KR102608311B1 - CRBN binding peptide and composition for preventing or treating Alzheimer's disease using the same - Google Patents

Abstract

The present invention relates to a peptide that binds to CRBN and inhibits the binding of CRBN and DJ2, and to a composition for preventing, treating or improving Alzheimer's disease using the same. The peptide of the present invention mimics the CRBN binding motif in DJ2 and thus binds to CRBN. This prevents the binding of DJ2 and CRBN, thereby suppressing the degradation of DJ2 by CRBN. As a result, the effect of DJ2 to inhibit tau protein aggregation is maintained and improved, so it can be usefully used for the prevention, improvement, or treatment of Alzheimer's disease.

Description

CRBN binding peptide and composition for preventing or treating Alzheimer's disease using the same {CRBN binding peptide and composition for preventing or treating Alzheimer's disease using the same}

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.

Recently, with the rapid development of medicine, the average human lifespan has increased, and as the elderly population increases, new social problems are emerging. In particular, geriatric neurological diseases such as stroke, Alzheimer's disease, and Parkinson's disease are caused by fatal nervous system dysfunction, and there is currently no effective method to treat them. In particular, the most common geriatric neurological disease is Alzheimer's disease (AD). Alzheimer's is the most common degenerative brain disease that causes dementia and causes problems with memory, thinking, and behavior. Dementia is a general term meaning loss of memory and other intellectual abilities severe enough to interfere with daily life. Alzheimer's accounts for 60 to 80% of dementia cases.

Although the pathogenesis and cause of Alzheimer's are unclear, the main causes are known to be neuronal damage due to decreased synthesis of the neurotransmitter acetylcholine, deposition of β-amyloid, and hyperphosphorylation of tau protein. Tau protein is a type of microtubule-binding protein with a molecular weight of 50,000 to 70,000 Da, and is mainly involved in the entanglement of nerve fibers. Tau proteins exhibit remarkable molecular diversity, the best known of which is proline-directed phosphorylation.

E3-ligase substrate recruiter cereblon (CRBN), a substrate receptor for CRL4 ^CRBN , is the therapeutic target of thalidomide and its derivatives and is known as an immunomodulatory drug (IMiD). CRBN mutations are associated with autosomal recessive, non-symptomatic mental retardation. CRBN has also been implicated in the regulation of AMP-activated protein kinase, glutamine synthetase, MEIS2, and ion channels.

The DNAJ family is involved in a variety of cellular activities, including protein folding/unfolding/refolding. Among them, DJ2 is known to react with DnaK and numerous Hsp70 and Hsp110, which attract and unfold misfolded proteins within stable aggregates. However, it is unclear whether DJ2 can prevent unfolding or aggregation in addition to its ability to recognize unfolded proteins, and its relationship with tau protein is not yet known, so research on this is required.

Republic of Korea registered patent 10-2033776

The object of the present invention is to provide a peptide containing the CRBN binding motif amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating Alzheimer's disease containing the above peptide.

The purpose of the present invention is to provide a food composition for preventing or improving Alzheimer's disease containing the above peptide.

The purpose of the present invention is to provide a screening method for a therapeutic agent for Alzheimer's disease.

In order to achieve the object of the present invention, the present invention provides a peptide containing the CRBN binding motif amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

Additionally, the present invention provides a pharmaceutical composition for preventing or treating Alzheimer's disease, comprising the peptide.

Additionally, the present invention provides a food composition for preventing or improving Alzheimer's disease, comprising the peptide.

In one embodiment of the present invention, Alzheimer's disease may be caused by tau phosphorylation.

Additionally, the present invention provides a screening method for an Alzheimer's drug, comprising the following steps.

a) synthesizing a peptide containing a CRBN binding motif represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3;

b) analyzing whether the peptide synthesized in step a) can bind to the DJ2 binding site of CRBN and inhibit the binding of CRBN and DJ2; and

c) determining that the peptide synthesized in step b) is a treatment for Alzheimer's disease if it inhibits the binding of CRBN and DJ2.

Additionally, the present invention provides a method for preventing or treating Alzheimer's disease, comprising administering to a subject a peptide containing the CRBN binding motif amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

Additionally, the present invention provides the use of a peptide containing the CRBN binding motif amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 for the prevention or treatment of Alzheimer's disease.

The peptide of the present invention is a peptide that mimics the CRBN binding motif in DJ2, binds to CRBN and interferes with the binding between DJ2 and CRBN, thus inhibiting the degradation of DJ2 by CRBN. As a result, the effect of DJ2 to inhibit tau protein aggregation is maintained and improved, so it can be usefully used for the prevention, improvement, or treatment of Alzheimer's disease.

Figure 1 identifies Hsp70, DJ1, and DJ2 as endogenous substrates of CRL4 ^CRBN . Specifically, Figure 1a shows the results of SH-SY5Y cells lysed, immunoprecipitated with rabbit IgG control or α-CRBN antibodies, and blotted with the indicated antibodies. Figures 1b to 1d show SH-SY5Y cells transiently transfected with scrambled SiRNA (Scr) or siRNA _CRBN and then 24 h later transfected with HA-Ub, followed by IP for α-Hsp70, α-DJ1, and α-Ub, respectively. This is the result of performing with DJ2 antibody and blotting with the indicated antibody. Figure 1E shows SH-SY5Y cells were transiently transfected with FLAG-CRBN and immunoprecipitated with Flag M2 agarose beads. In vitro ubiquitination of endogenous, coprecipitated chaperones was performed in the presence of E1 + E2 and Ub. Methylated ubiquitin (Me-Ub) was added where indicated, and the reaction was analyzed by SDS-PAGE and immunoblotting with the indicated antibodies.
Figure 1F shows the results of Crbn ^−/− and Crbn ^+/+ MEF cells treated with 2.5 μg/ml CHX and subjected to immunoblot analysis (statistical analyzes are shown in the order of Hsp70, DJ1, and DJ2 from top to bottom). Figure 1g shows the results of Crbn ^−/− and Crbn ^+/+ MEF cells treated with 0.5 μg/ml MG132 and subjected to immunoblot analysis (statistical analyzes are presented in the order of Hsp70, DJ1, and DJ2 from top to bottom).
Figure 2 is an experiment on the effect of CRBN and DJ2 on heparin-mediated aggregation of tau. Specifically, Figure 2A shows the results of incubating full-length hTau-44 (5 μM) with the indicated protein combinations for 48 hours, then adding 5 μM ThT and measuring fluorescence emission at 480 nm with excitation at 440 nm ( Heparin was included in all reactions except the control group at a concentration of 2.5 μM; Student's t-test was used to quantify data at a 95% significance level). Figure 2b shows the results of incubating monomeric hTau-K18 (5 μM) with the indicated combination (5 μM) and 5 μM ThT for 4 hours, confirming that the fluorescence emission was excited at 480 nm and 440 nm (heparin is 2.5 μM). (included in all reactions except the control group at a concentration of μM). Figure 2c shows the results of incubating monomer K18 with or without DJ2 for heparin-induced aggregation for 4 hours and performing TEM analysis. Figure 2d shows the results of treating SHSY5Y cells with monomeric K18, aggregating K18 in the presence or absence of DJ2 and CRBN, photographs were taken 48 hours after treatment, and MTT assay was performed to evaluate cell viability (error Bars represent SEM, Student's t-test was used to quantify data at 95% significance level)
Figure 3 confirms that the N-terminal Lon domain of CRBN binds to the C-terminal domain of DJ2. Specifically, Figures 3a and 3b show schematic diagrams of the full-length rCRBN and deletion constructs used in Figures 3c and 3d below (CULT (Cereblon domain of Unknown activity, binding cellular Ligands and Thalidomide), L (linker) , CTD (C-terminal domain)). Figures 3c and 3e show that SH-SY5Y cells were transiently cotransfected with Myc-DJ2 and the indicated plasmids, and cell extracts were immunoprecipitated with α-Myc antibody, sorted by SDS-PAGE, and immunoblotted with Myc and HA antibodies. The plotted results are shown (the band at ~55 kDa represents the IgG heavy chain (HC) and the band at ~25 kDa represents the IgG light chain (LC). Asterisks indicate non-specific bands). Figures 3d and 3f show the results of SH-SY5Y cells transiently co-transfected with HA-CRBN and the indicated primers, and then IP performed with HA antibody 24 hours later.
Figure 4 shows the results of an experiment confirming that K32 and K350 are the main ubiquitination sites of DJ2. Specifically, Figure 4a shows the results of immunoblot analysis performed by transfecting SH-SY5Y cells with wild-type (WT) DJ2 and lysine mutants, and treating the cells with 2.5 μg/ml CHX. Figure 4b is a graphical representation of the results of Figure 4a. Figure 4c confirms that ubiquitylation is impaired in the case of a double mutant of K32 and K350 residues.
Figure 5 confirms the effect of CRBN KO on CUMS (chronic ultra-mild stress) and tau pathology. Specifically, Figure 5A shows the results of exposing Crbn ^-/- (KO) and Crbn ^+/+ (WT) mice to the CUMS paradigm, followed by fractionation of WBLs by SDS-PAGE and immunoblotting with antibodies. Figures 5b and 5c show the results of Western blot analysis of WBL from WT and KO mice for selected pTau epitopes and taukinase. Figure 5d shows the results of statistical processing with n = 3 using 5 mice in each group of KO and WT mice. Figures 5e and 5f show the results of statistical analysis of the results of Figures 5b and 5c using a t-test, with P < 0.05.
Figure 6 confirms that CRBN KO suppresses the spread of tau pathology. Specifically, Figure 6A is a schematic diagram of the anatomy of the mouse brain, divided into anterior contralateral (AC), anterior ipsilateral (AI), posterior contralateral (PC), posterior ipsilateral (PI) and cerebellum (Cb) for analysis by Western blotting. The PI area includes the lateral tonsil injection site. Figure 6b shows the results of stereotactically injecting OA into the brains of WT and KO mice, preparing WBL in RIPA buffer, and blotting with antibodies from the pTau array. Figure 5C shows relative band intensity quantification results in each group of KO and WT mice n = 3 (error bars represent SEM).
Figure 7 shows the effect of CRBN KO on phosphorylation-mediated tau dimerization. Specifically, 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 both cell lines and Western blot was performed. am. Figure 7b shows the results of Figure 7a in color gradient scale. Figure 7c shows that SH-SY5Y cells were transiently transfected with siRNA or siRNA _CRBN , and then the cells were transfected with tau40-VN173 and tau-VC155 constructs and treated with OA (30 nM) to reveal BiFC using confocal microscopy. This is the measured result (Hoechst dye was used as a counter stain).
Figure 8 schematically shows the role of CRBN and DJ2 in tau pathology.
Figure 9 shows the contents of an experiment regarding the binding of DJ2 to CRBN and tau and thalidomide-independent degradation of DJ1. Specifically, Figure 9a shows the results of GST pull-down analysis performed on His-tagged DJ2 and GST-tagged CRBN and blotting with anti-DJ2 and anti-CRBN antibodies, and Figure 9b shows SH-SY5Y cells treated with HA-Ub This is the result of transient transfection and 30 hours later, cells were treated with thalidomide for 24 hours, and cell lysis and IP were performed with mouse IgG control or α-DJ2 antibody. Figure 9c shows that SH-SY5Y cells were transiently co-transfected with Myc-tagged tau and FLAG-tagged DJ2, and after 24 h, cell extracts were immunoprecipitated with α-Myc antibody and fractionated by SDS-PAGE and labeled with the indicated antibodies. This is the result of immunoblotting (SE: short exposure, LE: long exposure).
Figure 10 shows experimental details on the localization of the domain responsible for CRBN-DJ2 interaction. Specifically, 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, cell extracts were immunoprecipitated with α-HA antibody and sorted by SDS-PAGE. This is the result of immunoblotting with antibodies. Figures 10c and 10d show the results of Western blotting performed by transiently co-transfecting SH-SY5Y cells with HA-CRBN and Myc-tagged DJ2 digest, and 24 hours later, cell extracts were immunoprecipitated with α-HA antibody. Figure 10e shows the structural model of DJ2-CRBN interaction predicted by ClusPro.
Figure 11 shows the results of an experiment on the localization of a specific lysine residue responsible for ubiquitination of DJ2. Specifically, Figures 11A and 11B identify experimentally reported lysines ubiquitinated in DJ2 using PhosphositePlus and GGbase databases. Figure 11c shows that SH-SY5Y cells were transfected with wild-type (WT) DJ2 and various lysine mutants, confirming that K32 and K350 are the main ubiquitination sites of DJ2.
Figure 12 shows the results of 2D-polyacrylamide gel electrophoresis on whole brain lysates of Crbn ^-/- and Crbn ^+/+ , and the antioxidant level of Crbn ^-/- brain lysates compared to Crbn ^+/+ mice. The increase was confirmed.
Figure 13 confirms the effect of CRBN KO on tau pathology induced by injection of okadaic acid, densitometric quantification results of relative band intensity in each group (n = 3) of Crbn ^-/- and Crbn ^+/+ mice. is shown graphically.
Figure 14 shows the results of analyzing the activities of three tau kinases in the same brain sample.
Figure 15 confirms the effect of CRBN knock-out on tauopathy and the inhibitory effect of the prepared peptide on CRBN and DJ2 binding. Specifically, Figure 15a shows confirmation of APP plaques in a murine model of neuropathy. Brains were removed from 8-month-old APP knock-in mice, hippocampi were immunostained with CRBN and APP antibodies for 16 hours, and Alexa-infected mice were incubated for 1 hour. After incubation with conjugated secondary antibodies, they were imaged under a confocal microscope equipped with a 60X lens. Figures 15b and 15c confirm the high levels of CRBN and low DJ2 in brain samples from APP knock-in and 5XFAD mice. Brains were removed from 8-month-old APP knock-in and 5XFAD mice, and half of the brain was cryosectioned. The other half was used for Western blot analysis, and the hippocampus was immunostained with CRBN and DJ2 antibodies for 16 h, incubated with Alexa-conjugated secondary antibodies for 1 h, and imaged under a confocal microscope equipped with a 10X lens. It was done. Figure 15d shows the amino acid sequence of the peptide inhibitor of the present invention. Figure 15e confirms that the addition of the peptide inhibitor inhibits the binding of CRBN and DJ2. SH-SY5Y cells were transiently co-transfected with Myc-DJ2 and FLAG-CRBN, and 24 hours later, cell extracts were incubated with α-Myc antibody. This is the result of immunoprecipitation followed by immunoblotting with Myc and HA antibodies (the band of ~ 55 kDa represents the IgG heavy chain).
Figure 16 shows the ability to inhibit CRBN and DJ2 binding by immunoprecipitation using linear (SEQ ID NO: 1) and circular (SEQ ID NO: 3) peptide forms.
Figures 17a to 17d show the CRBN and DJ2 binding inhibition ability confirmed by immunoprecipitation based on the peptide of SEQ ID NO: 3, and show the results of an immunoprecipitation experiment in which peptides with randomly scrambled amino acid sequences were 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 Figure 17c).
Figure 18 shows the sequences and structures of the four peptide sequences (Tetra-Peptide sequence, SEQ ID NO: 2), which are important residues among the peptides of SEQ ID NO: 1, and the control group (scrambled tetra-peptide).
Figures 19a to 19c confirm that the peptide represented by SEQ ID NO: 2 significantly inhibits the binding of CRBN and DJ2.

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.

In the present invention, CRBN is a substrate receptor of the cillin-ring E3 ubiquitin ligase (CRL) complex, and the CRL complex ubiquitinates the substrate from ubiquitin-conjugation enzyme (E2) to induce degradation in the proteasome. It has also been reported as a target protein for thalidomide, lenalidomide, and pomalidomide, which are immunomodulators (IMiDs) with anticancer effects against multiple myeloma.

The term “peptide” used in the present invention refers to a polymer composed of amino acids linked by amide bonds or peptide bonds. For the purposes of the present invention, “peptide containing a CRBN binding motif amino acid sequence” refers to a peptide with a sequence that can specifically bind to CRBN and inhibit the function of CRBN. Specifically, the peptide is a peptide that has the activity to bind to the DJ2 binding site on CRBN, and acts as a competitive substrate for DJ2, showing the effect of inhibiting the binding of DJ2 and CRBN.

In the present invention, the peptide is included in the scope of the present invention without limitation as long as it is a peptide that can inhibit the function of CRBN by binding to the DJ2 binding site on CRBN. The peptide of the present invention may preferably be a peptide containing 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 another amino acid or another 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 within the scope of the present invention.

The amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 are as follows.

PISTLDNRTIV (SEQ ID NO: 1)

DNRT (SEQ ID NO: 2)

CPISTLDNRTIVC (SEQ ID NO: 3)

The present invention provides a pharmaceutical composition for preventing or treating Alzheimer's disease, comprising the above peptide.

In the present invention, the Alzheimer's disease may be caused by tau phosphorylation.

In the present invention, Alzheimer's disease is the most common degenerative brain disease that causes dementia, and it develops slowly and causes gradual deterioration of cognitive functions, including memory. The exact pathogenesis and cause of Alzheimer's disease are not exactly known. Currently, it is known that the core mechanism of the disease is that a small protein called beta-amyloid is excessively produced and deposited in the brain, which has a harmful effect on brain cells. In addition, tau protein (Tau protein), which plays an important role in maintaining the skeleton of brain cells, Hyperphosphorylation of tau protein is also known to contribute to brain cell damage and affect the development of the disease.

Tau protein is a cytoskeleton protein that supports microtubules (microtubule-associated protein). It is abundant in neurons of the central nervous system compared to other cells, and hyperphosphorylated tau Tauopathy occurs when fibrous aggregates of protein accumulate abnormally in inner nerve cells.

Tau disease is distinguished in that the accumulation of amyloid beta (Aβ, β-amyloid), which is considered the main cause of Alzheimer's dementia, occurs in external nerve cells, especially in patients with frontotemporal dementia, an inherited tau-related dementia (tau only dementia). It has been confirmed that dementia is induced solely by tau hyperphosphorylation and aggregation in inner nerve cells without accumulation of amyloid beta in outer nerve cells (van Swieten and Spillantini, 2007).

As used in the present invention, the term “prevention” refers to all actions that suppress or delay the onset of Alzheimer's disease by administering the pharmaceutical composition according to the present invention.

As used in the present invention, the term “treatment” refers to any action in which symptoms of 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 contains the peptide of the present invention as an active ingredient and may contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is commonly used in preparation and includes, but is limited to, saline solution, sterile water, Ringer's solution, buffered saline solution, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, etc. If necessary, other common additives such as antioxidants and buffers may be added. In addition, diluents, dispersants, surfactants, binders, lubricants, etc. can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets. Regarding suitable pharmaceutically acceptable carriers and formulations, the formulations can be preferably formulated according to each ingredient using the method disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in formulation, but can be formulated as an injection or oral ingestion.

The pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intravenously or subcutaneously) depending on the desired method, and the dosage depends on the patient's condition and weight, disease severity, drug form, It varies depending on the route and time of administration, but can be appropriately selected by a person skilled in the art.

The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, “pharmacologically effective amount” means an amount sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, and activity of the patient's disease. , can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the field of medicine. 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 singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

Specifically, the effective amount of the composition according to the present invention may vary depending on the patient's age, gender, and weight, and may increase or decrease depending on the route of administration, severity of disease, gender, weight, age, etc.

As used in the present invention, the term “pharmaceutically acceptable salt thereof” refers to one that can be prepared by methods common in the art, for example, hydrochloric acid, hydrogen bromide, sulfuric acid, sodium hydrogen sulfate, phosphoric acid, carbonic acid. Drugs with salts of 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). It forms scientifically acceptable salts of these acids, or reacts with alkali metal ions such as sodium and potassium to form metal salts, or reacts with ammonium ions to form another pharmaceutically acceptable salt thereof. means forming.

The present invention provides a food composition for preventing or improving Alzheimer's disease, comprising the above peptide.

The food composition includes a health functional food composition.

As used herein, the term “improvement” means any action that reduces at least the severity of a parameter, such as a symptom, related to the condition being treated.

In the food composition of the present invention, the active ingredient can be added as is to the food or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention or improvement). Generally, when producing food or beverages, 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 materials. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range.

The health functional food composition of the present invention has no particular restrictions on other ingredients other than containing the above-mentioned active ingredients as essential ingredients in the indicated proportions, and may contain various flavoring agents or natural carbohydrates as additional ingredients like ordinary beverages. there is. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of the natural carbohydrates can be appropriately determined by the selection of a person skilled in the art.

In addition to the above, the health functional food composition of the present invention contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, It may contain alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These ingredients can be used independently or in combination. The proportions of these additives can also be appropriately selected by those skilled in the art.

The present invention provides a screening method for an Alzheimer's drug, comprising the following steps.

a) synthesizing a peptide containing a CRBN binding motif represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3;

b) analyzing whether the peptide synthesized in step a) can bind to the DJ2 binding site of CRBN and inhibit the binding of CRBN and DJ2; and

c) determining that the peptide synthesized in step b) is a treatment for Alzheimer's disease if it inhibits the binding of CRBN and DJ2.

Step a) is a step of synthesizing any peptide containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 using the CRBN binding motif according to the present invention. At this stage, peptides can be obtained by chemical or recombinant methods using known peptide synthesizers.

Step b) is a step to confirm whether the synthesized peptide has binding activity to CRBN in the presence of DJ2. At this stage, whether the peptide of the present invention binds to CRBN can be confirmed by any method that can analyze peptide-peptide bonds known in the art, for example, it can be performed through chemical shift fluctuation analysis. .

Step c) is a step in which, if the synthesized peptide is confirmed to bind to CRBN as a result of the analysis in step b), the peptide is determined as a treatment for Alzheimer's disease.

The present invention provides a method for preventing or treating Alzheimer's disease, comprising administering to a subject a peptide containing the CRBN binding motif amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

The term “individual” used in the present invention refers to an animal, and may be a mammal that can exhibit beneficial effects through treatment using the peptide of the present invention. Preferred examples of such subjects may include primates such as humans. Additionally, such individuals may include all individuals who have symptoms of Alzheimer's disease or are at risk of having symptoms of Alzheimer's disease.

The present invention provides the use of a peptide containing the CRBN binding motif amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 for the prevention or treatment of Alzheimer's disease.

Descriptions related to the composition, treatment method, and treatment use may be applied in the same manner as long as they do not contradict each other.

Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.

<Example 1: Experimental method, materials and reagents>

Example 1-1. Reagents, DNA and Antibodies

Antibodies were purchased commercially from Sigma, Abnova, Abcam, CST, etc. Thalidomide, cycloheximide, MG132, okadaic acid, heparin, and ThT were purchased commercially from Sigma, Lipofectamine 2000 was purchased commercially from Invitrogen, ubiquitin, E1 , E2 and methylated ubiquitin were purchased commercially from BostonBiochem.

Example 1-2. Cell culture and materials

The human neuroblastoma cell line SH-SY5Y (Invitrogen), HEK-293T cells and mouse embryonic fibroblast (MEF) cells were purchased from ATCC and incubated with 10% fetal bovine serum (FBS; Invitrogen) at 37 °C with 5% _CO2 . Maintained in DMEM supplemented with 100U ml ^-1 antibiotic-antimycotic (Invitrogen).

Example 1-3. plasmid

For expression in mammalian cells, Myc-tagged hDJ2 and its mutants were cloned in pCS2+ MT vector. And HA-tagged rCRBN and its mutants were cloned into pEBB-3HA vector. Tau40-VN173 and Tau40-VC155 for BiFC analysis were provided by Dr. Yun-Kyung Kim. For bacterial expression, His-tagged Tau40 and Hsp70 were constructed in pET-28a and His-tagged DJ2-pET30b was provided by Dr. Ki-Seon Kwon. GST-tagged hCRBN in pGEX-4T-1 vector was provided by the laboratory of Raymond J. Deshaies. All cDNAs cloned into mammalian and bacterial expression vectors were confirmed by DNA sequence analysis.

Example 1-4. laboratory animals

Construction and screening of CRBN-knockout (KO) mice were performed using conventional techniques (Lee et al., 2013). Wild-type and CRBN-KO mice were provided standard chow diet and water under pathogen-free conditions with a 12-h light/dark cycle. All experiments and paradigms were approved by the Gwangju Science and Technology Animal Control Center. The animals' 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 equal amounts of protein were separated on SDS-PAGE and subjected to Western immunoblotting.

Examples 1-5. 2D-PAGE and image analysis

Whole brain tissue was flash frozen, ground to a fine powder in liquid N ₂ , and sonicated in urea lysis buffer (7M urea, 2M thiourea, 2% CHAPS, 1% DTT). The lysate was then removed by centrifugation at 45,000 RPM for 30 minutes and quantified by the Bradford method (Bio-Rad, Hercules, CA). Equal amounts of protein (300 μg/strip) were separated by IEF (GE Healthcare, pH 3–10, 7 cm) and then by SDS-PAGE. After silver staining, image analysis was performed at the Yonsei Proteome Research Institute, and spots with significant differences in Crbn ^-/- and Crbn ^+/+ were subjected to in-gel digestion and MS/MS analysis.

Example 1-6. In vitro ubiquitination assay

Ubiquitination analysis was performed as previously known. Briefly, SHSY5Y cells cultured in 6-well plates were transfected with ^Flag CRBN or empty vector, and 48 hours later, they were treated with MG132 (10 μM) for 4 hours. Cells were then harvested, lysed in RIPA buffer, and immunoprecipitated with Flag M2 agarose beads for 2–4 h at 4 °C. After washing twice with RIPA buffer and twice with ubiquitination buffer (50 mM Tris-HCl [pH 8.0], 10 mM MgCl ₂ , 0.2 mM CaCl ₂ , 1 mM DTT, and 100 nM MG132), the beads were incubated with E1 (0.5 µM), UbcH5a (0.5 µM), UbcH3 (1.67 µM), ubiquitin (60 µM) and ATP (4 mM) for 1 h at 30 °C. Methylated ubiquitin (Me-Ub) was also added where indicated, and the reaction was stopped by adding SDS sample buffer and immunoblot analysis was performed.

Example 1-7. Cell-based ubiquitination assay

SHSY5Y cells cultured in 6-well plates were transfected with siRNA or siRNA _CRBN . 24 hours later, cells were transfected with 1 μg of pRK5-HA-ubiquitin (HA-Ub). After 24 h, cells were harvested, washed twice with cold PBS and lysed in ubiquitination buffer (50mM Tris-HCl, 150mM NaCl, 1mM EDTA, 0.5% Triton X-100) supplemented with PMSF and protease inhibitor cocktail. Lysates were incubated with α-DJ2 antibody overnight at 4°C and immunoprecipitated with proteinG resin. Beads were washed thoroughly with ubiquitination buffer and PBS, the reaction was stopped by adding SDS sample buffer, and separated by SDS-PAGE and transferred to PVDF membrane for immunoblot analysis.

Examples 1-8. Identification of CRBN binding partners using LC-MS/MS

Whole brain tissue powdered in liquid nitrogen was lysed in lysis buffer (8 M urea, 75 mM NaCl, 50 mM Tris, pH 8.2, protease inhibitor cocktail, 1 mM NaF, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 10 mM sodium). pyrophosphate, 1 mM PMSF). Proteins were then reduced to a final concentration of 5mM dithiothreitol (DTT) for 30 min at 37 °C and alkylated using IAA to a final concentration of 25mM for 30 min in the dark at room temperature. After alkylation, the urea concentration was diluted to <2 μM using 25 mM Tris-HCl (pH 8.0) and digested with 1:50 diluted trypsin overnight at 37 °C. The digested proteins were subjected to multidimensional protein identification technology (MudPIT) analysis, which was a modification of a previously known method.

The MudPIT column is an analytical column fused silica capillary column (100-μm inner diameter) containing 7 cm of 5-μm Aqua C18 material and 2 cm of 5 μm Partisphere strong cation exchange material and 2 cm of 5-μm Aqua C18 reversed-phase column material. The trapping column was composed of a fused silica capillary column (250-μm inner diameter) packed with . After peptide elution from the microcapillary column, peptides were electrosprayed into an LTQ linear ion trap mass spectrometer and applied to the waste of the HPLC split with 2.3 kV spray voltage used distally. 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 throughout the multidimensional separation. It was repeated continuously.

MS/MS spectra obtained from LC/LC-ESI-MS/MS analysis were used to search the mouse International Protein Index (IPI) database. SEQUEST was searched with a fragment ion mass tolerance of 1.0 Da and a parent 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 were identified as variable modifications (methionine +16) with up to 3 modifications allowed per peptide in SEQUEST searches (maximum number of modifications per type is 5) and up to 2 missed cleavage sites for trypsin digestion. It has been designated.

Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the parsimony principle. BIOWORKS version 3.2 was used to filter the search results and the following 3.5. The requirement of both tryptic-digested ends was used in the filtering process.

Example 1-9. CRBN Knockdown

siRNA _CRBN and scrambled siRNA (control) were purchased from Invitrogen, and cell transfections were performed according to the manufacturer's protocol with minor modifications. Target cells were transfected with lipfectamine2000 and siRNA, and knockdown efficiency was analyzed by immunoblot.

Examples 1-10. Bimolecular Fluorescence Complementation (BiFC) analysis

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) 16 h later. The tau-BiFC fluorescence intensity in cells was analyzed using an FV1000 confocal laser scanning microscope (Olympus) equipped with a 100X objective.

Example 1-11. K18 agglutination assay

Aggregation of tau protein K18 (5 μM) was performed at 37 °C in the presence of polyanions (heparin, 2.5 μM) in PBS at pH 7.4. Assembly was performed qualitatively by TEM and quantitatively by fluorescence analysis using ThT. Samples were incubated at 37 °C for 6 h, and Dithiothreitol was added to a final concentration of 1 mM to avoid intramolecular disulfide bridges. For each state (pre-aggregates, aggregates, aggregates with DJ2), K18 (5 μM) was transferred to black 98-well plates with PBS containing 5 μM ThT (Sigma). ThT signals were measured at λ _ex = 430 nm and λ _em = 480 nm on a Synergy H1 hybrid reader, Biotek.

Examples 1-12. Transmission electron microscopy (TEM)

600-mesh carbon-coated copper grids (supplied by SPI) were used for TEM analysis of negatively stained Tau-K18 aggregates. Samples were incubated on a glow-discharge grid for 1 minute, then the solution was removed using filter paper. After washing three times with distilled water, the cells were stained with 1% (w/v) uranyl acetate for 2 min and a final washing step was performed with distilled water. Grids were analyzed by a Tecnai T12 electron microscope equipped with a CCD camera.

Example 1-13. Expression and purification of recombinant proteins

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).

Example 1-14. Immunoblot analysis

Cells were incubated in RIPA buffer (20 _mM Hepes, 150 mM NaCl, 1mM EDTA-EGTA, 1% Triton ₃ , dissolved in 100 mM NaF [pH 7.4]).

The lysate was then cleared by centrifugation at 12,500 RPM for 30 min and quantified by the Bradford method. Equal amounts of protein (10 μg/lane) were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blotted with the indicated antibodies. α-Rabbit or α-mouse antibodies conjugated to horseradish peroxidase were used as secondary antibodies, and signals were detected using ECL ^TM Western blotting detection reagent (Amersham).

Example 1-15. immunoprecipitation

Cells were dissolved in RIPA buffer and centrifuged at 12,500 RPM for 30 minutes. Proteins were immunoprecipitated with the indicated antibodies for 16 h at 4°C and then incubated with protein G Sepharose 4 Fast Flow (GE Healthcare) for 2.5 h at 4°C. Afterwards, the beads were washed twice in RIPA buffer and twice in PBS. Immunoprecipitated proteins were analyzed by SDS-PAGE, transferred to PVDF membrane, and analyzed by immunoblotting with specific antibodies.

Example 1-16. Cycloheximide tracking experiment

Cells were seeded overnight in complete medium in 6-well plates (1 × 10 ⁴ cells/wall), and then 2.5 μg/ml Cycloheximide (CHX) was added. Samples were then harvested at the indicated time points for immunoblot analysis.

Example 1-17. Generation of CRBN Knockout HEK293T cells by CRISPR/Cas9

CRBN CRISPR/Cas9 KO plasmid and CRBN HDR plasmid (sc-412142) were purchased from Santa Cruz and CRBN was knocked down 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). Puromycin was used to select cells with successful Cas-9-induced DSBs (DNA-containing double-strand breaks).

Example 1-18. stress paradigm

In the present invention, 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 three times. Specifically, the CUMS paradigm consisted of three daily exposures to one of the following aversive stressors: stroboscopic illumination (6 flashes of light/sec for 1 hour), cage tilting (30° position). , soiled cages (water was poured on the beds to wet the bedding), paired housing (two rats unfamiliar to each other were paired in each grouping session and the occupants were switched), loud noises (random noise generator (dB level <80; frequency 80-300 Hz) for 1 h), confinement (limiting the space available for movement by introducing sheets of cardboard along the width of the cage), and inverted light cycle (normal room lighting turned off during the day and turned on at night). Stressors were presented in a random, unpredictable order (see Table 1 below).

Example 1-19. Stereotactic Injections of okadaic acid (OA) into the mouse brain.

Stereotactic surgery was performed as follows. Mice were anesthetized with 6 130 nl of 100 μM OA (Sigma) dissolved in DMSO or DMSO alone was injected unilaterally in the lateral tonsil at an infusion rate of 60 nl/min (bregma coordinates: anterior/posterior -1.94, medial/lateral -3.15, dorsal/ ventral -4.5). Then, the needle was kept inserted for 5 minutes and then slowly withdrawn and the scalp was tightly stitched. 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 yield the region shown in Figure 6A, and snap frozen in liquid nitrogen.

Examples 1-20. statistical analysis

Data are presented as mean ± standard deviation (SD); P-values were calculated using an unpaired two-tailed Student's t-test in Microsoft Excel. P <0.05 was considered significant.

<Example 2: Identification of Hsp70 and its co-chaperone as endogenous substrates of CRBN>

To identify only CRBN-related substrates, we hypothesized that endogenous substrates of CRBN might be overexpressed in Crbn ^−/− mice and cell lines. To substantiate this hypothesis, we performed quantitative mass spectrometry of whole brain lysate (WBL) from Crbn ^−/− and Crbn ^+/+ mice. 3 Crbn ^−/− mice vs. Through experiments with Crbn ^+/+ mice, dozens of proteins with higher spectra (average > 2 times higher) were discovered. The most differentially expressed proteins are listed in Table 2 below.

Based on LC-MS/MS analysis of WBL (Crbn ^-/- Vs Crbn ^+/+ ), Hsp70 and co-chaperones DJ1 and DJ2 were predicted to be novel endogenous substrates of CRBN. Through pull-down analysis, we confirmed that Hsp70, DJ1, and DJ2 interact with CRBN in both endogenous and exogenous systems (Figures 1a and 9a). When the expression of CRBN was suppressed using CRBN-specific siRNA, the incorporation of HA-tagged ubiquitin into endogenous Hsp70, DJ1 and DJ2 was greatly reduced, indicating that DJ1 and others were degraded by CRBN-mediated ubiquitination.

Additionally, ^Flag CRBN increased co-precipitated endogenous chaperones upon in vitro ubiquitination when recombinant E1, E2, ubiquitin, and ATP were enriched (Figure 1e, line 5), and addition of methylated ubiquitin inhibited ubiquitination. (Figure 1e, line 4). Because thalidomide does not interfere with the ubiquitination of the chaperone, the association of the chaperone with IMiD was not studied further (Figure 9b).

Next, we tested whether CRBN knockout promoted chaperone accumulation in cells. Using WT-MEF and Crbn ^−/− MEF cells, we observed delayed degradation of Hsp70, DJ1 and especially DJ2 in the presence of cycloheximide (CHX) (Figure 1f), and these chaperones were activated in Crbn ^−/− upon treatment with MG132. accumulated in MEF cells (Figure 1g). These results indicate that Hsp70, DJ1, and DJ2 are novel endogenous substrates of ^CRL4 CRBN.

<Example 3: Effect of CRBN and DJ2 on heparin-mediated aggregation of tau>

Joint action of human Hsp70 and DJ1, but not DJ2, results in an effective reversal of α-synuclein fibril formation. This, in partnership with Hsp70, exhibits a well-regulated substrate-specific preference among J-proteins. Since the role of DJ1 and DJ2 in reducing the aggregation of α-synuclein has been studied, the present invention focused on their role in reducing the aggregation of the microtubule-associated protein tau (MAPT or tau).

First, in vitro aggregation assays were performed with full-length tau and truncated tau (repeat domain of tau or K18) in the presence of different combinations of chaperones and CRBN. Aggregation was induced by heparin and dithiothreitol (DTT) and assessed using thioflavin T (ThT)-fluorescence analysis and transmission electron microscopy (TEM) analysis (Figures 2a to 2d). Incubation of tau with the chaperone reduced aggregation, as evidenced by reduced binding of ThT dye, and Hsp70/DJ2 was more effective than Hsp70/DJ1 (Figure 2a). Interestingly, Hsp70 alone was not able to remove tau aggregation as efficiently as DJ2, indicating that DJ2 prevents aggregation by stabilizing the original form of tau.

Therefore, co-immunoprecipitation analysis was performed to confirm the binding of DJ2 to tau (Figure 9c). The binding of tau to DJ2 not only stabilizes the native form of tau, but can also limit the reaction with tau kinase in vivo, reducing phosphorylation and aggregation of tau. Meanwhile, removal of DJ2 by CRBN reduced the activity to prevent tau aggregation (Figure 2a), and it was confirmed that DJ2 effectively removes the aggregation of full-length tau, similar to K18, which aggregates faster than full-length tau (Figure 2b).

Previously, DJ2 was shown to have the function of stabilizing the native form of tau. Therefore, in vivo and in vitro studies were performed to confirm the effectiveness of DJ2 in eliminating tau aggregation and whether it is interfered with by CRBN. Tauopathies are characterized by significant accumulation of fibrillar tau inclusions in the CNS. However, extracellular tau plays an influential role in trans-cell proliferation. Extracellular monomers and seed-competent tau enter cells and aggregate strongly. To observe the effect of DJ2 on the toxicity of extracellular tau, heparin-induced K18 aggregates were treated with or without DJ2 for 48 h and added outside SH-SY5Y cells. Extensive cell death was observed in SH-SY5Y cells treated with K18 aggregates. However, K18 co-cultured with DJ2 prior to heparin-induced aggregation showed no cytotoxicity, as visualized through microscopy and MTT assay (Figure 2d). Interestingly, CRBN dissociated DJ2 from tau and interfered with the chaperone activity of DJ2. Relatively large fibrillar substances play an important role in the development of toxic effects of neurofibrillary tangles (NFTs), as they can accumulate inside neurons and cause direct physical interference with axonal movement. These results mean that while DJ2 interferes with tau aggregation, CRBN reduces the effect of DJ2 on interfering with tau aggregation through ubiquitination of DJ2.

<Example 4: C-terminal extension of DJ2 in CRBN-dependent ubiquitination>

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 (Figures 3a and 3b). Pull-down analysis showed that a stretch of ∼80 amino acids at the N-terminus of CRBN (Lon-N) interacted with a range of ∼100 amino acids at the C-terminus of DJ2 (Figure 3c and 3d). To pinpoint the sequences in DJ2 and CRBN that are important for the interaction, we constructed additional deletion mutants of both proteins, which contain a ß-turn that binds ß3 and ß4 of CRBN and a span of 40 amino acids from the C-terminus of DJ2. showed inability to form the -ß loop (Figures 10a to 10d).

We then analyzed several structural models of DJ2 docked to CRBN using the 3D coordinates of all atoms defined using ClusPro26, a fully automated algorithm for protein-protein docking. The crystal structure of CRBN (4TZ4) and the PDB file of the structural model of DJ2 generated by the I-Tasser27 server were used. Based on computer docking data as well as in vitro co-IP data, the crystal structure of CRBN (4TZ4) and the PDB files of the structural model of DJ2 generated by the I-Tasser27 server were used. Based on in vitro co-IP data and computational docking data, we manipulated six amino acids (T279-T284) of DJ2 and two amino acids (E152A, F153A, E152A/F153A) of CRBN, individually or in combination, to Mutants were constructed (Figure 10e). In the case of CRBN, binding affinity decreased when E152 and F153 were mutated to alanine (Figure 3e). Notably, all mutants of DJ2 retained binding to CRBN except D281A, N282A, R283A and T284A, indicating that these residues are involved in the binding of DJ2 to CRBN (Fig. 3f).

<Example 5: Confirmation of major ubiquitination sites of DJ2>

Because DJ2 is ubiquitinated by transfection of exogenous ubiquitin (Figure 1D), we sought to identify lysine (Lys or K) residues targeted by ^CRL4 CRBN for endogenous ubiquitination. According to PhosphoSitePlus28, a comprehensive database of experimentally observed mammalian post-translational modifications, Lys residues of DJ2 that undergo ubiquitination mainly accumulate in the J-domain and C-terminal domains (Figures 11A and 11B). These Lys residues were individually replaced with arginine (Arg or R) and ubiquitination analysis was performed. K32R and K350R mutants showed impaired ubiquitination (Figure 11c) and delayed degradation in CHX tracking assays (Figures 4a and 4b). The double mutant K32R/K350R had almost completely inhibited ubiquitination compared to WT-DJ2 (Figures 4A-4C). This means that K32 and K350 are the main ubiquitination sites of DJ2.

<Example 6: Confirmation of the effect of CRBN reduction on mild stress and tau pathology>

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. In Crbn ^+/+ mice, CUMS caused marked hyperactivity along with decreased feeding-related behavior. However, in Crbn ^−/− mice, these behavioral changes were prevented 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. Surprisingly, tau phosphorylation was significantly reduced in the brains of Crbn ^{−/− mice compared with Crbn +/+} mice, and DJ2 levels were increased in Crbn ^−/− mice (Figures 5A and 5B).

To verify whether the decrease in tau phosphorylation was a result of increased chaperonic activity of DJ2 alone or if the activity of the kinase targeting the tau protein was also impaired, the active form of tau kinase was further measured. As a result, it was confirmed that phosphorylation at Ser9 and Ser21 of glycogen synthase kinase 3 (GSK-3) was significantly increased in Crbn ^-/- WBL, thereby suppressing GSK-3α / β (Figure 5c). Likewise, the kinase activities of ERK1/2 and p38 were found to be reduced by decreased their phosphorylation (Figure 5c). Therefore, the improved co-chaperone activity and impaired tau kinase activity in Crbn ^−/− mice may together not only affect tau phosphorylation but also facilitate coping with stressors. 2D-PAGE analysis of WBL from Crbn ^-/- and Crbn ^+/+ mice revealed significantly increased levels of various antioxidant proteins, including thioredoxin, peroxiredoxin, and superoxide dismutase (see Table 4 below), which was consistent with stress. We demonstrate the characteristics of Crbn ^-/- mice that are resistant to factors (Figure 12).

<Example 7: Confirmation of the effect of CRBN reduction on tau-tau interaction and prion-like diffusion>

Local administration of okadaic acid (OA) can immediately induce phosphorylation and aggregation of tau at anatomically distal sites by inhibiting protein phosphatase 2A (PP2A). The prion-like spread of tau can be confirmed by analyzing the phosphorylation status of tau in various anatomical regions of the brain. In the present invention, we used the above strategy to further verify the response of Crbn ^−/− mice to tau phosphorylation and the spread of tau phosphorylation to other regions of the brain. OA was stereotactically injected into Crbn ^−/− and Crbn ^+/+ brains (M & M) and tau phosphorylation levels in the injected and non-injected hemispheres were analyzed ( Fig. 6A ). Although we observed a substantial increase in tau phosphorylation at multiple sites (e.g., pT181, pT231, and pS403/404) in Crbn ^+/+ brains, we found that tau phosphorylation was much less advanced or significantly prevented in Crbn ^−/− brains. (FIGS. 6b and 13).

Additionally, OA-induced inhibition of GSK3α/β was more pronounced in the WBL of Crbn ^−/− than Crbn ^+/+ mice (Figure 14). Inhibited tau phosphorylation was also observed when the CRBN locus was disrupted by CRISPR/Cas9 in HEK293 cell lines overexpressing Myc-tau (Figures 7A and 7B). Hsp70 has an inverse relationship with tau (as do Hsp70 and co-chaperones) in the Crbn ^+/+ HEK293 cell line overexpressing Myc-tau. Interestingly, chaperone levels were not reduced in CRISPR/Cas9-derived Crbn ^−/− cell lines (where GSK3α/β was inhibited by OA treatment).

Tau hyperphosphorylation leads to pathological tau aggregation. Venus-based BiFC technology was used to investigate the effect of CRBN on tau aggregation. SHSY5Y cells were transiently transfected with siRNA _CRBN (or control scrambled siRNA), with subsequent co-transfection of tau-BiFC constructs. Although OA-induced hyperphosphorylation increased tau assembly, resulting in activation of Venus fluorescence as expected, this effect was greatly reduced by CRBN inhibition (Figure 7c).

These results imply that the absence of CRBN induces resistance to the aggregation and proliferation of pathological tau.

<Example 8: Confirmation of suppressed activity of DJ2 in neuropathy murine model>

The roles of CRBN and DJ2 in disease development and progression in a mouse model of neuropathy were studied. Amyloid-β (Aβ) plaques are one of the triggers of tauopathy because they promote tauopathy by increasing the spread of pathological tau.

To determine whether CRBN is a tauopathy-inducing factor, brain samples from 8-month-old 5X-FAD and APP ^NL-GF knock-in mice exhibiting late-stage tauopathy were analyzed. APP antibody immunostaining confirmed the presence of Aβ plaques in brain samples from 5XFAD and APPNL-GF knock-in mice (Figure 15a).

Additionally, the hippocampus of an 8-month-old mouse was immunolabeled with CRBN and DJ2 antibodies. Elevated expression of CRBN was observed in the hippocampus and cortical regions of 5X-FAD and APP ^NL-GF knock-in mice, as confirmed by immunohistochemistry and Western blot (Figures 15b and 15c). On the other hand, the chaperone level was low at the same location (Figure 15c). These results imply that elevated CRBN is a biomarker for neurodegeneration. Therefore, CRBN-mediated attenuation of chaperone activity may be an additional neurotoxic mechanism in Alzheimer's disease.

<Example 9: Confirmation of inhibition of interaction between CRBN and DJ2 by the peptide of the present invention>

We attempted to design peptide inhibitors based on the CRBN-DJ2 interaction and the hotspot region of DJ2 that interacts with CRBN. The peptide was circularized by adding cysteine residues to both ends of the peptide (Figure 15d). This was done to obtain the conformation required for the interaction of CRBN with the DJ2 loop (Fig. 10e). As a result of varying the concentration of the designed peptide from 0 to 30 μM, treatment with a 10 μM concentration of peptide inhibitor completely blocked the interaction between CRBN and DJ2, as shown in the CoIP analysis results (FIG. 15e).

Chemical knockdown of CRBN may not be a desirable strategy for cells to maintain recruitment and ubiquitination of endogenous substrates other than DJ2. Therefore, utilizing peptides based on hotspot residues can mask CRBN, inhibit the ubiquitination of DJ2 by interfering with the interaction of CRBN with DJ2, and are useful for selective inhibition of pathological tau phosphorylation.

<Example 10: Confirmation of CRBN and DJ2 binding inhibition ability using linear and circular peptides>

Considering the above results of CRBN-DJ2 interaction, linear (SEQ ID NO: 1) and circular (SEQ ID NO: 3) peptides were designed based on the hotspot region of DJ2 that interacts with CRBN. In the case of circularity, the peptide was circularized by adding cysteine residues to both ends of the linear peptide. As a result of immunoprecipitation using the above peptide, both types of peptides showed significant inhibition of CRBN and DJ2 binding, as shown in Figure 16.

<Example 11: Confirmation of CRBN and DJ2 binding inhibition ability using circular peptide and its scrambled peptide>

In order to prove that the amino acid sequence of the peptide of the present invention has the ability to inhibit CRBN and DJ2 binding, immunoprecipitation was performed by preparing a scrambled peptide in which the peptide of SEQ ID NO: 3 and the amino acids constituting the peptide of SEQ ID NO: 3 were mixed in random order.

Specifically, SH-SY5Y cells were transiently co-transfected with Myc-DJ2 and FLAG-CRBN. Cell extracts were treated with scrambled peptide or peptide of SEQ ID NO: 3 for 24 hours. Cell extracts were immunoprecipitated with α-FLAG antibody from rabbit (Rb) and immunoblotted with Myc and FLAG antibodies from mouse (Ms) to remove bands of IgG heavy chains. Data were expressed as mean ± SD. n = 5. Student's t test was used.

As a result, as shown in Figures 17a to 17d, it was confirmed that the circular peptide (SEQ ID NO: 3, DJ2 peptide in Figure 17b) inhibited the binding of CRBN and DJ2 better than the scrambled peptide.

<Example 12: Confirmation of the ability of tetrapeptide to inhibit CRBN and DJ2 binding>

Since the most potent inhibitor is generally less than 500 Da, the experiment was repeated with a peptide covering the most important residue of the hotspot to reduce the size of the peptide inhibitor, and it was confirmed that the DNRT sequence (SEQ ID NO: 2) was critical (see FIG. 18). Accordingly, DNRT peptide and its scramble peptide were prepared and their ability to inhibit CRBN and DJ2 binding was confirmed.

Specifically, SH-SY5Y cells were transiently co-transfected with Myc-DJ2 and FLAG-CRBN. Cell extracts were treated with scrambled peptide or peptide of SEQ ID NO: 2 for 24 hours. Cell extracts were immunoprecipitated with α-Myc antibody from mouse (Ms) and immunoblotted with FLAG and Myc antibodies from rabbit (Rb). Data were expressed as mean ± SD. n = 5. Student's t test was used.

As a result, it was confirmed that a relatively higher concentration of ~30 μM tetra peptide significantly blocked the interaction between CRBN and DJ2, as seen in CoIP analysis, but the scrambled peptide could not (see FIGS. 19A to 19C).

<110> Gwangju Institute of Science and Technology <120> CRBN binding peptide and composition for preventing or treating Alzheimer's disease using the same <130>P210363P <150> KR 10-2020-0046909 <151> 2020-04-17 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Sequence No. One <400> 1 Pro Ile Ser Thr Leu Asp Asn Arg Thr Ile Val 1 5 10 <210> 2 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Sequence No. 2 <400> 2 Asp Asn Arg Thr One <210> 3 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Sequence No. 3 <400> 3 Cys Pro Ile Ser Thr Leu Asp Asn Arg Thr Ile Val Cys 1 5 10

Claims (6)

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.
A pharmaceutical composition for preventing or treating Alzheimer's disease, comprising the peptide of claim 1.
The pharmaceutical composition according to claim 2, wherein the Alzheimer's disease is caused by tau phosphorylation.
A food composition for preventing or improving Alzheimer's disease, comprising the peptide of claim 1.
The food composition according to claim 4, wherein the Alzheimer's disease is caused by tau phosphorylation.
A method for screening a therapeutic agent for Alzheimer's, comprising the following steps:
a) synthesizing a peptide containing a CRBN binding motif represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3;
b) analyzing whether the peptide synthesized in step a) can bind to the DJ2 binding site of CRBN and inhibit the binding of CRBN and DJ2; and
c) determining that the peptide synthesized in step b) is a treatment for Alzheimer's disease if it inhibits the binding of CRBN and DJ2.