CN118526480A - Application of USP4 inhibitor in preparation of leukemia treatment products - Google Patents

Abstract

The present invention discloses the use of USP4 inhibitors in the preparation of products for treating leukemia, and belongs to the field of biomedical technology. USP4 inhibitors inhibit USP4 activity, weaken protein synthesis of leukemia cells, inhibit leukemia cell growth, and then prevent the onset of leukemia, thereby achieving the therapeutic effect of leukemia. The use of USP4 inhibitors in the preparation of products for treating leukemia is disclosed for the first time.

Description

Application of USP4 inhibitors in the preparation of products for the treatment of leukemia

Technical Field

The present invention relates to the field of biomedicine technology, and in particular to the application of USP4 inhibitors in the preparation of products for treating leukemia.

Background Art

Leukemia is a common malignant tumor of the blood system and is listed as one of the top ten most common tumors in my country. The incidence of acute leukemia is significantly higher than that of chronic leukemia (about 5.5:1). Among acute leukemias, acute myeloid leukemia is the most common type. Finding an effective treatment strategy for acute myeloid leukemia is of great significance to alleviating the pressure on medical and health care in my country. The treatment of acute myeloid leukemia is difficult and has a high recurrence rate after treatment. Protein synthesis plays an important role in the growth of leukemia cells. USP4 protein is a deubiquitinating enzyme that plays an important role in the occurrence and development of many tumors, such as breast cancer, colon cancer, and lung cancer. Therefore, the study inhibits USP4 activity, weakens protein synthesis in leukemia cells, inhibits the growth of leukemia cells, and then prevents the onset of leukemia, thereby achieving the treatment of leukemia, which has important clinical application value.

Summary of the invention

The purpose of the present invention is to provide a method for inhibiting the activity of USP4 protein, thereby inhibiting the protein synthesis of leukemia cells and weakening the growth of leukemia cells, and to realize the use of USP4 inhibitors in the preparation of products for treating leukemia.

The USP4 inhibitor of the present invention includes Vialinin A, and the structure of Vialinin A is shown in formula (I):

Formula (I).

The inventors of the present application have found that the level of USP4 protein increases in leukemia patients. By knocking down the expression of the USP4 gene and reducing the USP4 activity in leukemia cells, the growth of leukemia cells can be inhibited. VialininA, an activity inhibitor of USP4 protein, can inhibit the growth of leukemia cells in a concentration-dependent manner. Inhibiting USP4 activity can significantly reduce the protein synthesis of leukemia cells and prolong the survival period of leukemia model mice, indicating that USP4 inhibitors can be used for the prevention and treatment of clinical leukemia.

The USP4 inhibitors involved in the present invention include Vialinin A (but not limited to this inhibitor), and their use in the preparation of products for treating leukemia is disclosed for the first time. Moreover, they can significantly reduce the protein synthesis of human chronic myeloid leukemia cells and acute monocytic leukemia cells. There is no possibility of any inspiration from other compounds. They have outstanding substantive characteristics and are obviously a significant improvement in the prevention and treatment of leukemia.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the expression level of USP4 protein in human leukemia sample (LAML) data analyzed through public databases (GEPIA databases);

FIG2 is a graph showing the results of knocking down the USP4 gene in human chronic myeloid leukemia cell line K562 and the cell growth;

FIG3 is a graph showing the results of knocking down the USP4 gene in the human acute monocytic leukemia cell line MV4-11 and the cell growth;

FIG4 is a graph showing the results of cell growth of human chronic myeloid leukemia cell line K562 cells treated with Vialinin A for 72 hours;

FIG5 is a graph showing the results of cell growth of human acute monocytic leukemia cell line MV4-11 cells treated with Vialinin A for 72 hours;

FIG6 is a graph showing the results of analyzing protein synthesis in K562 cells, a chronic myeloid leukemia cell line in which USP4 gene was knocked down, after incorporation of OP-Puro staining;

FIG7 is a graph showing the results of analyzing the protein synthesis of MV4-11 cells after OP-Puro staining was performed on MV4-11 cells, a human acute monocytic leukemia cell line in which the USP4 gene was knocked down;

FIG8 is a graph showing the results of analyzing protein synthesis in K562 cells after incorporating OP-Puro staining when human chronic myeloid leukemia cell line K562 cells were treated with Vialinin A;

FIG9 is a graph showing the results of analyzing the protein synthesis of MV4-11 cells after incorporating OP-Puro staining when human acute monocytic leukemia cell line MV4-11 cells were treated with Vialinin A;

FIG10 is a graph showing the survival curve of mice in a leukemia model induced by MLL-AF9 fusion gene treated with Vialinin A;

FIG. 11 is a graph showing the survival curve of leukemia model mice induced by the MLL-AF9 fusion gene in which the USP4 gene was knocked out.

DETAILED DESCRIPTION

The present invention is further described below with reference to specific embodiments and accompanying drawings, but the embodiments are not intended to limit the scope of the present invention.

Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

Example 1 The expression level of USP4 protein in leukemia patients was analyzed using public database data.

Using data from a public database (GEPIA databases), the inventors of the present application searched for the expression levels of USP4 protein in normal and leukemia patient samples in the database, and compared and analyzed the expression levels of USP4 protein in human leukemia sample data. The results are shown in FIG1 . The expression level of USP4 protein was significantly increased in human leukemia samples, indicating that increased levels of USP4 protein may play an important role in the occurrence and development of human leukemia, and that inhibiting USP4 activity can be used to treat human leukemia.

Example 2 Knockdown of USP4 gene in human chronic myeloid leukemia cell line K562 cells and human acute monocytic leukemia cell line MV4-11 cells.

Human chronic myeloid leukemia cell line K562 cells and human acute monocytic leukemia cell line MV4-11 cells were grown in 1640 medium containing 10% FBS. ShRNA targeting USP4 was used to knock down the gene expression level of USP4 in the cells, thereby reducing the activity of USP4 in the cells. Human chronic myeloid leukemia cell line K562 cells and human acute monocytic leukemia cell line MV4-11 cells with USP4 gene knockdown were grown in 1640 medium containing 10% FBS for 0, 24, 48, and 72 hours. The placenta blue staining cell counting method was used to count the number of live cells of human chronic myeloid leukemia cell line K562 cells and human acute monocytic leukemia cell line MV4-11 cells under a microscope to analyze the cell growth. The results are shown in Figures 2 and 3. Knocking down USP4 gene expression and reducing USP4 activity in cells can inhibit the growth of human chronic myeloid leukemia cell line K562 cells and human acute monocytic leukemia cell line MV4-11 cells. This proves that inhibiting USP4 activity can inhibit the growth of leukemia cells.

Example 3 Vialinin A was used to treat human chronic myeloid leukemia cell line K562 cells and human acute monocytic leukemia cell line MV4-11 cells.

Human chronic myeloid leukemia cell line K562 cells and human acute monocytic leukemia cell line MV4-11 cells were grown in 1640 medium containing 10% FBS and treated with 0, 2, 5, and 10 μM Vialinin A for 0, 24, 48, and 72 hours. The number of live cells of human chronic myeloid leukemia cell line K562 cells and human acute monocytic leukemia cell line MV4-11 cells was counted on a cell counting plate under a microscope using the placenta blue staining cell counting method. The growth of human chronic myeloid leukemia cell line K562 cells and human acute monocytic leukemia cell line MV4-11 cells treated with various concentrations of Vialinin A was analyzed. The results are shown in Figures 4 and 5, and Vialinin A exhibits a concentration-dependent inhibition of the growth of human chronic myeloid leukemia cell line K562 cells and human acute monocytic leukemia cell line MV4-11 cells. It was demonstrated that Vialinin A inhibits USP4 activity and can suppress the growth of leukemia cells.

Example 4 OP-Puro was incorporated into K562 cells, a chronic myeloid leukemia cell line, and MV4-11 cells, a human acute monocytic leukemia cell line, in which the USP4 gene was knocked down.

Protein synthesis is crucial to the growth and proliferation of leukemia cells and plays an important role in the treatment of leukemia. In order to further verify whether inhibiting USP4 activity can inhibit the growth of leukemia cells by inhibiting protein synthesis in leukemia cells. O-Propargyl-Puromycin (OP-Puro) is a puromycin analog that can be incorporated into new protein peptides. It can be used to label newly synthesized proteins in cells. The level of OP-Puro incorporated into cells can be analyzed by fluorescence colorimetry. The fluorescence intensity can be detected by flow cytometry to detect the protein synthesis level of cells.

OP-Puro was incorporated into K562 cells, a chronic myeloid leukemia cell line, and MV4-11 cells, a human acute monocytic leukemia cell line, in which the USP4 gene was knocked down, and the OP-Puro incorporated into the cells was fluorescently developed using the OP-Puro colorimetric kit. The fluorescence intensity of OP-Puro incorporated into K562 cells, a human chronic myeloid leukemia cell line, and MV4-11 cells, in which the USP4 gene was knocked down, was detected by flow cytometry to analyze the protein synthesis of the cells. The results are shown in Figures 6 and 7. Knockdown of the USP4 gene to reduce the level of USP4 protein in cells can significantly reduce the fluorescence intensity of OP-Puro incorporated into K562 cells, a human chronic myeloid leukemia cell line, and MV4-11 cells, a human acute monocytic leukemia cell line. This demonstrates that knocking down the USP4 gene to reduce the level of USP4 in cells can inhibit protein synthesis in leukemia cells.

Example 5 OP-Puro was incorporated into the human chronic myeloid leukemia cell line K562 cells/human acute monocytic leukemia cell line MV4-11 cells treated with Vialinin A.

Vialinin A (10 μM) was used to inhibit USP4 activity, and OP-Puro was incorporated into the cells. The OP-Puro colorimetric kit was used to perform fluorescence colorimetric analysis on OP-Puro incorporated into the cells of K562 cells, a human chronic myeloid leukemia cell line, and MV4-11 cells, a human acute monocytic leukemia cell line, treated with Vialinin A. The fluorescence intensity of OP-Puro incorporated into the cells was detected by flow cytometry to analyze the protein synthesis of the cells. As shown in Figures 8 and 9, Vialinin A treatment can significantly reduce the fluorescence intensity of OP-Puro incorporated into K562 cells, a human chronic myeloid leukemia cell line, and MV4-11 cells, a human acute monocytic leukemia cell line. This demonstrates that inhibiting USP4 activity using Vialinin A can inhibit protein synthesis in leukemia cells.

Example 6 Vialinin A was used to treat leukemia model mice induced by MLL-AF9 fusion gene.

Using leukemia model mice induced by the expression of MLL-AF9 fusion gene, Vialinin A was intraperitoneally injected at a dose of 5 mg/kg/2 day, and the control group was given an equal volume of phosphate buffer containing an equal amount of drug solvent. The survival time of the leukemia model mice was statistically analyzed. The results are shown in Figure 10. Vialinin A inhibits USP4 activity, and Vialinin A treatment of mice can significantly prolong the survival time of leukemia model mice.

Example 7 Knockout of USP4 gene in leukemia model mice induced by MLL-AF9 fusion gene.

Wild-type mouse leukemia cells expressing the MLL-AF9 fusion gene or mouse leukemia cells expressing the USP4 gene knockout of the MLL-AF9 fusion gene were transplanted into CD45.1 mice to construct wild-type leukemia model mice induced by the MLL-AF9 fusion gene and leukemia model mice induced by the USP4 gene knockout of the MLL-AF9 fusion gene. The survival time of the mice was statistically analyzed after transplantation, and the effect of reducing the USP4 activity in cells by using the USP4 gene knockout on the survival time of the leukemia model mice was analyzed. The results are shown in Figure 11, and reducing the USP4 activity in cells by the USP4 gene knockout can significantly prolong the survival time of the leukemia model mice.

The above results confirm that inhibiting USP4 activity can inhibit the growth of leukemia cells and delay the progression of leukemia in mice. Using USP4 inhibitors to reduce USP4 activity can be used for the prevention and treatment of clinical leukemia.

The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims (2)

1. Use of a USP4 inhibitor in the preparation of a product for treating leukemia, characterized in that the USP4 inhibitor comprises Vialinin A, and the structure of Vialinin A is shown in formula (I): Formula (I). 2. The use of the USP4 inhibitor according to claim 1 in the preparation of a product for treating leukemia, characterized in that the inhibitor inhibits the protein synthesis of leukemia cells and the growth of leukemia cells by inhibiting the activity of the USP4 protein, thereby ultimately achieving the use in a product for treating leukemia.