Application of obacarat mesylate in preparation of antituberculous drugs

Abstract

The invention provides application of ombracarb mesylate in preparation of antituberculosis drugs, and particularly belongs to the technical field of drug preparation. According to the present invention, the olbachia mesylate is used for preparing the anti-tuberculosis drug, and the olbachia mesylate has good inhibitory activity on mycobacterium tuberculosis standard strains, mycobacterium tuberculosis sensitive strains and clinical drug-resistant strains, such that the drug lead compound and the drug candidate are provided for the development of the novel anti-tuberculosis drug; the important significance is realized on the prevention and / or treatment of tuberculosis.

Description

Technical Field

[0001] The invention belongs to the technical field of drug preparation, and specifically relates to the application of obatoclax mesylate in the preparation of anti-tuberculosis drugs. Background Art

[0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis (MTB) infection that seriously endangers human health. In recent years, due to many factors such as unreasonable treatment plans, poor patient compliance, improper use of second-line anti-tuberculosis drugs, and metabolic and nutritional differences, multidrug-resistant Mycobacterium tuberculosis (MDR-MTB) and extensively drug-resistant Mycobacterium tuberculosis (XDR-MTB) have emerged and spread rapidly. At present, the treatment success rate of drug-resistant tuberculosis (DR-TB) is still low.

[0003] The speed at which bacteria develop drug resistance far exceeds the speed at which new anti-tuberculosis drugs are developed. Although the three new drugs, Bedaquiline, Delamanid and Pretomanid, have been used for a short time, they have all developed varying degrees of drug resistance. If new drugs cannot meet the demand, it will create a huge obstacle to the treatment of DR-TB. Therefore, there is an urgent need to develop new anti-tuberculosis drugs with new structures, new targets and new mechanisms to curb the spread of drug-resistant tuberculosis, especially drugs targeting new targets that do not have pre-existing clinical drug resistance. Summary of the invention

[0004] The purpose of the present invention is to provide an application of obakla mesylate in the preparation of anti-tuberculosis drugs. Obakla mesylate has good inhibitory activity against standard strains of Mycobacterium tuberculosis, sensitive strains of Mycobacterium tuberculosis and clinical drug-resistant strains, which provides drug lead compounds and drug candidates for the development of new anti-tuberculosis drugs.

[0005] The present invention also provides the use of ovaclazide mesylate in the preparation of anti-tuberculosis drugs.

[0006] The present invention also provides the use of obacla mesylate in preparing a product for inhibiting the growth of Mycobacterium tuberculosis.

[0007] Preferably, the Mycobacterium tuberculosis includes a standard strain of Mycobacterium tuberculosis, a sensitive strain of Mycobacterium tuberculosis or a drug-resistant strain of Mycobacterium tuberculosis.

[0008] The present invention provides the use of obakla mesylate in preparing a Pks13 (polyketide synthase 13) inhibitor.

[0009] The present invention also provides the use of obakla mesylate in the preparation of Pks13-TE inhibitors.

[0010] The present invention provides an application of obakla mesylate in the preparation of anti-tuberculosis drugs. The obakla mesylate of the present invention can significantly inhibit Pks13 and interact with the Pks13 protein. At the same time, the obakla mesylate has good inhibitory activity against standard strains of Mycobacterium tuberculosis, sensitive strains of Mycobacterium tuberculosis and clinical drug-resistant strains, which provides drug lead compounds and drug candidates for the development of new anti-tuberculosis drugs, and is of great significance for the prevention and / or treatment of tuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0012] Figure 1 A structural diagram of the compound Obatoclax Mesylate provided by the present invention;

[0013] Figure 2 This is a dose-effect curve diagram of the inhibitory effect of the compound Obatoclax Mesylate provided by the present invention on Pks13;

[0014] Figure 3 This is a graph showing the results of the interaction detection between the compound Obatoclax Mesylate provided by the present invention and the Pks13 protein;

[0015] Figure 4 This is a diagram showing the molecular docking results of the compound Obatoclax Mesylate provided by the present invention and the Pks13 protein. DETAILED DESCRIPTION

[0016] The present invention also provides the use of ovacarbamide mesylate in the preparation of anti-tuberculosis drugs. The structure of ovacarbamide mesylate is as follows: Figure 1 The present invention also provides the use of obakla mesylate in preparing a product for inhibiting the growth of Mycobacterium tuberculosis.

[0017] In a specific embodiment, the Mycobacterium tuberculosis includes a standard strain of Mycobacterium tuberculosis, a sensitive strain of Mycobacterium tuberculosis, or a drug-resistant strain of Mycobacterium tuberculosis.

[0018] The present invention provides an application of obakla mesylate in the preparation of a Pks13 inhibitor. Mycolic acid is a characteristic component necessary for the survival of Mycobacterium tuberculosis, and polyketide synthase 13 (Pks13) is mainly responsible for the enzyme in the last step of mycolic acid biosynthesis. The obakla mesylate of the present invention can inhibit the expression of Pks13 and achieve the inhibition of Mycobacterium tuberculosis.

[0019] The present invention also provides the use of obakla mesylate in the preparation of Pks13-TE inhibitors. The Pks13 protein is composed of 5 domains, wherein the TE domain has a cutting function, which is mainly responsible for catalyzing the release of polyketide compounds from the enzyme when they reach full length, which is necessary for the formation of mycolic acid precursors. The obakla mesylate of the present invention can inhibit the expression of the TE domain of the Pks13 protein, thereby achieving the inhibition of Mycobacterium tuberculosis.

[0020] In order to further illustrate the present invention, the use of the obakla mesylate provided by the present invention in the preparation of anti-tuberculosis drugs is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0021] Example 1

[0022] Determination of the dose-effect curve of Obatoclax Mesylate in the Pks13 inhibitor screening model

[0023] Using 4-methylumbelliferyl heptyl ester (4-MUH, purchased from Sigma, catalog number: M2514) as a substrate, under the catalysis of Pks13-TE (Pks13: Rv3800c, the constructed TE domain protein of Pks13 is the amino terminal sequence of 1464-1732 in the full length of Pks13, and the vector construction and protein expression methods are conventional methods), 4-MUH will be hydrolyzed into 4-MU, and the fluorescence value at 355nm of excitation light and 460nm of emission light can be detected to determine the amount of hydrolysis product 4-MU, so as to detect the IC value of the compound on Pks13-TE protein. 50 , determine the inhibitory activity. Detection method reference information: Wang Xiao, Meng Jianzhou, Guan Yan, Liu Sihan, Zhang Jiawei, Li Xiaohui, Liu Yishuang*. Discovery of new anti-tuberculosis lead compounds targeting polyketide synthase 13, Chinese Journal of Antibiotics, 2022, 47(10): 1038-1044.

[0024] The enzyme activity detection system is as follows: 0.1M Tris-HCl pH 7.0, 30μM 4-MUH, 1.8μM Pks13-TE protein, the total system is 100μL, and the reaction time is 20min. The specific operation is as follows: (1) The compound is prepared into a 10mM stock solution with DMSO, and then the compound is diluted in multiples and added to a 96-well black board to make the final concentrations of 0.78μM, 1.56μM, 3.125μM, 6.25μM, 12.5μM, 25μM, 50μM and 100μM, respectively. (2) Pks13-TE protein diluted with 0.1M Tris-HCl (final concentration of 1.8μM) is added and incubated with the compound for 15min. (3) Substrate 4-MUH (final concentration of 30μM) is added and incubated again for 20min. (4) The fluorescence value is detected at 355nm for excitation and 460nm for emission.

[0025] At the same time, a negative control group (containing the same amount of DMSO) and a positive control group (without adding Pks13-TE protein, ie, the enzyme activity was completely inhibited) were set up.

[0026] The inhibition rate was calculated as follows: enzyme inhibition rate = (fluorescence value of the negative control group - fluorescence value of the experimental group with added compounds) / (fluorescence value of the negative control group - fluorescence value of the positive control group) × 100%.

[0027] According to the above enzyme activity system, the inhibition rate under different concentration conditions of the compound was obtained, with the logarithm of the compound concentration as the horizontal axis and the inhibition rate as the vertical axis. The inhibition rate data were fitted using the inhibition dose dependence module in the data analysis of Graphpadprism 5 software to obtain the IC 50 .

[0028] The dose-effect curve of Obatoclax Mesylate is as follows Figure 2 As shown in the figure, with the increase of compound concentration, the inhibitory effect of Obatoclax Mesylate on Pks13-TE protein gradually increased, showing obvious concentration dependence. 50 The value is 19.93 μM.

[0029] Example 2

[0030] Detection of the interaction between Obatoclax Mesylate and Pks13 protein

[0031] Using the SPR experiment, the Pks13-TE protein was first fixed on a CM5 chip with dextran (purchased from Reichert, catalog number: 13206066), and then the compound was gradient diluted using PBST buffer to ensure that each concentration contained 5% DMSO. The diluted compound was then passed through the chip from low concentration to high concentration. The SPR experiment was completed using the Reichert2SPR system instrument, and the results were analyzed using TraceDrawer software. The results showed that Obatoclax Mesylate was able to bind to the Pks13-TE protein in a dose-dependent manner ( Figure 3 ), its combined K D The values ​​are 7.53×10 -5 M.

[0032] Example 3

[0033] Molecular docking analysis of Obatoclax Mesylate and Pks13 protein

[0034] In order to explore how the structure of positive compounds affects their inhibitory effect on Pks13-TE protein, the present invention used MOE 2020 to perform molecular docking analysis on the compounds and Pks13-TE. The crystal structure of Pks13-TE comes from the ProteinDataBank database (PDB: 5V3Y). Its protein structure consists of a core domain and a lid domain, and the active pocket is located at the interface between the two domains. The molecular docking software was used to optimize the Obatoclax Mesylate molecular structure and dock it with the active pocket, and the docking model with the highest score was selected to analyze its interaction. The results are as follows Figure 4 As shown, Obatoclax Mesylate can form a hydrogen bond between Asp1644 and Asn1640 of the Pks13-TE protein, and can also form an Arene-H interaction between the amino acids Tyr1674 and Asn1640, thereby improving the stability of the binding between the protein and the compound.

[0035] Example 4

[0036] Evaluation of the Antibacterial Activity of Obatoclax Mesylate

[0037] MTB standard strains, MTB sensitive strains and clinical drug-resistant strains (see Table 1 for details, MTB standard strain H37Rv, source: ATCC27294; MTB sensitive strains and drug-resistant strains, source: National Tuberculosis Clinical Laboratory) were cultured in 7H9 medium (containing 10% OADC, 0.05% Tween 80). In a 96-well plate, 200 μL of 7H9 culture medium inoculated with logarithmic phase strains (final concentration A600 ≈0.1), then add the compound in gradient dilution, and observe the results after static culture for 10 days. The minimum drug concentration that completely inhibits bacterial growth in the well is the minimum inhibitory concentration (MIC). At the same time, a negative control group (7H9 medium containing the same DMSO content) and a positive control group (isoniazid (INH) with the same concentration gradient as the compound) were set up.

[0038] The results showed that the compound Obatoclax Mesylate exhibited a strong inhibitory effect on MTB standard strains, sensitive strains and clinical strains, and its activity against clinical strains was better than that of positive drugs. The specific results are shown in Table 1.

[0039] Table 1 Anti-tuberculosis activity of Obatoclax Mesylate

[0040]

[0041]

[0042] Note: H37Rv: standard strain; STB: sensitive strain; MDR: multidrug-resistant strain; XDR: extensively drug-resistant strain.

[0043] MDR-30611: resistant to isoniazid and rifampicin.

[0044] MDR-30141: resistant to isoniazid and rifampicin.

[0045] XDR-28127: Resistant to isoniazid, rifampicin, ethambutol, streptomycin, levofloxacin, amikacin, clofazimine, capreomycin, and kanamycin.

[0046] XDR-27830: Resistant to isoniazid, rifampicin, ethambutol, streptomycin, levofloxacin, amikacin, capreomycin, and kanamycin.

[0047] Example 4

[0048] Acute toxicity test of Obatoclax Mesylate in mice

[0049] The experimental animals were 5 Kunming mice, weighing 18-22g. Weigh 50mg of the compound, dissolve in 1mL of 0.5% sodium carboxymethyl cellulose (CMC-Na) aqueous solution, vortex for 1min and ultrasonic for 10min to a final concentration of 50mg / mL. Each mouse was gavaged with 0.2mL, i.e. 500mg / kg body weight, and the behavioral state of the mice was observed at 0.5h, 1.0h, 2h, 4h, 8h, 12h, 24h, 48h and 72h to see if there was a significant change in the mice's behavior and whether they died. The experimental results showed that no mice died within 72h after administration, and no obvious toxic side effects occurred, indicating that the compound Obatoclax Mesylate has good safety.

[0050] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of obakla mesylate in the preparation of anti-tuberculosis drugs.

2. Application of obakla mesylate in the preparation of products for inhibiting the growth of Mycobacterium tuberculosis.

3. The use according to claim 2, characterized in that: The Mycobacterium tuberculosis includes a standard strain of Mycobacterium tuberculosis, a sensitive strain of Mycobacterium tuberculosis or a drug-resistant strain of Mycobacterium tuberculosis.

4. Application of obakla mesylate in the preparation of Pks13 inhibitors.

5. Application of obakla mesylate in the preparation of Pks13-TE inhibitors.

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