CN119033788B - Application of Mycobacterial Thiol Disulfide Reductase Inhibitors in the Preparation of Antimycobacterial Drugs - Google Patents
Application of Mycobacterial Thiol Disulfide Reductase Inhibitors in the Preparation of Antimycobacterial DrugsInfo
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- CN119033788B CN119033788B CN202411183084.7A CN202411183084A CN119033788B CN 119033788 B CN119033788 B CN 119033788B CN 202411183084 A CN202411183084 A CN 202411183084A CN 119033788 B CN119033788 B CN 119033788B
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- disulfide reductase
- thiol disulfide
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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Abstract
The invention discloses a new application of a pyridopyrimidine compound AK-968/11492032 as a mycobacterium thiol disulfide reductase inhibitor and an antimycobacterial drug, the invention screens out that the compound AK-968/11492032 can be combined with mycobacterium thiol disulfide reductase and reduce the enzyme activity thereof through a plurality of experiments. And the MIC of the compound to the mycobacterium smegmatis is 2 mug/ml, and the compound has obvious antibacterial effect. Therefore, AK-968/11492032 can be used as an inhibitor of mycobacterial thiol disulfide reductase, and is expected to be developed into a novel anti-pathogenic mycobacterial drug, and has important application value.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to application of a compound AK-968/11492032 serving as a mycobacterium thiol disulfide reductase inhibitor in preparation of anti-pathogenic mycobacterium medicines.
Background
Nontuberculous mycobacteria (Nontuberculous mycobacteria bacteria, NTM) refers to the collective term for mycobacteria other than mycobacterium tuberculosis (Mycobacterium tuberculosis, m.tuberculosis) and mycobacterium leprae (Mycobacterium leprae, m.lepra). The disease caused by NTM infection is called NTM disease, and in recent years, the global incidence of NTM disease is on the rise, and thus, it has become one of the important public health problems threatening human health. NTM is classified into fast-growing mycobacteria (rapidly growing mycobacteria, RGM) and slow-growing mycobacteria (slowly growingmycobacteria, SGM), and in china, mycobacterium abscessus (Mycobacterium abscessus subsp. The mycobacterium abscesses have high drug resistance to clinical first-line antibiotics and standard antituberculosis drugs, the existing antimusculism drugs have large toxic and side effects, and the drug sensitivity test results of different mycobacterium abscesses are greatly different, so the currently adopted treatment scheme mainly combines multiple drugs under the guidance of in-vitro drug sensitivity test, and great challenges are brought to clinical treatment, so that the search for new targets is urgently needed.
Mycobacteria are mainly present in host macrophages, which release active oxygen (Reactive Oxygen Species, ROS) and active nitrogen (Reactive nitrogen species, RNS) for survival and maintenance of redox balance and metabolic homeostasis, which use non-enzymatic defense systems including low molecular weight reduced mycobacterial thiols (mycothiol, MSH) and thioredoxin systems and other proteins (antioxidant enzyme systems such as superoxide dismutase (SOD), catalase (KatG) etc. to cope with oxidative stress pressure generated by the host MSH, which is an important intracellular antioxidant, to effect the clearance of ROS and RNS from host macrophages by the pool of MSH in mycobacterial thiol disulfide reductase (Mycothiol disulfide reductase, mtr), which is a homodimer NADPH dependent enzyme that can catalyze the recycling reaction of MSSM to MSH, is an important intracellular antioxidant to maintain redox homeostasis in the host as well as the same function of glutathione in high-class reducing organisms, and thus is a potential target for the development of mycobacterial drugs.
According to the invention, a large number of experiments are carried out to screen pyridopyrimidine compounds AK-968/11492032, the compounds have an inhibition effect on the growth of mycobacterium smegmatis, target mycobacterium thiol disulfide reductase and reduce the activity of the mycobacterium thiol disulfide reductase.
Disclosure of Invention
The invention aims to start from a crystal structure of mycobacterium thiol disulfide reductase, carry out molecular butt joint on a small molecular compound and a three-dimensional structure of the enzyme, and select candidate compounds with reasonable combination mode and high prediction score from a large number of small molecular compounds by researching interaction modes of the small molecular compounds. Through a large number of experiments, the pyridopyrimidine anti-mycobacterial compound AK-968/11492032 which has the function of inhibiting the growth of mycobacteria and targets the mycobacterial thiol disulfide reductase is screened out, and can be used as a mycobacterial bacteriostatic agent and a mycobacterial thiol disulfide reductase inhibitor.
The molecular formula of the compound AK-968/11492032 is C 22H19ClN4 O, and the structural formula is as follows:
The application discovers that the pyridopyrimidine compound AK-968/11492032 has antimycobacterial activity, can be combined with mycobacterium thiol disulfide reductase and effectively inhibit the activity thereof, and the mycobacterium thiol disulfide reductase has an important function on the maintenance of bacterial steady state. Therefore, the research shows that the pyridopyrimidine compounds AK-968/11492032 can be combined with the mycobacterium thiol disulfide reductase, and can effectively inhibit the activity of the pyridopyrimidine compounds AK-968/11492032 so as to exert the antibacterial activity on the mycobacterium, so that the pyridopyrimidine compounds AK-968/11492032 and derivatives thereof can be used for preparing antimycobacterial medicaments and mycobacterium thiol disulfide reductase inhibitors.
As a preferable scheme, the pyridopyrimidine compound AK-968/11492032 and its derivatives are prepared into inorganic acid salt or organic acid salt. As a preferable scheme, the pyridopyrimidine compounds AK-968/11492032 and derivatives thereof and pharmaceutically acceptable carriers are prepared into tablets, granules, capsules, pills, powder, oral liquid and injection.
The beneficial effects are described as follows:
The research of the invention shows that the compound AK-968/11492032 can effectively inhibit the activity of the mycobacterium tuberculosis thiol disulfide reductase of the abscess (IC 50 is 3.49 mu M) (as shown in figure 1). And the MIC value for the compound AK-968/11492032 for inhibiting Mycobacterium smegmatis was 2. Mu.g/ml. These results demonstrate that compound AK-968/11492032 binds to Mycobacterium thiol disulfide reductase and effectively inhibits its activity against Mycobacteria. Mycobacteria are causative bacteria of tuberculosis clinically, and therefore, the present invention has important clinical significance.
Drawings
FIG. 1 is a graph of the enzyme activity of compound AK-968/11492032 against Mycobacterium abscessum mycothiol disulfide reductase IC 50;
FIG. 2 is a schematic equation of the catalytic reaction principle of Mycobacterium thiol disulfide reductase.
Detailed Description
Through a large number of experimental screening, the compound AK-968/11492032 has better antibacterial activity on mycobacterium smegmatis and targets mycobacterium thiol disulfide reductase to inhibit the enzymatic activity. The invention is further described below in connection with specific embodiments, which should not be construed as limiting the invention.
Experimental materials of the following examples;
the compound AK-968/11492032 is a Spics brand, available from Shanghai Tao Shu Biotechnology Inc., and NADPH is derived from Shanghai Seiyaka Biotechnology Inc.
EXAMPLE 1 Compounds AK-968/11492032 inhibition of the enzymatic Activity of Mycobacterium abscessum thiol disulfide reductase IC 50 test
The compounds AK-968/11492032 were analyzed for their inhibitory effect on the activity of the thiol disulfide reductase of Mycobacterium IC 50.
Compounds tested Compound AK-968/11492032
The equation of the principle of the catalytic reaction of mycobacterial thiol disulfide reductase is shown in FIG. 2.
In vivo, NADPH catalyzes the reduction of MSSM to MSH by Mtr to maintain mycobacterial redox homeostasis. Since the natural substrate MSSM is difficult to synthesize, the experimental design refers to literature, doi: 10.1128/specrum.03723-23. In the enzyme activity measurement, a DTNB method is often adopted, the method is based on that an enzyme activity reaction product or a residual substrate can react with the DTNB to generate TNB, and the enzyme activity is measured by detecting the absorbance of the TNB. An asymmetric thiol disulfide BnMS-TNB (Asymmetric mycothiol disulfide) was designed as an alternative substrate in the experiments. After addition of NADPH, mtr reduced BnMS-TNB to Bn-MSH (Benzylated mycothiol) and TNB (5-thio-2-nitrobenzoic acid), the enzymatic activity of Mycobacterium thiol disulfide reductase could be determined by detecting the production of TNB, a product which has a characteristic absorption of TNB at 412 nm.
The principle equation of the catalytic reaction of mycobacterial thiol disulfide reductase is as follows:
The test method is as follows, 200. Mu.L of the reaction system comprising 50nM Mycobacterium abscess thiol disulfide reductase, bnMS-TNB final concentration fixed at 200. Mu.M and NADPH final concentration fixed at 100. Mu.M was prepared in a standard 100mM HEPES buffer (pH 7.2) purged with nitrogen (N 2) gas, the final concentration of the compound AK-968/11492032 was changed stepwise from 0 to 25. Mu.M, and the increase in TNB absorbance at 412nM was monitored at 25℃for 20 min. IC 50 was calculated from the change in TNB absorbance and analyzed using GRAPHPAD PRISM data, 3 replicates were run for each set of experiments. As shown in FIG. 1, the compound AK-968/11492032 can effectively inhibit activity of Mycobacterium smegmatis thiol disulfide reductase, and IC 50 is 3.409 mu M.
Example 2 Compound AK-968/11492032 has an anti-Mycobacterium smegmatis effect
Compounds tested Compound AK-968/11492032.
The determination method is to use a high-flux 96-well Alamar blue (Alamar blue) method to detect the lowest inhibitory concentration (MIC) of the compound AK-968/11492032, p-aminosalicylic acid (PAS) single and PAS combined compound AK-968/11492032. A series of PAS medicine (four times working concentration) solutions were added to 96-well plates (1. Mu.l for each well) at PAS final concentrations of 0.125 to 32. Mu.g/ml and compound AK-968/11492032 final concentrations of 0.125 to 16. Mu.g/ml, 179. Mu.l of bacterial solutions were added to each well, and bacterial solution wells and blank medium wells were used as controls. The bacterial liquid is completely cleared (representing that bacterial cells do not grow at all) as MIC interpretation standard. Experiments were independently repeated three times. As shown in Table 1, the compound AK-968/11492032 had an MIC of 2. Mu.g/ml for Mycobacterium smegmatis, a remarkable antibacterial effect, and was superior to PAS (MIC of 4. Mu.g/ml). MIC values did not significantly decrease after the combination.
TABLE 1PAS, AK-968/11492032 MIC against Mycobacterium smegmatis
The foregoing is merely a preferred embodiment of the present invention and should not be construed as limiting the scope of the invention. Various equivalent changes and modifications can be made by those skilled in the art without departing from the principles of the present invention, and it is intended that the present invention also be considered as the scope of the present invention.
Claims (3)
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004106538A1 (en) * | 2003-06-03 | 2004-12-09 | Leopold Flohe | Assay for identifying inhibitors of mycobacterium anti-oxidant defense system |
| CN103120689A (en) * | 2011-11-21 | 2013-05-29 | 华中农业大学 | Application of pyrazol compound as mycobacterium tuberculosis inhibitor |
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| US6833371B2 (en) * | 2001-11-01 | 2004-12-21 | Icagen, Inc. | Pyrazolopyrimidines |
| AU2005324492B2 (en) * | 2004-04-23 | 2012-06-07 | Paratek Pharmaceuticals, Inc. | Transcription factor modulating compounds and methods of use thereof |
| CN115006391B (en) * | 2022-04-29 | 2024-01-30 | 南京中医药大学 | Application of mycobacterium tetrahydrofolate reductase inhibitor in preparation of anti-pathogenic mycobacterium drug synergist |
| CN117122595A (en) * | 2022-05-27 | 2023-11-28 | 广州嘉越医药科技有限公司 | Uses of pyridine derivatives |
| CN115724833B (en) * | 2022-11-09 | 2025-06-24 | 中国海洋大学 | A NAT inhibitor and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004106538A1 (en) * | 2003-06-03 | 2004-12-09 | Leopold Flohe | Assay for identifying inhibitors of mycobacterium anti-oxidant defense system |
| CN103120689A (en) * | 2011-11-21 | 2013-05-29 | 华中农业大学 | Application of pyrazol compound as mycobacterium tuberculosis inhibitor |
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