A CD38 protein inhibitor and its application

Abstract

The present invention provides a CD38 protein inhibitor, namely dihydrocapsaicin, which is safe and non-toxic compared with traditional CD38 protein inhibitors, easy to obtain, and has great feasibility in application in health care products and medicines, and is of great significance to the subsequent research and development of anti-aging health care products and medicines.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a CD38 protein inhibitor and application thereof in the preparation of anti-aging drugs. Background Art

[0002] As a type II transmembrane glycoprotein with bifunctional extracellular enzyme activity, CD38 plays a key role in multiple physiological processes such as cell growth, signal transduction and immune regulation. In recent years, studies have found that overactivity or abnormal expression of CD38 may be closely related to the occurrence and development of various diseases. In particular, CD38 has been identified as the main consumption enzyme for reduced NAD+ levels. NAD+ is a molecule that is essential in many aspects such as cellular energy metabolism, DNA repair and signal transduction. With age, the level of NAD+ in the human body gradually decreases, which is mainly attributed to a variety of factors, including inhibition of the synthesis pathway, consumption by enzymes, and obstruction of transport and reuse. Among them, the increased activity of CD38 plays a key role in the consumption of NAD+. CD38 catalyzes NAD+ to generate nucleotide metabolites such as cyclic ADP ribose (cADPR), thereby regulating intracellular Ca2+ mobilization and various biological processes. However, this also means that overactivity of CD38 accelerates the consumption of NAD+, leading to a decrease in intracellular NAD+ levels.

[0003] The reduction of NAD+ levels not only affects the energy metabolism and DNA repair ability of cells, but is also associated with a variety of age-related diseases, such as neurodegenerative diseases, cardiovascular diseases, diabetes and cancer. In addition, the reduction of NAD+ will also aggravate cellular oxidative stress, leading to cell damage and aging, further accelerating the aging process. Therefore, slowing down the consumption of NAD+ by inhibiting the activity of CD38 has important theoretical value and practical significance for the treatment of anti-aging and metabolic diseases.

[0004] Dihydrokavain is a natural compound derived from the rhizome of Pipermethysticum Forst. It is one of the main ingredients of a traditional sedative and anti-anxiety drink circulated in the South Pacific, and is believed to have anti-inflammatory, sleep disorder and anti-anxiety effects. Dihydrokavain is a compound of the kavalactone family, and its mechanism of action is mainly through affecting the function of human GABA receptors, thereby affecting nerve conduction and mood regulation. This compound is widely used in traditional herbal medicine and is being valued by modern medical research institutes. The unique pharmacological effects of dihydrokavain make it a potential candidate for the treatment of anxiety and sleep disorders. So far, there has been no report on the use of dihydrokavain as a CD38 protein inhibitor, and there is no related patent application. Summary of the invention

[0005] The invention provides an application of dihydrocapsaicin, a CD38 protein inhibitor, in the preparation of anti-aging drugs.

[0006] Preferably, the CD38 protein inhibitor is used at a concentration of 2.5 μM.

[0007] The dihydrocapsaicin provided by the present invention has the following chemical structural formula:

[0008]

[0009] Molecular docking predicted the binding of dihydrocapsaicin to CD38 protein, and the docking binding energy was -5.88 kcal / mol. According to common knowledge in the art, if the binding energy is less than -1.2 kcal / mol, the docking result is considered feasible.

[0010] In vitro experiments found that dihydrocapsaicin can significantly reduce the substrate NAD + The degradation of CD38 is highly efficient.

[0011] In vivo experiments have found that dihydrocapsaicin can significantly increase NAD levels in multiple major organs + level.

[0012] The CD38 protein inhibitor provided by the present invention is used in the preparation of anti-aging drugs. The CD38 protein inhibitor is used as an active ingredient to directly interact with the CD38 protein to inhibit CD38 from degrading NAD + activity, reducing NAD + consumption, thereby achieving the effect of treating aging-related diseases.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention provides a CD38 protein inhibitor, namely dihydrocapsaicin. Compared with traditional CD38 protein inhibitors, dihydrocapsaicin is a natural product, safe and non-toxic, easy to obtain, and has great feasibility in drug application, which is of great significance to the subsequent research and development of anti-aging drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the inhibition rate of different concentrations of dihydrocapsaicin on CD38 protein activity.

[0016] Figure 2 This is the result of molecular docking prediction of the interaction between CD38 protein and dihydrocapsaicin.

[0017] Figure 3 The effect of dihydrocapsaicin on NAD in major organs of mice + Level improvement effect diagram. DETAILED DESCRIPTION

[0018] 1. Experimental Materials

[0019] (1) 293t cells were purchased from China Center for Type Culture Collection

[0020] (2) C57 mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0021] 2. Experimental instruments

[0022] (1) Multifunctional microplate reader, model Synergy H1, purchased from Boitek

[0023] (2) Biological X-ray irradiator, model RS2000Plus, purchased from RadSource Technologies, Inc.

[0024] 3. Main reagents

[0025] (1) Dihydrocapsaicin, product number S9446, purchased from selleck (in vitro)

[0026] (2) High glucose DMEM medium, product number C11995500BT, purchased from Gibco

[0027] (3) PBS pH = 7.4, product number C10010500BT, purchased from Gibco

[0028] (4) NAD+ standard, CAS 53-84-9, purchased from Roche

[0029] (5) Cell Counting kit 8 (CCK8), catalog number: C0040, purchased from Bio-Tech

[0030] (6) Glycine bis-Gly, CAS 556-50-3, purchased from Aladdin

[0031] (7) Alcohol dehydrogenase, CAS 9031-72-5, purchased from Sigma-Aldrich

[0032] (8) Niacinamide, CAS 98-92-0

[0033] (9) Anhydrous ethanol, CAS 64-17-5, purchased from Guangzhou

[0034] (10) 1,1,2-Trichlorotrifluoroethane, CAS 76-13-1, purchased from Nanjing Chemical Reagent Co., Ltd.

[0035] (11) Trioctylamine, product number T818834, purchased from Maclean

[0036] 4. Solution preparation

[0037] (1) NAD + Reaction solution preparation

[0038] 1ml reaction solution system 61mM Glycine 820μl Cell Counting kit8 100μl 2.6KU / ml alcohol dehydrogenase 5μl 3.5M Niacinamide 28.5μl Anhydrous ethanol 57μl

[0039] (2) NAD + Preparation of extract

[0040] 1,1,2-Trichlorotrifluoroethane and trioctylamine were prepared in a volume ratio of 3:1, mixed and shaken.

[0041] Prepare and use immediately.

[0042] Test Example 1: Test on the inhibition of CD38 activity by dihydrocapsaicin

[0043] 1. Use the obtained 293T-CD38 to degrade NAD + : In a 96-well plate, 3 wells were not seeded with cells and 1 μM NAD was added + PBS solution with a total volume of 150 μl was used as a blank control (mock); 1*10 4 After the cells adhered to the wall, 1 μM NAD + PBS solution with a total volume of 150 μl was used as a control, and 1*10 4 293T-CD38 cells were added after the cells adhered to the wall, respectively, containing 1 μM concentration of NAD + PBS solution was prepared, and dihydrocapsaicin was added to the solution to make the final concentrations of 2.5, 1.25, 0.625, and 0 μM, respectively, with a total volume of 150 μl. The plate was placed in a cell culture incubator and incubated for 20 minutes, and 100 μl of the supernatant was transferred to a new 96-well plate;

[0044] 2. Detection of NAD + The remaining amount was used to calculate the inhibitory efficiency of CD38: NAD was added to the 96-well plate containing the supernatant obtained above. + Reaction solution (can be used with NAD + The cells were incubated at 37°C for 30 min, and the OD450 was measured using a microplate reader. The inhibition efficiency of CD38 protein was calculated based on the OD450 value (inhibition rate = [(mock-control) - (mock-drug addition hole)] / (mock-control)).

[0045] Experimental Example 2: An experiment to predict the direct interaction between dihydrocapsaicin and CD38 was carried out according to the following steps:

[0046] 1. Receptor and ligand preparation: Download the 3D structure of CD38 protein from the PDB database (PDB DOI: https: / / www.rcsb.org / ), save it in pdb format, remove oxygen and small molecules in Pymol, and convert it to pdbqt format in AutoDock; download the 3D structure of dihydrocapsaicin from the PubChem database, save it in mol2 format, import it into AutoDock and save it in pdbqt format.

[0047] 2. Molecular docking of receptor ligands. Import the CD38 receptor prepared in S1 into the AutoDock4 software and use NAD + The binding pocket of CD38 was used as the docking pocket to calculate the grid energy. Then the dihydrocapsaicin ligand was imported and molecular docking was performed using the AutoDock4 software, where the number of dockings was set to 100 and the conformation search accuracy was set to Medium. The docking results were analyzed by clustering and binding energy, and a docking result with more clusters and lower binding energy was selected and saved in pdbqt format.

[0048] 3. Visualization of docking results. Import the results saved in S2 into Protein-Ligand InteractionProfiler sites, analyzed the interaction bonds, and imported into Pymol software for visualization.

[0049] Experimental Example 3: Dihydrocapsaicin increases NAD in major organs of mice + Horizontal experiment. Follow the steps below:

[0050] 1. Purchase 16 C57 mice aged 4-6 weeks and randomly divide them into 4 treatment groups, namely blank control group (YOUNG), irradiated aging control group (IR), low-dose experimental group (IR+S9446 (10 mg / kg)) and high-dose experimental group (IR+S9446 (20 mg / kg)). During the modeling stage, the YOUNG group was not treated, and the other 3 groups were irradiated twice, with a one-month interval between the two irradiations. The irradiation dose was 4.5 Gy, 25 mA.

[0051] 2. One month after the second irradiation, the mice were gavaged with normal saline in the YOUNG and IR groups, and with dihydrocapsaicin in the IR+S9446 (10 mg / kg) and IR+S9446 (20 mg / kg) groups, with the doses of 10 mg / kg and 20 mg / kg, respectively. The four groups were gavaged once a day for 6 consecutive days.

[0052] 3. Sacrifice the mouse, remove the main organs and extract their NAD + .

[0053] (1) On the 6th day, the mice were sacrificed, 10 mg of each of the heart and brain tissues were taken and placed in a 1.5 ml EP tube, and 1 ml of 10% TCA was quickly added and shaken to mix. Two small steel balls were added to the EP tube. The EP tube was placed in a tissue homogenizer and ground. (Set the frequency to 70 Hz, 1 min 15 s, interruption time 10 s, running 15 s, and the number of times 10)

[0054] (2) Place in a centrifuge at 12000g, 4°C, and centrifuge for 15 minutes.

[0055] (3) Take 900 μl of the supernatant and add 1800 μl of NAD + Extract solution (1:2), mix thoroughly, and let stand to wait for stratification.

[0056] (4) The liquid is divided into three layers. The middle layer is cell fragments. Carefully take 800 μl of the top layer of liquid and store it at 4°C.

[0057] 4. NAD + Standard product configuration

[0058] (1) Take out the prepared 1mM NAD standard and dilute it to 50μM with ultrapure water (ddH2O). (You can first add 100μl of 1mM standard to 900μl ddH2O to prepare 100μM NAD standard, and then take 500μl of 100μM NAD standard and add 500μl ddH2O to prepare 50μM NAD standard)

[0059] (2) Take out 8 5ml round-bottom EP tubes

[0060]

[0061] 1) Add 2940 μl 10% TCA (1g TCA powder + 10ml ddH2O) to each tube

[0062] 2) Add standard (50 μM) according to the sample volume, and then add water to 60 μl.

[0063] 3) Oscillate and mix.

[0064] (3) Prepare 8 more 5 ml EP tubes and divide 1 ml into sections from the original 8 tubes.

[0065] (4) Add 2 ml of NAD into the 8 tubes in step 3. + The extract (supernatant: NAD extract = 1:2) was mixed thoroughly and allowed to stand until the stratified liquid was divided into 3 layers. 800 μl of the top layer of liquid was carefully taken and stored at 4°C.

[0066] 5. Planking detection

[0067] (1) Add the above three sample preparations into 20 μl of 1 M Tris solution with a pH of 8.0.

[0068] (2) Add samples to a 96-well plate, 100 μl per well.

[0069] (3) Add 100 μl of NAD to each well. + Reaction liquid.

[0070] (4) Incubate in a 37°C incubator away from light for 30 min.

[0071] (5) Select OD450 scanning reading on the microplate reader.

[0072] (6) Calculate the NAD concentration of the sample according to the equation obtained from the standard. Note: The R2 of the standard curve should not be less than 0.995.

Claims

1. Use of a CD38 protein inhibitor in the preparation of an anti-aging drug, characterized in that: The CD38 protein inhibitor is dihydrocapsaicin.

2. The use according to claim 1, characterized in that: The dihydrocapsaicin, as an active ingredient, directly interacts with the CD38 protein, inhibits the activity of CD38 in degrading NAD+, and reduces the consumption of NAD+.

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