Purine compound and application thereof in preparation of drugs for preventing and treating brain aging

Abstract

The invention relates to purine compounds and application thereof in preparation of drugs for preventing and treating brain senescence, the screening efficiency is remarkably improved by combining structural biology and computer-aided drug design to quickly recognize candidate molecules strongly bound with AQP4 protein, a new method for AQP4 research is promoted, and reference is provided for drug screening of other related proteins. The binding force and affinity of the screened purine compound, namely the compound 61 and AQP4 protein, can be evaluated, so that more accurate targeted drug development can be realized, and the treatment effect can be improved. In addition, the compound 61 significantly enhances AQP4 polarity distribution, alleviates astrocyte senescence, and provides a new intervention target and a new drug design idea for delaying brain senescence and protecting brain health.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to purine compounds and their application in preparing drugs for preventing and treating brain aging. Background Art

[0002] Researching healthy aging and delaying the aging process is a common issue faced by the world. In the aging process, aging cells induce aging-related secretory phenotypes, which are manifested as the persistence of chronic low-grade inflammation in tissues, becoming a key risk factor for age-related neurodegenerative diseases such as Alzheimer's disease (AD). Removing aging glial cells in the brain can reduce brain aging and AD-related pathological changes and alleviate cognitive decline. The brain, as the most active organ in energy metabolism, is susceptible to aging. Astrocytes, as the most numerous glial cells in the brain parenchyma, are important maintainers of the homeostasis of the brain environment and are themselves prone to aging. Therefore, in-depth research on the aging mechanism of astrocytes and screening new potential compounds to slow down their aging are crucial to delaying brain aging and promoting brain health.

[0003] Aquaporin-4 (AQP4) is one of the most abundant water channel proteins expressed on the surface of astrocytes. It mainly has two subtypes, M1-AQP4 and M23-AQP4. The change in the ratio of these two subtypes leads to different supramolecular assembly states of AQP4 in the plasma membrane, namely, different orthogonal particle arrays. In particular, the M23-AQP4 subtype is closely related to the polar distribution of AQP4 on the terminal end feet of astrocytes. Previous studies have found that the disordered polar distribution of AQP4 leads to increased β-amyloid protein load in cortical neurons, astrocyte activation, and excitatory neuron activation, thereby causing hyperactive behavior in early APP / PS1 mice. In the brain edema model, reducing the membrane localization of AQP4 can effectively reduce astrocyte swelling. Minocycline reduces the activation and volume increase of astrocytes in the traumatic brain injury model, which also depends on the polar distribution of AQP4 at its end feet. In addition to the distribution of AQP4, the expression level of AQP4 is also indispensable for the maintenance of central homeostasis. AQP4 deficiency causes impaired drainage function of the glial glymphatic system, thereby exacerbating the accumulation of neurotoxic protein waste and neuroinflammatory response in neurodegenerative diseases such as AD and Parkinson's disease. The above studies show that AQP4 is a potential intervention target for nervous system-related diseases. In addition, studies have shown that the polarity distribution of AQP4 is closely related to the phenotype of astrocytes, and AQP4 depolarization is positively correlated with neurotoxic A1 astrocytes. This suggests that the polarity change of AQP4 is an important factor affecting the function and state of astrocytes.

[0004] Previous drug studies targeting AQP4 have achieved some results. In animal models of ischemic stroke, selective inhibition of AQP4 using AER-270 and AER-271 can reduce brain edema. The antipsychotic drug trifluoperazine can restore the ectopic distribution of AQP4, eliminate central nervous system edema, and accelerate functional recovery in rat spinal cord injury models. However, these drugs lack specific regulatory effects on AQP4 polarity and have limited neuroprotective effects in specific animal models.

[0005] Therefore, the present invention conducts virtual screening on the three-dimensional structure of mouse AQP4 protein, conducts molecular docking on a total of 33,600 compounds including the drug library listed by the U.S. Food and Drug Administration, and determines compound 61 as a candidate molecule that strongly binds to AQP4 after evaluating the binding force and affinity of the compound with mouse AQP4 purified protein. Further experiments found that compound 61 enhances the polarity of AQP4 and reduces astrocyte aging, providing a theoretical and experimental basis for the development of brain aging-related drugs. Summary of the invention

[0006] The inventors have discovered that purine compound 61 can be used to delay brain aging and prevent and treat related nervous system diseases.

[0007] The present invention provides a purine compound 61 represented by the following structural formula: .

[0008] The present invention also provides the use of purine compound 61 in preparing drugs for preventing and treating brain aging.

[0009] The dosage forms of drugs for preventing and treating brain aging are capsules, tablets, oral preparations, microcapsule preparations, injections, suppositories, sprays or ointments.

[0010] Pharmaceutically acceptable derivatives of purine compound 61 include solvates and salts, particularly acid addition salts such as hydrobromide.

[0011] The patient to be treated may suffer from a brain senescence disorder.

[0012] The present invention significantly improves the screening efficiency by quickly identifying candidate molecules that strongly bind to the AQP4 protein. Through the evaluation of binding force and affinity, compound 61 was accurately determined to be a candidate molecule that strongly binds to AQP4. Further experiments showed that compound 61 significantly enhanced the polarity of AQP4 and alleviated the aging of astrocytes, providing a practical theoretical and experimental basis for the research and development of brain aging-related drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is a flow chart of the targeted AQP4 virtual screening provided by the present invention; Figure 2 This is a graph showing the ranking of the affinity of the compounds to mouse AQP4 as shown in the affinity mass spectrometry experiment provided by the present invention; Figure 3 is the chemical structure diagram of the purine compound 61 provided by the present invention; Figure 4 It is a 3D diagram of the binding mode of the purine compound 61 provided by the present invention and the mouse AQP4 protein; Figure 5A is the dissociation constant in the surface plasmon resonance experiment provided by the present invention; Figure 5B The kinetic fitting diagram in the surface plasmon resonance technology experiment provided by the present invention; Fig. 6A This is a graph showing cell viability of aged astrocytes after 48 hours of treatment with the purine compounds provided by the present invention; Figure 6B This is a graph showing cell viability after aging astrocytes were treated with the compound provided by the present invention for 72 hours; Fig. 7A This is a flow chart of an in vitro experiment on the effect of compound 61 provided by the present invention on delaying astrocyte senescence; Figure 7B This is a Western blotting data diagram showing that compound 61 provided by the present invention delays astrocyte senescence. DETAILED DESCRIPTION

[0014] The invention is illustrated by the following examples.

[0015] like Figure 3 Shown is the structural formula of purine compound 61.

[0016] Screening method of purine compound 61 of the present invention: 1. Preparation of Mouse AQP4 Protein Structure and Compound Library The three-dimensional structure of mouse AQP4 protein was predicted using AlphaFold (AlphaFold ID: AF-P55088-F1), with a high overall confidence score and good structural reliability. The selected compounds were from MCE, which were a natural product analog library containing 13,200 compounds (Cat. No.: HY-L0107V) and 20,400 known active compounds (Cat. No.: HY-L001P).

[0017] 2. Affinity mass spectrometry Affinity verification experiment for affinity screening of mouse AQP4 protein and small molecule compounds. The experimental steps are as follows A. Preparation: The experiment was divided into two groups. The selected compounds were prepared into mixed solutions with a final concentration of 100 μM.

[0018] The concentration of mouse AQP4 protein is 100 μM. Prepare buffer: 20 mM Tris-HCl, pH 7.5.

[0019] B. Incubation: The two groups were: P+ group added with 5.5 μL compound mixed solution, 27.5 μL AQP4 protein, 517 μL Buffer; P- group added with 5.5 μL compound mixed solution, 27.5 μL PBS Buffer, 517 μL PBS Buffer. Incubated on ice for 0.5 h.

[0020] C.Ultrafiltration: 450 μL of the P+ and P- groups were taken as the R+ group, and 50 μL was taken as the R- group. The R+ group was ultrafiltered twice at 25°C and 13200 rpm.

[0021] D. Dissociation: After ultrafiltration, the R+ group sample was fixed to 50 μL. 200 μL of acetonitrile was added to the P+R+, P+R-, P-R+, and PR- groups respectively and mixed.

[0022] E. After 15 min of dissociation, centrifuge each group of samples for 10 min, and take the supernatant as the "dissociation 15 min group" sample and inject it into LC-MS.

[0023] F. Data processing: Use the P+R-group to determine the exact mass and retention time of all compounds and save the analytical method. Take the peak area data of each compound in the P+R-group sample. The relative content calculation formula of the compound that forms a complex with the protein is: B=A(P+R+) / A(P+R-)-A(P-R+ ) / A(PR-), Where A(X) refers to the chromatographic peak area of ​​small molecules in the sample marked as "X". The final result is expressed in the form of B*100.

[0024] 3. Surface Plasmon Resonance A. Prepare the sensor chip: Select a suitable sensor chip, usually a glass piece coated with a metal such as gold or silver. Clean the chip to remove impurities and contamination.

[0025] B. Functionalized Chip: The protein was fixed by amino coupling method, and the protein coupling buffer solution was 1.0× PBS-P+ (pH 7.4).

[0026] C. Prepare samples: The interaction buffer solution was 1.0× PBS-P+ (pH 7.4) containing 5% (v / v) DMSO.

[0027] D. Protein Coupling: (1) Place the running buffer (200 mL 1× PBS Buffer), water bottle, and waste bottle in the left and right trays respectively, and insert the corresponding liquid inlet tubes.

[0028] (2) Hold the CM5 chip with the printed side facing up. Gently push the chip into the card slot in the direction of the arrow on the chip, and then close the chip compartment door.

[0029] (3) Activate channel 2 of the chip with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) at a flow rate of 10 μL / min.

[0030] (4) The ligand protein was diluted to 50 μg / mL with sodium acetate and fixed on channel 2 of the chip at a flow rate of 10 μL / min to generate a coupling map.

[0031] (5) Block the channel with ethanolamine at a flow rate of 10 μL / min.

[0032] (6) For channel 1 as a reference, repeat steps (3)-(5), except that protein-free acetate buffer is used in step (4).

[0033] E. Analyte coupling: (1) Each analyte is diluted to several concentrations in a 96-well plate and coupled to the target protein from low concentration to high concentration through the chip at a flow rate of 30 μL / min for 150 seconds.

[0034] (2) After each concentration point has been run, the chip is regenerated with 10 mM glycine hydrochloride (pH 2.0) solution for 5 min. This process is repeated until all the corresponding concentrations of the analyte have been run.

[0035] F. Run the experiment: Data for samples were collected using BIAcore T200 Control software (v.2.0, GE Healthcare) and subtracted from the data for the reference channel. Association and dissociation constants were obtained by global fitting of the data to a 1:1 Langmuir binding model using BIAcore T200 Evaluation software (v.2.0, GE Healthcare).

[0036] 4. Western blotting Cell samples were lysed with RIPA lysis buffer (Beyotime biotechnology, catalog number: #P0013B) containing protease inhibitors (Beyotime biotechnology, catalog number: #st506) and phosphatase inhibitors (Roche, catalog number: #04906845001). After ultrasonic grinding, the samples were incubated on ice for 20 min to fully lyse the samples. The lysate solution was centrifuged at 13000 rpm for 20 min at 4°C, and the supernatant was collected. All protein supernatants were subjected to BCA protein concentration determination (Beyotime biotechnology, catalog number: #P0009). The protein supernatant was mixed with 6× loading buffer, boiled at 95°C for 10 min in a metal bath, and stored at -20°C for later use after aliquoting. 20-30 μg of protein sample was added to the sample wells of 8-15% polyacrylamide gel, and protein markers were added to indicate the band positions. After appropriate electrophoresis time, the samples were transferred to 0.20 μm PVDF membranes. After transfer, the bands were blocked in 5% skim milk in TBST at room temperature for 1 hour. The bands were then incubated with primary antibodies overnight at 4°C on a shaker. The next day, the membrane was washed 3 times with TBST for 10 min each time, and then incubated with HRP-conjugated secondary antibodies at room temperature for 1 hour. All bands were washed 5 times with TBST for 10 min each time, and then developed using an imaging system (ImageQuant™ LAS 4000 mini, version 1.2). The grayscale value of each band was analyzed using Image J.

[0037] 1. Virtual Screening Computer virtual screening was used to perform molecular docking of the three-dimensional structure of mouse AQP4 protein with 33,600 compounds with known and unknown activities. The software used for virtual screening was Schrödinger Maestro 11.4, and the 3D drawing software was PyMol.

[0038] 1. Protein preparation: The three-dimensional structure of mouse AQP4 was downloaded from the AlphaFold database (AlphaFold ID: AF-P55088-F1). The protein was hydrogenated using the Protein Preparation Wizard module, followed by energy optimization (OPLS2005 force field, RMSD of 0.30 Å). The processed protein was used to create a grid file using the Receptor Grid Generation module, with Site1 (nearby amino acids: SER55 / TRP59 / ASP69 / VAL147 / ASN206, etc.) as the center to generate the grid file, and the box size was set to 20 Å × 20 Å × 20 Å.

[0039] 2. Compound preparation: The 2D formats of HY-L0107V (natural product analog library, containing 13,200 compounds) and HY-L001P (biologically active compound library, containing 20,400 compounds) were processed by the Schrödinger software LigPrep Module for hydrogenation, energy optimization, etc., and the 3D structures were output for virtual screening.

[0040] 3. Molecular docking: The Virtual Screening Workflow module was used for virtual screening, and the prepared compounds were imported. The Glide module was used for molecular docking, that is, the receptor and ligand molecules were docked with each other through geometric matching and energy matching. First, the high-throughput screening mode in the Glide module was used to screen the prepared molecules in the natural product analog library, and the molecules with the top 10% of the scoring values ​​were selected for the second round of screening using the standard mode; then the molecules with the top 10% of the scoring values ​​were selected for the third round of screening using the high-precision mode, and all screening results were output. The high-throughput screening mode in the Glide module was used to screen the prepared molecules in the bioactive compound library, and the molecules with the top 15% of the scoring values ​​were selected for the second round of screening using the standard mode; then the molecules with the top 15% of the scoring values ​​were selected for the third round of screening using the high-precision mode, and all screening results were output. The binding force between the target and the compound, the compound structure, etc. were manually reviewed, and the top 200 compounds in the natural product analog library and the top 200 compounds in the bioactive compound library were selected and output as the final results. The higher the absolute value of the molecular docking score (docking_score), the stronger the binding force between the compound and the protein.

[0041] 2. Affinity mass spectrometry The binding affinity between mouse AQP4 protein and the top 30 small molecule compounds in docking scores was tested by affinity mass spectrometry experiments. The final results were expressed in the form of B*100, and the affinity was judged according to the value of B*100 of each compound.

[0042] 3. Surface Plasmon Resonance Technology The interaction affinity between mouse AQP4 protein and the analyte was determined by Biacore, and the protein was coupled to the CM5 chip. The association rate (kon) and dissociation rate (koff) between the molecules were measured to calculate the affinity constant (KD).

[0043] IV. In vitro experimental verification 1. Assessment of cell viability: The CCK-8 method was used to measure cell activity and evaluate the effect of compound 61 on cell viability.

[0044] 2. Detection of aging markers: The expression levels of relevant aging markers (such as p53, p21, p16, etc.) were detected by Western blotting.

[0045] 3. Cell function assessment: The effects of compounds on the polarity distribution of AQP4 (M1 / M23 ratio) in astrocyte endfeet were evaluated by Western blotting.

[0046] 4. Data Analysis ImageJ was used to calculate the gray value of the bands, and GraphPad Prism 8.0 was used for data statistics. Unpaired T test, one-way ANOVA combined with post hoc Tukey test were used to analyze the experimental data according to the treatment factors. The data are expressed as mean ± standard error, *p < 0.05 was considered statistically significant.

[0047] V. Experimental Results Molecular docking was performed based on the protein structure of AQP4 and 33,600 compounds (e.g. Figure 1 As shown in the figure), affinity mass spectrometry experiments were performed on the top virtual screening scores and mouse AQP4 purified protein to analyze the affinity interaction (as shown in the figure). Figure 2 As shown, it was preliminarily determined that compound 61 strongly binds to AQP4 (as shown Figure 3 As shown). Molecular docking analysis of the binding mode of compound 61 with mouse AQP4 protein. In the 3D figure, the C skeleton of mouse AQP4 protein is shown in blue, N atoms are shown in dark blue, O atoms are shown in bright red, H atoms are shown in white, and compound 61 is shown as a light blue stick. Hydrogen bonds are shown as rose-red dotted lines. The longer the bond length, the weaker the hydrogen bond. Compound 61 can form three hydrogen bonds with the target protein: the imino group of the compound can form three hydrogen bonds with SER55 / THR56 / PRO67 of the target protein, with distances of 2.1 Å, 1.9 Å, and 1.8 Å, respectively (as shown in Figure 2). Figure 4 Further, through the surface plasmon resonance experiment, KD(M)=5.32E-06, it was confirmed that compound 61 had a strong interaction affinity with AQP4 (as shown in Figure 5A , 5B as shown).

[0048] Using the primary astrocyte aging model established earlier, the aged astrocytes cultured in vitro were stimulated with different concentrations of compound 61 for 48h and 72h, and the cell viability was detected. The results of the CCK8 experiment showed that the treatment of aged astrocytes with 0.1, 1 and 10 μM concentrations of compound 61 for 48h and 72h had no significant effect on the cell viability (e.g. Fig. 6A ,6B Therefore, the subsequent experiments chose to use a longer treatment time of 72 h.

[0049] It has been reported previously that cyanamide (CYA) can significantly stimulate the expression of AQP4, but has little effect on the polarity distribution of AQP4. Therefore, we chose CYA as a positive reference for AQP4 agonists. Senescent astrocytes were treated with control, 10 μM CYA, and 10 μM compound 61 for 72 hours, and cell samples were collected to detect the levels of AQP4 polarity and aging-related markers. As shown in Figure 5, Western blotting data showed that compared with CYA, compound 61 significantly enhanced AQP4 polarity and reduced p16 levels. Compared with the control group, purine compound 61 significantly reduced p16, p21, and p53 levels (such as Fig. 7A , 6B It is shown in Figure 2). This suggests that it reduces the senescence of astrocytes and is more effective than CYA.

Claims

1. A purine compound 61 represented by the following structural formula: 。 2. Use of the purine compound 61 as shown in claim 1 in the preparation of drugs for preventing and treating brain aging.

3. The use according to claim 2, characterized in that: The drug dosage form for preventing and treating brain aging is capsule, tablet, oral preparation, microcapsule preparation, injection, suppository, spray or ointment.

4. The use according to claim 2, characterized in that: Purine compound 61 is in the form of a pharmaceutically acceptable salt.

5. The use according to claim 4, characterized in that: Purine compound 61 is in the form of a pharmaceutically acceptable acid addition salt.

6. The use according to claim 5, characterized in that: Purine compound 61 is in the form of its hydrobromide salt.

7. The use according to any one of claims 2 to 6, characterized in that: The brain aging drug is used to treat patients suffering from brain aging diseases.

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