Application of thiazolidone compound in preparation of CRM1 protein inhibitor

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of thiazolidinone compounds or pharmaceutically acceptable salts thereof in preparation of CRM1 protein inhibitors, and the thiazolidinone compounds have the following structure. Preferably, the CRM1 protein inhibitor is a medicine for treating chronic inflammatory diseases, and the chronic inflammatory diseases are inflammatory bowel diseases, rheumatic arthritis, gout, atherosclerosis or type II diabetes mellitus. The thiazolidone compound has high inhibitory activity on CRM1 protein, significantly reduces the expression quantity of key inflammatory factors and nitric oxide in an inflammatory cell model, has an anti-inflammatory effect superior to that of a clinical first-line anti-inflammatory drug mesalazine, has small side effects, is suitable for clinical application, and has broad application prospects. Therefore, the method has huge potential market space and economic benefits. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to an application of a thiazolidinone compound in the preparation of a CRM1 protein inhibitor. Background Art

[0002] The inflammatory response is a protective immune response of the body to invading foreign bodies. The inflammatory response involves the joint action of multiple inflammatory cells, inflammatory cytokines, inflammatory mediators, adhesion molecules and chemokines. Inflammatory cytokines include interleukin (IL), tumor necrosis factor-α (TNF-α), interferon-γ (INF-γ) and monocyte colony stimulating factor. Inflammatory mediators include platelet-activating factor, prostaglandins, leukotrienes and matrix metalloproteinases. Adhesion molecules include intercellular adhesion factors and integrins; chemokines include monocyte chemoattractant protein-1 and transforming growth factor-β. Chronic inflammation is caused by long-term exposure to inflammatory stimuli, and its effects can accumulate throughout life, eventually manifesting as many diseases, including inflammatory bowel disease, rheumatoid arthritis, gout, atherosclerosis, diabetes, arthritis, etc.

[0003] Chromosome region stabilizing protein CRM1 exists in all eukaryotic cells and is one of the most important nuclear export receptors. It is involved in the export of a variety of cargo proteins, including tumor suppressors, anti-inflammatory factors IκBα, and cycle regulatory proteins. CRM1 binds to the nuclear export signal (NES signal) of cargo proteins and RanGTP molecules to form a ternary complex. The concentration gradient of RanGTP between the nucleus and the cytoplasm provides energy for the transport of proteins, which then pass through the nuclear pore complex and enter the cytoplasm.

[0004] Relevant literature has reported that CRM1 protein inhibitors can be used to treat related chronic inflammation. CRM1 small molecule inhibitors can regulate the nuclear export pathway to cause the accumulation of anti-inflammatory factor IκBα in the cell nucleus, thereby regulating the NF-κB inflammatory signaling pathway and NLRP3 inflammasome, and inhibiting the production of proinflammatory cytokines, thereby exerting anti-inflammatory effects. These documents are: PMC2910824, PMC6482348, PMID: 32223194 and PMID: 35721113, etc.

[0005] Recently, the inventors have discovered a novel class of thiazolidinone compounds in preliminary studies, which can be used to prepare CRM1 protein inhibitors. These thiazolidinone compounds show good anti-inflammatory therapeutic effects for chronic inflammatory diseases such as inflammatory bowel disease, rheumatoid arthritis, gout, atherosclerosis, type II diabetes, etc., and their effects are better than the commonly used clinical anti-inflammatory drug mesalazine (5-ASA, 5-aminosalicylic acid). At present, there is no related report in the prior art about the use of these thiazolidinone compounds in the preparation of CRM1 protein inhibitors, and then potentially for the treatment of chronic inflammatory diseases. Summary of the invention

[0006] Based on the above research results, the present invention provides a new application of a class of thiazolidinone compounds in the preparation of CRM1 protein inhibitors and in the treatment of chronic inflammatory diseases such as inflammatory bowel disease, rheumatoid arthritis, gout, atherosclerosis, type II diabetes, etc.

[0007] In order to achieve the above-mentioned object of the present invention, the technical solution adopted by the present invention is:

[0008] The present invention provides a use of a thiazolidinone compound or a pharmaceutically acceptable salt thereof in the preparation of a CRM1 protein inhibitor, wherein the thiazolidinone compound (C-08) has the following structure of Formula I:

[0009]

[0010] In one embodiment, the present invention uses Fortebio Octet biofilm layer optical interferometry (BLI) to measure the interaction between CRM1 protein and small molecule compounds, and the affinity and binding dissociation constant can be obtained by real-time detection of binding and dissociation of analytes at different concentrations. The results show that the thiazolidinone compound (C-08) has a high affinity with the CRM1 protein.

[0011] In one embodiment, the present invention uses Western blotting (WB) technology to measure the effect of thiazolidinone compound (C-08) on the expression of CRM1 protein in cells. The results show that under the action of thiazolidinone compound (C-08), the expression of CRM1 protein in cells is dose-dependently downregulated.

[0012] In one embodiment, the present invention utilizes lipopolysaccharide (LPS) to stimulate mouse mononuclear macrophages (RAW264.7) to construct a RAW264.7 cell inflammation model. The effect of thiazolidinone compounds (C-08) on the levels of inflammatory cytokines in the LPS-stimulated RAW264.7 cell model was investigated. The results showed that at a safe dose, thiazolidinone compounds (C-08) can significantly reduce the expression of various key inflammatory factors (TNF-α, IL-6 and IL-1β) in the cell inflammation model.

[0013] In one embodiment, the present invention investigates the effect of thiazolidinone compounds (C-08) on the nitric oxide (NO) level of the RAW264.7 inflammatory cell model stimulated by LPS. The nitric oxide (NO) level in cells is an important indicator for measuring the level of cellular inflammation. The results show that as the dose increases, the content of NO decreases, inhibiting the development of inflammation, and the effect is significantly better than the clinical drug mesalazine.

[0014] As an alternative, in the above application, the CRM1 protein inhibitor is a drug for treating chronic inflammatory diseases.

[0015] In the present invention, the medicine comprises an effective dose of thiazolidinone compounds.

[0016] Furthermore, in the present invention, the medicine also contains pharmaceutically acceptable excipients.

[0017] As an option, in the above application, the chronic inflammatory disease includes but is not limited to: inflammatory bowel disease, rheumatoid arthritis, gout, atherosclerosis or type II diabetes.

[0018] As an alternative, in the above application, the CRM1 protein inhibitor is a tool drug for in vitro scientific research.

[0019] As an optional mode, in the above application, the pharmaceutically acceptable salt of the thiazolidinone compound is an acid addition salt.

[0020] As an optional mode, in the above application, the acid addition salt is a salt formed by the thiazolidinone compound and an inorganic acid or a salt formed by the thiazolidinone compound and an organic acid.

[0021] Those skilled in the art can directly or indirectly add the thiazolidinone compounds to various common pharmaceutically acceptable excipients required for preparing different dosage forms, and prepare common preparations using conventional pharmaceutical preparation methods.

[0022] As an optional mode, in the above application, the drug is administered orally, by injection, rectal perfusion, sublingual administration, inhalation, external topical administration or systemic transdermal administration.

[0023] As an optional mode, in the above application, the dosage form of the drug is one of injection, capsule, tablet, sustained release agent, suppository or spray.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] As can be seen from the above technical scheme, the present invention proposes a new application of a class of thiazolidinone compounds. Experimental results show that this class of thiazolidinone compounds can significantly reduce the content of inflammatory factors and nitric oxide in inflammatory cell models, and its anti-inflammatory effect is better than the clinical first-line anti-inflammatory drug mesalazine (5-ASA). Therefore, the present invention has a high potential clinical application value and development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention is attached Figure 6 Of which:

[0027] Figure 1 The affinity between CRM1 protein and thiazolidinone compound (C-08) was measured using Fortebio octet biofilm layer optical interferometry (BLI). The horizontal axis is the binding time (s) and the vertical axis is the binding signal (nm).

[0028] Figure 2 , Western blotting (WB) technology was used to measure the effect of thiazolidinone compounds (C-08) on the expression of CRM1 protein in cells.

[0029] Figure 3 , the effect of thiazolidinone compound (C-08) on nitric oxide level in LPS-stimulated RAW264.7 inflammatory cell model. The horizontal axis is the compound concentration (μM) and the vertical axis is the nitric oxide (NO) concentration.

[0030] Figure 4 ELISA was used to detect the effect of thiazolidinone compound (C-08) on the expression level of inflammatory cytokine TNF-α in LPS-stimulated RAW264.7 cells. The horizontal axis is the compound concentration (μM) and the vertical axis is the expression level of TNF-α cytokine.

[0031] Figure 5ELISA was used to detect the effect of thiazolidinone compounds (C-08) on the level of cytokine IL-6 in the RAW264.7 cell model stimulated by LPS. The horizontal axis is the compound concentration (μM) and the vertical axis is the expression level of IL-6 cytokine.

[0032] Figure 6 , ELISA technology was used to detect the effect of thiazolidinone compounds (C-08) on the level of inflammatory cytokine IL-1β in the LPS-stimulated RAW264.7 cell model, where the horizontal axis is the compound concentration (μM) and the vertical axis is the expression level of IL-1β inflammatory cytokine.

[0033] in, Figures 3 to 6 5-ASA in mesalazine is the first-line clinical anti-inflammatory drug. DETAILED DESCRIPTION

[0034] The present invention provides a new application of a class of thiazolidinone compounds. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. This discovery has been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods described herein without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention.

[0035] The following non-limiting embodiments can enable ordinary technicians in the field to understand the present invention more comprehensively. It must be noted that the following embodiments are used to illustrate the present invention rather than to limit the present invention. All improvements made to the present invention based on the essence of the present invention belong to the scope of protection required by the present invention.

[0036] Example

[0037] In order to more simply and intuitively demonstrate the technical solutions, purposes and advantages of the present invention, the technical solutions of the present invention are described in detail below in conjunction with specific embodiments. Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be purchased through commercial channels.

[0038] Example 1: Binding of thiazolidinone compounds to the target protein CRM1 protein

[0039] First, the target protein CRM1 protein was biotinylated and amino-coupled with the EZ-Llink biotin reagent; the target protein CRM1 protein was solidified, and 4 super streptavidin affinity sensors (SSA) were selected and pre-wetted in PBS for more than 10 minutes in two rows. Two sensors were used to solidify the biotinylated CRM1 protein, and the other two sensors were used as reference sensors, and no solidification of protein was required; the protein was bound to the small molecule, and small molecules of different concentrations were prepared, and dissociated and bound to the protein according to the procedure; the results were analyzed by double subtraction experimental analysis using Octet analysis software Data Analysis. The results showed that the binding constant K D =6.15×10 -7 μM (~610nM), indicating that C-08 can bind well to CRM1 protein and has a strong binding affinity. Figure 1 .

[0040] Example 2: Effects of thiazolidinone compounds on CRM1 protein expression in cells

[0041] Cells in the logarithmic growth phase were selected and inoculated in 6-well plates; after overnight, different concentrations of C-08 (10μM, 5μM, 2.5μM) were treated for 48 hours, and the whole cell protein was extracted by RIPA lysis buffer; the protein concentration was determined by BCA protein quantification kit; 40μg protein was taken for electrophoresis; gel transfer; blocking solution was used for one hour; primary antibody was used at 4℃ overnight; membrane was washed; secondary antibody was incubated at room temperature for 2 hours; membrane was washed; ECL development was performed. The results showed that C-08 can effectively inhibit the expression of CRM1 protein in cells. For specific results, see Figure 2 .

[0042] Example 3: Effects of Thiazolidinone Compounds on Nitric Oxide Levels in RAW264.7 Cells

[0043] Construction of inflammation model and drug administration: cells in logarithmic growth phase were selected and 5×10 4 / well were inoculated in a 96-well plate and cultured overnight in a 37°C, 5% CO2 incubator. The next day, the cell status was observed, and a blank control group, a model group, a treatment group, and a positive control group (mesalazine, 5-ASA) were set up. RAW264.7 cells were stimulated with 1μg / mL of LPS. Treatment group: After RAW264.7 cells were treated with C-08 (10μM, 5μM, 2.5μM) for 12 hours, 1μg / mL LPS was added to stimulate the cells for 12h. Blank control group: treated with an equal amount of culture medium at the same time point. Model group: After the cells were treated with an equal amount of culture medium for 12h, 1μg / mL LPS was added to stimulate the cells for 12h. Positive control group: RAW264.7 cells were treated with mesalazine (5μM) for 12 hours, and 1μg / mL LPS was added to stimulate the cells for 12h. After 24 hours, the cell culture supernatant was taken and centrifuged. Take the cell supernatant and detect the nitric oxide content according to the instructions of the total nitric oxide detection kit. The results showed that compared with the blank control group, the NO content in the model group showed a significant upward trend; compared with the model group, as the dose of C-08 increased, the NO content showed a downward trend, inhibiting the development of inflammation, and significantly better than the clinical drug mesalazine (5-ASA). Figure 3 .

[0044] Example 4: Effects of Thiazolidinone Compounds on the Expression Levels of Inflammatory Cytokines

[0045] The inflammatory cell model was constructed in the same manner as in Example 3, and the drug was administered. After 24 hours, the cell culture supernatant was taken and centrifuged. The supernatant was taken and the expression levels of inflammatory cytokines TNF-α, IL-6 and IL-1β were detected by ELISA. The results showed that after LPS stimulation of RAW264.7 cells, the model group significantly increased the expression of inflammatory cytokines TNF-α, IL-6 and IL-1β compared with the blank group; at a safe dose, C-08 significantly reduced the expression of various inflammatory cytokines, among which C-08 (5μM) was superior to the clinical drug mesalazine (5μM). For specific results, see Figures 4 to 6 ( Figure 4 :TNF-α; Figure 5 :IL-6; Figure 6 :IL-1β).

Claims

1. A use of a thiazolidinone compound or a pharmaceutically acceptable salt thereof in the preparation of a CRM1 protein inhibitor, characterized in that: The thiazolidinone compound has the following structure:

2. The use according to claim 1, characterized in that: The CRM1 protein inhibitor is a drug for treating chronic inflammatory diseases.

3. The use according to claim 2, characterized in that: The chronic inflammatory disease is inflammatory bowel disease, rheumatoid arthritis, gout, atherosclerosis or type II diabetes.

4. The use according to claim 1, characterized in that: The CRM1 protein inhibitor is a tool drug used in in vitro scientific research.

5. The use according to claim 1, characterized in that: The pharmaceutically acceptable salts of thiazolidinone compounds are acid addition salts.

6. The use according to claim 5, characterized in that: The acid addition salt is a salt formed by the thiazolidinone compound and an inorganic acid or a salt formed by the thiazolidinone compound and an organic acid.

7. The use according to claim 2, characterized in that: The drug is administered orally, by injection, by rectal perfusion, by sublingual administration, by inhalation, by topical administration in vitro, or by systemic transdermal administration.

8. The use according to claim 2, characterized in that: The dosage form of the drug is one of injection, capsule, tablet, sustained-release agent, suppository or spray.

Images