A small molecule that can inhibit clostridium difficile infection

Abstract

The present application relates to the technical field of medicine, specifically to a small molecule capable of inhibiting clostridium difficile infection, characterized by extracting alkaloids from white lotus seeds, wherein the alkaloids are lotus heart alkaloids. Experiments show that lotus heart alkaloids reduce the toxicity of TcdB by inhibiting the autophagy pathway and other effects, ultimately allowing actin to normally polymerize and depolymerize. In mechanism exploration, through cell surface binding experiments, it is found that the number of TcdB bound to cells is reduced in the presence of lotus heart alkaloids. These findings indicate that lotus heart alkaloids have an inhibitory effect on TcdB-induced cytotoxicity. Compared with metronidazole and vancomycin, which are traditionally used to inhibit clostridium difficile infection, lotus heart alkaloids have less frequent drug resistance and less negative impact on intestinal flora, ensuring the therapeutic efficacy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a small molecule capable of inhibiting Clostridium difficile infection. BACKGROUND

[0002] Clostridium difficile infection (CDI) is a gram-positive bacterium, strictly anaerobic, can produce spores, is a toxin-mediated intestinal disease, can cause from mild to severe diarrhea (i.e. antibiotic-associated diarrhea), and can even lead to pseudomembranous colitis, and is accompanied by severe complications such as toxic megacolon, intestinal perforation and death.

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

[0004] In clinical practice, CDI is an important pathogenic factor of antibiotic-associated diarrhea (AAD), antibiotic-associated colitis (AAC) and pseudomembranous enterocolitis (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 incidence, mortality and recurrence rate after cure of CDI have shown a significant upward trend, and the infected population has expanded to children and adolescent groups who have not long-term used antibiotics, and its treatment has become a major challenge in clinical practice.

[0005] Currently, CDI mainly infects the host through its exotoxins TcdA and TcdB, of 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 treatment methods, such as metronidazole and vancomycin, are first-line treatment drugs, but the frequent occurrence of drug resistance and the negative impact on intestinal flora limit their efficacy.

[0006] Lotus heart alkaloids is a kind of double benzyl tetrahydroisoquinoline alkaloids, extracted from embryo lotus, has been proved to have multiple protective effects on cardiovascular diseases, the inventors found that traditional Chinese medicine lotus extract has a significant inhibitory effect on the exotoxin TcdB of clostridium difficile, among which lotus heart alkaloids has the most significant inhibitory effect. In addition, lotus heart alkaloids 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 pathway, thereby reducing the production of inflammatory factors, but so far, the reports are mainly concentrated in pharmacology and extraction, analysis, and there is no report on its inhibitory effect on clostridium difficile infection. SUMMARY

[0007] To solve the above problems, the purpose of the present application is to provide a small molecule that can inhibit clostridium difficile infection, and to explore its inhibitory effect on TcdB cytotoxicity, so as to apply this extract to the treatment of CDI.

[0008] A small molecule that can inhibit clostridium difficile infection, alkaloids extracted from white lotus seed, the alkaloids are lotus heart alkaloids.

[0009] Further, the chemical structural formula of the lotus heart alkaloids is shown as formula I:

[0010]

[0011] Further, the lotus heart alkaloids can reduce the toxicity of TcdB by reducing TcdB-induced intracellular Rac1 glycosylation, inhibiting autophagy pathway, and ultimately making actin normal polymerization and depolymerization to achieve the purpose of treating clostridium difficile infection.

[0012] Application of a small molecule that can inhibit clostridium difficile infection, the application of the compound shown as formula I or its pharmaceutically acceptable salt in the preparation of a drug for treating clostridium difficile infection.

[0013] Further, it includes administering a therapeutically effective amount of iminosugar of formula I or its pharmaceutically acceptable salt to an individual, which includes, for example, isofagomine, norvancomycin or a combination thereof.

[0014] Further, the drug further comprises a pharmaceutically acceptable excipient.

[0015] Further, the drug is an oral preparation or an injection preparation.

[0016] Further, the oral preparation is a tablet, a capsule or a granule; the injection preparation is an injection solution or a powder injection.

[0017] Further, the drug is composed of 8%-92% lotus heart alkaloids and 8%-92% pharmaceutically acceptable excipients. Advantages

[0018] 1. The application provides a new application of a small molecule compound of nuciferine, in particular, an application of the small molecule compound of nuciferine in TcdB inhibition on Hutu and Hela cells. Experiments show that nuciferine can reduce TcdB-induced intracellular Rac1 glycosylation and decrease actin structures. In mechanism exploration, through cell surface binding experiments, it is shown that in the presence of nuciferine, the amount of TcdB combined with cells is reduced, which indicates that nuciferine has an inhibitory effect on TcdB-induced cytotoxicity. Meanwhile, compared with metronidazole and vancomycin, which are conventionally used to inhibit Clostridium difficile infection, nuciferine has less drug resistance and less negative impact on intestinal flora, thus ensuring the therapeutic efficacy.

[0019] 2. Nuciferine can regulate the autophagy process by affecting the expression of autophagy-related genes. In a cell model, nuciferine 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, nuciferine can also regulate the autophagy process by affecting the expression of autophagy-related proteins, such as LC3 and p62. The regulatory effect of nuciferine on autophagy in animal models has also been verified. Studies have shown that nuciferine can alleviate organ damage caused by autophagy abnormalities.

[0020] 3. In the research, the applicant identified two small molecule compounds PF-429242 and U18666A, which can inhibit the SREBP-2 pathway and cholesterol synthesis, and exhibit inhibitory effects on TcdB-induced cytotoxicity. Through analysis of multiple components in the traditional Chinese medicine Shenling Baizhu Powder, it is found that lotus seed extract can effectively inhibit the cytotoxicity of TcdB, a key pathogenic factor of Clostridium difficile infection. Further research shows that in an in vitro model, nuciferine can significantly inhibit the growth of Clostridium difficile and has a significant inhibitory effect on the exotoxin TcdB of Clostridium difficile. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is a schematic diagram of morphological changes of TcdB-induced Hela cell toxicity by nuciferine according to the application;

[0022] Figure 2 It is a schematic diagram of morphological changes of TcdB-induced Hutu cell toxicity by nuciferine according to the application;

[0023] Figure 3 It is a schematic diagram of Hela cell real-time label-free dynamic cell analysis technology (RTCA) detection of different concentrations of nuciferine anti-TcdB-induced Hela cell toxicity index (CI) curve according to the application;

[0024] Figure 4Differential diagram of the Hela cells of the experimental group of the application added with toxin for 6h;

[0025] Figure 5 Hutu cell real-time label-free dynamic cell analysis technology (RTCA) detection of different concentrations of lotus heart base anti-TcdB induced Hutu cell toxicity index (CI) curve schematic diagram of the application;

[0026] Figure 6 Differential diagram of the Hela cells of the application added with toxin for 6h;

[0027] Figure 7 Western Blot experiment schematic diagram of the protein extracted from the Hela cells treated with different concentrations of lotus heart base for 24h and then added with toxin for 6h of the application;

[0028] Figure 8 Gray scale calculation of the band of the Western Blot experiment of the Hela cells treated in the application, and the difference graph analyzed;

[0029] Figure 9 Schematic diagram of the application of 10μM lotus heart base, 10pM TcdB treated Hela cells, fixed cells after 6h, immunofluorescence staining, blue for nucleus, green for actin cytoskeleton, red for non-glycosylated Rac1 (40X);

[0030] Figure 10 Western Blot experiment schematic diagram of the protein extracted from the Hutu cells treated with different concentrations of lotus heart base for 24h and then added with TcdB for 6h;

[0031] Figure 11 Gray scale calculation of the band of the Western Blot experiment of the Hutu cells treated in the application, and the difference graph analyzed;

[0032] Figure 12 Schematic diagram of the application of 10μM lotus heart base, 10pM TcdB treated Hutu cells, fixed cells after 6h, immunofluorescence staining, blue for nucleus, green for actin cytoskeleton, red for non-glycosylated Rac1 (40X);

[0033] Figure 13 Caspase-1, Beclin-1, Atg16L1, Atg7, HMGB1 protein band diagram of the WB experiment of the protein extracted from the Hela cells treated with different concentrations of lotus heart base for 24h and then added with TcdB for 6h;

[0034] Figure 14Figure for quantitative analysis of difference in expression level of Atg7 protein in Hela cells of the present application;

[0035] Figure 15 Figure for quantitative analysis of difference in expression level of Atg16L1 protein in Hela cells of the present application;

[0036] Figure 16 Figure for quantitative analysis of difference in expression level of Beclin-1 protein in Hela cells of the present application;

[0037] Figure 17 Figure for quantitative analysis of difference in expression level of Caspase-1 protein in Hela cells of the present application;

[0038] Figure 18 Figure for quantitative analysis of difference in expression level of HMGB1 protein in Hela cells of the present application;

[0039] Figure 19 Figure for analysis of binding of TcdB (500 pM) to pre-cooled HuTu cells, with lotus plumule alkaloid or DMSO as control, to analyze the binding of TcdB;

[0040] Figure 20 Figure for SDS-PAGE and Coomassie color staining for analysis of effect of lotus plumule alkaloid on cysteine protease domain (CPD) of TcdB in vitro. DETAILED DESCRIPTION

[0041] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application. EMBODIMENT

[0042] A small molecule capable of inhibiting Clostridium difficile infection is extracted from white lotus seed, and the alkaloid is lotus plumule alkaloid. The chemical structural formula of the lotus plumule alkaloid is shown as formula I:

[0043]

[0044] The lotus plumule alkaloid reduces the toxicity of TcdB by inhibiting the autophagy pathway through reducing TcdB-induced intracellular Rac1 glycosylation, and ultimately achieves the purpose of treating Clostridium difficile infection by allowing actin to normally polymerize and depolymerize.

[0045] Use of a small molecule that can inhibit Clostridium difficile infection, a compound represented by Formula I or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating Clostridium difficile infection. The medicament comprises 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, nootropin, or a combination thereof. The medicament also includes a pharmaceutically acceptable excipient. The medicament is an oral preparation or an injection preparation. The oral preparation is a tablet, a capsule, or a granule; the injection preparation is an injection solution or a powder injection. The medicament consists of 8% to 92% of the lotus heart alkaloid compound and 8% to 92% of the pharmaceutically acceptable excipient.

[0046] The study shows that Shenling Baizhu Powder can significantly promote the growth of human probiotics, such as lactobacillus and bifidobacterium. The inventors studied the inhibitory effect of Shenling Baizhu Powder and its single herb extract on TcdB in HuTu and Hela cells.

[0047] The research results show that different parts of Shenling Baizhu Powder extract have no obvious inhibitory effect on TcdB in HuTu cells, but white lotus seed, yam, and tuckahoe in its single herb have obvious inhibitory effect on TcdB in HuTu cells.

[0048] Further research results show that lotus heart alkaloid small molecule compounds in white lotus seed have obvious inhibitory effect on TcdB in HuTu and Hela cells. Lotus heart alkaloid reduces TcdB-induced intracellular Rac1 glycosylation and reduces actin structure. In mechanism exploration, through cell surface binding experiment, the number of TcdB combined with cells is reduced in the presence of lotus heart alkaloid. Through analysis of intrinsic autoproteolysis of toxin in the presence of Insp6, it is tested in vitro whether lotus heart alkaloid has an impact on the cysteine protease domain (CPD) of TcdB and whether it affects the autocatalytic processing of TcdB induced by InsP6, indicating that the CPD-related activity of TcdB is not affected by lotus heart alkaloid.

[0049] The basic research results are as follows:

[0050] 1. Effect of lotus heart alkaloid on TcdB-induced cytotoxicity

[0051] Logarithmic growth Hela cells and HuTu cells were incubated with different concentrations of lotus heart alkaloid (0.1 μM, 1 μM, 10 μM) for 24 h, then 10 pM TcdB was added, and microscopic photography was performed at 3 h and 6 h after the addition of TcdB.

[0052] Results show: Hela cells and Hutu cells add toxin 3h after, compared with the blank group, the toxin group cells obviously round, 0.1 μM, 1 μM concentration of drug group only a few cells round, 10 μM group did not find obvious round cell; Add toxin 6h after, compared with the blank group, the toxin group has all round, with the increase of concentration, the number of cell rounding gradually reduced (see attached drawings in the description Figures 1-2 ).

[0053] The above description shows that lotus heart alkali has inhibitory effect on TcdB induced cytotoxicity.

[0054] 2, RTCA real-time label-free technology detects lotus heart alkali anti-TcdB cytotoxicity

[0055] Logarithmic growth of Hela cells and Hutu cells were incubated with different concentrations of lotus heart alkali (0.1 μM, 1 μM, 10 μM) for 24h, then 10pM TcdB was added, and RTCA method was used to monitor the real-time lotus heart alkali anti-TcdB cytotoxicity.

[0056] Results show: in Hela cell and Hutu cell line, compared with the blank group, only the TcdB curve has obvious downward trend, and with the increase of lotus heart alkali concentration, the curve downward speed gradually slows down; After adding toxin 6h, 10 μM lotus heart alkali group of Hela cell line (**P<0.01) and 10 μM lotus heart alkali group of Hutu cell line (*P<0.05) compared with toxin group, there is statistical significance (see attached drawings in the description Figures 3-6 ).

[0057] Among them: Figure 3 and Figure 5 In x axis is time, y axis is the real-time measured impedance value after adding toxin / the first measured impedance value after adding toxin, the larger the value represents more cell adhesion and less cell death;

[0058] Figure 4 and Figure 6 In n=3, compared with the blank group:####P<0.0001, compared with TcdB group: *P<0.05, **P<0.01;

[0059] 3, the effect of lotus heart alkali on TcdB induced intracellular Rac1 glycosylation expression

[0060] After confirming that lotus heart alkali has inhibitory effect on TcdB induced cytotoxicity, in order to further clarify the protective effect of lotus heart alkali on TcdB induced cytotoxicity, a specific antibody recognizing non-glycosylation was used for WB and immunofluorescence detection.

[0061] WB results showed that: compared with the Control group: the non-glycosylated Rac1 band in both Hela and Hutu cells treated with TcdB alone was lighter, with statistical significance; compared with TcdB: with the increase of the concentration of lotauagine, the expression level of intracellular Rac1 protein was significantly up-regulated, and the non-glycosylated Rac1 in Hela cells 10 μM group and Hutu cells 1 μM and 10 μM group (*P<0.05) had statistical significance. The same results were obtained in immunofluorescence. In addition, compared with the Control group, the actin structure of the cells treated with 10 μM lotauagine alone was not found to be damaged; while the actin structure of the cells treated with TcdB alone was severely damaged, and the actin structure of the cells treated with 10 μM lotauagine and then with toxin was still normal (see the attached drawings of the specification Figures 7-12 ).

[0062] 4. Effect of lotauagine on autophagy signaling pathway

[0063] In order to explore the inhibition mechanism of lotauagine on TcdB specific signaling pathway, the expression changes of key marker proteins in apoptosis, pyroptosis, autophagy, necrosis and ferroptosis signaling pathways were analyzed in detail by Western Blot technology.

[0064] The research results show that: after lotauagine pretreatment, the expression levels of autophagy-related key proteins including Caspase-1, Beclin-1, Atg16L1, Atg7, HMGB1, etc. show significant differential changes. Specifically, with the gradual increase of the concentration of lotauagine, the expression levels of these proteins all show different degrees of downward trend (see the attached drawings of the specification Figures 13-18 ). This finding indicates that lotauagine may effectively slow down the cytotoxicity response caused by TcdB by inhibiting the autophagy pathway.

[0065] 5. Effect of lotauagine on TcdB binding to cultured cells

[0066] In vitro experiment method was used to cool the cells to 4℃ to minimize endocytosis, and incubate with TcdB in the presence and absence of lotauagine. Then, the cells were washed, and the bound TcdB was analyzed by immunoblotting. Here, in the presence of lotauagine, the amount of TcdB bound to cells decreased slightly (see the attached drawings of the specification Figure 19 ).

[0067] 6. Effect of lotauagine on the cysteine protease domain (CPD) of TcdB in vitro

[0068] The steps of toxin uptake, such as intracellular processing of TcdB, were further investigated. Whether lotusin affected the cysteine protease domain (CPD) of TcdB in vitro was detected by analyzing the intrinsic autoproteolysis of the toxin in the presence of InsP6.

[0069] As can be seen, lotusin did not affect the InsP6-induced autocatalytic process of TcdB, indicating that the CPD-related activity of TcdB was not affected by lotusin (see the description of the accompanying drawings Figure 20 ). The positive drug n-ethylmaleimide (NEM), a mature CPD inhibitor, prevented the autocatalytic processing of TcdB in the same experiment.

[0070] wherein, Figure 14 Lotusin or NEM (1 mm) was added, respectively, and the cysteine protease activity was analyzed by SDS-PAGE and Coomassie staining after 1 h at 37 °C. After successful cleavage, the GTD (~63 kDa) was released from the full-length TcdB (~270 kDa), and a representative SDS-PAGE is described.

[0071] Experiments showed that lotusin could reduce the intracellular Rac1 glycosylation induced by TcdB and reduce the actin structure. In the mechanism exploration, through the cell surface binding experiment, it was found that the amount of TcdB bound to the cells in the presence of lotusin was slightly reduced, indicating that lotusin had an inhibitory effect on the cell toxicity induced by TcdB. Compared with metronidazole and vancomycin, which are used to inhibit Clostridium difficile infection, lotusin has less frequent drug resistance and less negative impact on intestinal flora, ensuring the therapeutic effect.

[0072] Although the embodiments of the present application have been shown and described, it can be understood by those skilled in the art that various changes, modifications, replacements and variations can be made to the embodiments without departing from the principles and spirits of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. Use of a chemical compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating Clostridium difficile infection, characterized in that: Alkaloids are extracted from white lotus seeds. The alkaloid is liesinine, and its chemical structure is shown in Formula I: ； The liensinine inhibits the autophagy pathway, reduces the toxicity of TcdB, and ultimately allows actin to polymerize and disaggregate normally to achieve the purpose of treating Clostridium difficile infection.

2. Use of a chemical compound represented by formula I or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for treating Clostridium difficile infection, characterized in that: The drug also includes pharmaceutically acceptable excipients.

3. Use of a chemical compound represented by formula I or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for treating Clostridium difficile infection, characterized in that: The medicine is an oral preparation or an injection preparation.

4. Use of a chemical compound represented by formula I or a pharmaceutically acceptable salt thereof as claimed in claim 3 in the preparation of a medicament for treating Clostridium difficile infection, characterized in that: The oral preparation is a tablet, capsule or granule; the injection preparation is an injection solution or powder injection.

5. Use of a chemical compound represented by formula I or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for treating Clostridium difficile infection, characterized in that: The medicine consists of 8%-92% of liensinine compounds and 8%-92% of pharmaceutically acceptable excipients.

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