Application of B-AP15 in preparation of antituberculous drugs

Abstract

The invention provides application of B-AP15 in preparation of antituberculosis drugs, and particularly belongs to the technical field of drug preparation. The B-AP15 is used for preparing the antituberculous drugs and has relatively good inhibitory activity on MTB standard strains, MTB sensitive strains and clinical drug-resistant strains, so that a drug lead compound and a drug candidate are provided for development of the novel antituberculous drugs, and the B-AP15 has important significance on prevention and / or treatment of tuberculosis.

Description

Technical Field

[0001] The invention belongs to the technical field of drug preparation, and particularly relates to application of B-AP15 in the preparation of anti-tuberculosis drugs. Background Art

[0002] Tuberculosis is a chronic infectious disease caused by infection with Mycobacterium tuberculosis (MTB). In recent years, due to problems such as non-standard treatment, poor patient compliance, and abuse of second-line drugs, the emergence and spread of drug-resistant Mycobacterium tuberculosis has become increasingly serious, especially multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains, which have become a major problem in the field of public health. The success rate of treatment of multidrug-resistant or rifampicin-resistant tuberculosis (MDR / RR-TB) also needs to be improved urgently.

[0003] With the accelerated evolution of bacterial drug resistance, the development of new anti-tuberculosis drugs has been unable to keep up with the speed of drug resistance. Although the emergence of new drugs such as Bedaquiline, Delamanid and Pretomanid has brought certain breakthroughs in treatment, drug resistance to these drugs has also begun to emerge, and there is an urgent need to develop new anti-tuberculosis drugs to cope with the spread of drug-resistant tuberculosis. Therefore, it is particularly critical to develop drugs that act on new targets without existing drug resistance. Summary of the invention

[0004] The purpose of the present invention is to provide the use of B-AP15 in the preparation of anti-tuberculosis drugs. The B-AP15 of the present invention has good inhibitory activity against Mycobacterium tuberculosis, which provides drug lead compounds and drug candidates for the development of new anti-tuberculosis drugs.

[0005] The present invention provides application of B-AP15 in preparing anti-tuberculosis drugs.

[0006] The present invention also provides the use of B-AP15 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 also provides the use of B-AP15 in preparing a Pks13 inhibitor.

[0009] The present invention also provides the use of B-AP15 in preparing a Pks13-TE inhibitor.

[0010] The present invention provides the use of B-AP15 in the preparation of anti-tuberculosis drugs. The B-AP15 of the present invention can significantly inhibit Pks13 and interact with the Pks13 protein, and at the same time, has good inhibitory activity against MTB standard strains, MTB sensitive strains 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 The structural diagram of the compound B-AP15 provided by the present invention;

[0013] Figure 2 This is a result diagram of the dose-effect relationship curve of the inhibitory effect of the compound B-AP15 provided by the present invention on Pks13;

[0014] Figure 3 This is a graph showing the results of the interaction detection between the compound B-AP15 provided by the present invention and the Pks13 protein;

[0015] Figure 4 This is a diagram showing the molecular docking results of the compound B-AP15 provided by the present invention and the Pks13 protein. DETAILED DESCRIPTION

[0016] The present invention provides the use of B-AP15 in the preparation of anti-tuberculosis drugs. The structure of B-AP15 of the present invention is as follows Figure 1 As shown. The present invention has no special limitation on the source of B-AP15, and conventional commercially available products can be used, such as those purchased from Taoshu Biotechnology Co., Ltd., item number: T1932. The present invention also provides the use of B-AP15 in the preparation of a product for inhibiting the growth of Mycobacterium tuberculosis. 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. The B-AP15 described in the present invention has good inhibitory activity against MTB standard strains, MTB sensitive strains 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.

[0017] The present invention also provides the use of B-AP15 in the preparation of Pks13 inhibitors. The survival of Mycobacterium tuberculosis depends on the synthesis of its unique component, mycolic acid. Polyketide synthase 13 (Pks13) is responsible for the last step of mycolic acid synthesis, and its absence will seriously damage the cell wall of Mycobacterium tuberculosis, causing it to be unable to survive. The B-AP15 described in the present invention inhibits Mycobacterium tuberculosis by inhibiting the Pks13 protein.

[0018] The present invention also provides the use of B-AP15 in the preparation of Pks13-TE inhibitors. The Pks13 protein is composed of multiple domains, among which the TE domain plays a key role in the cleavage reaction and is responsible for catalyzing the release of polyketide compounds from the enzyme when they reach full length, which is a necessary step in the formation of mycolic acid precursors. The activity of the TE domain regulates the function of the Pks13 protein. The present invention realizes the development of anti-tuberculosis drugs by acting on the TE domain of Pks13 through B-AP15.

[0019] In order to further illustrate the present invention, the application of B-AP15 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.

[0020] Example 1

[0021] Determination of the dose-effect curve of B-AP15 in the Pks13 inhibitor screening model

[0022] Pks13-TE protein (Pks13: Rv3800c, the constructed TE domain protein of Pks13 is the amino terminal sequence of the 1464-1732 interval in the full length of Pks13, and the vector construction and protein expression methods are conventional methods) can hydrolyze the substrate 4-methylumbelliferyl heptyl ester (4-MUH, purchased from Sigma, catalog number: M2514) into 4-MU, and 4-MU will emit fluorescence under the conditions of excitation light 355nm and emission light 460nm, so the protein activity can be judged by detecting the amount of 4-MU generated in the reaction system. According to the preliminary experiment, 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. 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.

[0023] The compound was diluted serially with DMSO, and 2 μL of the diluted compound was added to each well of a 96-well black board to make the final concentrations 0.78 μM, 1.56 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM and 100 μM, respectively.

[0024] 48 μL of Pks13-TE protein diluted with Tris-HCl was added (final concentration was 1.8 μM) and incubated with the compound for 15 min.

[0025] 50 μL of Tris-HCl-diluted substrate 4-MUH (final concentration 30 μM) was added and incubated again for 20 min.

[0026] The fluorescence value was detected at 355 nm for excitation light and 460 nm for emission light.

[0027] 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.

[0028] The inhibition rate was calculated as follows: enzyme inhibition rate = (negative control group - experimental group with added compound) / (negative control group - positive control group) × 100%.

[0029] 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 half IC 50 .

[0030] The dose-effect curve of B-AP15 is as follows Figure 2 As shown in the figure, with the increase of compound concentration, the inhibitory effect of B-AP15 on Pks13-TE protein gradually increased, showing obvious concentration dependence. 50 The value is 21.23 μM.

[0031] Example 2

[0032] Detection of interaction between B-AP15 and Pks13 protein

[0033] In the experiment to detect the interaction between compound B-AP15 and Pks13 protein, the buffer system used was PBST solution, which was completed by Reichert 2SPR system. The experimental steps are as follows:

[0034] Turn on the computer and open the software, install the CM5 chip (purchased from Reichert, catalog number: 13206066), select Prime, and rinse the chip surface with PBST buffer.

[0035] EDC / NHS were mixed in equal volumes to activate the carboxyl groups on the surface of the CM5 chip.

[0036] A sodium acetate solution with a pH value of 4.5 was selected as a buffer solution to dilute the Pks13 protein to 200 μg / mL, and it was coated on the surface of the CM5 chip by amino coupling. The target coupling amount was set to 5000 RU, and a blank channel was set as a control channel.

[0037] After the injection, ethanolamine was added to block the active carboxyl sites on the chip surface that were not bound to amino groups.

[0038] The gradient diluted B-AP15 was injected sequentially, and the small molecules were allowed to flow through the chip surface at a flow rate of 10 μL / min to detect the binding signal.

[0039] Binding signals were analyzed using TraceDrawer software.

[0040] The results showed that B-AP15 could bind to Pks13-TE protein in a dose-dependent manner ( Figure 3 ), and its binding KD value was 5.02×10 -9 M.

[0041] Example 3

[0042] Molecular docking analysis of B-AP15 and Pks13 protein

[0043] The crystal structure of Pks13-TE is derived from the Protein Data Bank database (PDB: 5V3Y). On this basis, a 3D model was established using MOE 2020 software. 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 software was used to optimize the B-AP15 molecular structure and dock it with the active pocket. The docking model with the highest score was selected to analyze its interaction. The results are as follows Figure 4 As shown, B-AP15 can form a hydrogen bond with His1664 of Pks13-TE protein, thereby improving the stability of the binding between the protein and the compound.

[0044] Example 4

[0045] Evaluation of antibacterial activity of B-AP15

[0046] Select Mycobacterium tuberculosis H37Rv and sensitive and resistant clinical isolates of Mycobacterium tuberculosis (see Table 1 for details, MTB standard strain H37Rv, source: ATCC27294; MTB sensitive strains and resistant strains, source: National Tuberculosis Clinical Laboratory) and culture for 2 to 3 weeks to make a bacterial suspension, which was inoculated into 7H9 medium containing 0.05% Tween 80 and 10% ADC. Add 200 μL of the above diluted bacterial suspension to each well of a 96-well plate, then add the gradient diluted compound, and observe the results by 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.

[0047] The results showed that compound B-AP15 exhibited an inhibitory effect superior to positive drugs on MTB standard strains, sensitive strains and clinical strains. The specific results are shown in Table 1.

[0048] Table 1B - Anti-tuberculosis activity of AP15

[0049]

[0050] Note: H37Rv: standard strain; STB: sensitive strain; MDR: multidrug-resistant strain; XDR: extensively drug-resistant strain.

[0051] MDR-30611: resistant to isoniazid and rifampicin.

[0052] MDR-30141: resistant to isoniazid and rifampicin.

[0053] XDR-28127: Resistant to isoniazid, rifampicin, ethambutol, streptomycin, levofloxacin, amikacin, clofazimine, capreomycin, and kanamycin.

[0054] XDR-27830: Resistant to isoniazid, rifampicin, ethambutol, streptomycin, levofloxacin, amikacin, capreomycin, and kanamycin.

[0055] Example 5

[0056] Acute toxicity test of B-AP15 in mice

[0057] The experimental animals were 5 Kunming mice weighing 18-22 g. 50 mg of the compound was weighed and dissolved in 1 mL of 0.5% sodium carboxymethylcellulose (CMC-Na) aqueous solution, vortexed for 1 min and sonicated for 10 min to a final concentration of 50 mg / mL.

[0058] Each mouse was given 0.2 mL (500 mg / kg body weight) by oral gavage, and the mice were observed at 0.5 h, 1.0 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h and 72 h to see whether there were obvious changes in their behavioral states and whether they died.

[0059] The experimental results showed that no mice died within 72 hours after administration, and no obvious toxic side effects occurred, indicating that compound B-AP15 has good safety.

[0060] 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 B-AP15 in the preparation of anti-tuberculosis drugs.

2. Application of B-AP15 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 B-AP15 in the preparation of Pks13 inhibitors.

5. Application of B-AP15 in the preparation of Pks13-TE inhibitors.

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