 amino acid sequence (ATXN3-Q27).
  • the polyQ abnormally amplified protein used in step (I) comprises polyQ abnormally amplified ATXN1 or a fragment containing a polyQ portion.
  • the normal polyQ protein used therein includes normal ATXN1 or a part containing polyQ.
  • the abnormally amplified polyQ protein used in step (I) is abnormally amplified polyQ ATXN1 or a fragment containing a polyQ portion, for example, ATXN1 with a polyQ length ⁇ 40 or a polyQ portion containing ATXN1 Fragments, such as the amino acid sequence of SEQ ID NO: 3 (ATXN1-Q92).
  • the normal polyQ protein used is ATXN1 with normal polyQ length or a fragment containing polyQ part, for example, ATXN1 with polyQ length ⁇ 40 or a fragment containing polyQ part.
  • the polyQ abnormally amplified protein used in step (I) comprises polyQ of abnormal length, which is labeled with a detectable molecule.
  • the abnormally amplified polyQ protein used in step (I) is a fusion protein of an abnormally long polyQ and a fluorescent protein (such as GFP or YFP).
  • the abnormally amplified polyQ protein used in step (I) is a polyQ-GFP protein containing polyQ of abnormal length, preferably selected from Q72-GFP, Q53-GFP, and Q46-GFP. According to the specific disease screened for, in an alternative embodiment, the polyQ abnormally amplified protein used in step (I) is Q38-GFP.
  • the normal polyQ protein used in step (D) contains polyQ of normal length, which is labeled with a detectable molecule.
  • the normal polyQ protein used in step (D) is a fusion protein of normal-length polyQ and a fluorescent protein (such as GFP or YFP).
  • the normal polyQ protein used in step (D) is a polyQ-GFP protein containing polyQ of normal length, preferably Q25-GFP. According to the specific disease screened for, in an alternative embodiment, the normal polyQ protein used in step (D) is Q38-GFP.
  • the LC3B protein or homologues or fragments thereof have 25% or higher, 30% or higher, 35% or higher, 40% or higher with the amino acid sequence of SEQ ID NO: 8 , 50% or higher, 60% or higher, 70% or higher, 80% or higher, 90% or higher, 95% or higher, or 100% sequence identity.
  • the LC3B protein homologue is from Drosophila or mouse.
  • the homolog of the LC3B protein is the Atg8 protein in Drosophila (31% sequence identity with human LC3B).
  • the present invention also provides a method for screening or identifying compounds for the treatment or prevention of polyQ-related neurodegenerative diseases, including
  • polyQ abnormally amplified protein its fragments containing polyQ part, and LC3B protein are as defined above.
  • step (1) selects a compound that can reduce the level of abnormally amplified polyQ protein in the cell by more than 10%, preferably more than 20%, such as about 20%, about 30%, about 40%, about 50%. %,Wait.
  • polypeptide refers to a polymer of amino acids of a certain length. Therefore, peptides, oligopeptides and proteins are included in the definition of “polypeptide”, and these terms are used interchangeably herein.
  • polypeptide or protein does not exclude post-translational modifications, which include but are not limited to phosphorylation, acetylation, glycosylation and the like.
  • the protein or protein fragment of the present invention can be produced by any technique known in the art per se, such as but not limited to any chemical, biological, genetic or enzymatic technique used alone or in combination.
  • those skilled in the art can easily prepare the protein or protein fragment by standard techniques for producing the protein or protein fragment. For example, they can be synthesized by using a well-known solid phase method, preferably using commercially available peptide synthesis equipment (such as those prepared by Applied Biosystems, Foster City, California) and according to the manufacturer's instructions.
  • the protein or protein fragment of the present invention can be synthesized by recombinant DNA technology well known in the art.
  • DNA sequence encoding the desired (poly)peptide is introduced into an expression vector and this vector is introduced into an appropriate eukaryotic or prokaryotic host for expressing the desired protein or protein fragment, these fragments can be obtained as DNA expression products, and then They can be separated from the host using well-known techniques.
  • Suitable vectors include, but are not limited to, derivatives of SV40 and pcDNA; and known bacterial plasmids, such as col EI, pCR1, pBR322, pMal-C2, pET, pGEX, pMB9 and their derivatives; plasmids, such as RP4; phage DNA , Such as numerous derivatives of phage I, such as NM989, and other phage DNA, such as M13 and filamentous single-stranded phage DNA; yeast plasmids, such as 2 micron plasmids or derivatives of 2 micron plasmids, and centromeres and integration Yeast shuttle vectors; vectors used in eukaryotic cells, such as vectors that can be
  • mammalian and typical human cells as well as bacterial, yeast, fungal, insect, nematode and plant cells can be used in the present invention, and can be transfected by the nucleic acid or recombinant vector as defined herein.
  • suitable cells include, but are not limited to, VERO cells; HELA cells, such as ATCC No. CCL2; CHO cell lines, such as ATCC No. CCL61; COS cells, such as COS-7 cells and ATCC No. CRL 1650 cells; W138, BHK , HepG2, 3T3, such as ATCC No. CRL6361; A549, PC12, K562 cells, 293T cells, Sf9 cells, such as ATCC No.
  • CRL1711 and Cv1 cells such as ATCC No. CCL70.
  • suitable cells include, but are not limited to, prokaryotic host cell strains, such as Escherichia coli (eg strain DH5-[ ⁇ ]), Bacillus subtilis, Salmonella typhimurium or Pseudomonas, Streptomyces and Staphylococcus Strains of the genus.
  • suitable cells include yeast cells, such as Saccharomyces cells, such as Saccharomyces cerevisiae.
  • protein or protein fragment of the present invention can be modified or modified for purposes such as facilitating test operations, increasing solubility, facilitating crystallization and purification, but does not affect the function of the protein or protein fragment.
  • Such a protein Or protein fragments and their applications also belong to the scope of the present invention.
  • polyQ abnormally amplified protein or fragments containing polyQ moieties or (2) LC3B protein or homologues or fragments thereof can be labeled with detectable molecules for screening purposes.
  • the detectable molecule can be composed of any compound or substance that can be detected by spectroscopic, photochemical, biochemical, immunochemical or chemical means.
  • useful detectable molecules include radioactive substances (including those containing 32P, 25S, 3H or 125I), fluorescent dyes (including 5-bromodeoxyuridine, fluorescein, acetamidofluorene or digitoxin), fluorescent protein (For example, GFP and YFP, where GFP may be sfGFP, for example).
  • the detectable label is located or bound to an amino acid residue located outside the sequence of (1) polyQ abnormally amplified protein or a fragment containing a polyQ portion or (2) LC3B protein or a homologue or a fragment thereof. Basically, thereby minimizing or preventing any artificial influence on the binding between the protein or protein fragment or between the candidate compound and any of the protein or protein fragment.
  • the protein or protein fragment of the present invention is fused to a fluorescent protein such as a GFP tag (green fluorescent protein).
  • a fluorescent protein such as a GFP tag (green fluorescent protein).
  • the protein or protein fragment of the present invention is respectively labeled with a suitable fluorescent group in a manner suitable for fluorescence energy transfer analysis.
  • step (1) or step (2) of the screening method of the present invention the complete polyQ abnormal expansion Increased protein and intact LC3B protein or homologs thereof can be used in the assay.
  • fragments of polyQ abnormally amplified protein including binding sites and fragments of LC3B protein or homologs thereof can be used in the assay.
  • the present invention provides a screening system comprising two or more parts, wherein part (A) comprises unlabeled or labeled polyQ abnormally amplified protein or a fragment containing polyQ part, and (B The) part contains LC3B protein or its homologues or fragments thereof that are unlabeled or labeled in an appropriate form, and each part is independently isolated from each other or not. Each part independently optionally contains suitable buffers and/or detection reagents.
  • the screening system of the present invention is in the form of a kit.
  • the prepared compound chip is placed in the kit of the present invention, and (1) polyQ abnormally amplified protein or a fragment containing polyQ portion thereof and (2) LC3B protein or its homologue Or after incubating their fragments, use a suitable plate reader to analyze the signal and screen to obtain compounds that can bind to both (1) and (2).
  • the invention also relates to compounds obtained by the method of the invention.
  • the compounds of the present invention encompass pharmaceutically acceptable salts, stereoisomers, solvates, polymorphs, tautomers, isotopic compounds, metabolites or prodrugs thereof.
  • pharmaceutically acceptable refers to contact with the patient's tissue within the scope of normal medical judgment without undue toxicity, irritation, allergic reactions, etc.
  • the pharmaceutically acceptable salts of the compounds of the present invention include acid addition salts and base addition salts thereof. Methods for preparing pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art.
  • the compounds of the present invention may exist in specific geometric or stereoisomeric forms, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-pairs Enantiomers, diastereomers, (D)-isomers, (L)-isomers, and their racemic mixtures and other mixtures, such as enantiomers or diastereomers enriched Mixtures, all these mixtures are within the scope of the present invention.
  • the compound of the present invention may exist in the form of a solvate (preferably a hydrate), wherein the compound of the present invention contains a polar solvent as a structural element of the compound crystal lattice, in particular, for example, water, methanol or ethanol.
  • a polar solvent as a structural element of the compound crystal lattice, in particular, for example, water, methanol or ethanol.
  • the amount of polar solvents, especially water can be present in stoichiometric or non-stoichiometric ratios.
  • the present invention also covers all possible crystalline forms or polymorphs of the compounds of the present invention, which can be a single polymorph or a mixture of more than one polymorph in any ratio.
  • metabolites of the compounds of the present invention that is, substances formed in the body when the compounds of the present invention are administered.
  • Such products can be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc. of the administered compound.
  • the present invention further includes within its scope the prodrugs of the compounds of the present invention, which are certain derivatives of the compounds of the present invention that may themselves have less pharmacological activity or no pharmacological activity when administered to or on the body It can be converted into the compound of the present invention having the desired activity by, for example, hydrolytic cleavage.
  • polymorph or “polymorph” refers to a single polymorph or a mixture of more than one polymorph in any ratio.
  • crystalline form or "crystalline” refers to any solid substance exhibiting a three-dimensional order, as opposed to an amorphous solid substance, which produces a characteristic X-ray powder diffraction pattern with well-defined peaks.
  • amorphous refers to any solid substance that is unordered in three dimensions.
  • the compounds of the present invention can be applied in the form of pharmaceutical compositions.
  • the pharmaceutical composition comprises the compound or a pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, tautomer, isotopic compound, metabolite or prodrug thereof, and at least one pharmaceutical Acceptable carrier.
  • pharmaceutically acceptable carrier refers to those substances that have no obvious stimulating effect on organisms and do not impair the biological activity and performance of the active compound.
  • “Pharmaceutically acceptable carriers” include, but are not limited to, glidants, sweeteners, diluents, preservatives, dyes/colorants, flavors, surfactants, wetting agents, dispersants, disintegrants, Stabilizer, solvent or emulsifier.
  • the present invention provides the use of the compound of the present invention or a pharmaceutical composition thereof in the preparation of a medicament for the treatment or prevention of polyQ-related neurodegenerative diseases.
  • the present invention provides compounds of the present invention for use in the treatment or prevention of polyQ-related neurodegenerative diseases.
  • the present invention provides a method of treating or preventing polyQ-related neurodegenerative diseases, comprising administering a compound of the present invention to an individual in need thereof.
  • the present invention provides a compound or a medicament thereof that positively regulates the binding between (1) polyQ abnormally amplified protein or a fragment containing polyQ portion and (2) LC3B protein or a homologue or a fragment thereof
  • the composition is used in the preparation of a medicine for treating or preventing polyQ-related neurodegenerative diseases.
  • the present invention provides a compound or a medicament that positively regulates the binding between (1) polyQ abnormally amplified protein or a fragment containing a polyQ portion and (2) LC3B protein or a homologue or a fragment thereof
  • the composition is used to treat or prevent polyQ-related neurodegenerative diseases.
  • the present invention provides a method for treating or preventing polyQ-related neurodegenerative diseases, which comprises administering to an individual in need thereof a positive regulation (1) polyQ abnormally amplified protein or a fragment containing a polyQ portion and ( 2) Compounds or pharmaceutical compositions of LC3B protein or its homologs or fragments thereof.
  • a compound that positively modulates the binding between (1) a polyQ abnormally amplified protein or a fragment containing a polyQ portion and (2) an LC3B protein or a homologue or a fragment thereof can be obtained, for example, by the method of the present invention.
  • the polyQ-related neurodegenerative disease is selected from the group consisting of spinocerebellar ataxia type 1 (SCA1), spinocerebellar ataxia type 2 (SCA2), spinocerebellar ataxia type 3 (SCA3), Spinocerebellar ataxia type 7 (SCA7), spinocerebellar ataxia type 12 (SCA12), spinocerebellar ataxia type 17 (SCA17), dentate nucleus-substantia nigra-red nucleus-globus pallidus-subthalamic nucleus atrophy Disease (DRPLA), Huntington’s disease (HD) and spinal bulbar muscular atrophy (SBMA), especially Huntington’s disease and spinocerebellar ataxia type 3.
  • the polyQ-related neurodegenerative disease is selected from Huntington's disease and SCA3; especially Huntington's disease.
  • the present invention provides an article of manufacture, for example, in the form of a kit.
  • the article of the present invention contains the compound or pharmaceutical composition of the present invention, and optionally includes a packaging box and instructions.
  • the present invention provides the use of the compounds of the present invention for screening or identifying target proteins that can be degraded through the autophagy pathway.
  • the compound obtained by screening can be used to find the target protein by the following method. 1) Screen or identify the protein capable of binding to the compound; 2) Determine the ability of the compound to modulate the binding between the protein obtained in 1) and LC3B protein or its homologues or fragments thereof, where if The compound can positively regulate the binding, and the protein obtained in 1) can be degraded through the autophagy pathway.
  • the present invention provides the use of the compound of the present invention or the pharmaceutical composition thereof in the preparation of a diagnostic reagent or kit for detecting subjects who are considered to have or are susceptible to polyQ-related neurodegenerative diseases.
  • the aforementioned test includes the step of analyzing a target sample obtained from the subject, including:
  • the target sample is selected from cell samples, blood, serum, spinal fluid; or any biopsy sample obtained from the subject's tissue.
  • the present invention provides a detection kit.
  • the kit includes the compound of the present invention or a pharmaceutical composition thereof, and optionally includes reagents required for detection, such as aqueous solutions, solvents, suitable detection reagents such as chemiluminescence reagents, and the like.
  • the present invention also relates to compounds selected from:
  • a method for screening or identifying compounds for the treatment or prevention of polyQ-related neurodegenerative diseases including
  • step (II) includes:
  • the binding amount of (1) a polyQ abnormally amplified protein or a fragment containing a polyQ portion and (2) an LC3B protein or a homologue or a fragment thereof is determined.
  • polyQ abnormally amplified protein comprises polyQ length ⁇ 40, ⁇ 33, ⁇ 41, ⁇ 19, ⁇ 46, ⁇ 44, ⁇ 39, ⁇ 36 or ⁇ 37 polyQ part;
  • the polyQ abnormally amplified protein comprises
  • AR with polyQ length ⁇ 37 or a fragment containing polyQ part AR with polyQ length ⁇ 37 or a fragment containing polyQ part.
  • the method for determining the binding is selected from the group consisting of in vitro pull-down test, Split-TEV, immunoprecipitation, affinity chromatography, complex co-purification, Enzyme-linked immunosorbent method, fluorescent molecular sieve, yeast two-hybrid, HIP-HOP method, time-resolved fluorescence resonance energy transfer, chemiluminescence method, surface plasmon resonance, isothermal titration calorimetry, microthermophoresis and any combination thereof; preferred Specifically, the method for determining the binding is selected from in vitro pull-down experiments, co-immunoprecipitation, enzyme-linked immunosorbent assay, fluorescent molecular sieves, time-resolved fluorescence resonance energy transfer and any combination thereof.
  • step (I) The method of any one of [1] to [5] above, wherein before step (I), it further includes a step of pre-screening the candidate compound for affinity activity, including:
  • step (A) and step (B) can be performed independently in any order.
  • Step (A) includes: determining the affinity reaction equilibrium dissociation constant of the candidate compound and (1) polyQ abnormally amplified protein or a fragment containing the polyQ portion, and selecting a compound whose affinity reaction equilibrium dissociation constant is less than 100 ⁇ M, Preferably 10 ⁇ M or less, particularly preferably 1 ⁇ M or less; and/or
  • Step (B) includes: determining the affinity reaction equilibrium dissociation constant of the candidate compound and (2) LC3B protein or its homologues or fragments thereof, and selecting a compound whose affinity reaction equilibrium dissociation constant is less than 100 ⁇ M, preferably 10 ⁇ M or less, particularly preferably 1 ⁇ M or less.
  • the step of pre-screening candidate compounds for affinity activity is performed by high-throughput screening using the oblique incident light reflectance method; including:
  • the target protein is (1) polyQ abnormally amplified protein or a fragment containing polyQ part or (2) LC3B protein or its homologue or their fragment;
  • the polyQ-related neurodegenerative disease is selected from the group consisting of spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 7, and spinocerebellar ataxia Ataxia type 12, spinocerebellar ataxia type 17, dentate nucleus-substantia nigra-red nucleus-globus pallidus-subthalamic atrophy, Huntington's disease and spinal bulbar muscular atrophy, especially Huntington's disease and spinocerebellar ataxia Type 3.
  • the compound or its pharmaceutical composition in the preparation of a medicament for the treatment or prevention of polyQ-related neurodegenerative diseases, or in the preparation of a drug for the detection of polyQ-related neurodegenerative diseases.
  • the compound is selected from:
  • a pharmaceutical composition, detection reagent or kit which comprises a compound obtained by the method of any one of [1] to [10] above.
  • a pharmaceutical composition, detection reagent, kit or screening system which comprises at least one compound selected from:
  • a screening system comprising
  • the screening system is in the form of a kit.
  • a compound or a pharmaceutical composition that positively regulates the binding between (1) polyQ abnormally amplified protein or a fragment containing polyQ portion and (2) LC3B protein or its homologue or their fragments is used in preparation
  • the utility model is used in drugs for treating or preventing polyQ-related neurodegenerative diseases.
  • the compound screened by the method of the present invention can selectively reduce the level of abnormally amplified polyQ protein in the cell by targeting autophagy, and has no effect on the autophagy function of the cell. Therefore, the method of the present invention can effectively screen compounds for treating or preventing polyQ-related neurodegenerative diseases.
  • Autophagy is a highly genetically conserved protein degradation pathway, which is ubiquitous in eukaryotic cells. It has strong protein degradation ability but low selectivity.
  • the current research mainly focuses on autophagy modulators, which have not solved the problem of global effects caused by non-specific regulation of autophagy function, so there are potential side effects.
  • the inventors unexpectedly discovered that the screening method of the present invention can obtain compounds that specifically and autophagy-dependently reduce the level of abnormally amplified polyQ proteins, which have lower side effects and good safety, are easy to pass through the BBB, and facilitate the passage Oral administration.
  • the screening method of the present invention has the advantages of easy implementation and high screening efficiency.
  • the compound library used in the examples is provided by Selleck, and contains 3375 biologically active compounds. These include 1,527 drugs approved by the U.S. Food and Drug Administration (FDA), 1,053 natural products from traditional Chinese medicine, and 795 known inhibitors. among them:
  • Compound 2 ispinesib, PubChem CID: 6851740, can be purchased from Selleck, catalog number S1452;
  • PubChem CID 5398649 can be purchased from ChemDiv, catalog number D715-2435;
  • Compound 5 Semaxanib, which can be purchased from Selleck, CAS No. 194413-58-6;
  • Compound 6 Su9516, which can be purchased from Selleck, CAS No. 377090-84-1;
  • Compound 7 can be purchased from TargetMol, CAS No.842-01-3;
  • Compound 8 Can be purchased from ChemDiv.
  • HTT antibody 2B7 (Weiss et al. Anal Biochem 2009, 395, 8-15), 4C9, ab1 (Sapp et al. J Biol Chem 2012, 287, 13487-13499) and MW1 (Ko et al. Brain research bulletin 2001, 56,319-329) were prepared by the method of the prior art; the antibody S830 used for immunostaining to detect HTT aggregates was obtained from Dr. Gillian Bates; other antibodies were purchased from companies such as Millipore and Sigma.
  • Verification of LC3B Verify the prepared human LC3B by MALDI-TOF-MS and X-ray diffraction.
  • the method for verifying LC3B by X-ray diffraction is: site-directed mutagenesis obtains the mutant LC3B ⁇ G120 protein lacking the G120 lipidation site, and uses this more stable protein for high-resolution X-ray diffraction.
  • site-directed mutagenesis obtains the mutant LC3B ⁇ G120 protein lacking the G120 lipidation site, and uses this more stable protein for high-resolution X-ray diffraction.
  • LC3 protein crystal structure PDB ID: 1UGM
  • the structure of the published LC3B ⁇ G120 (PDB ID: 6J04, ) To perform structural alignment.
  • the plasmid was transfected into HEK293T cells for expression with linearized polyethyleneimine (Polysciences, 24765).
  • the protein was purified with HisTrap HP column and Superose 6Increase 10/300GL size exclusion column.
  • the human HTT gene (GenBank: NM_002111.8) with (CAG) 23 or (CAG) 73 was synthesized de novo by Genewiz Inc.
  • the human HTT gene was cloned into a modified pCAG vector (from Addgene) with an N-terminal protein A tag.
  • PEI Polyethyleneimine
  • Hdh Q140/Q7 and Hdh Q7/Q7 neonatal mice (P0) were cultured after dissection, digestion, and dissociation.
  • Some primary patient fibroblasts and wild-type cells come from HD patients (Q47, Q55) and healthy controls (WT, Q19) of the Mongolian Huntington's disease family.
  • the SCA3 cell line comes from a patient (Q74).
  • the HD Q68 fibroblast cell line is from Corell Cell Repositories (Camden, NJ, USA).
  • Immortalized fibroblasts and iPS cells (iPSC) are prepared from primary fibroblasts.
  • Mouse striatum cells (STHdh) were from Coriell Cell Repositories (Camden, NJ, USA).
  • HEK293T cells and HeLa cells are from ATCC.
  • Nervous system driver elav-GAL4 (c155), HTT-expressing lines UAS-fl-HTT-Q16 and UAS-fl-HTT-Q128 are from Bloomington Drosophila Stock Center of Indiana University (http://flystocks.bio.indiana.edu /), and kept in a 25°C incubator.
  • Hdh Q7/Q7 mice expressing wild-type HTT gene
  • the Q140 gene knock-in heterozygous mouse was prepared according to the method of the prior art (Menalled et al., J Comp Neurol, 2003, 465:11-26).
  • HTRF Homogeneous time-resolved fluorescence
  • Determination of the amount of protein Determine the amount of protein by the above method. Perform background correction with blank samples. The protein concentration was measured for all samples to correct the amount of protein. Determine the different protein concentration or cell number in each well to ensure that the signal is within the linear range.
  • Immunofluorescence After the cells are washed, fixed, permeabilized, and blocked, they are incubated with the primary antibody overnight at 4°C, then washed three times with blocking buffer, and incubated with the secondary antibody for 1 hour at room temperature. After staining with DAPI, mounting, imaging with Zeiss Axio Vert A1 confocal microscope, and analyzing TUBB3 or colocalization with ImageJ.
  • the compound has no affinity binding with HTTexon1-Q25, and the affinity reaction parameters with HTTexon1-Q72 are shown in Table 1.
  • the chips were prepared with compounds 1, 2 and 4 according to the above method. Three copies of each compound are printed. OI-RD was used to measure the affinity reaction parameters of the compound with LC3B, full-length mHTT (flHTT-Q73) and full-length HTT-Q23 (flHTT-Q23).
  • the compound has no affinity binding with flHTT-Q23 (SEQ ID NO: 4), and the affinity reaction parameters with flHTT-Q73 and LC3B are shown in Table 2 and Table 3, respectively.
  • the affinity activity of the compound with full-length HTT and LC3B was verified with a microthermophoresis instrument (MST, in which Monolith NT.115 instrument comes from NanoTemper Technologies).
  • the reaction buffer is 20 mM HEPES, pH 7.4, 150 mM NaCl, and protein concentration of 500 nM.
  • the compound has no affinity binding with flHTT-Q23 (SEQ ID NO: 4), and the K d of flHTT-Q73 and LC3B are shown in Table 4, respectively.
  • the inventor also verified through MST that the compound has the respective affinity activities for both full-length HTT and LC3B in a system where both full-length HTT and LC3B are present (Figure 1).
  • the results of OI-RD detection and MST detection both show that the compound can bind to LC3B affinity; for HTT, the compound selectively binds to mHTT (HTTexon1-Q72 or flHTT-Q73). This indicates that the compound can positively regulate the binding effect between LC3B and mHTT.
  • compound 1 is in the range of 30-300nM (100nM is preferable)
  • compound 2 is in the range of 30-100nM (100nM is preferable)
  • compound 3 is in the range of 10-300nM (50nM is preferable)
  • compound 4 is in the range of 15-150nM The effect of selectively reducing mHTT levels was observed within (75nM is preferred).
  • the mHTT was detected with anti-polyQ antibodies MW1 and 3B5H10, and the bands of proteins with smaller molecular weights were observed. As a result, no increase in the N-terminal fragments of mHTT was observed ( Figure 5). The detected decrease in mHTT levels was not due to an increase in site-specific cleavage.
  • the viability of Hdh Q140/Q7 mouse cortical neuron cells after compound treatment was measured by CellTiter-glo (Promega, G7570) ( Figure 6).
  • the compound has no cytotoxicity to Hdh Q140/Q7 mouse cortical neurons at the test concentration described in 2.1.
  • the detected decrease in mHTT levels is not due to neuronal cell loss.
  • the compounds screened by the method of the present invention all show excellent allele-selective effects in reducing mHTT levels in cells.
  • 2 compounds that were verified to be active at the cellular level were screened from a structurally diverse compound library containing 3375 biologically active compounds, with a positive rate of 0.6 ⁇ .
  • Test method Treat HD patient fibroblasts (Q49, Q55, Q68) with 100nM compound, and detect mHTT (antibody pair: 2B7/MW1) and total HTT (antibody pair: 2B7/2166) by HTRF after 2 days.
  • mHTT antibody pair: 2B7/MW1
  • HTT antibody pair: 2B7/2166
  • HTRF antibody pair: 2B7/MW1
  • Figure 8 Using a test method similar to that described in 3.1, HTRF (antibody pair: 2B7/MW1) was used to detect the effect of the compound on the level of mHTT in immortalized fibroblasts of HD patients in the presence or absence of autophagy inhibitors ( Figure 8) .
  • the compound In the absence of autophagy inhibitors, the compound significantly reduced the mHTT level of immortalized fibroblasts from HD patients, consistent with the results observed on primary fibroblasts from HD patients.
  • autophagy inhibitors NH 4 Cl or chloroquine
  • compound 1 also has c-Raf inhibitor activity
  • compound 2 also has kinesin spindle kinase (KSP) inhibitory activity.
  • KSP kinesin spindle kinase
  • PLX-4720 c-Raf inhibitor
  • BAY1217389 KSP inhibitor
  • compound 4 for comparison, PLX-4720 and BAY1217389 could not reduce mHTT levels at concentrations below the micromolar level ( Figure 9). Therefore, the effect of the compound on mHTT is not caused by the inhibitory activity of c-Raf or KSP.
  • the compounds screened for the reduction of mHTT levels are autophagy-dependent.
  • Example 4 The effect of the compound on the level of neuronal mHTT and neuronal apoptosis induced by stem cell differentiation in HD patients
  • HTRF antibody pair: 2B7/MW1
  • iPSC induced stem cells
  • Figure 10 Decreased mHTT levels were observed on neuronal cells differentiated from induced stem cells (iPSC) in HD patients, and this effect was not observed in the presence of the autophagy inhibitor NH 4 Cl.
  • compound 5 reduced mHTT levels in neuronal cells differentiated from induced stem cells (iPSC) from HD patients
  • compound 5 reduced mHTT levels About 12.2%
  • compound 6 reduced mHTT levels by about 24.9%
  • compound 7 reduced mHTT levels by about 24.2%
  • compound 8 reduced mHTT levels by about 19.7%.
  • the compound rescues the disease-related phenotype in the neuronal cells differentiated from induced stem cells (iPSC) in HD patients through autophagy.
  • 100 nM compound 1, or 100 nM compound 2, or 100 nM compound 3 or 50 nM compound 4 were used to treat HD patients with induced stem cell (iPSC) differentiated neurons (Q47), and the cells were stressed (removal of BDNF) after 1 day.
  • iPSC induced stem cell
  • DAPI was used to stain the neuron-specific tubulin marker TUBB3.
  • the TUBB3 signal coverage area was normalized by the cell nucleus count, and the wild type was used as a control to normalize the data to analyze neuronal apoptosis ( Figure 11).
  • NucView 488 (Biotium, 30029) was used to detect active caspase-3. After removing BDNF, use Incucyte (Essen Bioscience, Incucyte FLR) to capture images every 3 hours in the incubator, and analyze with Incucyte2011A software. A total of three batches were tested and the results were consistent ( Figure 12). The compound significantly improves the progression of HD neurons and neuron contraction after BDNF is removed.
  • Example 5 The effect of compounds on Huntington's disease fruit flies
  • Fruit flies are kept in standard food at 25°C. Transfer the newly hatched fruit flies to a vial containing the positive drug (10 ⁇ M in 400 DMSO) or DMSO for control, and change the food every other day.
  • Drosophila head proteins were extracted on the 7th day, and mHTT levels were measured by HTRF (antibody pair: 2B7/MW1), where each sample included 5 Drosophila head proteins (Figure 13).
  • HTRF antibody pair: 2B7/MW1
  • Figure 13 The compound reduced mHTT levels in transgenic flies expressing the full-length human HTT protein (Q128).
  • mice were grouped and housed in individual ventilated cages with a 12-hour light/dark cycle, with a maximum of 5 adult mice per cage.
  • mice Hdh Q140/Q7 mice (3 months old), 4 in each group
  • intracerebroventricular injection is performed once a day, and each injection is administered with 2 ⁇ L artificial cerebrospinal fluid (ACSF: 1mM glucose, 119mM NaCl, 2.5mM KCl, 1.3mM MgSO 4 , 2.5mM CaCl 2 , 26.2mM) containing 25 ⁇ M compound NaHCO 3 , 1mM NaH 2 PO 4 ).
  • 2 ⁇ L ACSF containing the same amount of DMSO was used as a control.
  • Experimental method Dilute the compound or control DMSO with 0.9% NaCl intravenous infusion solution to 0.05 ⁇ g/ ⁇ L, intraperitoneal injection (0.5mg/kg) once a day, 14 days after injection, tissue extraction or behavioral experiment.
  • Compound 1 or 4 was administered according to the procedure described in 6.2.
  • the intraperitoneal injection was performed once a day, 14 days after the injection, the protein was extracted, and the levels of mHTT and wtHTT in the cortex of HD mice were detected by Western blotting ( Figure 17a).
  • mice brain striatum neuronal protein was administered and obtained according to the above method, and the levels of mHTT and wtHTT were detected by Western blotting ( Figure 17b).
  • the compound administered by intraperitoneal injection reduced the levels of mHTT in the cortex and striatum of Hdh Q140/Q7 mice, and showed mHTT selectivity relative to wtHTT. Therefore, the compound has the prospect of being developed as an oral drug.
  • Hdh Q140/Q7 mice a total of 42 mice, divided into 3 groups
  • Hdh Q7/Q7 mice a total of 48 mice, divided into 3 groups.
  • Rotating rod test The mice were pre-trained for 3 consecutive days (rotating the rod at 4 rpm for 2 minutes). The mice were then tested at an acceleration speed of 4 to 40 rpm within 2 minutes for 5 days. The result of each experiment is recorded as time on the rod (time on the rotating rod), until falling from the rod or until the end of the task. Each test consists of three repetitions, with an interval of 60 minutes to reduce stress and fatigue. The average of three trials for each mouse was analyzed ( Figure 19a).
  • Balance beam test a graduated rod with a thickness of 2 cm and a total length of 100 cm, suspended on the platform on both sides. There is a bright light at the beginning, and a dark box with food at the end. The total time for each mouse to walk across the balance beam was recorded (Figure 19b).
  • the compound of the present invention can improve the Huntington's disease-related behavioral defects in HD model mice, and has no effect on wild-type mice.
  • Example 7 Compound does not affect autophagy function and the level of control protein
  • the LC3B and other known autophagy selective substrate proteins including SQSTM1/p62, NBR1 and Ncoa4 in the cortical neurons of Hdh Q140/Q7 mice after the compound treatment were detected, with wild type
  • the compound did not affect the LC3B expression level in the primary cultured cortical neurons, and the levels of other proteins tested were not affected by the compound (change in protein level ⁇ 10%).
  • Hdh Q140/Q7 mice were injected and administered, and a method similar to 7.1 was used to detect other known autophagy selective substrate proteins in the cortical tissue of Hdh Q140/Q7 mice by intraperitoneal injection of the compound (SQSTM1/p62).
  • SQSTM1/p62 The results of western blotting are shown in Figure 21. After intraperitoneal injection of compound 1 or compound 4, the level of SQSTM1/p62 in the cortex of HD mice did not change.
  • the detected mHTT-lowering effect is not due to altered autophagy function.
  • Example 8 The effect of the compound on the binding between mHTT and LC3B
  • Example 9 The effect of compounds on the localization of mHTT and LC3B in cells
  • the HTTexon1 cDNA was subcloned into the mammalian expression vector pTT-MBP-His to prepare pTT-HTTexon1-Q72-MBP-His and pTT-HTTexon1-Q25-MBP-His.
  • pEX-GFP-hLC3WT (Addgene, 24987), pTT-HTTexon1-Q72-MBP-His and pTT-HTTexon1-Q25-MBP-His prepared in (1) were transiently transfected into HeLa cells to express HTTexon1- MBP-His (SEQ ID NO:1 and SEQ ID NO:5 with MBP-His tag added) and LC3B-GFP (based on SEQ ID NO:8).
  • the y-axis of the histogram in FIG. 23 represents the proportion of the red + (HTTexon1-MBP-His) aggregation points that are the same as the green + (LC3B-GFP) positioning to the total red + (HTTexon1-MBP-His) aggregation points.
  • the source of HD mouse striatal cells STHdh Q111/Q111 is as described above.
  • mHTT was detected by antibody 2166
  • endogenous LC3B was detected by an anti-LC3 antibody that specifically detects LC3B.
  • the y-axis of the histogram in FIG. 24 represents the proportion of the red + (HTTexon1-MBP-His) aggregation points that are the same as the green + (LC3B-GFP) positioning to the total red + (HTTexon1-MBP-His) aggregation points.
  • the compound screened by the method of the present invention can promote the co-localization of polyQ abnormally amplified protein and LC3B in cells, thus confirming that the compound can simultaneously bind to polyQ abnormally amplified protein and LC3B to promote polyQ abnormally amplified protein to have self-localization
  • the cellular substructure of phagocytic function is enriched, thereby reducing the level of the abnormally amplified polyQ protein in the cell through autophagy.
  • the screening method of the present invention can also use other known detection methods, such as fluorescence.
  • Example 10 Use of protein containing longer glutamine repeat region for affinity activity screening
  • the affinity activity of compound 1, compound 3, compound 4 and polyQ-GFP protein was tested with a microthermophoresis for verification.
  • the compound of the present invention has no affinity binding to Q25-GFP, and the K d of Q72-GFP is shown in Table 6.
  • the compound of the present invention selectively binds to a protein containing a longer glutamine repeat region.
  • the polyQ abnormally amplified protein or the fragment containing the polyQ part can be applied to the screening method of the present invention.
  • the screening method of the present invention can be effectively applied to screen or identify compounds for the treatment or prevention of diseases caused by proteins containing longer glutamine repeat regions, such as polyQ-related neurodegenerative diseases.
  • Example 11 The effect of the compound on the protein level containing the longer glutamine repeat region in the cell
  • the Q72-GFP expressing cells and Q25-GFP expressing cells obtained in step (1) of 10.1 were used.
  • the cells were treated with 100 nM compound 1, or 100 nM compound 3 or 50 nM compound 4, and the polyQ-GFP level was measured by Incucyte fluorescence counting after 2 days ( Figure 25).
  • the compounds screened according to the method of the present invention can selectively reduce the level of proteins containing longer glutamine repeat regions in cells. This indicates that the compounds obtained by screening have extended pharmacological effects and can be used to treat or prevent diseases caused by proteins containing longer glutamine repeat regions.
  • the following examples 12 to 14 further test the effects of the compounds screened by the method of the present invention on the levels of abnormally amplified polyQ ATXN3 protein and polyQ abnormally amplified ATXN1 protein, and further confirm that the screening method of the present invention can be used to contain longer grains.
  • the protein in the aminoamide repeat region serves as the target protein representing the disease state.
  • Example 12 Using polyQ abnormally amplified ATXN3 protein for affinity activity screening
  • the affinity activity of the compound with MBP-ATXN3-Q28 and MBP-ATXN3-Q68 protein was verified with a microthermophoresis.
  • the fibroblasts (Q74) and wild-type cells (Q27) of SCA3 patients were treated with 100nM compound 1, or 100nM compound 2, or 100nM compound 3 or 50nM compound 4.
  • the mutant ATXN3 protein (ATXN3) was determined by Western blotting.
  • -Q74, SEQ ID NO: 2), wild-type ATXN3 protein (ATXN3-Q27, SEQ ID NO: 7) levels Figure 26.
  • the decrease of mutant ATXN3 protein level was observed on SCA3 patient fibroblasts (Q74), but the decrease of wild-type ATXN3 protein level was not observed. Therefore, the compound of the present invention can be used to treat SCA3.
  • Example 14 Using polyQ abnormally amplified ATXN1 protein for affinity activity screening
  • polyQ is Q92
  • pcDNA vector was transfected into HEK293T cells (ATCC, CRL-3216) to obtain cells expressing His-ATXN1-Q92 (SEQ ID NO: 3 with a His tag at the N end).
  • step (1) of 1.1 Follow the method similar to step (1) of 1.1, but replace the target protein HTTexon1-Q72-MBP in step (1) of 1.1 with His-ATXN1-Q92, and screen for those that can bind to the abnormally amplified ATXN1 protein of polyQ. Compound.
  • the His-ATXN1-Q92-expressing cell obtained in 14.1 was used.
  • the cells were treated with 100 nM compound (compound 1, or compound 2, or compound 3, or compound 4, or compound 6, or compound 7).
  • the level of mutant ATXN1 protein was detected by HTRF (antibody pair : Anti-His-Tb/MW1-D2, Figure 27.
  • HTRF antibody pair : Anti-His-Tb/MW1-D2, Figure 27.
  • a decrease in the level of variant ATXN1 protein was observed on SCA1 patient fibroblasts (Q92). Therefore, the compounds of the present invention can be used to treat or prevent spinocerebellar ataxia type 1 .
  • the compounds screened by the method of the present invention can reduce the levels of a variety of proteins containing longer glutamine repeat regions, such as mutant mHTT protein, mutant ATXN3 protein, and mutant ATXN1 protein. Therefore, the screening method of the present invention is effectively applied to screening or identifying compounds. Such compounds can be used to treat or prevent diseases caused by proteins containing longer glutamine repeat regions, such as polyQ-related neurodegenerative diseases.

Abstract

涉及一种筛选或鉴定用于治疗或预防polyQ相关的神经退行性疾病的化合物的方法以及由该方法获得的化合物和用途。

Description

一种筛选用于治疗或预防polyQ相关的神经退行性疾病的化合物的方法 技术领域
本发明涉及生物医药领域，并且具体地涉及一种筛选或鉴定用于治疗或预防polyQ相关的神经退行性疾病的化合物的方法以及由该方法获得的化合物和用途。
背景技术
许多疾病是由细胞或组织内出现特定的蛋白或特定蛋白水平的水平过高引起的，其中一些影响疾病的特定蛋白的活性未知，因而针对这样的疾病，常见的治疗策略为控制蛋白水平。目前一些用于控制蛋白水平的方法例如RNAi或CRIPSR等生物工具在体内的递送困难，因此，一种可行的治疗策略是通过低分子量化合物(简称化合物)来控制影响疾病的蛋白的水平。通过蛋白裂解靶向嵌合体(PROTAC)技术增强疾病蛋白的泛素化并将其靶向蛋白酶体降解途径是一种新兴方法，但这种方法依赖于某些E3连接酶，这些连接酶可能不存在于疾病细胞中。并且，蛋白酶体的蛋白降解能力有限，对某些大型疾病蛋白或聚集体降解效率较低。
神经退行性疾病(Neurodegenerative Disorder)是指中枢神经元不正常死亡引起神经系统功能障碍而导致的疾病，迄今为止没有可以减缓其发展进程的根本性治疗方法。以其中最常见的亨廷顿病(HD)为例，这是一种单基因遗传的疾病，患者的四号染色体所含基因HTT的exon1外显子的CAG重复区域变异导致合成的变异蛋白(mHTT)的谷氨酰胺重复区域(polyQ)扩增。mHTT易被剪切、聚集并产生毒性，最终导致特定神经元功能障碍和死亡。目前通过低分子量化合物控制mHTT水平的方法缺乏特异性，可能造成副作用，并且是非等位选择性的，无法区分mHTT与野生型HTT蛋白(wtHTT)，会导致具有重要生物学功能的wtHTT水平降低。
因此，本领域亟需有效的筛选或鉴定方法，以获得能够用于治疗或预防polyQ相关的神经退行性疾病的化合物。
发明内容
在一方面，本发明提供一种筛选或鉴定用于治疗或预防polyQ相关的神经退行性疾病的化合物的方法，包括
(I)：使候选化合物与待测体系接触，所述待测体系包含
(1)polyQ异常扩增蛋白或其含有polyQ部分的片段，和
(2)LC3B蛋白或其同源物或它们的片段；和
(II)：测定所述候选化合物调节(1)polyQ异常扩增蛋白或其含有polyQ部分的片段 与(2)LC3B蛋白或其同源物或它们的片段之间的结合的能力，并且选择正向调节所述结合的化合物。
在另一方面，本发明提供通过本发明的方法获得的化合物或其药物组合物在制备用于治疗或预防polyQ相关的神经退行性疾病的药物中的用途，或者在制备用于检测被认为患有或易患polyQ相关的神经退行性疾病的受试者的诊断试剂或试剂盒中的用途。
在又一方面，本发明提供正向调节(1)polyQ异常扩增蛋白或其含有polyQ部分的片段与(2)LC3B蛋白或其同源物或它们的片段之间的结合的化合物或其药物组合物在制备用于治疗或预防polyQ相关的神经退行性疾病的药物中的用途。
附图说明
图1：在同时存在全长HTT和LC3B的体系中检测化合物的亲和活性。
图2：化合物对Hdh Q140/Q7小鼠皮质神经元HTT水平的影响。
图3：化合物1和2对Hdh Q7/Q7小鼠皮质神经元HTT水平的影响。
图4：多种抗体检测化合物处理后Hdh Q140/Q7小鼠皮质神经元HTT水平。
图5：用抗体MW1和3B5H10检测化合物处理后Hdh Q140/Q7小鼠皮质神经元中mHTT的N-端碎片。
图6：化合物处理后Hdh Q140/Q7小鼠皮质神经元细胞活力检测结果。
图7：化合物在100nM浓度下对HD患者原代成纤维细胞mHTT水平的影响。
图8：化合物对HD患者永生化成纤维细胞mHTT水平的影响。
图9：化合物4降低永生化的HD患者成纤维细胞的mHTT水平
图10：化合物对HD患者诱导干细胞(iPSC)分化的神经元的mHTT水平的影响。
图11：化合物对HD患者诱导干细胞(iPSC)分化的神经元细胞凋亡的影响。纵坐标跨度50μM。
图12：化合物对HD患者诱导干细胞(iPSC)分化的神经元细胞凋亡的影响。其中I、II、III、IV分别代表化合物1、化合物2、化合物3、化合物4。
图13：化合物对亨廷顿病果蝇mHTT水平的影响。
图14：化合物对亨廷顿病果蝇生存率的影响。其中I、II、III、IV分别代表化合物1、化合物2、化合物3、化合物4。
图15：化合物对亨廷顿病果蝇爬行能力的影响。其中I、II、III、IV分别代表化合物1、化合物2、化合物3、化合物4。
图16：脑室内注射化合物对亨廷顿病小鼠皮质mHTT和wtHTT水平的影响。
图17：腹腔注射化合物对亨廷顿病小鼠皮质和纹状体mHTT和wtHTT水平的影响。
图18：腹腔注射化合物后，检测亨廷顿病小鼠皮质中的mHTT聚集物。
图19a和图19b：腹腔注射化合物对亨廷顿病小鼠行为缺陷的影响。
图20-1、图20-2、图20-3和图20-4：化合物不影响培养的Hdh Q140/Q7小鼠皮质神经 元细胞自噬功能和对照蛋白的水平。
图21：腹腔注射化合物不影响小鼠皮质中SQSTM1/p62水平。
图22：化合物选择性增强mHTT与LC3B之间的结合。
图23：化合物影响下mHTT与LC3B在Hela细胞中的共定位。白色箭头表示代表性的共定位点。部分图像放大显示共定位点。纵坐标跨度10μM。
图24：化合物影响下mHTT与LC3B在STHdh Q111/Q111细胞中的共定位。
图25：化合物在HEK293T细胞中对含有谷氨酰胺重复区域的蛋白水平的影响。
图26：化合物对脊髓小脑共济失调3型患者成纤维细胞的ATXN3蛋白水平的影响。
图27：化合物在HEK293T细胞中对变异ATXN1蛋白水平的影响。
具体实施方式
以下通过特定的具体实施例说明本发明的技术内容，本领域技术人员可由本说明书公开的内容容易地了解本发明的其他优点与功效。本发明也可以通过其他不同的具体实施例加以施行或应用。本领域技术人员能够在不背离本发明的精神前提下，进行各种修饰与变更。
一般术语和定义
除非另有定义，本文所用所有技术和科学术语与本发明所属领域的普通技术人员通常理解的含义相同。若存在矛盾，则以本申请提供的定义为准。当本文中出现商品名时，意在指代其对应的商品或其活性成分。本文引用的所有专利、已经公开的专利申请和出版物均通过引用并入到本文中。
术语“包括”、“包含”、“具有”、“含有”或“涉及”及其在本文中的其它变体形式为包含性的或开放式的，且不排除其它未列举的元素或方法步骤。本领域技术人员应当理解，上述术语如“包括”涵盖“由…组成”的含义。
术语“一个(种)或多个(种)”或者类似的表述“至少一个(种)”可以表示例如1、2、3、4、5、6、7、8、9、10个(种)或更多个(种)。
当公开了数值范围的下限和上限时，落入该范围中的任何数值和任何包括的范围都被具体公开。特别地，本文公开的值的每个取值范围应理解为表示涵盖于较宽范围中的每个数值和范围。例如，表述“polyQ长度≥40的ATXN1”可以涵盖polyQ长度≥41的情况，并且可以涵盖例如polyQ长度为92的情况。例如，“polyQ长度≥33的ATXN2”可以涵盖polyQ长度≥34的情况。例如，“polyQ长度≥41的ATXN3”可以涵盖polyQ长度≥62的情况，并且可以涵盖例如polyQ长度为74或68的情况。例如，“polyQ长度<41的ATXN3”可以涵盖polyQ长度为27或28的情况。例如，“polyQ长度≥19的ATXN7”可以涵盖polyQ长度≥38的情况。例如，“polyQ长度≥44的TBP”可以涵盖polyQ长度≥45的情况。例如，“polyQ长度≥39的ATN1”可以涵盖polyQ长度≥49的情况。例如，“polyQ长度≥36的HTT”可以涵盖polyQ长度为47、49、55、68、72、73、111、128或 140等的情况。又例如，“polyQ长度<36的HTT”可以涵盖polyQ长度为7、16、19、23或25等的情况。例如，“polyQ长度≥37的AR”可以涵盖polyQ长度≥38的情况。
术语“任选”或“任选地”是指随后描述的事件或情况可能发生或可能不发生，该描述包括发生所述事件或情况和不发生所述事件或情况。
术语“神经退行性疾病”指神经元和/或其髓鞘的丧失或病变所致的疾病。神经退行性疾病患者的脑神经元内可以观察到特征性的病理结构物，例如蛋白组成的不溶性聚集体。不溶性聚集体可能产生细胞毒性，进而导致神经元丧失和疾病发生。
术语“polyQ”或“polyglutamine”指蛋白中的谷氨酰胺重复区域。谷氨酰胺由基因中的胞嘧啶-腺嘌呤-鸟嘌呤(CAG)编码，谷氨酰胺重复区域的长度与基因外显子中的CAG重复数目有关，因此基因外显子中CAG重复数目增多会造成合成的蛋白谷氨酰胺重复区域扩增。已知polyQ异常扩增蛋白与一些神经退行性疾病相关。如本文中所使用的，基因名称中可以通过“Q+数字”的形式来表示外显子中CAG重复数目，例如Q25或Q72，分别表示外显子中CAG的25个重复或72个重复。蛋白名称中可以通过如上“Q+数字”的形式来表示谷氨酰胺重复区域的长度，例如Q27或Q73，分别表示谷氨酰胺重复区域的长度为27个Q(glutamine)或73个Q。本文中“Q+数字”的形式标示的CAG重复或谷氨酰胺重复均为连续重复。除非特别指明，本文中的polyQ长度均指连续重复的谷氨酰胺区域长度。
术语“polyQ相关的神经退行性疾病”指与polyQ异常扩增相关的神经退行性疾病，或对含扩增的polyQ的蛋白水平响应的神经退行性疾病，是一组临床和遗传上异质性的神经退行性疾病。
“正常polyQ”是指正常生理状态下的蛋白具有长度小于特定数目的polyQ。与之对应地，“polyQ异常扩增”是指蛋白的polyQ长度大于正常长度。对于疾病或病理状态，polyQ长度会更长。
如本文所用，“正常polyQ蛋白”包含长度正常的polyQ(即其polyQ部分)。
如本文所用，“polyQ异常扩增蛋白”包含长度异常的polyQ(即其polyQ部分)。例如，polyQ长度≥40、≥33、≥41、≥19、≥46、≥44、≥39、≥36或≥37的polyQ部分。
作为示例，正常或异常polyQ蛋白的片段的长度可以是对应的蛋白全长的1％或更高、2％或更高、3％或更高、4％或更高、5％或更高、10％或更高、15％或更高、20％或更高、25％或更高、30％或更高、35％或更高、40％或更高、50％或更高、60％或更高、70％或更高、80％或更高、90％或更高或者95％或更高，但包含完整的长度正常或异常的polyQ部分。
在优选的实施方案中，当用于本发明的方法，特别是高通量方法时，可以使用polyQ异常扩增蛋白的片段。其含有该蛋白的polyQ部分，并且长度可以仅为对应蛋白的1％或更高、2％或更高、3％或更高、4％或更高。在示例性实施方案中，所用的“polyQ异常扩增蛋白”或其片段可以包含SEQ ID No:1、2或3的氨基酸序列。在另一示例性方案 中，所用的“正常polyQ蛋白”或其片段可以包含SEQ ID No:4或5的氨基酸序列。在又一示例性方案中，所用的“正常polyQ蛋白”或其片段可以包含SEQ ID No:7的氨基酸序列。在另一示例性方案中，所用的“正常polyQ蛋白”或其片段可以包含SEQ ID No:6的氨基酸序列。
“正常polyQ蛋白”或“polyQ异常扩增蛋白”可以是天然存在的蛋白，其中包含长度正常或异常的polyQ部分，例如可以来自人(例如为ATXN1、ATXN2、ATXN3、ATXN7、ATXN12、TBP、ATN1、HTT或AR)或来自其他动物，例如来自小鼠(例如小鼠HTT蛋白，其polyQ长度可以例如为140或111)，或来自昆虫、鱼类、啮齿类、偶蹄类、灵长类动物。
“正常polyQ蛋白”或“polyQ异常扩增蛋白”也可以是人工改造或修饰的蛋白，但仍然带有其相应的polyQ部分。这样的改造或修饰可以是为了加工、纯化、表征、示踪等目的，例如加入MBP标签或His标签或与绿色荧光蛋白融合，或者引入各种氨基酸取代、增加或缺失。这样的技术为本领域技术人员所熟知。优选地，这样的蛋白包含对应的正常或异常polyQ部分，并且与对应的正常或polyQ异常扩增蛋白的具有15％或更高、20％或更高、25％或更高、30％或更高、35％或更高、40％或更高、50％或更高、60％或更高、70％或更高、80％或更高、90％或更高或者95％或更高的序列相同性。这样的蛋白可以是由对应的正常或polyQ异常扩增蛋白的全长或片段截取或拼接得到的，并且任选地包含改造或修饰，也可以是从头设计的。
如本文中使用的，“含有较长谷氨酰胺重复区域的蛋白”是指特定蛋白中的谷氨酰胺重复区域较长。在一些情况下，对于特定的疾病，“较长谷氨酰胺重复区域”可以指“polyQ异常扩增”，在这一情况下，“含有较长谷氨酰胺重复区域的蛋白”可以与本文所述的“polyQ异常扩增蛋白”具有相同的含义。然而在另一些情况下，“较长谷氨酰胺重复区域”的polyQ长度可以同时满足：(1)针对某些特定疾病而言，这样的polyQ大于正常长度，属于异常扩增；(2)针对另一些特定疾病而言，这样的polyQ长度为正常长度，不属于异常扩增。这样的“含有较长谷氨酰胺重复区域的蛋白”同样可以理解为“polyQ异常扩增蛋白”，涵盖于本发明的范围内，并且可以由技术人员根据实际需要来使用。
作为示例，polyQ相关的神经退行性疾病包括但不限于脊髓小脑共济失调(spinocerebellar ataxia,SCA)1型(polyQ长度≥41)、2型(polyQ长度≥34)、3型(polyQ长度≥62)、7型(polyQ长度≥38)、12型(polyQ长度≥46)、17型(polyQ长度≥45)；以及齿状核-黑质-红核-苍白球-丘脑底核萎缩症(dentatorubral-pallidoluysian atrophy,DRPLA，polyQ长度≥49)、亨廷顿病(Huntington's Disease,HD，polyQ长度≥36)和脊髓延髓肌肉萎缩(spinal-bulbar muscular atrophy,SBMA，polyQ长度≥38)。这些疾病分别由在ATXN1、ATXN2、ATXN3、ATXN7、ATXN12、TBP、ATN1、HTT和AR基因上的CAG重复区域的扩增引起(Lesley Jones等，DNA repair in the trinucleotide repeat disorders,Lancet Neurol.2017；16:88–96)。其中脊髓小脑共济失调3型(SCA3，又称为Machado-Joseph  disease,MJD)是全世界最常见的常染色体显性脊髓小脑共济失调和仅次于HD的常见polyQ相关疾病，由Ataxin-3基因(ATXN3；又称为MJD1基因)CAG重复数目增多导致编码蛋白ATXN3的C末端形成异常扩增的polyQ引起。本文所述的正常polyQ蛋白的实例包括但不限于polyQ长度<40的ATXN1、polyQ长度<33的ATXN2、polyQ长度<41的ATXN3、polyQ长度<19的ATXN7、polyQ长度<46的ATXN12、polyQ长度<44的TBP、polyQ长度<39的ATN1、polyQ长度<36的HTT，和polyQ长度<37的AR。与之对应地，本文所述的polyQ异常扩增蛋白的实例包括但不限于polyQ长度≥40的ATXN1、polyQ长度≥33的ATXN2、polyQ长度≥41的ATXN3、polyQ长度≥19的ATXN7、polyQ长度≥46的ATXN12、polyQ长度≥44的TBP、polyQ长度≥39的ATN1、polyQ长度≥36的HTT，和polyQ长度≥37的AR。
人微管相关蛋白1轻链3β(MAP1LC3B,LC3B)属于Atg8蛋白家族中的微管相关蛋白1轻链3(LC3)家族，其定位于前自噬体和自噬体的膜上，并能控制自噬体形成，是自噬过程中的关键蛋白。
可以使用的LC3B蛋白的形式例如有LC3B-I和LC3B-II，但不限于此。对于LC3B蛋白，例如可以参见Uniprot Accession:Q9GZQ8。
在本文中，可以使用LC3B蛋白的同源物，只要其能够用于本发明的方法，例如与polyQ异常扩增蛋白或其含有polyQ部分的片段相互作用。
本文所用的LC3B蛋白的同源物可以来自真核生物，例如酵母或非人的其他动物，例如昆虫(例如果蝇)、鱼类、啮齿类、偶蹄类、灵长类等。LC3B蛋白的同源物也可以来自于其他类似结构功能的蛋白，例如LC3A、LC3C、GABARAP和GABARAPL1，但不限于此。例如，可以参见LC3A(Uniprot Accession:Q9H492-1和Q9H492-2)、LC3C(Uniprot Accession:Q9BXW4)、GABARAP(Uniprot Accession:O95166)和GABARAPL1(Uniprot Accession:Q9H0R8-1和Q9H0R8-2)，但不限于此。
在本文中，还可以使用LC3B蛋白或其同源物的片段，只要其能够用于本发明的方法，例如与polyQ异常扩增蛋白或其含有polyQ部分的片段相互作用。这样的片段，例如可以与LC3B蛋白或其同源物具有25％或更高、30％或更高、35％或更高、40％或更高、50％或更高、60％或更高、70％或更高、80％或更高、90％或更高、95％或更高或者100％的相同性。
可以使用与LC3B蛋白或其同源物具有相同性或同源性的蛋白或其片段来实现本发明的目的，只要其能够用于本发明的方法。例如，这样的与polyQ异常扩增蛋白或其含有polyQ部分的片段相互作用。例如，这样的相同性可以是25％或更高、30％或更高、35％或更高、40％或更高、50％或更高、60％或更高、70％或更高、80％或更高、90％或更高、95％或更高或者100％。
在示例性实施方案中，LC3B蛋白或其同源物或者它们的片段与SEQ ID NO:8的氨基酸序列具有25％或更高、30％或更高、35％或更高、40％或更高、50％或更高、60％或 更高、70％或更高、80％或更高、90％或更高、95％或更高或者100％的序列相同性。本领域技术人员应当理解，这也可以表示所用的LC3B蛋白或其同源物或者它们的片段可包含这样的序列。
本领域技术人员还应当理解，可以使用LC3B蛋白或其同源物或者它们的片段和/或polyQ异常扩增蛋白或其含有polyQ部分的片段的成熟形式。例如，LC3B蛋白如SEQ ID NO:8的C端的氨基酸121-125切除后可以获得其成熟形式。这样的方案同样在本发明的范围内。
本领域技术人员应当理解，可以根据实际需要，对所用的LC3B蛋白或其同源物或它们的片段和/或polyQ异常扩增蛋白或其含有polyQ部分的片段进行修饰或改造，来用于本发明。这样的方案同样涵盖在本发明的范围内。这样的修饰或改造例如可以包括但不限于，增加标签(如GST或HIS)或进行标记等，或者对部分氨基酸进行取代、缺失、增加或替换。例如为了剪切标签方便，可以对SEQ ID NO:8的序列的末端进行少量修饰或改造，例如可以将SEQ ID NO:8的N末端的M替换为GG，然后可以任选地将改造或未改造的序列添加诸如GST的标签，从而用于本发明。这样的修饰或改造后的序列或与其具有25％或更高、30％或更高、35％或更高、40％或更高、50％或更高、60％或更高、70％或更高、80％或更高、90％或更高、95％或更高或者100％的序列相同性的序列所对应的方案也在本发明的范围内。
术语“亲和活性筛选”是检测样品与靶标之间的亲和结合的过程。亲和活性筛选采用的检测方法可以例如是吸光度法、放射法(例如接近闪烁分析)、荧光法(例如荧光共振能量转移、荧光偏振检测，尤其例如时间相关荧光技术)、化学发光法(例如放大化学发光亲和均相检测，ALPHAScreen)、表面等离子体共振(SPR，可以例如采用GE公司的Biacore系列实施)、等温滴定量热法(ITC)、微量热泳动(MST)或斜入射光反射差法。
如本文所用，两个氨基酸序列之间的“序列相同性”表示所述序列之间相同的氨基酸的百分比。“序列同源性”表示相同或代表保守氨基酸取代的氨基酸的百分比。对于序列比较，通常一条序列作为参考序列，将测试序列与之比较。当使用序列比较算法时，将测试和参考序列输入到计算机中，如果需要，指定子序列坐标，并指定序列算法程序参数。基于指定的程序参数，序列比较算法计算测试序列相对于参考序列的序列相同性百分数。适合用于确定序列相同性和序列相似性百分比的算法实例包括但不限于BLAST和BLAST 2.0算法。实施BLAST分析的软件可从美国国家生物技术信息中心(NCBI)获得。
术语“目标样品”包括含有从受试者获得的并可用于诊断分析的各种样品类型。本文中的目标样品可以例如是任何细胞样品、生物流体(包括血液、血清、脊髓液，等)或从受试者的组织获得的任何活检样品。
筛选方法
本发明的概念至少是部分地基于以下新机制的发现：能够与自噬过程中的关键蛋白LC3B以及polyQ异常扩增蛋白亲和结合的化合物可以促进polyQ异常扩增蛋白经由细胞自噬而降解，从而达到降低polyQ异常扩增蛋白水平、治疗或预防相应疾病的目的。
因此，在一方面，本发明提供一种筛选或鉴定用于治疗或预防polyQ相关的神经退行性疾病的化合物的方法，包括
(I)：使候选化合物与待测体系接触，所述待测体系包含
(1)polyQ异常扩增蛋白或其含有polyQ部分的片段，和
(2)LC3B蛋白或其同源物或它们的片段；和
(II)：测定所述候选化合物调节(1)polyQ异常扩增蛋白或其含有polyQ部分的片段与(2)LC3B蛋白或其同源物或它们的片段之间的结合的能力，并且选择正向调节所述结合的化合物。
所述的体系包括但不限于溶液体系、亚细胞体系、细胞(细胞培养物)体系、组织体系、器官体系、或动物体系。
在一实施方案中，候选化合物可以以化合物库的方式提供。这样的库可以是商购的或者设计合成的。候选化合物可以包括肽、拟肽和有机小分子等。例如以选自设计合成的化合物、数据库(例如Pubmed)中结构确定的化合物或从头合成的化合物。在一个具体的实施方式中，候选化合物选自有机小分子。
术语“有机小分子”或“低分子量化合物”是指大小与通常用于药物的有机分子相当的分子。该术语排除生物大分子(例如蛋白质、核酸等)，但是涵盖小分子量蛋白或其衍生物，例如二肽、三肽、四肽、五肽等。
在一具体的实施方案中，有机小分子的大小为约100-约2000Da，优选为约200-约1000Da，例如约200-约900Da、约200-约800Da、约200-约700Da、约200-约600Da、约200-约500Da。
本发明的方法使用的polyQ异常扩增蛋白或其含有polyQ部分的片段由针对的polyQ相关的神经退行性疾病决定。
在一实施方案中，根据针对的polyQ相关的神经退行性疾病，polyQ异常扩增蛋白包含polyQ的长度≥40、≥33、≥41、≥19、≥46、≥44、≥39、≥36或≥37的polyQ部分。在一优选的实施方案中，polyQ异常扩增蛋白包含polyQ长度≥40的ATXN1或其含有polyQ部分的片段。在一优选的实施方案中，polyQ异常扩增蛋白包含polyQ长度≥33的ATXN2或其含有polyQ部分的片段。在另一优选的实施方案中，polyQ异常扩增蛋白包含polyQ长度≥41的ATXN3或其含有polyQ部分的片段。在又一优选的实施方案中，polyQ异常扩增蛋白包含polyQ长度≥19的ATXN7或其含有polyQ部分的片段。在一优选的实施方案中，polyQ异常扩增蛋白包含polyQ长度≥46的ATXN12或其含有polyQ部分的片段。在另一优选的实施方案中，polyQ异常扩增蛋白包含polyQ长度≥44的TBP或其含 有polyQ部分的片段。在又一优选的实施方案中，polyQ异常扩增蛋白包含polyQ长度≥39的ATN1或其含有polyQ部分的片段。在一优选的实施方案中，polyQ异常扩增蛋白包含polyQ长度≥36的HTT或其含有polyQ部分的片段。在另一优选的实施方案中，polyQ异常扩增蛋白包含polyQ长度≥37的AR或其含有polyQ部分的片段。
对结合的“正向调节”是指在相同条件下进行测定，与化合物不存在时相比，化合物存在时，所述结合涉及的蛋白或其片段之间的结合强度变大，或结合量变大。在一实施方案中，可以在分子水平测定化合物对所述结合的正向调节，例如测定蛋白或其片段之间结合亲和力在化合物存在的条件下的增大。正向调节也可以通过分别测定化合物对结合涉及的蛋白或其片段之间的结合强度(如亲和活性)来验证。如果化合物对结合涉及的蛋白或其片段均具有亲和活性，而对相应的参照不具有或具有较低的亲和活性，则可以确定为正向调节。
本发明对于步骤(II)的实施方法没有特别的限制。在一实施方案中，步骤(II)通过以下方式实施：分别在存在/不存在候选化合物的条件下，测定表征(1)polyQ异常扩增蛋白或其含有polyQ部分的片段与(2)LC3B蛋白或其同源物或它们的片段之间的结合强度的参数，并将存在/不存在候选化合物时进行的相同试验中获得的参数相比较，选择正向调节(1)polyQ异常扩增蛋白或其含有polyQ部分的片段与(2)LC3B蛋白或其同源物或它们的片段的结合的化合物。取决于所进行的结合试验，上述“参数”可以是各种各样的，但特别可以例如是吸光度值、放射性信号和/或在样品中的分布、荧光信号强度和/或在样品中的分布、热量变化、反射光强度、反射光相位变化，等。
在一实施方案中，(1)polyQ异常扩增蛋白或其含有polyQ部分的片段与(2)LC3B蛋白或其同源物或它们的片段之间的结合为亲和结合，步骤(II)包括：分别在存在/不存在所述候选化合物的条件下，测定(1)与(2)之间的结合亲和力大小，和/或，分别在存在/不存在所述候选化合物的条件下，测定(1)与(2)的结合量；对存在/不存在候选化合物时得到的测定值进行比较，如果当存在候选化合物时，上述蛋白或其片段之间的结合出现正向变化，则选择该候选化合物。
在一实施方案中，步骤(II)中测定蛋白或其片段之间的结合的方法选自体外拉下实验(pull-down assay)、Split-TEV、免疫共沉淀(co-IP)、亲和层析(AP)、复合物共纯化(Copurification)、酶联免疫吸附法(ELISA)、荧光分子筛(Fluorescence-SEC)、酵母双杂交(yeast two-hybrid,Y2H)、HIP-HOP(haploinsufficiency profiling and homozygous deletion profiling)法、时间分辨荧光共振能量转移(TR-FRET)、化学发光法、表面等离子体共振(surface plasmon resonance,SPR)、等温滴定量热法、微量热泳动及其任意组合。在一优选的实施方案中，测定所述结合的方法选自体外拉下实验、免疫共沉淀、酶联免疫吸附法、荧光分子筛和时间分辨荧光共振能量转移及其任意组合。
在另一实施方案中，在步骤(I)之前还包括对候选化合物进行亲和活性预筛选的步骤；包括：
(A)：检测候选化合物与(1)polyQ异常扩增蛋白或其含有polyQ部分的片段的亲和结合，和/或(B)：检测候选化合物与(2)LC3B蛋白或其同源物或它们的片段的亲和结合；
以及(C)：选择与(1)polyQ异常扩增蛋白或其含有polyQ部分的片段和/或(2)LC3B蛋白或其同源物或它们的片段能够亲和结合的化合物。
其中步骤(A)和步骤(B)可以以任何顺序独立地进行。例如步骤(A)和步骤(B)可以同时分别在不同的体系中进行。可以先进行步骤(A)，然后进行步骤(B)，反之亦然。在优选的方案中，选择与(1)polyQ异常扩增蛋白或其含有polyQ部分的片段和(2)LC3B蛋白或其同源物或它们的片段都能够亲和结合的化合物。
按需要，步骤(A)和步骤(B)可以独立地进行或不进行，也可以以各自独立的方式进行至少一次(例如各自独立地1次、2次、3次或更多次)。这样的方式，根据需要可以各自独立地是定性或定量的。
在一优选的实施方案中，步骤(A)可以包括：测定候选化合物与(1)polyQ异常扩增蛋白或其含有polyQ部分的片段的亲和反应平衡解离常数(K d)，选择亲和反应平衡解离常数为100μM以下的化合物，优选10μM以下，特别优选1μM以下，例如600nM以下、500nM以下、400nM以下、300nM以下、200nM以下、100nM以下，等。
在一优选的实施方案中，步骤(B)可以包括：测定候选化合物与(2)LC3B蛋白或其同源物或它们的片段的亲和反应平衡解离常数，选择亲和反应平衡解离常数为100μM以下的化合物，优选10μM以下，特别优选1μM以下，例如600nM以下、500nM以下、400nM以下、300nM以下、200nM以下、100nM以下，等。
在一个替代方案中，步骤(A)和步骤(B)同时在同一体系中进行，即，使候选化合物同时与(1)polyQ异常扩增蛋白或其含有polyQ部分的片段和(2)LC3B蛋白或其同源物或它们的片段接触，检测候选化合物分别与所述蛋白或其片段的亲和结合。
在一实施方案中，步骤(A)或步骤(B)用于测定亲和结合的方法选自接近闪烁分析、荧光共振能量转移、荧光偏振检测、荧光分子筛、微量热泳动、化学发光法、表面等离子体共振、等温滴定量热法、斜入射光反射差法及其任意组合。在一优选的实施方案中，步骤(A)或步骤(B)用于测定亲和结合的方法选自荧光共振能量转移、荧光偏振检测、斜入射光反射差法及其任意组合。在一特别优选的实施方案中，步骤(A)或步骤(B)用于测定亲和结合的方法为斜入射光反射差法。
在一优选的实施方案中，对候选化合物进行亲和活性预筛选的步骤通过采用斜入射光反射差法的高通量筛选进行；包括：
(a)对候选化合物进行固定化以制备化合物芯片，扫描芯片，得到孵育前的图像；
(b)将化合物芯片与靶蛋白进行孵育，扫描芯片，得到孵育后的图像；
其中靶蛋白为(1)polyQ异常扩增蛋白或其含有polyQ部分的片段或者(2)LC3B蛋白或其同源物或它们的片段；和
(c)对差异图像(孵育后的图像-孵育前的图像)进行分析，选择与(1)和(2)都能够亲 和结合的化合物。
在另一实施方案中，步骤(A)或步骤(B)用于测定平衡解离常数的方法选自表面等离子体共振、等温滴定量热法和微量热泳动。
在另一实施方案中，本发明的方法还可以包括步骤(D)：测定候选化合物与正常polyQ蛋白或其含有polyQ部分的片段的结合。该步骤例如可以在步骤(II)之后，或在步骤(C)之后进行。
在一实施方案中，所述结合为亲和结合。在一实施方案中，选择不与正常polyQ蛋白或其含有polyQ部分的片段亲和结合的化合物。步骤(D)可以按需要进行或不进行，也可以进行至少一次(例如1次、2次、3次或更多次)。当进行多于一次时，每次所用的正常polyQ多肽可以独立地为正常polyQ蛋白或其含有polyQ部分的片段。
步骤(D)所用的正常polyQ蛋白为步骤(I)中所用的polyQ异常扩增蛋白对应的正常polyQ蛋白。在一实施方案中，步骤(I)中所用的polyQ异常扩增蛋白包含polyQ异常扩增的HTT或其含有polyQ部分的片段。与之相应地，如果存在步骤(D)，其中所用的正常polyQ蛋白包含正常HTT或其含有polyQ的部分。在一示例性的实施方案中，步骤(I)中所用的polyQ异常扩增蛋白为polyQ异常扩增的HTT或其含有polyQ部分的片段，例如为polyQ长度≥36的HTT或其含有polyQ部分的片段，例如SEQ ID NO:1的氨基酸序列(HTTexon1-Q72)。与之相应地，如果存在步骤(D)，其中所用的正常polyQ蛋白为polyQ长度正常的HTT或其含有polyQ部分的片段，例如为polyQ长度<36的HTT或其含有polyQ部分的片段，例如SEQ ID NO:4的氨基酸序列(HTT-Q23)或SEQ ID NO:5的氨基酸序列(HTTexon1-Q25)。
在又一实施方案中，步骤(I)中所用的polyQ异常扩增蛋白包含polyQ异常扩增的ATXN3或其含有polyQ部分的片段。与之相应地，如果存在步骤(D)，其中所用的正常polyQ蛋白包含正常ATXN3或其含有polyQ的部分。在一示例性的实施方案中，步骤(I)中所用的polyQ异常扩增蛋白为polyQ异常扩增的ATXN3或其含有polyQ部分的片段，例如为polyQ长度≥41的ATXN3或其含有polyQ部分的片段，例如SEQ ID NO:2的氨基酸序列(ATXN3-Q74)。与之相应地，如果存在步骤(D)，其中所用的正常polyQ蛋白为polyQ长度正常的ATXN3或其含有polyQ部分的片段，例如为polyQ长度<41的ATXN3或其含有polyQ部分的片段，例如SEQ ID NO:7的氨基酸序列(ATXN3-Q27)。
在又一实施方案中，步骤(I)中所用的polyQ异常扩增蛋白包含polyQ异常扩增的ATXN1或其含有polyQ部分的片段。与之相应地，如果存在步骤(D)，其中所用的正常polyQ蛋白包含正常ATXN1或其含有polyQ的部分。在一示例性的实施方案中，步骤(I)中所用的polyQ异常扩增蛋白为polyQ异常扩增的ATXN1或其含有polyQ部分的片段，例如为polyQ长度≥40的ATXN1或其含有polyQ部分的片段，例如SEQ ID NO:3的氨基酸序列(ATXN1-Q92)。与之相应地，如果存在步骤(D)，其中所用的正常polyQ蛋白为polyQ长度正常的ATXN1或其含有polyQ部分的片段，例如为polyQ长度<40的 ATXN1或其含有polyQ部分的片段。
在另一实施方案中，步骤(I)中所用的polyQ异常扩增蛋白包含长度异常的polyQ，其被可检测的分子标记。在一实施方案中，步骤(I)中所用的polyQ异常扩增蛋白为长度异常的polyQ与荧光蛋白(例如GFP或YFP)的融合蛋白。在一优选的实施方案中，步骤(I)中所用的polyQ异常扩增蛋白为含有长度异常的polyQ的polyQ-GFP蛋白，优选地选自Q72-GFP、Q53-GFP、Q46-GFP。根据筛选针对的特定疾病，在一备选的实施方案中，步骤(I)中所用的polyQ异常扩增蛋白为Q38-GFP。
在又一实施方案中，步骤(D)中所用的正常polyQ蛋白为包含长度正常的polyQ，其被可检测的分子标记。在一实施方案中，步骤(D)中所用的正常polyQ蛋白为长度正常的polyQ与荧光蛋白(例如GFP或YFP)的融合蛋白。在一优选的实施方案中，步骤(D)中所用的正常polyQ蛋白为含有长度正常的polyQ的polyQ-GFP蛋白，优选地为Q25-GFP。根据筛选针对的特定疾病，在一备选的实施方案中，步骤(D)中所用的正常polyQ蛋白为Q38-GFP。
在一实施方案中，LC3B蛋白或其同源物或它们的片段与SEQ ID NO:8的氨基酸序列具有25％或更高、30％或更高、35％或更高、40％或更高、50％或更高、60％或更高、70％或更高、80％或更高、90％或更高、95％或更高或者100％的序列相同性。在一优选的实施方案中，LC3B蛋白的同源物来自果蝇或小鼠。
在一特别的实施方案中，LC3B蛋白的同源物为果蝇中的Atg8蛋白(与人LC3B的序列相同性为31％)。
在又一方面，本发明还提供一种筛选或鉴定用于治疗或预防polyQ相关的神经退行性疾病的化合物的方法，包括
(1)：测定候选化合物在细胞中对polyQ异常扩增蛋白的水平的影响，选择能降低所述蛋白水平的化合物；或
(2)：测定候选化合物在细胞中对polyQ异常扩增蛋白或其含有polyQ部分的片段的亚细胞定位的影响，选择能促进polyQ异常扩增蛋白或其含有polyQ部分的片段定位至具有自噬功能的细胞亚结构例如自噬小体的化合物；优选地，选择能促进polyQ异常扩增蛋白或其含有polyQ部分的片段与LC3B蛋白共定位的化合物，
其中polyQ异常扩增蛋白、其含有polyQ部分的片段、LC3B蛋白等术语如上文所定义。
在一实施方案中，步骤(1)选择在细胞中能够使polyQ异常扩增蛋白的水平降低10％以上的化合物，优选20％以上，例如约20％、约30％、约40％、约50％，等。
术语“多肽”是指一定长度的氨基酸的聚合物。因此，肽、寡肽和蛋白质包括在“多肽”的定义中，且这些术语在本文中可互换使用。术语“多肽”或“蛋白”不排除翻译后修饰，其包括但不限于磷酸化、乙酰化、糖基化等。本发明的蛋白或蛋白片段可通过本身是本领域已知的任何技术产生，例如但不限于单独或组合使用的任何化学、生物学、遗传学 或酶学技术。
知道所需序列的氨基酸序列后，本领域技术人员可以容易地通过产生蛋白或蛋白片段的标准技术来制备所述蛋白或蛋白片段。例如，它们可以通过用众所周知的固相法，优选用可商购的肽合成仪器(例如由Applied Biosystems，Foster City，California制备的那些)并根据厂商指导来合成。
或者，本发明的蛋白或蛋白片段可通过本领域众所周知的重组DNA技术来合成。例如，在将编码所需(多)肽的DNA序列引入表达载体并将这种载体引入表达所需蛋白或蛋白片段的适当的真核或原核宿主后，这些片断可作为DNA表达产物获得，随后可以用众所周知的技术将它们从宿主中分离出来。
多种宿主/表达载体组合可用于表达编码本发明蛋白或蛋白片段的核酸。可使用的表达载体包括例如染色体片断、非染色体和合成DNA序列。适当的载体包括但不限于SV40和pcDNA的衍生物；和已知的细菌质粒，例如col EI、pCR1、pBR322、pMal-C2、pET、pGEX、pMB9及其衍生物；质粒，例如RP4；噬菌体DNA，例如噬菌体I的众多衍生物，例如NM989，以及其他的噬菌体DNA，例如M13和细丝状单链噬菌体DNA；酵母质粒，例如2微米质粒或2微米质粒的衍生物，以及着丝粒和整合酵母穿梭载体；用于真核细胞中的载体，例如可用于昆虫或哺乳动物细胞中的载体；来源于质粒和噬菌体DNA的组合的载体，例如已修饰以使用噬菌体DNA或表达控制序列的质粒；等等。
因此，哺乳动物和典型的人细胞，以及细菌、酵母、真菌、昆虫、线虫和植物细胞可用于本发明中，且可通过此处定义的核酸或重组载体来转染。适当细胞的实例包括但不限于，VERO细胞；HELA细胞，例如ATCC No.CCL2；CHO细胞系，例如ATCC No.CCL61；COS细胞，例如COS-7细胞和ATCC No.CRL 1650细胞；W138、BHK、HepG2、3T3，例如ATCC No.CRL6361；A549、PC12、K562细胞、293T细胞、Sf9细胞，例如ATCC No.CRL1711和Cv1细胞，例如ATCC No.CCL70。可用于本发明的其他适当的细胞包括但不限于原核宿主细胞菌株，例如大肠杆菌(例如菌株DH5-[α])、枯草杆菌、鼠伤寒沙门氏菌或假单胞菌属、链霉菌属和葡萄球菌属的菌株。可用于本发明的其他适当的细胞包括酵母细胞，例如酵母属细胞，例如酿酒酵母。
本领域技术人员可以理解，为了例如方便试验操作、增加溶解度、便于结晶、纯化等目的，可以对本发明的蛋白或蛋白片段进行修饰或改造，但并不影响蛋白或蛋白片段的功能，这样的蛋白或蛋白片段及其应用同样属于本发明的范畴。
在一实施方案中，(1)polyQ异常扩增蛋白或其含有polyQ部分的片段或者(2)LC3B蛋白或其同源物或它们的片段可以被可检测的分子标记以用于筛选目的。
可检测的分子可以由能通过分光镜、光化学、生物化学、免疫化学或化学手段检测的任何化合物或物质组成。例如，有用的可检测分子包括放射性物质(包括含有32P、25S、3H或125I的那些)、荧光染料(包括5-溴脱氧尿苷、荧光素、乙酰氨基芴或毛地黄毒苷)、 荧光蛋白(例如GFP和YFP，其中GFP例如可以为sfGFP)。
在一实施方案中，可检测标记位于或结合至位于(1)polyQ异常扩增蛋白或其含有polyQ部分的片段或者(2)LC3B蛋白或其同源物或它们的片段的序列外部的氨基酸残基上，从而最小化或阻止任何对所述蛋白或蛋白片段之间或者候选化合物和任何所述蛋白或蛋白片段之间的结合产生人为影响。
在一具体实施方案中，本发明的蛋白或蛋白片段与荧光蛋白例如GFP标签(绿色荧光蛋白)融合。在又一具体实施方案中，本发明的蛋白或蛋白片段按照适用于荧光能量转移分析的方式，分别被合适的荧光基团标记。
不管本发明的筛选方法的步骤(I)、(II)、(A)、(B)、(C)、(b)、步骤(1)或步骤(2)的实施方式，完整的polyQ异常扩增蛋白和完整的LC3B蛋白或其同源物可用于所述测定。或者，包括结合位点的polyQ异常扩增蛋白片段和LC3B蛋白或其同源物的片段可用于所述测定。
在进一步的方面，本发明提供一种筛选系统，包含两个以上的部分，其中(A)部分包含非标记或以适当形式标记的polyQ异常扩增蛋白或其含有polyQ部分的片段，并且(B)部分包含非标记或以适当形式标记的LC3B蛋白或其同源物或它们的片段，每个部分各自独立地相互隔离或不隔离。每个部分各自独立地任选包含合适的缓冲液和/或检测试剂。在一具体实施方案中，本发明的筛选系统为试剂盒的形式。在另一具体实施方案中，将制备好的化合物芯片放置于本发明的试剂盒中，与(1)polyQ异常扩增蛋白或其含有polyQ部分的片段和(2)LC3B蛋白或其同源物或它们的片段孵育后，用合适的读板仪器分析信号，筛选得到与(1)和(2)都能够结合的化合物。
本发明的化合物
本发明还涉及通过本发明的方法获得的化合物。本发明的化合物涵盖其药学上可接受的盐、立体异构体、溶剂化物、多晶型、互变异构体、同位素化合物、代谢产物或前药。
术语“药学上可接受的”是指在正常的医学判断范围内与患者的组织接触而不会有不适当毒性、刺激性、过敏反应等。
本发明的化合物的药学上可接受的盐包括其酸加成盐及碱加成盐。用于制备本发明的化合物的药学上可接受的盐的方法为本领域技术人员已知的。
本发明的化合物可以存在特定的几何或立体异构体形式，包括顺式和反式异构体、(-)-和(+)-对对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本发明的范围之内。
本发明的化合物可以溶剂化物(优选水合物)的形式存在，其中本发明的化合物包含作为所述化合物晶格的结构要素的极性溶剂，特别是例如水、甲醇或乙醇。极性溶剂特 别是水的量可以化学计量比或非化学计量比存在。
本发明还涵盖本发明的化合物的所有可能的结晶形式或多晶型物，其可为单一多晶型物或多于一种多晶型物的任意比例的混合物。
在本发明的范围内还包括本发明的化合物的代谢产物，即在给药本发明的化合物时体内形成的物质。这样的产物可由例如由给药的化合物的氧化、还原、水解、酰胺化、脱酰胺化、酯化、酶解等产生。
本发明在其范围内进一步包括本发明的化合物的前药，其为自身可具有较小药理学活性或无药理学活性的本发明的化合物的某些衍生物当被给药至身体中或其上时可通过例如水解裂解转化成具有期望活性的本发明的化合物。
术语“多晶型”或“多晶型物”是指单一多晶型物或多于一种多晶型物的任意比例的混合物。
术语“晶型”或“晶体”是指呈现三维排序的任意固体物质，与无定形固体物质相反，其产生具有边界清楚的峰的特征性X-射线粉末衍射图谱。
术语“无定形”是指三维上无排序的任意固体物质。
本发明的化合物可以以药物组合物的方式应用。所述药物组合物包含所述化合物或其药学上可接受的盐、立体异构体、溶剂化物、多晶型、互变异构体、同位素化合物、代谢产物或前药，以及至少一种药学上可接受的载体。
术语“药学上可接受的载体”是指对有机体无明显刺激作用，而且不会损害该活性化合物的生物活性及性能的那些物质。“药学上可接受的载体”包括但不限于助流剂、增甜剂、稀释剂、防腐剂、染料/着色剂、矫味剂、表面活性剂、润湿剂、分散剂、崩解剂、稳定剂、溶剂或乳化剂。
在一方面，本发明提供本发明的化合物或其药物组合物在制备用于治疗或预防polyQ相关的神经退行性疾病的药物中的用途。
在又一方面，本发明提供本发明的化合物，用于治疗或预防polyQ相关的神经退行性疾病。
在另一方面，本发明提供治疗或预防polyQ相关的神经退行性疾病的方法，包括向有此需要的个体给药本发明的化合物。
在又一方面，本发明提供正向调节(1)polyQ异常扩增蛋白或其含有polyQ部分的片段与(2)LC3B蛋白或其同源物或它们的片段之间的结合的化合物或其药物组合物在制备用于治疗或预防polyQ相关的神经退行性疾病的药物中的用途。
在另一方面，本发明提供正向调节(1)polyQ异常扩增蛋白或其含有polyQ部分的片段与(2)LC3B蛋白或其同源物或它们的片段之间的结合的化合物或其药物组合物，用于治疗或预防polyQ相关的神经退行性疾病。
在又一方面，本发明提供治疗或预防polyQ相关的神经退行性疾病的方法，包括向有此需要的个体给药正向调节(1)polyQ异常扩增蛋白或其含有polyQ部分的片段与(2) LC3B蛋白或其同源物或它们的片段之间的结合的化合物或其药物组合物。
正向调节(1)polyQ异常扩增蛋白或其含有polyQ部分的片段与(2)LC3B蛋白或其同源物或它们的片段之间的结合的化合物可以例如通过本发明的方法获得。
在一优选的实施方案中，polyQ相关的神经退行性疾病选自脊髓小脑共济失调1型(SCA1)、脊髓小脑共济失调2型(SCA2)、脊髓小脑共济失调3型(SCA3)、脊髓小脑共济失调7型(SCA7)、脊髓小脑共济失调12型(SCA12)、脊髓小脑共济失调17型(SCA17)、齿状核-黑质-红核-苍白球-丘脑底核萎缩症(DRPLA)、亨廷顿病(HD)和脊髓延髓肌肉萎缩(SBMA)，特别是亨廷顿病和脊髓小脑共济失调3型。在一更优选的实施方案中，polyQ相关的神经退行性疾病选自亨廷顿病和SCA3；特别是亨廷顿病。
在进一步的方面，本发明提供一种制品，例如以试剂盒形式提供。本发明的制品包含本发明的化合物或药物组合物，并任选地包括包装盒和说明书。
在又一方面，本发明提供本发明的化合物用于筛选或鉴定可以通过自噬途径降解的靶蛋白的用途。在一实施方案中，例如可以通过以下方法用筛选得到的化合物寻找靶蛋白。1)筛选或鉴定能够与所述化合物亲和结合的蛋白；2)测定所述化合物调节1)中得到的蛋白与LC3B蛋白或其同源物或它们的片段之间的结合的能力，其中如果所述化合物能够正向调节所述结合，则1)中得到的蛋白可以通过自噬途径降解。
在另一方面，本发明提供本发明的化合物或其药物组合物在制备用于检测被认为患有或易患polyQ相关的神经退行性疾病的受试者的诊断试剂或试剂盒中的用途。在一实施方案中，上述测试包括分析从所述受试者获得的目标样品的步骤，包括：
i)将所述化合物用可检测标记例如荧光基团进行标记；
ii)用i)中得到的化合物处理目标样品；
iii)检测目标样品中可检测信号的位置和/或强度，获得目标样品中polyQ异常扩增蛋白或其含有polyQ部分的片段的位置和/或含量。
在一优选的实施方案中，目标样品选自细胞样品、血液、血清、脊髓液；或从受试者的组织中获得的任何活检样品。
在进一步的方面，本发明提供一种检测试剂盒。所述试剂盒包含本发明的化合物或其药物组合物，并且任选地包含检测需要的试剂，例如水溶液、溶剂、合适的检测试剂例如化学发光试剂，等。
本发明还涉及化合物，选自：
Figure PCTCN2020078775-appb-000001
Figure PCTCN2020078775-appb-000002
如前所述，本发明的实施方案可以列举如下：
[1]一种筛选或鉴定用于治疗或预防polyQ相关的神经退行性疾病的化合物的方法，包括
(I)：使候选化合物与待测体系接触，所述待测体系包含
(1)polyQ异常扩增蛋白或其含有polyQ部分的片段，和
(2)LC3B蛋白或其同源物或它们的片段；和
(II)：测定所述候选化合物调节(1)polyQ异常扩增蛋白或其含有polyQ部分的片段与(2)LC3B蛋白或其同源物或它们的片段之间的结合的能力，并且选择正向调节所述结合的化合物。
[2]上述[1]的方法，其中步骤(II)包括：
测定存在/不存在所述候选化合物的条件下，(1)polyQ异常扩增蛋白或其含有polyQ部分的片段与(2)LC3B蛋白或其同源物或它们的片段之间的结合亲和力大小，和/或
测定存在/不存在所述候选化合物的条件下，(1)polyQ异常扩增蛋白或其含有polyQ部分的片段与(2)LC3B蛋白或其同源物或它们的片段的结合量。
[3]上述[1]或[2]的方法，其中所述polyQ异常扩增蛋白包含polyQ长度≥40、≥33、≥41、≥19、≥46、≥44、≥39、≥36或≥37的polyQ部分；
优选地，所述polyQ异常扩增蛋白包含
polyQ长度≥40的ATXN1或其含有polyQ部分的片段，
polyQ长度≥33的ATXN2或其含有polyQ部分的片段，
polyQ长度≥41的ATXN3或其含有polyQ部分的片段，
polyQ长度≥19的ATXN7或其含有polyQ部分的片段，
polyQ长度≥46的ATXN12或其含有polyQ部分的片段，
polyQ长度≥44的TBP或其含有polyQ部分的片段，
polyQ长度≥39的ATN1或其含有polyQ部分的片段，
polyQ长度≥36的HTT或其含有polyQ部分的片段，或
polyQ长度≥37的AR或其含有polyQ部分的片段。
[4]上述[1]-[3]中任一项的方法，其中(2)LC3B蛋白或其同源物或它们的片段与SEQ ID NO:8的氨基酸序列具有25％或更高、30％或更高、35％或更高、40％或更高、50％或更高、60％或更高、70％或更高、80％或更高、90％或更高、95％或更高或者100％ 的序列相同性。
[5]上述[1]-[4]中任一项的方法，其中测定所述结合的方法选自体外拉下实验、Split-TEV、免疫共沉淀、亲和层析、复合物共纯化、酶联免疫吸附法、荧光分子筛、酵母双杂交、HIP-HOP法、时间分辨荧光共振能量转移、化学发光法、表面等离子体共振、等温滴定量热法、微量热泳动及其任意组合；优选地，测定所述结合的方法选自体外拉下实验、免疫共沉淀、酶联免疫吸附法、荧光分子筛和时间分辨荧光共振能量转移及其任意组合。
[6]上述[1]-[5]中任一项的方法，其中在步骤(I)之前还包括对候选化合物进行亲和活性预筛选的步骤，包括：
(A)：检测候选化合物与(1)polyQ异常扩增蛋白或其含有polyQ部分的片段的亲和结合，和/或(B)：检测候选化合物与(2)LC3B蛋白或其同源物或它们的片段的亲和结合；以及
(C)：选择与(1)polyQ异常扩增蛋白或其含有polyQ部分的片段和/或(2)LC3B蛋白或其同源物或它们的片段能够亲和结合的化合物；
其中步骤(A)和步骤(B)可以以任何顺序独立地进行。
[7]上述[6]的方法，其中
步骤(A)包括：测定候选化合物与(1)polyQ异常扩增蛋白或其含有polyQ部分的片段的亲和反应平衡解离常数，选择所述亲和反应平衡解离常数为100μM以下的化合物，优选10μM以下，特别优选1μM以下；和/或
步骤(B)包括：测定候选化合物与(2)LC3B蛋白或其同源物或它们的片段的亲和反应平衡解离常数，选择所述亲和反应平衡解离常数为100μM以下的化合物，优选10μM以下，特别优选1μM以下。
[8]上述[6]或[7]的方法，其中用于测定亲和结合的方法选自接近闪烁分析、荧光共振能量转移、荧光偏振检测、荧光分子筛、微量热泳动、化学发光法、表面等离子体共振、等温滴定量热法、斜入射光反射差法及其任意组合；优选地选自荧光共振能量转移、荧光偏振检测、斜入射光反射差法及其任意组合；特别优选为斜入射光反射差法。
[9]上述[6]-[8]中任一项的方法，其中
所述对候选化合物进行亲和活性预筛选的步骤通过采用斜入射光反射差法的高通量筛选进行；包括：
(a)对候选化合物进行固定化以制备化合物芯片，扫描芯片，得到孵育前的图像；
(b)将化合物芯片与靶蛋白进行孵育，扫描芯片，得到孵育后的图像；
其中靶蛋白为(1)polyQ异常扩增蛋白或其含有polyQ部分的片段或者(2)LC3B蛋白或其同源物或它们的片段；和
(c)对差异图像(孵育后的图像-孵育前的图像)进行分析；选择与(1)polyQ异常扩增蛋白或其含有polyQ部分的片段和(2)LC3B蛋白或其同源物或它们的片段都能够亲和 结合的化合物。
[10]上述[1]-[9]中任一项的方法，还包括以下步骤：
(D)：测定候选化合物与正常polyQ蛋白或其含有polyQ部分的片段的结合，选择不与正常polyQ蛋白或其含有polyQ部分的片段亲和结合的化合物。
[11]通过上述[1]-[10]中任一项的方法获得的化合物或其药物组合物在制备用于治疗或预防polyQ相关的神经退行性疾病的药物中的用途，或者在制备用于检测被认为患有或易患polyQ相关的神经退行性疾病的受试者的诊断试剂或试剂盒中的用途；
优选地，其中所述polyQ相关的神经退行性疾病选自脊髓小脑共济失调1型、脊髓小脑共济失调2型、脊髓小脑共济失调3型、脊髓小脑共济失调7型、脊髓小脑共济失调12型、脊髓小脑共济失调17型、齿状核-黑质-红核-苍白球-丘脑底核萎缩症、亨廷顿病和脊髓延髓肌肉萎缩，特别是亨廷顿病和脊髓小脑共济失调3型。
[12]上述[11]的用途，其中所述检测包括分析从所述受试者获得的目标样品的步骤，包括：
i)将所述化合物用可检测标记例如荧光基团进行标记；
ii)用i)中得到的化合物处理目标样品；
iii)检测目标样品中可检测信号的位置和/或强度，获得目标样品中polyQ异常扩增蛋白或其含有polyQ部分的片段的位置和/或含量。
[13]化合物或其药物组合物在制备用于治疗或预防polyQ相关的神经退行性疾病的药物中的用途，或者在制备用于检测被认为患有或易患polyQ相关的神经退行性疾病的受试者的诊断试剂或试剂盒中的用途，所述化合物选自：
Figure PCTCN2020078775-appb-000003
[14]一种药物组合物、检测试剂或试剂盒，其包含通过上述[1]-[10]中任一项的方法获得的化合物。
[15]一种药物组合物、检测试剂、试剂盒或筛选系统，其包含至少一种选自以下的化合物：
Figure PCTCN2020078775-appb-000004
[16]一种筛选系统，其包含
(1)polyQ异常扩增蛋白或其含有polyQ部分的片段，和
(2)LC3B蛋白或其同源物或它们的片段；
优选地，所述筛选系统为试剂盒的形式。
[17]正向调节(1)polyQ异常扩增蛋白或其含有polyQ部分的片段与(2)LC3B蛋白或其同源物或它们的片段之间的结合的化合物或其药物组合物在制备用于治疗或预防polyQ相关的神经退行性疾病的药物中的用途。
有益效果
本发明的方法筛选得到的化合物可以通过靶向自噬，选择性地降低polyQ异常扩增蛋白在细胞内的水平，并且对细胞的自噬功能没有影响。因此，本发明的方法能够有效地筛选治疗或预防polyQ相关的神经退行性疾病的化合物。
自噬是一种高度遗传保守的蛋白降解途径，普遍存在于真核细胞中，蛋白降解能力强，但选择性较低。目前的研究主要为自噬调节剂，没有解决非特异性调节自噬功能带来的全局效应问题，因而存在潜在的副作用。发明人出人意料地发现，本发明的筛选方法可以得到特异性和自噬依赖性降低polyQ异常扩增蛋白水平的化合物，其具有较低的副作用和良好的安全性，易于透过BBB，有利于通过口服给药。并且，本发明的筛选方法还具有容易实施、筛选效率高的优点。
实施例
除非特别说明，本文使用的仪器和试剂材料均为可商购的，或者可以通过本领域已知的常规方法进行制备。
除非特别说明，本文的所有统计分析中，*表示p<0.05；**表示p<0.01；***表示p<0.001,****表示p<0.0001。对两组之间的比较，所使用的统计分析方法为双尾非配对t检验。对三组或以上组之间的比较，在只有一个变量影响的情况下，所使用方法为双尾单向方差分析，在有两个变量影响的情况下，所使用方法为双尾双向方差分析。
缩写
Figure PCTCN2020078775-appb-000005
实验材料、试剂与方法步骤
化合物
实施例中所用化合物库由Selleck公司提供，含有3375种生物活性化合物。其中包括美国食品药品管理局(FDA)批准的1527种药物、1053种来自中药的天然产物和795种已知的抑制剂。其中：
化合物1：GW5074，可以购自DC Chemicals，目录号DC8810；
化合物2：ispinesib，PubChem CID:6851740，可以购自Selleck，目录号S1452；
化合物3：PubChemID 1759437，可以购自Specs，目录号AN-655/15003575；
化合物4：PubChem CID 5398649，可以购自ChemDiv，目录号D715-2435；
化合物5：Semaxanib，可以购自Selleck，CAS No.194413-58-6；
化合物6：Su9516，可以购自Selleck，CAS No.377090-84-1；
化合物7：可以购自TargetMol，CAS No.842-01-3；
化合物8：可以购自ChemDiv。
抗体
HTT抗体2B7(Weiss et al.Anal Biochem 2009,395,8-15)，4C9，ab1(Sapp et al.J Biol Chem 2012,287,13487-13499)和MW1(Ko et al.Brain research bulletin 2001,56,319-329)采用现有技术的方法制备；用于免疫染色检测HTT聚集体的抗体S830获赠自Gillian Bates博士；其他抗体购自Millipore、Sigma等公司。
重组人LC3B的制备
(1)在pGEX-6P1(来自GE Healthcare)中添加His8标签和TEV蛋白酶切割位点，制 备原核表达载体pGHT。编辑LC3B基因(GenBank:NM_022818.4)以在LC3B的N端移除Met并增加两个Gly，扩增基因并克隆到制备的pGHT中。
(2)将表达质粒pGHT-LC3B导入大肠杆菌BL21(DE3)pLsyS中表达。用HisTrap HP柱(GE Healthcare，17524701)纯化。然后与TEV蛋白酶(Sigma，T4455)混合，透析过夜。依次用HisTrap HP柱、Superose 6 Increase 10/300GL尺寸排阻柱(GE Healthcare)纯化。
(3)LC3B的验证：通过MALDI-TOF-MS和X射线衍射，验证制备得到的人LC3B。X射线衍射验证LC3B的方法为：定点突变得到缺失G120脂化位点的突变体LC3BΔG120蛋白，将该更稳定的蛋白用于高分辨X射线衍射。用已知LC3蛋白晶体结构(PDB ID:1UGM)作为分子置换的搜索模型，通过分子置换来解析公开的LC3BΔG120的结构(PDB ID:6J04,
Figure PCTCN2020078775-appb-000006
)，进行结构对齐。
重组人HTTexon1-MBP蛋白的制备
(1)从头合成25Q或72Q的HTTexon1cDNA，克隆到哺乳动物表达载体pTT5SH8Q2中，在HTTexon1序列后加入C末端MBP标签，得到pTT-HTTexon1-Q72-MBP和pTT-HTTexon1-Q25-MBP质粒。
(2)用线性化聚乙烯亚胺(Polysciences,24765)将质粒转染到HEK293T细胞中表达。用HisTrap HP柱和Superose 6Increase 10/300GL尺寸排阻柱纯化蛋白。
(3)通过Superose 6Increase尺寸排阻柱将纯化的HTTexon1-MBP蛋白透析到5mM NH 4Ac中，用Bruker FLEX MALDI-TOF验证。
重组人全长HTT蛋白的制备
(1)具有(CAG) 23或(CAG) 73的人HTT基因(GenBank:NM_002111.8)由Genewiz Inc.从头合成。将人HTT基因克隆到具有N末端蛋白A标签的修饰的pCAG载体(来自Addgene)中。
(2)使用聚乙烯亚胺(PEI，来自Polysciences，23966)将质粒转染到人胚胎肾E293细胞中表达。用IgG单抗-琼脂糖(Smart-lifesciences,SA030010)纯化，用TEV蛋白酶消化去除蛋白A标签，用来自GE healthcare的Mono Q和Superose 6(5/150GL)柱进一步纯化蛋白。通过考马斯蓝染色和蛋白印迹验证。
重组人MBP-ATXN3的制备
(1)在pMal-C2x质粒(来自New England Biolabs)中添加His8标签和TEV蛋白酶切割位点，制备原核表达载体pMBP。编辑ATXN3基因(GenBank:NM_001127696.2)以控制谷氨酰胺重复区域的长度，扩增基因并克隆到制备的pMBP中，得到polyQ分别重复28次和68次的pMBP-ATXN3-Q28和pMBP-ATXN3-Q68质粒。
(2)将表达质粒pMBP-ATXN3-Q28和pMBP-ATXN3-Q68导入大肠杆菌Rosetta(DE3)pLsyS中表达，用HisTrap HP柱(GE Healthcare，17524701)初步纯化，产物超滤浓缩后，继续用Superose 6 Increase 10/300GL尺寸排阻柱(GE Healthcare)纯化。
(3)通过SDS-PAGE验证，制备得到的人MBP-ATXN3-Q28和MBP-ATXN3-Q68纯 度超过98％。
用于试验的细胞
原代培养的皮质神经元：Hdh Q140/Q7和Hdh Q7/Q7新生小鼠(P0)的大脑解剖后，消化、解离后培养得到。
一些原发性患者成纤维细胞和野生型细胞来自蒙古亨廷顿病家族的HD患者(Q47,Q55)和健康对照(WT,Q19)。SCA3细胞系来自患者(Q74)。HD Q68成纤维细胞系来自Coriell Cell Repositories(Camden,NJ,USA)。永生化的成纤维细胞和iPS细胞(iPSC)由原代成纤维细胞制备得到。小鼠纹状体细胞(STHdh)来自Coriell Cell Repositories(Camden,NJ,USA)。HEK293T细胞和HeLa细胞来自ATCC。
用于试验的动物
亨廷顿病果蝇
神经系统驱动品系elav-GAL4(c155)、表达HTT的品系UAS-fl-HTT-Q16和UAS-fl-HTT-Q128来自印第安纳大学的Bloomington Drosophila Stock Center(http://flystocks.bio.indiana.edu/)，并保持在25℃培养箱中。
通过elav-GAL4处女蝇和UAS-fl-HTT-Q16或UAS-fl-HTT-Q128雄性果蝇杂交，获得由elav-GAL4驱动在神经系统中表达人HTT全长蛋白(Q16)或(Q128)的转基因果蝇。
亨廷顿病小鼠
表达野生型HTT基因的小鼠(Hdh Q7/Q7)，来自哈佛大学麻省总医院Marian Difiglia实验室。按照现有技术(Menalled等，J Comp Neurol,2003,465:11-26)的方法制备Q140基因敲入杂合小鼠(Hdh Q140/Q7)。
蛋白分析
均相时间分辨荧光(HTRF)分析：用原始裂解缓冲液PBS+1％(v/v)Triton X-100+1×cOmplete TM蛋白酶抑制剂稀释细胞或组织裂解物，裂解样品，然后用HTRF测定缓冲液(50mM NaH 2PO 4,400mM NaF,0.1％BSA,0.05％(v/v)Tween-20,1％(v/v)Triton X-100,pH 7.4)稀释的指定的抗体对，进行检测。在HTRF缓冲液中，供体抗体浓度为0.023ng/μL，受体抗体浓度为1.4ng/μL。
蛋白的量的测定：通过上述方法测定蛋白的量。通过空白样品进行背景校正。对于所有样品均测定蛋白的浓度，以校正蛋白的量。测定每孔的不同蛋白浓度或细胞数以确保信号在线性范围内。
细胞分析
免疫荧光：将细胞洗涤固定、透化、封闭后，在4℃下与一抗孵育过夜，然后用封闭缓冲液洗涤三次，在室温下与二抗孵育1小时。用DAPI染色，封固后，用Zeiss Axio Vert A1共聚焦显微镜成像，用ImageJ分析TUBB3或共定位情况。
实施例1 化合物对蛋白亲和活性的高通量筛选
1.1 亲和活性筛选
(1)使用接触式微阵列点印机(SmartArrayer 136,CapitalBio Corporation)，按照现有技术(Zhu et al.,Sensors(Basel)2016,16(3),378；Fei et al.,J Biomed Opt 2010,15(1),016018)的方法制备化合物芯片。每个化合物各点印两份。洗去未固定在芯片表面的化合物，封闭、清洗芯片。用OI-RD显微镜扫描图像。以HTTexon1-Q72-MBP(添加了MBP标签的SEQ ID NO:1，其中使用MBP标签以增加溶解度)为靶蛋白。将化合物芯片与靶蛋白(浓度为238nM)一起孵育2小时。清洗、扫描芯片。
(2)以HTTexon1-Q25-MBP(添加了MBP标签的SEQ ID NO:5)为靶蛋白(浓度454nM)，按照(1)的方法，获取与靶蛋白孵育前、孵育后的OI-RD图像。
(3)以LC3B(SEQ ID NO:8)为靶蛋白(浓度680nM)，按照(1)的方法，获取与靶蛋白孵育前、孵育后的OI-RD图像。
分别分析以上步骤(1)、(2)、(3)的差异图像(孵育后的图像-孵育前的图像)，选择与HTTexon1-Q72-MBP和LC3B结合、不与HTTexon1-Q25-MBP结合的化合物(OI-RD显微镜检测限为约0.001mV)。得到能够与LC3B和具有异常扩增的polyQ的HTTexon1结合、不与具有正常polyQ的HTTexon1结合的化合物：化合物1(GW5074)和化合物2(ispinesib)。
Figure PCTCN2020078775-appb-000007
按照上述方法用化合物1和2制备芯片。每个化合物各点印三份。用OI-RD测量化合物与HTTexon1-Q72、HTTexon1-Q25的亲和反应参数。
化合物与HTTexon1-Q25没有亲和结合，与HTTexon1-Q72的亲和反应参数如表1所示。
表1.化合物与HTTexon1-Q72的亲和反应的结合速率常数、解离速率常数和平衡解离常数
  K on(min·nM) -1 K off(min) -1 K d(nM)
化合物1 9.07×10 -5 1.43×10 -2 157.6
化合物2 2.13×10 -4 6.23×10 -3 29.2
由可商购的已知化合物中，选取数目不大于20个的具备如化合物1和2的双环结构和含苯环的侧链，以及包含制备小分子芯片所需的反应官能团(例如OH，SH，NH等)的化合物，按照上文的方法，筛选得到两个能够与HTTexon1-Q72和LC3B结合，并且不与HTTexon1-Q25结合的化合物：化合物3和化合物4。
Figure PCTCN2020078775-appb-000008
按照上述方法用化合物1、2、4制备芯片。每个化合物各点印三份。用OI-RD测量化合物与LC3B、全长mHTT(flHTT-Q73)和全长HTT-Q23(flHTT-Q23)的亲和反应参数。
化合物与flHTT-Q23(SEQ ID NO:4)没有亲和结合，与flHTT-Q73和LC3B的亲和反应参数分别如表2和表3所示。
表2.化合物与flHTT-Q73的亲和反应的结合速率常数、解离速率常数和平衡解离常数
  K on(min·nM) -1 K off(min) -1 K d(nM)
化合物1 5.43×10 -5 7.97×10 -3 14.7
化合物2 6.43×10 -5 2.07×10 -2 321.9
化合物4 4.81×10 -4 1.19×10 -2 24.7
表3.化合物与LC3B的亲和反应的结合速率常数、解离速率常数和平衡解离常数
  K on(min·nM) -1 K off(min) -1 K d(nM)
化合物1 3.72×10 -5 1.74×10 -2 467.7
化合物2 2.46×10 -5 5.86×10 -3 238.2
化合物4 4.61×10 -5 2.49×10 -2 540.1
1.2 化合物与全长HTT、LC3B的亲和活性的MST检测
用微量热泳动仪(MST，其中Monolith NT.115仪器来自NanoTemper Technologies)对化合物与全长HTT和LC3B的亲和活性进行验证。反应缓冲液为20mM HEPES,pH 7.4,150mM NaCl，蛋白浓度500nM。化合物与flHTT-Q23(SEQ ID NO:4)没有亲和结合，与flHTT-Q73和LC3B的K d分别如表4所示。
表4.化合物与待测蛋白的亲和反应平衡解离常数
Figure PCTCN2020078775-appb-000009
发明人还通过MST验证了化合物在同时存在全长HTT和LC3B的体系中，对二者分别的亲和活性(图1)。
综上，OI-RD检测和MST检测结果均表明化合物能与LC3B亲和结合；对于HTT，化合物选择性地亲和结合mHTT(HTTexon1-Q72或flHTT-Q73)。这表明，化合物能够正向调节LC3B与mHTT之间的结合作用。
实施例2 化合物对HD模型小鼠皮质神经元的作用
2.1 对mHTT和wtHTT水平的影响
(1)用化合物处理原代培养的Hdh Q140/Q7和Hdh Q7/Q7小鼠皮质神经元细胞，2天后通过蛋白印迹(2166抗体)检测mHTT、wtHTT水平(图2和图3)。化合物使Q140基因敲入杂合小鼠(Hdh Q140/Q7)皮质神经元mHTT水平降低，而几乎不影响wtHTT水平。化合物1和2不降低Hdh Q7/Q7小鼠皮质神经元wtHTT水平。其中化合物1在30-300nM范围内(100nM较佳)、化合物2在30-100nM范围内(100nM较佳)、化合物3在10-300nM范围内(50nM较佳)、化合物4在15-150nM范围内(75nM较佳)均观察到选择性降低mHTT水平的效果。
(2)使用与(1)相似的方法，分别用多种抗体(2166、ab1、D7F7、3B5H10)检测化合物处理后Hdh Q140/Q7小鼠皮质神经元细胞中的mHTT水平(图4)。多种抗体检测得到相对一致的结果，因此检测到的mHTT水平降低不是由于mHTT对特定抗体的亲和力的变化。
用抗polyQ抗体MW1和3B5H10检测mHTT，并且观察分子量较小的蛋白的条带，结果没有观察到mHTT的N-端碎片的增加(图5)。检测到的mHTT水平降低不是由于位点特异性剪切的增加。
2.2 细胞毒性测试
按照试剂盒中提供的方案，通过CellTiter-glo(Promega，G7570)测定化合物处理后Hdh Q140/Q7小鼠皮质神经元细胞的活力(图6)。化合物在2.1所述试验浓度下，对Hdh Q140/Q7小鼠皮质神经元没有细胞毒性。检测到的mHTT水平降低不是由于神经元细胞丧失。
综上，本发明的方法筛选得到的化合物在细胞内均表现出优异的等位基因选择性地降低mHTT水平的作用。其中从含有3375个生物活性化合物的结构多样性化合物库中筛选得到2个经验证在细胞水平具有活性的化合物，阳性率0.6‰。
实施例3 化合物对亨廷顿病患者成纤维细胞HTT水平的影响
3.1 对HD患者原代成纤维细胞的mHTT和wtHTT水平的影响
进行预实验，确定化合物在100nM浓度下表现出最佳的降低mHTT水平的效果。
试验方法：用100nM浓度的化合物处理HD患者成纤维细胞(Q49、Q55、Q68)，2天后通过HTRF检测mHTT(抗体对：2B7/MW1)和总HTT(抗体对：2B7/2166)，结果如 图7所示。在HD患者原代成纤维细胞(Q49、Q55、Q68)上观察到mHTT水平降低。在野生型原代成纤维细胞上没有观察到HTT水平降低。
3.2 对HD患者永生化成纤维细胞mHTT水平的影响
采用与3.1中所述相似的试验方法，通过HTRF(抗体对：2B7/MW1)检测化合物在存在/不存在自噬抑制剂的条件下对HD患者永生化成纤维细胞mHTT水平的影响(图8)。不存在自噬抑制剂的条件下，化合物显著降低HD患者永生化成纤维细胞的mHTT水平，与在HD患者原代成纤维细胞上观察到的结果一致。而在存在自噬抑制剂(NH 4Cl或氯喹)的条件下，没有观察到mHTT水平的降低。
据报道，化合物1还具有c-Raf抑制剂活性，化合物2还具有驱动蛋白纺锤体激酶(KSP)抑制活性。为此发明人对一些c-Raf抑制剂和KSP抑制剂进行了检测(包括GSK923295、BAY1217389、PLX-4720、Dabrafenib、Sorafenib Tosylate)。使用100nM浓度的上述c-Raf抑制剂和KSP抑制剂分别处理HD患者永生化成纤维细胞，没有观察到mHTT水平的降低。使用PLX-4720(c-Raf抑制剂)、BAY1217389(KSP抑制剂)与化合物4进行对照，PLX-4720和BAY1217389在低于微摩尔级的浓度下不能降低mHTT水平(图9)。因此，化合物对mHTT的作用不是由c-Raf或KSP抑制活性带来的。
综上，筛选得到的化合物对mHTT水平的降低是自噬依赖性的。
实施例4 化合物对HD患者诱导干细胞分化神经元mHTT水平和神经元凋亡的影响
4.1 对mHTT和wtHTT水平的影响
采用与3.1中所述相似的试验方法，通过HTRF(抗体对：2B7/MW1)检测化合物1、化合物、2化合物3、化合物4对HD患者诱导干细胞(iPSC)分化的神经元(Q47)的mHTT水平的影响(图10)。在HD患者诱导干细胞(iPSC)分化的神经元细胞上观察到mHTT水平降低，并且在存在自噬抑制剂NH 4Cl时没有观察到该效果。
用上文同样的方法，筛选获得并观察到以下化合物5、化合物6、化合物7、化合物8使HD患者诱导干细胞(iPSC)分化的神经元细胞中的mHTT水平降低，其中化合物5使mHTT水平降低约12.2％，化合物6使mHTT水平降低约24.9％，化合物7使mHTT水平降低约24.2％，化合物8使mHTT水平降低约19.7％。化合物通过自噬作用拯救了HD患者诱导干细胞(iPSC)分化的神经元细胞中的疾病相关表型。
Figure PCTCN2020078775-appb-000010
4.2 对神经元凋亡的影响
(1)免疫染色
用100nM化合物1，或100nM化合物2，或100nM化合物3或50nM化合物4处理HD患者诱导干细胞(iPSC)分化的神经元(Q47)，1天后对细胞进行应激(去除BDNF)。
用DAPI对神经元特异性微管蛋白标记物TUBB3染色。以细胞核计数对TUBB3信号覆盖面积进行归一化，并以野生型为对照，对数据进行归一化，分析神经元凋亡(图11)。
(2)胱天蛋白酶-3活性检测
在去除BDNF后，观察到HD神经元停滞，并出现了神经元收缩。
用NucView 488(Biotium,30029)检测活性胱天蛋白酶-3。去除BDNF后，使用Incucyte(Essen Bioscience,IncuCyte FLR)在培养箱内每3小时捕获图像，用Incucyte2011A软件分析。共测试三批，结果一致(图12)。化合物对于去除BDNF后HD神经元的停止进展和神经元收缩有明显的改善。
实施例5 化合物对亨廷顿病果蝇的作用
5.1 对mHTT水平的影响
实验方法：将Q128果蝇和Q16果蝇分别随机分为阴性对照组、阳性药物组(化合物1、化合物2、化合物3、化合物4)，每组75只。阴性对照组给药相应的溶剂DMSO，阳性药物组给药相应的阳性药物。
果蝇保持在25℃的标准食物中。将新孵化的果蝇转移到所分配的食物含有阳性药物(10μM在400的DMSO中)或对照用的DMSO的小瓶中，每隔一天更换一次食物。
连续喂养6天。第7天提取果蝇头部蛋白，通过HTRF(抗体对：2B7/MW1)测量mHTT水平，其中每个样品包括5只果蝇头部提取的蛋白(图13)。化合物使表达人HTT全长蛋白(Q128)的转基因果蝇mHTT水平降低。
5.2 化合物对生存率的影响
将75只年龄匹配的处女蝇放入含有标准食物的空的塑料小瓶中，并每天记录每个小瓶的存活情况，以测量其寿命(图14)。Q128果蝇阳性药物组相对于对照组的生存率改善。
5.3 对爬行能力的影响
将15只年龄匹配的处女蝇放入空瓶中并轻拍使它们处于瓶底。记录15秒后爬过7厘米高的果蝇的百分比。每天对每个小瓶记录五个观察值的平均值，并且记录来自包含不同批次的果蝇的多个小瓶的数据并分析(图15)。括号中的数字表示测试的小瓶的数量。Q128果蝇阳性药物组相对于对照组的爬行能力改善。
在以上5.2和5.3的试验中，没有观察到化合物对Q16果蝇有影响。
实施例6 化合物对HD模型小鼠的作用
将小鼠分组饲养于具有12小时光照/黑暗循环的单独通风笼中，每笼最多5只成年小鼠。
6.1 脑室内注射化合物对HD小鼠皮质mHTT和wtHTT水平的影响
实验动物：Hdh Q140/Q7小鼠(3月龄)，每组4只
实验方法：每天进行一次脑室内注射，每次注射给药2μL含有浓度为25μM的化合物的人造脑脊液(ACSF：1mM葡萄糖，119mM NaCl，2.5mM KCl，1.3mM MgSO 4，2.5mM CaCl 2，26.2mM NaHCO 3，1mM NaH 2PO 4)。将含有等量DMSO的2μL ACSF用作对照。
注射10天后，提取小鼠脑皮质神经元蛋白，通过蛋白印迹(2166抗体)检测mHTT和wtHTT水平。每个样品重复检测3次，计算平均值(图16)。化合物1、3和4显著降低Hdh Q140/Q7小鼠皮质mHTT水平，并相对于wtHTT表现出mHTT选择性。
6.2 腹腔注射给药化合物1或4
实验方法：将化合物或对照用的DMSO用0.9％NaCl静脉内输注溶液稀释至0.05μg/μL，每天进行一次腹腔注射(0.5mg/kg)，注射14天后，进行组织提取或行为学实验。
腹腔注射小鼠脑组织的体内化合物检测：小鼠腹腔注射给药DMSO或化合物(化合物1或化合物4)之后2小时，解剖取脑，称重，提取脑组织内的化合物，用UPLC-MS(Acquity超高效液相色谱系统、Acquity UPLC BEH C18(1.7μm,2.1×50mm)柱和Xevo TQ-S质谱仪，Waters Corporation,Milford,MA,USA)进行LC-MS/MS分析。结果表明到达脑组织的化合物1水平为81.0ng/g。到达脑组织的化合物4水平为9.48ng/g。对照(DMSO)组未观测到化合物质谱信号。化合物能顺利透过BBB递送至小鼠的脑，有利于通过口服给药化合物。
6.3 腹腔注射化合物对HD小鼠皮质和纹状体mHTT和wtHTT水平的影响
(1)取Hdh Q140/Q7小鼠(5月龄)，共20只，分为3组。
按照6.2所述步骤给药化合物1或4。每天进行一次腹腔注射，注射14天后，提取蛋白，通过蛋白印迹检测HD小鼠皮质中的mHTT和wtHTT水平(图17a)。
(2)取Hdh Q140/Q7小鼠(10月龄)，共21只，每组6-8只。
按照上述方法给药并获取小鼠脑纹状体神经元蛋白，通过蛋白印迹检测mHTT和wtHTT水平(图17b)。
(3)通过dot-blot实验(抗体：4C9，条形图示出重复检测两次的结果)和HTRF(抗体对：4C9/4C9)检测Hdh Q140/Q7小鼠皮质中的mHTT聚集物。每只小鼠平均重复取样两到三次(图18)。没有观察到mHTT聚集物的增加。化合物给药组的mHTT水平降低不是由于mHTT溶解度变化。
综上，腹腔注射给药化合物降低Hdh Q140/Q7小鼠皮质和纹状体mHTT水平，并相对 于wtHTT表现出mHTT选择性。因此化合物具有开发为口服药物的前景。
6.4 腹腔注射化合物对HD小鼠行为缺陷的影响
实验动物：Hdh Q140/Q7小鼠，共42只，分为3组；Hdh Q7/Q7小鼠，共48只，分为3组。
实验方法：按照6.2所述步骤给药化合物1或4。每天进行一次腹腔注射，注射14天后，进行行为学实验。
所有的行为学实验均在光照阶段进行。在开始实验之前，将所有小鼠在昏暗的红光下保持在行为测试室中一小时。
旋转棒试验：连续3天对小鼠进行预训练(旋转棒上以4rpm旋转2分钟)。然后在2分钟内以4至40rpm的加速速度测试小鼠5天。每次实验结果记录为在棒时间(旋转棒上的时间)，直到从棒上跌落或直到任务结束。每次测试包括三次重复，试验间隔60分钟以减少压力和疲劳。分析每只小鼠三次试验的平均值(图19a)。
平衡梁试验：2厘米厚、总长度为100厘米的带刻度的棒，两侧悬挂于平台上。起点处有一道明亮的灯光，终点处有一个装有食物的暗盒子。记录每只小鼠走过平衡梁的总时间(图19b)。
旋转棒试验中，F(2,195)＝4.963；平衡梁试验中，F(2,195)＝37.31。本发明化合物能够改善HD模型小鼠的亨廷顿病相关行为缺陷，并且对野生型小鼠不产生影响。
实施例7 化合物不影响自噬功能和对照蛋白的水平
7.1 化合物对培养的Hdh Q140/Q7小鼠皮质神经元细胞的影响
按照与2.1相似的方法，检测化合物处理后Hdh Q140/Q7小鼠皮质神经元细胞中的LC3B、其他已知的自噬选择性底物蛋白(包括SQSTM1/p62，NBR1和Ncoa4)、具有野生型长度polyQ的其他polyQ蛋白(Atxn3和Tbp)和几种对照管家蛋白的水平，蛋白印迹的结果如图20-1、图20-2、图20-3、图20-4所示。化合物不影响原代培养的皮质神经元中的LC3B表达水平，被测的其他蛋白的水平也不受化合物影响(蛋白水平变化<10％)。
7.2 检测腹腔注射化合物的影响
使用与6.2相似的方法，对Hdh Q140/Q7小鼠注射给药，使用与7.1相似的方法，检测腹腔注射化合物对Hdh Q140/Q7小鼠皮质组织中其他已知的自噬选择性底物蛋白(SQSTM1/p62)的影响。蛋白印迹的结果如图21所示。腹腔注射化合物1或化合物4后，HD小鼠的皮质中SQSTM1/p62水平没有改变。
综上，检测到的mHTT降低作用不是由于改变了自噬功能。
实施例8 化合物对mHTT和LC3B之间的结合的影响
8.1 化合物对HTTexon1与LC3B之间的结合的影响
蛋白质体外结合实验(in vitro pull-down assay)
(1)在直链淀粉树脂(New England BioLabs，E8021L)上分别亲和固化纯化的重组人HTTexon1-MBP(添加了MBP标签的SEQ ID NO:1或SEQ ID NO:5)和用于对照的MBP。
(2)取含有约10μg MBP-融合蛋白或MBP的(1)中得到的树脂，分别与化合物(1μM的化合物1或100nM的化合物4)或相应的溶剂DMSO孵育。然后加入40μg纯化的LC3B(对应SEQ ID NO:8)，孵育。然后去除未结合蛋白。
(3)洗脱树脂结合的蛋白质，用抗-LC3抗体通过SDS-PAGE和蛋白印迹进行分析(图22a)。化合物1和化合物4能够增强HTTexon1-Q72-MBP与LC3B之间的结合，而不增强HTTexon1-Q25-MBP与LC3B之间的结合。
8.2 化合物对flHTT-Q73和LC3B之间的结合的影响
将GST-LC3B和用于对照的GST固定在小鼠抗GST抗体偶联磁珠(Cell Signaling Technology,11847S)上，使用与8.1相似的方法，将全长HTT(flHTT-Q73或flHTT-Q23)分别与GST-LC3B或GST进行拉下实验(图22b)。其中上样量为：GST-LC3B或GST上样量为100％，全长HTT上样量为10％。用SDS-PAGE蛋白上样缓冲液洗脱。化合物能够增强全长mHTT和LC3B之间的结合。
实施例9 化合物对mHTT和LC3B在细胞中的定位的影响
9.1 化合物影响下HTTexon1和LC3B在Hela细胞中的共定位
(1)将HTTexon1cDNA亚克隆到哺乳动物表达载体pTT-MBP-His中，制备pTT-HTTexon1-Q72-MBP-His和pTT-HTTexon1-Q25-MBP-His。
(2)将pEX-GFP-hLC3WT(Addgene,24987)、(1)中制备的pTT-HTTexon1-Q72-MBP-His和pTT-HTTexon1-Q25-MBP-His瞬时转染到HeLa细胞中表达HTTexon1-MBP-His(添加了MBP-His标签的SEQ ID NO:1和SEQ ID NO:5)和LC3B-GFP(基于SEQ ID NO:8)。
(3)用100nM浓度的化合物1，或50nM化合物4处理(1)或(2)中制备的细胞，4小时后封固细胞。使用Zeiss Axio Vert A1共聚焦显微镜，分别通过抗His免疫荧光检测HTTexon1-MBP-His，通过GFP荧光检测LC3B-GFP。其中前者荧光信号为红色，后者荧光信号为绿色。在His通道中检测了单独转染LC3B-GFP的细胞，在GFP通道中检测了单独转染HTTexon1-MBP-His的细胞，以验证检测方法的特异性。对两个通道得到的图像中的聚集点进行计数，分析共定位(图23)。图23柱状图的y轴表示与绿色 +(LC3B-GFP)定位相同的红色 +(HTTexon1-MBP-His)聚集点占全部红色 +(HTTexon1-MBP-His)聚集点的比例。
9.2 化合物影响下mHTT和LC3B在HD小鼠纹状体细胞中的共定位
HD小鼠纹状体细胞STHdh Q111/Q111的来源如上文所述。
使用与9.1相似的方法，通过抗体2166检测mHTT，并且通过特异性检测LC3B的抗LC3抗体检测内源性LC3B。分别测量各种荧光的像素并分析STHdh Q111/Q111细胞中内源性mHTT和LC3B之间的共定位(图24)。图24柱状图的y轴表示与绿色 +(LC3B-GFP)定位相同的红色 +(HTTexon1-MBP-His)聚集点占全部红色 +(HTTexon1-MBP-His)聚集点的比例。本发明的方法筛选得到的化合物在细胞中能促进polyQ异常扩增蛋白与LC3B共定位，由此证实了化合物能够通过同时与polyQ异常扩增蛋白和LC3B结合，促进polyQ异常扩增蛋白向具有自噬功能的细胞亚结构富集，从而通过自噬降低所述polyQ异常扩增蛋白在细胞中的水平。
发明人还出人意料地发现，常见的可检测标记例如GFP等可以有效地用于本发明的筛选方法中。因此，虽然非标记的检测方法，例如非标记的斜入射光反射差法具备对检测对象无干扰的优点，但本发明的筛选方法也可以采用已知的其他检测方法，例如荧光法等。
实施例10 使用含有较长谷氨酰胺重复区域的蛋白进行亲和活性筛选
10.1 polyQ-GFP蛋白的制备
(1)从头合成polyQ-GFP cDNA序列(表达Met-polyQ-GFP，其中polyQ为Q72或Q25)并亚克隆到pcDNA载体中。将其正向转染到HEK293T细胞(ATCC，CRL-3216)中，分别得到表达Q72-GFP的细胞和表达Q25-GFP的细胞。并且，用同样的方法获得表达Q53-GFP的细胞、表达Q46-GFP的细胞、表达Q38-GFP的细胞。
(2)分别纯化通过(1)表达的polyQ-GFP蛋白，以获取Q72-GFP、Q53-GFP、Q46-GFP、Q38-GFP和Q25-GFP(SEQ ID NO:6)。其中Q72-GFP、Q53-GFP、Q46-GFP、Q38-GFP与SEQ ID NO:6的氨基酸序列相比，区别在于谷氨酰胺重复区域的长度。
10.2 亲和活性筛选
按照与1.1相似的方法，但将1.1的步骤(1)中的靶蛋白HTTexon1-Q72-MBP替换为10.1制备得到的polyQ-GFP蛋白，将1.1的步骤(2)中的靶蛋白HTTexon1-Q25-MBP替换为GFP，筛选能够选择性地亲和结合含有较长谷氨酰胺重复区域的蛋白的化合物。
其中，通过分析化合物1、化合物3、化合物4与polyQ-GFP蛋白孵育后与孵育前的差异图像(孵育后的图像-孵育前的图像)，发现这些化合物不与Q25-GFP或GFP结合，但能够与具有较长谷氨酰胺重复区域的蛋白结合。例如，化合物1、化合物3、化合物4与具有38个或更多个谷氨酰胺重复的蛋白具有一定的亲和结合。按照与1.1相似的方法，用OI-RD测量化合物1、化合物3、化合物4与Q72-GFP、Q53-GFP、Q46-GFP、Q38-GFP的亲和反应参数。结果见表5-1至5-3。
表5-1.化合物1与Q72-GFP、Q53-GFP、Q46-GFP、Q38-GFP的亲和反应的结合速率常数、解离速率常数和平衡解离常数
  K on(min·nM) -1 K off(min) -1 K d(nM)
Q72-GFP 8.37×10 -5 1.51×10 -2 180.4
Q53-GFP 1.51×10 -4 3.78×10 -3 25.0
Q46-GFP 3.04×10 -4 1.83×10 -2 60.2
Q38-GFP 1.06×10 -5 2.03×10 -2 1915.1
表5-2.化合物3与Q72-GFP、Q53-GFP、Q46-GFP、Q38-GFP的亲和反应的结合速率常数、解离速率常数和平衡解离常数
  K on(min·nM) -1 K off(min) -1 K d(nM)
Q72-GFP 2.23×10 -5 7.94×10 -3 356.1
Q53-GFP 1.25×10 -4 1.35×10 -2 108.0
Q46-GFP 1.29×10 -4 7.67×10 -3 59.5
Q38-GFP 8.72×10 -6 2.62×10 -2 3004.6
表5-3.化合物4与Q72-GFP、Q53-GFP、Q46-GFP、Q38-GFP的亲和反应的结合速率常数、解离速率常数和平衡解离常数
  K on(min·nM) -1 K off(min) -1 K d(nM)
Q72-GFP 6.20×10 -5 1.92×10 -2 309.7
Q53-GFP 1.08×10 -4 4.65×10 -3 43.1
Q46-GFP 7.65×10 -6 2.57×10 -2 3359.5
Q38-GFP 2.99×10 -6 1.5×10 -2 5016.7
10.3 化合物对含有较长谷氨酰胺重复区域的蛋白的亲和活性的MST检测
按照2.2的方法，用微量热泳动仪检测化合物1、化合物3、化合物4与polyQ-GFP蛋白(Q25-GFP和Q72-GFP)的亲和活性进行验证。本发明的化合物与Q25-GFP没有亲和结合，与Q72-GFP的K d如表6所示。
表6.化合物与待测蛋白的亲和反应平衡解离常数
Figure PCTCN2020078775-appb-000011
由以上试验可知，本发明的化合物选择性地与含有较长谷氨酰胺重复区域的蛋白结 合。polyQ异常扩增蛋白或其含有polyQ部分的片段都可以应用于本发明的筛选方法。本发明的筛选方法可以有效地应用于筛选或鉴定化合物，其用于治疗或预防含有较长谷氨酰胺重复区域的蛋白引起的疾病，例如polyQ相关的神经退行性疾病。
实施例11 化合物在细胞中对含有较长谷氨酰胺重复区域的蛋白水平的影响
使用10.1的步骤(1)中获得的表达Q72-GFP的细胞和表达Q25-GFP细胞。用100nM浓度的化合物1，或100nM化合物3或50nM浓度的化合物4处理细胞，2天后通过Incucyte检测荧光计数测量polyQ-GFP水平(图25)。按照本发明的方法筛选得到的化合物能够在细胞中选择性地降低含有较长谷氨酰胺重复区域的蛋白的水平。说明筛选得到的化合物具有扩展的药理作用，能够用于治疗或预防含有较长谷氨酰胺重复区域的蛋白引起的疾病。
以下实施例12至14进一步检测本发明的方法筛选得到的化合物对于polyQ异常扩增的ATXN3蛋白、polyQ异常扩增的ATXN1蛋白水平的影响，并且进一步证实本发明的筛选方法可以使用含有较长谷氨酰胺重复区域的蛋白作为代表疾病状态的靶蛋白。
实施例12 使用polyQ异常扩增的ATXN3蛋白进行亲和活性筛选
12.1 亲和活性筛选
按照与1.1相似的方法，但将1.1的步骤(1)中的靶蛋白HTTexon1-Q72-MBP替换为MBP-ATXN3-Q68，将1.1的步骤(2)中的靶蛋白HTTexon1-Q25-MBP替换为MBP-ATXN3-Q28，筛选能够选择性地亲和结合polyQ异常扩增的ATXN3蛋白(MBP-ATXN3-Q68)的化合物。
其中，通过分析化合物1、化合物3、化合物4与MBP-ATXN3-Q68和MBP-ATXN3-Q28分别孵育后与孵育前的差异图像(孵育后的图像-孵育前的图像)，发现这些化合物不与MBP-ATXN3-Q28结合，但能够与MBP-ATXN3-Q68结合。
12.2 化合物对polyQ异常扩增的ATXN3蛋白的亲和活性的MST检测
按照2.2的方法，用微量热泳动仪验证化合物与MBP-ATXN3-Q28和MBP-ATXN3-Q68蛋白的亲和活性。
结果表明，化合物1、化合物3、化合物4与正常ATXN3蛋白(MBP-ATXN3-Q28)没有亲和结合，与变异ATXN3蛋白(MBP-ATXN3-Q68)的K d如表7所示。
表7.化合物与待测蛋白的亲和反应平衡解离常数
Figure PCTCN2020078775-appb-000012
实施例13 化合物对脊髓小脑共济失调3型患者成纤维细胞的ATXN3蛋白水平的影响
用100nM浓度的化合物1，或100nM化合物2，或100nM化合物3或50nM浓度 的化合物4处理SCA3患者成纤维细胞(Q74)、野生型细胞(Q27)，2天后通过蛋白印迹测定变异ATXN3蛋白(ATXN3-Q74,SEQ ID NO:2)、野生型ATXN3蛋白(ATXN3-Q27,SEQ ID NO:7)水平(图26)。在SCA3患者成纤维细胞(Q74)上观察到变异ATXN3蛋白水平降低，而没有观察到野生型ATXN3蛋白水平降低，因此本发明的化合物可用于治疗SCA3。
实施例14 使用polyQ异常扩增的ATXN1蛋白进行亲和活性筛选
14.1 polyQ异常扩增的ATXN1蛋白的制备
(1)从头合成polyQ-ATXN1cDNA(其中polyQ为Q92)并亚克隆到pcDNA载体中。将其转染至HEK293T细胞(ATCC，CRL-3216)中，得到表达His-ATXN1-Q92(N端带有His标签的SEQ ID NO:3)的细胞。
(2)纯化通过(1)表达的His-ATXN1-Q92，获取polyQ异常扩增的ATXN1蛋白。
14.2 亲和活性筛选
按照与1.1的步骤(1)相似的方法，但将1.1的步骤(1)中的靶蛋白HTTexon1-Q72-MBP替换为His-ATXN1-Q92，筛选能够亲和结合polyQ异常扩增的ATXN1蛋白的化合物。
其中，通过分析化合物1、化合物2、化合物3、化合物4与His-ATXN1-Q92孵育后与孵育前的差异图像(孵育后的图像-孵育前的图像)，发现这些化合物能够与His-ATXN1-Q92结合。
实施例15 化合物在细胞中对polyQ异常扩增的ATXN1蛋白水平的影响
使用14.1中获得的表达His-ATXN1-Q92的细胞。用100nM浓度的化合物(化合物1，或化合物2，或化合物3，或化合物4，或化合物6，或化合物7处理细胞，2天后通过HTRF检测变异ATXN1蛋白(His-ATXN1-Q92)水平(抗体对：anti-His-Tb/MW1-D2，图27。在SCA1患者成纤维细胞(Q92)上观察到变异ATXN1蛋白水平降低。因此本发明的化合物可以用于治疗或预防脊髓小脑共济失调1型。
综上，已经通过实验证明本发明的方法筛选得到的化合物能够降低多种含有较长谷氨酰胺重复区域的蛋白的水平，例如变异mHTT蛋白、变异ATXN3蛋白、变异ATXN1蛋白的水平。因此，本发明的筛选方法有效地应用于筛选或鉴定化合物。这样的化合物可用于治疗或预防含有较长谷氨酰胺重复区域的蛋白引起的疾病，例如polyQ相关的神经退行性疾病。
序列表
[SEQ ID NO:1]HTTexon1-Q72:
Figure PCTCN2020078775-appb-000013
Figure PCTCN2020078775-appb-000014
[SEQ ID NO:2]ATXN3-Q74:
Figure PCTCN2020078775-appb-000015
[SEQ ID NO:3]ATXN1-Q92:
Figure PCTCN2020078775-appb-000016
[SEQ ID NO:4]HTT-Q23:
Figure PCTCN2020078775-appb-000017
Figure PCTCN2020078775-appb-000018
Figure PCTCN2020078775-appb-000019
[SEQ ID NO:5]HTTexon1-Q25:
Figure PCTCN2020078775-appb-000020
[SEQ ID NO:6]Q25-GFP:
Figure PCTCN2020078775-appb-000021
[SEQ ID NO:7]ATXN3-Q27:
Figure PCTCN2020078775-appb-000022
[SEQ ID NO:8]LC3B:
Figure PCTCN2020078775-appb-000023

Claims (19)

  1. 一种筛选或鉴定用于治疗或预防polyQ相关的神经退行性疾病的化合物的方法，包括
    (I)：使候选化合物与待测体系接触，所述待测体系包含
    (1)polyQ异常扩增蛋白或其含有polyQ部分的片段，和
    (2)LC3B蛋白或其同源物或它们的片段；和
    (II)：测定所述候选化合物调节(1)polyQ异常扩增蛋白或其含有polyQ部分的片段与(2)LC3B蛋白或其同源物或它们的片段之间的结合的能力，并且选择正向调节所述结合的化合物。
  2. 权利要求1的方法，其中步骤(II)包括：
    分别存在/不存在所述候选化合物的条件下，测定(1)polyQ异常扩增蛋白或其含有polyQ部分的片段与(2)LC3B蛋白或其同源物或它们的片段之间的结合亲和力大小，和/或
    分别存在/不存在所述候选化合物的条件下，测定(1)polyQ异常扩增蛋白或其含有polyQ部分的片段与(2)LC3B蛋白或其同源物或它们的片段的结合量。
  3. 权利要求1或2的方法，其中所述polyQ异常扩增蛋白包含polyQ长度≥40、≥33、≥41、≥19、≥46、≥44、≥39、≥36或≥37的polyQ部分；
    优选地，所述polyQ异常扩增蛋白包含
    polyQ长度≥40的ATXN1或其含有polyQ部分的片段，
    polyQ长度≥33的ATXN2或其含有polyQ部分的片段，
    polyQ长度≥41的ATXN3或其含有polyQ部分的片段，
    polyQ长度≥19的ATXN7或其含有polyQ部分的片段，
    polyQ长度≥46的ATXN12或其含有polyQ部分的片段，
    polyQ长度≥44的TBP或其含有polyQ部分的片段，
    polyQ长度≥39的ATN1或其含有polyQ部分的片段，
    polyQ长度≥36的HTT或其含有polyQ部分的片段，或
    polyQ长度≥37的AR或其含有polyQ部分的片段；
    特别优选地，所述polyQ异常扩增蛋白包含
    polyQ长度≥40的ATXN1或其含有polyQ部分的片段，
    polyQ长度≥33的ATXN2或其含有polyQ部分的片段，
    polyQ长度≥41的ATXN3或其含有polyQ部分的片段，
    polyQ长度≥19的ATXN7或其含有polyQ部分的片段，或
    polyQ长度≥46的ATXN12或其含有polyQ部分的片段；
    尤其是polyQ长度≥40的ATXN1或其含有polyQ部分的片段，或者polyQ长度≥41的ATXN3或其含有polyQ部分的片段。
  4. 权利要求1-3中任一项的方法，其中(2)LC3B蛋白或其同源物或它们的片段包含与SEQ ID NO:8的氨基酸序列具有25％或更高、30％或更高、35％或更高、40％或更高、50％或更高、60％或更高、70％或更高、80％或更高、90％或更高、95％或更高或者100％的序列相同性的序列。
  5. 权利要求1-4中任一项的方法，其中测定所述结合的方法选自体外拉下实验、Split-TEV、免疫共沉淀、亲和层析、复合物共纯化、酶联免疫吸附法、荧光分子筛、酵母双杂交、HIP-HOP法、时间分辨荧光共振能量转移、化学发光法、表面等离子体共振、等温滴定量热法、微量热泳动及其任意组合；优选地，测定所述结合的方法选自体外拉下实验、免疫共沉淀、酶联免疫吸附法、荧光分子筛和时间分辨荧光共振能量转移及其任意组合。
  6. 权利要求1-5中任一项的方法，其中在步骤(I)之前还包括对候选化合物进行亲和活性预筛选的步骤，包括：
    (A)：检测候选化合物与(1)polyQ异常扩增蛋白或其含有polyQ部分的片段的亲和结合，和/或(B)：检测候选化合物与(2)LC3B蛋白或其同源物或它们的片段的亲和结合；以及
    (C)：选择与(1)polyQ异常扩增蛋白或其含有polyQ部分的片段和/或(2)LC3B蛋白或其同源物或它们的片段能够亲和结合的化合物；
    其中步骤(A)和步骤(B)可以以任何顺序独立地进行。
  7. 权利要求6的方法，其中
    步骤(A)包括：测定候选化合物与(1)polyQ异常扩增蛋白或其含有polyQ部分的片段的亲和反应平衡解离常数，选择所述亲和反应平衡解离常数为100μM以下的化合物，优选10μM以下，特别优选1μM以下；和/或
    步骤(B)包括：测定候选化合物与(2)LC3B蛋白或其同源物或它们的片段的亲和反应平衡解离常数，选择所述亲和反应平衡解离常数为100μM以下的化合物，优选10μM以下，特别优选1μM以下。
  8. 权利要求6或7的方法，其中用于测定亲和结合的方法选自接近闪烁分析、荧光共振能量转移、荧光偏振检测、荧光分子筛、微量热泳动、化学发光法、表面等离子体 共振、等温滴定量热法、斜入射光反射差法及其任意组合；优选地选自荧光共振能量转移、荧光偏振检测、斜入射光反射差法及其任意组合；特别优选为斜入射光反射差法。
  9. 权利要求6-8中任一项的方法，其中
    所述对候选化合物进行亲和活性预筛选的步骤通过采用斜入射光反射差法的高通量筛选进行；包括：
    (a)对候选化合物进行固定化以制备化合物芯片，扫描芯片，得到孵育前的图像；
    (b)将化合物芯片与靶蛋白进行孵育，扫描芯片，得到孵育后的图像；
    其中靶蛋白为(1)polyQ异常扩增蛋白或其含有polyQ部分的片段或者(2)LC3B蛋白或其同源物或它们的片段；和
    (c)对差异图像(孵育后的图像-孵育前的图像)进行分析；选择与(1)polyQ异常扩增蛋白或其含有polyQ部分的片段和(2)LC3B蛋白或其同源物或它们的片段都能够亲和结合的化合物。
  10. 权利要求1-9中任一项的方法，还包括以下步骤：
    (D)：测定候选化合物与正常polyQ蛋白或其含有polyQ部分的片段的结合，选择不与正常polyQ蛋白或其含有polyQ部分的片段亲和结合的化合物。
  11. 通过权利要求1-10中任一项的方法获得的化合物或其药物组合物在制备用于治疗或预防polyQ相关的神经退行性疾病的药物中的用途，或者在制备用于检测被认为患有或易患polyQ相关的神经退行性疾病的受试者的诊断试剂或试剂盒中的用途。
  12. 权利要求11的用途，其中所述polyQ相关的神经退行性疾病选自脊髓小脑共济失调1型、脊髓小脑共济失调2型、脊髓小脑共济失调3型、脊髓小脑共济失调7型、脊髓小脑共济失调12型和脊髓小脑共济失调17型，特别是脊髓小脑共济失调1型和脊髓小脑共济失调3型。
  13. 权利要求11的用途，其中所述polyQ相关的神经退行性疾病选自齿状核-黑质-红核-苍白球-丘脑底核萎缩症、亨廷顿病和脊髓延髓肌肉萎缩，优选为亨廷顿病。
  14. 权利要求11的用途，其中所述检测包括分析从所述受试者获得的目标样品的步骤，包括：
    i)将所述化合物用可检测标记例如荧光基团进行标记；
    ii)用i)中得到的化合物处理目标样品；
    iii)检测目标样品中可检测信号的位置和/或强度，获得目标样品中polyQ异常扩增 蛋白或其含有polyQ部分的片段的位置和/或含量。
  15. 化合物或其药物组合物在制备用于治疗或预防polyQ相关的神经退行性疾病的药物中的用途，或者在制备用于检测被认为患有或易患polyQ相关的神经退行性疾病的受试者的诊断试剂或试剂盒中的用途，所述化合物选自：
    Figure PCTCN2020078775-appb-100001
  16. 一种药物组合物、检测试剂或试剂盒，其包含通过权利要求1-10中任一项的方法获得的化合物。
  17. 一种药物组合物、检测试剂、试剂盒或筛选系统，其包含至少一种选自以下的化合物：
    Figure PCTCN2020078775-appb-100002
  18. 一种筛选系统，其包含
    (1)polyQ异常扩增蛋白或其含有polyQ部分的片段，和
    (2)LC3B蛋白或其同源物或它们的片段；
    优选地，所述筛选系统为试剂盒的形式。
  19. 正向调节(1)polyQ异常扩增蛋白或其含有polyQ部分的片段与(2)LC3B蛋白或其同源物或它们的片段之间的结合的化合物或其药物组合物在制备用于治疗或预防polyQ相关的神经退行性疾病的药物中的用途。
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