CN118750475A - Application of small molecule compounds in the preparation of antioxidant drugs and immunomodulatory drugs - Google Patents

Abstract

The present invention relates to the use of a small molecule compound in the preparation of an antioxidant drug and an immunomodulatory drug, the main feature of which is the following compound or a pharmaceutically acceptable salt thereof: The small molecule compound provided by the present invention regulates the production of reactive oxygen species, affects the body's immune response, and has important activity and medicinal value in the treatment of aging, tumors, immune-related diseases, metabolic diseases, etc.

Description

Application of small molecule compounds in the preparation of antioxidant drugs and immunomodulatory drugs

Technical Field

The present invention belongs to the field of biomedical technology, and specifically relates to the application of a small molecule compound in the preparation of antioxidant drugs and immunomodulatory drugs.

Background Art

Reactive oxygen species (ROS) such as hydrogen peroxide, singlet oxygen, hydroxyl radicals and superoxide anions are natural byproducts of normal oxygen metabolism in the body. ROS can act as signaling molecules to regulate various physiological functions of cells and play an important role in cell signal transduction and homeostasis. ROS can directly affect the activity of pathogens, cancer cells and various immune cells.

The generation and clearance of ROS are strictly regulated to maintain homeostasis. The appropriate level of ROS in the body is a key signaling molecule that regulates various physiological functions (including inflammatory responses), and can also activate the immune system to produce immune protection against pathogens or tumor cells. However, excessive and uncontrolled ROS may trigger harmful oxidative stress responses in the body, including lipid peroxidation, oxidative damage to DNA, protein oxidation, and monosaccharide oxidation, and destroy cell structures such as mitochondrial structures, leading to various diseases. Reactive oxygen species have been widely found to play an important role in a variety of diseases that threaten human health, including cancer, cardiovascular disease, diabetes, neurodegeneration (such as Parkinson's disease and Alzheimer's disease), autoimmune diseases, stroke, aging, inflammatory diseases, etc. Therefore, finding ways to remove intracellular reactive oxygen species has become an important solution to restore ROS homeostasis, limit inflammatory responses, immune imbalance, and treat diseases.

The function of the immune system is closely related to human health, such as inflammation, infection, tumors, aging, reproduction, allergic diseases, autoimmune diseases, etc. The pathogenesis of most human diseases involves immune function. With the accelerated pace of modern life, irregular work and rest, unbalanced diet, lack of exercise, and air pollution, the incidence of human immune system dysfunction is on the rise, and the ability to resist pathogens is also declining. Therefore, strengthening immunity is of great significance for preventing diseases, responding to viral infections, and promoting overall health.

Macrophages are important innate immune cells in the immune system. They are specialized cells of the natural immune system that have a long survival time and phagocytic function. They are the first responders to infection. Macrophages mediate a bridge between innate and adaptive immune responses. Macrophages participate in nonspecific defense (innate immunity) and specific defense (cellular immunity) in vivo. Macrophages participate in the recognition, phagocytosis and degradation of cell fragments and pathogens. Macrophages also play a role in presenting antigens to T cells and inducing other antigen-presenting cells to express co-stimulatory molecules, thereby initiating adaptive immune responses. In addition, in the early stages of inflammation, they play an important role by releasing cytokines and chemokines, which in turn recruit other immune cells to the site of inflammation. In addition to initiating immune and inflammatory responses to pathogens, macrophages also play a role in maintaining tissue homeostasis and repairing and remodeling tissues. Unfortunately, this function is associated with many diseases, including metabolic and autoimmune diseases, cancer, infection, obesity and fibrosis. Therefore, macrophages have become therapeutic targets for a variety of diseases, and the development of drugs that regulate macrophage function has important clinical value.

ROS plays an important role in regulating macrophage function. However, excessive ROS can cause mitochondrial structural damage, lipid peroxidation, promote macrophage immunosuppression or inflammatory response, and lead to the generation and development of diseases such as tumors, aging, and inflammation. Therefore, targeted regulation of ROS levels in cells is an important strategy for the treatment of various diseases. However, currently, there are limited reagents and drugs that regulate the production or clearance of intracellular ROS.

Small molecule drugs play an important regulatory role in disease treatment, and small molecule compounds have the advantage of entering cells to regulate ROS levels. The present invention screens new natural small molecule compounds that can eliminate intracellular reactive oxygen species and regulate macrophage function.

Summary of the invention

In order to solve the above problems in the prior art, the present invention provides an application of a small molecule compound in the preparation of antioxidant drugs and immunomodulatory drugs.

In order to achieve the above-mentioned object, the present invention provides a small molecule compound for use in the preparation of antioxidant drugs and immunomodulatory drugs, the main feature of which is the following compound or a pharmaceutically acceptable salt thereof

Preferably, the small molecule compound achieves antioxidant and immunomodulatory effects by reducing the generation of reactive oxygen species (ROS) in cells.

Preferably, the small molecule compound achieves anti-oxidation and immunomodulation by increasing the production of glutathione.

Preferably, the small molecule compound regulates the polarization direction of macrophages to achieve antioxidant and immune regulation.

Preferably, the small molecule compound regulates macrophage redox homeostasis.

Preferably, the small molecule compound regulates the level of ROS in macrophages.

Preferably, the small molecule compound reduces the level of ROS in macrophage mitochondria.

Preferably, the small molecule compound regulates the level of glutathione GSH, a redox substance in macrophages.

Preferably, the small molecule compound protects the plasma membrane of macrophages from lipid peroxidation caused by reactive oxygen species.

Preferably, the small molecule compound protects the structure and function of macrophage mitochondria.

Preferably, the small molecule compound regulates the production of macrophage cytokines.

Preferably, the small molecule compound regulates the phagocytic ability of macrophage cytokines.

The small molecule compounds provided by the present invention are used in the preparation of antioxidant drugs and immunomodulatory drugs. The small molecule compounds regulate the redox homeostasis, phenotype and function of macrophages, affect the body's immune response, and have important activity and medicinal value in the treatment of tumors, immune-related diseases and metabolic diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the regulatory effect of small molecule compounds on ROS levels in macrophages after H ₂ O ₂ treatment.

FIG. 2A is a representative picture showing the regulation of macrophage mitochondrial ROS levels by the small molecule compound IM502; FIG. 2B is a corresponding statistical analysis graph.

FIG. 3A is a representative picture of the effect of the cellular antioxidant small molecule compound IM502 on the glutathione (GSH) level in macrophages; FIG. 3B is a corresponding statistical analysis graph.

FIG. 4A is a representative picture of the effect of the cellular antioxidant small molecule compound IM502 on lipid peroxidation in macrophages; FIG. 4B is a corresponding statistical analysis graph.

Figure 5A is a representative picture of the effect of the antioxidant small molecule IM502 on the mitochondrial membrane potential in mouse macrophages; Figure 5B is a statistical analysis of the effect of the small molecule compound IM502 on the mitochondrial membrane potential in mouse macrophages; Figure 5C is a representative picture of the effect of the small molecule compound IM502 on the mitochondrial membrane potential in human macrophages; Figure 5D is a statistical analysis of the effect of the small molecule compound IM502 on the mitochondrial membrane potential in human macrophages.

FIG6A is a schematic diagram of the experimental design for evaluating the phagocytic ability of the antioxidant small molecule compound IM502 on mouse macrophages; FIG6B is a representative picture of the effect of IM502 treatment on the phagocytic ability of macrophages; and FIG6C is a corresponding statistical analysis graph.

FIG. 7A is a statistical analysis of the effects of the antioxidant small molecule compound IM502 on the expression of anti-inflammatory and pro-inflammatory factors in mouse macrophages; FIG. 7B is a statistical analysis of the effects of the antioxidant small molecule compound IM502 on the expression of anti-inflammatory and pro-inflammatory factors in human macrophages.

DETAILED DESCRIPTION

In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments.

Intracellular reactive oxygen species have an important regulatory effect on cell function and the development of diseases. Macrophages are an important immune cell subset that participates in a variety of inflammatory-related diseases. The maintenance of redox homeostasis in macrophages is the key to the normal physiological function of macrophages. Under stress conditions, macrophages become dysfunctional, leading to the development of diseases. Intracellular reactive oxygen species can regulate the polarization of macrophages, and polarized macrophages have different abilities to produce reactive oxygen species. The present invention evaluates the antioxidant activity and immunomodulatory activity of small molecule compounds after treating macrophages _{with H2O2} .

Tumor-associated macrophages (TAMs) in the tumor microenvironment inhibit anti-tumor immune responses under the regulation of reactive oxygen species. The present invention treats macrophages with tumor cell culture supernatant (TCM) to induce the generation of reactive oxygen species in macrophages, and further evaluates the inhibitory activity of the compound on the level of reactive oxygen species, its ability to regulate the redox homeostasis of macrophages, and the activity of small molecule compounds in protecting macrophages from damage by reactive oxygen species, resulting in the destruction of cytoplasmic membrane structure and mitochondrial structure.

Macrophages regulate the type and intensity of immune responses and affect the progression of diseases by expressing various cytokines. Furthermore, the changes in the expression of different cytokines in macrophages were evaluated after treating macrophages with small molecule compounds, and the ability of small molecule compounds to regulate inflammatory responses was evaluated.

The preparation methods of the small molecule compounds IM16, IM17, IM18, and IM502 involved in each embodiment of the present invention can be referred to: Construction of a meroterpenoid-like compound collection by precursor-assisted biosynthesis[J].Organic&Biomolecular Chemistry,2020,18.DOI:10.1039/D0OB01235A.

Example 1

Small molecules have antioxidant activity

The specific experimental steps for the antioxidant activity of small molecule compounds are as follows:

1.1 Preparation and treatment of mouse macrophages

1.1.1 Take C57BL/6J wild-type adult mice, kill them and soak them in 75% alcohol for 2-3 minutes for disinfection. Make an incision in the abdomen, separate the muscles on the bones with scissors, take the mouse tibia and femur and then soak them in PBS buffer. The subsequent steps are completed in a sterile operating table.

1.1.2 After removing the tibia and femur with sterile forceps, cut off small pieces on both sides with scissors, and repeatedly flush the bone marrow up and down with a 20mL syringe filled with PBS. Collect the flushed bone marrow cells through a 70μm cell sieve into a 50mL sterile centrifuge tube and centrifuge at 2000rpm for 4min at 4℃.

1.1.3 Add 1 mL of 1× red blood cell lysis buffer to the cell pellet to suspend the cells. After standing for 2 min, add 10 mL of PBS for washing and centrifuge at 2000 rpm at 4°C for 4 min.

1.1.4 The cells were resuspended in RPMI 1640 culture medium (containing 5% L929 supernatant) and the cell density was adjusted to 2×10 ⁶ /well, and plated into untreated 6-well cell culture plates for culture at 37°C and 5% CO _2. On the fourth day of cell culture, bone marrow-differentiated macrophages (BMDM) were observed under a microscope.

1.1.5 BMDM cells were plated in 6-well plates and treated as follows: Control (0.1% DMSO), small molecule compound treatment: IM16, IM17, IM18, IM502 (10 μM) for 12 h.

Group 1.1.6 was treated with H ₂ O ₂ (0.25 μM) for 1 h.

1.1.7. Discard the supernatant, add PBS to wash once, resuspend in 200ul 2% FACS Buffer per well in a 96-well U-bottom plate, and centrifuge at 2000rpm for 4min.

1.1.8. Measure ROS: discard the supernatant, add PBS to wash once, add ROS probe (1000x) and treat at 37 degrees for 30 minutes (1640 serum-free and double antibody-free)

1.1.9. Wash once with PBS, add 150ul of 7AAD (400x) to each well of 2% FACS Buffer, mix well, filter into flow tube, detect on Canto II flow cytometer, and analyze with Flowjo software.

The results are shown in Figure 1. The small molecule compounds IM16, IM17, IM18, and IM502 can effectively reduce the level of reactive oxygen species (ROS) in macrophages after H ₂ O ₂ treatment. This indicates that IM16, IM17, IM18, and IM502 have strong antioxidant activity and are effective antioxidant molecules.

Example 2

Small molecule compounds reduce mitochondrial ROS levels

Under the action of various cytokines, the phenotype and function of macrophages change, and the metabolic function of mitochondria is abnormally regulated. For example, different cytokines released by tumor cells in the tumor microenvironment can promote changes in mitochondrial energy metabolism and the production of reactive oxygen species in macrophage mitochondria, which causes changes in the function of macrophages and promotes the development of the disease. For details, please refer to the literature NMDAR antagonists suppress tumor progression by regulating tumor-associated macrophages, PNAS, 2023, Dongcheng Yuan et al.

This example evaluates the effect of small molecule compounds on mitochondrial ROS levels in cells by the following experimental steps:

2.1 The induced differentiated BMDM cells (prepared in the same manner as step 1.1 of Example 1) were plated in a 96-well flat-bottom plate and treated as follows: BMDM cells were pre-cultured in fresh culture medium (FM) or Hepa1-6BL liver cancer cell culture supernatant (TCM) for 12 hours, and then IM502 (10 μM) was added for 6 hours, and a control group (Control) was set up: Hepa1-6BL liver cancer cell culture supernatant (TCM) was pre-cultured for 12 hours, and then 0.1% DMSO (10 μM) was added for 6 hours. Among them, the TCM preparation method is: 3×10 ⁶ Hepa1-6LM cells were mixed in 10 ml culture medium, plated in a 10 cm culture dish, and the cell culture supernatant was collected after culturing for 36 hours.

2.2 Measure mitochondrial ROS: Collect BMDM cells, discard the supernatant, add PBS to wash once, add MitoSOX probe (mitochondrial superoxide red fluorescent probe) and treat at 37 degrees for 30 minutes (1640 serum-free and without double antibody).

2.3 Wash once with PBS, add 150ul of 7AAD (400x) to each well of 2% FACS Buffer, mix well, filter into flow tube, detect by flow cytometer, and analyze by Flowjo software.

The results are shown in Figures 2A and 2B. Tumor cell culture supernatant (TCM) promotes the production of reactive oxygen species in macrophage mitochondria. IM502 can significantly reduce the level of mitochondrial reactive oxygen species (ROS) in macrophages treated with tumor cell culture supernatant, restoring it to the level of mitochondrial ROS in freshly cultured macrophages. Mitochondria are one of the main sources of ROS in cells, indicating that IM502 has strong antioxidant activity and is an effective method for reducing the production of reactive oxygen species in mitochondria.

Example 3

Small molecule compounds promote the production of glutathione, a cell-reducing active substance

The corresponding experimental steps are as follows:

3.1 The induced differentiated BMDM cells (prepared as in step 1.1 of Example 1) were plated into a 96-well flat-bottom plate and treated as follows: BMDM cells were pre-cultured in fresh culture medium (FM) or Hepa1-6BL liver cancer cell culture supernatant for 12 h, and then IM502 (10 μM) was added for treatment for 6 h. A control group (Control) was set up: Hepa1-6BL liver cancer cell culture supernatant (TCM) was pre-cultured for 12 h, and then 0.1% DMSO (10 μM) was added for treatment for 6 h.

3.2 Measure mitochondrial ROS: Collect BMDM cells, discard the supernatant, add PBS to wash once, add glutathione probe (1000x) and treat at 37 degrees for 30 minutes (1640 serum-free and without double antibody).

3.3 Wash once with PBS, add 150ul of 7AAD (400x) to each well of 2% FACS Buffer, mix well, filter into flow tube, detect by flow cytometer, and analyze by Flowjo software.

As shown in Figures 3A and 3B, tumor cell culture supernatant (TCM) reduces the production of glutathione (GSH) in macrophages, and IM502 can significantly promote the GSH level in macrophages treated with tumor cell culture supernatant, restoring it to the GSH level in freshly cultured macrophages. GSH is an important antioxidant in cells, which can effectively neutralize reactive oxygen and maintain redox homeostasis in cells. This shows that IM502 can promote the production of antioxidant substances in cells and has the function of maintaining redox in cells.

Example 4

Small molecule compounds reduce lipid peroxidation

Lipids are important components of the cytoplasmic membrane structure. Lipid peroxidation leads to the destruction of the cytoplasmic membrane structure and affects the function of cells. Cellular lipid peroxidation is closely related to the production of intracellular ROS and redox homeostasis. The corresponding experimental steps are as follows:

4.1 The induced differentiated BMDM cells (prepared as in step 1.1 of Example 1) were plated into a 96-well flat-bottom plate and treated as follows: BMDM cells were pre-cultured in fresh culture medium (FM) or Hepa1-6BL liver cancer cell culture supernatant for 12 h, and then IM502 (10 μM) was added for treatment for 6 h. A control group (Control) was set up: Hepa1-6BL liver cancer cell culture supernatant (TCM) was pre-cultured for 12 h, and then 0.1% DMSO (10 μM) was added for treatment for 6 h.

4.2 Measure mitochondrial ROS: Collect BMDM cells, discard the supernatant, wash once with PBS, add lipid peroxidation detection probe, and incubate at 37 degrees for 30 minutes.

4.3 Wash once with PBS, add 150ul of 7AAD (400x) to each well of 2% FACS Buffer, mix well, filter into flow tube, detect by flow cytometer, and analyze by Flowjo software.

The results are shown in Figures 4A and 4B. Tumor cell culture supernatant (TCM) promotes the occurrence of macrophage lipid peroxidation, and IM502 treatment can significantly inhibit the lipid peroxidation of macrophages after TCM treatment, restoring it to the level of freshly cultured macrophages. This shows that IM502 can protect cells from lipid peroxidation and maintain the integrity of cell membrane structure.

Example 5

Small molecule compounds inhibit the decrease of mitochondrial membrane potential

The decrease in mitochondrial membrane potential is a landmark event in the early stages of apoptosis. It occurs before the appearance of nuclear apoptosis characteristics (chromatin condensation, DNA fragmentation). Once the mitochondrial membrane potential collapses, apoptosis is irreversible. JC-1 is an ideal fluorescent probe widely used to detect mitochondrial membrane potential AΨ'm, which can detect the mitochondrial membrane potential of cells, tissues or purified mitochondria. JC-1 exists in two forms, monomers and polymers, and the emission spectra of the two are different. In normal cells, the mitochondrial membrane potential is high, and JC-1 exists in the mitochondrial matrix in the form of polymers, producing red fluorescence; in the early stages of apoptosis, the mitochondrial membrane potential decreases, and JC-1 exists in the mitochondrial matrix in the form of monomers, producing green fluorescence. The change in JC-1 from red fluorescence to green fluorescence can reflect the decrease in cell membrane potential, and the change in JC-1 fluorescence color can be used as a detection indicator of cell membrane potential. The corresponding experimental steps are as follows:

5.1 The method for preparing BMDM is the same as step 1.1 of Example 1; human macrophages are induced by treating THP-1 monocytes with PMA.

The induced differentiated macrophages were plated into 96-well flat-bottom plates and treated as follows: the cells were pre-cultured in fresh culture medium (FM) or liver cancer cell culture supernatant (TCM) for 12 h, and then IM502 (10 μM) was added for 6 h. A control group (Control) was set up: TCM was pre-cultured for 12 h, and then 0.1% DMSO (10 μM) was added for 6 h.

Mouse macrophages BMDM and human macrophages were treated with mouse Hepa1-6BL cell TCM and HepG2 cell TCM, respectively.

5.2 Measure mitochondrial ROS: Collect BMDM cells, discard the supernatant, wash once with PBS, add membrane potential detection probe (JC-1 working solution), and incubate at 37 degrees for 30 minutes.

Prepare the probe: For every 50ul system, add 0.2ul JC-1 (500×) to 70ul JC-1 Assay Buffer (10×) and then add 630ul ddH ₂ O. Mix well to obtain the JC-1 working solution.

5.3 Wash once with PBS, add 150 μl 2% FACS Buffer to each well, mix well, filter into flow tube, detect by flow cytometer, and analyze with Flowjo software.

As shown in Figures 5A to 5D, tumor cell culture supernatant (TCM) promoted the decrease of macrophage membrane potential, and IM502 treatment could significantly inhibit the decrease of macrophage membrane potential after TCM treatment, restoring it to the level of macrophage membrane potential treated with fresh culture. This indicates that IM502 can protect cells from lipid peroxidation, maintain the integrity of cell membrane structure, effectively inhibit the decrease of macrophage membrane potential, and prevent cell apoptosis.

Example 6

IM502 enhances the phagocytic ability of macrophages

The experimental method for the phagocytic ability of IM502 on macrophages is shown in FIG6A , and the specific steps are as follows:

6.1 CFSE-labeled Hepa1-6BL tumor cells were heat treated at 55°C for 10-15 min.

6.2 BMDM (prepared in the same manner as step 1.1 of Example 1) were co-cultured with heat-treated tumor cells. Three groups were set up: fresh culture medium (FM), tumor cell culture supernatant (TCM), and TCM+IM502.

6.3 Flow cytometry was used to detect the proportion of CFSE+BMDM cells to characterize the phagocytic function of macrophages.

As shown in Figure 6B, IM502 treatment can significantly promote the phagocytic ability of macrophages against tumor cells.

Example 7

Small molecules regulate the expression of inflammatory molecules

The corresponding specific steps are as follows:

7.1 Preparation and treatment of mouse macrophages

7.1.1 The preparation method of mouse macrophages is the same as step 1.1 of Example 1.

7.1.2 BMDM cells were plated in 6-well plates and treated as follows: control (0.1% DMSO) and IM502 (10 μM) for 6 h.

7.1.3 Extract mRNA, convert it into cDNA and detect the expression of different factors by qPCR.

7.2 Preparation and treatment of human macrophages

7.2.1 Resuscitated human THP-1 monocytes were plated in 6-well plates and treated with PMA to induce macrophages.

7.2.2 The following treatments were performed: control (0.1% DMSO), IM502 (10 μM) for 6 h.

7.2.3 Extract mRNA, convert it into cDNA and detect the expression of different factors by qPCR.

The qPCR primer sequences used are shown in the following table:

The results are shown in Figures 7A and 7B. IM502 significantly changed the expression levels of different inflammation-related factors in mouse and human macrophages, indicating that IM502 is a potential inflammatory regulatory active molecule. The expression levels of genes NOS2, IL-6, TNFα, and CXCL9 that characterize M1 macrophages increased, while the expression levels of genes Arg1, TGFβ, and IL-10 that characterize M2 macrophages decreased, indicating that small molecule treatment promotes macrophages to present an M1 phenotype and polarize to the M1 type.

In this specification, the present invention has been described with reference to specific embodiments thereof. However, it is apparent that various modifications and variations may be made without departing from the spirit and scope of the present invention. Therefore, the specification and drawings should be regarded as illustrative rather than restrictive.

Claims (4)

1. Use of a small molecule compound in the preparation of antioxidant drugs and immunomodulatory drugs, characterized in that it is a compound as shown below or a pharmaceutically acceptable salt thereof 2. The use according to claim 1, characterized in that the small molecule compound achieves antioxidant and immunomodulatory effects by reducing the production of reactive oxygen species in cells. 3. The use according to claim 1, characterized in that the small molecule compound achieves antioxidant and immunomodulatory effects by increasing the production of glutathione. 4. The use according to claim 1, characterized in that the small molecule compound regulates the polarization direction of macrophages to achieve antioxidant and immune regulation.