Small molecule capable of inhibiting clostridium difficile infection

Abstract

The invention relates to the technical field of medicines, in particular to a small molecule capable of inhibiting clostridium difficile infection, which is characterized in that alkaloid is extracted from white lotus seeds, and the alkaloid is liensinine. Experiments show that liensinine reduces the toxicity of TcdB through the effects of inhibiting autophagy pathways and the like, finally actin is normally polymerized and depolymerized, in mechanism exploration, the number of TcdB combined with cells is reduced by combining cell surfaces with experimental surfaces in the presence of liensinine, and the discoveries show that liensinine has an inhibiting effect on TcdB-induced cytotoxicity, and can be used for preparing a medicine for treating actin. Meanwhile, compared with metronidazole and vancomycin which are used for inhibiting clostridium difficile infection in the prior art, the drug resistance is frequent, the negative influence on intestinal flora is small, and the treatment effect is guaranteed.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a small molecule capable of inhibiting Clostridium difficile infection. Background Art

[0002] Clostridium difficile infection (CDI) is a Gram-positive, strictly anaerobic, spore-forming, toxin-mediated intestinal disease that can cause diarrhea ranging from mild to severe (i.e., antibiotic-associated diarrhea). In severe cases, it may even lead to pseudomembranous colitis with serious complications such as toxic megacolon, intestinal perforation, and death.

[0003] Autophagy is an important degradation and recycling process inside cells, which plays an important role in maintaining the stability of the intracellular environment and responding to inflammation. In recent years, the role of the autophagy pathway in antibacterial and anti-inflammatory research has gradually attracted attention. Studies have found that autophagy can not only prevent cell damage by degrading damaged organelles and proteins, but also affect inflammatory responses by regulating the activity of immune cells. In intestinal immune regulation, autophagy can help maintain the integrity of the intestinal barrier and prevent the invasion of pathogens. At present, drug research targeting autophagy is actively underway, aiming to develop therapeutic drugs that can effectively regulate autophagy.

[0004] Clinically, CDI is an important pathogenic factor of antibiotic-associated diarrhea (AAD), antibiotic-associated colitis (AAC), and pseudomembranous colitis (PMC). Specifically, about 15% to 25% of AAD, 50% to 75% of AAC, and up to 95% to 100% of PMC are caused by CDI. In recent years, the morbidity, mortality, and recurrence rate of CDI after cure have shown a significant upward trend, and the infected population has expanded to children and adolescents who have not used antibiotics for a long time. Its treatment has become a major clinical challenge.

[0005] Currently, CDI mainly infects the host through its exotoxins TcdA and TcdB, among which TcdB is the main cause of severe pathological effects. Due to the inherent insensitivity of Clostridium difficile to most antibiotics, there has been a lack of specific means for the treatment of CDI. Traditional treatments, such as metronidazole and vancomycin, are first-line treatment drugs, but the frequent occurrence of drug resistance and negative effects on intestinal flora limit their efficacy.

[0006] Liensinine is a dibenzyltetrahydroisoquinoline alkaloid extracted from lotus seed embryos. It has been shown to have multiple protective effects on cardiovascular diseases. The inventors have found that lotus seed extracts have a significant inhibitory effect on the exotoxin TcdB of Clostridium difficile, among which lisinine has the most significant inhibitory effect. In addition, lisinine can also reduce the inflammatory response caused by infection. Its mechanism may involve inhibiting inflammatory signaling pathways, such as NF-κB (transcription factor protein family) and MAPK pathways, thereby reducing the production of inflammatory factors. However, so far, most reports on it have focused on pharmacology, extraction, and analysis, and it has not yet been found to have an inhibitory effect on Clostridium difficile infection. Summary of the invention

[0007] To solve the above problems, the purpose of the present invention is to provide a small molecule that can inhibit Clostridium difficile infection and explore its inhibitory effect on TcdB cytotoxicity, in order to be able to apply this extract to the treatment of CDI.

[0008] A small molecule capable of inhibiting Clostridium difficile infection is obtained by extracting an alkaloid from white lotus seeds, wherein the alkaloid is liensinine.

[0009] Furthermore, the chemical structural formula of liesinine is as shown in Formula I:

[0010] Furthermore, the liensinine reduces the toxicity of TcdB by reducing the intracellular Rac1 glycosylation induced by TcdB and inhibiting the autophagy pathway, ultimately achieving the purpose of treating Clostridium difficile infection by allowing actin to polymerize and depolymerize normally.

[0011] An application of a small molecule capable of inhibiting Clostridium difficile infection, and an application of a compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating Clostridium difficile infection.

[0012] Further, it includes administering to the individual a therapeutically effective amount of an iminosugar of Formula I or a pharmaceutically acceptable salt thereof, which includes, for example, isofagomine, nooromycin, or a combination thereof.

[0013] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0014] Furthermore, the drug is an oral preparation or an injectable preparation.

[0015] Furthermore, the oral preparation is a tablet, capsule or granule; the injection preparation is an injection solution or powder injection.

[0016] Furthermore, the drug is composed of 8%-92% of liesinine compounds and 8%-92% of pharmaceutically acceptable excipients.

[0017] Beneficial effects: 1. The present invention provides a new application of a small molecule compound of liensin, especially the application of a small molecule compound of liensin in inhibiting TcdB in Hutu and Hela cells. Experiments have shown that liensin can reduce the intracellular Rac1 glycosylation induced by TcdB, while reducing the actin structure. In the mechanism exploration, cell surface binding experiments have shown that in the presence of liensin, the number of cell-bound TcdB is reduced, indicating that liensin has an inhibitory effect on TcdB-induced cytotoxicity. At the same time, compared with the traditional metronidazole and vancomycin used to inhibit Clostridium difficile infection, its frequent occurrence of drug resistance and negative impact on intestinal flora are less, ensuring the efficacy of treatment.

[0018] 2. Liensinine can regulate the autophagy process by affecting the expression of autophagy-related genes. In cell models, lisinine can inhibit the activity of key proteins in the autophagy pathway, such as the PI3K / AKT / mTOR signaling pathway, thereby inhibiting the occurrence of autophagy. In addition, lisinine can also regulate the autophagy process by affecting the expression of autophagy-related proteins, such as LC3 and p62. The role of lisinine in regulating autophagy has also been verified in animal models. Studies have shown that lisinine can reduce organ damage caused by abnormal autophagy.

[0019] 3. In the study, the applicant identified two small molecule compounds, PF-429242 and U18666A, which showed inhibitory effects on TcdB-induced cytotoxicity by inhibiting the SREBP-2 pathway and cholesterol synthesis. Analysis of multiple components in the traditional Chinese medicine Shenling Baizhu San found that lotus seed extract can effectively inhibit the cytotoxicity of TcdB, a key pathogenic factor of Clostridium difficile infection. Further studies have shown that in an in vitro model, lianxinine can significantly inhibit the growth of Clostridium difficile and has a significant inhibitory effect on Clostridium difficile exotoxin TcdB. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram of the morphological changes of the toxicity of liensinine to TcdB-induced Hela cells of the present invention; Figure 2 This is a schematic diagram of the morphological changes of the toxicity of liesinine to TcdB-induced Hutu cells of the present invention; Figure 3 It is a schematic diagram of the cytotoxicity index (CI) curve of Hela cells induced by TcdB detected by the real-time label-free dynamic cell analysis technology (RTCA) of Hela cells of the present invention; Figure 4 This is a schematic diagram of the difference in Hela cells in the experimental group of the present invention after adding toxin for 6 hours; Figure 5It is a schematic diagram of the cytotoxicity index (CI) curve of Hutu cells induced by TcdB detected by the real-time label-free dynamic cell analysis technology (RTCA) of Hutu cells of the present invention; Figure 6 This is a schematic diagram of the difference in Hela cells of the present invention when toxins are added for 6 hours; Figure 7 This is a schematic diagram of Western Blot experiments of proteins extracted from Hela cells treated with different concentrations of liesinine for 24 hours and then added with toxin for 6 hours; Figure 8 The grayscale of the bands in the Western Blot experiment of Hela cells treated by the present invention is calculated and the difference diagram of the analysis is analyzed; Fig. 9 The present invention treats Hela cells with 10 μM liensinine and 10 pM TcdB, fixes the cells after 6 hours, and performs immunofluorescence staining. The blue represents the nucleus, the green represents the actin cytoskeleton, and the red represents the non-glycosylated Rac1 (40X); Fig.10 Schematic diagram of Western Blot experiment of proteins extracted from Hutu cells treated with different concentrations of liesinine for 24 hours and then added with TcdB for 6 hours; Fig.11 The grayscale of the bands in the Western Blot experiment was calculated for the Hutu cells treated by the present invention, and the difference map of the analysis was analyzed; Fig.12 The present invention treats Hutu cells with 10 μM liensinine and 10 pM TcdB, fixes the cells after 6 hours, and performs immunofluorescence staining. Blue represents the cell nucleus, green represents the actin cytoskeleton, and red represents the non-glycosylated Rac1 (40X); Fig.13 The protein band diagrams of Caspase-1, Beclin-1, Atg16L1, Atg7 and HMGB1 in the WB experiment were shown in the figure after Hela cells were treated with different concentrations of liesinine for 24 h and TcdB was added for 6 h. Fig.14 This is a quantitative analysis difference diagram of the expression level of Atg7 protein in Hela cells of the present invention; Fig.15 This is a quantitative analysis difference diagram of the expression level of Atg16L1 protein in Hela cells of the present invention; Fig.16 This is a quantitative analysis difference diagram of the Beclin-1 protein expression level in Hela cells of the present invention; Fig.17This is a quantitative analysis difference diagram of the expression level of Caspase-1 protein in Hela cells of the present invention; Fig.18 This is a quantitative analysis difference diagram of the HMGB1 protein expression level in Hela cells of the present invention; Fig.19 This is a schematic diagram of treating pre-cooled HuTu cells with TcdB (500 pM) to allow the toxin to bind, and using liensinine or DMSO as a control to analyze the binding of TcdB; Fig. 20 The SDS-PAGE and Coomassie staining diagrams are used to analyze the effect of liensinine on the cysteine ​​protease domain (CPD) of TcdB in vitro. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Example: A small molecule that can inhibit Clostridium difficile infection, an alkaloid extracted from white lotus seeds, the alkaloid is liesinine. The chemical structure of liesinine is shown in Formula I:

[0023] Among them, the said liesinine reduces the intracellular Rac1 glycosylation induced by TcdB, and liesinine reduces the toxicity of TcdB by inhibiting the autophagy pathway, and finally achieves the purpose of treating Clostridium difficile infection by allowing actin to polymerize and depolymerize normally.

[0024] An application of a small molecule that can inhibit Clostridium difficile infection, and an application of a compound shown in Formula I or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating Clostridium difficile infection. It includes administering to an individual a therapeutically effective amount of an iminosugar of Formula I or a pharmaceutically acceptable salt thereof, which includes, for example, isofagomine, neunomycin or a combination thereof. The drug also includes a pharmaceutically acceptable excipient. The drug is an oral preparation or an injectable preparation. The oral preparation is a tablet, a capsule or a granule; the injectable preparation is an injectable solution or a powder injection. The drug is composed of 8%-92% of a liensinine compound and 8%-92% of a pharmaceutically acceptable excipient.

[0025] Studies have shown that Shenling Baizhu powder can significantly promote the growth of probiotics in the human body, such as lactobacillus and bifidobacterium. The inventors studied the inhibitory effect of Shenling Baizhu powder compound and its single herbal extracts on Clostridium difficile TcdB in HuTu and Hela cells.

[0026] The research results show that the extracts from different parts of the Shenling Baizhu San compound have no obvious inhibitory effect on TcdB in HuTu cells, but the white lotus seeds, yam and Poria cocos in the single Chinese medicine have a significant inhibitory effect on TcdB in Hutu cells.

[0027] Further research results showed that the small molecule compound of liensinine in white lotus seeds also had a significant inhibitory effect on TcdB in Hutu and Hela cells. Liensinine reduced TcdB-induced intracellular Rac1 glycosylation and reduced actin structure. In the mechanism exploration, the cell surface binding experiment showed that in the presence of liensinine, the number of cell-bound TcdB was reduced. By analyzing the inherent autoprotein hydrolysis of the toxin in the presence of Insp6, it was tested in vitro whether liensinine had an effect on the cysteine ​​protease domain (CPD) of TcdB and did not affect the autocatalytic processing of TcdB induced by Insp6, indicating that the CPD-related activity of TcdB was not affected by liensinine.

[0028] The basic results of the research are as follows: 1. Effect of liensinine on TcdB-induced cytotoxicity Logarithmically growing Hela cells and Hutu cells were incubated with different concentrations of liensinine (0.1 μM, 1 μM, 10 μM) for 24 h, and then 10 pM TcdB was added. Microscopic images were taken 3 h and 6 h after the addition of TcdB.

[0029] The results showed that 3 hours after the addition of the toxin to Hela cells and Hutu cells, compared with the blank group, the cells in the toxin group became rounded obviously, only a small number of cells in the 0.1μM and 1μM concentration drug groups became rounded, and no obvious rounded cells were found in the 10μM group; 6 hours after the addition of the toxin, compared with the blank group, all the cells in the toxin group had become rounded, and the number of rounded cells in the drug group gradually decreased with the increase of the concentration (see the attached manual for details). Figure 1-Figure 2 ).

[0030] The above results indicate that liensinine has an inhibitory effect on TcdB-induced cytotoxicity.

[0031] 2. RTCA real-time label-free technology to detect the effect of liensinine on TcdB cell toxicity Logarithmically growing Hela cells and Hutu cells were incubated with different concentrations of liesinine (0.1μM, 1μM, 10μM) for 24h, and then 10pM TcdB was added. The cytotoxicity of liesinine against TcdB was monitored in real time by RTCA method.

[0032] The results showed that in Hela cells and Hutu cell lines, compared with the blank group, only the TcdB curve showed an obvious downward trend, and with the increase of liesinine concentration, the curve decline rate gradually slowed down; 6 hours after the addition of toxin, the 10μM liesinine group of Hela cell line (**P<0.01) and the 10μM liesinine group of Hutu cell line (*P<0.05) were statistically significant compared with the toxin group (see the appendix of the instruction manual for details). Figure 3-Figure 6 ).

[0033] in: Figure 3 and Figure 5 The x-axis is time, and the y-axis is the impedance value measured in real time after the toxin is added / the first impedance value measured after the toxin is added. The larger the value, the more cells adhere to the wall and the less cell death. Figure 4 and Figure 6 n = 3, compared with the blank group: ####P < 0.0001, compared with the TcdB group: *P < 0.05, **P < 0.01; 3. Effect of Liensinine on TcdB-induced Rac1 glycosylation expression in cells After confirming that liesinine has an inhibitory effect on TcdB-induced cytotoxicity, in order to further clarify the protective effect of liesinine on TcdB-induced cytotoxicity, WB and immunofluorescence detection were performed using an antibody that specifically recognizes non-glycosylated glycosylation.

[0034] WB results showed that compared with the Control group, the non-glycosylated Rac1 bands in Hela cells and Hutu cells treated with TcdB alone were lighter, which was statistically significant; compared with TcdB, with the increase of the concentration of liesinine, the expression level of intracellular Rac1 protein was significantly upregulated, and the non-glycosylated Rac1 in the cells of the 10μM group of Hela cells and the 1μM and 10μM groups of Hutu cells (*P < 0.05) was statistically significant. The same results were obtained in immunofluorescence. In addition, compared with the Control group, no damage was found in the actin structure of cells treated with 10μM liesinine alone; while the actin structure of cells treated with TcdB alone was severely damaged, and the actin structure of some cells treated with 10μM liesinine and then added with toxins was normal (see the attached manual for details). Figure 7-Figure 12 ).

[0035] 4. Effects of Liensinine on the Autophagy Signaling Pathway In order to explore the inhibitory mechanism of liensinine on the specific signaling pathway of TcdB, this study used Western Blot technology to conduct a detailed analysis of the expression changes of key marker proteins in signaling pathways such as cell apoptosis, pyroptosis, autophagy, necrosis and ferroptosis.

[0036] The results showed that after pretreatment with liensinine, the expression levels of key autophagy-related proteins including Caspase-1, Beclin-1, Atg16L1, Atg7, HMGB1, etc. showed significant differences. Specifically, with the gradual increase of liensinine concentration, the expression levels of these proteins showed a decreasing trend to varying degrees (see the attached manual for details). Figure 13-Figure 18 This finding suggests that liensinine may effectively slow down the cytotoxic response caused by TcdB by inhibiting the autophagy pathway.

[0037] 5. Effect of Liensinine on the Binding of TcdB to Cultured Cells Using an in vitro assay, cells were cooled to 4°C to minimize endocytosis and incubated with TcdB in the presence and absence of liesinine. Subsequently, cells were washed and bound TcdB was analyzed by immunoblotting. Here, the amount of cell-bound TcdB was slightly reduced in the presence of liesinine (see Appendix for details). Fig.19 ).

[0038] 6. Effect of liensinine on the cysteine ​​protease domain (CPD) of TcdB in vitro We further investigated the steps of toxin uptake, such as the intracellular processing of TcdB. We tested whether liensinine has an effect on the cysteine ​​protease domain (CPD) of TcdB in vitro by analyzing the intrinsic autoproteolysis of the toxin in the presence of InsP6.

[0039] It can be seen that liensinine does not affect the autocatalytic process of TcdB induced by InsP6, indicating that the CPD-related activity of TcdB is not affected by liensinine (see the attached manual for details). Fig. 20 The cationic drug n-ethylmaleimide (NEM), a well-established CPD inhibitor, blocked the autocatalytic processing of TcdB in the same experiment.

[0040] in, Fig.14 In the figure, liensinine or NEM (1 mM) was added, and the cysteine ​​protease activity was analyzed by SDS-PAGE and Coomassie staining after 1 h at 37°C. After successful cleavage, GTD (~63 kDa) was released from full-length TcdB (~270 kDa), and a representative SDS-PAGE is depicted.

[0041] Experiments have shown that lianxinine can reduce the intracellular Rac1 glycosylation induced by TcdB and reduce the actin structure. In the mechanism exploration, the cell surface binding experiment showed that in the presence of lianxinine, the number of cell-bound TcdB was slightly reduced, indicating that lianxinine has an inhibitory effect on TcdB-induced cytotoxicity. At the same time, compared with the traditional metronidazole and vancomycin used to inhibit Clostridium difficile infection, its frequent drug resistance and negative impact on intestinal flora are less, ensuring the efficacy of treatment.

[0042] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A small molecule that can inhibit Clostridium difficile infection, characterized in that: Alkaloids are extracted from white lotus seeds, and the alkaloids are liesinine.

2. A small molecule capable of inhibiting Clostridium difficile infection according to claim 1, characterized in that: The chemical structural formula of the liensinine is shown in Formula I: A small molecule capable of inhibiting Clostridium difficile infection as described in claim 1, characterized in that the liensinine reduces the toxicity of TcdB by inhibiting the autophagy pathway, and ultimately allows normal polymerization and depolymerization of actin to achieve the purpose of treating Clostridium difficile infection.

3. An application of a small molecule capable of inhibiting Clostridium difficile infection, characterized in that: Use of the compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating Clostridium difficile infection.

4. The use of a small molecule capable of inhibiting Clostridium difficile infection as claimed in claim 4, characterized in that: It includes administering to the individual a therapeutically effective amount of an iminosugar of Formula I or a pharmaceutically acceptable salt thereof, including, for example, isofagomine, nooromycin, or a combination thereof.

5. The use of a small molecule capable of inhibiting Clostridium difficile infection as claimed in claim 4, characterized in that: The drug also includes pharmaceutically acceptable excipients.

6. The use of a small molecule capable of inhibiting Clostridium difficile infection as claimed in claim 4, characterized in that: The medicine is an oral preparation or an injection preparation.

7. The use of a small molecule capable of inhibiting Clostridium difficile infection as claimed in claim 7, characterized in that: The oral preparation is a tablet, a capsule or a granule; the injection preparation is an injection solution or a powder injection.

8. The use of a small molecule capable of inhibiting Clostridium difficile infection as claimed in claim 4, characterized in that: The medicine consists of 8%-92% of liesinine compounds and 8%-92% of pharmaceutically acceptable auxiliary materials.

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