WO2021210878A1 - Peptide de liaison au crbn et composition pour la prévention ou le traitement de la maladie d'alzheimer faisant appel à celui-ci - Google Patents

Peptide de liaison au crbn et composition pour la prévention ou le traitement de la maladie d'alzheimer faisant appel à celui-ci Download PDF

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WO2021210878A1
WO2021210878A1 PCT/KR2021/004627 KR2021004627W WO2021210878A1 WO 2021210878 A1 WO2021210878 A1 WO 2021210878A1 KR 2021004627 W KR2021004627 W KR 2021004627W WO 2021210878 A1 WO2021210878 A1 WO 2021210878A1
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crbn
peptide
seq
tau
disease
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PCT/KR2021/004627
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Korean (ko)
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박철승
아크버우르스
전승제
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광주과학기술원
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • 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
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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 a peptide that binds to CRBN and inhibits the binding of CRBN and DJ2, and a composition for preventing, treating or improving Alzheimer's disease using the same.
  • Alzheimer's disease is the most common among geriatric neurological diseases.
  • Alzheimer's is the most common degenerative brain disease that causes dementia and is a brain disease that causes problems with memory, thinking and behavior.
  • Dementia is a general term that refers to loss of memory and other intellectual abilities severe enough to interfere with daily life, and Alzheimer's accounts for 60 to 80% of dementia cases.
  • Tau protein is a kind of microtubule binding protein having a molecular weight of 50,000 to 70,000 Da, and is mainly involved in entanglement of nerve fibers. Tau proteins exhibit significant molecular diversity, the most well known of which is due to proline-directed phosphorylation.
  • CRL4 The substrate receptor of CRBN , E3-ligase substrate recruiter cereblon (CRBN), is a therapeutic target for thalidomide and its derivatives, and is known as an immunomodulatory drug (IMiD). CRBN mutations are associated with autosomal recessive, non-symptomatic mental retardation. CRBN is also implicated in the regulation of AMP-activated protein kinase, glutamine synthetase, MEIS2 and ion channels.
  • IMD immunomodulatory drug
  • the DNAJ family is involved in a variety of cellular activities, including protein folding/unfolding/refolding.
  • DJ2 is known to react with DnaK and numerous Hsp70 and Hsp110, which attract and unfold misfolded proteins within stable aggregates.
  • DJ2 can prevent unfolding or aggregation in addition to the ability to recognize unfolded proteins, and the relationship with tau protein is still unknown, and research on this is required.
  • the present invention provides a peptide comprising a CRBN binding motif amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
  • the present invention provides a pharmaceutical composition for preventing or treating Alzheimer's disease, comprising the peptide.
  • the present invention provides a food composition for preventing or improving Alzheimer's disease, comprising the peptide.
  • the Alzheimer's disease may be caused by phosphorylation of tau.
  • the present invention provides a method for screening a therapeutic agent for Alzheimer's disease, comprising the following steps.
  • step b) analyzing whether the peptide synthesized in step a) can inhibit the binding of CRBN and DJ2 by binding to the DJ2 binding site of CRBN;
  • step c) determining as a therapeutic agent for Alzheimer's disease when the peptide synthesized in step b) inhibits the binding of CRBN and DJ2.
  • the present invention provides a method for preventing or treating Alzheimer's disease, comprising administering to an individual a peptide comprising a CRBN binding motif amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
  • the present invention provides the use of a peptide comprising the CRBN binding motif amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 for preventing or treating Alzheimer's disease.
  • the peptide of the present invention is a peptide mimicking the CRBN binding motif in DJ2, and since it binds to CRBN and interferes with the binding of DJ2 and CRBN, the degradation of DJ2 by CRBN is suppressed. As a result, since the effect of DJ2 inhibiting the aggregation of tau protein is maintained and improved, it can be usefully used for preventing, improving or treating Alzheimer's disease.
  • FIG. 1 shows the identification of Hsp70, DJ1 and DJ2 as endogenous substrates of CRL4 CRBN.
  • Figure 1a shows the results of lysing SH-SY5Y cells, immunoprecipitation with rabbit IgG control or ⁇ -CRBN antibody, and blotting with the indicated antibodies.
  • 1b to 1d show that SH-SY5Y cells were transiently transfected with scrambled SiRNA (Scr) or siRNA CRBN and then transfected with HA-Ub 24 h after IP, respectively, with ⁇ -Hsp70, ⁇ -DJ1 and ⁇ - Results performed with the DJ2 antibody and blotted with the indicated antibodies.
  • Scr scrambled SiRNA
  • siRNA CRBN siRNA
  • Figure 1e shows SH-SY5Y cells transiently transfected with FLAG-CRBN and immunoprecipitated with FLAG M2 agarose beads.
  • In vitro ubiquitination of endogenous, co-precipitated chaperones was performed in the presence of E1 + E2 and Ub; Methylated ubiquitin (Me-Ub) was added at the indicated sites, and the reaction was analyzed by SDS-PAGE and the result of immunoblotting with the indicated antibody.
  • Fig. 1f shows the results of treating Crbn ⁇ /- and Crbn +/+ MEF cells with 2.5 ⁇ g/ml CHX and performing immunoblot analysis (statistical analysis is shown from top to bottom in the order of Hsp70, DJ1 and DJ2).
  • Figure 1g shows the results of Crbn ⁇ /- and Crbn +/+ MEF cells treated with 0.5 ⁇ g/ml MG132 and subjected to immunoblot analysis (statistical analysis is shown from top to bottom in the order of Hsp70, DJ1 and DJ2).
  • Figure 2a is an experiment on the effect of CRBN and DJ2 on heparin-mediated aggregation of tau.
  • Figure 2a shows the results of incubating full-length hTau-44 (5 ⁇ M) with the indicated protein combinations for 48 hours, then adding 5 ⁇ M ThT and measuring the fluorescence emission at 480 nm with excitation at 440 nm ( Heparin was included in all reactions except control at a concentration of 2.5 ⁇ M, Student's t-test was used to quantify the data at 95% significance level).
  • Figure 2b is the result of confirming that the fluorescence emission is excited at 480 nm and excited at 440 nm by incubating the monomer hTau-K18 (5 ⁇ M) with the indicated combination (5 ⁇ M) and 5 ⁇ M ThT for 4 hours (heparin is 2.5 (included in all reactions except control at a concentration of ⁇ M)
  • Figure 2d shows the results of treating SHSY5Y cells with monomer K18, aggregating K18 in the presence or absence of DJ2 and CRBN, pictures were taken 48 hours after treatment, and MTT analysis was performed to evaluate cell viability (errors) Bars represent SEM, Student's t-test used to quantify data at 95% significance level)
  • FIGS. 3A and 3B show schematics of the full-length rCRBN and deletion constructs used in FIGS. 3C and 3D below (Cereblon domain of Unknown activity, binding cellular Ligands and Thalidomide), L (linker) , C-terminal domain (CTD)).
  • Figures 3c and 3e show that SH-SY5Y cells were transiently co-transfected with Myc-DJ2 and the indicated plasmids, the cell extracts were immunoprecipitated with ⁇ -Myc antibody, followed by SDS-PAGE sorting, and immunoblotting with Myc and HA antibodies.
  • FIG. 4 is an experimental result confirming that K32 and K350 are major ubiquitination sites of DJ2.
  • FIG. 4a shows the results of immunoblot analysis by transfecting SH-SY5Y cells with wild-type (WT) DJ2 and lysine mutants, and treating the cells with 2.5 ⁇ g/ml CHX.
  • Figure 4b is a graph showing the result of Figure 4a.
  • Figure 4c confirms that ubiquitylation is impaired in the case of the double mutant of K32 and K350 residues.
  • Figure 5 confirms the effect of CRBN KO on CUMS (chronic ultra-mild stress) and tau pathology.
  • Figure 5a shows the results of exposing Crbn ⁇ /- (KO) and Crbn +/+ (WT) mice to the CUMS paradigm, then fractionating WBL by SDS-PAGE and performing immunoblotting with antibodies.
  • Figures 5b and 5c show the results of Western blot analysis of the selected pTau epitope and taukinase in WBL of WT and KO mice.
  • Figures 5e and 5f show the results of statistical analysis of the results of Figures 5b and 5c by the t-test with P ⁇ 0.05.
  • Figure 6 confirms that CRBN KO inhibits the spread of tau pathology.
  • Figure 6a is an anatomical schematic diagram of the mouse brain, divided into anterior contralateral (AC), anterior ipsilateral (AI), posterior contralateral (PC), posterior ipsilateral (PI) and cerebellar (Cb) for analysis of western blotting. and the PI region includes the lateral tonsil injection site.
  • 6B shows the results of three-dimensional injection of OA into the brains of WT and KO mice, preparation of WBL in RIPA buffer, and blotting with antibodies of the pTau array.
  • FIG. 7 shows the effect of CRBN KO on phosphorylation-mediated tau dimerization.
  • Figure 7a shows that HEK293T cells were subjected to CRISPR/Cas9-mediated knockout (KO) of CRBN or used as a negative control (NC), and Myc-tau was transiently transfected into all cell lines and Western blot was performed. am.
  • FIG. 7B shows the result of FIG. 7A on a color gradant scale.
  • Figure 7c shows SH-SY5Y cells were transiently transfected with siRNA or siRNA CRBN , then the cells were transfected with tau40-VN173 and tau-VC155 constructs and treated with OA (30 nM) to generate BiFCs using confocal microscopy. This is the result of the measurement (Hoechst dye is used as counterstain).
  • Fig. 9 is an experiment on whether DJ2 binds to CRBN and tau and thalidomide-independent degradation of DJ1.
  • Fig. 9a shows the results of GST pull-down analysis on His-tagged DJ2 and GST-tagged CRBN and blotting with anti-DJ2 and anti-CRBN antibodies
  • Fig. 9b shows SH-SY5Y cells in HA-Ub This is the result of transient transfection with , 30 hours later, cells were treated with thalidomide for 24 hours, and cell lysis and IP were performed with mouse IgG control or ⁇ -DJ2 antibody.
  • Figure 9c shows SH-SY5Y cells were transiently co-transfected with Myc-tagged Tau and FLAG-tagged DJ2, and 24 hours later, the cell extracts were fractionated by immunoprecipitation and SDS-PAGE with ⁇ -Myc antibody and SDS-PAGE with the indicated antibodies. Results from immunoblotting (SE: short exposure, LE: long exposure).
  • FIG. 10 is an experiment for localization of a domain responsible for CRBN-DJ2 interaction.
  • Figures 10a and 10b show that SH-SY5Y cells were transiently co-transfected with Myc-DJ2 and HA-tagged CRBN digests and after 24 h, the cell extracts were immunoprecipitated with ⁇ -HA antibody, sorted by SDS-PAGE. and the results of immunoblotting with antibodies.
  • 10c and 10d show the results of western blotting by transiently co-transfecting SH-SY5Y cells with HA-CRBN and Myc-tagged DJ2 cleavage and 24 hours later, immunoprecipitating the cell extract with ⁇ -HA antibody.
  • Figure 10e shows a structural model of the DJ2-CRBN interaction predicted by ClusPro.
  • FIG. 11 is an experimental result for the localization of a specific lysine residue responsible for ubiquitination of DJ2.
  • Figures 11a and 11b confirm the experimentally reported lysine ubiquitinated in DJ2 using PhosphositePlus and GGbase database.
  • 11c shows that SH-SY5Y cells were transfected with wild-type (WT) DJ2 and various lysine mutants, confirming that K32 and K350 are major ubiquitination sites of DJ2.
  • FIG. 15 shows the effect of CRBN knock-out on tauopathy and the inhibitory effect of the prepared peptide on CRBN and DJ2 binding.
  • Fig. 15a shows the confirmation of APP plaques in a murine neuropathic model. Brains were removed from 8-month-old APP knock-in mice, the hippocampus was immunostained with CRBN and APP antibodies for 16 hours, and Alexa- After incubation with conjugated secondary antibody, imaged under a confocal microscope equipped with a 60X lens.
  • Figures 15b and 15c confirmed high levels of CRBN and low DJ2 in brain samples from APP knock-in and 5XFAD mice.
  • 16 is a graph showing the inhibition of CRBN-DJ2 binding by immunoprecipitation using linear (Linear, SEQ ID NO: 1) and circular (SEQ ID NO: 3) peptide forms.
  • 17a to 17d show the results of immunoprecipitation experiments in which CRBN and DJ2 binding inhibition ability was confirmed by immunoprecipitation based on the peptide of SEQ ID NO: 3, and a randomly mixed (Scrambled) peptide of amino acid sequence was added as an experimental control group.
  • DJ2-peptide is the peptide of SEQ ID NO: 3 and the structure of each peptide is shown in FIG. 17C).
  • the present invention provides a peptide comprising the CRBN binding motif amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
  • CRBN is a substrate receptor of the cillin-ring E3 ubiquitin ligase (CRL) complex, and the CRL complex ubiquitinates the substrate from the ubiquitin-conjugation enzyme (E2) to induce degradation in the proteasome. It has also been reported as a target protein of thalidomide, lenalidomide, and pomalidomide, which are immunomodulators (IMiDs) with anticancer effects on multiple myeloma.
  • IiDs immunomodulators
  • peptide refers to a polymer composed of amino acids linked by amide bonds or peptide bonds.
  • peptide comprising a CRBN binding motif amino acid sequence refers to a peptide having a sequence capable of specifically binding to CRBN and inhibiting the function of CRBN.
  • the peptide is a peptide having an activity capable of binding to the DJ2 binding site on CRBN, and acts as a competitive substrate for DJ2, thereby exhibiting an effect of inhibiting the binding of DJ2 and CRBN.
  • the peptide is included in the scope of the present invention without limitation as long as it is a peptide capable of inhibiting the function of CRBN by binding to the DJ2 binding site on CRBN.
  • the peptide of the present invention may preferably be a peptide comprising an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, but is not limited thereto, and at least one amino acid in the amino acid sequence is a different amino acid or other compound Even if substituted with , if the effect of inhibiting the function of CRBN by binding to the DJ2 binding site on CRBN is maintained, it is included in the scope of the present invention.
  • amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 are as follows.
  • the present invention provides a pharmaceutical composition for preventing or treating Alzheimer's disease, comprising the peptide.
  • the Alzheimer's disease may be caused by tau phosphorylation.
  • Alzheimer's disease is the most common degenerative brain disease causing dementia, and the onset gradually deteriorates cognitive functions including memory.
  • the exact pathogenesis and causes of Alzheimer's disease are not precisely known.
  • a small protein called beta-amyloid is excessively produced and deposited in the brain and has a detrimental effect on brain cells.
  • hyperphosphorylation of tau protein also contributes to brain cell damage and affects the pathogenesis.
  • Tau protein is a cytoskeleton protein, which is a microtubule-associated protein, and is abundantly present in neurons of the central nervous system compared to other cells, and hyperphosphorylated tau The fibrous aggregation of proteins abnormally accumulates in internal neurons, resulting in tauopathy.
  • Tau disease is distinguished in that the accumulation of amyloid beta (A ⁇ , ⁇ -amyloid), which is considered the main cause of Alzheimer's dementia, occurs in external nerve cells.
  • a ⁇ amyloid beta
  • ⁇ -amyloid amyloid beta
  • prevention refers to any act of inhibiting or delaying the onset of Alzheimer's disease by administering the pharmaceutical composition according to the present invention.
  • treatment refers to any action in which symptoms due to Alzheimer's disease are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.
  • the pharmaceutical composition according to the present invention includes the peptide of the present invention as an active ingredient, and may include a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is commonly used in formulation, and includes, but is not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and the like. It does not, and may further include other conventional additives such as antioxidants and buffers, if necessary. In addition, diluents, dispersants, surfactants, binders, lubricants, etc.
  • compositions can be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules, or tablets.
  • an injectable formulation such as an aqueous solution, suspension, emulsion, etc.
  • pills, capsules, granules, or tablets Regarding suitable pharmaceutically acceptable carriers and formulations, formulations can be preferably made according to each component using the method disclosed in Remington's literature.
  • the pharmaceutical composition of the present invention is not particularly limited in formulation, but may be formulated as an injection or oral ingestion.
  • the pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenously or subcutaneously) according to a desired method, and the dosage may vary depending on the patient's condition and weight, the degree of disease, drug form, although it depends on the route and time of administration, it may be appropriately selected by those skilled in the art.
  • composition according to the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and drug activity of the patient. , sensitivity to drugs, administration time, administration route and excretion rate, duration of treatment, factors including concurrent drugs, and other factors well known in the medical field.
  • the composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.
  • the effective amount of the composition according to the present invention may vary depending on the age, sex, and weight of the patient, and may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, and the like.
  • the term "pharmaceutically acceptable salt thereof” may be prepared by a conventional method in the art, for example, hydrochloric acid, hydrogen bromide, sulfuric acid, sodium hydrogen sulfate, phosphoric acid, carbonic acid
  • Drugs with salts with inorganic acids such as formic acid, acetic acid, oxalic acid, benzoic acid, citric acid, tartaric acid, gluconic acid, gestisic acid, fumaric acid, lactobionic acid, salicylic acid, or acetylsalicylic acid (aspirin)
  • forms pharmaceutically acceptable salts of these acids or reacts with alkali metal ions such as sodium or potassium to form metal salts thereof, or reacts with ammonium ions to form another pharmaceutically acceptable salt thereof means to form
  • the present invention provides a food composition for preventing or improving Alzheimer's disease, comprising the peptide.
  • the food composition includes a health functional food composition.
  • the term “improvement” refers to any action that at least reduces a parameter related to the condition being treated, for example, the degree of symptoms.
  • the active ingredient may be added to food as it is or used together with other food or food ingredients, and may be appropriately used according to a conventional method.
  • the mixing amount of the active ingredient may be appropriately determined depending on the purpose of its use (for prevention or improvement).
  • the composition of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material.
  • the amount may be less than or equal to the above range.
  • the health functional food composition of the present invention is not particularly limited in other ingredients other than containing the active ingredient as an essential ingredient in the indicated ratio, and may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional beverage.
  • natural carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol.
  • natural flavoring agents such as taumatine, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.
  • the proportion of the natural carbohydrate can be appropriately determined by the selection of a person skilled in the art.
  • the health functional food composition of the present invention contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and salts thereof, Alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like may be contained. These components may be used independently or in combination. The proportion of these additives may also be appropriately selected by those skilled in the art.
  • the present invention provides a method for screening a therapeutic agent for Alzheimer's disease, comprising the following steps.
  • step b) analyzing whether the peptide synthesized in step a) can inhibit the binding of CRBN and DJ2 by binding to the DJ2 binding site of CRBN;
  • step c) determining as a therapeutic agent for Alzheimer's disease when the peptide synthesized in step b) inhibits the binding of CRBN and DJ2.
  • Step a) is a step of synthesizing any peptide comprising the sequence using a CRBN binding motif having an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 according to the present invention.
  • the peptide can be obtained by a chemical method or a recombinant method using a known peptide synthesizer.
  • Step b) is a step of confirming whether the synthesized peptide has binding activity to CRBN in the presence of DJ2.
  • whether the peptide of the present invention binds to CRBN can be confirmed by any method capable of analyzing peptide-peptide bonds known in the art, for example, it can be carried out through chemical shift fluctuation analysis. .
  • Step c) is a step of determining the peptide as a therapeutic agent for Alzheimer's disease when it is confirmed that the synthesized peptide binds to CRBN as a result of the analysis of step b).
  • the present invention provides a method for preventing or treating Alzheimer's disease, comprising administering to a subject a peptide comprising a CRBN binding motif amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
  • the term “individual” refers to an animal, and may be a mammal capable of exhibiting beneficial effects by treatment using the peptide of the present invention.
  • Preferred examples of such subjects may include primates such as humans.
  • such individuals may include all individuals who have or are at risk of having symptoms of Alzheimer's disease.
  • the present invention provides the use of a peptide comprising a CRBN binding motif amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 for preventing or treating Alzheimer's disease.
  • compositions, treatment method, and therapeutic use may be equally applied as long as they do not contradict each other.
  • Antibodies were commercially purchased from Sigma, Abnova, Abcam, CST, etc. Thalidomide, cycloheximide, MG132, okadaic acid, heparin and ThT were commercially purchased from Sigma, Lipofectamine 2000 was commercially purchased from Invitrogen, ubiquitin, E1 , E2 and methylated ubiquitin were purchased commercially from Boston Biochem.
  • Human neuroblastoma cell line SH-SY5Y (Invitrogen), HEK-293T cells, and mouse embryonic fibroblast (MEF) cells were purchased from ATCC, and supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 10% fetal bovine serum (FBS; Invitrogen) at 37 °C with 5% CO 2 . It was maintained in DMEM supplemented with 100 U ml ⁇ 1 antibiotic-antifungal (Invitrogen).
  • hDJ2 and its mutants were cloned into the pCS2+ MT vector.
  • HA-tagged rCRBN and its mutants were cloned into pEBB-3HA vector.
  • Tau40-VN173 and Tau40-VC155 for BiFC analysis were provided by Dr. Yunkyung Kim.
  • His-tagged Tau40 and Hsp70 were constructed from pET-28a and His-tagged DJ2-pET30b was provided by Dr. Kiseon Kwon.
  • GST-tagged hCRBN in the pGEX-4T-1 vector was provided by Raymond J. Deshaies' laboratory. All cDNAs cloned into mammalian expression vectors and bacterial expression vectors were confirmed by DNA sequence analysis.
  • CRBN-knockout mice The preparation and screening of CRBN-knockout (KO) mice was done using the prior art (Lee et al., 2013). Wild-type and CRBN-KO mice were given standard chow diet and water in pathogen-free conditions with a light-dark cycle of 12 h. All experiments and paradigms were approved by the Gwangju Science and Technology Animal Care Center. The animal's exposure to various stressors is listed in Table 1 below. Protein extracts were prepared from the brain and quantified by the Bradford method (Bio-Rad Laboratories). Samples containing the same amount of protein were separated by SDS-PAGE and subjected to western immunoblotting.
  • Example 1-6 In vitro ubiquitination assay
  • the ubiquitination assay was performed as previously known. Briefly, SHSY5Y cells cultured in 6-well plates were transfected with Flag CRBN or empty vector and treated with MG132 (10 ⁇ M) for 4 hours after 48 hours. Cells were then harvested, lysed in RIPA buffer, and immunoprecipitated with Flag M2 agarose beads at 4 °C for 2-4 h.
  • Example 1-7 Cell-based ubiquitination assay
  • SHSY5Y cells cultured in 6-well plates were transfected with siRNA or siRNA CRBN . After 24 hours, cells were transfected with 1 ⁇ g of pRK5-HA-ubiquitin (HA-Ub). After 24 h, cells were harvested, washed twice with cold PBS and lysed in ubiquitination buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100) supplemented with PMSF and protease inhibitor cocktail. Lysates were incubated with ⁇ -DJ2 antibody overnight at 4 °C and immunoprecipitated with proteinG resin. Beads were thoroughly washed with ubiquitination buffer and PBS, the reaction was stopped by adding SDS sample buffer, and SDS-PAGE was separated and transferred to PVDF membrane for immunoblot analysis.
  • ubiquitination buffer 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA
  • lysis buffer 8 M urea, 75 mM NaCl, 50 mM Tris, pH 8.2, protease inhibitor cocktail, 1 mM NaF, 1 mM ⁇ -glycerophosphate, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 1 mM PMSF.
  • the protein was then reduced to a final concentration of 5 mM dithiothreitol (DTT) for 30 min at 37 °C and alkylated using IAA to a final concentration of 25 mM for 30 min in the dark at room temperature.
  • DTT dithiothreitol
  • urea concentration was diluted to ⁇ 2 ⁇ M with 25 mM Tris-HCl (pH 8.0) and digested overnight at 37° C. with 1:50 dilution trypsin.
  • the digested protein was subjected to multidimensional protein identification technology (MudPIT) analysis, which is a modification of the known method.
  • MudPIT column is an analytical column fused silica capillary column containing 7 cm of 5- ⁇ m Aqua C18 material (100- ⁇ m inner diameter) and 2 cm of 5 ⁇ m Partisphere strong cation exchange and 2 cm of 5- ⁇ m Aqua C18 reversed-phase column material.
  • the trapping column consisted of a fused silica capillary column (250- ⁇ m inner diameter) packed with After elution of the peptides from the microcapillary column, the peptides were electrosprayed with an LTQ linear ion trap mass spectrometer and applied to the waste of the HPLC split with the 2.3 kV atomization voltage used remotely. Cycles of full-scan mass spectra followed by nine data-dependent tandem MS (MS/MS) spectra (400-1400 m/z (mass-to-charge ratio)) at 35% normalized collision energies span the multidimensional separation repeated successively.
  • MS/MS data-dependent tandem MS
  • the mouse International Protein Index (IPI) database was searched using MS/MS spectra obtained from LC/LC-ESI-MS/MS analysis. SEQUEST was retrieved with a fragment ion mass tolerance of 1.0 Da and a parental mass tolerance of 3.0 Da.
  • the iodoacetamide derivative of cysteine was accepted as a fixed modification (cysteine +57) in SEQUEST.
  • Oxidation of methionine and acetylation of lysine are variable modifications (methionine +16) allowing up to 3 modifications per peptide (maximum number of modifications per type is 5) and missed cleavage sites for up to 2 trypsin digestions in SEQUEST searches. has been designated
  • BIOWORKS version 3.2 was used to filter the search results and apply the following Xcorr values and delta Cn values of 0.08 to the different charge states of the peptides: 1.8 for single charged peptides, 2.5 for double charged peptides, and triple charged peptides. 3.5. The requirement of both tryptic-digested ends was used in the filtering process.
  • siRNA CRBN and scrambled siRNA were purchased from Invitrogen, and cell transfection was performed with minor modifications according to the manufacturer's protocol. Target cells were transfected with lipfectamine2000 and siRNA, and knockdown efficiency was analyzed by immunoblot.
  • SH-SY5Y cells were seeded on poly-L-lysine-coated coverslips in complete medium in 6-well plates (1 x 10 4 cells/well) and transfected with siRNA or siRNA CRBN . After 24 h, cells were co-transfected with Tau40-VN173 and Tau-VC155 constructs and then treated with 30 nM okadaic acid (OA) after 16 h.
  • the tau-BiFC fluorescence intensity in cells was analyzed using a FV1000 confocal laser scanning microscope (Olympus) equipped with a 100X objective.
  • a 600-mesh carbon-coated copper grid (supplied by SPI) was used for TEM analysis of negatively stained Tau-K18 aggregates. Samples were incubated for 1 minute on a glow-discharge grid, then the solution was removed using filter paper. After washing with distilled water three times, the dye was stained with 1% (w / v) uranyl acetate for 2 minutes, and the final washing step was performed with distilled water. Grids were analyzed by a Tecnai T12 electron microscope equipped with a CCD camera.
  • CRBN-GST was purified from glutathione resin and digested with TEV protease. Tau-6xHis, DJ2-6xHis and Hsp70-6xHis were purified on Ni-NTA affinity resin (Clontech).
  • Cells were cultured in RIPA buffer (20 mM Hepes, 150 mM NaCl, 1 mM EDTA-EGTA, 1% Triton X-100, 1% NP40, 1% sodium deoxycholate, 2 mM Na 4 VO) supplemented with PMSF and protease inhibitor cocktail (Roche). 3 , 100 mM NaF [pH 7.4]).
  • the lysates were then removed by centrifugation at 12,500 RPM for 30 min and quantified by the Bradford method. Equal amounts of protein (10 ⁇ g/lane) were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blotted with the indicated antibodies. An ⁇ -rabbit or ⁇ -mouse antibody conjugated to Horseradish peroxidase was used as a secondary antibody, and the signal was detected using ECLTM Western Blotting Detection Reagent (Amersham).
  • CRBN CRISPR/Cas9 KO plasmid and CRBN HDR plasmid were purchased from Santa Cruz and knocked out CRBN according to the manufacturer's protocol. Briefly, HEK293T cells were co-transfected with CRBN CRISPR/Cas9 KO plasmid and CRBN HDR plasmid or control CRISPR plasmid (sc-418922). Cas-9-induced DSB (DNA-containing double-strand break) using Puromycin selected cells that were successful.
  • a multimodal short-term stress paradigm was used.
  • One group of Crbn +/+ C57BL/6 animals and one group of Crbn ⁇ /- C57BL/6 animals were exposed to CUMS for 5 weeks, and the experiment was repeated 3 times.
  • the CUMS paradigm consisted of three daily exposures to one of the following aversive stressors: stroboscopic illumination (6 times per hour / 6 light per sec.), cage tilting (30° position).
  • soiled cages (beds with bedding soaked with water), paired houses (in each grouping session paired two unfamiliar rats and switched occupants), loud noises (random noise generators (dB level ⁇ 80; generated for 1 hour at a frequency of 80-300 Hz), confinement (introducing a corrugated sheet along the width of the cage to limit the movable space), and inverted lighting cycle (general indoor lighting off during the day and on at night).
  • the stressors were presented in an unpredictable random order (see Table 1 below).
  • mice were anesthetized with 6X ketamine (0.1mL/10 g body weight), hair removed by giving local anesthesia (lidocaine 0.3mL), and the mice were placed firmly on the earbars.
  • OA 100 ⁇ M OA (Sigma) dissolved in DMSO or DMSO alone was unilaterally injected in the lateral amygdala (bregma coordinates: anterior/posterior -1.94, medial/lateral -3.15, dorsal/ ventral -4.5). Then, the needle was held in the inserted state for 5 minutes, then slowly pulled out and the scalp was sewn tightly.
  • Postoperative analgesics and antibiotics were administered intramuscularly once reflexes were restored (septazol 0.05 mL, ketapro 0.05 mL) and mice were placed in their cages and allowed to recover for 24 h.
  • the brain was removed from the skull, perfused with PBS, dissected to obtain the area shown in Figure 6a and snap frozen in liquid nitrogen.
  • Hsp70 and co-chaperones DJ1 and DJ2 were expected to be novel endogenous substrates of CRBN. Pull-down analysis confirmed that Hsp70, DJ1 and DJ2 interact with CRBN in both endogenous and exogenous systems (Fig. 1a, Fig. 9a).
  • CRBN expression was suppressed using CRBN-specific siRNA, the introduction of HA-tagged ubiquitin into endogenous Hsp70, DJ1 and DJ2 was greatly reduced, which means that DJ1 and the like are degraded by CRBN-mediated ubiquitination.
  • Flag CRBN increased co-precipitated endogenous chaperones in in vitro ubiquitination when enriched with recombinant E1, E2, ubiquitin and ATP (Fig. 1e, line 5), and the addition of methylated ubiquitin inhibited ubiquitination. (Fig. 1e, line 4). Since thalidomide does not interfere with the ubiquitination of chaperones, the association of chaperones with IMiDs was not further studied (Fig. 9b).
  • DJ2 has the ability to stabilize the native conformation of tau. Accordingly, in vivo and in vitro studies were performed to determine the effect of DJ2 on removing tau aggregation and whether it was interfered with by CRBN. Tauopathy is characterized by a marked accumulation of fibrillar tau inclusion bodies in the CNS. However, extracellular tau plays an influential role in trans-cell proliferation. Extracellular monomers and seed-competent tau enter the cell and strongly aggregate. To observe the effect of DJ2 on the toxicity of extracellular tau, heparin-induced K18 aggregates were treated for 48 h in the presence or absence of DJ2 and added outside SH-SY5Y cells.
  • FIGS. 3A and 3B To specify the amino acid sequence of DJ2 recognized by CRBN, deletion mutants for Myc-tagged DJ2 (Myc-DJ2) and HA-tagged CRBN (HA-CRBN) were constructed ( FIGS. 3A and 3B ). Pull-down analysis showed that a stretch of ⁇ 80 amino acids (Lon-N) at the N-terminus of CRBN interacted with a range of ⁇ 100 amino acids at the C-terminus of DJ2 (Figs. 3c and 3d). To pinpoint the sequences of DJ2 and CRBN important for the interaction, we constructed additional deletion mutants of both proteins, which were ß-turn binding to a span of 40 amino acids at the C-terminus of DJ2 and ß3 and ß4 of CRBN. -ß showed the integrity of the loop (Figs. 10a to 10d).
  • the present inventors manipulated 6 amino acids of DJ2 (T279-T284) and 2 amino acids of CRBN (E152A, F153A, E152A/F153A) individually or in combination. Mutations were constructed ( FIG. 10E ). In the case of CRBN, when E152 and F153 were mutated to alanine, binding strength was reduced (FIG. 3e). Notably, all mutants of DJ2 retained binding to CRBN with the exception of D281A, N282A, R283A and T284A, indicating that these residues are involved in the binding of DJ2 to CRBN (Fig. 3f).
  • DJ2 was selected for further analysis as a potential co-chaperone for the prevention of tauopathies, and a multimodal chronic ultra-mild stress (CUMS) paradigm was used.
  • CUMS chronic ultra-mild stress
  • Crbn +/+ mice CUMS produced marked hyperactivity with reduced feeding-related behavior.
  • these behavioral changes were prevented in Crbn ⁇ / ⁇ mice during the entire stress process (Table 3).
  • Tau phosphorylation was also measured in the WBL of control and stressed mice at the end of CUMS.
  • Crbn + / + mice as compared to Crbn - / - were markedly reduced to the tau phosphorylation in mouse brain, Crbn - the DJ2 levels were increased in mice (Fig. 5a and 5b) - /.
  • Topical administration of okadaic acid can immediately induce phosphorylation and aggregation of tau at anatomically distal sites by inhibiting protein phosphatase 2A (PP2A).
  • P2A protein phosphatase 2A
  • the prion-like diffusion of tau can be confirmed by analyzing the phosphorylation status of tau in various anatomical regions of the brain.
  • the response of Crbn ⁇ /- mice to tau phosphorylation and diffusion of tau phosphorylation to other regions of the brain were further verified using the above strategy.
  • OA was sterically injected into Crbn ⁇ /- and Crbn +/- and Crbn +/+ brains (M & M) and tau phosphorylation levels in the injected and non-injected hemispheres were analyzed (Fig.
  • Amyloid- ⁇ (A ⁇ ) plaques are one of the triggers of tauopathy because they promote tauopathy by increasing the spread of pathological tau.
  • Chemical knockdown of CRBN may not be a desirable strategy for cells for the recruitment and maintenance of ubiquitylation of endogenous substrates other than DJ2. Therefore, the use of peptides based on hotspot residues can mask CRBN, inhibit the interaction of DJ2 with CRBN, thereby inhibiting ubiquitylation of DJ2 and useful for selective inhibition of pathological tau phosphorylation.
  • linear (SEQ ID NO: 1) and circular (SEQ ID NO: 3) peptides were designed based on the hotspot region of DJ2 interacting with CRBN.
  • cysteine residues were added to both ends of the linear peptide to circularize the peptide.
  • both types of peptides showed significant CRBN and DJ2 binding inhibition ability as shown in FIG. 16 .
  • immunoprecipitation was performed by preparing a scrambled peptide in which the peptide of SEQ ID NO: 3 and amino acids constituting the peptide of SEQ ID NO: 3 were mixed in random order.
  • SH-SY5Y cells were transiently co-transfected with Myc-DJ2 and FLAG-CRBN.
  • Cell extracts were treated with scrambled peptides or peptides of SEQ ID NO: 3 for 24 hours.
  • the most potent inhibitors are generally less than 500 Da, so to reduce the size of the peptide inhibitor, the experiment was repeated with a peptide covering the most important residues of the hotspot, confirming that the DNRT sequence (SEQ ID NO: 2) was key (see Fig. 18). Accordingly, DNRT peptide and its scrambled peptide were prepared, and the ability to inhibit CRBN and DJ2 binding was confirmed.
  • SH-SY5Y cells were transiently co-transfected with Myc-DJ2 and FLAG-CRBN.
  • Cell extracts were treated with the scrambled peptide or the peptide of SEQ ID NO: 2 for 24 hours.
  • the peptide of the present invention is a peptide mimicking the CRBN binding motif in DJ2, and since it binds to CRBN and interferes with the binding of DJ2 and CRBN, the degradation of DJ2 by CRBN is suppressed. As a result, since the effect of DJ2 inhibiting the aggregation of tau protein is maintained and improved, it can be usefully used for preventing, improving or treating Alzheimer's disease.

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Abstract

La présente invention concerne un peptide qui se lie au CRBN et inhibe la liaison du CRBN et de DJ2, et une composition pour la prévention, le traitement ou l'atténuation de la maladie d'Alzheimer faisant appel à celui-ci. Le peptide selon la présente invention imite un motif de liaison au CRBN dans DJ2, et se lie ainsi au CRBN et interfère avec la liaison de DJ2 et du CRBN, ce qui permet d'inhiber la dégradation de DJ2 par le CRBN. Par conséquent, étant donné que l'effet de DJ2 inhibant l'agrégation de la protéine Tau est maintenu et amélioré, DJ2 peut être utilisée utilement pour la prévention, l'atténuation ou le traitement de la maladie d'Alzheimer.
PCT/KR2021/004627 2020-04-17 2021-04-13 Peptide de liaison au crbn et composition pour la prévention ou le traitement de la maladie d'alzheimer faisant appel à celui-ci WO2021210878A1 (fr)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
US6001598A (en) * 1997-06-03 1999-12-14 Incyte Pharmaceuticals, Inc. Two new human DnaJ-like proteins
KR20180123339A (ko) * 2017-05-08 2018-11-16 가톨릭대학교 산학협력단 β-아밀로이드 단백질의 응집을 억제하는 생체친화적 펩타이드
JP2020506922A (ja) * 2017-01-31 2020-03-05 アルビナス・オペレーションズ・インコーポレイテッドＡｒｖｉｎａｓ Ｏｐｅｒａｔｉｏｎｓ， Ｉｎｃ． セレブロンリガンド、およびセレブロンリガンドを含有する二官能性化合物

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US6001598A (en) * 1997-06-03 1999-12-14 Incyte Pharmaceuticals, Inc. Two new human DnaJ-like proteins
JP2020506922A (ja) * 2017-01-31 2020-03-05 アルビナス・オペレーションズ・インコーポレイテッドＡｒｖｉｎａｓ Ｏｐｅｒａｔｉｏｎｓ， Ｉｎｃ． セレブロンリガンド、およびセレブロンリガンドを含有する二官能性化合物
KR20180123339A (ko) * 2017-05-08 2018-11-16 가톨릭대학교 산학협력단 β-아밀로이드 단백질의 응집을 억제하는 생체친화적 펩타이드

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JOSE F ABISAMBRA; UMESH K JINWAL; AMIRTHAA SUNTHARALINGAM; KARTHIK ARULSELVAM; SARAH BRADY; MATTHEW COCKMAN; YING JIN; BO ZHANG; C: "DnaJA1 Antagonizes Constitutive Hsp70-Mediated Stabilization of Tau", JOURNAL OF MOLECULAR BIOLOGY, vol. 421, 2 February 2012 (2012-02-02), pages 653 - 661, XP028433439, ISSN: 0022-2836, DOI: 10.1016/j.jmb.2012.02.003 *

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