Application of KY1220 in the preparation of drugs for inhibiting Mycobacterium tuberculosis

Abstract

The present invention relates to the field of medical technology. The present invention provides an application of a small molecule compound in the preparation of an anti-tuberculosis mycobacterium preparation, wherein the small molecule compound is KY1220 or a composition containing KY1220; the composition containing KY1220 is a composition containing KY1220 and other active ingredients, and the other active ingredients are active substances that can promote the body's anti-tuberculosis and / or drugs that improve tuberculosis symptoms. The present invention uses KY1220 to show good antibacterial activity against tuberculosis bacilli and can significantly inhibit Mycobacterium tuberculosis. In addition, KY1220 has a relatively high survival rate for U937 and THP-1 cells.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of a small molecule compound KY1220 in the preparation of a drug for inhibiting mycobacterium tuberculosis. Background Art

[0002] Tuberculosis (TB) is one of the most lethal chronic infectious diseases in the world caused by Mycobacterium tuberculosis (Mtb). It is also one of the major challenges facing global public health, the "number one killer" of people infected with human immunodeficiency virus (HIV), and the main lethal infectious disease associated with antimicrobial resistance. The mortality rate is very high among HIV carriers who are also infected with multidrug-resistant Mycobacterium tuberculosis. This mixed infection is also the cause of the prevalence of multidrug-resistant tuberculosis. According to estimates by the World Health Organization, in 2021, there will be more than 10.6 million new cases of tuberculosis and more than 1.6 million deaths worldwide. Only 36% of patients who need treatment will receive treatment. The prevalence of drug-resistant strains and co-infection with HIV have further exacerbated the global TB burden. The number of patients with rifampicin resistance-tuberculosis (RR-TB) and multiple drug resistant tuberculosis (MDR-TB) who started treatment in 2021 was 161 and 746, covering only one-third of all patients in need of treatment. However, 187,000 people infected with HIV died of tuberculosis. Effective control of tuberculosis remains a major public health challenge facing the world.

[0003] Although the drugs for the treatment of tuberculosis are continuously being improved and developed, and the world has been actively developing a vaccine that can effectively prevent the spread of Mycobacterium tuberculosis, it is still challenging to develop a new vaccine that is more applicable and durable than Bacillus Calmette-Guerin (BCG). Mycobacterium tuberculosis has the ability to regulate the host immune response and evade drugs, which can lead to the failure of BCG vaccination and eventually form drug-resistant Mycobacterium tuberculosis.

[0004] In recent years, great progress has been made in the treatment of tuberculosis. For example, isoniazid (INH), rifampicin (RFP), bedaquiline, delamanid, etc. are very effective against Mycobacterium tuberculosis, but tuberculosis cannot be completely cured. When INH becomes resistant, it usually leads to the emergence of multidrug-resistant strains, which has become a major obstacle to the control of tuberculosis. Bedaquiline and delamanid have limited activity in killing persisters, and both drugs have strong cardiotoxicity. More importantly, clinically, mutant strains of Mycobacterium tuberculosis that are resistant to these two drugs were quickly discovered. Fluoroquinolones are effective second-line anti-tuberculosis drugs for the treatment of drug-resistant tuberculosis and are widely used to treat multidrug-resistant tuberculosis. However, it has been found that fluoroquinolones are easily resistant to multidrug-resistant tuberculosis. Although fluoroquinolones have a good inhibitory effect on DNA gyrase, mutations in the gyrA domain produce resistance to these drugs during the treatment of tuberculosis. With the emergence of drug resistance in Mycobacterium tuberculosis, new therapies that can shorten the treatment cycle are crucial for the prevention and treatment of tuberculosis, especially some therapies with new mechanisms of action that provide effective methods for the treatment of drug-resistant tuberculosis.

[0005] Based on the above problems, the urgent need to develop new highly effective and low-toxic anti-tuberculosis drugs to effectively control tuberculosis has become an important research direction for many researchers. Summary of the invention

[0006] In a first aspect, the present invention provides a use of a small molecule compound in the preparation of an anti-Mycobacterium tuberculosis preparation.

[0007] Furthermore, the small molecule compound is KY1220 or a composition containing KY1220.

[0008] Furthermore, the structural formula of KY1220 is as shown in I:

[0009]

[0010] Furthermore, the composition containing KY1220 is a composition comprising KY1220 and other active ingredients.

[0011] Furthermore, the other active ingredients are active substances that can promote the body's anti-tuberculosis and / or drugs that improve tuberculosis symptoms.

[0012] Furthermore, the preparation may be a biological preparation or a pharmaceutical preparation.

[0013] Furthermore, the dosage forms of the anti-tuberculosis mycobacterium preparation include: sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, lozenges, granules, granules, pills, pills, suspensions, powders, wine preparations, preparations, drops, injections, powder injections, creams, sustained-release preparations, targeted agents, etc.

[0014] Furthermore, the administration methods of the anti-tuberculosis mycobacterium preparation include oral administration, injection, implantation, external application, spraying, and inhalation.

[0015] Furthermore, the anti-Mycobacterium tuberculosis preparation can be used to prevent or treat tuberculosis caused by Mycobacterium tuberculosis infection.

[0016] Furthermore, the Mycobacterium tuberculosis infection includes: primary infection, secondary infection, and extrapulmonary infection.

[0017] Furthermore, the tuberculosis includes but is not limited to drug-resistant tuberculosis, non-drug-resistant tuberculosis, pulmonary tuberculosis, extrapulmonary tuberculosis, etc.

[0018] Furthermore, the drug-resistant tuberculosis includes but is not limited to mono-drug-resistant tuberculosis, multi-drug-resistant tuberculosis, multidrug-resistant tuberculosis, and extensively drug-resistant tuberculosis.

[0019] Furthermore, the pulmonary tuberculosis includes but is not limited to primary pulmonary tuberculosis, secondary pulmonary tuberculosis, blood type disseminated pulmonary tuberculosis, tracheobronchial tuberculosis, tuberculous pleurisy, septic tuberculosis, etc.

[0020] Furthermore, the extrapulmonary tuberculosis includes but is not limited to intestinal tuberculosis, renal tuberculosis, bone and joint tuberculosis, etc.

[0021] In a second aspect, the present invention provides use of the compound described in the first aspect in the preparation of an anti-tuberculosis preparation.

[0022] Furthermore, the small molecule compound is KY1220 or a composition containing KY1220.

[0023] Furthermore, the structural formula of KY1220 is as shown in I:

[0024]

[0025] Furthermore, the combination of KY1220 is a composition comprising KY1220 and other active ingredients.

[0026] Furthermore, the other active ingredients are active substances that can promote the body's anti-tuberculosis and / or drugs that improve tuberculosis symptoms.

[0027] Furthermore, the preparation may be a biological preparation or a pharmaceutical preparation.

[0028] Furthermore, the anti-tuberculosis preparations and / or drug dosage forms include: sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, lozenges, granules, granules, pills, pills, suspensions, powders, wine preparations, preparations, drops, injections, powder injections, creams, sustained-release agents, targeted agents, etc.

[0029] Furthermore, the administration methods of the anti-tuberculosis drug include oral administration, injection, implantation, external application, spraying, inhalation and / or their combination.

[0030] Furthermore, the tuberculosis includes but is not limited to drug-resistant tuberculosis, non-drug-resistant tuberculosis, pulmonary tuberculosis, extrapulmonary tuberculosis, etc.

[0031] Furthermore, the drug-resistant tuberculosis includes but is not limited to mono-drug-resistant tuberculosis, multi-drug-resistant tuberculosis, multidrug-resistant tuberculosis, and extensively drug-resistant tuberculosis.

[0032] Furthermore, the pulmonary tuberculosis includes but is not limited to primary pulmonary tuberculosis, secondary pulmonary tuberculosis, blood type disseminated pulmonary tuberculosis, tracheobronchial tuberculosis, tuberculous pleurisy, septic tuberculosis, etc.

[0033] Furthermore, the extrapulmonary tuberculosis includes but is not limited to intestinal tuberculosis, renal tuberculosis, bone and joint tuberculosis, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 MIC test results of KY1220 on standard strains of Mycobacterium tuberculosis and drug-resistant Mycobacterium tuberculosis

[0035] Figure 2 The effect of KY1220 on the survival rate of U937 and THP-1 cells DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with the accompanying drawings and specific examples so that those skilled in the art can better understand the present invention and implement it, but the examples are not intended to limit the present invention. The experimental methods in the examples, unless otherwise specified, all adopt conventional techniques in the art, and the experimental reagents are all purchased commercially.

[0037] The chemical structure of KY1220 is shown in I:

[0038]

[0039] It is a small molecule compound that targets the Wnt / β-catenin pathway to disrupt the stability of β-catenin and Ras, inhibiting a series of reactions including epithelial-mesenchymal transition (EMT) and apoptosis. Abnormal activation of the Wnt / β-catenin and Ras pathways has been found to be associated with metastatic colorectal cancer and to act synergistically in the process of tumor metastasis. KY1220 disrupts the binding affinity of Ras and β-catenin complex by binding to the G protein signaling domain (Regulator of G protein Signaling, RGS) domain of axin, causing β-catenin and Ras to lose stability simultaneously through GSK3β activation. This mechanism of action is an ideal strategy for the treatment of metastatic colorectal cancer.

[0040] Cell lines: The standard strain of Mycobacterium tuberculosis H37Rv was from the cell bank (ATCC: 27294), two drug-resistant strains MDR-1 and MDR-2 were from clinical isolates of Beijing Chest Hospital (MDR-1 strain was resistant to rifampicin, isoniazid, and streptomycin; MDR-2 strain was resistant to rifampicin, isoniazid, streptomycin, and ethambutol), human histiocytic lymphoma cells (U937 cells), and human monocytic leukemia cells (THP-1 cells).

[0041] Monoresistant tuberculosis: Monoresistant tuberculosis refers to the Mycobacterium tuberculosis infected in tuberculosis patients, which has been confirmed to be resistant to one anti-tuberculosis drug in vitro.

[0042] Multidrug-resistant tuberculosis: Multidrug resistance means that the Mycobacterium tuberculosis infected by tuberculosis patients has been confirmed in vitro to be resistant to more than one anti-tuberculosis drug, but does not include cases where the Mycobacterium tuberculosis is resistant to both isoniazid and rifampicin at the same time.

[0043] Multidrug-resistant tuberculosis: Tuberculosis that is resistant to both isoniazid and rifampicin is called multidrug-resistant tuberculosis.

[0044] Extensively drug-resistant tuberculosis: In addition to multidrug-resistant tuberculosis, tuberculosis is also resistant to fluoroquinolones and one of the second-line injectable anti-tuberculosis drugs, including kanamycin, amikacin, capreomycin and streptomycin. It is called extensively drug-resistant tuberculosis.

[0045] Example 1 Drug sensitivity test of KY1220 against tuberculosis in vitro

[0046] The in vitro anti-TB standard strain H37Rv of KY1220 was determined by 96-well plate microdilution method.

[0047] Add 200 μL of sterile PBS twice around the perimeter of a sterile 96-well plate to prevent evaporation. 120 μL of 7H9 liquid culture medium containing 10% OADC was added to wells C3 and D3 (except rows A and H), and 100 μL of 7H9 liquid culture medium containing 10% OADC was added to the wells in the remaining columns C4-C10. 80 uL of KY1220 (powder dissolved in DMSO and stored at -80°C) with a diluted concentration of 100 μM was added to the experimental wells C3 and D3. The liquid in the wells in the third column was pipetted evenly, and 100 μL was drawn into the corresponding wells in the fourth column. Then, the liquid was pipetted evenly and the above operation was repeated to the corresponding wells in the tenth column until all drugs were diluted to a final concentration of 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.312 μM, and 0.156 μM (200 μL volume). Add INH and RIF stock solutions with a final concentration of 1 μg / ml to the positive drug wells in row E, and set up 4 replicate wells. Add 200 μL of 7H9 liquid culture medium containing 10% OADC to rows F3-F6 as negative control wells, and add 100 μL of 7H9 liquid culture medium containing 10% OADC to rows F7-F10 as positive control wells (see Table 1).

[0048] Table 1. Distribution of concentrations of MTB standard strains in 96-well plates

[0049]

[0050]

[0051] The activity of KY1220 against drug-resistant strains MDR-1 and MDR-2 was determined by 96-well plate microdilution assay.

[0052] Add 200 μl of sterile PBS around a sterile 96-well plate (to prevent evaporation), add 120 μl of 7H9 liquid culture medium containing 10% OADC to each well of B2, C2, D2, and E2, add 100 μl of 7H9 liquid culture medium containing 10% OADC to each of the remaining wells, and add 80 μl of KY1220 stock solution (powder dissolved in DMSO and stored at -80°C) diluted to 100 μM to wells B2, C2, D2, and E2, respectively. The liquid in the B2, C2, D2, and E2 wells was pipetted evenly and 100 μL was drawn into the wells corresponding to the 3rd column, and then continued to pipet evenly and repeat the above operation to the wells corresponding to the 9th column until all drugs were diluted to make the final drug concentrations 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.312 μM, and 0.156 μM (200 μL volume). B2-11 / C2-11 is the MDR-1 group, and D2-11 / E2-11 is the MDR-2 group. 100 μL of 7H9 liquid culture medium containing 10% OADC was added to the 12th column as a negative control well, the 10th column as a positive control well, and 2 wells each for the positive control of 2 strains of drug-resistant bacteria (see Table 2).

[0053] Table 2 Distribution of concentrations of MTB resistant strains in 96-well plates

[0054]

[0055] The Roche culture medium of the two MTB strains cultured to the logarithmic growth phase was taken out from the 37°C constant temperature incubator, and the colonies were scraped and placed in a 2mL ultrasonic dispersion tube containing 10% OADC 7H9 liquid culture medium. The tube was ultrasonicated for 1 minute in an ultrasonic dispersion instrument, and the ratio of the bacterial solution to the liquid culture medium was adjusted in a colorimetric cup, and the cup was placed in a spectrophotometer. The detection wavelength was set to 600nm, and the absorbance value of each strain was adjusted to 0.3. The concentration of the bacterial solution of each strain was calculated based on the absorbance value. Then, the two bacterial solutions were diluted 10 times with 7H9 liquid culture medium, and 100 μL of the diluted bacterial solution was pipetted and added to two 96-well plates, and the sterile 96-well culture plates with the bacterial solution were sealed with sealing film and placed in a 37°C constant temperature incubator for static culture for 10 days.

[0056] After 10 days, take out the two 96-well culture plates from the constant temperature incubator, carefully remove the sealing film in the biosafety cabinet, and add 70 μL of resazurin indicator to each well. The sterile 96-well culture plates with the indicator added are placed in a 37°C constant temperature incubator for 24-48 hours.

[0057] After the incubation, take out the two 96-well culture plates, observe and record the color changes of each well in the two 96 microplates (blue indicates no strain growth, pink indicates strain growth), and determine the MIC (minimum inhibitory concentration is defined as the lowest drug concentration that inhibits visible bacterial growth). The MIC value is defined as the drug concentration value at which the color changes from blue to pink after adding the resazurin indicator 24-48 hours after culturing the 96-well plate in a 37°C constant temperature incubator for 10 days, and pink indicates bacterial growth.

[0058] GraphPad 8.0.2 software was used to draw graphs and perform statistical analysis on the results.

[0059] The experimental results showed that the MIC of KY1220 against MTB H37Rv was 2.5 μM, and the MIC of KY1220 against two clinical multidrug-resistant strains, MDR1 and MDR1, was 10 μM. KY1220 had a good inhibitory effect on Mycobacterium tuberculosis in vitro (see Figure 1 ).

[0060] Example 2 Experimental study on the inhibition of cytotoxicity of Mycobacterium tuberculosis by KY1220

[0061] The present invention adopts CCK-8 method to determine the cell viability. The CCK-8 method utilizes WST-8 [(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt], which can be reduced to a water-soluble orange-yellow formazan dye by dehydrogenase in the mitochondria of living cells under the action of the electron carrier 1-methoxy-5-methylphenazine dimethyl sulfate, and the amount of the generated formazan is proportional to the number of living cells.

[0062] Human histiocytic lymphoma cells (U937 cells) and human monocytic leukemia cells (THP-1 cells) were taken out of liquid nitrogen and revived to the logarithmic phase. The concentration of the cell suspension was adjusted to about 2×105 cells / ml and added to a 96-well cell culture plate, 100 μL per well. Phorbol 12-myristate 13-acetate (PMA) was added to differentiate the cells into macrophages at a final concentration of 50 ng / mL. The plates were placed in a cell culture incubator (5% CO 2 , 37°C) overnight. Then, the old culture medium was discarded, and the culture medium was added to the culture plate to dilute the final concentration of KY1220 to 20μM, 10μM, 5μM, 2.5μM, and 1.25μM, with 2 replicates for each concentration. The negative control wells did not contain drugs, and the cell plate was placed in the incubator for another 24h. Then the culture medium was discarded, and the cytotoxicity of the drug was detected according to the instructions of the CCK-8 Cell Proliferation / Cytotoxicity Kit (Cat. No.: CK04, Dojin Chemical Research Institute, Japan).

[0063] The experimental results showed that the survival rates of U937 and THP-1 cells were 107.6% and 85.0% when the concentration of KY1220 was 20 μM, and 109.2% and 92.0% when the concentration of KY1220 was 2.5 μM, respectively. When the drug concentration was 20 μM and 2.5 μM, the cell survival rate was not significantly different from that of the control group (see Figure 2 ).

[0064] The above experimental results show that KY1220 exhibits good antibacterial activity against Mycobacterium tuberculosis and can significantly inhibit Mycobacterium tuberculosis. In addition, KY1220 has a relatively high survival rate on U937 and THP-1 cells. Therefore, KY1220 can be used as a drug for the preparation of anti-Mycobacterium tuberculosis drugs.

Claims

1. Use of a small molecule compound in the preparation of an anti-tuberculosis Mycobacterium tuberculosis preparation, wherein the small molecule compound is KY1220, and the structural formula of the small molecule compound KY1220 is as shown in I: （I）。 2. The use according to claim 1, wherein the preparation further comprises other active ingredients.

3. The use according to claim 2, characterized in that The other active ingredients are drugs that promote the body's resistance to tuberculosis and / or improve tuberculosis symptoms.

4. The use according to claim 1, characterized in that The preparation can be used for treating tuberculosis caused by infection with Mycobacterium tuberculosis.

5. Use of a small molecule compound in the preparation of a preparation for treating tuberculosis, wherein the small molecule compound is KY1220, and the structural formula of the small molecule compound KY1220 is as shown in I: （I）。 6. The use according to claim 5, characterized in that The formulations may also contain other active ingredients.

7. The use according to claim 6, characterized in that The other active ingredients are drugs that promote the body's resistance to tuberculosis and / or improve tuberculosis symptoms.

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