Polypeptide inhibitors targeting rna methyltransferase dimers and uses thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses a polypeptide inhibitor targeting RNA methyltransferase dimer and application thereof. The polypeptide inhibitor provided by the application targets a binding site required for METTL3-METTL14 heterodimer assembly. The polypeptide inhibitor of the application effectively inhibits the formation of the heterodimer and its functional activity, for example, can significantly reduce the total m 6 A modification level in acute myeloid leukemia (AML) cells, thereby inhibiting cell proliferation and inducing apoptosis. Experimental results show that the polypeptide inhibitor of the application exhibits good antitumor activity in AML cell lines and can significantly reduce the marker protein level related to AML.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polypeptide inhibitor targeting RNA methyltransferase dimers and its application. Background Technology

[0002] N 6 -Methyladenosine (N) 6 -methyladenosine,m 6 A) is the most abundant form of chemical modification in mammalian messenger RNA (mRNA). 6 A modification is dynamically regulated by methyltransferases and demethylases, with methylation reading proteins specifically recognizing and binding to m. 6 Site A mediates its biological function. Among them, m 6 The A-methyltransferase complex is a heterodimer composed of the catalytic subunit METTL3 and the substrate recognition subunit METTL14, and its functional stability is maintained by auxiliary components such as WTAP and ZC3H13.

[0003] High expression of METTL3 and METTL14 is associated with a variety of diseases. For example, in recent years, numerous studies have shown that m 6 Abnormal 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 acute leukemia in adults. It is characterized by uncontrolled proliferation, impaired differentiation, and arrested apoptosis of leukemia cells in the bone marrow, often accompanied by clinical manifestations such as anemia, bleeding, infection, and fever. Currently, commonly used treatments include chemotherapy and hematopoietic stem cell transplantation. While these have improved patient survival rates to some extent, problems such as high drug resistance and significant side effects remain. Therefore, developing novel drug therapeutic targets is of great significance. Notably, METTL3 and METTL14 are significantly overexpressed in AML patients. These two components not only drive leukemia development and progression by post-transcriptionally regulating the expression of key oncogenes such as MYC and MYB, but also influence disease progression by maintaining the self-renewal capacity of leukemia stem cells. Furthermore, studies have confirmed that these two components play crucial roles in the development of AML and the regulation of chemotherapy drug response, suggesting they may be potential targets for AML treatment.

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

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

[0006] Firstly, the present invention aims to provide a polypeptide inhibitor targeting RNA methyltransferase dimers. The polypeptide inhibitor is a synthetically produced polypeptide, denoted in this invention as polypeptide M14P1. The amino acid sequence of polypeptide M14P1 comprises the sequence shown in SEQ ID NO.1 of the sequence listing. Preferably, the amino acid sequence of polypeptide M14P1 is the sequence shown in SEQ ID NO.1. Specifically, the C-terminal sequence of the sequence shown in SEQ ID NO.1 from the N-terminus is as follows:

[0007] RRRRRRRRGGGLDLGRVCLRKWGYRRCEDI.

[0008] The crystal structure of the METTL3-METTL14 heterodimer reveals two key interaction interfaces. Interface 1 consists of residues 238-258 of the METTL14 protein, exhibiting an α-helix structure; interface 2 spans residues 278-312, forming a β-sheet structure. Based on interface 1, the inventors of this application designed the aforementioned peptide inhibitor M14P1, which can inhibit the formation of this dimer complex. Specifically, by targeting the binding sites on the METTL14 protein related to the assembly of the METTL3 complex, the peptide M14P1 was designed based on the α-helix structure of METTL14. To enhance cell penetration, an R8 cell-penetrating peptide was fused to the N-terminus of the peptide.

[0009] Secondly, the present invention aims to provide the use of the peptide inhibitor targeting the RNA methyltransferase dimer and a compound formulation containing the peptide inhibitor in the preparation of drugs for treating or preventing diseases caused by high expression of METTL3-METTL14. The peptide M14P1 exhibits significant anti-leukemic activity by targeting and interfering with the translational assembly of the METTL3-METTL14 heterodimer and its substrate expression levels associated with AML, for example, by significantly inhibiting the proliferation and survival of leukemia cells.

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

[0011] Furthermore, the acute leukemia includes L1 type acute lymphoblastic leukemia, L2 type acute lymphoblastic leukemia, L3 type 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 concentration of the polypeptide M14P1 used is 10-20 μM.

[0015] As used in this invention, the term "prevention" refers to the prophylactic administration of the polypeptide to healthy patients to prevent the occurrence of the diseases and conditions described herein. Furthermore, the term "prevention" can also refer to the prophylactic administration of the polypeptide to patients in the pre-treatment stage of a disease to be treated. This prophylactic administration specifically refers to high-risk individuals with the METTL3 / 14 overexpression biomarker.

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

[0017] This invention has been experimentally verified to show that treating cells with high expression of METTL3 and METTL14 using the peptide M14P1 can inhibit the interaction between the RNA methyltransferase dimer subunits METTL3 and METTL14, for example, significantly reducing the m-expression of poly(A)RNA in AML cells. 6 Level A; In AML cell lines MOLM-13 and NOMO-1, treatment with peptide M14P1 significantly inhibited cell proliferation and led to a substantial decrease in the levels of some AML marker proteins. Unlike traditional small-molecule competitive inhibitors, M14P1 interferes with METTL3 and METTL14 assembly by targeting an interaction interface. This target differs from the target of existing inhibitors, such as FLT3 and IDH1 inhibitors, and even drugs targeting RNA methyltransferase complexes like STM2457, whose interference targets are different from the specific interaction interface of peptide M14P1 provided in this application. Therefore, peptide M14P1 provided in this application has a unique mechanism for inhibiting METTL3-METTL14 heterodimer activity. Attached Figure Description

[0018] To more clearly illustrate the technical solution of the present invention, the accompanying 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 Representative laser confocal microscopy images, scale bar 10 μm;

[0020] Figure 2The results of Western blotting using anti-METTL3 antibody for immunoprecipitation.

[0021] Figure 3 The results of Western blotting using anti-METTL14 antibody for immunoprecipitation.

[0022] Figure 4 mRNA m in MOLM-13 cells treated with 15 μM M14P1 for 4 hours 6 A-level dot blots were used as a control, stained with methylene blue.

[0023] Figure 5 The dose-response curves of M14P1 treatment in MOLM-13 and NOMO-1 cells are shown.

[0024] Figure 6 The percentage of apoptosis in MOLM-13 and NOMO-1 cells after treatment with a specified concentration of M14P1;

[0025] Figure 7 The effects of R8 and M14P1 peptide treatment on cell cycle distribution in MOLM-13 cells;

[0026] Figure 8 The effects of R8 and M14P1 peptide treatment on NOMO-1 cell cycle distribution;

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

[0028] The following is a detailed description of specific embodiments of the present invention. The provided embodiments are for clarification only and do not limit the scope of the invention. These embodiments can serve as a reference for further improvements by those skilled in the art and do not constitute a limitation on the present invention in any way.

[0029] Unless otherwise specified in the examples, the techniques or conditions described in the literature in this field shall be followed, for example, by referring to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press, or by following the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0030] All peptides in the embodiments of this invention were synthesized by Nanjing Genscript Biotech Co., Ltd.

[0031] MOLM-13 and NOMO-1 were purchased from Wuhan Punosei Life Science Technology Co., Ltd.

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

[0033] The following examples use acute myeloid leukemia cell lines as an example to verify the inhibitory effect of the peptides provided by the present invention, but this does not mean that the peptides 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 the M14P1 peptide with an N-terminus FITC label for 4 hours, followed by PBS washing and nuclear staining with Hoechst. Images were acquired using a laser confocal microscope in the FITC channel, DAPI channel, and bright field.

[0036] Experimental results are as follows Figure 1 As shown, the distribution characteristics of FITC-labeled M14P1 in MOLM-13 and NOMO-1 cells are displayed, and the nuclei exhibit blue fluorescence after Hoechst counterstaining. Figure 1 The scale bar is 10 μm; it can be seen that M14P1 in MOLM-13 and NOMO-1 cells exhibits cytoplasmic localization characteristics, confirming that it successfully transmembrane and accumulates in the cytoplasm.

[0037] Example 2: Protein immunoprecipitation detection of METTL3-METTL14 interaction

[0038] Cells were collected and lysed on ice for 30 minutes using polyribosome lysis buffer. Insoluble debris was then removed by centrifugation at 13,000 rpm for 10 minutes, and the supernatant was collected. The supernatant was gently incubated with the target antibody overnight at 4°C using a rotating incubator. The next day, protein A / G magnetic beads were added, and incubation continued at 4°C for 2–4 hours. The magnetic beads were then thoroughly washed with high-salt buffer to remove non-specific bindings. The magnetic beads were transferred to 2×SDS sample buffer and boiled at 100°C for 10 minutes to elute the bound proteins. Proteins were detected by Western blotting as follows: equal volumes of protein samples were separated by SDS-PAGE, and the proteins were then transferred to a PVDF membrane. The PVDF membrane was blocked for 1 hour at room temperature using TBST solution containing 5% skim milk. After blocking, the membrane was incubated with the primary antibody overnight at 4°C. The next day, the membrane was washed, and 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] Experimental results are as follows Figure 2 and Figure 3As shown, the results of immunoprecipitation using anti-METTL3 antibody and anti-METTL14 antibody are presented respectively, with R8 peptide serving as a negative control; the results show that the interaction between METTL3 and METTL14 is significantly weakened when M14P1 peptide is added.

[0040] Example 3: m 6 A dot blot method for detecting poly(A)RNA m 6 A

[0041] Total RNA was extracted from MOLM-13 cells using Trizol reagent, and poly(A) RNA was enriched using oligo-dT magnetic beads to remove ribosomal RNA (rRNA). The isolated poly(A) RNA was then transferred to a positively charged nylon membrane, and an RNA concentration gradient was constructed (500 ng, 250 ng, and 125 ng). After air-drying, the membrane was fixed using a UV crosslinker (256 nm wavelength) at 0.3 J to ensure stable RNA binding. Subsequently, the membrane was incubated in PBST solution (containing 0.5% Tween-20 phosphate buffer) with 5% skim milk at room temperature for 1 hour on a shaker at 50 rpm to complete blocking. After blocking, the membrane was then incubated with m... 6 A specific primary antibody was incubated 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 shaking conditions. Finally, the signal was detected using a chemiluminescence imaging system for visual analysis.

[0042] like Figure 4 The image shows a dot blot of m6A levels of poly(A)RNA in MOLM-13 cells treated with 15 μM M14P1 for 4 hours. Methylene blue staining was used as a control. The results show that M14P1 treatment significantly reduced the m6A level of poly(A)RNA in MOLM-13 cells. 6 A modifies the level.

[0043] Example 4: Cell viability assay

[0044] MOLM-13 and NOMO-1 cells were seeded at a density of 10,000 cells per well in 96-well plates and cultured overnight at 37°C in a 5% CO2 incubator. Then, working solutions of different concentration gradients of R8 peptide and M14P1 stock solutions were prepared: 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM, 64 μM, and 128 μM, and added to the cell culture wells for incubation 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 450 nm were recorded using a microplate reader. Background noise was corrected by subtracting the absorbance value of the negative control group, and cell viability was calculated using the formula: Cell viability (%) = (Absorbance of peptide-treated group / Absorbance of untreated control group) × 100%. Finally, cytotoxicity curves were fitted using a nonlinear regression model with GraphPad Prism 8.0.

[0045] Figure 5 Dose-response curves for M14P1 treatment in MOLM-13 and NOMO-1 cells are shown. Data are expressed as mean ± sem (n = 3), and the half-maximal inhibitory concentration (IC50) of M14P1 in each cell line is displayed. Figure 5 The experimental results showed that the half-maximal inhibitory concentrations (IC50) of M14P1 against MOLM-13 and NOMO-1 cells were 15.53 μM and 19.63 μM, respectively, indicating that M14P1 has significant anti-tumor proliferation activity.

[0046] Example 5: Apoptosis Detection

[0047] MOLM-13 and NOMO-1 cells were collected and washed with PBS to remove serum interference. Subsequently, cells were treated with R8 peptide (5 μM) and M14P1 peptide (5 μM and 15 μM), respectively, in DMEM medium for 3 hours. After treatment, apoptosis levels were detected using the Annexin V-FITC / PI apoptosis detection kit, and data were analyzed using FlowJo software.

[0048] Figure 6 Percentage of apoptosis in MOLM-13 and NOMO-1 cells after treatment with a specified concentration of M14P1. Data are expressed as mean ± sem (n = 3) and analyzed using a two-tailed Student's t-test (***P < 0.001). Figure 6 The experimental results shown indicate that M14P1 treatment can induce dose-dependent apoptosis in a human AML model.

[0049] Example 6: Cell cycle detection

[0050] Cells treated with R8 and M14P1 peptides were collected by centrifugation, resuspended in 75% ethanol, and fixed at 4°C for 12 hours. Cells were stained with propidium iodide (PI) and analyzed by flow cytometry. The distribution of cell cycle phases (G1, S, G2) was analyzed using FlowJo software.

[0051] Figure 7 The effects of R8 and M14P1 peptide treatment on cell cycle distribution in MOLM-13 cells; Figure 8 The effects of R8 and M14P1 peptide treatment on NOMO-1 cell cycle distribution; Figure 7 and Figure 8 All data are expressed as mean ± sem (n = 3) and are analyzed using the Two-tailed Student's t-test (***P < 0.001). Figure 7 and Figure 8 The results showed 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: Western blot analysis for detecting AML biomarker expression levels

[0053] Cells were collected by centrifugation, and 2×SDS sample buffer was added and mixed thoroughly. The mixture was then boiled at 100°C for 10 minutes. Proteins were detected using Western blotting.

[0054] Experimental results are as follows Figure 9 As shown, M14P1 treatment significantly reduced the expression levels of AML-related proteins SP1, MYC, and BRD4, whose transcripts have been confirmed as substrates for METTL3 / 14.

[0055] The results of the above embodiments demonstrate that the present invention has developed a peptide inhibitor M14P1 targeting RNA methyltransferase dimers, successfully inducing dose-dependent apoptosis and inhibiting proliferation in acute myeloid leukemia (AML) cells, exhibiting significant antitumor activity. This discovery not only provides an important research direction for leukemia treatment but also offers insights for the development of novel drugs based on METTL3-METTL14 heterodimers.

[0056] The above embodiments are preferred embodiments of the present invention, but the scope of protection of the present invention is not limited to the described embodiments. Any improvements, modifications, substitutions, combinations, or simplifications made within the spirit and principle of the present invention are considered to be the same as the present invention in substance and fall within the scope of protection of the present invention.

Claims

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

1.

2. A complex formulation comprising the polypeptide inhibitor targeting RNA methyltransferase dimers as described in claim 1.

3. The use of the polypeptide inhibitor of claim 1 or the compound formulation of claim 2 in the preparation of a medicament for treating acute myeloid leukemia.

4. The application according to claim 3, characterized in that, The acute myeloid leukemia mentioned includes granulocytic leukemia, erythroleukemia, and megakaryocytic leukemia.

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