Application of small molecule D6 and its derivatives in the preparation of anti-tuberculosis drugs

Abstract

This application relates to the field of biomedical technology, and in particular to the application of a small molecule D6 and its derivatives in the preparation of anti-tuberculosis drugs. Through research, this application has discovered that small molecule D6 can specifically target methionine synthase in Mycobacterium tuberculosis to block the methionine synthesis pathway, thereby causing metabolic disorders and cell death in the bacteria. Furthermore, this small molecule D6 exhibits significant antibacterial activity against various Mycobacterium tuberculosis strains, including drug-resistant and dormant strains. Therefore, this small molecule D6 can be well-suited for the preparation of anti-tuberculosis drugs.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, and in particular relates to the application of a small molecule D6 and its derivatives in the preparation of anti-tuberculosis drugs. Background Technology

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb). Controlling TB remains a significant challenge due to its long treatment duration, poor patient adherence, and the prevalence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains.

[0003] Currently, anti-tuberculosis drugs mainly target bacterial cell wall synthesis, energy metabolism, or RNA polymerase, but most of these drugs are only effective against Mycobacterium tuberculosis in the active replication phase, and have limited killing effect on persistent bacteria in the non-replicating or slow-metabolizing phase. Summary of the Invention

[0004] The purpose of this application is to provide an application of small molecule D6 and its derivatives in the preparation of anti-tuberculosis drugs, aiming to solve the technical problem of applying a new target of Mycobacterium tuberculosis to better address tuberculosis.

[0005] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows:

[0006] In a first aspect, this application provides the use of small molecule D6 and its derivatives in the preparation of anti-tuberculosis drugs; wherein the chemical structure of the small molecule D6 is shown below:

[0007] .

[0008] In some embodiments, the derivatives of small molecule D6 include at least one of the following: a deacetylated product of small molecule D6, an esterified product of small molecule D6, and a pharmaceutically acceptable salt of small molecule D6.

[0009] In some embodiments, the small molecule D6 targets and inhibits methionine synthase in Mycobacterium tuberculosis.

[0010] In some embodiments, the anti-tuberculosis drug includes drugs for treating or preventing active or latent tuberculosis;

[0011] Alternatively, the anti-tuberculosis drugs may include drugs for treating or preventing multidrug-resistant or extensively drug-resistant tuberculosis.

[0012] In some embodiments, the minimum inhibitory concentration of the small molecule D6 is 1~5 μM.

[0013] Secondly, this application provides the application of small molecule D6 and its derivatives in the preparation of methionine synthase inhibitors; wherein the chemical structure of the small molecule D6 is shown below:

[0014] .

[0015] In some embodiments, the derivatives of small molecule D6 include at least one of the following: a deacetylated product of small molecule D6, an esterified product of small molecule D6, and a pharmaceutically acceptable salt of small molecule D6.

[0016] In some embodiments, the methionine synthase inhibitor inhibits methionine synthesis in Mycobacterium tuberculosis.

[0017] In some embodiments, the methionine synthase inhibitor is used as a drug for treating or preventing active or latent tuberculosis.

[0018] In some embodiments, the methionine synthase inhibitor is used as a drug for treating or preventing multidrug-resistant tuberculosis or extensively drug-resistant tuberculosis.

[0019] The application of small molecule D6 and its derivatives in the preparation of anti-tuberculosis drugs, as provided in the first aspect of this application, is based on the research finding that small molecule D6 can specifically target methionine synthase in Mycobacterium tuberculosis, blocking the methionine synthesis pathway, thereby causing metabolic disorders and inducing cell death. Furthermore, this small molecule D6 exhibits significant antibacterial activity against various tuberculosis strains, including drug-resistant and dormant Mycobacterium tuberculosis. Therefore, this small molecule D6 and its derivatives can be well used in the preparation of anti-tuberculosis drugs.

[0020] The application of small molecule D6 and its derivatives in the preparation of methionine synthase inhibitors provided in the second aspect of this application is based on the research finding that small molecule D6 can specifically target methionine synthase in Mycobacterium tuberculosis, and therefore can be made into methionine synthase inhibitors, providing a new molecular tool and strategy for the development of anti-tuberculosis drugs that target methionine metabolism. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 These are the results of the in vitro antibacterial activity assay of small molecule D6 against Mycobacterium tuberculosis in the embodiments of this application;

[0023] Figure 2These are the results of the antibacterial activity assay of small molecule D6 in a macrophage model in the embodiments of this application;

[0024] Figure 3 These are the results of the antibacterial activity assay of small molecule D6 in a mouse model in the embodiments of this application;

[0025] Figure 4 This is the result of the pull-down assay measuring the binding between small molecule D6 and methionine synthase MetE in the embodiments of this application. Detailed Implementation

[0026] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0027] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0028] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of a single item or a plurality of items.

[0029] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0030] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0031] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application can be a well-known unit of mass in the chemical industry, such as µg, mg, g, or kg.

[0032] The terms "first" and "second" are used for descriptive purposes only, to distinguish objects, such as substances, from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. For example, without departing from the scope of the embodiments of this application, "first XX" may also be referred to as "second XX," and similarly, "second XX" may also be referred to as "first XX." Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of that feature.

[0033] Currently, anti-tuberculosis drugs mainly target bacterial cell wall synthesis, energy metabolism, or RNA polymerase. However, most of these drugs are only effective against Mycobacterium tuberculosis in its active replication phase, and have limited killing effect on persistent bacteria in a non-replicating or metabolically slow state. Therefore, developing anti-tuberculosis drugs with novel mechanisms of action, especially those targeting bacterial metabolic weaknesses, is an important direction in current anti-tuberculosis drug research.

[0034] Methionine is a sulfur-containing amino acid essential for the growth and survival of Mycobacterium tuberculosis (Mtb). Since Mtb cannot directly obtain sufficient methionine from the host, it relies heavily on endogenous biosynthesis to meet its metabolic needs during infection. Methionine is not only the starting amino acid for protein synthesis in Mtb metabolism but also serves as a precursor to S-adenosylmethionine (SAM), participating in a wide range of methylation reactions and influencing key processes such as DNA / RNA modification, lipid synthesis, and redox balance. In this biosynthetic pathway, the methionine synthase MetE is the Mtb homocysteine ​​methyltransferase responsible for catalyzing the terminal reaction in methionine synthesis. Multiple studies have confirmed that MetE is crucial for the growth of Mtb in both in vitro culture and the host environment, and is an essential metabolic enzyme for its survival. Therefore, methionine biosynthesis, especially the maintenance of MetE activity, is considered an important target for addressing Mtb metabolic vulnerability and has potential drug development value.

[0035] The applicant previously designed and synthesized a series of small molecule compounds from the structures of some traditional Chinese medicine-related natural products, and discovered that the cis isomer of small molecule D6 has a good therapeutic effect on non-small cell lung cancer (NSCLC) (see: A small-molecule compound D6 overcomes EGFR T790M-mediated resistance in non-small cell lung cancer; https: / / doi.org / 10.1038 / s42003-021-02906-4). This application further discovered through experiments that small molecule D6 and its derivatives have significant antibacterial and bactericidal activity against Mycobacterium tuberculosis. Based on this, the technical solution provided in the embodiments of this application is as follows.

[0036] The first aspect of this application provides an application, namely, the application of small molecule D6 and its derivatives with the following chemical structure in the preparation of anti-tuberculosis drugs:

[0037] .

[0038] Since small molecule D6 contains an ester bond in its structure, and ester bonds are usually broken in vivo, the deacetylated products of small molecule D6 and the various esters and other derivatives formed thereafter are all within the scope of protection of this application.

[0039] The embodiments of this application reveal that small molecule D6 can specifically target methionine synthase in Mycobacterium tuberculosis, blocking the methionine synthesis pathway, thereby causing metabolic disorders and cell death. Furthermore, this small molecule D6 exhibits significant antibacterial activity against various tuberculosis strains, including drug-resistant and dormant Mycobacterium tuberculosis. Therefore, this small molecule D6 and its derivatives can be well-suited for the preparation of anti-tuberculosis drugs.

[0040] In some embodiments, the derivatives of small molecule D6 include at least one of the following: a deacetylated product of small molecule D6, an esterified product of small molecule D6 (the esterified product may be a methyl esterified product, a propyl esterified product, a butyl esterified product, etc.), and a pharmaceutically acceptable salt of small molecule D6.

[0041] In some embodiments, the use of small molecule D6 in the preparation of anti-tuberculosis drugs that can treat or prevent active tuberculosis, latent tuberculosis, multidrug-resistant tuberculosis, and extensively drug-resistant tuberculosis.

[0042] Active tuberculosis, also known as active tuberculosis disease, is a progressive infectious disease caused by Mycobacterium tuberculosis breaching the immune barrier. It is characterized by clear infectivity and refers to a disease state in which Mycobacterium tuberculosis actively replicates in the body, causing symptoms and being infectious. Patients with active tuberculosis experience a large proliferation of Mycobacterium tuberculosis in their bodies, which can cause symptoms such as cough, fever, night sweats, and weight loss. The disease requires standardized anti-tuberculosis treatment to control it. The key is early diagnosis and complete medication treatment to prevent transmission and the development of drug resistance.

[0043] Latent tuberculosis refers to a special state of infection in which the body is infected with Mycobacterium tuberculosis but does not develop the clinical symptoms of active tuberculosis, and the bacteria remain dormant. In this state, the infected person is not infectious, but the pathogen may be activated when the immune system is weakened, leading to active tuberculosis. Early screening and preventative treatment are crucial for reducing the risk of developing the disease. Active tuberculosis requires immediate treatment, while latent infection may require prophylactic medication.

[0044] Drug-resistant tuberculosis (DRT) is a type of tuberculosis in which Mycobacterium tuberculosis develops resistance to one or more anti-tuberculosis drugs. Its transmission routes are the same as ordinary tuberculosis, but treatment is more difficult, takes longer, and is more expensive. Risk reduction requires standardized medication, early diagnosis, and strict management.

[0045] Among drug-resistant tuberculosis, multidrug-resistant tuberculosis (MDR-TB) refers to tuberculosis that is resistant to both isoniazid and rifampin, two first-line drugs, and requires second-line treatment. Extensively drug-resistant tuberculosis (XDR-TB), on the other hand, is in addition to MDR-TB, also resistant to second-line injectable drugs (such as amikacin) and fluoroquinolones.

[0046] This application's embodiments reveal that small molecule D6 can target not only active tuberculosis but also latent tuberculosis, preventing latent tuberculosis infection or recurrence. Furthermore, small molecule D6 can target not only multidrug-resistant tuberculosis but also extensively drug-resistant tuberculosis. Therefore, the anti-tuberculosis drugs prepared from small molecule D6 according to this application's embodiments include drugs for treating or preventing active or latent tuberculosis; or, include drugs for treating or preventing multidrug-resistant or extensively drug-resistant tuberculosis.

[0047] In some embodiments, small molecule D6 targets and inhibits methionine synthase in Mycobacterium tuberculosis. Specifically, the methionine synthase is methionine synthase MetE. Small molecule D6 can directly bind to the MetE protein of Mtb, inhibiting its catalytic activity.

[0048] The embodiments of this application reveal that the small molecule D6 can specifically target the methionine synthase MetE in Mycobacterium tuberculosis, blocking the methionine synthesis pathway by inhibiting its enzyme activity, leading to metabolic disorders and bacterial death. It is suitable for treating infections of various tuberculosis strains, including multidrug-resistant (MDR), extensively drug-resistant (XDR), and dormant Mycobacterium tuberculosis.

[0049] In some embodiments, an effective dose of small molecule D6 is 0.1 to 50 mg / kg of body weight per day, for example, 1 to 10 mg / kg. The therapeutically effective dose is administered to mammalian individuals suffering from tuberculosis. Mammals may include humans.

[0050] In some embodiments, antituberculosis drugs prepared from small molecule D6, pharmaceutical compositions comprising small molecule D6, may also include, in addition to small molecule D6, pharmaceutically acceptable carriers.

[0051] "Effective amount" refers to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals. "Pharmaceutically acceptable carrier" refers to a carrier used for the administration of a therapeutic agent, including pharmaceutical excipients such as various excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity after administration. Suitable carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in a composition may contain liquids such as water, saline, or buffer solutions. Additionally, these carriers may contain auxiliary substances such as fillers, lubricants, flow aids, wetting agents or emulsifiers, pH buffers, etc.

[0052] In some embodiments, the minimum inhibitory concentration (MIC) of small molecule D6 is 1-5 μM. Small molecule D6 has a low micromolar MIC and exhibits strong antibacterial activity against Mtb H37Rv, Mtb H37Ra, and clinical MDR strains.

[0053] A second aspect of this application provides an application of small molecule D6 and its derivatives with the following chemical structure in the preparation of methionine synthase inhibitors:

[0054] .

[0055] The study of the embodiments of this application found that small molecule D6 can specifically target methionine synthase in Mycobacterium tuberculosis, and therefore can be made into a methionine synthase inhibitor, providing a new molecular tool and strategy for the development of anti-tuberculosis drugs that target methionine metabolism.

[0056] In some embodiments, the derivatives of small molecule D6 include at least one of the following: a deacetylated product of small molecule D6, an esterified product of small molecule D6 (the esterified product may be a methyl esterified product, a propyl esterified product, a butyl esterified product, etc.), and a pharmaceutically acceptable salt of small molecule D6.

[0057] In some embodiments, the methionine synthase inhibitor is a methionine synthase MetE inhibitor. Therefore, small molecule D6 can be formulated as an inhibitor targeting the methionine synthase MetE in Mycobacterium tuberculosis.

[0058] In some embodiments, methionine synthase inhibitors inhibit methionine synthesis in Mycobacterium tuberculosis.

[0059] Specifically, the small molecule D6 in this application embodiment can directly target the methionine synthase MetE protein in Mtb, thereby inhibiting methionine synthesis, causing homocysteine ​​accumulation and metabolic disorders, and ultimately leading to cell death, thus expanding the research space of the methionine synthesis pathway as a target for antibacterial drugs.

[0060] In some embodiments, methionine synthase inhibitors are used as drugs for the treatment or prevention of active or latent tuberculosis. Alternatively, methionine synthase inhibitors are used as drugs for the treatment or prevention of multidrug-resistant or extensively drug-resistant tuberculosis.

[0061] In summary, the embodiments of this application have shown that the small molecule D6 exhibits significant bactericidal activity against various tuberculosis strains, including drug-resistant (MDR, XDR) and dormant strains. Furthermore, in in vitro culture, macrophage infection models, and mouse pulmonary tuberculosis models, the small molecule D6 demonstrated good antibacterial activity and biocompatibility. Further surface plasmon resonance and molecular docking studies confirmed that the small molecule D6 can bind to the methionine synthase MetE with high affinity. Therefore, this small molecule D6 compound and its derivatives provide new molecular tools and strategies for the development of anti-tuberculosis drugs targeting methionine metabolism.

[0062] The following description is based on specific embodiments.

[0063] Example 1: Determination of the in vitro antibacterial activity of small molecule D6 against Mtb

[0064] M. smegmatis (abbreviated M. smeg), Mtb H37Rv (abbreviated H37Rv), and three clinically derived MDR resistant strains (#S1, #S2, and #S3) were inoculated into 96-well plates containing 7H9 medium (BD 271310). Different concentrations of small molecule D6 (0.625–20 μM) were added to each well. After 7 days of incubation, the growth inhibition rate was determined by turbidimetric assay.

[0065] The results are as follows Figure 1As shown, the small molecule D6 exhibited a concentration-dependent antibacterial effect against all the above-mentioned strains, with MIC values ​​ranging from 2.5 to 5 μM. Figure 1 In the figure, A represents the effect of different concentration treatments on the growth rate of M. smegmatis, and the results are expressed as a percentage relative to the control group (small molecule D6 concentration: 0 μg / ml). Figure 1 B in the figure represents the effect of different concentrations of treatment on the growth rate of Mtb H37Rv, and the results are expressed as a percentage relative to the control group (small molecule D6 concentration: 0 μg / ml). Figure 1 The C in the figure represents the change in surviving colony-forming units (CFU / ml) of Mtb H37Rv after treatment with different concentrations. Figure 1 In the figure, D represents the inhibitory effect of different concentrations of treatment on clinical MDR resistant strains (#S1, #S2, #S3), and mock represents the blank control group. Figure 1 E in the figure represents the effect of different concentrations of treatment on the growth of dormant *M. tuberculosis* H37Rv. These results indicate that the small molecule D6 has significant antibacterial activity against a variety of tuberculosis strains.

[0066] Example 2: Determination of the bactericidal activity of small molecule D6 in a macrophage model

[0067] THP1 macrophages were infected with Mycobacterium tuberculosis H37Rv strain at MOI=5. After infection, small molecule D6 was added for treatment, while the control group (Mock) was treated with DMSO. On the third day after infection, cells were collected and lysed with 0.1% SDS. After mixing by pipetting, the lysate was diluted 10-fold and plated on 7H10 culture plates. After incubation at 37°C for 2-3 weeks, the number of Mycobacterium tuberculosis colonies (CFU) was calculated.

[0068] The results are as follows Figure 2 As shown, the number of CFUs in the small molecule D6 treatment group was significantly lower than that in the control group (p<0.01), indicating that small molecule D6 can penetrate host cells and kill intracellular tuberculosis bacteria.

[0069] Example 3: In vivo anti-tuberculosis activity assay of small molecule D6 in a mouse model

[0070] C57 mice were infected with Mtb H37Rv via aerosol. After 14 days, they were intraperitoneally injected with small molecule D6 at a dose of 10 mg / kg, once every two days for 14 days. After the experiment, lung tissue from the mice was isolated for CFU counting.

[0071] The results are as follows Figure 3 As shown, the lung CFU in the small molecule D6 treatment group decreased significantly, indicating that small molecule D6 has clear in vivo anti-tuberculosis activity.

[0072] Example 4: Verification of the binding of small molecule D6 to methionine synthase MetE

[0073] 1. Synthesize a biotin-labeled small molecule D6 probe, the specific steps of which include: dissolving small molecule D6 in 100mM carbonate buffer (pH=8.3); the concentration of small molecule D6 is 0.1-1mM. Then add 3-10 equivalents of Biotin-NHS (total organic solvent volume percentage ≤20%). After standing at room temperature (25-27℃) for 30-60 min, add 50mM Tris-HCl solution (pH=8.0), and let stand for another 5-10 min; then desalt via gel electrophoresis (NAP-5) to obtain the probe biotin-D6.

[0074] 2. Protein preparation, specifically including: expressing and purifying the His-tagged target protein (His-MetE), using a Ni-NTA column for affinity purification. After purification, dialyzing with a suitable buffer (such as PBS or binding buffer) to remove free small molecules.

[0075] 3. Binding reaction: In the binding buffer, mix His-MetE with biotin-D6 (or biotin control), shake and incubate to allow the probe to fully bind to the protein. The groups include: His-MetE + biotin (non-specific binding control) and His-MetE + biotin-D6 (experimental group).

[0076] 4. Pull-down capture: Add the binding reaction solution to pre-equilibrated streptavidin magnetic beads or agarose beads. Mix and incubate to allow the biotinylated probe-protein complex to bind to the beads.

[0077] 5. Washing: Wash the beads multiple times with binding buffer to remove unbound proteins and small molecules and reduce nonspecific background.

[0078] 6. Elution and Detection: Add SDS-PAGE loading buffer and heat at 95°C to lyse the probe-protein complex on the beads. Separate the eluted product by SDS-PAGE and transfer to a membrane for Western blot detection. Develop the sample using a primary antibody against MetE (e.g., anti-MetE antibody) and an HRP-labeled secondary antibody.

[0079] The results are as follows Figure 4As shown in the embodiments of this application, a biotin-labeled small molecule D6 (biotin-D6) was synthesized, and biotin was used as a negative control. Pull-down analysis using streptavidin magnetic beads was performed to verify the binding ability of small molecule D6 to MetE. Western blot results showed that MetE protein was detected in all input samples; in the pull-down products, only the biotin-D6 treatment group showed significant enrichment of MetE protein, while no signal was detected in the biotin control group. These results indicate that small molecule D6 can specifically bind to MetE, and this interaction can be captured by biotinylated probes.

[0080] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. The application of a small molecule D6 and its derivatives in the preparation of anti-tuberculosis drugs; wherein, The chemical structure of the small molecule D6 is shown below: ； The derivative of the small molecule D6 is a pharmaceutically acceptable salt of the small molecule D6.

2. The application as described in claim 1, characterized in that, The small molecule D6 targets and inhibits methionine synthase in Mycobacterium tuberculosis.

3. The application as described in any one of claims 1-2, characterized in that, The anti-tuberculosis drugs are those used to treat or prevent active or latent tuberculosis.

4. The application as described in any one of claims 1-2, characterized in that, The anti-tuberculosis drugs mentioned are those used to treat or prevent multidrug-resistant tuberculosis.

5. The application as described in any one of claims 1-2, characterized in that, The anti-tuberculosis drugs mentioned are those used to treat or prevent extensively drug-resistant tuberculosis.

6. The application as described in any one of claims 1-2, characterized in that, The minimum inhibitory concentration of the small molecule D6 against Mycobacterium tuberculosis is 1~5 μM.

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