Polypeptide inhibitor of targeted RNA methyltransferase dimer and application of polypeptide inhibitor

Abstract

The invention belongs to the technical field of biological medicine, and discloses a polypeptide inhibitor of a targeted RNA methyltransferase dimer and application of the polypeptide inhibitor. The polypeptide inhibitor provided by the invention effectively inhibits the formation and functional activity of the heterodimer by targeting a binding site required by the assembly of the METTL3-METTL14 heterodimer, for example, the polypeptide inhibitor can significantly reduce the total m6A modification level in acute myelogenous leukemia (AML) cells, thereby inhibiting cell proliferation and inducing apoptosis. Experimental results show that the polypeptide inhibitor disclosed by the invention shows good anti-tumor activity in an AML (acute myeloid leukemia) cell line, and the level of marker protein related to AML can be remarkably reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a polypeptide inhibitor targeting an RNA methyltransferase dimer and its application. Background Art

[0002] N 6 -methyladenosine (N 6 -methyladenosine, m 6 A) is the most abundant chemical modification form in mammalian messenger RNA (mRNA). m 6 A modification is dynamically regulated by methyltransferases and demethylases. Methylation reader proteins specifically recognize and bind to the m 6 A site and mediate its biological functions. Among them, the m 6 A methyltransferase complex consists of a catalytic subunit METTL3 and a substrate recognition subunit METTL14 to form a heterodimer, and depends on auxiliary components such as WTAP and ZC3H13 to maintain its functional stability.

[0003] The high expression of METTL3 and METTL14 is related to various diseases. For example, in recent years, a large number of studies have shown that abnormal m 6 A modification is closely related to the pathogenesis of acute myeloid leukemia (AML). AML is a malignant proliferative disease originating from myeloid hematopoietic stem cells / progenitor cells in the bone marrow and is the most common type of adult acute leukemia. It is characterized by the uncontrolled proliferation of leukemia cells in the bone marrow, dysfunction of the differentiation function, and obstruction of the apoptosis process, often accompanied by clinical manifestations such as anemia, bleeding, infection, and fever. Currently, the commonly used treatment methods mainly include chemotherapy, hematopoietic stem cell transplantation, etc. Although the survival rate of patients has been improved to a certain extent, there are still problems such as high drug resistance and large side effects. Therefore, it is of great significance to develop new drug treatment targets. It is worth noting that METTL3 and METTL14 are significantly overexpressed in AML patients. The two not only drive the occurrence and development of leukemia by post-transcriptionally regulating the expression of key oncogenes such as MYC and MYB, but also affect the disease process by maintaining the self-renewal ability of leukemia stem cells. In addition, studies have confirmed that both of these components play key roles in the development of AML and the regulation of chemotherapy drug response, suggesting that they may be potential targets for AML treatment.

[0004] Polypeptide inhibitors have attracted much attention in the field of drug research and development due to their high specificity, low toxicity, and good biocompatibility. In recent years, with the progress of screening technologies and design methods, their development efficiency and application scope have been significantly improved. Summary of the Invention

[0005] The present invention provides a polypeptide inhibitor targeting an RNA methyltransferase dimer. The polypeptide inhibitor targets the binding site on the METTL14 protein related to the assembly with METTL3. Therefore, the polypeptide inhibitor can be used to inhibit diseases caused by upregulation of METTL14 or METTL3 expression.

[0006] In a first aspect, the object of the present invention is a polypeptide inhibitor targeting an RNA methyltransferase dimer. The polypeptide inhibitor is a synthetic polypeptide, denoted as polypeptide M14P1 in the present invention. The amino acid sequence of the polypeptide M14P1 contains the sequence shown in SEQ ID NO.1 in the sequence listing. Preferably, the amino acid sequence of the polypeptide M14P1 is exactly the sequence shown in SEQ ID NO.1. The specific sequence of SEQ ID NO.1 from the N-terminus to the right (C-terminus) is:

[0007] RRRRRRRRGGGLDLGRVCLRKWGYRRCEDI.

[0008] The crystal structure of the METTL3-METTL14 heterodimer reveals two key interaction interfaces. Interface 1 is composed of residues 238-258 of the METTL14 protein and presents an α-helical structure; Interface 2 spans residues 278-312 and forms a β-sheet structure. Based on Interface 1, the inventors of the present application designed the above-mentioned polypeptide inhibitor M14P1, which can inhibit the formation of this dimer complex. Specifically, by targeting the binding site on the METTL14 protein related to the assembly of the METTL3 complex, polypeptide M14P1 was designed based on the α-helical structure of METTL14. To enhance cell penetration, the R8 cell-penetrating peptide was fused to the N-terminus of the polypeptide.

[0009] In a second aspect, the object of the present invention is to provide the use of the polypeptide inhibitor targeting an RNA methyltransferase dimer and a composite preparation containing the polypeptide inhibitor in the preparation of a drug for treating or preventing diseases caused by high expression of METTL3-METTL14. The polypeptide M14P1 can significantly inhibit the proliferation and survival of leukemia cells by targeting and interfering with the translational assembly of the METTL3-METTL14 heterodimer and its expression level of AML-related substrates, showing significant anti-leukemia activity.

[0010] In the above-mentioned use, the leukemia includes acute leukemia, chronic leukemia, hairy cell leukemia, and prolymphocytic leukemia.

[0011] Furthermore, the acute leukemia includes L1 acute lymphoblastic leukemia, L2 acute lymphoblastic leukemia, L3 acute lymphoblastic leukemia, and acute myeloid leukemia.

[0012] Furthermore, the acute myeloid leukemia includes granulocytic leukemia, erythroleukemia, and megakaryocytic leukemia.

[0013] Furthermore, the chronic leukemia includes chronic lymphocytic leukemia and chronic myeloid leukemia.

[0014] Furthermore, the use concentration of the polypeptide M14P1 is 10 - 20 μM.

[0015] As used herein, the term "prevention" refers to prophylactically administering the polypeptide to a healthy patient to prevent the occurrence of the diseases and disorders described herein. Additionally, the term "prevention" can also refer to prophylactically administering the polypeptide to a patient in the early stage of a disease to be treated. The prophylactic administration specifically refers to high-risk populations with the METTL3 / 14 overexpression biomarker.

[0016] Compared with the prior art, the present invention has the following advantages and effects:

[0017] The present invention has been verified through experiments that treating cells with high expression of METTL3 and METTL14 with the polypeptide M14P1 can inhibit the interaction between the RNA methyltransferase dimer subunits METTL3 and METTL14. For example, it can significantly reduce the m 6 A level of poly(A)RNA in AML cells; in the AML cell lines MOLM-13 and NOMO-1, treatment with the polypeptide M14P1 significantly inhibits cell proliferation and causes a significant decrease in the levels of some AML marker proteins; different from traditional small molecule competitive inhibitors, M14P1 exerts an interfering effect by targeting an interaction interface during the assembly of METTL3 and METTL14. This target is different from the target sites of currently existing inhibitor drugs. For example, for inhibitor drugs targeting FLT3 and IDH1, and even for drugs targeting the RNA methyltransferase complex such as STM2457, the target sites of their interfering effects are different from the target site of the polypeptide M14P1 provided in this application, that is, the specific interaction interface. Therefore, the polypeptide M14P1 provided in this application has a unique mechanism in inhibiting the activity of the METTL3-METTL14 heterodimer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] To more clearly illustrate the technical solutions of the present invention, the drawings used in the embodiments of the present invention will be described below. These drawings only show some embodiments of the present invention.

[0019] Figure 1 It is a representative laser confocal microscopy image with a scale bar of 10 μm;

[0020] Figure 2Western blotting results of immunoprecipitation using anti-METTL3 antibody;

[0021] Figure 3 Western blotting results of immunoprecipitation using anti-METTL14 antibody;

[0022] Figure 4 Dot blot of m 6 A levels of poly(A)RNA in MOLM-13 cells treated with 15 μM M14P1 for 4 hours, with methylene blue staining as a control.

[0023] Figure 5 Dose-response curves of MOLM-13 and NOMO-1 cells to M14P1 treatment;

[0024] Figure 6 Apoptosis percentages of MOLM-13 and NOMO-1 cells after treatment with M14P1 at the specified concentrations;

[0025] Figure 7 Effect of R8 and M14P1 peptide treatments on the cell cycle distribution of MOLM-13 cells;

[0026] Figure 8 Effect of R8 and M14P1 peptide treatments on the cell cycle distribution of NOMO-1 cells;

[0027] Figure 9 Western blotting of AML-related proteins SP1, MYC, and BRD4 in MOLM-13 cells treated with M14P1. Detailed Description of the Specific Embodiments

[0028] The following is a detailed description of the specific embodiments of the present invention. The provided examples are only for clarifying the present invention and do not limit the scope of the present invention. The examples can be used as a reference for further improvement by those of ordinary skill in the art and do not constitute any limitation to the present invention in any way.

[0029] For those where specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in this field are followed, such as referring to "Molecular Cloning: A Laboratory Manual" by J. Sambrook et al., translated by Huang Peitang et al., 3rd Edition, Science Press, or according to the product instructions. For reagents or instruments where the manufacturer is not indicated, they are all conventional products that can be obtained commercially.

[0030] The polypeptides in the embodiments of the present invention are all synthesized by Nanjing Genscript Biotech Co., Ltd.;

[0031] MOLM-13 and NOMO-1 were purchased from Wuhan Pusai Biotechnology Co., Ltd.

[0032] The R8 peptide sequences involved in the following examples are all RRRRRRRR;

[0033] The following examples take the acute myeloid leukemia cell line as an example to verify the inhibitory effect of the polypeptide provided by the present invention on it, but it does not mean that the polypeptide provided by the present invention can only inhibit the activity of acute myeloid leukemia cells.

[0034] Example 1: Subcellular localization of M14P1

[0035] MOLM-13 and NOMO-1 cells were treated with M14P1 peptide labeled with FITC at the N-terminus for 4 hours, washed with PBS, and nuclear stained with Hoechst. Images were collected using a laser confocal microscope in the FITC channel, DAPI channel, and bright field, respectively.

[0036] The experimental results are as Figure 1 shown, showing the distribution characteristics of FITC-labeled M14P1 in MOLM-13 and NOMO-1, and the nuclei were counterstained with Hoechst to show blue fluorescence. Figure 1 The scale bar in

[0037] is 10 μm; it can be seen that M14P1 shows cytoplasmic localization characteristics in MOLM-13 and NOMO-1 cells, confirming that it has successfully crossed the membrane and accumulated in the cytoplasm.

[0038] Cells were collected and lysed on ice for 30 minutes using a polysome lysis buffer. Subsequently, it was centrifuged at 13,000 rpm for 10 minutes to remove insoluble debris, and the supernatant was collected. The supernatant was gently rotated and incubated overnight with the target antibody at 4 °C. The next day, protein A / G magnetic beads were added and incubated at 4 °C for another 2 - 4 hours. Then, the magnetic beads were thoroughly washed with a high-salt buffer to remove non-specific binders. The magnetic beads were transferred to 2×SDS sample buffer and boiled at 100 °C for 10 minutes to elute the bound proteins. The proteins were detected by Western blotting. The specific operations were as follows: Equal amounts of protein samples were separated by SDS-PAGE, and then the proteins were transferred to a PVDF membrane. At room temperature, the PVDF membrane was blocked with a TBST solution containing 5% skim milk for 1 hour. After blocking, the membrane was incubated with the primary antibody overnight at 4 °C. The next day, after washing the membrane, the corresponding secondary antibody was added and incubated at room temperature for 1 hour. Finally, the protein bands were developed using an enhanced chemiluminescence detection system.

[0039] The experimental results are as Figure 2 and Figure 3As shown, the results of co-immunoprecipitation using anti-METTL3 antibody and anti-METTL14 antibody are respectively shown, where the R8 peptide serves as a negative control; the results show that when the M14P1 peptide is added, the interaction between METTL3 and METTL14 is significantly weakened.

[0040] Example 3: m 6 A Dot blot assay for poly(A)RNA m 6 A

[0041] After extracting the total RNA of MOLM-13 cells using Trizol reagent, poly(A)RNA was enriched by oligo-dT magnetic beads to remove ribosomal RNA (rRNA). Subsequently, the isolated poly(A)RNA was transferred to a positively charged nylon membrane, and an RNA concentration gradient (500 ng, 250 ng, and 125 ng respectively) was constructed. After air-drying the membrane, it was fixed using a UV crosslinker (256 nm wavelength) under the condition of 0.3 J to ensure stable binding of RNA. Subsequently, the membrane was soaked in a PBST solution containing 5% skim milk (phosphate buffer containing 0.5% Tween-20) at room temperature and incubated on a shaker at 50 rpm for 1 hour to complete blocking. After blocking, the membrane was incubated with m 6 A specific primary antibody overnight in a shaker at 50 rpm. The next day, the membrane was transferred to room temperature and incubated with the corresponding secondary antibody for 1 hour under the same oscillation conditions. Finally, signal detection was performed using a chemiluminescence imaging system for visualization analysis.

[0042] As Figure 4 Shown is the dot blot of the m6A level of poly(A)RNA in MOLM-13 cells treated with 15 μM of M14P1 for 4 hours, with methylene blue staining as a control. From the experimental results, it can be seen that M14P1 treatment significantly reduced the m 6 A modification level in MOLM-13 cells.

[0043] Example 4: Cell viability assay

[0044] MOLM-13 and NOMO-1 cells were seeded in 96-well plates at a density of 10,000 cells per well and cultured overnight in a 37 °C incubator with 5% CO2. Subsequently, the stock solutions of R8 peptide and M14P1 were respectively formulated into working solutions with different concentration gradients, and the working solutions with different concentration gradients included: 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM, 64 μM, 128 μM, which were respectively added to the cell culture wells and incubated for 3 hours. After incubation, CCK-8 working solution was added to each well and incubated for 1 hour under the same conditions. The absorbance values of each well at a wavelength of 450 nm were recorded using a microplate reader. The background noise was corrected by subtracting the absorbance value of the negative control group, and the cell viability was calculated using the formula: Cell viability (%) = (Absorbance of the polypeptide treatment group / Absorbance of the untreated control group) × 100%. Finally, a nonlinear regression model was used with GraphPad Prism 8.0 to fit the cytotoxicity curve.

[0045] Figure 5 The dose-response curves of MOLM-13 and NOMO-1 cells to M14P1 treatment are shown, and the data are presented as mean ± s.e.m. (n = 3), and the half-maximal inhibitory concentration (IC50) of M14P1 in each cell line is shown. As Figure 5 The experimental results shown indicate that the half-maximal inhibitory concentrations (IC50) of M14P1 for MOLM-13 and NOMO-1 cells are 15.53 μM and 19.63 μM, respectively, indicating that M14P1 has significant anti-tumor proliferation activity.

[0046] Example 5: Detection of cell apoptosis

[0047] After collecting MOLM-13 and NOMO-1 cells, they were washed with PBS to remove serum interference. Subsequently, the cells were treated with R8 peptide (5 μM) and M14P1 peptide (5 μM and 15 μM) in DMEM medium for 3 hours. After treatment, the Annexin V-FITC / PI cell apoptosis detection kit was used to detect the cell apoptosis level, and data analysis was performed using FlowJo software.

[0048] Figure 6 The apoptotic percentages of MOLM-13 and NOMO-1 cells after treatment with M14P1 at the specified concentrations are shown. The data are presented as mean ± s.e.m. (n = 3), and Two-tailed Student’s t-test was used (***P < 0.001). As Figure 6 The experimental results shown indicate that M14P1 treatment can induce dose-dependent apoptosis in a human AML model.

[0049] Example 6: Detection of cell cycle

[0050] After the cells treated with R8 and M14P1 peptides were collected by centrifugation, they were resuspended in 75% ethanol and fixed at 4 °C for 12 hours. They were stained with propidium iodide (PI) and detected by flow cytometry. FlowJo software was used to analyze the distribution of cell cycle phases (G1, S, G2).

[0051] Figure 7 Shows the effects of R8 and M14P1 peptide treatments on the cell cycle distribution of MOLM-13 cells; Figure 8 Shows the effects of R8 and M14P1 peptide treatments on the cell cycle distribution of NOMO-1 cells; Figure 7 and Figure 8 The data in are expressed as mean ± s.e.m. (n = 3) and analyzed using Two-tailed Student’s t-test (***P < 0.001). Figure 7 and Figure 8 The results of and show that the proportion of S-phase cells in the MOLM-13 and NOMO-1 cell lines was significantly lower than that in the control group, indicating that M14P1 can inhibit the proliferation activity of AML cells.

[0052] Example 7: Detection of the expression levels of AML markers by Western blotting

[0053] Cells were collected by centrifugation, 2×SDS sample buffer was added and mixed well, and then boiled at 100 °C for 10 minutes. Western blotting was used to detect proteins.

[0054] The experimental results are as Figure 9 shown. M14P1 treatment significantly reduced the expression levels of AML-related proteins SP1, MYC, and BRD4, and the transcripts of these genes have been confirmed as enzyme substrates of METTL3 / 14.

[0055] The results of the above examples show that the polypeptide inhibitor M14P1 targeting the RNA methyltransferase dimer was developed in this invention, successfully inducing dose-dependent apoptosis and inhibiting the proliferation of acute myeloid leukemia (AML) cells, and showing significant anti-tumor activity. This discovery not only provides an important research direction for leukemia treatment, but also provides ideas for the development of new drugs based on the METTL3-METTL14 heterodimer.

[0056] The above examples are the preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the described embodiments. Any improvement, deformation, substitution, combination, or simplification carried out within the spirit and principle of the present invention shall be regarded as a substitution method substantially the same as the present invention and fall within the protection scope of the present invention.

Claims

1. A polypeptide inhibitor targeting an RNA methyltransferase dimer, characterized in that, The amino acid sequence of the polypeptide inhibitor comprises the sequence shown in SEQ ID NO.

1.

2. A composite preparation comprising the polypeptide inhibitor targeting the RNA methyltransferase dimer described in claim 1.

3. Use of the polypeptide inhibitor described in claim 1 or the composite preparation described in claim 3 in the preparation of a medicament for treating or preventing a disease caused by high expression of METTL3-METTL14.

4. Use of the polypeptide inhibitor described in claim 1 or the composite preparation described in claim 3 in the preparation of a medicament for treating or preventing leukemia.

5. The application according to claim 4, characterized in that, The use concentration of the polypeptide M14P1 is 10-20 μM.

6. The application according to claim 4, characterized in that, The leukemia includes acute leukemia, chronic leukemia, hairy cell leukemia, and prolymphocytic leukemia.

7. The application according to claim 6, characterized in that, The chronic leukemia includes chronic lymphocytic leukemia and chronic myeloid leukemia.

8. The application according to claim 6, characterized in that, The acute leukemia includes acute lymphoblastic leukemia L1, acute lymphoblastic leukemia L2, acute lymphoblastic leukemia L3, and acute myeloid leukemia.

9. The application according to claim 8, characterized in that The acute myeloid leukemia includes granulocytic leukemia, erythroleukemia, and megakaryocytic leukemia.

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