Application of Mycobacterial Thiol Disulfide Reductase Inhibitors in the Preparation of Antimycobacterial Drugs

Abstract

This invention discloses a novel application of the pyridopyrimidine compound AK-968 / 11492032 as an inhibitor of mycobacterial thiol disulfide reductase and in anti-mycobacterial drugs. Through extensive experimental screening, this invention has demonstrated that compound AK-968 / 11492032 can bind to mycobacterial thiol disulfide reductase and reduce its enzyme activity. Furthermore, this compound exhibits a significant antibacterial effect against *Mycobacterium smegmae* with a MIC of 2 μg / ml. Therefore, AK-968 / 11492032 can serve as an inhibitor of mycobacterial thiol disulfide reductase and holds promise for development into a novel anti-pathogenic mycobacterial drug, possessing significant application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine. Specifically, this invention relates to the application of compound AK-968 / 11492032 as a mycobacterial thiol disulfide reductase inhibitor in the preparation of drugs against pathogenic mycobacteria. Background Technology

[0002] Nontuberculous mycobacteria (NTM) refer to all mycobacteria except Mycobacterium tuberculosis (M. tuberculosis) and Mycobacterium leprae (M. leprae). Diseases caused by NTM infection are called NTM diseases. In recent years, the global incidence of NTM diseases has been on the rise, becoming a significant public health problem threatening human health. NTMs are divided into rapidly growing mycobacteria (RGM) and slowly growing mycobacteria (SGM). In China, Mycobacterium abscessus subsp. abscessus (Mab) is the most pathogenic and drug-resistant pathogen among RGMs. Mycobacterium abscessis exhibits high resistance to first-line clinical antibiotics and standard anti-tuberculosis drugs. Existing anti-abscessis drugs have significant toxic side effects, and drug sensitivity test results vary greatly among different Mycobacterium abscessis isolates. Therefore, current treatment regimens mainly rely on multi-drug combinations guided by in vitro drug sensitivity tests, which poses a great challenge to clinical treatment. Thus, there is an urgent need to explore new targets.

[0003] Mycobacteria are primarily found within host macrophages. Host macrophages release reactive oxygen species (ROS) and reactive nitrogen species (RNS). To survive and maintain redox balance and metabolic homeostasis, mycobacteria utilize non-enzymatic defense systems, including low-molecular-weight reduced mycothiol (MSH), as well as thioredoxin systems and antioxidant enzyme systems such as superoxide dismutase (SOD) and catalase (KatG), to cope with oxidative stress from the host. MSH, as an important intracellular antioxidant, is oxidized to oxidized mycothiol disulfide (MSSM), thereby clearing ROS and RNS from host macrophages. The intracellular MSH pool in mycobacteria is maintained by mycothiol disulfide reductase. In addition to glutathione reductase (Mtr), Mtr is a homodimer NADPH-dependent enzyme that catalyzes the recycling reaction of MSSM to MSH, thereby helping to maintain redox homeostasis in mycobacteria. Mtr functions similarly to glutathione reductase in higher organisms and is also specific to mycobacteria, therefore Mtr is a potential target for the development of anti-mycobacterial drugs.

[0004] This invention screened out the pyridine-pyrimidine compound AK-968 / 11492032 through a large number of experiments. This compound has an inhibitory effect on the growth of Mycobacterium smegmatis and targets mycobacterial thiol disulfide reductase, reducing the activity of mycobacterial thiol disulfide reductase. Summary of the Invention

[0005] The purpose of this invention is to start with the crystal structure of mycobacterial thiol disulfide reductase, and to perform molecular docking between small molecule compounds and the three-dimensional structure of the enzyme. By studying their interaction modes, candidate compounds with reasonable binding modes and high prediction scores are selected from a large number of small molecule compounds. Through extensive experimental screening, AK-968 / 11492032, a pyridopyrimidine anti-mycobacterial compound, was identified as having the effect of inhibiting the growth of mycobacteria and targeting mycobacterial thiol disulfide reductase. It can be used as a mycobacterial antibacterial agent and an inhibitor of mycobacterial thiol disulfide reductase.

[0006] The molecular formula of compound AK-968 / 11492032 is C 22 H 19 ClN4O, with the following structural formula:

[0007]

[0008] This application has discovered that the pyridopyrimidine compound AK-968 / 11492032 possesses anti-mycobacterial activity, binding to and effectively inhibiting the activity of mycobacterial thiol disulfide reductase, which plays a crucial role in maintaining bacterial homeostasis. Therefore, this application's research demonstrates that the pyridopyrimidine compound AK-968 / 11492032 can bind to mycobacterial thiol disulfide reductase and effectively inhibit its activity, thereby exerting antibacterial activity against mycobacteria. Thus, this invention has screened out the pyridopyrimidine compound AK-968 / 11492032 and its derivatives for use in the preparation of anti-mycobacterial drugs and mycobacterial thiol disulfide reductase inhibitors.

[0009] As a preferred embodiment, pyridopyrimidine compound AK-968 / 11492032 and its derivatives are prepared into inorganic or organic acid salts. Alternatively, pyridopyrimidine compound AK-968 / 11492032 and its derivatives are prepared into tablets, granules, capsules, pills, powders, oral liquids, or injections using pharmaceutically acceptable carriers.

[0010] Explanation of beneficial effects:

[0011] This invention has found that compound AK-968 / 11492032 can effectively inhibit the activity of thiol disulfide reductase in Mycobacterium abscessus (IC50). 50 (3.49μM) Figure 1 Furthermore, the MIC value of compound AK-968 / 11492032 for inhibiting Mycobacterium smegma was 2 μg / ml. These results indicate that compound AK-968 / 11492032 can bind to and effectively inhibit the activity of Mycobacterium thiol disulfide reductase, thereby exerting an anti-mycobacterial effect. Mycobacteria are the pathogens that cause tuberculosis in clinical practice; therefore, this invention has important clinical significance. Attached Figure Description

[0012] Figure 1 The IC50 value of compound AK-968 / 11492032 was used to inhibit the enzymatic activity of mycothiol disulfide reductase in Mycobacterium abscessis. 50 ;

[0013] Figure 2 This is the equation for the reaction principle catalyzed by mycobacterial thiol disulfide reductase. Detailed Implementation

[0014] Through extensive experimental screening, this invention has discovered that compound AK-968 / 11492032 exhibits good antibacterial activity against Mycobacterium smegma and targets and inhibits the activity of mycobacterial thiol disulfide reductase. The invention is further described below with reference to specific embodiments, but these embodiments should not be construed as limiting the invention.

[0015] The experimental materials used in the following examples;

[0016] Compound AK-968 / 11492032 is from the Specs brand and was purchased from Shanghai Taoshu Biotechnology Co., Ltd., while NADPH is from Shanghai Yuanye Biotechnology Co., Ltd.

[0017] Example 1: Compound AK-968 / 11492032 inhibits the enzymatic activity (IC50) of thiol disulfide reductase in Mycobacterium abscessis. 50 test

[0018] Analysis of the inhibitory effect of compound AK-968 / 11492032 on the activity of mycobacterial thiol disulfide reductase (IC50) 50 .

[0019] The compound tested: Compound AK-968 / 11492032

[0020] The principle equation for the catalytic reaction of mycobacterial thiol disulfide reductase is as follows: Figure 2 .

[0021] In vivo, NADPH, catalyzed by Mtr, reduces MSSM to MSH, thus maintaining redox homeostasis in mycobacteria. Since the natural substrate MSSM is difficult to synthesize, this experimental design follows the literature (doi:10.1128 / spectrum.03723-23). ​​The DTNB method is commonly used for enzyme activity assays. This method is based on the fact that enzyme activity reaction products or residual substrates can react with DTNB to generate TNB, and enzyme activity is determined by detecting the absorbance of TNB. In this experiment, an asymmetric mycothiol disulfide, BnMS-TNB, was designed as a substitute substrate. After the addition of NADPH, Mtr reduces BnMS-TNB to generate Bn-MSH (Benzylated mycothiol) and TNB (5-thio-2-nitrobenzoic acid). The enzyme activity of mycobacterial thiol disulfide reductase can be determined by detecting the formation of the product TNB (TNB has a characteristic absorption at 412 nm).

[0022] The equation for the reaction catalyzed by mycobacterial thiol disulfide reductase is as follows:

[0023]

[0024] The test method is as follows: A 200 μL reaction system containing 50 nM Mycobacterium abscessis thiodisulfide reductase was prepared in a standard 100 mM HEPES buffer (pH 7.2) purged with nitrogen (N2) gas. The final concentration of BnMS-TNB was fixed at 200 μM, and the final concentration of NADPH was fixed at 100 μM. The final concentration of compound AK-968 / 11492032 was varied from 0 to 25 μM. The increase in TNB absorbance at 412 nm was monitored over 20 min at room temperature (25°C). The IC50 was calculated based on the change in TNB absorbance. 50 Data analysis was performed using GraphPad Prism, with each experiment conducted in triplicate. The experimental results are as follows: Figure 1 As shown, compound AK-968 / 11492032 can effectively inhibit the activity of thiol disulfide reductase in Mycobacterium smegma, IC50. 50 The value is 3.409 μM.

[0025] Example 2: Compound AK-968 / 11492032 has antibacterial activity against Mycobacterium smegma.

[0026] The compound tested was AK-968 / 11492032.

[0027] Assay Method: The minimum inhibitory concentration (MIC) of compound AK-968 / 11492032, para-aminosalicylic acid (PAS) alone, and PAS in combination with compound AK-968 / 11492032 was determined using a high-throughput 96-well Alamar blue assay. A series of PAS solutions (four times the working concentration) were added to 96-well plates (1 μl per well), with final PAS concentrations ranging from 0.125 to 32 μg / ml, and final AK-968 / 11492032 concentrations ranging from 0.125 to 16 μg / ml. 179 μl of bacterial culture was added to each well. Wells with bacterial culture and blank culture medium served as controls. Complete clarity of the bacterial culture (indicating no bacterial growth) was used as the MIC interpretation criterion. The experiment was independently repeated three times. The experimental results are shown in Table 1. Compound AK-968 / 11492032 had a MIC of 2 μg / ml against Mycobacterium smegma, exhibiting significant antibacterial activity, which was superior to that of PAS (MIC of 4 μg / ml). The MIC value did not decrease significantly after combination therapy.

[0028] Table 1. MICs of PAS, AK-968 / 11492032, and their combination against Mycobacterium smegma.

[0029]

[0030]

[0031] The above description is merely a preferred embodiment of the present invention and should not be considered as a limitation on the scope of the present invention. For those skilled in the art, various possible equivalent changes or modifications can be made based on the technical solutions and preferred embodiments of the present invention without departing from the principles of the invention, and these changes and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of compound AK-968 / 11492032 and its salt in the preparation of drugs against Mycobacterium smegma. The structural formula of compound AK-968 / 11492032 is as follows: 。 2. The application according to claim 1, characterized in that, The salts mentioned include inorganic acid salts or organic acid salts.

3. The application according to claim 1 or 2, characterized in that, Compound AK-968 / 11492032 was prepared into tablets, granules, capsules, pills, powders, oral liquids or injections using a pharmaceutically acceptable carrier.

Images