CN110652515A - Application of AMPK inhibitor Compound C in tumor treatment drug - Google Patents

Abstract

The invention discloses a new application of Compound C, an AMPK inhibitor. Specifically, the compound C inhibitor is used in the preparation of a tumor drug. Compund C can play an anti-hematological tumor effect by inhibiting tumor cell activity and inducing tumor cell apoptosis. The new application of the AMPK inhibitor Compound C of the present invention will provide a new drug for clinical treatment of hematological tumors.

Description

Application of AMPK inhibitor Compound C in the treatment of tumor drugs

technical field

The invention belongs to the technical field of medicine, and in particular relates to the application of an AMPK inhibitor Compound C in the treatment of tumors.

Background technique

Tumor (neoplasm) is a non-hereditary genetic disease. Under the action of tumorigenic factors, the genes of normal cells are changed, and the normal regulation of their growth is lost, resulting in abnormal proliferation. Tumor cells have three striking basic characteristics namely: immortality, migration and loss of contact inhibition. The core idea of tumor therapy is to kill and remove tumor cells.

At present, one of the mechanisms of tumor treatment commonly used in clinical treatment, such as chemotherapy, radiotherapy, hormone therapy and some biological therapies, is to induce tumor cell apoptosis. For example, apoptosis induced by radiotherapy is mainly mediated by Fas, and some cell cycle-related proteins, Bcl-2 and Bax also play a key role in this process. Chemotherapy induces tumor cells to undergo apoptosis through some chemical drugs to achieve the therapeutic purpose of inhibiting tumors. Drugs such as doxorubicin and 5-fluorouracil can effectively cause damage to cellular DNA, resulting in cell G1 cycle arrest to complete repair or enter apoptosis. Paclitaxel and colchicine, which act on microtubules, mediate apoptosis through the Bcl-2 family. At present, chemotherapy is still one of the main strategies for cancer treatment at this stage and even in the future. Toxic side effects and drug resistance are the main obstacles facing tumor chemotherapy.

Therefore, whether it is radiotherapy or chemotherapy, its inducing tumor cell apoptosis lacks targeting, killing tumor cells and harvesting the life of normal cells at the same time, with great toxic and side effects. In addition, after radiotherapy and chemotherapy, tumor recurrence and drug resistance often occur, which also seriously limits the clinical treatment effect. At present, the effect of tumor treatment is poor, and the five-year survival rate of patients is very low. New treatments with high efficiency and low toxicity are urgently needed.

Compound C (Dorsomorphin) is a potent, reversible and selective AMPK inhibitor that competes for its ATP binding site. Ki was 109 nM in cell-free assay. Studies have shown that Compound C selectively inhibits AMPK activity while inhibiting some other structurally related kinases, including ZAPK, SYK (Spleen Tyrosine Kinase), PKCθ (Protein Kinase C-θ), PKA (Protein Kinase A), and JAK3 (Janus Kinase). 3) There is no significant inhibitory effect. Therefore, currently in the art, Compound C is often used as a specific AMPK inhibitor, and no other new uses of Compound C have been found, such as its use in the treatment of tumors.

SUMMARY OF THE INVENTION

The object of the present invention is to overcome the above-mentioned deficiencies of the prior art, and to provide a method for the application of Compound C, an AMPK inhibitor, to solve the side effects that Compound C is only used for specific AMPK inhibitors and current methods and drugs for treating cancer. Large, prone to technical problems such as drug resistance.

Another object of the present invention is to provide a drug for the treatment of tumors, so as to solve the technical problems of using the existing drugs for the treatment of tumors, such as toxic and side effects, easy occurrence of drug resistance, and easy tumor recurrence.

In order to achieve the above purpose of the invention, one aspect of the present invention provides an application method of the AMPK inhibitor Compound C. Specifically, the application of the AMPK inhibitor Compound C in the preparation of a tumor drug.

Another aspect of the present invention provides a medicament for treating tumors. The medicament for treating tumors includes an effective dose of active ingredients for treating tumors, and the active ingredients include Compound C, an AMPK inhibitor.

Compared with the prior art, the application of the AMPK inhibitor Compound C of the present invention in the preparation of a tumor medicine has the following beneficial effects:

Experiments have shown that the AMPK inhibitor Compound C has relatively strong effects of inhibiting tumor cell activity and inducing tumor cell apoptosis, and exerts an anti-blood system tumor effect. Therefore, after the AMPK inhibitor Compound C is used as an active ingredient to prepare a tumor-treating drug, the tumor-treating drug can be endowed with significant effects of inhibiting tumor cell activity and inducing tumor cell apoptosis.

The medicament for treating tumors of the present invention uses the AMPK inhibitor Compound C as an active ingredient. Therefore, the medicament for treating tumors can effectively inhibit the activity of tumor cells and induce and promote apoptosis of tumor cells, so as to alleviate and reduce the effects of malignant tumors on the human body. harm, relieve clinical symptoms, improve quality of life, and prolong life.

Description of drawings

The present invention will be further described below in conjunction with the accompanying drawings and embodiments, in which:

FIG. 1 is a graph showing the effect of different concentrations of Compound C and Rapamycin reagents on the activity of Jurkat lymphoma cells in Example 11 of the present invention;

Figure 2 is a graph showing the effect of different concentrations of Compound C and Rapamycin reagents on the activity of K562 lymphoma cells in Example 11 of the present invention;

3 is a graph showing the effect of Compound C and a control reagent (DMSO) on apoptosis of Jurkat lymphoma cells in Example 12 of the present invention;

Figure 4 is a graph showing the effect of Compound C and a control reagent (DMSO) on apoptosis of K562 lymphoma cells in Example 12 of the present invention.

Detailed ways

In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and the attached table. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention.

Explanation of Proprietary Names:

Tumor (neoplasm): refers to the new organism (neogrowth) formed by the proliferation of local tissue cells under the action of various tumorigenic factors, because this new organism is mostly space-occupying massive protrusions, also known as neoplasms (neoplasm).

Compound C (Dorsomorphin): a potent, reversible, and selective AMPK inhibitor with Ki of 109nM in cell-free assays. Its molecular structural formula is as follows:

AMPK (Adenosine Monophosphate-activated Protein Kinase): is a key sensor of cellular energy. The protein kinase can maintain the balance of ATP production and consumption in eukaryotic cells by sensing the energy state of cells, that is, energy homeostasis. Its activation helps to correct metabolic disorders and make cell metabolism tend to physiological balance, thus becoming a treatment for metabolic disorders. and possible targets of cancer. Many studies have shown that the activation of AMPK can stimulate the body to produce ATP to provide energy for physiological activities; at the same time, the activation of AMPK can strongly inhibit the synthesis of protein and fat to reduce the consumption of ATP. Among them, inhibition of AMPK activity by Dorsomorphin almost completely inhibited the proteolysis of autophagy in HT-29 cells.

In one aspect, the embodiments of the present invention provide a method for applying the AMPK inhibitor Compound C. Specifically, the application of the AMPK inhibitor Compound C in the preparation of a tumor drug. According to relevant experiments, the AMPK inhibitor Compound C is an active ingredient, which can effectively inhibit tumor cell activity and induce and promote tumor cell apoptosis. Specifically, as in Example 1 below, the effect of different concentrations of Compound C on the activity of blood tumor cells and the experimental results. Therefore, the AMPK inhibitor Compound C can be used as an effective anti-tumor ingredient to inhibit tumor cell activity and induce and promote tumor cell apoptosis, so as to alleviate and reduce the harm of malignant tumors to the human body and relieve clinical symptoms , improve the quality of life, prolong the clinical effect of life.

In one embodiment, the tumor that the AMPK inhibitor Compound C can inhibit the activity of tumor cells and promote tumor cell apoptosis can be a hematological tumor, that is, the AMPK inhibitor Compound C can be used in the preparation of a drug for the treatment of hematological tumors. . In a specific embodiment, the hematological tumor includes at least one of lymphoma, myeloid leukemia, and myeloma. Wherein, the lymphoma includes T/B lymphoma.

Of course, the AMPK inhibitor Compound C can be other types of tumors besides inhibiting the activity of blood tumor cells and promoting the apoptosis of blood tumor cells.

In addition, when the AMPK inhibitor Compound C is used to prepare a drug for treating tumors, a corresponding dosage form can be prepared according to different administration modes. Then, the AMPK inhibitor Compound C can be compounded with the pharmaceutically acceptable components of the corresponding dosage form, such as excipients, etc., and can also be compounded with other anti-tumor active components to form an anti-tumor compound active component.

In addition, according to the molecular structural formula of the Compound C, it is also within the scope of the present disclosure to appropriately modify the molecular body to introduce other groups, such as the substitution and modification of the hydrogen of the carbon atom.

On the other hand, based on the application of the AMPK inhibitor Compound C described above, the embodiments of the present invention also provide a drug for treating tumors. The tumor-treating drug includes an effective dose of the active ingredient for treating the tumor, and of course, there may be further pharmaceutically acceptable auxiliary ingredients. The effective dose refers to an effective amount for treating a tumor, and refers to an amount of a compound of the invention sufficient to show benefit or clinical significance in an individual. It will be understood by those skilled in the art that the actual amount or dose administered, as well as the schedule of administration, will depend on the nature and severity of the disease being prevented or treated, the age and general condition of the subject being prevented or treated, and the administration medicine, etc.

Wherein, the active ingredient includes Compound C, an AMPK inhibitor; of course, as mentioned above, the active ingredient may also include other active ingredients that are effective against tumors. The effective component may also be a component that can improve the anti-tumor clinical effect of the AMPK inhibitor Compound C after being compounded with the AMPK inhibitor Compound C. In one embodiment, the content of the AMPK inhibitor Compound C in the drug for treating tumors is 5-20 μM.

The auxiliary components can be related auxiliary materials of corresponding dosage forms prepared according to the drug administration mode of the tumor treatment. In one embodiment, the adjuvant is a pharmaceutically acceptable carrier of the AMPK inhibitor Compound C. In a specific embodiment, the carrier includes at least one of an antibody capable of coupling to the AMPK inhibitor Compound C, a targeted recombinant protein, a nanoparticle, and an exosome. Through the carrier, the stability of the AMPK inhibitor Compound C can be improved, the targeted drug delivery effect in vivo can be improved, and the purpose of targeted killing of tumor cells can even be achieved.

Therefore, the drug for the treatment of tumors is based on the AMPK inhibitor Compound C mentioned above as an active ingredient. Therefore, the drug for the treatment of tumors can effectively inhibit the activity of tumor cells and induce and promote the apoptosis of tumor cells, so as to achieve remission and reduction of malignancy. The harm of tumors to the human body can relieve clinical symptoms, improve the quality of life and prolong life.

In addition, the drug for treating tumors can be prepared according to conventional pharmaceutical preparation methods. For example, when the drug for treating tumors contains an antibody capable of coupling with AMPK inhibitor Compound C, the antibody can be coupled with AMPK inhibitor compound C according to conventional methods. Agent Compound C.

With reference to specific examples, the application of Compound C in the preparation of tumor medicines will be further described in detail.

1. Example of the effect of Compound C on tumor cell activity and apoptosis

Example 11

The effect of different concentrations of Compound C on the activity of blood tumor cells detected by MTS

S11: Cell Pretreatment

1) Two kinds of lymphoma cells (Jurkat, K562) were resuspended to 5×10 ⁴ cells/mL and seeded in 96-well plates;

2) Compound C was added to the 96-well plate, and the concentration gradient was set to 0 μM, 0.5 μM, 1 μM, 5 μM, 10 μM and 20 μM, and after culturing for 48 hours, the cell survival was detected;

S12: MTS detects cell viability

1) Gently mix 2mL MTS and 100μL PMS evenly;

2) Add 20 μL MTS mixture to each well, mix well, place in a carbon dioxide cell incubator, and incubate in the dark for 3 hours;

3) Detect the wavelengths at OD490nm and OD630nm with a microplate reader;

4) Using the OD value at the compound C concentration of 0 μM as a control, analyze the cell viability under each concentration gradient.

Using Rapamycin reagent as a control, the same comparative example was done according to the method of Example 11. After experimental inspection, it is known that the effects of Compound C and Rapamycin reagents on the activity of Jurkat lymphoma cells are shown in Figure 1, and the effects of Compound C and Rapamycin reagents on the activity of K562 lymphoma cells are shown in Figure 2. It can be seen from Figure 1 and Figure 2 that Compound C has a significant inhibitory effect on the activity of blood tumor cells, and Compound C has a stronger ability to inhibit the activity of tumor cells than Rapamycin at the same dose.

Example 12

Analysis of the effect of Compound C on tumor cell apoptosis by flow cytometry

S11: Cell Pretreatment

1) Lymphoma cells (Jurkat, K562) were resuspended to 3×10 ⁵ cells/mL and seeded in 12-well plates;

2) After culturing with Compound C for 48 hours, the apoptosis of cells was detected;

S12: Flow cytometry analysis of apoptosis

1) Collect cell suspension;

2) Centrifuge the cell suspension at 800 g at 4°C for 5 min, discard the supernatant, and collect the cells in the pellet;

3) Wash the cells twice with pre-cooled PBS, and then resuspend the cells with 100 μL of 1×AnnexinV Binding Buffer;

4) Add 5 μL of Annexin V-FITC and 5 μL of PI staining solution to each sample, mix gently, and react in the dark at room temperature for 15 minutes; pay attention to setting single staining and blank control, and subsequent flow detection adjustment needs;

5) After the incubation, add 400 μL of pre-cooled 1×Annexin V Binding Buffer to each sample, mix gently; detect within 1 h;

6) Detection of apoptosis by flow cytometry: Annexin V-FITC single positive cells are early apoptotic cells, Annexin V-FITC/PI double positive cells are late apoptotic cells.

Using DMSO reagent as the control group (Ctrl), the same comparative example was carried out according to the method of Example 12. After experimental inspection, it is known that the apoptosis of Compound C and the control group (Ctrl) on Jurkat lymphoma cells is shown in Figure 3, and the effect of Compound C and the control group (Ctrl) on the apoptosis of K562 lymphoma cells is shown in Figure 4. It can be seen from Figure 1 and Figure 2 that Compound C has a significant apoptotic effect on Jurkat and K562.

The above-mentioned embodiments are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included in the present invention. within the scope of protection of the invention.

Claims (7)

1. Application of Compound C, an AMPK inhibitor, in the preparation of medicaments for treating tumors. 2 . The application according to claim 1 , wherein the drug for treating tumors is a drug for treating blood tumors. 3 . 3. The use according to claim 2, wherein the blood tumor comprises at least one of T/B lymphoma, myeloid leukemia, and myeloma. 4. A medicament for treating tumors, comprising an effective dose of an active ingredient for treating tumors, wherein the active ingredient comprises the AMPK inhibitor Compound C according to any one of claims 1-3. 5 . The medicine according to claim 4 , wherein the content of the AMPK inhibitor Compound C is 5-20 μM. 6 . 6. The medicine according to claim 4 or 5, characterized in that: further comprising a pharmaceutically acceptable carrier of the AMPK inhibitor Compound C. 7 . The medicine according to claim 6 , further comprising a pharmaceutically acceptable carrier of the AMPK inhibitor Compound C. 8 . The carrier includes at least one of an antibody coupled with the AMPK inhibitor Compound C, a targeted recombinant protein, a nanoparticle and an exosome.

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