A small molecule compound and its use in the preparation of a medicament for treating tuberculosis infection

Abstract

The application discloses a small-molecule compound and application of the small-molecule compound in preparation of a medicine for treating tuberculosis infection, relates to the field of biochemistry, and the small-molecule compound is a small-molecule compound 105, chemical formula is C 25 H 17 N5O2S. The application uses the small-molecule compound to increase the occurrence of copper death of macrophages, exposes the tuberculosis bacteria hidden in the macrophages in advance, and makes the tuberculosis bacteria more easily killed by host immune cells or medicines.

Description

Technical Field

[0001] This invention relates to the field of biochemistry, and more particularly to a small molecule compound and its application in the preparation of drugs for treating tuberculosis infection. Background Technology

[0002] Elesclomol (ES) is a copper ion carrier that induces oxidative stress by specifically binding to and transporting copper ions into cells, thereby inducing copper death in tumor cells. It is currently used in the treatment of various cancers, including melanoma.

[0003] Macrophages, as the first line of defense against pathogens, can kill pathogens by delivering large amounts of copper ions into the cell. Our research found that ES (extracellular substances) can introduce excessive copper into macrophages, thereby inducing copper death. Tuberculosis bacteria can often hide in macrophages to grow and replicate, evading the immune system, leading to latent tuberculosis. Furthermore, tuberculosis bacteria hiding in macrophages are prone to developing antibiotic resistance. Intracellular bacteria hiding in macrophages can easily leave small amounts of bacteria, leading to incomplete treatment and reinfection, which also contributes to the development of antibiotic resistance.

[0004] Therefore, those skilled in the art are dedicated to developing a method that uses small molecule compounds to increase the occurrence of copper death in macrophages, thereby exposing tuberculosis bacteria hidden in macrophages earlier and making them more easily killed by host immune cells or drugs. Summary of the Invention

[0005] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to develop a method for using small molecule compounds to increase the occurrence of copper death in macrophages, thereby exposing the tuberculosis bacteria hidden in macrophages in advance and making them more easily killed by host immune cells or drugs.

[0006] To achieve the above objectives, this invention provides the application of a small molecule compound in the preparation of a drug for treating tuberculosis infection. The small molecule compound is small molecule compound 105, with the chemical formula C. 25 H 17 N5O2S.

[0007] Furthermore, the drug for treating tuberculosis infection consists of: irismo, copper chloride, and small molecule compound 105.

[0008] Furthermore, the application includes the following steps: Based on a solution containing irismo, copper chloride was added, followed by the addition of a small molecule compound 105, to induce copper death in macrophages.

[0009] Furthermore, the concentration of the ilismoxol solution was 200 nM.

[0010] Furthermore, the concentration of copper chloride is 10, 30, or 60 μM.

[0011] Furthermore, the concentration of small molecule compound 105 was 10 μM.

[0012] Furthermore, by using the small molecule compound 105 to increase the occurrence of copper death in macrophages, tuberculosis bacteria hidden in macrophages are exposed earlier and are more easily killed by host immune cells or drugs.

[0013] Furthermore, FDX2 is a key molecule regulating copper death in macrophages, and overexpression of FDX2 increases copper death in macrophages. The small molecule compound 105 can increase copper death in macrophages by activating FDX2.

[0014] Furthermore, the drugs for treating tuberculosis infection are administered via intraperitoneal injection.

[0015] Furthermore, the strain causing the tuberculosis infection was Mycobacterium tuberculosis H37Rv.

[0016] Furthermore, the steps also include the detection of bacterial load in the lungs and / or spleen.

[0017] In a preferred embodiment 1 of the present invention, it is described in detail that small molecule compound 105 can be used as an activator of FDX2; In a preferred embodiment 2 of the present invention, the effects of irismo combined with copper (ES+Cu) on macrophages are described in detail; In a preferred embodiment 3 of the present invention, the effect of combined administration of ES and Cu on the viral load of Mycobacterium tuberculosis in a mouse infection model is described in detail. In a preferred embodiment 4 of the present invention, FDX2 is highly expressed and FDX1 is lowly expressed in macrophages. In a preferred embodiment 5 of the present invention, it is described in detail that overexpression of FDX2 in macrophages increases copper death, while knockdown of FDX2 reduces copper death. In a preferred embodiment 6 of the present invention, the effect of small molecule compound 105 on cell growth is described in detail; In a preferred embodiment 7 of the present invention, the effect of small molecule compound 105 on copper death-related proteins is described in detail. In a preferred embodiment 8 of the present invention, the effects of small molecule compound 105 on mice are described in detail; In a preferred embodiment 9 of the present invention, the effect of combined administration of 105 and ES+Cu on the viral load of Mycobacterium tuberculosis in a mouse infection model is described in detail.

[0018] The beneficial technical effects of this invention are as follows: Macrophages are the first line of defense against bacteria, but Mycobacterium tuberculosis can resist macrophage killing, using macrophages as a hiding place to resist the killing of immune cells and also affecting the therapeutic effect of antibacterial drugs. The combined use of esophageal extract (ES) and copper can induce copper death in macrophages, and the small molecule compound 105 disclosed in this application can increase the occurrence of copper death in macrophages. ES can kill tumor cells and has been used to treat various cancers, so the safety of this drug in clinical use is guaranteed. Small molecule compound 105 has no significant effect on mouse body weight and has a certain safety profile, but it can significantly increase the copper concentration in lung tissue, exacerbating the occurrence of copper death in macrophages. Therefore, the combination of small molecule compound 105 and ES could be a drug for treating tuberculosis. It can promote the killing of Mycobacterium tuberculosis, thereby treating tuberculosis infection and reducing the probability of recurrence.

[0019] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0020] Figure 1 This is a diagram showing the experimental results of the effect of ES+Cu on macrophages in a preferred embodiment 2 of the present invention; Figure 2 This is a graph showing the experimental results of the tuberculosis bacterial load in the lungs of mice in a preferred embodiment 3 of the present invention; Figure 3 This is a graph showing the experimental results of the tuberculosis bacterial load in the spleen of mice in a preferred embodiment 3 of the present invention; Figure 4 This is a diagram showing the experimental results of FDX2 and FDX1 expression in different cell lines in a preferred embodiment 4 of the present invention. Figure 5 This is a diagram showing the experimental results of overexpressing FDX2 in macrophages to increase copper cell death, according to a preferred embodiment 5 of the present invention. Figure 6 This is a diagram showing the experimental results of knocking down FDX2 in macrophages to reduce copper cell death, which is a preferred embodiment 5 of the present invention. Figure 7 This is a diagram showing the experimental results of a preferred embodiment 6 of the present invention, in which the small molecule compound 105 can increase copper death in macrophages. Figure 8 The figure shows the experimental results of how small molecule compound 105, a preferred embodiment 7 of the present invention, can further reduce the decrease in expression of lipophilized proteins caused by copper death and increase the expression of HSP70. Figure 9 This is a diagram showing the experimental results of a preferred embodiment 8 of the present invention, in which the small molecule compound 105 had no significant effect on mouse body weight. Figure 10 This is a diagram showing the experimental results of a preferred embodiment 8 of the present invention, in which the small molecule compound 105 significantly increases the copper concentration in mouse lung tissue.

[0021] Figure 11 This is a graph showing the experimental results of the tuberculosis bacterial load in the lungs of mice in a preferred embodiment 9 of the present invention; Figure 12 This is a graph showing the experimental results of the tuberculosis bacterial load in the spleen of mice in a preferred embodiment 9 of the present invention. Detailed Implementation

[0022] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein. Among them, strain H37Rv, deposited at the American Center for Type Culture Collection (ATCC) 25618, is published in the Patent Gazette with publication number CN119185269A and application date of September 20, 2024. Strain H37Ra, deposited at the American Center for Type Culture Collection (ATCC) 25177, is published in the Patent Gazette with publication number CN112899329A and application date of February 2, 2021.

[0023] Example 1: 105 can be used as an activator for FDX2.

[0024] We found that ilismo combined with copper (ES+Cu) can induce copper death in macrophages, such as Figure 1As shown, this disrupts the "hiding place" of Mycobacterium tuberculosis. To confirm this hypothesis, we infected Balb / c mice with H37Rv (ATCC 25618) via nebulization. One week later, we started intraperitoneal injections of copper chloride (Cu) 1.25 mg / kg, irismodium (ES) 10 mg / kg, and ES+Cu three times a week into Balb / c mice. We found that compared with the control group (NC), the bacterial load in the lungs and spleen of the ES+Cu group was significantly reduced. The molecule that plays a key role in the copper death pathway in tumor cells is FDX1. We found that FDX1 is lowly expressed in macrophages, while FDX2, which is responsible for the source of iron-sulfur cluster synthesis, is highly expressed. Overexpression of FDX2 in macrophages can increase macrophage copper death, while knockdown of FDX2 in macrophages can reduce macrophage copper death. The experimental results indicate that the molecule that plays a key role in macrophage copper death is FDX2. Since concentrations exceeding 10 mg / kg ES and 2.5 mg / kg copper significantly reduce mouse body weight, we screened for small molecule compounds that activate FDX2 to further increase copper-induced mortality without affecting mouse body weight. First, using a ChemDiv library of approximately 2 million small molecules, we initially eliminated a large number of irrelevant molecules through ultra-rapid sequence-based virtual screening. Then, we performed precise co-structure prediction on the remaining compounds, ultimately obtaining 111 candidate small molecules for subsequent experimental validation. Based on cell viability assays and Western blot analysis, compound 105 (chemical formula C105) was identified as capable of activating FDX2. 25 H 17 (N5O2S). The addition of 105 significantly increased ES+Cu-induced copper death in macrophages. The small molecule compound 105 increased macrophage copper death and further reduced the expression of lipoylated proteins and increased HSP70 expression. Animal experiments confirmed that 20 mg / kg of 105 significantly affected mouse body weight. While the small molecule compound 105 had no significant effect on mouse body weight, it significantly increased copper content in mouse lung tissue. These results indicate that 105 can act as an activator of FDX2 and can control tuberculosis infection by increasing copper death.

[0025] Example 2: Effects of ES+Cu on macrophages

[0026] This embodiment used the classic intracellular bacterium Mycobacterium tuberculosis H37Ra (ATCC 25177) as the test strain and the J774A.1 mouse macrophage cell line. H37Ra was cultured in Middlebrook 7H9 liquid medium (supplemented with OADC and 0.05% Tween-80) to the logarithmic growth phase. 1 OD of H37Ra is approximately 1 x 10^8 CFU. Macrophages were infected with MOI10, and the number of viable cells was detected at 0, 24, 48, 72, and 96 hours using the Cell Counting Lite 2.0 Luminescent Cell Viability Assay Kit (Nanjing Novizan, dd1101-02) according to the manufacturer's instructions. Four groups were set up: a control group (NC) without drug treatment, 50 nM irismo (ES), 50 μM copper chloride (Cu), and 50 nM ES + 50 μM Cu (ES + Cu). The results are as follows: Figure 1 As shown, compared with the NC group, ES+Cu can significantly induce copper death in macrophages.

[0027] Example 3: Effect of combined administration of ES and Cu on in vivo tuberculosis viral load in a mouse infection model

[0028] In this embodiment, an aerosol infection model was established using 6–8 week old SPF-grade Balb / c mice: each mouse was infected with approximately 100–200 CFU of Mycobacterium tuberculosis H37Rv using a standardized aerosol generator; after infection, two control mice were randomly euthanized, lung tissue was collected to prepare tissue homogenates, and diluted 10-fold serially. 100 μL of the diluted solution was plated onto Middlebrook 7H11 (supplemented with OADC) solid culture plates to confirm successful aerosol infection and estimate the initial implantation volume; the remaining mice began intraperitoneal injection treatment (3 times a week) 7 days after infection, with groups including a PBS control group, Cu group, ES group, and ES+Cu group (injection dose was ES 10 mg / kg, CuCl2 1.25 mg / kg, which was the maximum dose that did not affect mouse body weight); animals were sacrificed 3 weeks after the start of administration, lung and spleen tissue homogenates were collected and prepared, and diluted 10-fold serially. 100 μL of the diluted solution was plated onto Middlebrook 7H11 (supplemented with OADC) solid culture plates to confirm successful aerosol infection and estimate the initial implantation volume; the remaining mice began intraperitoneal injection treatment (3 times a week) 7 days after infection, with groups including a PBS control group, a Cu group, an ES group, and an ES+Cu group (injection dose was ES 10 mg / kg, CuCl2 1.25 mg / kg, which was the maximum dose that did not affect mouse body weight); animals were sacrificed 3 weeks after the start of administration, lung and spleen tissue homogenates were collected and prepared, and diluted 10-fold serially. μL was spread onto solid culture medium and incubated at 37°C until colonies were countable to determine CFU per organ. Experimental results are as follows: Figure 2 and Figure 3As shown: After 3 weeks of treatment, the bacterial load in both the ES group and the ES+Cu group in the lungs was significantly lower than that in the control group, and the reduction in the ES+Cu group was greater than that of ES alone. There was no significant difference between the Cu group and the control group. In the spleen, only the ES+Cu group showed a significant difference in bacterial load compared to the control group, exhibiting a decrease in bacterial count; there was no difference between the ES group and the Cu group. These results indicate that in a mouse model infected by aerosols, systemic intraperitoneal administration of ES combined with low-dose Cu produces a superior antibacterial effect compared to single-drug therapy, and this combination regimen showed significant effects in both the lungs and spleen after a longer treatment period (3 weeks).

[0029] Example 4: FDX2 is highly expressed and FDX1 is lowly expressed in macrophages.

[0030] In this embodiment, the expression of FDX2 and FDX1 in macrophages and other cells was compared. The macrophages used included mouse macrophages J774A.1 and RAW 264.7, human histiocytic lymphoma cells U937, and macrophages induced from U937 cells with 50 ng / mL PMA, human lung cancer adenocarcinoma cells ABC1, human embryonic kidney cells HEK293T, and human lung adenocarcinoma cells A549. 40 μg of total protein was collected from each cell type, and the expression of FDX2 and FDX1 proteins in different cells was compared. The experimental results are as follows: Figure 4 As shown, in macrophages J774A.1, RAW 264.7, and U937 (after PMA induction), FDX1 was lowly expressed while FDX2 was highly expressed, even with unchanged expression of the internal control microtubule protein Tubulin. FDX1 is a key molecule in copper death in cancer cells, but its low expression in macrophages indicates that it is not a key molecule for copper death in macrophages. The high expression of FDX2 in macrophages is a source of iron-sulfur cluster synthesis, suggesting a possible involvement in macrophage copper death.

[0031] Example 5: Overexpression of FDX2 in macrophages increased copper death, while knockdown of FDX2 decreased copper death.

[0032] In this embodiment, FDX2 was overexpressed in J774A.1. Solutions containing 200 nM ilismo were supplemented with 0, 1, 3, 5, 7, 10, 30, and 50 μM CuCl2, respectively. Viable cell counts were determined after 24 hours using the method described in the instructions of the Cell Counting Lite 2.0 luminescent cell viability assay kit (Nanjing Novizan, dd1101-02). Results are as follows... Figure 5As shown, compared with the control group, the FDX2 overexpression group significantly induced a significant increase in copper death in macrophages. In J774A.1, after FDX2 knockdown with lentivirus, compared with the control group, FDX2 knockdown reduced macrophage copper death, as shown in... Figure 6 As shown.

[0033] Example 6: Effect of 105 on cell growth

[0034] In this embodiment, J774A.1 cells were used. The control group was treated with 200 nM ilismo solution, followed by the addition of 0, 10, 30, and 60 μM CuCl2 to induce copper death in cells. The experimental group (10⁵ cells) was treated with the control group plus an additional 10 μM 10⁵ CuCl2. After 24 hours of treatment, the number of viable cells was detected according to the instructions of the Cell Counting Lite 2.0 Luminescent Cell Viability Assay Kit (Nanjing Novizan, dd1101-02). The results are as follows: Figure 7 As shown, compared with the control group, 105 significantly increased copper death in macrophages.

[0035] Example 7: Effect of 105 on copper death-related proteins

[0036] In this embodiment, four groups were set up: a control group (NC), a group with only 10⁵ added (10⁵), an ES+Cu group, and a 10⁵+ES+Cu group. NC served as the control group, 10⁵ was at a concentration of 10 μM, ES at 50 nM, and CuCl₂ at 50 μM. J774A.1 cells were treated for 24 hours, and protein expression was detected by Western blot. The experimental results are as follows: Figure 8 As shown, ES+Cu-induced copper death resulted in decreased expression of lipoylated proteins Lip-DLAT and Lip-DLST, increased FDX2, and increased HSP70 in macrophages. ES+Cu+105 further decreased the expression of lipoylated proteins Lip-DLAT and Lip-DLST and further increased HSP70 expression. These results indicate that 105 exacerbated copper death in cells.

[0037] Example 8: Effects of 105 on mice

[0038] In this embodiment, 8-week-old SPF-grade Balb / c mice were used, and four groups were set up: a PBS control group, an ilismo plus copper group (ES+Cu), a small molecule compound 105 (105), and a small molecule compound combined with ilismo and copper (105 + ES+Cu). The mice were injected intraperitoneally three times a week at doses of ES 10 mg / kg, CuCl2 1.25 mg / kg, and 105 20 mg / kg. Body weight was monitored continuously for 3 weeks. Results are as follows: Figure 9 As shown, 10⁵ alone or 10⁵+ES+Cu had no significant effect on the body weight of mice. Lung tissue was collected from mice after 3 weeks, and the copper concentration in the lung tissue was detected by mass spectrometry. The results are as follows... Figure 10 As shown, 105 alone can significantly increase the copper concentration in lung tissue, and the combination of 105 with ES and copper can further significantly increase the copper concentration in lung tissue.

[0039] Example 9: Effect of combined administration of 105 and ES+Cu on in vivo tuberculosis viral load in a mouse infection model.

[0040] In this embodiment, an aerosol infection model was established using 6–8 week old SPF-grade Balb / c mice: each mouse was infected with approximately 100–200 CFU of Mycobacterium tuberculosis H37Rv using a standardized aerosol generator; after infection, two control mice were randomly euthanized, lung tissue was collected to prepare tissue homogenates, and the homogenates were serially diluted tenfold. 100 μL of the diluted solution was spread on Middlebrook 7H11 (supplemented with OADC) solid culture plates to confirm successful aerosol infection and estimate the initial implantation volume; the remaining mice received intraperitoneal injection treatment (3 times a week) starting 7 days after infection, with four groups: PBS control group (NC), ilismo plus copper group (ES+Cu), small molecule compound 105 (105), and small molecule compound combined with ilismo and copper (105 + ES+Cu). Intraperitoneal injections were administered 3 times a week, with injection doses of ES 10 mg / kg, Cu 1.25 mg / kg, and 105 20 mg / kg. The experimental results are as follows. Figure 11 and Figure 12 As shown, after 3 weeks of treatment, the bacterial load in the lungs and spleen of the ES+Cu group was significantly lower than that of the control group; the bacterial load in the lungs and spleen of the combined 105 and ES+Cu group (105+ES+Cu group) was further significantly lower than that of the ES+Cu group, and 3 weeks of treatment with 105 alone also significantly reduced the bacterial load in the lungs compared with the control group. These results indicate that in a mouse model infected by aerosols, systemic intraperitoneal administration of 105 combined with ES+Cu can significantly enhance the killing effect of Mycobacterium tuberculosis.

[0041] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. The application of a small molecule compound in the preparation of a drug for treating tuberculosis infection, characterized in that, The small molecule compound is small molecule compound 105, with the chemical formula C. 25 H 17 N5O2S.

2. The application as described in claim 1, wherein the drug for treating tuberculosis infection comprises: irismo, copper chloride, and the small molecule compound 105.

3. The application as described in claim 1, comprising the following steps: Adding copper chloride to a solution containing ilismo, followed by the addition of the small molecule compound 105, induces copper death in macrophages.

4. The application as described in claim 3, wherein the concentration of the ilismo solution is 200 nM.

5. The application as described in claim 3, wherein the concentration of copper chloride is 10, 30, or 60 μM.

6. The application as described in claim 3, wherein the concentration of the small molecule compound 105 is 10 μM.

7. The application as described in claim 1, wherein the drug for treating tuberculosis infection is administered via intraperitoneal injection.

8. The application as described in claim 1, wherein the tuberculosis infection strain is Mycobacterium tuberculosis H37Ra.

9. The application as described in claim 1, wherein the tuberculosis infection strain is Mycobacterium tuberculosis H37Rv.

10. The application as described in claim 3, wherein the step further includes the detection of bacterial load in the lungs and / or spleen.

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