Application of coptisine sulfate in preparation of medicine for preventing and / or treating viral intestinal inflammation

Abstract

The invention relates to application of coptisine sulfate in preparation of a medicine for preventing and / or treating viral enteritis. The application of coptisine sulfate in treatment of viral intestinal inflammation is provided for the first time, and the application range of the medicine is widened. It is found that coptisine sulfate can relieve intestinal inflammation caused by papain-like protein on an animal model, and can play a role in treatment of intestinal inflammation caused by novel coronavirus caused by SARS-CoV-2.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the application of coptisine sulfate in the preparation of a drug for preventing and / or treating viral intestinal inflammation. Background Art

[0002] Novel coronavirus infection (COVID-19) is a global pandemic disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The RNA genome of SARS-CoV-2 is approximately 30,000 bp in length, of which the replicase gene occupies two-thirds of the 5' end of the viral genome. The replicase gene encodes two long overlapping polyproteins, pp1a and pp1ab. In the life cycle of SARS-CoV-2, the processing of pp1a and the conversion of pp1ab into 16 nonstructural proteins (Nsps) are key steps required for RNA transcription and genome replication. Papain-like protein (PLpro) and major protease (Mpro) play a proteolytic role, thereby participating in the process of viral replication and translation. At the same time, SARS-CoV-2 PLpro has the function of removing ubiquitin and ubiquitin-like interferon-stimulated gene 15 (ISG15) post-translational modifications, inhibiting the host antiviral interferon signaling pathway, and plays an important role in the innate immune response during viral infection.

[0003] The human upper respiratory tract is a site of high expression of ACE2 receptors that mediate SARS-CoV-2 infection of host cells, so the clinical symptoms of COVID-19 patients are mainly manifested as upper respiratory tract reactions. In addition to common respiratory symptoms, gastrointestinal infection and digestive system symptoms of SARS-CoV-2 have attracted attention as early as the beginning of the epidemic. Early studies have shown that approximately 10%-50% of COVID-19 patients in China have reported gastrointestinal reactions during infection with the new coronavirus. And in a study of 318 COVID-19 patients in the United States, digestive symptoms were widely reported. COVID-19-related gastrointestinal symptoms mainly include diarrhea (24.2%), anorexia (17.9%), and nausea (17.9%), and there are also COVID-19 patients with acute abdomen. Most importantly, some clinical studies have shown that digestive symptoms are closely related to the severity of COVID-19, such as severe and critical COVID-19, dyspnea, etc. In fact, SARS-CoV-2 has a high replication efficiency in intestinal epithelial cells, which may be one of the causes of digestive system symptoms.

[0004] Treatments for viral intestinal inflammation are currently limited. Some antiviral drugs, such as Remdesivir and Favipiravir, are used to treat COVID-19, but they are mainly used to treat respiratory infections and have no efficacy against intestinal symptoms. Therefore, finding new drugs to treat intestinal symptoms is very necessary. Summary of the invention

[0005] In order to solve the deficiencies in the prior art, the present invention provides a method for preparing a drug for preventing and / or treating viral enteritis using coptisine sulfate.

[0006] The molecular formula of Coptisine sulfate described in the present invention is C 19 H 15 NO8S, molecular weight is 417.39, and the structural formula is as follows:

[0007]

[0008] On the one hand, the present invention provides the use of coptisine sulfate or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and / or treating viral enteritis, wherein the virus is a coronavirus.

[0009] Among them, coronaviruses include HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, severe acute respiratory syndrome coronavirus SARS-CoV, severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 and Middle East respiratory syndrome coronavirus MERS-CoV. Preferably, the coronavirus is SARS-CoV-2.

[0010] On the other hand, the present invention also provides the use of coptisine sulfate or its pharmaceutically acceptable salt in the preparation of enteritis drugs induced by papain-like protein PLpro.

[0011] Preferably, the papain-like protein is a papain-like protein of SARS-CoV-2.

[0012] The present invention also provides a drug for combating intestinal inflammation caused by the new coronavirus, the active ingredient of which includes coptisine sulfate.

[0013] The present invention also provides the use of coptisine sulfate or a pharmaceutically acceptable salt thereof in the preparation of a drug that targets the binding interface between coronavirus PLpro and ISG15 protein, wherein the targeting effect is a targeted inhibitory effect.

[0014] Further, the coronavirus includes HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, severe acute respiratory syndrome coronavirus SARS-CoV, severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 and Middle East respiratory syndrome coronavirus MERS-CoV. Preferably, the coronavirus is SARS-CoV-2.

[0015] The present invention also provides a drug for targeted inhibition of the binding of PLpro of the new coronavirus to ISG15 protein, the active ingredient of which includes coptisine sulfate.

[0016] The beneficial effects of the present invention include:

[0017] The present invention discovered for the first time the use of coptisine sulfate and its pharmaceutically acceptable salts in the preparation of drugs for preventing and / or treating viral enteritis, thereby expanding their scope of use. Specifically: papain-like protein (PLpro) is involved in the replication and translation process of SARS-CoV-2 virus. SARS-CoV-2 PLpro has the function of removing ubiquitin and ubiquitin-like interferon-stimulated gene 15 (ISG15) post-translational modifications, inhibiting the host antiviral interferon signaling pathway. The embodiments of the present invention show that the papain-like protein of the new coronavirus SARS-CoV-2 can cause colon inflammation in mice. Furthermore, in vitro experiments show that coptisine sulfate can effectively inhibit the activity of papain-like protein PLpro binding to ISG15, and its half effective concentration IC 50 It is 4.48μM; the results of animal experiments showed that coptis sulfate can effectively relieve colon inflammation caused by the new coronavirus PLpro, which indicates that coptis sulfate has a positive effect in the treatment of viral intestinal inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results of establishing a mouse colon inflammation model using papain-like protein, an important immunomodulatory protein of the new coronavirus SARS-CoV-2, in Example 1 of the present invention. A in the figure is an H&E stained section, and the red arrow indicates an obvious inflammatory cell infiltration phenomenon; B in the figure is the real-time fluorescence quantitative PCR detection results of the inflammatory factors TNFα, IL-1α and ISG15 mRNA levels in the corresponding colon parts of the papain-like protein treatment group and the blank control.

[0019] Figure 2The result of the half-inhibitory concentration IC50 value of coptis sulfate inhibiting the interaction between PLpro and ISG15 determined based on the fluorescence polarization method in Example 2 of the present invention. The horizontal axis is the concentration of coptis sulfate, and the vertical axis is the inhibition rate of coptis sulfate inhibiting the binding of papain-like protein to ISG15, and the half-effective concentration IC of coptis sulfate inhibiting the binding of PLpro to ISG15 is calculated based on the inhibition rate. 50 value.

[0020] Figure 3 The results of using coptis sulfate in Example 3 of the present invention to apply to the mouse colon inflammation model stimulated by papain-like protein, an important immunomodulatory protein of the new coronavirus SARS-CoV-2. Figure A is an H&E stained section, showing that coptis sulfate can inhibit inflammatory cell infiltration; Figure B is the real-time fluorescence quantitative PCR test result showing that the levels of inflammatory factors TNFα, IL-1α and ISG15 were significantly reduced after treatment with coptis sulfate. Specific implementation method:

[0021] In order to better understand the content of the present invention, the content of the present invention is further described below in conjunction with the accompanying drawings and specific implementation methods, but the protection content of the present invention is not limited to the following embodiments.

[0022] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. The terms used in the specification of the present invention are for the purpose of describing specific embodiments and are not limited to the present invention.

[0023] The following examples mainly use papain-like protein, an important immunomodulatory protein of the new coronavirus SARS-CoV-2, to establish a mouse colon inflammation model, and based on the function of papain-like protein to remove the post-translational modification of the ubiquitin-like interferon-stimulated gene 15 (ISG15) protein, establish an inhibitor screening method targeting the interface of papain-like protein and ISG15. This example uses a fluorescence polarization method to evaluate the activity of coptis sulfate in blocking the interface between papain-like protein and ISG15. At the same time, coptis sulfate was applied to a mouse colon inflammation model stimulated by papain-like protein to observe its anti-inflammatory effect.

[0024] The C57BL / 6J mice used in the examples of the present invention were purchased from the Animal Experiment Center of Southern Medical University.

[0025] The coptisine sulfate used in the embodiments of the present invention was purchased from Shanghai Taosu Biochemical Technology Co., Ltd. with a purity of >99%.

[0026] The ISG15-FITC used in the examples of the present invention was purified and prepared by our laboratory.

[0027] The papain-like protein used in the examples of the present invention was recombinantly expressed and purified in Escherichia coli by our laboratory.

[0028] The acetic acid solution, chloroform, anhydrous ethanol and isopropanol used in the examples of the present invention were purchased from Guangzhou Chemical Reagent Company.

[0029] The reverse transcription kit and qPCR SYBR Green Master Mix used in the examples of the present invention were purchased from Shanghai Yisheng Biotechnology.

[0030] The physiological saline used in the embodiments of the present invention was purchased from Sevier Biotechnology.

[0031] PEG300 and Tween 80 used in the examples of the present invention were purchased from MacLean Chemical Reagents.

[0032] The DMSO used in the examples of the present invention was purchased from Bio-Tech Biotechnology.

[0033] Example 1: Establishment of a mouse colon inflammation model stimulated by papain-like protein

[0034] 1. Establishment of intestinal inflammation model

[0035] Eight-week-old C57BL / 6J mice were allowed to eat and drink freely in an environment with a constant temperature of 26°C and a 14 / 10-hour periodic light and dark period. The mice were divided into a blank control group, an acetic acid control group, and a model group in advance, with 5 mice in each group.

[0036] 1) Animals were fasted for 24 hours before modeling.

[0037] 2) 1% (v / v) acetic acid was prepared with normal saline as an agent to promote intestinal inflammation.

[0038] 3) Anesthetize mice by intraperitoneal injection of 40mg / kg 1% sodium pentobarbital. About 5 minutes later, apply glycerol to the surface of the hose and insert the hose from the mouse anus to a depth of 4 cm. Connect the other end of the hose to a syringe and inject 500μL of 1% acetic acid solution. The blank control group is injected with an equal volume of saline. The mouse is inverted for 1min and irrigated twice with 500μL PBS. After the operation is completed, the mouse is returned to the cage and provided with food.

[0039] 4) After 16 hours, mice in the protein group were intraperitoneally injected with 2.5 mg / kg of papain-like protein of the new coronavirus, prepared with PBS as the solvent to the required concentration. The blank group and the acetic acid group were injected with an equal volume of PBS.

[0040] 5) After 10 hours, the mice were observed for diarrhea. The mice were anesthetized by intraperitoneal injection of 40 mg / kg 1% sodium pentobarbital, and the chest and abdominal cavities were opened to collect liver, kidney, lung and intestinal tissues.

[0041] 2. Tissue RNA Extraction

[0042] 1) Take out the mouse tissue stored at -80℃ and thaw it on ice. Subsequent operations were performed in a clean bench to avoid interference from RNase.

[0043] 2) Transfer the intestinal tissues of each group to a homogenization tube, add grinding beads and 200 μL Trizol at the same time, place the sample in a biological sample homogenizer, operate at 60 Hz for 60 seconds, grind the sample thoroughly and evenly, and then let it stand for 5 minutes.

[0044] 3) Add 40 μL of chloroform, mix quickly and evenly, and let stand for 2 minutes.

[0045] 4) Place the sample in a centrifuge at 4°C and centrifuge at 11,000 rpm for 15 minutes. After centrifugation, the sample can be observed to be clearly layered, from top to bottom, into aqueous phase, protein, and organic phase. Carefully transfer the aqueous phase containing tissue RNA to a new centrifuge tube.

[0046] 5) Add 0.5 mL of isopropanol, shake well immediately, and let stand for 10 minutes.

[0047] 6) Centrifuge at 11,000 rpm at 4°C for 10 minutes. The white precipitate observed at the bottom of the centrifuge tube is RNA. After discarding the supernatant, add 1 mL of 75% anhydrous ethanol to wash the precipitate, centrifuge at 6,700 rpm at 4°C for 5 minutes, repeat this step three times, and finally retain the precipitate.

[0048] 7) After drying the precipitate at room temperature for 5-10 minutes, add an appropriate amount of 55°C enzyme-free water to dissolve the precipitate to obtain a tissue RNA solution.

[0049] 3. Reverse transcription and real-time fluorescence quantitative PCR

[0050] According to the instructions of the reverse transcription kit, the extracted tissue RNA was quantitatively reverse transcribed to obtain cDNA.

[0051] Real-time fluorescence quantitative PCR (qRT-PCR) was performed according to the instructions of qPCR SYBR Green Master Mix. Each sample was tested in triplicate. β-actin was used as an internal control. The relative expression of RNA was calculated using the 2-ΔΔCt method.

[0052] 4. Results

[0053] like Figure 1As shown, H&E staining showed that 500 μg of PLpro could induce significant intestinal inflammatory response. qRT-PCR results showed that compared with the control group, the expression levels of TNF-α, IL-1α mRNA and ISG15 mRNA in the PLpro protein treatment group were significantly increased, indicating an inflammatory response in the intestine.

[0054] Example 2: Detection of the interfacial activity of coptisine sulfate blocking the binding of papain-like protein to ISG15

[0055] 1. Drug Inhibitory Activity Experiment

[0056] 1) Prepare the buffer required for the experiment: 25 mM NaH2PO4, 150 mM NaCl, 0.1 mg / ml BSA and 7.5 μM ZnCl2.

[0057] 2) Take papain-like protein PLpro, with a molecular weight of 36.8 kD, and dilute PLpro with buffer to a concentration of 2 μM.

[0058] 3) Take ISG15-FITC protein with a molecular weight of 9.5 kD and dilute ISG15-FITC with buffer to a concentration of 200 nM.

[0059] 4) In this experiment, the initial drug concentration was 1 μM (the solvent was dimethyl sulfoxide (DMSO)). The drug was diluted in half, and a total of 8 concentration gradients were set, with 3 replicate wells for each concentration.

[0060] 5) Take 50 μL of diluted PLpro and place it in a 96-well black plate.

[0061] 6) Add 1 μL of coptisine sulfate to the well.

[0062] 7) In the control wells, 1 μL DMSO was used instead of coptisine sulfate.

[0063] 8) Mix well and incubate for 10 minutes.

[0064] 9) Take 50 μL of diluted ISG15-FITC and put it in the same well, so that the final concentrations of PLpro and ISG15-FITC are 1 μM and 100 nM, respectively.

[0065] 10) Incubate at room temperature for 1 hour.

[0066] 11) Place the sample in a microplate reader to read the fluorescence value, with an excitation wavelength of 483 nm and an emission wavelength of 535 nm.

[0067] 2. Results

[0068] like Figure 2As shown in the figure, the copy number of the DMSO group was used as a reference, and the drug inhibition rate curve was fitted according to the inhibition rate of different drug concentrations using GraphPad Prism8.4.2 software, and the half-inhibitory effective concentration IC of coptis sulfate on the binding of SARS-CoV-2 PLpro to ISG15 was calculated. 50 It is 4.48μM.

[0069] Example 3: Effect of Coptisine Sulfate on Alleviating Colon Inflammation Produced by Papain-like Protein of SARS-CoV-2 at the Animal Level

[0070] 1. Experimental design

[0071] 8-week-old C57BL / 6J mice were housed in an environment with a constant temperature of 26°C, a 14 / 10 hour periodic change of light and dark time, and the animals were free to eat and drink water in this environment. The dosage was set at 25 mg / kg and 100 mg / kg, and each drug dosage was used as a dosage group. The drug solvent was used as a control group, with 5 mice in each group. The compound was dissolved in a solvent of 10% DMSO, 40% PEG300, 5% Tween 80 and 45% saline. In this experiment, the pre-treatment method was adopted, and the drug was administered continuously for 3 days by gavage before animal modeling. After the administration was completed, the animal model was constructed according to the method of Example 1.

[0072] 2. Experimental methods

[0073] 2.1 Modeling method

[0074] Follow the method in Example 1.

[0075] 2.2 RNA extraction and reverse transcription

[0076] Follow the method in Example 1.

[0077] 2.3 Real-time fluorescence quantitative PCR

[0078] Follow the method in Example 1.

[0079] 3. Results

[0080] like Figure 3 As shown in the figure, after treatment with coptisine sulfate, the expression levels of TNFα, IL-1α mRNA and ISG15 mRNA in the intestine of mice were significantly reduced, and the intestinal inflammation of mice was significantly alleviated.

[0081] The above is only a specific embodiment of the present invention, not all embodiments. Any equivalent changes made to the technical solution of the present invention by a person skilled in the art after reading the present specification shall fall within the scope of the claims of the present invention.

Claims

1. Use of coptisine sulfate or its pharmaceutically acceptable salt in the preparation of a drug for preventing and / or treating viral enteritis, characterized in that: The virus is a coronavirus, and the molecular formula of the coptisine sulfate is C 19 H 15 NO8S, molecular weight is 417.39, and the structural formula is as follows:

2. The use according to claim 1, characterized in that: The coronaviruses include HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, severe acute respiratory syndrome coronavirus SARS-CoV, severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 and Middle East respiratory syndrome coronavirus MERS-CoV.

3. Use of coptisine sulfate or its pharmaceutically acceptable salt in the preparation of a drug for preventing and / or treating enteritis induced by papain-like protein, characterized in that: The molecular formula of the coptisine sulfate is C 19 H 15 NO8S, molecular weight is 417.39, and the structural formula is as follows:

4. The use according to claim 3, characterized in that: The papain-like protein is the papain-like protein of SARS-CoV-2.

5. Use of coptisine sulfate or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting the binding of papain-like protein of coronavirus to ISG15, characterized in that: The molecular formula of the coptisine sulfate is C 19 H 15 NO8S, molecular weight is 417.39, and the structural formula is as follows:

6. The use according to claim 5, characterized in that: The coronaviruses include HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, severe acute respiratory syndrome coronavirus SARS-CoV, severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 and Middle East respiratory syndrome coronavirus MERS-CoV.

Images