Use of pulegone as an active ingredient in immunosuppressants

Abstract

The application discloses application of pulegone as an effective component in an immunosuppressant, and the pulegone with significant calcineurin (CN) inhibiting activity is screened from the Schleichera oleosa Kurz, the immunosuppressive effect of the pulegone is equivalent to that of cyclosporine A which is widely used in clinic, mechanism research shows that the pulegone targets CN / NFAT signal path to play the immunosuppressive activity, and the pulegone has very weak cytotoxicity to spleen cells, and is a high-efficiency and low-toxicity immunosuppressant.

Description

Technical Field

[0001] This invention relates to the use of tebufenol, and particularly to its use as an active ingredient in immunosuppressants. Background Technology

[0002] Calcineurin (CN) is a Ca2+-dependent enzyme. 2+ Calmodulin-specific serine / threonine phosphatases, extracted from yeast fermentation broth, are often referred to as calmodulin phosphatase due to their high concentration in nerve tissue (Klee et al., 1979). While biochemists and neurobiologists were studying this new protein, they inadvertently discovered the immunosuppressant cyclosporine A (CsA). CsA exerts a significant immunosuppressive effect by binding to intracellular cytosolic proteins to form a complex, and has been widely used in organ transplantation surgery (Borel et al., 1976; Handschumacher et al., 1984).

[0003] Protein phosphorylation and dephosphorylation are crucial regulatory mechanisms in life processes, permeating almost every life activity. This process is the result of the dynamic co-regulation of protein kinases (PKases) and protein phosphatases (PPases). Compared to protein kinases, fewer types of protein phosphatases are currently known, and their regulatory mechanisms in cellular life activities are poorly understood. Calcineurin (CN), as a member of the protein phosphatase family, has its activity directly regulated by the intracellular second messenger Ca2+. 2+ Calmodulin (CaM) plays a crucial role in immune regulation, learning and memory, and cell signal transduction. Due to its unique properties, structure, and function, it has attracted significant interest and attention from researchers. Extensive studies have been conducted on its substrate discovery, physiological functions, and enzyme activity inhibitors. The most groundbreaking achievement was the identification of calmodulin phosphatase as a specific target enzyme of the immunosuppressive drugs cyclosporin A (CsA) and FK506 in 1990. However, existing calmodulin phosphatase inhibitors, such as cyclosporin A and tacrolimus, are commonly used immunosuppressants in clinical practice, but they have various adverse reactions, including nephrotoxicity, hyperglycemia, and non-specific effects on cells.

[0004] This invention targets calmodulin phosphatase and screens compounds from hornberry trees that inhibit calmodulin phosphatase (CN) activity in vitro. It also conducts in-depth research on the mechanism of action of calmodulin phosphatase, which is of great significance for studying and revealing the function and mechanism of action of calmodulin phosphatase in life activities and provides a new approach for developing new, highly effective and low-toxicity immunosuppressants. Summary of the Invention

[0005] In view of this, the present invention proposes the application of fumarate as an active ingredient in immunosuppressants.

[0006] The technical solution of this invention is implemented as follows:

[0007] The application of tebufenol as an active ingredient in immunosuppressants, wherein the inhibitor is an immunosuppressive drug.

[0008] This invention uses immunomodulatory calmodulin phosphatase (CN) as the target enzyme and pNPP as the substrate for initial immunosuppressive activity screening. Monomeric compounds isolated from *Corylus siliqueurus* were screened for immunosuppressive activity, and tebufenol exhibited good immunosuppressive activity during the initial screening process. Further research on tebufenol was conducted by establishing a PMA / Io-induced Jurkat cell activation model, detecting changes in CN activity, IL-2 levels, and NFAT protein expression within Jurkat cells, thus elucidating the mechanism of action of tebufenol.

[0009] Etodiol showed no cytotoxic activity, nor did it promote cell proliferation or inhibit cell growth in Jurkat cells at concentrations ranging from 1 to 40 μm.

[0010] In the toxicity evaluation of tebufenozide on Jurkat cells, Jurkat cells were treated with different concentrations of tebufenozide for 48 hours. It was found that under different treatment time conditions and within the concentration range (1–40 μM), the survival rate of Jurkat cells did not differ significantly, and the cell survival rate remained above 90%. With further increases in the concentration of the compound, high-dose groups (75, 100 μM) of tebufenozide showed a slight inhibitory effect on Jurkat cells, with the cell survival rate decreasing to below 90%. This indicates that tebufenozide has no cytotoxic activity against Jurkat cells within the concentration range of 1–40 μM and does not promote cell proliferation or inhibit cell growth.

[0011] Ephedrine was used to inhibit the activity of Jurkat cells that were stimulated to proliferate and differentiate by PMA / Io.

[0012] This invention utilizes a calmodulin phosphatase activity assay, using pNPP as a substrate, to screen for the activity of active components in *Siamese algae*, revealing that emetol has a strong inhibitory effect on nifedipine (CN). Further analysis of its mechanism of action was conducted using Jurkat cells. PMA / Io stimulation of Jurkat cell proliferation and differentiation simulated the physiological environment of T cells after receiving external antigen stimulation. Jurkat cells were first stimulated by PMA / Io for proliferation and differentiation, and then co-cultured with different concentrations of emetol. The results showed that emetol had a certain inhibitory effect on PMA / Io-stimulated Jurkat cell proliferation and differentiation. This indicates that emetol can, to a certain extent, inhibit cellular immune activity.

[0013] Ethamnosine is used to inhibit the activity of calmodulin phosphatase.

[0014] Ettoretin is an immunosuppressant that inhibits the dephosphorylation of intracellular phosphorylated NFAT protein.

[0015] Ephedrine is used to inhibit the production of interleukin-2 (IL-2) in Jurkat cells.

[0016] Calmodulin phosphatase (CN) is an enzyme closely related to NFAT activation. Under normal circumstances, intracellular NFAT protein exists only in the cytoplasm. When cells are activated by external stimuli, intracellular CN binds to NFAT protein, dephosphorylating it and allowing it to enter the nucleus. There, it binds to downstream transcription factors to produce corresponding immune molecules (such as interleukin-2). This invention not only screened compounds through extracellular calmodulin phosphatase (CN) inhibition experiments and preliminarily confirmed the immunosuppressive activity of emetol, but also established a PMA / Io-induced Jurkat cell proliferation and differentiation model. Activated cells were treated with emetol, and the intracellular calmodulin phosphatase (CN) activity and intracellular IL-2 content were detected. Western blot experiments were used to study the cellular pathway levels. The results showed that cells not treated with emetol were significantly activated, with increased intracellular interleukin-2 (IL-2) content and increased intracellular calmodulin phosphatase (CN) activity. Western blot results showed that the level of NFAT in Jurkat cells treated with emetol was significantly reduced. This indicates that tebufenol reduces the dephosphorylation of NF-κB by inhibiting the activity of intracellular calmodulin phosphatase (CN), thereby reducing NF-κB entry into the nucleus to bind downstream proteins and consequently lowering IL-2 levels. Therefore, this invention successfully elucidates the mechanism of action of tebufenol, providing a theoretical basis for its development into a novel, highly effective, and low-toxicity immunosuppressant.

[0017] Eupatorol is a compound isolated and purified from hornberry.

[0018] The molecular formula of the eczematenol is C1 13 H 20 O3: The structural formula is:

[0019]

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention screened tebufenol, which has significant calmodulin phosphatase (CN) inhibitory activity, from hornberry and conducted in-depth research on its mechanism. Its immunosuppressive effect is comparable to that of cyclosporine A, an immunosuppressant widely used in clinical practice. The mechanism of action study shows that tebufenol exerts immunosuppressive activity by targeting the CN / NFAT signaling pathway and has very low toxicity to spleen cells. The application of tebufenol as an active ingredient in immunosuppressants provides a new approach for developing novel, highly effective, and low-toxicity immunosuppressants. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the principle of the initial screening of calmodulin phosphatase (CN) activity according to the present invention.

[0022] Figure 2 This is a schematic diagram of an enzyme activity test tube according to an embodiment of the present invention.

[0023] Figure 3 A bar chart illustrating the toxicity of emamectin to Jurkat cells in an embodiment of the present invention;

[0024] Figure 4 This is a bar chart illustrating the inhibitory effect of emamectin on the proliferation of PMA / Io-stimulated Jurkat cells according to an embodiment of the present invention.

[0025] Figure 5 A bar chart illustrating the inhibitory effect of emamectin tartrate on calmodulin phosphatase (CN) activity in an embodiment of the present invention;

[0026] Figure 6 A bar chart illustrating the inhibitory effect of emamectin on intracellular IL-2 in Jurkat cells, as described in an embodiment of the present invention.

[0027] Figure 7 Electrophoretic band diagram of the effect of fumarate on NFAT in an embodiment of the present invention;

[0028] Figure 8 A bar chart showing the effect of emamectin on NFAT in an embodiment of the present invention;

[0029] Figure 9 The above is a hydrogen spectrum of compound HZ-2 obtained by isolating and purifying it from hornberry in an embodiment of the present invention;

[0030] Figure 10The carbon spectrum of compound HZ-2 obtained by isolating and purifying it from hornberry in an embodiment of the present invention; Detailed Implementation

[0031] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0032] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0033] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0034] I. Experimental Materials and Instruments

[0035] 1. Cell origin

[0036] The Jurkat cell line used in this experiment was purchased from the Stem Cell Bank of the Chinese Academy of Sciences and was passaged and cryopreserved in liquid nitrogen in our laboratory.

[0037] 2. Materials and reagents: Vomifoliol (Vom), purity ≥ 90%.

[0038] Table 1 Main Reagents

[0039] Table 1 Main reagents

[0040]

[0041] 3. Main instruments and equipment

[0042] Table 2 Main Experimental Instruments

[0043] Table 2Main experimental instruments

[0044]

[0045]

[0046] II. Experimental Methods and Results Analysis

[0047] Example 1 - Initial screening of calmodulin phosphatase target enzyme activity using pNPP as a substrate

[0048] In this experiment, the expression, extraction, and purification of CN were completed by Professor Wei Qun's research group at Beijing Normal University. Referring to their enzyme activity assay method, a molecular reaction model for drug screening using CN as the target enzyme was established using modern molecular biology techniques. To reduce the labor intensity of drug screening and improve its safety and reliability, considering the characteristics of CN and the relevant properties of drug-target enzyme binding, and by comparing the activity assay results of p-NPP and RII multiple times, it was found that for small molecules directly acting on CN, the results measured using p-NPP as a substrate and the results measured using RII as a substrate showed a consistent trend in their effects. Therefore, p-NPP was chosen as the substrate for drug screening, and the design concept is described below. Figure 1 As shown.

[0049] from Figure 1 As we know, this molecular model primarily evaluates the drug's effect on the target enzyme by measuring the amount of products from CN hydrolysis of the substrate, thereby examining changes in CN activity. Therefore, the accuracy of enzyme activity measurement is crucial to this molecular model. Although previous experiments have shown that small molecules directly acting on CN show similar trends when measured using p-NPP as a substrate and when measured using RII as a substrate, this experiment selected calmodulin phosphatase (CN) as the target enzyme and p-NPP as the substrate for initial screening.

[0050] (1) Prepare a stock solution of 20 mg / mL of monomer compound using DDMSO for later use.

[0051] (2) Preparation of the solution required for activity assay

[0052]

[0053]

[0054] Stop solution: 0.5M Na2CO3

[0055] 20mL EDTA

[0056] (3) Test methods

[0057] Dilute CNA to a suitable enzyme solution and set aside. Place several 5ml test tubes on ice. Figure 2 As shown, 10 μL of the drug and 10 μL of the enzyme solution were added sequentially and incubated on ice for 5 min. Then, 180 μL of the activity test solution was added and the reaction was carried out in a water bath at 30 °C for 20 min. Finally, 1800 μL of the stop solution was added to terminate the reaction, and the OD410 value was measured on a 720 spectrophotometer.

[0058] Blank control: 10 μL enzyme dilution buffer + 10 μL Buffer

[0059] Enzyme: 10 μL enzyme + 10 μL buffer

[0060] Control group: 10 μL enzyme dilution + 10 μL drug

[0061] Enzyme + Drug: 10μL enzyme + 10μL drug

[0062] Note: Buffer is the solution used to dissolve the medication.

[0063] The relative inhibition rate of the drug on CNA is calculated using the following formula:

[0064] Relative inhibition rate (%) = [1 - (OD drug + enzyme - OD drug control) / OD enzyme] × 100%

[0065] (ODdrug+enzyme represents the absorbance of the solution obtained after the reaction of the enzyme and drug at 410 nm; ODdrug represents the absorbance of the drug control at 410 nm;)

[0066] (4) Screening of immunosuppressive monomers targeting calmodulin phosphatase (CN).

[0067] The compounds isolated and purified from *Cornus horn* in this experiment were initially screened for immunosuppressive activity targeting calmodulin phosphatase (CN). The compounds were named according to their laboratory numbers, and the results are shown in the table below.

[0068] Table 3. Preliminary screening results of compounds in hornberry.

[0069] Table3 PreliminaryscreeningresultsofcompoundisolatedfromC.tagal

[0070]

[0071] As shown in Table 3 above, among the 15 compounds isolated from *Siamese algae*, two compounds, HZ-2 and J9-a15-3, exhibited inhibition rates exceeding 50% against calmodulin phosphatase (CN), with inhibition rates of 87.30% and 65.23%, respectively. The compound with the best initial activity, HZ-2, was selected for further investigation. Structural comparison of HZ-2 revealed it to be vomifoliol.

[0072] The structural formula of compound HZ-2 is:

[0073]

[0074] White needle-like crystals (chloroform), melting point 108-110℃, cation ESI-MS m / z 224.1 [M+H] + (calcdfor C 21 H 33O3, 225.1412). Therefore, the molecular formula of this compound is C. 13 H 20 O3, 1 H NMR (400MHz, CDCl3)δ H 5.90(1H,s,H-4),5.84(1H,dd,J=15.60,5.27Hz,H-8),5.78(1H,d,J=15.7 3Hz,H-7),4.40(1H,m,H-9),2.44(2H,d,J=17.06Hz,H-2),2.25,2.21(each 1H,brs,OH),1.89(3H,d,J=0.83Hz,H-13),1.29(3H,d,J=6.41Hz,H-10),1.24(3H,s,H-13),1.07,1.00(each 3H,s,H-12,11or H-11,12); 13 C NMR (125MHz, CDCl3)δ C 198.9 (C-3), 163.9 (C-5), 136.4 (C-8), 129.6 (C-4), 127.5 (C-7), 79.7 (C-6), 68.7 (C-9), 50.3 (C-2), 41.8 (C-1), 24.7 (C-10), 24.4, 23.6 (C-11, 12 or C-12, 11), 19.7 (C-13). Based on spectral data analysis and comparison with reported data in the literature (Zhang et al., 2000), the compound was identified as vomifoliol. Figure 9 and 10 The proton and carbon spectra of the compound shown.

[0075] Example 2 - Culture of human leukemia T lymphocytes (Jurkat)

[0076] The biggest difference between Jurkat cells and HepG2 cells is that Jurkat cells are suspension cells while HepG2 cells are adherent cells. Therefore, their culture media and methods differ slightly, as detailed below:

[0077] (1) Jurkat cell resuscitation

[0078] Cell resuscitation method: Carefully remove the cryovial containing cells from the liquid nitrogen container using tweezers and place it in a 37°C constant temperature water bath. Hold the cap of the cryovial with tweezers and gently shake it in the water to allow the solid inside the cryovial to thaw completely as quickly as possible, following the "slow freeze, rapid thaw" principle to prevent intracellular water crystallization and damage to the cells. After disinfecting the cryovial by spraying it with 75% ethanol, place it in a laminar flow hood. Use a pipette to remove the cell suspension and add it to a centrifuge tube containing 9 mL of complete RPMI 1640 medium (preheated to 37°C) containing 10% fetal bovine serum. Centrifuge at 1000 rpm for 5 min.

[0079] (2) Passage of Jurkat cells

[0080] Jurkat cells were passaged using the "half-medium replacement method." The T25 culture flask was removed from the cell culture incubator, and cells were pipetted to ensure even distribution. 2.5 mL of the old RPMI 1640 medium in the flask was discarded, and then 2 mL of fresh RPMI 1640 medium and 0.5 mL of fetal bovine serum were added, bringing the total volume to 5 mL. The cells were then pipetted to form a single-cell suspension, and the flask was placed back into a CO2 incubator for further culture. The cell passage time was determined based on cell growth; cells were passaged when they covered 80% of the bottom surface of the culture flask.

[0081] (3) Cryopreservation of Jurkat cells

[0082] The purpose of this step is to preserve surplus Jurkat cells for long-term use. Cells in good growth condition and in the logarithmic growth phase are selected for cryopreservation experiments. Following the experimental procedures described above, cells are prepared into a suspension, counted, and diluted with RPMI 1640 medium to a concentration of 5 × 10⁻⁶ cells / mL. 6 Cells / mL: Take 0.85 mL of the diluted cell suspension into a cryovial, then add 0.1 mL of fetal bovine serum and 0.05 mL of DMSO solution. Label the cryovial with information such as the person performing the cryopreservation, the cryopreservation time, and the type of cells. Place the cryovial in a programmed cooling box and keep it at -80°C for 24 hours. Then, quickly remove the cryovial and store it in a liquid nitrogen tank. Regularly replenish the liquid nitrogen tank to prevent cell damage caused by liquid nitrogen loss.

[0083] Example 3 - The toxic effects of vomifoliol on Jurkat cells

[0084] (1) Take a 96-well cell culture plate and seed each well with 90 μL of Jurkat cell suspension at a concentration of 2*10. 6Cells / mL were cultured at 37°C in a 5% CO2 incubator for 4 hours.

[0085] (2) Add 10 μL of different concentrations of tebufenol solution to each well, so that the final concentrations of tebufenol are 1, 5, 10, 15, 30, 40, 50, 75, and 100 μM, respectively. Add 10 μL of culture medium to the blank control. Set up 3 replicates for each group. (If the drug itself has color, which will affect the absorbance, then set up a background control.)

[0086] (3) Place the culture plate in an incubator and incubate for 44 hours.

[0087] (4) Take out the cell culture plate, observe it under an inverted microscope, and then centrifuge it in a 96-well plate centrifuge at 2500 rpm for 5 min. Use a pipette to remove the supernatant, add 100 μL of fresh culture medium to each well, add 20 μL of MTT working solution, and the final concentration is 0.5 g / L.

[0088] (5) Continue incubation in the dark for 4 hours, centrifuge at 2500 rpm for 5 minutes, carefully discard the culture medium with a pipette, add 200 μL DMSO, let stand in the dark for 30 minutes to fully dissolve the blue formazan, and record the absorbance (OD) of the solution at 570 nm using an ELISA reader.

[0089] (6) Calculate cell viability and plot the cell viability against different drug concentrations. Repeat each experiment three times.

[0090] Cell viability = (Average OD of experimental group / Average OD of blank control group) * 100%

[0091] Jurkat cells were treated with different concentrations of tebufenozide for 48 hours. Figure 3 It was found that under different treatment time conditions, within the concentration range (1–40 μM) used in this experiment, there was no significant difference in the survival rate of Jurkat cells, and the cell survival rate was consistently above 90%. With further increases in the concentration of the compound, the high-dose groups (75, 100 μM) of tebufenol showed a slight inhibitory effect on Jurkat cells, with the cell survival rate decreasing to below 90%. This indicates that tebufenol has no cytotoxic activity against Jurkat cells within the concentration range of 1–40 μM and does not promote cell proliferation or inhibit cell growth. This result eliminates the possibility of interference from non-specific factors in the experiment.

[0092] Example 4 - Inhibitory effect of vomifoliol on Jurkat cells (PMA / Io-induced proliferation)

[0093] Take a 96-well cell culture plate and seed each well with 100 μL of Jurkat cell suspension at a concentration of 2*10⁻⁶. 6Cells / mL. Add 50 μL PMA / Io (Phorbol 12-myristate 13-acetate / Ionomycin) mixed solution to make the final concentrations of PMA and Io 25 ng / mL and 1 μg / mL, respectively, and stimulate for 4 h. Then add different final concentrations of tebufenol (5, 10, 15, 30, 40 μM) and cyclosporine A solution (15 μM), and add 50 μL of blank culture medium for the blank control. Incubate the culture plate in an incubator for 44 h. Remove the cell culture plate, observe it under an inverted microscope, and then centrifuge it in a 96-well plate centrifuge at 2500 rpm for 5 min. Discard the supernatant with a pipette, add 100 μL of fresh culture medium to each well, and add 20 μL of MTT working solution to make the final concentration 0.5 g / L. Continue incubation in the dark for 4 hours, centrifuge at 2500 rpm for 5 minutes, carefully discard the culture medium with a pipette, add 200 μL LDMSO, and let stand in the dark for 30 minutes to fully dissolve the blue formazan. Record the absorbance (OD) of the solution at 570 nm using a microplate reader. Calculate the cell viability and plot the cell viability against different drug concentrations. Repeat each experiment three times.

[0094] Cell viability = (Average OD of experimental group / Average OD of blank control group) * 100%

[0095] Depend on Figure 4 It was found that etanercept had a certain inhibitory effect on Jurkat cells co-stimulated by PMA / Io, and the cell survival rate decreased with higher concentrations, showing a concentration-dependent effect. At the same concentration of 15 μM, etanercept and the positive control cyclosporine A (CsA) had comparable inhibitory effects on Jurkat cells, which to some extent indicates that the immunosuppressive activity of etanercept is comparable to that of CsA. The half-maximal inhibitory concentration (IC50) of etanercept for Jurkat cells co-stimulated by PMA / Io was 20.167 ± 0.09 μM.

[0096] Example 5 - Jurkat Cell Calmodulin Phosphatase (CN) Activity Assay

[0097] (1) Prepare the required reagents

[0098] a. Cell lysis buffer used for malachite green viability assay

[0099]

[0100] b.2×Ca 2+ Activity test solution

[0101]

[0102]

[0103] c. 2×EGTA activity assay solution

[0104]

[0105] d. Solutions used in cell experiments

[0106]

[0107] e. Proteasome assay for viability of cell lysate

[0108] 10mMHEPESBuffer: HEPES 2.383g

[0109] Adjust the pH to 7.6 with 5M NaOH, and bring the volume to 500mL.

[0110] (2) Culture of the cells to be tested

[0111] Take a 6-well cell culture plate and seed each well with 1 mL of Jurkat cell suspension at a concentration of 2*10⁻⁶. 6 PMA / mL was added. 50 μL of a mixed solution of PMA / Io (Phorbol 12-myristate 13-acetate / Ionomycin) was added to achieve final concentrations of 25 ng / mL for PMA and 1 μg / mL for Io, and stimulation was performed for 4 h. Then, different final concentrations of tebufenozide (5, 10, 15, 30, 40 μM) and cyclosporine A solution (15 μM) were added, with an equal volume of blank culture medium added to the blank control. The culture plates were incubated in an incubator for 44 h.

[0112] Collect the treated Jurkat cells in sterile EP tubes and centrifuge at 1000 rpm for 5 min. Discard the supernatant, resuspend the cells in fresh cell culture medium, count them, and centrifuge at 1000 rpm for 5 min at 4°C using a low-temperature centrifuge. Wash the cells twice with TBS buffer as described above, then centrifuge at 1×10⁻⁶ cells per tube. 6 Add 100 μL of pre-prepared cell lysis buffer to each cell, lyse on ice for 10 min, centrifuge at 12000 rpm for 15 min at 4°C, and transfer the supernatant to a new sterile EP tube for later use.

[0113] (3) Pretreatment of P6-DG cell dephosphorization column

[0114] The cell lysate obtained after the above steps contains a large amount of free intracellular phosphate ions. Without dephosphaterization, the concentration of these phosphate ions will affect the accuracy of subsequent experiments. Therefore, pretreatment of the cell lysate with a P6-DG cell dephosphater column can eliminate interference from intracellular phosphate ions. The specific steps are as follows:

[0115] a. Place the P6-DG dephosphorization resin into a 50mL centrifuge tube, add 20mL of dd water, and vortex to mix. Let stand at room temperature for 4 hours or at 4℃ overnight.

[0116] b. Carefully remove ddH2O, then add new ddH2O in a 1:1 ratio to hydrate the dephosphorization resin.

[0117] c. Place 5 mL of the hydrated dephosphorizing resin into the dephosphorizing column, remove the dephosphorizing column stopper, and remove ddH2O from the medium by gravity.

[0118] d. Add 8 ml of lysis buffer (without enzyme inhibitors) to the P6-DG cell dephosphorization column to equilibrate the P6-DG cell dephosphorization column.

[0119] e. Insert the P6-DG cell dephosphorization column into a 15ml centrifuge tube and centrifuge at 800g for 3min in a 4℃ low-temperature centrifuge to remove the buffer solution in the medium.

[0120] f. Place the desalting column into a new 15ml centrifuge tube and add 350μL of supernatant sample obtained from high-speed centrifugation into the desalting column.

[0121] g. Centrifuge at 800g for 3 minutes in a low-temperature centrifuge at 4℃, and save the sample obtained from centrifugation in the tube. This sample can be used for the next step of CN activity determination (if it cannot be detected immediately, the sample should be stored in an ultra-low temperature freezer at -70℃).

[0122] Note: P6-DG cell dephosphater resin can be reused. When using new samples, wash the medium with 4-5 times the amount of ddH2O for dephosphaterization, then add 8 ml of lysis buffer to the P6-DG cell dephosphater column to equilibrate the medium. One P6-DG cell dephosphater column can be used for desalting 4-5 samples.

[0123] (4) Detection of free phosphate ions in cell extracts after dephosphorization via P6-DG cell dephosphorization column

[0124] a. Take 1 μL of ddH2O and 1 μL of cell lysate after dephosphorization and add them to a 96-well plate.

[0125] b. Add 100 μL of malachite green reagent to each.

[0126] c. React at room temperature in the dark for 30 minutes.

[0127] d. Record the wavelength of the solution at 630 nm after the reaction using an enzyme-linked immunosorbent assay (ELISA) reader.

[0128] (5) Determination of phosphoric acid standard curve

[0129] 500 μL of 2×Ca 2+ The activity test solution was diluted to 1×Ca 2+ Assay solutions. The 80 μM phosphate standard was diluted using a fractional dilution method to prepare corresponding concentration gradients. The corresponding phosphate concentrations in the 40, 20, 10, 5, 2.5, 1.25, and 0.625 μM phosphate standard solutions were 2, 1, 0.5, 0.25, 0.125, 0.063, and 0.031 nmol, respectively.

[0130] A <![CDATA[2nmol PO4]]> B <![CDATA[1nmol PO4]]> C <![CDATA[0.5nmol PO4]]> D <![CDATA[0.25nmol PO4]]> E <![CDATA[0.125nmol PO4]]> F <![CDATA[0.063nmol PO4]]> G <![CDATA[0.031nmol PO4]]> H <![CDATA[0nmol PO4]]>

[0131] Add 50 μl of 2×Ca to A 2+ Assay buffer, add 50 μl of 1×Ca to BH. 2+ The activity buffer was prepared. Then, 50 μl of 80 μM phosphate standard was added to A and mixed thoroughly by repeated pipetting. 50 μl of the mixture from A was added to B and mixed thoroughly by repeated pipetting. 50 μl of the mixture from B was added to C and mixed thoroughly by repeated pipetting. This process was repeated until the solution in G was thoroughly mixed, at which point the 50 μl of mixture from G was discarded. No operation was performed on H. Then, 100 μl of malachite green reagent was added to each well, and the mixture was allowed to stand at room temperature for 30 min. The absorbance at 630 nm was measured. The values ​​were plotted with PO4 on the x-axis and OD on the y-axis. 630 Plot a standard curve with the numerical values ​​on the ordinate. This yields the dephosphorylation formula.

[0132] (6) Malachite green assay for intracellular CN activity in Jurkat cells

[0133] The determination of intracellular CN activity was performed using Ca 2+ -EGTA method. Four experimental groups will be set up in the experiment: Ca 2+ EGTA, Ca 2+ +OA and EGTA+OA groups. A blank control group was also set up for instrument zeroing, and the experimental control group (background) was used for experimental analysis. The reagents added to each well and the specific procedures are shown in Table 3 below:

[0134] Table 4. Methods for Assaying Intracellular Calmodulin Phosphatase (CN) Activity

[0135] Table4 Procedure for testing intracellularCNenzymeactivity

[0136]

[0137] (7) CN activity calculation

[0138] Under conditions of pH 7.4 and 37°C, the CN activity was defined as the amount of substrate catalyzed by each milligram of enzyme in one minute (pmol / min / mg).

[0139] CN relative vitality calculation formula (OD) 630 express:)

[0140] Formula 1: CN = Ca 2 +buffer-EGTAbuffer

[0141] Formula 2: CN=(Ca 2 ++OA)buffer-(EGTA+OA)buffer

[0142] OD 630 Convert the value to the amount of phosphate released:

[0143] Phosphate released (nmol) = (OD) 630 -Yint) / Slope

[0144] Calculate the specific activity of CN:

[0145] CN activity (pmol / min / mg) = Amount of phosphate released by CN / (protein content per pore × enzyme activity reaction time)

[0146] (8) Inhibitory effect of emetol on calmodulin phosphatase (CN)

[0147] Using pNPP as a substrate, the effect of the monomer compound tebufenol on CN activity was investigated. Figure 5 It can be seen that the higher the concentration of tebufenol, the higher the inhibition rate of CN, which shows a concentration-dependent effect, and the half-inhibitory concentration is IC50 = 9.66 ± 0.150 μM.

[0148] Example 6 - Determination of intracellular interleukin-2 content in Jurkat cells

[0149] (1) Take a 6-well cell culture plate and seed each well with 1 mL of Jurkat cell suspension at a concentration of 2*10⁻⁶. 6PMA / mL was added. 50 μL of a mixed solution of PMA / Io (Phorbol 12-myristate 13-acetate / Ionomycin) was added to achieve final concentrations of 25 ng / mL for PMA and 1 μg / mL for Io, and stimulation was performed for 4 h. Then, different final concentrations of tebufenozide (5, 10, 15, 30, 40 μM) and cyclosporine A solution (15 μM) were added, with an equal volume of blank culture medium added to the blank control. The culture plates were incubated in an incubator for 44 h.

[0150] Transfer the treated cells to 2 mL EP tubes, centrifuge at 2000 rpm for 5 min to collect the cells, and wash once with PBS solution. Centrifuge at 2000 rpm for 5 min to collect the cells, discard the supernatant, add 150-200 μL of lysis buffer containing PMSF, then mix well, vortex for 30 s, and incubate on ice for 15 min (vortex the sample for 30 s every 5 min). Centrifuge at 12000 rpm for 5 min at 4 °C, carefully transfer the supernatant from the cell lysis to a new 1.5 mL EP tube, take a certain amount of the supernatant, and determine the protein concentration using a BCA protein quantification kit; the remainder is reserved.

[0151] Set up blank wells (blank control wells contain no sample or enzyme-labeled reagent, all other steps are the same), standard wells, and sample wells. Accurately add 50 μl of standard to the enzyme-labeled plate. Add 40 μl of sample diluent to the sample wells, then add 10 μl of the sample to be tested (final sample dilution is 5-fold). Add the sample to the bottom of the wells, avoiding contact with the well walls, and gently shake to mix. Seal the plate with sealing film and incubate at 37°C for 30 minutes. Dilute the 30-fold concentrated washing buffer 30 times with distilled water. Carefully remove the sealing film, discard the liquid, and shake dry. Fill each well with washing buffer, let stand for 30 seconds, then discard. Repeat this process 5 times, then pat dry. Add 50 μl of enzyme-labeled reagent to each well, except for the blank wells. Seal the plate with sealing film and incubate at 37°C for 30 minutes. Carefully peel off the sealing film, discard the liquid, and shake dry. Fill each well with washing solution, let stand for 30 seconds, then discard. Repeat this process 5 times, and pat dry. Add 50 μL of colorimetric reagent A to each well, followed by 50 μL of colorimetric reagent B, and gently shake to mix. Incubate at 37°C in the dark for 15 minutes. Add 50 μL of stop solution to each well to stop the reaction (the blue color will immediately turn yellow). Zero the microscope using a blank well and measure the absorbance (OD value) of each well sequentially at a wavelength of 450 nm. Measurements should be performed within 15 minutes of adding the stop solution.

[0152] (2) Effects of emetol on interleukin-2 (IL-2)

[0153] Jurkat cells, after co-induction with PMA / Io, rapidly activated, with a sharp increase in IL-2 levels. Treatment with different concentrations of emetol and a positive control (cyclosporine A) showed that emetol significantly reduced cellular interleukin-2 levels; the inhibitory effect was stronger at higher concentrations, exhibiting a concentration-dependent pattern. At a concentration of 15 μM, Vom and CsA showed comparable inhibitory activity. At an emetol concentration of 40 μM, the intracellular IL-2 level was close to the unstimulated IL-2 concentration. Emetol can inhibit IL-2 production in Jurkat cells, thus exhibiting good immunosuppressive activity.

[0154] Example 7 - Western blot (WB) assay to detect intracellular NFAT protein expression levels

[0155] (1) Electrophoresis: Cells were incubated with the drug, collected, and lysed according to the method described above for "determination of interleukin-2 content" to obtain total cell protein. Total cell protein was subjected to SDS-PAGE electrophoresis.

[0156] (2) Transfer: Transfer the protein onto a PVDF membrane by 250mA ice bath for 1.5h in electrophoresis buffer.

[0157] (3) Blocking: Transfer the PVDF membrane into the blocking solution prepared with PBS buffer containing 5% skim milk powder and block at room temperature for 4 hours or overnight at 4°C.

[0158] (4) Washing: Wash with PBS with 0.1% Tween-20 for 3 times × 10 min.

[0159] (5) Primary antibody conjugation: Dilute the primary antibody with TBST containing 5% skim milk powder (weigh 1.0g of skim milk powder and add 20mL of TBST and mix well). The dilution ratio is 1:1000, that is, 1μL of primary antibody is added to 1000μL of 5% skim milk powder. Fix the sealing film on the table with water, peel off the sealing film, and use a pipette to transfer the prepared primary antibody onto the sealing film. Be careful not to generate air bubbles. The PVDF membrane covers the diluted antibody.

[0160] (6) Secondary antibody binding: Wash the PVDF membrane three times with TBST for 10 min each time at room temperature; dilute the secondary antibody with TBST containing 5% skim milk powder (1:5000), that is, add 0.1 μL of secondary antibody to 1000 μL of TBST containing 5% skim milk powder, fix the sealing film on the table with water, peel off the sealing film, use a pipette to aspirate the prepared secondary antibody into the center of the sealing film, do not generate air bubbles, cover the diluted antibody with the PVDF membrane face down, cover with the box, and seal the edge of the basin with water, incubate at room temperature for 1 h, and then recover the secondary antibody.

[0161] (7) Color development: Place the filter membrane in a plastic box or petri dish of appropriate size (drain off excess liquid), add the chemiluminescent solution onto the PVDF membrane, shake gently, and react for 3-5 minutes. Wrap the PVDF membrane with plastic wrap and squeeze off the excess chemiluminescent solution onto filter paper.

[0162] (8) Development and fixing: In a darkroom, expose the PVDF membrane printed with protein to the X-ray film. After exposure, remove the X-ray film and place the exposed film in the developing solution. After the film shows bands, transfer it to the fixing solution. After fixing is complete, remove the film, rinse it with tap water and air dry it. Take a picture (scan) and use ImageJ to perform quantitative analysis of the results.

[0163] (9) Inhibitory effect of tebufenol on dephosphorylation of cellular NFAT protein

[0164] NFATs are nuclear transcription proteins that regulate the expression of IL-2 and other early T cell differentiation cytokine genes during T cell activation. Activated CN can dephosphorylate NFATs, thereby activating them. After entering the nucleus, they work with other transcription factors to bind to the promoter DNA of immune factor genes, initiating the transcription of these genes. (The text then abruptly shifts to a description of banding.) Figure 6 It can be seen that the degree of dephosphorylation in Jurkat cells significantly increased after PMA / Io induction. Treatment with different concentrations of emetol and the positive control (cyclosporine A) reduced the degree of dephosphorylation, and the degree of dephosphorylation in Jurkat cells decreased with increasing drug concentration. Figure 7 It is known that NFAT activation can be inhibited by Vom. Therefore, Vom is a potential immunosuppressant that can inhibit the dephosphorylation of intracellular phosphorylated NFAT protein.

[0165] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of fumarate as an active ingredient in the preparation of immunosuppressive drugs.

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