Application of FBP enzyme inhibitor in resisting mycobacterium tuberculosis infection

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of an inhibitor taking cell FBP enzyme as a target spot in resisting mycobacterium tuberculosis infection. The invention finds that the FBP enzyme inhibitor has a good inhibition effect on the survival of mycobacterium tuberculosis in macrophages, and has good safety to cells. The effect of the FBP enzyme inhibitor for resisting intracellular survival of mycobacterium tuberculosis is superior to that of other types of currently reported diabetes drugs, the FBP enzyme inhibitor can be used as a candidate drug to be applied to a host-oriented tuberculosis treatment scheme, and the drug can play a role in reducing blood sugar and can also help a body to resist mycobacterium tuberculosis infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of an inhibitor targeting cell FBP enzyme in anti - Mycobacterium tuberculosis infection. Background Art

[0002] Tuberculosis is a primary or secondary chronic infectious disease caused by Mycobacterium tuberculosis invading the body. It is the main cause of death globally due to a single infectious pathogen, seriously endangering public safety and human health (Global tuberculosis report 2024. Geneva: World Health Organization; 2024.). Currently, the incidence of tuberculosis is still increasing year by year. In particular, drug - resistant Mycobacterium tuberculosis has been included in the WHO's list of priority pathogens (Williams PM et.al. Tuberculosis - United States, 2023. MMWR Morb Mortal Wkly Rep, 2024, 73(12):265 - 270.; WHO bacterial priority pathogens list, 2024: Bacterial pathogens of public health importance to guide research, development and strategies to prevent and control antimicrobial resistance. Geneva: World Health Organization; 2024). In recent years, the treatment regimens for tuberculosis have made great progress, but the existing treatment regimens still face many problems such as a severe drug - resistant situation, a long treatment course, and poor patient prognosis. The treatment and prognosis of diabetes - tuberculosis co - morbidity are another difficult problem in tuberculosis treatment. Type 2 diabetes patients are more likely to contract tuberculosis (the incidence is about 2 - 8 times that of normal people) and often have worse treatment effects and prognosis responses (Baker MA et.al. The impact of diabetes on tuberculosis treatment outcomes: a systematic review. BMC Med. 2011 Jul 1; 9:81.,).

[0003] Host-Directed Therapy (HDT) can make up for the shortcomings of traditional antibiotic therapies and has become a forefront and hot research direction in tuberculosis treatment in recent years. HDT aims to resist Mycobacterium tuberculosis infection by regulating the host immune response. HDT usually uses strategies such as suppressing inflammation, regulating immunity, repairing tissue damage, and improving metabolism, which have great advantages and application prospects in reducing side effects, treating drug-resistant tuberculosis, shortening the treatment course, and improving treatment outcomes for tuberculosis patients (Smith SG et.al. Host-directed therapy in diabetes and tuberculosis comorbidity, towards global TB elimination. Int J Infect Dis. 2025 Mar 9:107877.).

[0004] Metformin is currently the most promising HDT drug discovered for diabetes-tuberculosis comorbidity and has been preliminarily confirmed in basic and clinical studies (Singhal A et.al. Metformin as adjunctant tuberculosis therapy. Sci Transl Med. 2014 Nov 19;6(263):263ra159.;Cornejo-Báez AA et.al. Association Between Diabetes Mellitus-Tuberculosis and the Generation of Drug Resistance. Microorganisms. 2024 Dec 20;12(12):2649.). Metformin can induce autophagy in host cells by activating the AMPK signaling pathway, enhancing the immune defense ability against Mycobacterium tuberculosis; when combined with first-line anti-tuberculosis drugs (rifampicin, isoniazid), it can reduce the drug resistance risk of Mycobacterium tuberculosis and shorten the treatment cycle; moreover, metformin can also significantly improve the disease progression and prognosis of patients through mechanisms such as inhibiting inflammatory damage and tissue destruction and improving the metabolic microenvironment, which is a successful case of using diabetes drugs to treat tuberculosis and diabetes-tuberculosis comorbidity.

[0005] Regrettably, at present, other types of diabetes drugs that are promising for treating diabetes-tuberculosis co-infection are very limited, and new and potent drugs remain to be developed. Due to severe side effects, sulfonylurea drugs (glyburide, glimepiride) should be avoided in combination with rifampicin and isoniazid; thiazolidinedione drugs such as (pioglitazone, rosiglitazone, troglitazone) may exacerbate the risk of edema or heart failure and need to be used with caution when tuberculosis causes malnutrition or organ dysfunction; SGLT2 inhibitors (empagliflozin, dapagliflozin) may also carry the risk of genitourinary tract infections and may increase the immune burden on tuberculosis patients (Xu Xinyue, Zhao Xiaolong. Progress in the Pharmacological Treatment of Tuberculosis and Diabetes Comorbidity [J]. Electronic Journal of Emerging Infectious Diseases, 2023, 8(2): 74-77.).

[0006] FBPase (fructose-1,6-bisphosphatase) is a key rate-limiting enzyme in glucose metabolism and is mainly involved in the gluconeogenesis pathway. Recent research reports have shown that FBPase not only has classical metabolic functions but also plays a role in regulating inflammation, immunity, and metabolic homeostasis (Hirata H et.al. Decreased Expression of Fructose-1,6-bisphosphatase Associates with Glucose Metabolism and Tumor Progression in Hepatocellular Carcinoma. Cancer Res. 2016 Jun 1; 76(11): 3265-76.). FBPase inhibitors were first used in the treatment of diabetes and can significantly reduce blood glucose levels and improve glucose tolerance in diabetic patients. In recent years, they have also been preliminarily applied in cancer research. MB05032 is a compound with high inhibitory potency against FBPase [IC 50= 16 ± 1.5 nM] and specific AMP mimetics, first reported in 2005, have been shown to inhibit the ability of rat and human liver cells to produce glucose in a concentration-dependent manner (Erion MD et al. MB06322 (CS-917): A potent and selective inhibitor of fructose 1,6-bisphosphatase for controlling gluconeogenesis in type 2 diabetes. Proc Natl Acad Sci U S A. 2005 May 31;102(22):7970-5.). MB05032 is achieved by using the orally administered diaminoester prodrug MB06322 (CS-917), which is converted to MB05032 in two steps through the action of esterase and phosphorylase, and ultimately has a hypoglycemic effect in vivo (Hunter RW et al. Metformin reduces liver glucose production by inhibition of fructose-1-6-bisphosphatase. Nat Med. 2018 Sep;24(9):1395-1406.). Currently, the role of FBP enzyme inhibitors in anti-tuberculosis infection and the formation of tuberculous granulomas has not been reported. SUMMARY OF THE INVENTION

[0007] The present invention discovers that the FBP enzyme inhibitor MB05032 has good antibacterial activity against Mycobacterium tuberculosis in vitro, and thus completes the present invention.

[0008] In a first aspect, the present invention provides the use of an FBP enzyme inhibitor in the preparation of a product for inhibiting Mycobacterium tuberculosis.

[0009] Furthermore, the FBP enzyme inhibitor is selected from MB05032, FBPase-IN-1, and / or FBPase-1-IN.

[0010] Preferably, the FBP enzyme inhibitor is MB05032.

[0011] Furthermore, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis clinical isolates, Mycobacterium tuberculosis standard strains, or Mycobacterium tuberculosis carried by patients infected with Mycobacterium tuberculosis.

[0012] Furthermore, the product for inhibiting Mycobacterium tuberculosis includes medical products or non-medical products.

[0013] Even further, the medical product refers to a medical drug, and the non-medical product refers to an experimental reagent or an antibacterial agent.

[0014] Furthermore, one or more pharmaceutically acceptable carriers or excipients may also be added to the product.

[0015] Furthermore, the carrier material includes, but is not limited to, one or more of water-soluble carrier materials, poorly water-soluble carrier materials, and / or enteric-soluble carrier materials.

[0016] Furthermore, the water-soluble carrier materials include, but are not limited to, one or more of polyethylene glycol, polyvinylpyrrolidone, and / or organic acids.

[0017] Furthermore, the poorly water-soluble carrier materials include, but are not limited to, one or more of ethylcellulose and / or cholesteryl stearate.

[0018] Furthermore, the enteric-soluble carrier materials include, but are not limited to, one or more of cellulose acetate phthalate and / or carboxymethyl ethyl cellulose.

[0019] Furthermore, if necessary, coloring agents, preservatives, fragrances, flavoring agents, sweetening agents, or other materials may also be added to the pharmaceutical preparations.

[0020] In a second aspect, the present invention provides the use of an FBP enzyme inhibitor in the preparation of a drug for preventing and / or treating diseases caused by Mycobacterium tuberculosis infection, and the FBP enzyme inhibitor exerts an anti-tuberculosis infection effect by promoting macrophages to clear intracellular survival of Mycobacterium tuberculosis.

[0021] Furthermore, the FBP enzyme inhibitor is selected from MB05032, FBPase-IN-1, and / or FBPase-1-IN.

[0022] Preferably, the FBP enzyme inhibitor is MB05032.

[0023] Furthermore, the Mycobacterium tuberculosis includes clinical isolates of Mycobacterium tuberculosis, standard strains of Mycobacterium tuberculosis, or Mycobacterium tuberculosis carried by patients infected with Mycobacterium tuberculosis.

[0024] Furthermore, one or more pharmaceutically acceptable carriers or excipients may also be added to the drug for preventing and / or treating diseases caused by Mycobacterium tuberculosis infection.

[0025] Furthermore, the carrier material includes, but is not limited to, one or more of water-soluble carrier materials, poorly water-soluble carrier materials, and / or enteric-soluble carrier materials.

[0026] Furthermore, the water-soluble carrier materials include, but are not limited to, one or more of polyethylene glycol, polyvinylpyrrolidone, and / or organic acids.

[0027] Furthermore, the poorly soluble carrier material includes, but is not limited to, one or more of ethylcellulose and / or cholesteryl stearate.

[0028] Furthermore, the enteric-soluble carrier material includes, but is not limited to, one or more of cellulose acetate phthalate and / or carboxymethyl ethyl cellulose.

[0029] Furthermore, the drug can be formulated into various dosage forms, including, but not limited to, one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, and / or suppositories.

[0030] Furthermore, the preparation can be one or more of an ordinary preparation, a sustained-release preparation, a controlled-release preparation, and / or various particulate drug delivery systems.

[0031] Furthermore, if necessary, coloring agents, preservatives, fragrances, flavoring agents, sweetening agents, or other materials can also be added to the pharmaceutical preparation.

[0032] Furthermore, the drug can be administered by injection, via a body cavity, via the respiratory tract, or via the mucosa.

[0033] Furthermore, the injection administration includes subcutaneous injection, intravenous injection, intramuscular injection, and intracavitary injection, etc.; the body cavity administration includes rectal or vaginal administration; the respiratory tract administration includes nasal administration.

[0034] In a third aspect, the present invention provides a pharmaceutical composition, which comprises an FBP enzyme inhibitor and another active ingredient against Mycobacterium tuberculosis infection, and the pharmaceutical composition has at least one of the following effects:

[0035] a) inhibiting the activity of Mycobacterium tuberculosis;

[0036] b) anti-Mycobacterium tuberculosis infection;

[0037] c) preventing and / or treating diseases caused by Mycobacterium tuberculosis.

[0038] Furthermore, the FBP enzyme inhibitor is selected from MB05032, FBPase-IN-1, and / or FBPase-1-IN.

[0039] Preferably, the FBP enzyme inhibitor is MB05032.

[0040] Furthermore, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis clinical isolates, Mycobacterium tuberculosis standard strains, or Mycobacterium tuberculosis carried by patients infected with Mycobacterium tuberculosis.

[0041] Furthermore, the other anti-Mycobacterium tuberculosis infection active ingredient includes one or more of antibiotics and other active ingredients that can help inhibit or kill Mycobacterium tuberculosis or provide resistance to the patient.

[0042] Furthermore, the antibiotics include one or more of rifampicin, ethambutol, isoniazid, streptomycin, pyrazinamide, thioacetazone, para-aminosalicylic acid, prothionamide, amikacin, capreomycin, rifapentine, rifabutin, bedaquiline, delamanid, linezolid, and / or clofazimine.

[0043] Furthermore, the active ingredients that are beneficial for inhibiting or killing Mycobacterium tuberculosis or providing resistance to the patient include one or more of vitamins, amino acids, proteins, and / or minerals.

[0044] Furthermore, one or more pharmaceutically acceptable carriers can be added to the pharmaceutical composition.

[0045] Furthermore, the carrier materials include, but are not limited to, one or more of water-soluble carrier materials, poorly water-soluble carrier materials, and / or enteric-soluble carrier materials.

[0046] Preferably, the carrier material is a water-soluble carrier material.

[0047] Furthermore, the pharmaceutical composition can be made into various dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, and / or suppositories.

[0048] Furthermore, the preparation can be one or more of an ordinary preparation, a sustained-release preparation, a controlled-release preparation, and / or various particulate drug delivery systems.

[0049] Furthermore, if necessary, coloring agents, preservatives, fragrances, flavoring agents, sweetening agents, or other materials can also be added to the pharmaceutical preparation for various preparations.

[0050] Furthermore, the pharmaceutical composition can be administered by injection, via body cavities, by inhalation, or through mucous membranes.

[0051] Furthermore, the injection administration includes subcutaneous injection, intravenous injection, intramuscular injection, and intracavitary injection, etc.; the administration via body cavities includes rectal or vaginal administration; the administration by inhalation includes nasal administration.

[0052] Beneficial effects

[0053] 1. The present invention discovers that the FBP enzyme inhibitor has a good inhibitory effect on the survival of Mycobacterium tuberculosis in macrophages, and the drug MB05032 can significantly inhibit the survival of Mycobacterium tuberculosis in macrophages at a relatively low concentration (200 nM - 500 nM).

[0054] 2. At the same concentration of 5 μM, the FBP enzyme inhibitor has a better effect on resisting the intracellular survival of Mycobacterium tuberculosis than other reported diabetes drugs such as metformin and troglitazone.

[0055] 3. The FBP enzyme inhibitor in the present invention has good safety for cells.

[0056] 4. The FBP enzyme inhibitor, especially MB05032, can be used as a candidate drug in host-directed tuberculosis treatment regimens. For patients with diabetes-tuberculosis co-infection, MB05032 can be preferentially considered. This type of drug can not only play a role in lowering blood sugar but also help the body resist Mycobacterium tuberculosis infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Detection of the antibacterial activity of the FBP enzyme inhibitor against the standard strain of Mycobacterium tuberculosis.

[0058] Figure 2 Intracellular survival count of the standard strain of Mycobacterium tuberculosis in macrophages by the FBP enzyme inhibitor at different times.

[0059] Figure 3 Comparison of the intracellular survival numbers of MB05032, metformin, and troglitazone against the standard strain of Mycobacterium tuberculosis in macrophages.

[0060] Figure 4 Evaluation of the cytotoxicity of MB05032 against THP-1 macrophages. DETAILED DESCRIPTION OF THE INVENTION

[0061] The present invention will be further described in detail below in conjunction with the specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not limit the present invention in any way.

[0062] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can all be obtained from commercial sources unless otherwise specified.

[0063] Material description:

[0064] Bacteria: The standard strain of Mycobacterium tuberculosis H37Rv (ATCC 27294) was purchased from the American Type Culture Collection (ATCC).

[0065] MB05032: Molecular formula C 11 H 15N2O4PS, with a molecular weight of 302.29 and a CAS number of 261365-11-1, has the following structural formula:

[0066]

[0067] FBPase-IN-1: with a molecular formula of C6H4N2S4, a molecular weight of 232.37, and a CAS number of 20362-54-3, has the following structural formula:

[0068]

[0069] FBPase-1-IN: with a molecular formula of C 13 H7Cl3N2O3S, a molecular weight of 377.63, and a CAS number of 883973-99-7, has the following structural formula:

[0070]

[0071] Metformin: with a molecular formula of C4H 11 N5, a molecular weight of 129.17, and a CAS number of 657-24-9, has the following structural formula:

[0072]

[0073] Troglitazone: with a molecular formula of C 24 H 27 NO5S, a molecular weight of 441.54, and a CAS number of 97322-87-7, has the following structural formula:

[0074]

[0075] Example 1 Detection of the antibacterial activity of FBP enzyme inhibitors against Mycobacterium tuberculosis standard strains and the number of Mycobacterium tuberculosis surviving in macrophages

[0076] 1.1 Preparation of drugs

[0077] Dissolve the drugs MB05032, FBPase-IN-1, and FBPase-1-IN (purchased from MedChemExpress Co., Ltd., Shanghai, with product numbers HY-16307, HY-146725, and HY-136717) in DMSO to prepare a stock solution with a concentration of 10 mM.

[0078] 1.2 Preparation of bacterial solutions

[0079] Culture Mycobacterium tuberculosis H37Rv (Mycobacterium tuberculosis, ATCC 27294) in Middlebrook 7H9 medium until the logarithmic growth phase, scrape the colonies and grind them. When evaluating the in vitro antibacterial activity, dilute the bacterial suspension with 7H9 medium and adjust the turbidity to OD 600 = 0.3, and then add it to the 7H9 medium at a ratio of 1:20 and mix well to obtain the Mycobacterium tuberculosis suspension. When detecting the number of viable bacteria in macrophages, dilute the bacterial suspension with 7H9 medium and adjust the turbidity to OD 600 = 0.6, and at this time, the bacterial concentration is regarded as 1×10 8 / mL.

[0080] 1.3 Detection of the minimum inhibitory concentration of FBP enzyme inhibitor against Mycobacterium tuberculosis standard strain

[0081] 1. Add the Mycobacterium tuberculosis suspension to each well in columns 3 - 10 respectively, so that the final concentration of the bacterial suspension in each well is 4×10 5 CFU / mL. At this time, the final drug concentrations in each well in columns 2 - 10 are 40, 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.31 μM, and 0.16 μM respectively. At the same time, set up negative control wells (medium without bacterial suspension and drug) and positive control wells (bacterial-containing medium without drug).

[0082] 2. Incubate the microplate for 7 days.

[0083] 3. Add the premixed chromogenic solution of Alamar Blue and 5% Tween-8 to the microplate, and continue to incubate for 24 h, then observe the color change of the microplate.

[0084] 4. The blue wells indicate no bacterial growth, and the pink wells indicate bacterial growth. The lowest drug concentration that prevents the color from changing from blue to pink is recorded as the minimum inhibitory concentration (MIC) that can inhibit the growth of Mycobacterium tuberculosis H37Rv.

[0085] The results are as Figure 1 shown. None of the three FBP enzyme inhibitors (MB05032, FBPase-IN-1, FBPase-1-IN) can directly inhibit the growth of H37Rv at a maximum concentration of 40 μM.

[0086] 1.4 Detection of the effect of FBP enzyme inhibitor on the number of viable Mycobacterium tuberculosis in macrophages

[0087] 1. Cell culture: The human blood-derived monocyte cell line THP-1 was purchased from the Cell Bank of the Chinese Academy of Sciences. THP-1 cells were cultured in RPMI-1640 complete medium containing 10% FBS and subcultured every 2 days. Cells in good growth condition were taken and PMA was added at a final concentration of 100 ng / mL, and inoculated at 5×10 5 cells / well in a well plate and cultured for 48 hours to induce adherent macrophages. After induction, the cells were continued to be cultured in complete medium without PMA to restore the resting state.

[0088] 2. Infection with Mycobacterium tuberculosis: The prepared Mycobacterium tuberculosis suspension (1×10 8 / mL) was added to each well of the cells at a ratio of MOI = 5:1. After continued culture for 4 hours, the cell culture supernatant was aspirated, washed with PBS buffer, and then added with RPMI-1640 complete medium containing different concentrations of MB05032 (200 nM, 500 nM), FBPase-IN-1 (1 μM, 10 μM), and FBPase-1-IN (1 μM, 10 μM) drugs for continued culture.

[0089] 3. Counting the viable intracellular bacteria: At 4, 24, 48, and 72 hours after H37Rv infection, the cell culture supernatant was aspirated, the cells were lysed with 0.1% Triton-X, and the cell lysate was serially diluted. The diluted liquid was inoculated onto Middlebrook 7H10 solid medium (containing 10% Middlebrook OADC enrichment broth), and finally the culture plates were cultured for 2 - 3 weeks and then the colony counts were performed.

[0090] 4. Statistical analysis: GraphPad Prism 9 software was used for statistical analysis and graphing. The comparison of Mycobacterium tuberculosis colony counts after co-culture with different concentrations of drugs was analyzed by Two-way ANOVA method, and P < 0.05 was considered statistically significant (****, P < 0.0001).

[0091] The results are as Figure 2As shown, at 4 and 24 hours after infection, there was no significant difference in the number of viable bacteria in cells between the group with the FBP enzyme inhibitor added and the normal infection control group (P>0.05). At 48 hours after infection, the number of viable bacteria in cells with the FBP enzyme inhibitor added was slightly reduced compared to the normal infection control group; after 72 hours, at this time, Mycobacterium tuberculosis H37Rv gradually survived and proliferated in macrophages, while the FBP enzyme inhibitors MB05032, FBPase-IN, and FBPase-1-IN could significantly inhibit the number of bacteria surviving in cells (P<0.05). Especially for MB05032, even at concentrations of 200 nM and 500 nM, the number of viable bacteria of H37Rv in cells was still inhibited to about 15% of the non-drug-added group, showing good anti-tuberculosis infection effect.

[0092] Example 2 Comparison of the number of Mycobacterium tuberculosis standard strains surviving in macrophages between MB05032, metformin, and troglitazone

[0093] 1.1 Preparation of drugs

[0094] Dissolve MB05032, metformin, and troglitazone drugs (purchased from Shanghai MedChemExpress Co., Ltd., product numbers HY-16307, HY-B0627, HY-50935) using DMSO.

[0095] 1.2 Comparison of the effects of three inhibitors on the number of Mycobacterium tuberculosis surviving in macrophages

[0096] 1. Cell culture: Culture THP-1 cells and induce them to adhere to macrophages as in Example 1.4.

[0097] 2. Infection with Mycobacterium tuberculosis: Use the prepared Mycobacterium tuberculosis suspension (1×10 8 / mL) and add it to each well of cells at a ratio of MOI = 5:1. After continuing to culture for 4 hours, aspirate the cell culture supernatant, wash with PBS buffer, and then add RPMI-1640 complete medium containing different concentrations of MB05032 (0.5 μM, 5 μM, 50 μM) and 5 μM of metformin and troglitazone drugs for continued culture.

[0098] 3. Counting the number of viable bacteria in cells: Lyse the cells at the corresponding time points as in Example 1.4 to release Mycobacterium and count the number of viable bacteria in cells.

[0099] 4. Statistical analysis: GraphPad Prism 9 software was used for statistical analysis and graphing. The Two-way ANOVA method was used to analyze the comparison of Mycobacterium tuberculosis colony counts after co-culture with different concentrations of drugs. P<0.05 was considered statistically significant (****, P<0.0001).

[0100] The results were as Figure 3 shown. At 4 hours after infection, there was no significant difference in the number of viable intracellular bacteria between the group treated with the FBP enzyme inhibitor and the normal infection control group (P>0.05). At 72 hours after infection, Mycobacterium tuberculosis H37Rv gradually survived and proliferated in macrophages, while the FBP enzyme inhibitor MB05032 could significantly inhibit the number of viable intracellular Mycobacterium tuberculosis in a concentration-gradient manner (P<0.05). At the concentration of 5 μM, the inhibitory effect of MB05032 on the intracellular survival of Mycobacterium tuberculosis was significantly better than that of metformin and troglitazone, which have been reported to be effective.

[0101] Example 3 CCK-8 assay to detect the cytotoxicity of MB05032 on THP-1 cells

[0102] 2.1 Cell culture

[0103] THP-1 cells were cultured with reference to Example 1.4. Cells in good growth condition were taken and PMA with a final concentration of 100 ng / mL was added. The cells were seeded in a well plate at 5×10 5 cells / well and cultured for 48 hours to induce adherent macrophages. After induction, the cells were continued to be cultured with complete medium without PMA to restore the resting state.

[0104] 4.2 Verification of the cytotoxicity of MB05032 on THP-1 cells

[0105] THP-1 cells were stimulated with DMSO and different concentrations of the FBP enzyme inhibitor MB05032 (final concentrations of 1.5625 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM) for 24 and 48 hours respectively. In addition, THP-1 cells after infection (MOI = 5) were stimulated with DMSO and different concentrations of MB05032 and the cell viability was observed. After culturing for the corresponding time, the culture supernatant was discarded and the cells were washed with PBS buffer (to remove the influence of the original drug on absorbance). RPMI-1640 complete medium containing 10% CCK-8 was added to the corresponding wells to incubate the cells, and the OD 450nm absorbance was measured with an enzyme-linked immunosorbent assay reader.

[0106] The survival rate (%) of THP-1 cells after stimulation with MB05032 was calculated as = (OD 450nm [experimental group] - OD450nm [Negative control group] / OD 450nm [Positive control group] - OD 450nm [Negative control group]) × 100%.

[0107] As Figure 4 shown, MB05032 had no toxicity to THP-1 cells when stimulated at concentrations of 1.5 - 100 μM for 24 and 48 hours, and the cell viability was greater than 80%. For THP-1 cells infected with Mycobacterium tuberculosis (MOI = 5), MB05032 drug also had no obvious virulence in the concentration range of 0.78 - 100 μM (the cell viability was greater than 80%).

Claims

1. Application of FBP enzyme inhibitors in the preparation of products for inhibiting Mycobacterium tuberculosis.

2. The use according to claim 1, wherein the FBPase inhibitor is selected from MB05032, FBPase-IN-1 or FBPase-1-IN.

3. The use according to claim 1, wherein the product for inhibiting Mycobacterium tuberculosis comprises a medical product or a non-medical product.

4. The use as claimed in claim 1, wherein the medical product refers to a medical drug, and the non-medical product refers to an experimental reagent or an antibacterial agent.

5. Use of an FBPase inhibitor in the preparation of a drug for preventing and / or treating diseases caused by Mycobacterium tuberculosis infection, wherein the FBPase inhibitor promotes macrophages to clear the intracellular survival of Mycobacterium tuberculosis, thereby exerting an anti-tuberculosis infection effect.

6. The use according to claim 5, wherein the FBPase inhibitor is selected from MB05032, FBPase-IN-1 or FBPase-1-IN.

7. The use according to claim 5, wherein the Mycobacterium tuberculosis comprises a clinical isolate of Mycobacterium tuberculosis, a standard strain of Mycobacterium tuberculosis or Mycobacterium tuberculosis carried by a patient infected with Mycobacterium tuberculosis.

8. The use as claimed in claim 5, wherein the drug can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents and / or suppositories.

9. A pharmaceutical composition comprising a FBPase inhibitor and another active ingredient against Mycobacterium tuberculosis infection, wherein the pharmaceutical composition has at least one of the following effects: a) inhibiting the activity of Mycobacterium tuberculosis; b) Anti-Mycobacterium tuberculosis infection; c) Prevention and / or treatment of diseases caused by Mycobacterium tuberculosis.

10. The use according to claim 9, wherein the FBPase inhibitor is selected from MB05032, FBPase-IN-1 or FBPase-1-IN; and the other active ingredient against Mycobacterium tuberculosis infection comprises one or more of antibiotics and other active ingredients that can help inhibit or kill Mycobacterium tuberculosis or provide resistance to patients.

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