CN111358778A - Application of MCC950 in preparation of medicine for preventing or treating Alzheimer disease - Google Patents

Abstract

The invention relates to the application of MCC950 in the preparation of medicines for preventing or treating Alzheimer's disease, and belongs to the technical field of medicine preparation. The medicine prepared by MCC950 can inhibit the expression of amyloid precursor protein secretase and inhibit the aggregation of β-amyloid, so as to realize the prevention or treatment of Alzheimer's disease.

Description

Application of MCC950 in the preparation of drugs for preventing or treating Alzheimer's disease

technical field

The invention relates to the technical field of drug preparation, in particular to the application of MCC950 in the preparation of drugs for preventing or treating Alzheimer's disease.

Background technique

Alzheimer's Disease (AD) has a long course of disease and brings a huge economic burden to the patients themselves, their families and the society. For the pathogenesis of AD, there are currently β-amyloid (Aβ) cascade hypothesis and Tau protein hyperphosphorylation hypothesis. However, until now, the exact etiology and mechanism of AD are not well understood.

Currently FDA-approved drugs for AD treatment include: cholinesterase inhibitors, tacrine (Shinogi), donepezil (Eisai), rivastigmine (Novartis), galantamine (Johnson &Johnson); stimulant Amino acid receptor inhibitor: Memantine (Mermandarin), and the combination therapy of donepezil and memantine was approved in 2014. In addition to the above five domestically approved therapeutic drugs, there are cerebrolysin (cerebral protein hydrolyzate) similar to nerve growth factor, cholinesterase inhibitor Huperzine A, and nicergot that dilates blood vessels and enhances nerve conduction. Forest.

At present, AD therapeutic drugs that have entered clinical research mainly focus on three directions: tau protein antibody, β-amyloid protein antibody and BACE1 inhibitor, such as Eli Lilly's Solanezumab, Pfizer and Johnson & Johnson's monoclonal antibody Bapineuzumab and Roche's Gantenerumab All of them failed in phase III clinical trials, and there was no significant improvement in the cognitive dysfunction of the patients.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide the application of MCC950 in the preparation of medicaments for preventing or treating Alzheimer's disease. MCC950 and the prepared medicine can inhibit the expression of amyloid precursor protein secretase and inhibit the aggregation of β-amyloid, so as to realize the prevention or treatment of Alzheimer's disease.

The invention provides the application of MCC950 in the preparation of medicaments for preventing or treating Alzheimer's disease.

The present invention also provides the use of MCC950 in the preparation of a medicine for reducing β-amyloid cleavage.

The invention also provides the application of MCC950 in the preparation of medicines for inhibiting the aggregation of beta-amyloid.

The present invention also provides the application of MCC950 in preparing a medicine for inhibiting the expression of amyloid precursor protein secretase.

Preferably, the amyloid precursor protein secretase comprises a beta-secretase and/or a gamma-secretase complex.

The present invention also provides the application of MCC950 in the preparation of a medicine for reducing the expression level of amyloid precursor protein and/or amyloid precursor carbon-terminal fragment protein.

The invention provides the application of MCC950 in the preparation of medicaments for preventing or treating Alzheimer's disease. MCC950 can inhibit β-secretase BACE1, γ-secretase complex (PS, NCT), amyloid precursor protein (amyloid precursor full-length protein, APP full length, fl-APP), amyloid precursor carbon The expression level of APP C-terminal fragments (APP-CTFs) protein, so MCC950 can reduce the cleavage of Aβ by specifically inhibiting the expression of amyloid precursor protein and its related secretase; MCC950 can inhibit the expression of Aβ Aggregation, thereby reducing the formation of senile plaques and alleviating the pathological process of AD. The test results showed that, as verified by Western Blot, when 50nM MCC950 was administered to Sw cells, β-secretase BACE1, γ-secretase complex (PS, NCT), amyloid precursor protein (fl-APP), amyloid precursor The protein expression level of carbon-terminal fragments (APP-CTFs) was significantly decreased. Verified by SDD-Age, 5μM and 50μM MCC950 can significantly inhibit the aggregation of Aβ.

Description of drawings

Fig. 1 is the molecular mechanism schematic diagram that MCC950 provided by the present invention improves AD pathological process;

2 is a schematic diagram of AD-related targets provided by the present invention;

3 is a schematic diagram of MCC950-related targets provided by the present invention;

Fig. 4 is the MCC950 target point provided by the present invention and the schematic diagram of the existing interaction gene network;

Fig. 5 is the common potential target Venn diagram of MCC950 and AD provided by the present invention;

Fig. 6 is the biological process diagram that MCC950 provided by the present invention and AD molecular target participates;

Fig. 7 is the signal pathway diagram that MCC950 provided by the present invention and AD molecular target participate in;

Figure 8 is a statistical diagram of the protein expression level and relative protein expression level of APP amyloid metabolic pathway-related secretase and APP digestion products provided by the present invention;

Figure 9 is a graph showing the results of Aβ protein aggregation provided by the present invention.

Detailed ways

The invention provides the application of MCC950 in the preparation of medicaments for preventing or treating Alzheimer's disease. Through the gene screening and biological process analysis of MCC950 and AD in the present invention, it is found that the biological processes with high correlation with MCC950 and AD mainly include: protein modification, mitochondrial regulation, cytokine production, programmed cell death, DNA damage, oxidative stress Figure 1 is a schematic diagram of the molecular mechanism of MCC950 provided by the present invention to improve the pathological process of AD. There are two distinct pathways for APP cleavage (Figure 1), the non-amyloid pathway and the amyloid pathway. Non-amyloid pathway: APP is cleaved by α-secretase to generate an 83 amino acid C-terminal fragment (C83) that remains in the membrane and an N-terminal extracellular domain (sAPPα) that is released into the extracellular matrix. C83 can be further cleaved by γ-secretase, resulting in a short fragment called "P3 peptide" and a C-terminal fragment (CTF). Amyloid pathway: APP is cleaved by β-secretase (BACE1) to produce a C-terminal fragment (C99) of 99 amino acids, which remains in the membrane. This fragment (between residues 38 and 43) continues to be cleaved by γ-secretase, releasing the neurotoxic Aβ peptide. Gamma-secretase is an enzymatic complex consisting of presenilin 1 or 2 (PS1 and PS2), natinin, propharyngeal defect protein (APH-1), and presenilin enhancer 2 (PEN2).

The cleavage and aggregation of Aβ are the main pathological features of AD. MCC950 can reduce the cleavage of Aβ and inhibit its aggregation, which provides a sufficient theoretical basis for the development of MCC950 as a drug for alleviating the pathological characteristics of AD.

The present invention also provides the use of MCC950 in the preparation of a medicine for reducing β-amyloid cleavage.

The invention also provides the application of MCC950 in the preparation of medicines for inhibiting the aggregation of beta-amyloid.

The invention also provides the application of MCC950 in the preparation of inhibiting the expression of amyloid precursor protein secretase. In the present invention, the amyloid precursor protein secretase includes β-secretase and/or γ-secretase complex. The present invention shows through specific experiments that MCC950 can inhibit β-secretase (β-site precursor-like protein cleaving enzyme-1, Beta-site amyloidprecursor protein cleaving enzyme-1, BACE1), γ-secretase complex (PS, NCT) protein expression level. Therefore, MCC950 can reduce the cleavage of Aβ by specifically inhibiting the expression of amyloid precursor protein secretase.

The present invention also provides the application of MCC950 in preparing the expression level of amyloid precursor protein and/or amyloid precursor carbon-terminal fragment protein. The present invention shows through specific experiments that MCC950 can inhibit the expression levels of amyloid precursor protein (fl-APP) and amyloid precursor carbon-terminal fragments (APP-CTFs). APP is cleaved by the non-amyloid pathway (α-secretase) or the amyloid pathway (β-secretase) to generate CTF-83 (C83) or CTF-99 (C99), respectively, and further by γ-secretase, respectively. P3 peptide or Aβ, the expression levels of the two CTF fragments were reduced, and the expression levels of APP were also significantly reduced, indicating that APP production and cleavage were reduced.

The present invention selects SW cells as a research model, detects the influence of MCC950 on the expression of amyloid precursor protein and its related secretase, and studies the molecular mechanism of MCC950 participating in the process of Aβ cleavage and aggregation. Drugs for the treatment of sexually transmitted diseases provide sufficient theoretical basis.

The application of MCC950 of the present invention in the preparation of drugs for preventing or treating Alzheimer's disease will be further described in detail below with reference to specific examples. The technical solutions of the present invention include but are not limited to the following examples.

Example 1

Bioinformatics analysis of MCC950 and AD-related targets

(1) Virtual screening of MCC950 and AD targets: The PharmMapper workstation was used to screen the targets of MCC950, based on the "pharmacodynamic characteristic elements" that play an important role in the activity of the active molecules and their spatial arrangement, namely Pharmacophore conducts virtual screening of MCC950 targets. For the screening of AD targets (Figure 2, schematic diagram of AD-related targets), the Therapeutic Target Database (TTD), GeneticAssociationDatabase (GAD), PharmGkb and DiGSeE databases were used to search for AD-related targets and perform statistical analysis. The DIP, BIDGRID, HPRD, INTACT, MINT, and BIND databases in the Bisogenet plugin in Cytoscape were used to perform virtual screening for proteins that interacted with MCC950 targets (Figure 3, schematic diagram of MCC950-related targets) to obtain MCC950 targets. The interacting protein network (Fig. 4, the schematic diagram of MCC950 target and existing interacting gene network), namely the (ProteinProtein Interaction, PPI) network.

(2) Analyze the biological process of the common targets of AD and MCC950 obtained by statistical analysis, and draw a Venn diagram (Fig. 5, Venn diagram of common potential targets of MCC950 and AD). Using the GO BiologicalProcessGOA in Cytoscape to analyze the relevant biological processes (Figure 6, the biological process map involved in MCC950 and AD molecular targets), the results include programmed cell death, protein modification, development of the nervous system, regulation of mitochondria, metal ion stabilization state and other biological processes.

The analysis results are shown in the figures (Figures 2 to 7). There are 3238 target genes of AD and 115 target genes of MCC950. The protein interaction network prediction of the target genes was carried out, and a total of 4117 genes that interacted with the target genes were obtained. Statistical analysis was carried out between the genes that interacted with AD and AD target genes, and a Venn diagram was drawn to obtain 1105 genes in common, accounting for 17.7% of the total number of genes, signaling pathways that were highly correlated with MCC950 and AD (Figure 7, MCC950 and the signaling pathways involved in AD molecular targets) mainly include protein modification, programmed cell death, cytokine production, and nervous system development.

Example 2

MCC950 affects the expression levels of amyloid precursor protein metabolic secretase and amyloid precursor protease cleavage product (CTF)

SW cell protein extraction:

The SW was inoculated into a six-well cell culture plate at a density of 5×10 ⁵ cells/well, and after culturing for 24 h until the cells adhered to the wall, the fresh serum-free medium was replaced, and DMSO and MCC950 were added respectively at a dose of 10 nM and 50 nM, and then the cells were placed Incubate at 37°C for 24h in a 5% _CO2 incubator.

Use a negative pressure device to aspirate the culture in the six-well petri dish, rinse the cells once with PBS, add 0.5 mL of cell lysate to each well, and pipet repeatedly until it becomes thick, then add it to the corresponding numbered EP tube, 98-99°C Heat in a water bath for 5-10 min, and store at -80°C.

Use a negative pressure device to aspirate the culture in the six-well petri dish, rinse the cells once with PBS, scrape the cells with a cell scraper, add them to the corresponding numbered EP tube, and put in 100 μL of lysis solution (1/25, V/ V, MPER/PMSF), ice bath, vortexed once for 10 min for 1 h, centrifuged at 14000 rpm for 15 min at 4°C, pipet the supernatant into the corresponding numbered EP tube, use the BCA method to determine the concentration, after aliquoting, 98-99 °C water bath. Heated for 5-10min and stored at -80℃.

Determination of protein concentration by BCA method:

Add 15 μL of Nuclease-Free H ₂ O to wells A1-A8 of the 96-well plate, add 15 μL of BSA solution to the A1 position of the 96-well plate, and after thorough mixing, pipette 15 μL of the mixture into well A2, and so on to well A8 . Add 5 μL from wells A1-A8 to wells B1-B8 respectively. Add 5 μL of the sample to be tested to each well of C1-C8 of the 96-well plate in turn, add the mixture of solution A and solution B (1/49, V/V, B/A), mix thoroughly, and place in a 37°C incubator Incubate for 30 min, and measure the absorbance (OD) of the sample at a wavelength of 570 nm with a multifunctional microplate reader. According to the relationship between the concentration gradient of the BSA standard solution and the absorbance, the concentration standard curve is obtained, and the concentration of the sample to be tested can be obtained by calculation. Generally, the total protein of the Western blot sample is 4-8 μg/well.

WesternBlot:

Use a 1.0mm board, pay attention to the alignment clamp. Prepare the lower layer separating gel, adjust the corresponding lower layer gel concentration according to the molecular weight of the sample protein, add deionized water to level the glue surface, and after the lower layer gel solidifies, pour out deionized water, add a layer of stacking gel, fill it up, and insert a comb. After the upper layer of gel has solidified, take out the gel plate and put it into the electrophoresis tank, pour 1×RunningBuffer into the middle, shake it from side to side, and carefully remove the air bubbles. To ensure that there is no leakage, the red line is enough. Pull out the comb in parallel, add 20 μL of the protein to be tested into each well of a microsyringe, dilute the marker with 1:4, and fill the blank wells with LoadingBuffer. The voltage of the sample when running the stacking gel electrophoresis is 90V, and the voltage is raised to 110V when entering the separating gel, and the electrophoresis is stopped when the blue Loading line runs to the bottom of the gel.

Before transferring the membrane, fully soak the PVDF membrane in methanol, and then press the anode carbon plate, 1 piece of sponge, 2 pieces of filter paper, 1 piece of PVDF membrane, gel, 2 pieces of filter paper, 1 piece of sponge, Align the cathode carbon plates in order, put them in the electrophoresis tank, turn on the power supply, constant current 90V, transfer the membrane for 2-3 hours, and adjust the time required for the transfer according to the molecular weight of the target protein. After transferring the membrane, take the transfer device out of the electrophoresis tank, select the membrane with the molecular weight of the target protein according to the instructions of the Marker strip, mark it, put it in the incubation box, put it on the shaker, and wash it with 1×TBST. 1 time, discard 1×TBST, block with 5% skim milk at room temperature for 1 h, discard the blocking solution, wash 3 times with 1× TBST, 5 min each time, add primary antibody (1:3000, V/V, primary antibody: 1× TBST, the side of the PVDF membrane that is close to the adhesive surface is placed upward, the mixture must be completely immersed in the PVDF membrane), and incubated at room temperature on a shaker for 2h or 4°C overnight. Recover the primary antibody, wash it with 1×TBST for 5 min×3 times, add the secondary antibody conjugated with horseradish peroxidase (1:3000, V/V, secondary antibody: 1×TBST, the side close to the adhesive surface) The PVDF membrane is placed upward, and the mixture must be completely immersed in the PVDF membrane) and incubated at room temperature for 1 h. The secondary antibody was recovered and washed with 1×TBST for 5 min×6 times. The luminescent solution was added dropwise, and luminescence was performed on the gel imaging analysis system. All Western blot experiments were repeated at least 3 times, and each experiment was performed using a different batch of cells with the same treatment. β-actin was used as the control group.

Marker: 10kDa, 15kDa, 25kDa, 35kDa, 40kDa, 55kDa, 70kDa, 100kDa, 130kDa, 170kDa.

Target proteins: β-actin, β-secretase BACE1, γ-secretase complex (PS, NCT), amyloid precursor protein (fl-APP), and amyloid precursor carbon-terminal fragments (APP-CTFs) proteins.

Figure 8 is a statistical chart of the protein expression level and relative protein expression level of APP amyloid metabolic pathway-related secretase and APP digestion products, wherein A is a Western Blot result chart of protein expression, and B is a relative protein expression statistical chart. Western Blot results showed that when SW cells were administered 50nM MCC950, β-secretase BACE1, γ-secretase complex (presenilin (PS), nicastrin (NCT)), amyloid precursor full-length protein (fl-APP), the protein expression level of amyloid precursor carbon-terminal fragments (APP-CTFs) was significantly decreased. Aβ is formed by the cleavage of amyloid precursor protein through the amyloid pathway, among which the related β-secretase BACE1, γ-secretase complex (PS, NCT), amyloid precursor protein (fl-APP), The protein expression level of amyloid precursor carbon-terminal fragments (APP-CTFs) was significantly reduced, thereby reducing the cleavage of Aβ.

Example 4

MCC950 can inhibit the aggregation of Aβ

In vitro investigation of Aβ aggregation:

Aβ (0.02 μmol) was dissolved in 100 μL of sterile deionized water, then 100 μL of HCl (10 mM), MCC950 (50 μM) and MCC950 (5 μM) were added, vortexed for 30 s, sealed with parafilm, and incubated at 37 °C for 8 d.

Preparation of sample buffer (Loading Buffer):

Take 10 mL of 1×TAE, add 10 mg of bromophenol blue, 0.4 g of SDS, and 1 mL of 10% glycerol, and mix well.

SDD-Age:

Prepare SDD-Age separation gel (1.5% Agarose+1×TAE+0.1%SDS), insert a comb, after the separation gel solidifies, take out the gel plate and put it into the electrophoresis tank, pour 1×TAE+0.1%SDS in the middle, and then pour it into the electrophoresis tank. Shake, be careful to remove air bubbles, make sure there are no leaks, and place the electrophoresis tank in an ice bath. Pull out the comb in parallel, and add 20 μL of the protein to be tested into each well of the microsyringe. When the sample enters the separation gel, adjust the current to 60mA, and stop electrophoresis when the blue Loading line runs to the bottom of the gel.

Before transferring the membrane, fully soak the PVDF membrane in methanol, and place the membrane transfer device in the order of 24 dry filter papers, 1 wet filter paper, 1 PVDF membrane, gel, and 3 wet filter papers from bottom to top. Flatten and transfer the membrane for 4-5h, and adjust the time required for the membrane transfer according to the molecular weight of the target protein. After the membrane transfer, take out the PVDF membrane, select the membrane with the molecular weight of the target protein according to the instructions of the Marker strip, mark it, put it in the incubation box, put it on the shaker, wash it once with 1×TBST, 5min, Discard 1×TBST, add primary antibody (1:3000, V/V, Aβ: 1×TBST, place the PVDF membrane on the side close to the adhesive surface upward, the mixture must completely submerge the PVDF membrane), shake at 4°C. Incubate overnight in bed. Recover the primary antibody, wash it with 1×TBST for 5 min×3 times, add the secondary antibody conjugated with horseradish peroxidase (1:3000, V/V, secondary antibody: 1×TBST, the side close to the adhesive surface) The PVDF membrane is placed upward, and the mixture must be completely immersed in the PVDF membrane) and incubated at room temperature for 1 h. The secondary antibody was recovered and washed with 1×TBST for 5 min×6 times. The luminescent solution was added dropwise, and luminescence was performed on the gel imaging analysis system. All SDD-Age experiments were repeated at least 3 times, and each time experiments were performed using different batches of Aβ with the same treatment. The Aβ fibrils were used as the control group.

Target protein: Aβ

Figure 9 is a graph showing the aggregation of Aβ protein, the results show that MCC950 can inhibit the aggregation of Aβ.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Claims (6)

1. Application of MCC950 in the preparation of a drug for preventing or treating Alzheimer's disease. 2. The use of MCC950 in the preparation of a drug for reducing β-amyloid cleavage. 3. The application of MCC950 in the preparation of a drug for inhibiting the aggregation of beta-amyloid. 4. The application of MCC950 in the preparation of a drug for inhibiting the expression of amyloid precursor protein secretase. 5. The use according to claim 4, wherein the amyloid precursor protein secretase comprises a β-secretase and/or a γ-secretase complex. 6. The use of MCC950 in the preparation of a medicament for reducing the expression level of amyloid precursor protein and/or amyloid precursor carbon-terminal fragment protein.

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