WO2023284782A1 - 筛选用于治疗或预防mHTT相关的神经退行性疾病的化合物的方法，靶蛋白，以及化合物 - Google Patents

筛选用于治疗或预防mHTT相关的神经退行性疾病的化合物的方法，靶蛋白，以及化合物 Download PDF

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WO2023284782A1
WO2023284782A1 PCT/CN2022/105437 CN2022105437W WO2023284782A1 WO 2023284782 A1 WO2023284782 A1 WO 2023284782A1 CN 2022105437 W CN2022105437 W CN 2022105437W WO 2023284782 A1 WO2023284782 A1 WO 2023284782A1
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polyq
compound
htt
mhtt
desonide
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French (fr)
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鲁伯埙
费义艳
丁澦
党永军
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复旦大学
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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/12—Radicals substituted by oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
    • 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

Definitions

  • the present invention relates to the field of biomedicine, and specifically relates to a method for screening compounds for treating or preventing mHTT-related neurodegenerative diseases, target proteins for screening, and corresponding compounds.
  • Neurodegenerative diseases refer to diseases caused by the abnormal death of central neurons and the dysfunction of the nervous system. For neurodegenerative diseases, so far there is no fundamental treatment that can slow down the progression of the disease.
  • Variations in the gene HTT are associated with neurodegenerative diseases.
  • Huntington's Disease is a monogenic neurodegenerative disease caused by mutations in the CAG repeat region of HTT exon 1.
  • the increased number of CAG repeats in the mutated gene leads to the expansion of the glutamine repeat region (polyQ) of the synthesized mutated protein (mutant Huntingtin, mHTT), which eventually leads to the death of neurons mainly concentrated in the striatum, and causes A spectrum of neurological, psychosocial, and metabolic symptoms characterized by abnormalities in extremity movement.
  • Wild-type HTT may function as a scaffold protein (a scaffold protein/a multiprotein scaffold) in cells.
  • the function of mHTT is unknown, and the exact cause of HD is also unknown. Due to the lack of the "binding pocket" of traditional small molecule drugs, and the lack of measurable biochemical readout, mHTT has been considered “undruggable”. That is, candidate drugs for treating HD cannot be obtained by screening "inhibitors" of mHTT.
  • the present invention provides a method of screening or identifying compounds for the treatment or prevention of mHTT-related neurodegenerative diseases, comprising
  • test system comprising polyQ abnormally amplified HTT or mutants thereof or fragments thereof, wherein
  • the polyQ abnormally amplified HTT contains a polyQ length ⁇ 36;
  • the fragment comprises the N17 region and the polyQ region of polyQ aberrantly amplified HTT or a mutant thereof, and optionally comprises a PRD;
  • the amino acid sequence of the polyQ aberrantly amplified HTT or a mutant or fragment thereof comprises K6. In some embodiments, the amino acid sequence of polyQ aberrantly amplified HTT or mutants thereof or fragments thereof comprises one or more amino acid substitutions selected from the group consisting of K9X, K15X, E12X, S13X and S16X, wherein X represents Any natural amino acid that differs from the wild-type sequence.
  • the present invention provides the use of a compound obtained by the method of the present invention in the preparation of a PROTAC compound for the treatment or prevention of mHTT-related neurodegenerative diseases.
  • the present invention provides a compound obtained by the method of the present invention, or a PROTAC compound thereof, or a pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, tautomer, isotope thereof
  • a compound obtained by the method of the present invention or a PROTAC compound thereof, or a pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, tautomer, isotope thereof
  • the compounds obtained by the methods of the present invention are selected from
  • Q+number in the figure represents the protein or fragment containing polyQ of the corresponding length used. des stands for desonide.
  • Figures 6 to 8 Figure 17, Figure 24, Figure 25, Figure 26 to Figure 29, Figure 31, Figure 32, all data for each figure were normalized to the DMSO control and statistical analyzes were performed using two-tailed unpaired t test.
  • Figure 1 Effects of compounds on the apoptosis of HD mouse striatal cells STHdh Q7/Q111 induced by mHTT. Data are from ⁇ 7 batches of experiments. Statistical analysis used two-way ANOVA analysis and Dunnett's post-hoc analysis compared with DMSO group.
  • Fig. 2A, 2B The effect of compounds on the apoptosis of HD mouse striatal cells STHdh Q7/Q111 induced by mHTT is dose-dependent.
  • Figure 3 Effects of compounds on apoptosis of WT mouse striatal cells STHdh Q7/Q7 independent of mHTT. Data are from ⁇ 7 batches of experiments. Statistical analysis used two-way ANOVA analysis and Dunnett's post-hoc analysis compared with DMSO group.
  • Figure 4 OI-RD detection of the binding activity of compounds to mHTT exon1.
  • the graph shows the association-dissociation curves of the compounds, with vertical dashed lines showing the onset of the association phase and the dissociation phase.
  • Figure 5 MST assay for desonide binding to proteins in standard capillaries.
  • FIG. 6 Effect of desonide on HTT levels in STHdh Q7/Q111 cells.
  • Figure 7 desonide dose-dependently reduces mHTT levels in STHdh Q7/Q111 cells.
  • FIG. 8A HTRF measurement of mHTT (antibody pair: 2B7/MW1; n ⁇ 9) and total HTT (antibody pair: 2B7/2166; n ⁇ 9) in HD patient fibroblasts (Q47, Q55, Q68).
  • Figure 8B Dose effect curve in HD patient fibroblasts (Q47).
  • Figure 9 Effect of desonide on HTT levels in neuronal cells differentiated from HD patient iPSCs. Statistical analysis was performed using a two-tailed unpaired t-test.
  • Figure 10 Effect of compounds on apoptosis of neurons differentiated from HD patient iPSCs.
  • Figure 11 Effects of compounds on crawling ability of Drosophila Huntington's disease. Statistical analysis used two-way ANOVA analysis and Dunnett's post-hoc analysis compared with DMSO group.
  • Figure 12 Effect of desonide on mHTT levels in HD Drosophila. n indicates the number of vials. Statistical analysis was performed using a two-tailed unpaired t-test.
  • Figures 13A, 13B Effects of compounds on behavior in HD mice.
  • Figure 13A Open field test.
  • Figure 13B Hindlimb stance behavior.
  • Statistical analysis was performed using a two-tailed unpaired t-test.
  • Figures 14A, 14B, 14C, 14D, 14E Effect of ip injection of desonide on behavior of HD mice.
  • Figures 14A, 14B, 14C, 14D Statistical analysis was performed by one-way ANOVA.
  • Figure 14E Statistical analysis using two-way ANOVA analysis.
  • Figure 15 Effect of intracerebroventricular injection of desonide on mHTT body and DARPP-32 signaling in HD mice. n indicates the number of sections from three mice. Statistical analysis was performed using a two-tailed unpaired t-test.
  • FIG. 16 Concentration of desonide in the brain of mice injected ip with desonide. Three mice were tested at each time point.
  • Figure 17 Effect of ip injection of desonide on mHTT levels in striatum of HD mice.
  • Figures 18A, 18B, 18C, 18D, 18E Effect of ip injection of desonide on behavior of HD mice. Statistical analysis used two-way ANOVA analysis and Turkey's post hoc test.
  • Figure 18F Effect of ip injection of desonide on the level of mHTT aggregates in the striatum of HD mice. n indicates the number of striatal slices from 3 mice per group. Statistical analysis was performed using a two-tailed unpaired t-test.
  • Figure 19 Effect of ip injection of desonide on DARPP-32 and NFL levels in HD mice. All data are normalized to DMSO-treated wild-type mice. n indicates the number of striatal slices from 3 mice per group. Scale bar 100 ⁇ m. Statistical analysis was performed using a two-tailed unpaired t-test.
  • Figure 20 Effect of desonide on mHTT levels in SCA3 patient fibroblasts. Statistical analysis was performed using one-way ANOVA analysis and Dunnett's post hoc test.
  • Figure 21 MST detection of affinity binding of desonide to GFP-polyQ fusion protein.
  • Figure 22 MST detection of affinity binding of desonide to HTTexon1-Q72 mutant.
  • Figure 23 Effects of desonide on mHTT and mutant (K6R, K9R, K15R or K6,9,15R) levels of transfected HEK293T cells or STHdh Q7/Q7 cells.
  • Figure 24 Effect of desonide on mHTT and mutant (E12A or S13, 16A) levels in transfected HEK293T cells.
  • Figure 25 Effect of desonide on apoptosis of transfected HEK293T cells induced by mHTT and mutants (K6R, K9R, K15R or K6, 9, 15R). Data were normalized to HTTexon1-Q72 transfected cells and DMSO control. Statistical analysis was performed using a two-tailed unpaired t-test.
  • Figure 26 Effect of desonide on apoptosis of transfected HEK293T cells induced by mHTT and mutants (E12A or S13,16A).
  • Figure 27 Effect of desonide on apoptosis of transfected STHdh Q7/Q7 cells induced by mHTT and mutants (K6R, K9R, K15R or K6, 9, 15R).
  • Figure 28 Effect of desonide on mHTT levels in STHdh Q7/Q111 cells in the presence or absence of proteasome inhibitors or autophagy inhibitors.
  • Figure 29 Effect of desonide on cellular mHTT levels in the presence or absence of proteasome inhibitors or autophagy inhibitors.
  • Left panel HD patient fibroblasts (Q47) endogenously expressing mHTT.
  • Right panel transfected HEK293T.
  • Figure 30 IP-western assay to determine the effect of desonide on polyubiquitination of mHTT and mutants (K6R, K9R or K15R) in transfected HEK293T cells. Results are from 5 batch tests. In the figure, the position of Q25 protein is slightly higher than IgG light chain.
  • Figure 31 GR agonists do not affect mHTT levels in STHdh Q7/Q111 cells, nor do they bind mHTT with affinity.
  • Figure 32 Effect of desonide on the mHTT level of STHdh Q7/Q111 cells with knockdown of GR.
  • one (species) or more (species) may mean, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 (species) or more (species).
  • the expression "HTT with polyQ length ⁇ 36" may cover polyQ lengths 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,
  • neurodegenerative disease refers to a disease caused by loss or disease of neurons and/or their myelin sheath. Characteristic pathological structures, such as insoluble aggregates composed of proteins, can be observed in brain neurons of patients with neurodegenerative diseases. Insoluble aggregates can be cytotoxic, leading to neuronal loss and disease.
  • polyQ or "polyglutamine” refers to a glutamine repeat region in a protein.
  • Glutamine is encoded by cytosine-adenine-guanine (CAG) in the gene, and the length of the glutamine repeat region is related to the number of CAG repeats in the exons of the gene, so an increase in the number of CAG repeats in the exons of the gene will Causes an expansion of the glutamine repeat region of the synthesized protein.
  • CAG cytosine-adenine-guanine
  • the number of CAG repeats in the exon can be represented in the form of "Q+number" in the gene name, such as Q25 or Q72, respectively representing 25 repeats or 72 repeats of CAG in the exon.
  • the length of the glutamine repeat region can be expressed in the form of "Q+number” as above in the protein name, such as Q23 or Q73, respectively indicating that the length of the glutamine repeat region is 23 Q (glutamine) or 73 Q.
  • 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.
  • Human HTT protein sequence for example, the protein sequence encoded by GenBank: BAA36753.1.
  • mHTT refers to polyQ aberrantly amplified HTT.
  • mHTT-associated neurodegenerative disease refers to a neurodegenerative disease associated with abnormally expanded polyQ HTT, or a neurodegenerative disease responsive to HTT levels containing expanded polyQ, such as Huntington's disease.
  • Normal polyQ means that HTT under normal physiological conditions has a polyQ length less than a specified number.
  • polyQ abnormal amplification means that the polyQ length of the protein is larger than the normal length. For diseases or pathological states, the polyQ length will be longer.
  • normal polyQ HTT comprises a polyQ (ie, its polyQ region) of normal length.
  • the expression "normal polyQ HTT” has the same meaning as HTT containing polyQ of normal length” and can be used interchangeably.
  • normal polyQ HTT comprises the sequence of SEQ ID No: 1.
  • the sequence of SEQ ID No: 1 The length of polyQ in the sequence is 23, which belongs to the category of normal polyQ. On this basis, the number of polyQ can be increased or decreased appropriately, but if it is less than a specific number, such as 36, it still belongs to "normal polyQ HTT".
  • a "polyQ abnormally amplified HTT” comprises a polyQ of abnormal length (ie, its polyQ region) based on a "normal polyQ HTT". For example, polyQ regions with polyQ length ⁇ 36.
  • the length of a fragment of a normal or abnormal polyQ protein can be 0.1% or higher, 1% or higher, 2% or higher, 3% or higher, 4% or higher, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 90% or higher, or 95% or higher, but contains an intact polyQ region of normal or abnormal length and contains the N17 region , and optionally contains PRD.
  • polyQ can be used to amplify fragments of HTT or mutants thereof aberrantly when used in the methods of the invention, especially in high-throughput methods. It contains the N17 region and polyQ region of mHTT, and optionally contains a PRD.
  • the fragments may be only about 0.1% or more, about 0.15% or more, about 0.16% or more, about 0.168% or more, about 0.2% or more, about 0.3% or more of the mHTT in length High, about 0.4% or higher, about 0.5% or higher, about 0.6% or higher, about 0.7% or higher, about 0.8% or higher, about 0.9% or higher, about 1% or higher , about 2% or higher, about 3% or higher, about 4% or higher, about 5% or higher, about 6% or higher, about 7% or higher.
  • Normal polyQ HTT or “polyQ abnormally amplified HTT” may be naturally occurring HTT comprising a polyQ region of normal or abnormal length, e.g. may be from a human or from another animal, e.g. from a mouse (e.g. mouse HTT, which The polyQ length can eg be 140 or 111), or from insects, fish, rodents, artiodactyls, primates.
  • modified forms of HTT or fragments thereof may be used.
  • phosphorylated, acetylated, benzoylated and palmitoylated modified forms especially phosphorylated or acetylated modified forms.
  • Such schemes are also within the scope of "normal polyQ HTT” or “polyQ abnormally amplified HTT” as described in the present invention.
  • Normal polyQ HTT or “polyQ abnormally amplified HTT” can also be artificially engineered or modified HTT, but still with its corresponding N17 region and polyQ region.
  • modification or modification can be for processing, purification, characterization, tracing and other purposes, such as adding MBP tag or HA tag or fusion with green fluorescent protein, or introducing various amino acid substitutions, additions or deletions.
  • Such techniques are well known to those skilled in the art.
  • proteins comprise a corresponding normal or abnormal polyQ region and have a 15% or higher, 20% or higher, 25% or higher, 30% or more High, 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 sequence identity.
  • Such proteins can be truncated or spliced from the full-length or fragments of corresponding normal or polyQ abnormally amplified proteins, and optionally include transformation or modification, and can also be designed from scratch.
  • HTT containing a longer glutamine repeat region refers to a longer glutamine repeat region in a particular protein.
  • Longer glutamine repeat region may refer to “polyQ aberrant expansion”, in which case “HTT containing longer glutamine repeat region” may have the same expression as “polyQ aberrant expansion HTT” described herein same meaning.
  • mHTT-related neurodegenerative diseases include, but are not limited to, Huntington's Disease (HD, polyQ length ⁇ 36).
  • affinity activity screening is the process of detecting affinity binding between a sample and a target.
  • the detection method used for affinity activity screening can be, for example, absorbance method, radiation method (such as proximity scintillation analysis), fluorescence method (such as fluorescence resonance energy transfer, fluorescence polarization detection, especially such as time-correlated fluorescence technique), chemiluminescence method (such as amplification Chemiluminescent Affinity Homogeneous Detection, ALPHAScreen), Surface Plasmon Resonance (SPR, which can be implemented, for example, with the Biacore series from GE), Isothermal Titration Calorimetry (ITC), Microthermophoresis (MST) or Oblique Incident Light Reflectance difference method.
  • radiation method such as proximity scintillation analysis
  • fluorescence method such as fluorescence resonance energy transfer, fluorescence polarization detection, especially such as time-correlated fluorescence technique
  • chemiluminescence method such as amplification Chemiluminescent A
  • sequence identity between two amino acid sequences means the percentage of amino acids that are identical between the sequences.
  • Sequence homology indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • sequence comparison algorithm test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Based on the designated program parameters, the sequence comparison algorithm calculates the percent sequence identities for the test sequences relative to the reference sequence. Examples of algorithms suitable for use in determining percent sequence identity and sequence similarity include, but are not limited to, the BLAST and BLAST 2.0 algorithms. Software for performing BLAST analyzes is available from the National Center for Biotechnology Information (NCBI).
  • sample of interest includes various sample types that are obtained from a subject and that can be used for diagnostic analysis.
  • a sample of interest herein can be, for example, any cell sample, biological fluid (including blood, serum, spinal fluid, etc.), or any biopsy sample obtained from a subject's tissue.
  • the concept of the present invention is based at least in part on the discovery of a new mechanism: compounds capable of affinity binding to polyQ aberrantly amplified HTT or fragments thereof can reduce the levels of polyQ aberrantly amplified proteins, treat or prevent corresponding diseases, wherein said fragments comprise Its N17 region and polyQ region, and optionally contains a PRD.
  • the present invention provides a method of screening or identifying compounds for the treatment or prevention of mHTT-related neurodegenerative diseases, comprising
  • test system comprising polyQ abnormally amplified HTT or mutants thereof or fragments thereof, wherein
  • the polyQ abnormally amplified HTT contains a polyQ length ⁇ 36;
  • the fragment comprises the N17 region and the polyQ region of polyQ aberrantly amplified HTT or a mutant thereof, and optionally comprises a PRD;
  • 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.
  • the system described in (I) is a solution system.
  • candidate compounds may be provided as a compound library. Such libraries can be commercially available or engineered.
  • Candidate compounds may include peptides, peptidomimetics, and small organic molecules, among others. For example, it can be selected from a compound synthesized by design, a compound whose structure is determined in a database (such as Pubmed), or a compound synthesized de novo. In a specific embodiment, candidate compounds are selected from small organic molecules.
  • small organic molecule or “low molecular weight compound” refers to a molecule comparable in size to organic molecules commonly used in medicine.
  • the small organic molecule has a size of about 100-about 2000 Da, preferably about 200-about 1000 Da, such as about 200-about 900 Da, about 200-about 800 Da, about 200-about 700 Da, about 200- About 600 Da, about 200-about 500 Da.
  • step (II) is carried out by the following method: determining parameters characterizing the binding strength of polyQ aberrantly amplified HTT or its mutants or their fragments containing N17 region and polyQ region to the candidate compound.
  • the aforementioned "parameters" can be determined by a person skilled in the art, but can in particular be, for example, absorbance values, radioactive signal and/or distribution in the sample, fluorescence signal intensity and/or distribution in the sample, heat changes, etc.
  • step (II) selects a compound having said binding ability. In another embodiment, step (II) selects compounds for which said binding reaches a certain strength.
  • the method for determining the combination in step (II) is selected from in vitro pull-down assay, Split-TEV, co-immunoprecipitation, affinity chromatography, complex co-purification, enzyme-linked immunosorbent assay, fluorescent molecular sieve, Yeast two-hybrid, HIP-HOP, time-resolved fluorescence resonance energy transfer, chemiluminescence, surface plasmon resonance, isothermal titration calorimetry, microcalorimetry and any combination thereof.
  • the method for determining the binding in step (II) is selected from in vitro pull-down assay, co-immunoprecipitation, enzyme-linked immunosorbent assay, fluorescent molecular sieve, time-resolved fluorescence resonance energy transfer and any combination thereof.
  • step (II) determines the affinity binding of said candidate compound to said polyQ aberrantly amplified HTT or a fragment thereof.
  • the method used in step (II) to determine said 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 reflectance difference method and any combination thereof.
  • the method used in step (II) to determine said binding is selected from fluorescence resonance energy transfer, fluorescence polarization detection, oblique incident light reflectance method and any combination thereof; particularly preferably oblique incident light reflectance difference Law.
  • step (II) selects a compound with an affinity reaction equilibrium dissociation constant below 100 ⁇ M, preferably below 10 ⁇ M, particularly preferably below 1 ⁇ M, such as below 600 nM, below 500 nM, below 400 nM, below 300 nM, below 200 nM, or below 100 nM Next, wait.
  • an affinity reaction equilibrium dissociation constant below 100 ⁇ M, preferably below 10 ⁇ M, particularly preferably below 1 ⁇ M, such as below 600 nM, below 500 nM, below 400 nM, below 300 nM, below 200 nM, or below 100 nM Next, wait.
  • step (II) determining said binding is performed using high-throughput screening; comprising:
  • the target protein comprises polyQ aberrantly amplified HTT or a mutant thereof or a fragment thereof, wherein said fragment comprises the N17 region and the polyQ region of polyQ aberrantly amplified HTT or a mutant thereof, and optionally comprises a PRD;
  • a compound with an affinity reaction equilibrium dissociation constant of 100 ⁇ M or less is selected, preferably 10 ⁇ M or less, particularly preferably 1 ⁇ M or less, such as 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, Wait.
  • the high-throughput screening is performed using a fluorescent method
  • the target protein comprises (1) a detectable molecule (such as a fluorescent protein, such as a GFP tag) and (2) polyQ aberrantly amplified HTT or a mutation thereof bodies or their fragments.
  • the target protein is polyQ aberrantly amplified HTT or mutants thereof or fragments thereof fused to a fluorescent protein such as a GFP tag.
  • analyzing the image in step (c) refers to analyzing the fluorescence signal intensity.
  • the high-throughput screening is carried out using the oblique incident light reflectance difference method, and the step (a) further includes: scanning the chip to obtain an image before incubation; the step (c) includes: analyzing the difference image (image after incubation - image before incubation) for analysis; select compounds capable of affinity binding to the target protein.
  • the method further includes step (III): selecting a compound that has the following effects on mHTT-related neurodegenerative diseases: preventing, alleviating, alleviating or improving mHTT-induced lesions.
  • step (III) selects compounds that reduce mHTT-dependent apoptosis.
  • step (III) selects a compound that reduces the level of polyQ aberrantly amplified HTT in the cell.
  • step (III) is performed on a cell or animal model of mHTT-related neurodegenerative diseases.
  • step (III) comprises: measuring mHTT-dependent apoptosis of neurons comprising polyQ aberrantly amplified HTT, such as striatal cells.
  • step (III) includes: measuring the apoptosis of mouse striatal cells (STHdh) containing polyQ aberrantly amplified HTT under stress conditions.
  • the mouse striatal cell comprising polyQ aberrantly amplified HTT is STHdhQ7 /Q111 .
  • the stress condition is serum starvation.
  • cell apoptosis is measured by detecting apoptotic signal, such as caspase (eg, caspase-3 and/or caspase-7) activity.
  • mHTT-dependent apoptosis is measured by observing the morphology of cells or subcellular structures, such as neuronal arrest or neuronal shrinkage.
  • step (III) includes: determining the level of mHTT-related neurodegenerative disease biomarkers.
  • the biomarker is selected from mHTT aggregates, Tuj1, GABA and DARPP-32, in particular mHTT aggregates or DARPP-32.
  • the cell model of step (III) is a cell containing polyQ abnormally amplified HTT from a patient or an animal disease model, or a cell expressing polyQ abnormally amplified HTT transformed with recombinant DNA.
  • Exemplary models or experimental conditions include but are not limited to this application and our prior research (Li, Z. et al. Allele-selective lowering of mutant HTT protein by HTT-LC3 linker compounds. Nature 575,203-209 (2019) and Those described in Yao Y,.et al.A striatal-enriched intronic GPCR modulates huntingtin levels and toxicity.Elife.2015 Mar 4;4:e05449).
  • the method further comprises step (IV): determining the binding of the candidate compound to normal polyQ HTT or its mutants or their fragments, and selecting for affinity not to normal polyQ HTT or its mutants or their fragments combined compounds, where
  • the polyQ length contained in the normal polyQ HTT is ⁇ 36;
  • the fragment comprises the N17 region and the polyQ region of a normal polyQ HTT or a mutant thereof, and optionally comprises a PRD.
  • step (I) and step (II) are performed sequentially.
  • step (III) and/or step (IV) there is no particular limitation on the sequence between step (III), step (IV) and step (I) and step (II).
  • step (III) is performed after step (I) and step (II).
  • step (IV) there is no particular limitation on the sequence between step (IV) and step (I) and step (II).
  • step (IV) is performed after step (I) and step (II).
  • step (III) and step (IV) exist, the order between step (III) and step (IV) is not particularly limited.
  • the two or more steps can be carried out independently, for example in the same or different systems; and can be carried out simultaneously or sequentially or in sequence; and its sequence and sequence can be arranged arbitrarily.
  • polypeptide refers to a polymer of amino acids of a certain length.
  • peptides, oligopeptides and proteins are included within the definition of “polypeptide” and these terms are used interchangeably herein.
  • the terms “polypeptide” or “protein” do not exclude post-translational modifications, which include, but are not limited to, phosphorylation, acetylation, benzoylation, palmitoylation, glycosylation, and the like.
  • a protein or protein fragment of the invention may be produced by any technique known per se in the art, such as, but not limited to, any chemical, biological, genetic or enzymatic technique used alone or in combination.
  • amino acid sequence of a desired sequence one skilled in the art can readily prepare said protein or protein fragment by standard techniques for producing proteins or protein fragments. For example, they can be synthesized by using well-known solid-phase methods, preferably using commercially available peptide synthesis instruments (such as those manufactured by Applied Biosystems, Foster City, California) and according to the manufacturer's instructions.
  • proteins or protein fragments of the invention can be synthesized by recombinant DNA techniques well known in the art. For example, after introducing a DNA sequence encoding a desired (poly)peptide into an expression vector and introducing this vector into an appropriate eukaryotic or prokaryotic host expressing the desired protein or protein fragment, these fragments can be obtained as DNA expression products, followed by They can be isolated from the host by well known techniques.
  • a variety of host/expression vector combinations can be used to express nucleic acids encoding proteins or protein fragments of the invention.
  • Expression vectors that can be used include, for example, chromosomal fragments, non-chromosomal and synthetic DNA sequences. 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, as well as centromeres and integrated Yeast shuttle vectors; vectors for use in eukaryotic cells, such
  • mammalian and typically human cells as well as bacterial, yeast, fungal, insect, nematode and plant cells can be used in the present invention and can be transfected with nucleic acids or recombinant vectors 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 (e.g. strain DH5-[ ⁇ ]), Bacillus subtilis, Salmonella typhimurium or Pseudomonas, Streptomyces and Staphylococcus strains of the genus.
  • yeast cells such as Saccharomyces cells, such as Saccharomyces cerevisiae.
  • the protein or protein fragment of the present invention can be modified or transformed without affecting the function of the protein or protein fragment.
  • Such protein Or protein fragments and applications thereof also belong to the scope of the present invention.
  • HTT or mutants thereof or fragments thereof containing the N17 region and polyQ region may be detectably molecularly labeled for screening purposes.
  • a detectable molecule may consist of any compound or substance capable of detection 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 proteins (such as GFP and YFP, wherein GFP can be, for example, sfGFP).
  • the detectable label is located or bound to amino acid residues located outside the sequence of HTT or mutants thereof or their fragments containing the N17 region and polyQ region, thereby minimizing or preventing any damage to the protein or Binding artifacts between protein fragments or between a candidate compound and any such protein or protein fragment.
  • the protein or protein fragment of the 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 invention is labeled with a suitable fluorophore, respectively, in a manner suitable for fluorescence energy transfer analysis.
  • step (I) or step (II) of the screening method of the present invention intact polyQ aberrantly amplified HTT can be used for the assay.
  • polyQ aberrantly amplified HTT fragments that include binding sites can be used in the assay.
  • the degradation pathways of HTT in cells mainly include ubiquitin-proteasome system (UPS) and autophagy.
  • UPS ubiquitin-proteasome system
  • autophagy the shorter N-terminal fragment of mHTT was more toxic and produced a more pronounced phenotype. This may suggest that the shorter form of HTT is more toxic, possibly due to reduced interactions with proteins key to autophagy.
  • Post-translational modifications of HTT may affect HD disease states. Possible post-translational modifications such as ubiquitination, acetylation, small ubiquitin-related modifier (SUMO) modification, ie SUMOylation, as well as benzoylation and palmitoylation.
  • ubiquitination ubiquitination
  • acetylation small ubiquitin-related modifier (SUMO) modification
  • ie SUMOylation ie SUMOylation
  • benzoylation and palmitoylation as benzoylation and palmitoylation.
  • HTT exon1 The amino acid sequence encoded by exon 1 of the HTT gene (called HTT exon1) mainly includes the N-terminal 17 amino acids (N17, also known as the N17 region), a glutamine repeat region (polyQ) and a proline-rich structure domain (PRD, or PRD domain).
  • N17 contains possible phosphorylation sites T3, S13 and S16. Oxidation of M8 may promote phosphorylation of S13 and S16.
  • N17 contains possible acetylation, sumoylation and ubiquitination sites K6, K9 and K15.
  • the length of the polyQ region can significantly alter the overall conformation and phosphorylation pattern of HTT proteins. Thus, overall there is a marked difference between polyQ aberrantly amplified full-length HTT and normal full-length HTT.
  • the PRD contains 49 amino acid residues. It mediates the binding of various proteins to HTT.
  • the target protein used in step (I) is polyQ abnormally amplified HTT, and its polyQ length is ⁇ 36.
  • the target protein used in step (IV) is normal polyQ HTT.
  • the normal polyQ HTT comprises the amino acid sequence of SEQ ID NO:1, and the length of its polyQ region can be the same as or different from SEQ ID NO:1, as long as the polyQ length ⁇ 36.
  • the target protein used in step (I) is a polyQ abnormally amplified HTT mutant. In one embodiment, the target protein used in step (IV) is a mutant of normal polyQ HTT.
  • the amino acid sequence of polyQ aberrantly amplified HTT or a mutant or fragment thereof comprises K6. In one embodiment, the amino acid sequence of the mutant retains K6. In one embodiment, the amino acid sequence of the mutant comprises one or more of the following, preferably contains an amino acid substitution selected from K9X, K15X, E12X, S13X and S16X, wherein X represents the same as wild Any natural amino acid that differs in type sequence.
  • the natural amino acids include glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, semi Cystine, Methionine, Asparagine, Glutamine, Threonine, Aspartic Acid, Glutamic Acid, Lysine, Arginine, and Histidine.
  • X represents a conservative amino acid substitution.
  • the amino acid sequence of the mutant comprises one or more of the following, preferably one amino acid substitution selected from K9R, K15R, E12A, S13A and S16A.
  • the amino acid sequence of the mutant comprises one or more of the following, preferably one amino acid substitution selected from: K9R, K15R, E12A and S13,16A.
  • the target protein used in step (I) is a fragment of polyQ aberrantly amplified HTT or a mutant thereof.
  • the polyQ aberrantly amplified HTT mutant is as described above.
  • the target protein used in step (IV) is a fragment of normal polyQ HTT or a mutant thereof.
  • the amino acid sequence of the mutant of normal polyQ HTT may comprise the amino acid substitutions contained in the mutant of abnormally amplified polyQ HTT described above, but is not limited thereto.
  • the fragment is the amino acid sequence encoded by exon 1 of the HTT gene (exemplary sequences such as SEQ ID NO: 2 and SEQ ID NO: 4), or the N17 region of exon 1 of the HTT gene and the amino acid sequence encoded by the polyQ region (exemplary sequences such as SEQ ID NO: 3 and SEQ ID NO: 5).
  • the invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding a protein or protein fragment of the invention.
  • the invention also provides a vector comprising a nucleic acid molecule of the invention.
  • the invention also relates to compounds obtained by the process of the invention.
  • the present invention also relates to compounds selected from:
  • the compound of the invention is selected from in particular
  • the compounds of the present invention encompass pharmaceutically acceptable salts, stereoisomers, solvates, polymorphs, tautomers, isotopes, metabolites or prodrugs thereof.
  • pharmaceutically acceptable means contact with a patient's tissue without undue toxicity, irritation, allergic reaction, etc. within the scope of normal medical judgment.
  • 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 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 (+)-pairs of enantiomers, (R)- and (S)-pairs Enantiomers, diastereoisomers, (D)-isomers, (L)-isomers, and racemic and other mixtures thereof, e.g. enantiomerically or diastereomerically enriched mixtures, all such mixtures are within the scope of the present invention.
  • the compounds of the invention may exist in the form of solvates, preferably hydrates, wherein the compounds of the invention comprise a polar solvent, such as in particular water, methanol or ethanol, as a structural element of the crystal lattice of the compound.
  • a polar solvent such as in particular water, methanol or ethanol
  • the amount of polar solvent, especially water, may 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 may be a single polymorph or a mixture of more than one polymorph in any proportion.
  • metabolites of the compounds of the invention ie substances formed in vivo upon administration of the compounds of the invention. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc. of the administered compound.
  • the present invention further includes within its scope prodrugs of the compounds of the invention, which are certain derivatives of the compounds of the invention which themselves may have little or no pharmacological activity when administered into or on the body. can be converted to a compound of the 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 proportion.
  • crystalline form or “crystal” refers to any solid material that exhibits a three-dimensional order, as opposed to amorphous solid material, which produces a characteristic X-ray powder diffraction pattern with well-defined peaks.
  • amorphous refers to any solid substance that is not ordered in three dimensions.
  • the compounds of the present invention can be used 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 the organism and will not impair the biological activity and performance of the active compound.
  • “Pharmaceutically acceptable carrier” includes, but is not limited to, glidants, sweeteners, diluents, preservatives, dyes/colorants, flavoring agents, surfactants, wetting agents, dispersants, disintegrants, Stabilizer, solvent or emulsifier.
  • the invention provides a PROTAC compound comprising a compound of the invention, an E3 ubiquitin ligase binding ligand, and a linker linking the compound of the invention and the E3 ubiquitin ligase binding ligand.
  • the compound of the present invention is used as a ligand to bind to polyQ abnormally amplified HTT or its mutant or their fragments.
  • the E3 ubiquitin ligase is selected from VHL, CRBN, MDM2, ⁇ -TRCP, cIAP, RNF4, RNF14, DCAF16, preferably selected from VHL, CRBN, MDM2 and cIAP1, especially VHL and CRBN .
  • the PROTAC compound can be used to promote the degradation of polyQ aberrantly amplified HTT or its fragments containing polyQ region and N17 region by UPS.
  • inactive PROTAC compounds inactive PROTAC can also be prepared, that is, the molecules produced do not bind to E3 ubiquitin ligase, but the compounds of the present invention therein are still capable of binding to polyQ abnormal expansion Increase the binding of HTT or its fragments containing polyQ region and N17 region.
  • compositions, formulations and kits are provided.
  • the invention provides an article of manufacture, for example in kit form.
  • An article of manufacture of the invention comprises a compound or pharmaceutical composition of the invention, and optionally includes a box and instructions.
  • the present invention provides the use of a compound of the present invention, or a pharmaceutical composition thereof, in the manufacture of a diagnostic reagent or kit for detecting a subject believed to have or be susceptible to an mHTT-related neurodegenerative disease.
  • the detection described above comprises the step of analyzing a sample of interest obtained from a subject, comprising:
  • the sample of interest is selected from a cell sample, blood, serum, spinal fluid; or any biopsy sample obtained from a subject's tissue.
  • kits comprise a compound of the present invention or a pharmaceutical composition thereof, and optionally reagents required for detection, such as aqueous solutions, solvents, suitable detection reagents such as chemiluminescent reagents, and the like.
  • the present invention provides a compound of the present invention, or a PROTAC compound thereof, or a pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, tautomer, isotopic compound, metabolite or precursor thereof Use of the medicament or its pharmaceutical composition in the preparation of medicaments for treating or preventing mHTT-related neurodegenerative diseases.
  • the present invention provides a compound of the present invention, or a PROTAC compound thereof, or a pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, tautomer, isotopic compound, metabolite thereof Or a prodrug, or a pharmaceutical composition thereof, for the treatment or prevention of mHTT-related neurodegenerative diseases.
  • the present invention provides a method for treating or preventing mHTT-related neurodegenerative diseases, comprising administering a compound of the present invention, or a PROTAC compound thereof, or a pharmaceutically acceptable salt, stereo Isomers, solvates, polymorphs, tautomers, isotopic compounds, metabolites or prodrugs, or pharmaceutical compositions thereof.
  • the mHTT-associated neurodegenerative disease is Huntington's disease (HD).
  • the present invention provides a compound of the present invention, or a PROTAC compound thereof, or a pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, tautomer, isotopic compound, metabolite or precursor thereof Use of a medicine or a pharmaceutical composition thereof for reducing the level of abnormally amplified polyQ HTT in cells, tissues or organs.
  • HTT Huntingtin
  • HTT belongs to the larger protein in the animal proteome. It is currently believed that HTT has multiple sites that can bind to other proteins, and in physiological environments, HTT may achieve some functions by binding to these proteins.
  • the mode of action of small molecules on HTT and the "binding pocket" of small molecules and HTT are unknown, so for the development of traditional small molecule drugs, HTT is an "undruggable" target.
  • the screening method obtains the target compound by screening the compound that interacts with the target, and can verify the therapeutic effect of the compound through phenotypic screening.
  • the screening process is fast and simple, with high efficiency, low cost, and easy to achieve high-throughput screening.
  • the compound library used in the examples is provided by Selleck Company and contains 3375 biologically active compounds. These included 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. in:
  • Compound 1 desonide, PubChem CID: 5311066, Selleck cat.no.S1701;
  • Compound 4 Loratadine, PubChem CID: 3957, Selleck cat. no. S1358.
  • HTT antibody 2B7 (Weiss et al.Anal Biochem 2009,395,8-15) and MW1 (Ko et al.Brain research bulletin 2001,56,319-329) were prepared using prior art methods; used for immunostaining to detect HTT aggregates
  • the antibody S830 was donated from Dr. Gillian Bates; other antibodies were purchased from Millipore, Sigma and other companies.
  • HTT antibody 2166 (Millipore, cat.no.MAB2166), 3B5H10 (Sigma, #P1874), anti- ⁇ -tubulin (Abcam, cat.no.ab6046), anti-TUBB3 (Covance, cat.no.MMS- 435P), anti-DARPP-32 (Abcam, cat.no.ab40801), anti-ATXN3 (Millipore, cat.no.MAB5360), anti-spectrin (Millipore, cat.no.MAB1622), anti-GR (Abmart, cat.no.T56612), anti-HA (Abmart, cat.no.M20003), anti-ubiquitin (ProteinTech, cat.no.10201-2-AP), anti-Actin (Abmart, cat.no.M20011).
  • the exon 1 sequence of human HTT gene (GenBank: NM_002111.8) with 72Q or 25Q fragment (encoded by CAGCAA mixed sequence) was cloned into the pTT5SH8Q2 vector with sfGFP tag to obtain pTT-HTTexon1-Q72-sfGFP and pTT -HTTexon1-Q25-sfGFP plasmid.
  • the plasmid was transfected into HEK293T cells for expression using polyethyleneimine (PEI, from Polysciences, 23966). Protein was purified with HisTrap HP column (GE Healthcare, 17524701). The buffer was exchanged to 50mM HEPES buffer pH 7.0 with 150mM NaCl.
  • the plasmid was transfected into Escherichia coli BL21(DE3)pLsyS for expression. Purify with HisTrap HP column (GE Healthcare, 17524701) and Superose 6 Increase 10/300GL column.
  • the mHTT exon1 construct with the HA tag at the C-terminus was cloned into a mammalian expression vector (pSG5), and the mammalian cells were transfected for 24 hours to express mHTT exon1.
  • Human HTT gene (GenBank: NM_002111.8) with (CAG) 23 or (CAG) 73 was de novo synthesized by Genewiz Inc.
  • the human HTT gene was cloned into a modified pCAG vector (from Addgene) with an N-terminal protein A tag.
  • Primary cultured cortical neurons The brains of Hdh Q7/Q140 and Hdh Q7/Q7 neonatal mice (P0) were dissected, digested, dissociated and cultured.
  • Some primary patient fibroblasts and wild-type cells were from HD patients (Q47, Q49, Q55) and healthy controls (WT, Q19) of the Mongolian Huntington's disease family.
  • the SCA3 cell line is from a patient (Q74).
  • the HD Q68 fibroblast cell line was from Coriell Cell Repositories (Camden, NJ, USA).
  • Immortalized fibroblasts and iPS cells (iPSCs) were prepared from primary fibroblasts.
  • Mouse striatal cells STHdh Q111/Q7 were from Coriell Cell Repositories (Camden, NJ, USA).
  • HEK293T cells were from ATCC.
  • the nervous system driver line elav-GAL4(c155), the HTT-expressing lines UAS-fl-HTT-Q16 and UAS-fl-HTT-Q128 were obtained from the Bloomington Drosophila Stock Center at Indiana University (http://flystocks.bio.indiana.edu /) and kept in a 25°C incubator.
  • Hdh Q7/Q7 mice expressing the wild-type HTT gene
  • Hdh Q7/Q7 mice expressing the wild-type HTT gene
  • Q140 gene knock-in heterozygous mice mice expressing the wild-type HTT gene (Hdh Q7/Q7 ) were from the laboratory of Marian Difiglia, Massachusetts General Hospital, Harvard University.
  • Q140 gene knock-in heterozygous mice (Hdh Q7Q140 ) were prepared according to the method of the prior art (Mealled et al., J Comp Neurol, 2003, 465:11-26).
  • Compounds were formulated as DMSO stock solutions. Compound stocks were diluted in culture medium to the indicated concentrations of working solutions prior to use in treating cells. Compounds were diluted 10-fold in culture medium and added to plated cells unless otherwise stated. Compounds were added 5 days after plating for primary cultured neurons and iPS cell-derived neurons and 1 day after plating for patient fibroblasts and other cell lines. After 2 days of compound treatment at 37°C in an incubator with 5% CO 2 , cells were harvested to measure mHTT levels.
  • HTRF Homogeneous Time-Resolved Fluorescence
  • the amount of protein was measured by the method described above. Background correction was performed with a blank sample. Protein concentrations were determined for all samples to correct for sample size. Measure different protein concentrations or cell numbers per well to ensure the signal is in the linear range.
  • Immunofluorescence After the cells were washed, fixed, permeabilized, and blocked, they were incubated with the primary antibody at 4°C overnight, then washed three times with blocking buffer, and incubated with the secondary antibody for 1 hour at room temperature. After staining with DAPI, after mounting, image with confocal microscope, and analyze TUBB3 or co-localization with ImageJ.
  • siRNA target sequence and/or source information Negative control (Neg siRNA): non-targeting siRNA (Generalbiol, #RX028810); Glucocorticoid receptor siRNA: targeting GGUAAUUAAGCAAGAGAAATT.
  • mice were intraperitoneally injected (ip) with compounds or DMSO for control.
  • a small animal anesthesia machine (MSS-3, MSS International, Keighley, UK) was anesthetized with isoflurane, and blood was collected from the heart with a vacuum blood collection tube.
  • Heart blood samples were centrifuged at 10,000 r.p.m for 5 minutes to obtain heart plasma.
  • mice were perfused with 1 ⁇ PBS to remove blood. Euthanize the mouse and remove the brain.
  • Add 5 volumes of methanol:acetonitrile (50:50, vol/vol) to each brain sample, and then homogenize. After sonicating for 15 minutes, the homogenate was centrifuged for 5 minutes, and 20 ⁇ L of the supernatant was mixed with 20 ⁇ L of water for 30 s, and then injected into LC-MS/MS.
  • LC-MS/MS analysis an Acquity ultra-high performance liquid chromatography (UPLC) system (Waters Corporation) connected to a Xevo TQ-S mass spectrometer (Waters Corporation) was used.
  • Chromatographic column Acquity UPLC BEH C18 (1.7 ⁇ m 2.1 ⁇ 50 mm).
  • the compound chip was image-scanned with a fluorescence scanner (from Molecular Device) with an excitation wavelength of 488 nm. Bright spots in the fluorescence image indicate compounds bound to the target protein. Select compounds that bind to the target protein.
  • STHdh Q7/Q111 cells under stress conditions such as serum starvation, mHTT-dependent apoptosis signal.
  • apoptotic signals have been widely used as indicators of mHTT toxicity.
  • STHdh Q7/Q111 cells were used as HD cell models to test whether the 21 compounds specifically binding to mHTT obtained in Example 1 could improve mHTT-induced cytotoxicity.
  • Compounds were added at the onset of serum starvation.
  • DMSO and the pan-caspase inhibitor z-vad-fmk were used as negative and positive controls, respectively.
  • Apoptosis was measured by detecting caspase-3 activity with a green fluorescent dye (NucView 488) at various time points after serum depletion (start of serum starvation).
  • the five compounds with phenotype rescue effect obtained in 2.1 were tested against the proteasome inhibitor MG132 induced by serum-free conditions, which may be caused by the unfolded protein response (UPR )-induced apoptosis of wild-type (WT) striatal cells.
  • URR unfolded protein response
  • Embodiment 3 The determination of compound to mHTT affinity activity
  • the compound chip was prepared with the four compounds desonide, apeledoxifene, Loratadine and iloperidone obtained in 2.2, and the compound was detected by OI-RD Affinity activity with HTTexon1-Q72 and HTTexon1-Q25, respectively. These 4 compounds were observed to bind mHTT exon1 with allele-selective affinity (Figure 4).
  • the K on (association rate constant), K off (dissociation rate constant) and K d (reaction equilibrium dissociation constant) of the affinity reaction are shown in Fig. 4, respectively.
  • the reaction buffer is 20mM HEPES, pH 7.4, 150mM NaCl, protein concentration 500nM.
  • the compound has no affinity binding to HTTexon1Q25-MBP and flHTT-Q23 (K d >100 ⁇ M), and the K d of HTTexon1Q72-MBP and flHTT-Q73 are shown in Table 1, respectively.
  • ITC Isothermal titration calorimetry
  • the same buffer solution of desonide was injected into the pool 20 times (0.4 ⁇ L for the first drop, 2 ⁇ L for the 2nd-20th drop), with an interval of 180s between each injection.
  • the heat (H) released by the protein binding to the compound is recorded in real time.
  • the heat released or absorbed by molecular binding is proportional to the number of molecules bound. When the system is saturated, only the heat of dilution is observed.
  • the mutant and wild-type HTT genes share the same promoter. Therefore, the effect of desonide on reducing mHTT levels may be achieved by reducing mHTT stability rather than inhibiting mHTT expression.
  • Our experiments show that desonide can allele-selectively bind to mHTT exon1 or full-length mHTT, which is also consistent with the above theory.
  • Example 4 Effect of desonide on the HTT level of HD mouse striatal cells, HD patient fibroblasts, and HD patient induced stem cell differentiation neuron cells
  • Drosophila flies expressing mHTT exon1 or full-length mHTT in neurons as described above were used as HD flies.
  • the experiment of this embodiment was carried out with reference to the experimental method in the previous research (see Li, Z. et al. literature mentioned above).
  • Drosophila were maintained on a compound diet.
  • the administration group was fed with food containing the compound desonide, apeledoxifene, loratadine or iloperidone obtained in 2.2.
  • the crawling ability of Drosophila was measured in vials to evaluate the motor function of HD Drosophila. 4 vials per group.
  • the percentage of flies that climbed the 7 cm high line after 15 seconds was recorded and plotted against the number of days since eclosion (day age). The compound was observed to significantly improve the crawling ability of HD flies.
  • Drosophila expressing HTTexon1-Q25 fed food containing the compound showed no significant decrease in performance (Figure 11).
  • the elav-GAL4:UAS-flHTTQ128 flies were fed with food containing the compound for 6 days after eclosion, and the HTT protein in the head of the flies was extracted and measured by HTRF (antibody pair: 2B7/MW1; n ⁇ 9). It was observed that the desonide allele selectively reduces the level of mHTT (Figure 12).
  • mice were housed in groups of up to 5 adult mice per cage in individually ventilated cages with a 12-h light/dark cycle.
  • DMSO used for the compound or control was diluted to 0.5 mg/mL with 0.9% NaCl intravenous infusion solution, and intraperitoneally injected (0.5 mg/kg) once a day. After 4 weeks of injection, tissue extraction or behavioral experiments were performed .
  • intracerebroventricular injection 2 ⁇ L of artificial cerebrospinal fluid (ACSF: 1 mM glucose, 119 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO 4 , 2.5 mM CaCl 2 , 26.2mM NaHCO 3 , 1mM NaH 2 PO 4 ), where the compound concentration was 2mM.
  • ASF artificial cerebrospinal fluid
  • the concentration of the compound in the brain is about 4 ⁇ M.
  • 2 ⁇ L of ACSF containing an equal amount of DMSO was used as a control.
  • Intraventricular injections were given once daily. Four weeks after injection, open field behavior and rearing behavior were measured. No effect was observed in the Loratadine group. A slight rescue effect was observed in the apeledoxifene group. A more significant rescue effect was observed in the desonide group (Fig. 13A, 13B).
  • mice Hdh Q140/Q140 mice, 12 months old at the start of administration. Littermates of wild-type mice were used as a reference to rule out behavioral manifestations unrelated to HD.
  • Hindlimb standing frequency mice were placed in a three-dimensional frame for 5 minutes, and the three-dimensional frame was formed by a pen holder (high 98mm x diameter 91mm) with uneven surface. The total number of events in which the animal raised at least two forelimbs was the number of hindlimb stances.
  • Open field test the mice were placed in a 30x30x40cm white plexiglass chamber in the behavior room, and the movement (locomotion) of the mice was recorded for 15 minutes through a camera on the top of the chamber. Tracks and distance traveled are then analyzed with an activity monitoring program.
  • mice were pre-trained (rotating on a rotarod for 2 min at 4 rpm) for 3 consecutive days. The mice were then tested for 5 days at an accelerated speed of 4 to 40 rpm within 2 minutes. Each trial outcome was recorded as on-rod time (time on the spinning rod) until dropped from the rod or until the end of the task. Each test consisted of three repetitions with 60-minute intervals between trials to reduce stress and fatigue. The average of three trials per mouse was analyzed.
  • Grip strength test (1) First, each mouse is given a grip training to stimulate the grip reflex, and train it to grasp the force measuring bar of the measuring instrument to ensure that it can grasp stably.
  • the training method is: gently lift the mouse from the tail, and when the two forelimbs of the mouse are close to the cross bar, it can be induced to actively grasp the cross bar with its paws.
  • Acceptable grip criteria are mice actively extending their paws and holding the bar steadily without ducking, limb twisting or resistance. Grasp training for each mouse was carried out for approximately 5-10 min per day for 3-5 days until it was able to easily and tamely perform a double forelimb grasp.
  • Equilibrium beam test a graduated rod 2 cm thick and 100 cm in total length, suspended on both sides from a platform. There is a bright light at the beginning and a dark box with food at the end. The total time each mouse spent walking across the balance beam was recorded.
  • molecular biomarkers of HD including mHTT aggregates and the marker DARPP-32 of medium spiny neurons, were detected by immunostaining with S830 and anti-DARPP-32. A significant decrease in the level of mHTT aggregates and an increase in DARPP-32 signal was observed ( Figure 15).
  • Hdh Q7/Q140 mice 14 months old at the beginning of administration, 9 mice in each group. Hdh Q7/Q7 mice were used as controls.
  • desonide is an FDA-approved drug for the treatment of atopic dermatitis, the safety of topical desonide has been proven. According to examples 7 and 8, intracerebroventricular injection of desonide is effective, but other feasible modes of administration are still needed.
  • mice Inject ip at a dose of 5 mg/kg, anesthetize mice at designated time points, and collect cardiac blood to prepare cardiac plasma. Mice were then perfused with 1 ⁇ PBS to remove blood. Mice were euthanized, and in vivo compound concentrations were determined as described above. It was found that the brain concentration of desonide can reach about 2 to 3 ⁇ M ( FIG. 16 ). Also, ip injection of the desonide allele selectively and significantly reduced striatal mHTT levels (Fig. 17).
  • HTTexon1-Q72 K6R, K9R, K15R, K6,9,15R, E12A or S13,16A, where the numbers are Based on the sequence in the wild type sequence (SEQ ID NO: 1). Sequences were verified by sequencing. The construct was prepared and transformed into a suitable host to express the HTTexon1-Q72 mutant containing the desired point mutation (hereinafter referred to as the mutant).
  • a truncated mHTT exon1 sequence ⁇ PRD (deletion of PRD, see SEQ ID NO: 3 for an exemplary sequence, i.e. deletion of amino acids 90 to 138 in HTTexon1-Q72) was prepared using a method similar to that described above for the preparation of recombinant human HTT exon1 protein .
  • K6 is the key to the affinity activity of desonide for mHTT
  • the polyQ length might regulate the proximity between N17 and PRD on its flanks.
  • the PRD may be spatially close to N17 and shield or partially shield the site from which the compound can bind.
  • the K d value of ⁇ PRD and desonide is 1.6 ⁇ M. It is possible that PRD shields or partially shields the binding site of wtHTT exon1 to desonide, while having no such effect on mHTT exon1.
  • HEK293T cells or STHdh Q7/Q7 cells transfected with HTTexon1-Q72 or its mutants or HTTexon1-Q25 were treated with desonide at a concentration of 3 ⁇ M. Detection was performed with HTRF (antibody pair: 2B7/MW1). It was observed that desonide could reduce the levels of HTTexon1-Q72 and its K9R, K15R, E12A or S13,16A mutants in cells, but not HTTexon1-Q72K6R mutants ( FIG. 23 , FIG. 24 ). At the functional level, it was confirmed that the action of desonide was dependent on K6.
  • HEK293T cells or STHdh Q7/Q7 cells transfected with HTTexon1-Q72 or its mutants or HTTexon1-Q25 were treated with desonide at a concentration of 3 ⁇ M.
  • the cytotoxicity of mHTT was determined by measuring the apoptotic signal (caspase-3 activity) under serum starvation conditions.
  • desonide can improve the apoptosis caused by HTTexon1-Q72 (Figure 25) or its K9R, K15R, E12A or S13,16A mutants ( Figure 25, Figure 26), but cannot improve the apoptosis caused by HTTexon1-Q72K6R ( Figure 25 ).
  • Example 12 The reducing effect of desonide on mHTT depends on the proteasome-ubiquitin system (UPS)
  • HEK293T cells were transiently transfected with HTTexon1Q72-HA or HTTexon1Q25-HA constructs and control plasmids for 24 hours, and then cells were treated with 100 nM epoxymycin for 24 hours.
  • test results showed that treatment of HEK293T cells overexpressing mHTT exon1 with desonide resulted in a significant increase in the polyubiquitination (poly-ub) of mHTT exon1. Also, no increase in polyubiquitination of wtHTT exon1 was observed ( FIG. 30 ).
  • Example 13 The effect of desonide on mHTT without the participation of glucocorticoid receptors
  • Desonide is known to be a weak glucocorticoid receptor (GR) agonist.
  • HD cells STHdh Q7/Q111 were treated with GR agonists (prednisolone, ciclesonide, hydrocortisone or fludroxone) at a concentration of 3 ⁇ M for 48 hours.
  • GR agonists prednisolone, ciclesonide, hydrocortisone or fludroxone
  • mHTT and wtHTT were detected and quantified by Western blot (antibody: 2166) (antibody: D7F7 or 2166).
  • Affinity binding was detected by MST in a similar manner to 3.2. It was observed that these agonists did not bind mHTT with affinity nor reduce mHTT levels (Figure 31).
  • siRNA transfection for 24 hours to knock down GR in STHdh Q7/Q111 cells and then use the method similar to 13.1 to test the effect of desonide on mHTT levels in cells.
  • desonide can still reduce their mHTT levels (Fig. 32).

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Abstract

一种筛选用于治疗或预防mHTT相关的神经退行性疾病的化合物的方法，用于筛选的靶蛋白，以及相应的化合物。

Description

筛选用于治疗或预防mHTT相关的神经退行性疾病的化合物的方法，靶蛋白，以及化合物 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用于治疗或预防mHTT相关的神经退行性疾病的化合物的方法，用于筛选的靶蛋白，以及相应的化合物。
背景技术
神经退行性疾病(Neurodegenerative Disorders)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。针对神经退行性疾病，迄今为止没有可以减缓疾病发展进程的根本性治疗方法。
人类四号染色体所含基因HTT(曾被命名为IT15)的变异与神经退行性疾病有关。例如，亨廷顿病(Huntington's Disease,HD)是HTT外显子1的CAG重复区域的变异引起的一种单基因遗传的神经退行性疾病。变异基因CAG重复数目增多(大于等于36)，导致合成的变异蛋白(mutant Huntingtin,mHTT)的谷氨酰胺重复区域(polyQ)扩增，最终导致主要集中在纹状体的神经元死亡，并引起以肢体运动能力异常为特征的一系列神经功能、心理、以及代谢相关症状。
野生型HTT在细胞内有可能作为支架蛋白(a scaffold protein/a multiprotein scaffold)起作用，然而mHTT的功能是未知的，其导致HD的确切病因也是未知的。由于缺乏传统小分子药物的“结合口袋”，以及缺乏可测量的生化读数，mHTT一直被认为“无可成药性”。即，不能通过筛选mHTT的“抑制剂”得到治疗HD的候选药物。
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mHTT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mHTT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polyQ异常扩增HTT或其突变体或它们的片段，其中
所述polyQ异常扩增HTT包含的polyQ长度≥36；
所述片段包含polyQ异常扩增HTT或其突变体的N17区域和polyQ区域，并任选地包含PRD；
(II)：测定所述候选化合物与所述polyQ异常扩增HTT或其突变体或它们的片段结 合的能力，选出目标化合物。
在一些实施方案中，所述polyQ异常扩增HTT或其突变体或它们的片段的氨基酸序列包含K6。在一些实施方案中，所述polyQ异常扩增HTT或其突变体或它们的片段的氨基酸序列包含选自以下的一种或多种氨基酸替换：K9X、K15X、E12X、S13X和S16X，其中X代表与野生型序列中不同的任意天然氨基酸。
在另一方面，本发明提供通过本发明的方法获得的化合物在制备用于治疗或预防mHTT相关的神经退行性疾病的PROTAC化合物中的用途。
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PROTAC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，或其药物组合物在制备用于治疗或预防mHTT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mHTT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。
在一些实施方案中，本发明的方法获得的化合物选自
Figure PCTCN2022105437-appb-000001
Figure PCTCN2022105437-appb-000002
附图说明
图中“Q+数字”代表使用的含相应长度的polyQ的蛋白或片段。des表示desonide。在图6至图8、图17、图24、图25、图26至图29、图31、图32中，每张图的所有数据相对于DMSO对照归一化，统计分析采用双尾非配对t检验。
图1：化合物对mHTT引起的HD小鼠纹状体细胞STHdh Q7/Q111的凋亡的影响。数据来自≥7批实验。统计分析采用双向ANOVA分析及与DMSO组比较的Dunnett’s post-hoc分析。
图2A、2B：化合物对mHTT引起的HD小鼠纹状体细胞STHdh Q7/Q111的凋亡的影响是剂量依赖性的。
图3：化合物对与mHTT无关的WT小鼠纹状体细胞STHdh Q7/Q7的凋亡的影响。数据来自≥7批实验。统计分析采用双向ANOVA分析及与DMSO组比较的Dunnett’s post-hoc分析。
图4：OI-RD检测化合物与mHTT exon1的结合活性。图中显示化合物的结合-解离曲线，垂直虚线示出结合阶段和解离阶段的开始。
图5：MST测定标准毛细管中的desonide与蛋白的结合。
图6：desonide对STHdh Q7/Q111细胞中HTT水平的影响。
图7：desonide剂量依赖性地降低STHdh Q7/Q111细胞中的mHTT水平。
图8A、8B：desonide对永生化的HD患者成纤维细胞中HTT水平的影响。图8A：HTRF测定HD患者成纤维细胞(Q47、Q55、Q68)的mHTT(抗体对：2B7/MW1；n≥9)和总HTT(抗体对：2B7/2166；n≥9)。图8B：在HD患者成纤维细胞(Q47)中的剂量效应曲线。
图9：desonide对HD患者iPSC分化的神经元细胞中HTT水平的影响。统计分析采用双尾非配对t检验。
图10：化合物对HD患者iPSC分化的神经元凋亡的影响。
图11：化合物对亨廷顿病果蝇爬行能力的影响。统计分析采用双向ANOVA分析及与DMSO组比较的Dunnett’s post-hoc分析。
图12：desonide对HD果蝇mHTT水平的影响。n表示小瓶的数量。统计分析采用双尾非配对t检验。
图13A、13B：化合物对HD小鼠行为的影响。图13A：旷场试验。图13B：后肢站立行为。统计分析采用双尾非配对t检验。
图14A、14B、14C、14D、14E：ip注射desonide对HD小鼠行为学的影响。图14A、14B、14C、14D：统计分析采用单向ANOVA分析。图14E：统计分析采用双向ANOVA分析。
图15：脑室内注射desonide对HD小鼠体内mHTT体和DARPP-32信号的影响。n表示来自三只小鼠的切片数量。统计分析采用双尾非配对t检验。
图16：ip注射desonide的小鼠脑内desonide浓度。每个时间点检测3只小鼠。
图17：ip注射desonide对HD小鼠纹状体mHTT水平的影响。
图18A、18B、18C、18D、18E：ip注射desonide对HD小鼠行为学的影响。统计分析采用双向ANOVA分析和Turkey’s post hoc检验。图18F：ip注射desonide对HD小鼠纹状体中mHTT聚集体的水平的影响。n表示每组3只小鼠的纹状体切片数。统计分析采用双尾非配对t检验。
图19：ip注射desonide对HD小鼠DARPP-32和NFL水平的影响。所有数据均相对于DMSO处理的野生型小鼠归一化。n表示每组3只小鼠的纹状体切片数。比例尺100μm。统计分析采用双尾非配对t检验。
图20：desonide对SCA3患者成纤维细胞mHTT水平的影响。统计分析采用单向ANOVA分析和Dunnett’s post hoc检验进行分析。
图21：MST检测desonide与GFP-polyQ融合蛋白的亲和结合。
图22：MST检测desonide与HTTexon1-Q72突变体的亲和结合。
图23：desonide对转染的HEK293T细胞或STHdh Q7/Q7细胞mHTT及突变体(K6R、K9R、K15R或K6,9,15R)水平的影响。
图24：desonide对转染的HEK293T细胞中mHTT及突变体(E12A或S13,16A)水平的影响。
图25：desonide对mHTT及突变体(K6R、K9R、K15R或K6,9,15R)引起的转染的HEK293T细胞凋亡的影响。数据相对于HTTexon1-Q72转染细胞和DMSO对照进行归一化。统计分析采用双尾非配对t检验。
图26：。desonide对mHTT及突变体(E12A或S13,16A)引起的转染的HEK293T细胞凋亡的影响。
图27：desonide对mHTT及突变体(K6R、K9R、K15R或K6,9,15R)引起的转染的STHdh Q7/Q7细胞凋亡的影响。
图28：desonide在存在或不存在蛋白酶体抑制剂或自噬抑制剂的情况下对STHdh Q7/Q111细胞的mHTT水平的影响。
图29：desonide在存在或不存在蛋白酶体抑制剂或自噬抑制剂的情况下对细胞mHTT水平的影响。左图：内源性表达mHTT的HD患者成纤维细胞(Q47)。右图：转染的HEK293T。
图30：IP-western试验测定desonide对转染的HEK293T细胞中mHTT及突变体(K6R、K9R或K15R)的多聚泛素化的影响。结果来自5批试验。图中，Q25蛋白的位置略高于IgG轻链。
图31：GR激动剂不影响STHdh Q7/Q111细胞mHTT水平，也不与mHTT亲和结合。
图32：desonide对敲低了GR的STHdh Q7/Q111细胞mHTT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≥36的HTT”可以涵盖polyQ长度为36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139或140的情况；特别是46、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1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34或35的情况；特别是7、16、19、23或25等的情况。
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异常扩增的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Q23或Q73，分别表示谷氨酰胺重复区域的长度为23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。
人HTT蛋白序列例如GenBank:BAA36753.1编码的蛋白序列。
术语“mHTT”指polyQ异常扩增的HTT。
术语“mHTT相关的神经退行性疾病”指与polyQ异常扩增的HTT相关的神经退行性疾病，或对含扩增的polyQ的HTT水平响应的神经退行性疾病，例如亨廷顿病。
“正常polyQ”是指正常生理状态下的HTT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 HTT”包含长度正常的polyQ(即其polyQ区域)。表述“正常polyQ HTT”与含有正常长度polyQ的HTT”具有相同的意义并可以互换使用。在一示例性实施方案中，正常polyQ HTT包含SEQ ID No:1的序列。SEQ ID No:1的序列中polyQ长度为23，属于正常的polyQ的范畴。在此基础上，polyQ的数目可以适当增加或减少，但小于特定数目，例如36，仍然属于“正常polyQ HTT”。
如本文所用，“polyQ异常扩增HTT”在“正常polyQ HTT”基础上包含长度异常的polyQ(即其polyQ区域)。例如，polyQ长度≥36的polyQ区域。
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0.1％或更高、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区域，并且包含N17区域，并且任选地包含PRD。
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HTT或其突变体的片段。其含有mHTT的N17区域和polyQ区域，并且任选地包含PRD。所述片段的长度可以仅为mHTT的约0.1％或更高、约0.15％或更高、约0.16％或更高、约0.168％或更高、约0.2％或更高、约0.3％或更高、约0.4％或更高、约0.5％或更高、约0.6％或更高、约0.7％或更高、约0.8％或更高、约0.9％或更高、约1％或更高、约2％或更高、约3％或更高、约4％或更高、约5％或更高、约6％或更高、约7％或更高。
“正常polyQ HTT”或“polyQ异常扩增HTT”可以是天然存在的HTT，其中包含长度正常或异常的polyQ区域，例如可以来自人或来自其他动物，例如来自小鼠(例如小鼠HTT，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。
本领域技术人员还应当理解，可以使用HTT或其片段的修饰形式。例如磷酸化、乙酰化、苯甲酰化和棕榈酰化修饰形式，特别是磷酸化或乙酰化修饰形式。这样的方案同样在本发明所述的“正常polyQ HTT”或“polyQ异常扩增HTT”范围内。
“正常polyQ HTT”或“polyQ异常扩增HTT”也可以是人工改造或修饰的HTT，但仍然带有其相应的N17区域和polyQ区域。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HA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区域，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HTT”是指特定蛋白中的谷氨酰胺重复区域较长。“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HTT”可以与本文所述的“polyQ异常扩增HTT”具有相同的含义。
作为示例，mHTT相关的神经退行性疾病包括但不限于亨廷顿病(Huntington's Disease,HD，polyQ长度≥36)。
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polyQ异常扩增HTT或其片段亲和结合的化合物可以降低polyQ异常扩增蛋白水平、治疗或预防相应疾病，其中所述片段包含其N17区域和polyQ区域，并任选地包含PRD。
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mHTT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polyQ异常扩增HTT或其突变体或它们的片段，其中
所述polyQ异常扩增HTT包含的polyQ长度≥36；
所述片段包含polyQ异常扩增HTT或其突变体的N17区域和polyQ区域，并任 选地包含PRD；
(II)：测定所述候选化合物与所述polyQ异常扩增HTT或其突变体或它们的片段结合的能力，选出目标化合物。
(I)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。在一优选的实施方案中，(I)所述的体系为溶液体系。
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：测定表征polyQ异常扩增HTT或其突变体或它们的含有N17区域和polyQ区域的片段与候选化合物的结合强度的参数。取决于所进行的结合试验，上述“参数”可以由本领域技术人员确定，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，等。
在一实施方案中，步骤(II)选择具备所述结合能力的化合物。在另一实施方案中，步骤(II)选择所述结合达到一定强度的化合物。
在一实施方案中，步骤(II)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。在一优选的实施方案中，步骤(II)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。
在一实施方案中，步骤(II)测定所述候选化合物与所述polyQ异常扩增HTT或其片段的亲和结合。在一实施方案中，步骤(II)用于测定所述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(II)用于测定所述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；特别优选为斜入射光反射差法。
在一实施方案中，步骤(II)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。
在一实施方案中，步骤(II)测定所述结合采用高通量筛选进行；包括：
(a)对候选化合物进行固定化以制备化合物芯片；
(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；
其中靶蛋白包含polyQ异常扩增HTT或其突变体或它们的片段，其中所述片段包含polyQ异常扩增HTT或其突变体的N17区域和polyQ区域，并任选地包含PRD；和
(c)对图像进行分析；选择与靶蛋白能够亲和结合的化合物。在一优选的实施方案中，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。
在一实施方案中，所述高通量筛选采用荧光法进行，所述靶蛋白包含(1)可检测的分子(例如荧光蛋白，比如GFP标签)以及(2)polyQ异常扩增HTT或其突变体或它们的片段。在一实施方案中，所述靶蛋白是与荧光蛋白例如GFP标签融合的polyQ异常扩增HTT或其突变体或它们的片段。在一实施方案中，步骤(c)对图像进行分析是指分析荧光信号强度。
在一实施方案中，所述高通量筛选采用斜入射光反射差法进行，所述步骤(a)还包括：扫描芯片，得到孵育前的图像；所述步骤(c)包括：对差异图像(孵育后的图像-孵育前的图像)进行分析；选择与靶蛋白能够亲和结合的化合物。
在一实施方案中，所述方法还包括步骤(III)：选择对mHTT相关的神经退行性疾病具有以下作用的化合物：阻止、减轻、缓解或改善mHTT引起的病变。在一实施方案中，步骤(III)选择减轻mHTT依赖性的细胞凋亡的化合物。在一实施方案中，步骤(III)选择能降低细胞中的polyQ异常扩增HTT的水平的化合物。在一实施方案中，步骤(III)在mHTT相关的神经退行性疾病的细胞或动物模型上进行。在一实施方案中，步骤(III)包括：测定包含polyQ异常扩增HTT的神经元，例如纹状体细胞的mHTT依赖性的细胞凋亡。在一实施方案中，步骤(III)包括：测定包含polyQ异常扩增HTT的小鼠纹状体细胞(STHdh)在应激条件下的凋亡。在一实施方案中，所述包含polyQ异常扩增HTT的小鼠纹状体细胞为STHdh Q7/Q111。在一实施方案中，所述应激条件是血清饥饿。在一实施方案中，测定细胞凋亡的方式为检测凋亡信号，例如caspase(例如caspase-3和/或caspase-7)活性。在一实施方案中，测定mHTT依赖性的细胞凋亡的方式为观察细胞或亚细胞结构的形态，例如神经元的停止进展或神经元收缩。在一实施方案中，步骤(III)包括：测定mHTT相关的神经退行性疾病生物标记物的水平。在一实施方案中，所述生物标记物选自mHTT聚集体、Tuj1、GABA和DARPP-32，特别是mHTT聚集体或DARPP-32。
在一实施方案中，步骤(III)的细胞模型为来自患者或动物疾病模型的包含polyQ异常扩增HTT的细胞，或来自重组DNA转化的表达polyQ异常扩增HTT的细胞。示例 性的模型或实验条件包括但不限于本申请和我们的在先研究(Li,Z.et al.Allele-selective lowering of mutant HTT protein by HTT–LC3 linker compounds.Nature 575,203-209(2019)和Yao Y,.et al.A striatal-enriched intronic GPCR modulates huntingtin levels and toxicity.Elife.2015 Mar 4；4:e05449)中描述的那些。
在一实施方案中，所述方法还包括步骤(IV)：测定候选化合物与正常polyQ HTT或其突变体或它们的片段的结合，选择不与正常polyQ HTT或其突变体或它们的片段亲和结合的化合物，其中
所述正常polyQ HTT包含的polyQ长度<36；
所述片段包含正常polyQ HTT或其突变体的N17区域和polyQ区域，并任选地包含PRD。
在一实施方案中，步骤(I)和步骤(II)顺序进行。在一实施方案中，当存在步骤(III)和/或步骤(IV)时，对步骤(III)、步骤(IV)与步骤(I)和步骤(II)之间的顺序没有特别的限制。在一实施方案中，步骤(III)在步骤(I)和步骤(II)之后进行。在一实施方案中，当存在步骤(IV)时，对步骤(IV)与步骤(I)和步骤(II)之间的顺序没有特别的限制。在一实施方案中，步骤(IV)在步骤(I)和步骤(II)之后进行。当存在步骤(III)和步骤(IV)时，步骤(III)和步骤(IV)之间的顺序没有特别的限制。当对二个或二个以上的步骤之间的顺序不做特别的限制时，所述二个或二个以上的步骤可以独立地进行，例如在相同或不同体系中进行；并且可以同时、先后或顺序进行；并且其先后、顺序可以任意排列。
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、苯甲酰化、棕榈酰化、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，HTT或其突变体或它们的含有N17区域和polyQ区域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HTT或其突变体或它们的含有N17区域和polyQ区域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)或步骤(II)的实施方式，完整的polyQ异常扩增HTT可用于所述测定。或者，包括结合位点的polyQ异常扩增HTT片段可用于所述测定。
用于筛选的蛋白
HTT在细胞中的降解途径主要有泛素-蛋白酶体系统(UPS)和自噬。在HD模型中，mHTT较短的N端片段具有更强的毒性，并产生更显著的表型。这可能表明，较短形式 的HTT毒性更大，原因可能是与自噬关键蛋白的相互作用减少。
HTT的翻译后修饰可能影响HD疾病状态。可能的翻译后修饰例如泛素化、乙酰化、小泛素相关修饰物(small ubiquitin-related modifier,SUMO)修饰，即SUMO化(SUMOylation)，以及苯甲酰化和棕榈酰化。
HTT基因外显子1编码的氨基酸序列(称为HTT exon1)主要包括N-末端17个氨基酸(N17，也称为N17区域)、谷氨酰胺重复区域(polyQ)和富含脯氨酸的结构域(PRD，或称为PRD结构域)。N17包含可能的磷酸化位点T3、S13和S16。M8被氧化可能促进S13和S16磷酸化。N17包含可能的乙酰化、SUMO化和泛素化位点K6、K9和K15。polyQ区域的长度可以显著改变HTT蛋白的整体构象和磷酸化模式。因此，polyQ异常扩增的全长HTT与正常的全长HTT之间，整体上存在显著的区别。PRD含有49个氨基酸残基。其介导多种蛋白与HTT的结合。
在一实施方案中，步骤(I)使用的靶蛋白是polyQ异常扩增HTT，其polyQ长度≥36。
在一实施方案中，步骤(IV)使用的靶蛋白是正常polyQ HTT。在示例性实施方案中，所述正常polyQ HTT包含SEQ ID NO:1的氨基酸序列，并且其polyQ区域的长度可以与SEQ ID NO:1相同或不同，只要polyQ长度<36。
在一实施方案中，步骤(I)使用的靶蛋白是polyQ异常扩增HTT的突变体。在一实施方案中，步骤(IV)使用的靶蛋白是正常polyQ HTT的突变体。
在一实施方案中，polyQ异常扩增HTT或其突变体或它们的片段的氨基酸序列包含K6。在一实施方案中，所述突变体的氨基酸序列保留K6。在一实施方案中，所述突变体的氨基酸序列包含选自以下的一种或多种，优选含有选自以下的一种氨基酸替换：K9X、K15X、E12X、S13X和S16X，其中X代表与野生型序列中不同的任意天然氨基酸。在一实施方案中，所述天然氨基酸包括甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸和组氨酸。在一实施方案中，X代表保守的氨基酸替换。在一实施方案中，所述突变体的氨基酸序列包含选自以下的一种或多种，优选含有选自以下的一种氨基酸替换：K9R、K15R、E12A、S13A和S16A。在另一实施方案中，所述突变体的氨基酸序列包含选自以下的一种或多种，优选含有选自以下的一种氨基酸替换：K9R、K15R、E12A和S13,16A。
在一实施方案中，步骤(I)使用的靶蛋白是polyQ异常扩增HTT或其突变体的片段。其中，polyQ异常扩增HTT突变体如上文所述。在一实施方案中，步骤(IV)使用的靶蛋白是正常polyQ HTT或其突变体的片段。其中，正常polyQ HTT的突变体的氨基酸序列可以包含上文所述polyQ异常扩增HTT的突变体包含的氨基酸替换，但不限于此。在一实施方案中，所述片段为HTT基因外显子1编码的氨基酸序列(示例性的序列例如SEQ ID NO:2和SEQ ID NO:4)，或者为HTT基因外显子1的N17区域和polyQ区域编码的氨基酸序列(示例性的序列例如SEQ ID NO:3和SEQ ID NO:5)。
在另一方面，本发明还提供包含核苷酸序列的核酸分子，所述核苷酸序列编码本发明的蛋白或蛋白片段。
在另一方面，本发明还提供包含本发明的核酸分子的载体。
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。
在一示例性实施方案中，本发明还涉及化合物，选自：
Figure PCTCN2022105437-appb-000003
Figure PCTCN2022105437-appb-000004
在一实施方案中，本发明的化合物选自
Figure PCTCN2022105437-appb-000005
Figure PCTCN2022105437-appb-000006
特别是
Figure PCTCN2022105437-appb-000007
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。
PROTAC化合物
在又一方面，本发明提供一种PROTAC化合物，其包含本发明的化合物、E3泛素连接酶结合配体，以及将本发明的化合物和E3泛素连接酶结合配体连接起来的接头。本发明的化合物作为配体，结合至polyQ异常扩增HTT或其突变体或它们的片段。在一实施方案中，所述E3泛素连接酶选自VHL、CRBN、MDM2、β-TRCP、cIAP、RNF4、RNF14、DCAF16，优选地选自VHL、CRBN、MDM2和cIAP1，特别是VHL和CRBN。在一实施方案中，所述PROTAC化合物可以用于促进polyQ异常扩增HTT或其含有polyQ区域和N17区域的片段经UPS降解。在一备选的实施方案中，也可以制备非活性的PROTAC化合物(inactive PROTAC)，即所产生的分子不与E3泛素连接酶结合，但在其中的本发明的化合物仍然能与polyQ异常扩增HTT或其含有polyQ区域和N17区域的片段结合。
药物组合物、制剂和试剂盒
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mHTT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，上述检测包括分析从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HTT或其含有polyQ区域和N17区域的片段的位置和/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。
治疗方法和用途
在一方面，本发明提供本发明的化合物，或其PROTAC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、代谢产物或前药或其药物组合物在制备用于治疗或预防mHTT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，或其PROTAC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，或其药物组合物，用于治疗或预防mHTT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mHTT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，或其PROTAC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，或其药物组合物。
在一优选的实施方案中，mHTT相关的神经退行性疾病为亨廷顿病(HD)。
在另一方面，本发明提供本发明的化合物或其PROTAC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、代谢产物或前药或其药物组合物用于降低细胞、组织或器官中的polyQ异常扩增HTT水平的用途。
有益效果
亨廷顿蛋白(Huntingtin,HTT)属于动物蛋白质组中较大的蛋白。目前认为，HTT具有多个可以与其他蛋白结合的位点，并且在生理环境中，HTT可能通过与这些蛋白结合来实现一些功能。然而，小分子对HTT的作用方式和小分子与HTT的“结合口袋”均为未知的，因此对于传统小分子药物开发而言，HTT属于“无可成药性”的靶点。
对于“无可成药性”的靶点，现有技术尝试了多种方式来降低其最终致病功能的影响，例如基因疗法、降解剂、增强自噬、其它用于调节靶标水平的基因或化学手段，等，但缺乏筛选直接作用于靶点的小分子的方法。本发明提供的筛选方法通过筛选与靶点相互 作用的化合物得到目标化合物，并且可以通过表型筛选验证化合物的治疗效果。筛选过程快速简便，效率高，成本低，并且易于实现高通量筛选。
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 PCTCN2022105437-appb-000008
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：desonide，PubChem CID:5311066，Selleck cat.no.S1701；
化合物2：bazedoxifene HCl，PubChem CID:154257，Selleck cat.no.S2128；
化合物3：iloperidone，PubChem CID:71360，Selleck cat.no.S1483；
化合物4：Loratadine，PubChem CID:3957，Selleck cat.no.S1358。
抗体
HTT抗体2B7(Weiss et al.Anal Biochem 2009,395,8-15)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。其中：HTT antibody 2166(Millipore,cat.no.MAB2166),3B5H10(Sigma,#P1874),anti-β-tubulin(Abcam,cat.no.ab6046),anti-TUBB3(Covance,cat.no.MMS-435P),anti-DARPP-32(Abcam,cat.no.ab40801),anti-ATXN3(Millipore,cat.no.MAB5360),anti-spectrin(Millipore,cat.no.MAB1622),anti-GR(Abmart,cat.no.T56612),anti-HA(Abmart,cat.no.M20003),anti-ubiquitin(ProteinTech,cat.no.10201-2-AP),anti-Actin(Abmart,cat.no. M20011)。
重组人HTTexon-sfGFP蛋白的制备
将具有72Q或25Q片段(由CAGCAA混合序列编码)的人HTT基因(GenBank:NM_002111.8)外显子1序列克隆到带有sfGFP标签的pTT5SH8Q2载体上，得到pTT-HTTexon1-Q72-sfGFP和pTT-HTTexon1-Q25-sfGFP质粒。使用聚乙烯亚胺(PEI，来自Polysciences，23966)将质粒转染到HEK293T细胞中表达。用HisTrap HP柱(GE Healthcare,17524701)纯化蛋白。将缓冲液置换为50mM HEPES buffer pH 7.0 with 150mM NaCl。
重组人HTTexon1-MBP蛋白的制备
(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。
(2)将具有72Q或25Q片段(由CAGCAA混合序列编码)的人HTT基因外显子1序列克隆到pGHT中，得到HTTexon1Q72-MBP和HTTexon1Q25-MBP质粒。
(3)将质粒转染到大肠杆菌BL21(DE3)pLsyS中表达。用HisTrap HP柱(GE Healthcare，17524701)和Superose 6 Increase 10/300GL柱纯化。
表达mHTT exon1的哺乳动物细胞的制备
将C-末端带有HA标签的mHTT exon1构建体克隆到哺乳动物表达载体(pSG5)中，转染所述的哺乳动物细胞24小时，以表达mHTT exon1。
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。
用于试验的细胞
原代培养的皮质神经元：Hdh Q7/Q140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、Q49、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 Q111/Q7来自Coriell Cell Repositories(Camden,NJ,USA)。HEK293T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 Q7Q140)。
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。
用化合物处理细胞的一般试验方法
将化合物配制DMSO贮存液。在用于处理细胞之前，用培养基将化合物贮存液稀释为指定浓度的工作溶液。除非另有说明，使用培养基将化合物稀释10倍，加入到铺好板的细胞中。对于原代培养的神经元和iPS细胞来源的神经元，在铺板后5天加入化合物；对于患者成纤维细胞和其他细胞系，在铺板后1天加入化合物。在37℃、5％CO 2的培养箱培养下用化合物处理2天后，收集细胞以测量mHTT水平。
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，以校正样品量。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，共聚焦显微镜成像，用ImageJ分析TUBB3或共定位情况。
siRNA转染
用lipofectamine 2000(Life Technologies,#11668)将siRNA反向转染到STHdh细胞中。所有转染均按照制造商提供的方案进行。3天后收集细胞进行Western印迹、HTRF或免疫荧光检测。siRNA靶序列和/或来源信息：阴性对照(Neg siRNA)：非靶向siRNA(Generalbiol,#RX028810)；糖皮质激素受体siRNA：靶向GGUAAUUAAGCAAGAGAAATT。
小鼠体内的化合物浓度测定
由SIM-Servier联合实验室进行实验。
(1)小鼠腹腔注射(ip)化合物或对照用的DMSO。
(2)于指定的时间点使用小动物麻醉机(MSS-3,MSS International,Keighley,UK)用异氟烷麻醉，用真空采血管采集心脏血。心脏血样品以10,000r.p.m离心5分钟，得到心脏血浆。采集心脏血后用1×PBS灌流小鼠以除去血液。将小鼠安乐死，取出脑。向每个脑样品中加入5倍体积的甲醇：乙腈(50:50,vol/vol)，然后匀浆。超声15分钟后，将匀浆离心5分钟，取20μL上清液与20μL水混合30s，然后进样LC-MS/MS。
(3)LC-MS/MS分析：采用与Xevo TQ-S质谱仪(Waters Corporation)连接的Acquity超高效液相色谱(UPLC)系统(Waters Corporation)。色谱柱：Acquity UPLC BEH C18(1.7μm 2.1×50mm)。流速：0.5mL/min的流量加水。使用溶剂A(含有0.1％甲酸和5mM NH 4Ac的水)和溶剂B(乙腈：甲醇＝9/1，vol/vol，含0.1％甲酸)进行梯度洗脱。
实施例1 等位基因选择性mHTT结合化合物的高通量筛选
(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)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，用PBS清洗芯片。以重组的GFP标记的mHTT蛋白片段HTTexon1-Q72-sfGFP(与sfGFP融合的SEQ ID NO:2)为靶蛋白。将化合物芯片与靶蛋白(浓度为0.002mg/mL)在室温下一起孵育120分钟。用PBS漂洗20分钟后，用激发波长为488nm的荧光扫描仪(来自Molecular Device)对化合物芯片进行图像扫描。荧光图像中的亮点表示与靶蛋白结合的化合物。选取与靶蛋白结合的化合物。
(2)以重组的GFP标记的wtHTT片段HTTexon1-Q25-sfGFP(与sfGFP融合的SEQ ID NO:4)为靶蛋白。按照与(1)相似的方法进行反筛，即，选取不与靶蛋白结合的化合物。
重复以上(1)和(2)步骤两次，识别出21个特异性结合mHTT的化合物(本文中未全部示出)。
实施例2 表型筛选获取mHTT细胞毒性化合物
2.1检测化合物对mHTT引起的细胞毒性的影响
STHdh Q7/Q111细胞在诸如血清饥饿的应激条件下，mHTT依赖性地发出凋亡信号。这样的凋亡信号已被广泛地用作mHTT毒性的指征。
使用高容量成像技术，用STHdh Q7/Q111细胞作为HD细胞模型，检测实施例1中得到的21个特异性结合mHTT的化合物是否可以改善mHTT诱导的细胞毒性。血清饥饿开始时加入化合物。以DMSO和泛caspase抑制剂z-vad-fmk分别作为阴性、阳性对照。在去除血清(血清饥饿开始)后的不同时间点，用绿色荧光染料(NucView 488)检测 caspase-3活性，以测定凋亡。其中5个化合物在微摩尔浓度下表现出剂量依赖性的显著的和可重复的拯救作用(图1、图2A、2B)，其中图1的化合物浓度为3μM。图2A、2B的化合物浓度如图所示。
2.2检测化合物对非mHTT引起的细胞毒性的影响
为了排除与mHTT无关的非特异性效应引起的细胞毒性，检测2.1中得到的5个具有表型拯救作用的化合物对蛋白酶体抑制剂MG132在去血清条件下诱导的，可能由未折叠蛋白反应(UPR)引起的野生型(WT)纹状体细胞的凋亡的影响。
实验方法：使用与2.1相似的方法，但采用野生型(WT)纹状体细胞STHdh Q7/Q7细胞，并在去除血清的同时，加入用3μM浓度的化合物以及0.5μM蛋白体抑制剂MG132。在2.1中得到的5个化合物中，gossypol显示出对于WT细胞的细胞凋亡的显著的挽救作用，提示可能存在非特异性效应。其他4个化合物无效应(desonide)或轻微加重了MG132诱导的细胞毒性(bazedoxifene、Loratadine和iloperidone)，说明这4个化合物是mHTT依赖毒性的特异性抑制剂(图3)。
实施例3 化合物对mHTT亲和活性的测定
3.1化合物与HTT exon1的亲和活性的OI-RD检测
参照我们在先研究(见上文所述Li,Z.et al.文献)中的实验方法，用2.2中得到的4个化合物desonide、bazedoxifene、Loratadine和iloperidone制备化合物芯片，并用OI-RD检测化合物分别与HTTexon1-Q72和HTTexon1-Q25的亲和活性。观察到这4个化合物能够等位基因选择性地与mHTT exon1亲和结合(图4)。亲和反应的K on(结合速率常数)、K off(解离速率常数)和K d(反应平衡解离常数)分别如图4所示。
3.2 desonide与全长HTT或HTT exon1的亲和活性的MST检测
用微量热泳动仪(其中Monolith NT.115仪器来自NanoTemper Technologies)对desonide与HTTexon1Q72-MBP、HTTexon1Q25-MBP、全长mHTT(flHTT-Q73)和全长HTT-Q23(flHTT-Q23)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HTTexon1Q25-MBP和flHTT-Q23没有亲和结合(K d>100μM)，与HTTexon1Q72-MBP和flHTT-Q73的K d分别如表1所示。
表1.Desonide与待测蛋白的亲和反应平衡解离常数
  K d(μM)
HTTexon1Q72-MBP 1.9
flHTT-Q73 1.8
MST检测结果验证了desonide能选择性地亲和结合mHTT(HTTexon1-Q72或flHTT-Q73)。(图5)
3.3 desonide与HTT exon1的亲和活性的等温滴定量热法(ITC)检测
使用等温滴定量热法(其中PEAQ-ITC仪器来自Malvern)对desonide与HTTexon1-Q72和全长HTTexon1-Q25)的亲和活性进行验证。对于每次滴定，在25℃恒温下，将280μL含有浓度为10μM的纯化的蛋白的ITC缓冲液(20mM HEPES pH 7.0,100mM NaCl,0.2％DMSO)注射到池中，并将含有浓度为20μM的desonide的相同缓冲液，分20次注射到池中(第一滴0.4μL，第2-20滴为2μL)，每次注射之间间隔180s。实时记录蛋白与化合物结合释放的热(H)。分子结合释放或吸收的热量与结合的分子的数量成正比。当体系饱和时，只能观察到稀释热。
重复进行滴定。使用Origin软件的One Sites模型分析数据，以最后5次注射的数据作为基线。观察到desonide能选择性地亲和结合HTTexon1-Q72，K d为5.32μM。
突变型和野生型HTT基因有相同的启动子。因此，desonide降低mHTT水平的作用可能是通过降低mHTT稳定性而不是抑制mHTT表达来实现的。我们的实验表明desonide能等位基因选择性地与mHTT exon1或全长mHTT亲和结合，这一现象也与上述理论一致。
实施例4 desonide对HD小鼠纹状体细胞、HD患者成纤维细胞、HD患者诱导干细胞分化神经元细胞HTT水平的影响
用3μM浓度的desonide处理细胞48h后，进行检测：
(1)通过蛋白印迹检测到desonide等位基因选择性地显著降低STHdh Q7/Q111细胞中的mHTT水平(图6)。进一步检测发现desonide对STHdh Q7/Q111细胞中mHTT水平的降低作用是剂量依赖性的(图7)。
(2)通过HTRF测定mHTT(抗体对：2B7/MW1；n≥9)和总HTT(抗体对：2B7/2166)。观察到desonide等位基因选择性并且剂量依赖性地降低HD患者成纤维细胞中的mHTT水平(图8A、8B)。
(3)通过HTRF(抗体对：2B7/MW1；n≥9)测定，观察到desonide等位基因选择性地降低HD患者iPSC分化的神经元细胞中的mHTT水平(图9)。
实施例5 化合物对HD患者诱导干细胞分化神经元凋亡的影响
对细胞进行应激(去除BDNF)，同时，加入2.2中得到的化合物desonide、bazedoxifene、Loratadine或iloperidone(化合物浓度：3μM)，通过TUBB3免疫荧光染色，以及采用与2.1相似的方法定量检测caspase-3活性(用绿色荧光染料(NucView 488)检测，图片由InCucyte生成)，检测HD患者iPSC分化的神经元的凋亡情况。观察到这4个化合物对HD患者iPSC分化的神经元细胞具有表型拯救作用(图10)。
实施例6 化合物对亨廷顿病果蝇的作用
使用上文所述的在神经元中表达mHTT exon1或全长mHTT的果蝇作为HD果蝇。 参照在先研究(见上文所述Li,Z.et al.文献)中的实验方法进行本实施例的实验。
果蝇保持在复合饲料中。给药组用含有2.2中得到的化合物desonide、bazedoxifene、Loratadine或iloperidone的食物喂养。在小瓶中测定果蝇的爬行能力，以评价HD果蝇的运动功能。每组4个小瓶。记录15秒后爬过了7厘米高的线的果蝇的百分比，将其相对于羽化后的天数(日龄)作图。观察到化合物显著改善了HD果蝇的爬行能力。用含有化合物的食物喂养的表达HTTexon1-Q25的果蝇能力没有明显下降(图11)。
elav-GAL4:UAS-flHTTQ128果蝇羽化后用含有化合物的食物喂养6天，提取果蝇头部的HTT蛋白，通过HTRF(抗体对：2B7/MW1；n≥9)测定。观察到desonide等位基因选择性地降低mHTT的水平(图12)。
实施例7 化合物对HD模型小鼠作用的初步试验
将小鼠分组饲养于具有12小时光照/黑暗循环的单独通风的笼中，每笼最多5只成年小鼠。
实验动物：Hdh Q140/Q140小鼠(10月龄)。
7.1腹腔注射iloperidone对HD小鼠的影响
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0.5mg/mL，每天进行一次腹腔注射(0.5mg/kg)，注射4周后，进行组织提取或行为学实验。
初步的实验数据表明iloperidone显著减少了小鼠在旷场的活动和后肢站立行为(图13A、13B)。
7.2脑室内注射化合物对HD小鼠的影响
实验方法：脑室内注射(icv)：每次注射给药2μL含有化合物(desonide、Loratadine或bazedoxifene)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)，其中化合物浓度为2mM。以脑体积1mL估算，脑中化合物浓度约4μM。将含有等量DMSO的2μL ACSF用作对照。
每天进行一次脑室内注射。注射4周后，测定了旷场行为和后肢站立(rearing)行为。Loratadine组没有观察到效果。bazedoxifene组观察到轻微的拯救效果。desonide组观察到较显著的拯救效果(图13A、13B)。
实施例8 脑室内注射desonide对HD模型小鼠的影响
8.1脑室内注射desonide对HD小鼠行为学的影响
实验动物：Hdh Q140/Q140小鼠，开始给药时12月龄。用同窝出生的野生型小鼠作为参比，以排除与HD无关的行为表现。
实验方法：按照7.2所述步骤给药desonide。连续注射4周后，进行行为学实验。
后肢站立频率：将小鼠置于立体框架中5分钟，所述立体框架是由表面凹凸不平的 笔筒(高98mm x直径91mm)形成。动物至少抬起两条前肢的事件总数为后肢站立次数。
旷场试验：小鼠被放置在行为室中的30x 30x 40cm的白色有机玻璃室中，通过室顶的摄像机记录小鼠15分钟的移动(locomotion)。然后用活动监测程序分析行进轨迹和距离。
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。
握力测试：(1)首先对每只小鼠进行一次抓握训练，刺激抓握反射，训练其抓住测量仪的测力横杆，确保其能够稳定抓握。训练方法为：从尾巴轻轻抬起小鼠，当小鼠两前肢靠近横杆时，可诱导其主动用爪子抓住横杆。可接受的握持标准为小鼠能主动伸展爪子并平稳握杆，无闪避、肢体扭曲或抵抗。每只小鼠的抓握训练每天进行大约5-10分钟，持续3-5天，直到能够容易和驯服地执行双前肢抓握。(2)在小鼠能够进行满意的抓握动作后，从尾巴轻轻抬起小鼠，靠近横杆，诱导其主动用爪子抓住横杆，测量小鼠处于水平位置、爪子抓握横杆时的握力，作为基线。然后将小鼠由水平方向向回拉，直到将其拉离横杆，此时横杆处的握力反映小鼠在抵抗拉扯时的最大双前肢握力。测量3次并取平均值。每次测量间隔1小时，以避免小鼠疲劳。
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。
在旷场试验、握力测试、后肢站立频率、平衡梁试验和平衡梁试验中，均观察到小鼠的运动障碍被显著改善。此外，desonide对野生型小鼠没有影响，说明desonide对HD有特异性的治疗作用(图14A、14B、14C、14D、14E)。
8.2脑室内注射desonide对HD小鼠体内生物标记物的影响
为进一步在分子水平上证实desonide的体内作用，用S830和抗-DARPP-32通过免疫染色检测了HD的分子生物标记物，包括mHTT聚集体和中型多棘神经元的标记物DARPP-32。观察到mHTT聚集体的水平显著降低，DARPP-32信号增加(图15)。
实施例9 外周给药desonide对HD小鼠的影响
实验动物：Hdh Q7/Q140小鼠，开始给药时14月龄，每组9只。以Hdh Q7/Q7小鼠为对照。
由于desonide是FDA批准的治疗特应性皮炎的药物，因此外用desonide的安全性已被证实。根据实施例7和8，脑室内注射desonide是有效的，但仍需要其他可行的给药方式。
9.1初步的药代动力学和药理学实验
以5mg/kg剂量进行ip注射，在指定时间点麻醉小鼠，采集心脏血制备心脏血浆。然后用1×PBS灌流小鼠以除去血液。对小鼠进行安乐死，按照上文所述方法检测体内化合物浓度。发现desonide的脑内浓度可以达到约2至3μM(图16)。并且，ip注射desonide等位基因选择性地显著降低了纹状体mHTT水平(图17)。
9.2腹腔注射desonide对HD小鼠行为学的影响
进一步地，我们测试了ip注射desonide对不同年龄Hdh Q7/Q140小鼠的影响。
按照8.1所述的行为学试验方法进行测试。9月龄(n＝15)、11月龄(n＝13)、14月龄(n＝13)月龄的HD小鼠经过4周的desonide注射给药之后，行为缺陷得到恢复。此外，desonide对野生型小鼠没有影响，说明desonide对HD有特异性的治疗作用(图18A、18B、18C、18D、18E)。
9.3腹腔注射desonide对HD小鼠体内生物标记物的影响
按照8.2所述的试验方法进行测试。观察到在15个月龄的HD小鼠(14个月龄注射4周)纹状体中，mHTT聚集体的水平降低(图18F)。HD分子生物标记物的水平部分恢复，其中，显著缓解了HD纹状体DARPP-32和NFL水平的降低。(图19)，证实腹腔注射desonide能够治疗HD。
实施例10 desonide对mHTT exon1突变体的亲和活性的测定
10.1点突变或截短HTTexon1-Q72突变体的制备
采用与上述制备重组人HTT exon1蛋白相似的方法，使用定点诱变PCR在HTTexon1-Q72中引入以下点突变：K6R、K9R、K15R、K6,9,15R、E12A或S13,16A，其中的编号是基于野生型序列(SEQ ID NO:1)中的顺序。通过测序来验证序列。制备构建体并转化入合适的宿主中，表达包含所需点突变的HTTexon1-Q72突变体(以下简称突变体)。
采用与上述制备重组人HTT exon1蛋白相似的方法，制备截短的mHTT exon1序列ΔPRD(缺失PRD，示例性序列参见SEQ ID NO:3，即，缺失HTTexon1-Q72中的第90至138个氨基酸)。
10.2 polyQ的影响
我们的在先研究(见上文所述Li,Z.et al.文献)表明，某些化合物同时与LC3B以及扩增的polyQ相互作用，从而降低含有扩增的polyQ的几种不同的蛋白的水平，所述蛋白包括突变的ATXN3。
为此，我们测量了desonide在细胞内对含有扩增的polyQ的蛋白水平的影响。用浓度为3μM的desonide处理SCA3患者成纤维细胞，并以上述在先研究报道的AN2(5,7-二羟基-4-苯基香豆素)作为阳性对照。蛋白印迹检测观察到desonide不能降低突变ATXN3蛋白水平。(图20)。
此外，用与3.2相似的方法进行MST检测，观察到与GFP标签融合的扩增的polyQ 片段也不与desonide亲和结合(图21)。因此，试验结果表明仅含有扩增的polyQ并不足以使多肽与desonide亲和结合。
10.3 K6是desonide对mHTT亲和活性的关键
用与3.2相似的方法通过MST检测desonide与K6R、K9R、K15R、E12A的亲和结合。结果表明desonide不与K6R突变体亲和结合，但能与N17中的另两个赖氨酸发生突变的突变体(K9R和K15R)或者N17中带负电的谷氨酸发生突变的突变体(E12A)亲和结合(图22)。
为了进一步证实desonide-mHTT相互作用的K6依赖性，我们按照实施例3的方法进行了等温滴定量热法(ITC)测定，得到了与MST测定一致的结果(K6R突变体不与desonide结合)。
由于mHTT固有的不稳定构象，对于mHTT中的polyQ，一直缺乏晶体或冷冻电镜数据来对其进行结构生物学解析。发明人出人意料地发现，K6位点是desonide对mHTT exon1的亲和活性的关键。
10.4 PRD的影响
polyQ长度可能调节其两侧的N17和PRD之间的接近程度。因此，在一些情况下，PRD可能在空间上接近N17，并屏蔽或部分屏蔽能够与化合物结合的位点。
用与3.2相似的方法，观察到desonide能够与缺失PRD的HTTexon1-Q72(ΔPRD)亲和结合(图22)。
ΔPRD与desonide的K d值为1.6μM。PRD可能屏蔽或部分屏蔽了wtHTT exon1与desonide的结合位点，而对mHTT exon1没有这样的影响。
本实施例测定的desonide与mHTT exon1及其突变体的亲和反应平衡解离常数如表2所示。
表2.desonide与mHTT exon1及其突变体(K6R、K9R、K15R、E12A或ΔPRD)的亲和反应平衡解离常数
Figure PCTCN2022105437-appb-000009
实施例11 desonide对mHTT的作用依赖K6
11.1 desonide降低mHTT水平的作用依赖K6
用3μM浓度的desonide处理转染了HTTexon1-Q72或其突变体或HTTexon1-Q25的HEK293T细胞或STHdh Q7/Q7细胞。用HTRF(抗体对：2B7/MW1)进行检测。观察到desonide能够降低细胞中HTTexon1-Q72及其K9R、K15R、E12A或S13,16A突变体的水平，但不能降低HTTexon1-Q72K6R突变体的水平(图23、图24)。在功能水平上证实了desonide的作用依赖K6。
11.2 desonide对mHTT引起的细胞毒性的改善作用依赖K6
用3μM浓度的desonide处理转染了HTTexon1-Q72或其突变体或HTTexon1-Q25的HEK293T细胞或STHdh Q7/Q7细胞。采用与2.1相似的方法，通过在血清饥饿条件下测量凋亡信号(caspase-3活性)，测定mHTT的细胞毒性。
desonide能够改善HTTexon1-Q72(图25)或其K9R、K15R、E12A或S13,16A突变体引起的细胞凋亡(图25、图26)，但不能改善HTTexon1-Q72K6R引起的细胞凋亡(图25)。
用相似的方法，在转染mHTT exon1的野生型小鼠纹状体细胞STHdh中也观察到有相似的结果(图27)。
实施例12 desonide对mHTT的降低作用依赖蛋白酶体-泛素系统(UPS)
12.1 desonide对mHTT的降低作用被蛋白酶体抑制剂阻断
在存在或不存在蛋白酶体抑制剂MG132(浓度2μM)或环氧霉素(浓度100nM)，或者自噬抑制剂NH 4Cl(浓度10mM)或氯喹(浓度25μM)的情况下，用desonide处理细胞，进行以下测试：
(1)用蛋白印迹测试desonide对HD细胞(STHdh Q7/Q111)中mHTT的降低作用。观察到desonide对mHTT的降低作用被MG132显著阻断，但不被氯喹(CQ)阻断(图28)，表明desonide降低mHTT的作用可能是经由蛋白酶体实现的。
(2)用HTRF(抗体对：2B7/MW1)进行检测。在HD患者成纤维细胞(图29)和过表达外源性mHTT exon1的HEK293T细胞(图29)中进一步验证了desonide对mHTT的降低作用被蛋白酶体抑制剂阻断，但不被自噬抑制剂阻断。
12.2 desonide增强了mHTT的K6多聚泛素化
样品制备：
(1)用HTTexon1Q72-HA或HTTexon1Q25-HA构建体和对照质粒瞬时转染HEK293T细胞24小时，然后用100nM环氧霉素处理细胞24小时。
(2)按照上文所述化合物处理细胞的一般试验方法，用desonide处理(1)中得到的细胞。
(3)用蛋白酶体抑制剂epoxomicin处理细胞，使多聚泛素化蛋白保留在细胞中。收集细胞，在冰上裂解和超声处理，4℃下>20000g离心10分钟，取上清液进行下一步试验。
进行IP-Western试验，包括：
(1)使用SureBeads TM磁珠免疫沉淀系统(Bio-Rad,#161-4023)拉下带有HA标签的HTTexon1-Q25和HTTexon1-Q72(用α-HA和α-actin作为对照)。
(2)洗脱磁珠结合的蛋白质。
(3)将(1)的输入物和(2)中得到洗脱液用抗-HA和抗-泛素抗体进行蛋白印迹分析， 其中使用仅具有轻链的IgG作为二抗(Abmart，#M21004)，以避免重链干扰。
试验结果表明，用desonide处理过表达mHTT exon1的HEK293T细胞导致mHTT exon1的多聚泛素化(poly-ub)明显增加。并且，没有观察到wtHTT exon1的多聚泛素化增加(图30)。
用相似的方法测定desonide对mHTT exon1突变体的多聚泛素化的影响。观察到desonide能够使K9R或K15R突变体的多聚泛素化增加(图30)，但不能使K6R突变体的多聚泛素化增加(图30)。因此，是K6是desonide使多聚泛素化增加的作用位点。
实施例13 desonide对mHTT的影响没有糖皮质激素受体参与
已知desonide为弱的糖皮质激素受体(GR)激动剂。
13.1 desonide对mHTT的降低作用被蛋白酶体抑制剂阻断
用3μM浓度的GR激动剂(泼尼松龙、环索奈德、氢化可的松或氟羟泼尼松龙)处理HD细胞STHdh Q7/Q111 48小时。用蛋白印迹(抗体：2166)检测mHTT和wtHTT，并进行了定量(抗体：D7F7或2166)。用与3.2相似的方法通过MST检测亲和结合。观察到这些激动剂不与mHTT亲和结合，也不降低mHTT的水平(图31)。
13.2 desonide对mHTT的降低作用被蛋白酶体抑制剂阻断
使用siRNA转染24小时以敲低STHdh Q7/Q111细胞中的GR，然后用与13.1相似的方法测试desonide对细胞中mHTT水平的影响。对于敲低了GR的HD细胞，desonide仍然能够降低其mHTT水平(图32)。
因此，desonide对mHTT的影响与其糖皮质激素受体激动活性无关。
序列表
Figure PCTCN2022105437-appb-000010
Figure PCTCN2022105437-appb-000011
Figure PCTCN2022105437-appb-000012

Claims (14)

  1. 一种筛选或鉴定用于治疗或预防mHTT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polyQ异常扩增HTT或其突变体或它们的片段，其中
    所述polyQ异常扩增HTT包含的polyQ长度≥36；
    所述片段包含polyQ异常扩增HTT或其突变体的N17区域和polyQ区域，并任选地包含PRD；
    (II)：测定所述候选化合物与所述polyQ异常扩增HTT或其突变体或它们的片段结合的能力，选出目标化合物。
  2. 权利要求1的方法，其中步骤(II)选择具备所述结合能力的化合物，优选地，选择所述结合达到一定强度的化合物；
    更优选地，步骤(II)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。
  3. 权利要求1或2的方法，其中步骤(II)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；
    或者
    其中步骤(II)用于测定所述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。
  4. 权利要求1-3中任一项的方法，其中
    步骤(II)测定所述结合采用高通量筛选进行；包括：
    (a)对候选化合物进行固定化以制备化合物芯片；
    (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；
    其中靶蛋白包含polyQ异常扩增HTT或其突变体或它们的片段，其中所述片段包 含polyQ异常扩增HTT或其突变体的N17区域和polyQ区域，并任选地包含PRD；和
    (c)对图像进行分析；选择与靶蛋白能够亲和结合的化合物；优选地，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；
    优选地，
    所述靶蛋白包含(1)可检测的分子以及(2)polyQ异常扩增HTT或其突变体或它们的片段；更优选地，所述靶蛋白是与荧光蛋白例如GFP标签融合的polyQ异常扩增HTT或其突变体或它们的片段；或者
    所述高通量筛选采用斜入射光反射差法进行，所述步骤(a)还包括：扫描芯片，得到孵育前的图像；所述步骤(c)包括：对差异图像(孵育后的图像-孵育前的图像)进行分析；选择与靶蛋白能够亲和结合的化合物。
  5. 权利要求1-4中任一项的方法，还包括以下步骤
    (III)：选择对mHTT相关的神经退行性疾病具有以下作用的化合物：阻止、减轻、缓解或改善mHTT引起的病变，例如，减轻mHTT依赖性的细胞凋亡。
  6. 权利要求1-5中任一项的方法，还包括以下步骤：
    (IV)：测定候选化合物与正常polyQ HTT或其突变体或它们的片段的结合，选择不与正常polyQ HTT或其突变体或它们的片段亲和结合的化合物，其中
    所述正常polyQ HTT包含的polyQ长度<36；
    所述片段包含正常polyQ HTT或其突变体的N17区域和polyQ区域，并任选地包含PRD。
  7. 权利要求1-6中任一项的方法，其中所述片段包含HTT基因外显子1编码的氨基酸序列，或者包含HTT基因外显子1的N17区域和polyQ区域编码的氨基酸序列。
  8. 权利要求1-7中任一项的方法，其中所述polyQ异常扩增HTT或其突变体或它们的片段的氨基酸序列包含K6。
  9. 权利要求8的方法，其中所述polyQ异常扩增HTT或其突变体或它们的片段的氨基酸序列包含选自以下的一种或多种氨基酸替换：K9X、K15X、E12X、S13X和S16X，其中X代表与野生型序列中不同的任意天然氨基酸；优选地，包含选自以下的一种或多种氨基酸替换：K9R、K15R、E12A、S13A和S16A。
  10. 通过权利要求1-9中任一项的方法获得的化合物在制备用于治疗或预防mHTT相关的神经退行性疾病的PROTAC化合物中的用途。
  11. 通过权利要求1-9中任一项的方法获得的化合物，或其PROTAC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，或其药物组合物在制备用于治疗或预防mHTT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mHTT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mHTT相关的神经退行性疾病为亨廷顿病。
  12. 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区域和N17区域的片段的位置和/或含量。
  13. 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，或其PROTAC化合物，或其药物组合物在制备用于治疗或预防mHTT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mHTT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 PCTCN2022105437-appb-100001
    Figure PCTCN2022105437-appb-100002
    优选地选自
    Figure PCTCN2022105437-appb-100003
    Figure PCTCN2022105437-appb-100004
    特别是
    Figure PCTCN2022105437-appb-100005
    优选地，其中所述mHTT相关的神经退行性疾病为亨廷顿病。
  14. 一种检测试剂、试剂盒或筛选系统，其包含至少一种选自以下的化合物，或其PROTAC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：
    Figure PCTCN2022105437-appb-100006
    Figure PCTCN2022105437-appb-100007
    优选地选自
    Figure PCTCN2022105437-appb-100008
    Figure PCTCN2022105437-appb-100009
    特别是
    Figure PCTCN2022105437-appb-100010
PCT/CN2022/105437 2021-07-14 2022-07-13 筛选用于治疗或预防mHTT相关的神经退行性疾病的化合物的方法，靶蛋白，以及化合物 WO2023284782A1 (zh)

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