A polypeptide targeting mitochondrial ribonuclease P and its applications

Abstract

This invention discloses a polypeptide targeting mitochondrial ribonuclease P and its application in the preparation of antitumor drugs. Based on the principle of the inhibition of mtRNA P by the endogenous natural peptide AC027045.3, this invention designs a novel polypeptide based on the amino acid sequence of the binding functional domain of AC027045.3. This polypeptide is designed based on the natural polypeptide sequence and functions by mimicking the endogenous physiological regulatory pathway. Compared to the natural peptide, this novel polypeptide has a shorter sequence, which is beneficial for chemical synthesis. Given the high energy dependence of actively proliferating tumor cells, this invention uses mtRNA P as a novel target for tumor therapy, designing a targeting peptide to inhibit the mitochondrial oxidative phosphorylation process and exert an anti-cancer effect.

Description

(I) Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to PMHR peptide drugs for targeting mitochondrial energy metabolism to achieve tumor treatment. (II) Background Technology

[0002] According to the annual report of the China Cancer Registry, approximately 4.064 million new cancer cases are diagnosed annually in China, and the overall crude cancer incidence rate continues to rise, reflecting the heavy actual burden of cancer in my country. Through concerted efforts, my country's 5-year cancer survival rate rose from 40.5% in 2015 to 43.7% in 2022, achieving the phased goals of the Healthy China Initiative as scheduled. However, there is still a significant gap compared to developed countries. The main reason is the difference between the cancer spectrum in my country and that in developed countries, with a higher incidence of digestive system cancers with poor prognoses, such as liver cancer, stomach cancer, and esophageal cancer. Cancer treatment methods, in addition to traditional surgical resection, radiotherapy, and chemotherapy, include immunotherapy and targeted therapy. Targeted therapy is a drug therapy that inhibits cancer cell growth by interfering with specific molecules required for cancerous changes or tumor proliferation. Compared to traditional chemotherapy, which interferes with all continuously dividing cells, it has a more specific effect and less toxicity to normal cells. Currently, numerous targeted therapies are used clinically, such as osimertinib and bevacizumab targeting the EGFR signaling pathway, trastuzumab and T-DM1 targeting Her-2, and cetuximab targeting the Ras signaling pathway. These drugs have been widely used in first- and second-line treatment of various cancers, including lung cancer, colorectal cancer, and liver cancer, with significant success. However, the development of targeted cancer therapies remains challenging, with a high failure rate. Natural resistance due to patient gene mutations and acquired resistance due to bypass activation further limit the efficacy of targeted therapies. Therefore, discovering more potential targets for cancer treatment is crucial for the development of targeted cancer drugs.

[0003] Mitochondrial ribonuclease P (mtRnase P) is a ternary protein complex in the mitochondrial matrix that cleaves mitochondrial precursor RNA and plays a crucial role in mitochondrial RNA processing. Since all mt-mRNAs encode subunits of the respiratory chain complex, dysfunction of mtRnase P leads to impaired mitochondrial RNA maturation and translation, and may further result in various oxidative phosphorylation (OXPHOS) deficiencies. The mitochondrial OXPHOS pathway plays a vital role in liver cancer; liver cancer cells can upregulate OXPHOS to produce ATP and ROS, promoting tumor growth, drug resistance, and metastasis. Therefore, targeting mitochondrial function is one of the important strategies for liver cancer treatment.

[0004] Micropeptides, as an emerging research subject in biology and clinical medicine, have been proven to play important roles in various cellular life activities such as energy metabolism and signal transduction, and exert important biological functions in the regulation of cellular homeostasis. However, as novel biological functional molecules, the functions of micropeptides in tumor regulation have not yet been fully elucidated, and their applications in the field of cancer therapy remain to be explored. Therefore, identifying novel endogenous micropeptides and their functional sequences, and developing micropeptide drugs, will provide new perspectives and strategies for cancer treatment. (III) Summary of the Invention

[0005] The purpose of this invention is to provide a polypeptide targeting mitochondrial ribonuclease P and its application. This invention is based on the polypeptide PMHR, which is an interaction domain between human endogenous expressed micropeptide AC027045.3 (chr17:9808841_9809020_180) and the HSD17B10 subunit of mtRNA P. It can inhibit tumor proliferation by inhibiting mitochondrial energy metabolism in tumor cells, and thus be used for targeted tumor therapy.

[0006] The technical solution adopted in this invention is:

[0007] This invention provides a polypeptide that targets mitochondrial ribonuclease P, the polypeptide comprising one or more of the following:

[0008] (a) A polypeptide with the amino acid sequence LEPRRMALVSIRTISRTSPP (SEQ ID NO.1);

[0009] (b) A polypeptide with more than 90% identity to the amino acid sequence shown in (a);

[0010] (c) A polypeptide comprising one or more amino acid sequences, including the amino acid sequence shown in (a), through substitution, deletion, or insertion of one or more amino acids.

[0011] (d) A polypeptide comprising one or more amino acid sequences modified by acetylation, phosphorylation, glycosylation, succinylation, or ubiquitination of the amino acid sequence shown in (a).

[0012] Preferably, the amino acid sequence of the polypeptide is as shown in LEPRRMALVSIRTISRTSPP.

[0013] This invention also provides the application of the aforementioned polypeptide in the preparation of antitumor drugs. The tumor is selected from: lung cancer (such as non-small cell lung cancer), melanoma, breast cancer, ovarian cancer, prostate cancer, liver cancer, kidney cancer, colorectal cancer, head and neck cancer, skin cancer, bladder cancer, and pancreatic cancer.

[0014] Preferably, the formulation of the drug includes: solution, emulsion, and suspension.

[0015] The inhibitors described in this invention, or drugs containing said inhibitors, can be used in combination with known chemotherapy or targeted therapies in the art. Such combinations include: simultaneous, sequential, separate, or individual administration of the substances or products of this invention, as well as other known drugs or therapies.

[0016] Compared with existing technologies, the beneficial effects of this invention are mainly reflected in the following aspects: This invention is based on the principle of the binding and inhibition of mtRNA P by the endogenous natural micropeptide AC027045.3. A novel polypeptide is designed based on the amino acid sequence of the AC027045.3 binding functional domain. This polypeptide is designed based on the natural polypeptide sequence and functions by mimicking endogenous physiological regulatory pathways. Compared with the natural micropeptide, this novel polypeptide sequence is shorter, which is beneficial for chemical synthesis. Based on the high energy dependence of actively proliferating tumor cells, this invention uses mtRNA P as a novel target for tumor therapy, designing a targeting peptide to inhibit the mitochondrial oxidative phosphorylation process and exert an anti-cancer effect. (iv) Description of the attached drawings

[0017] Figure 1 The bar chart shows the tRNA expression levels of the Huh7 cell line overexpressing AC027045.3 and the control in Example 1.

[0018] Figure 2 The bar chart shows the ATP content of the Huh7 cell line overexpressing AC027045.3 and the control in Example 1.

[0019] Figure 3 This is the OD570 curve of the Huh7 cell line overexpressing AC027045.3 and the control in Example 1 as a function of time.

[0020] Figure 4 This is a schematic diagram and gel image of the amino acid sequence and the mtRnase P functional region regulated by AC027045.3 in Example 2.

[0021] Figure 5 This is a schematic diagram of the amino acid sequence of the polypeptide PMHR-FITC in Example 2.

[0022] Figure 6 This is a confocal fluorescence micrograph of PMHR-FITC entering tumor cells in Example 2.

[0023] Figure 7 This is a bar graph showing the tRNA expression level of liver cancer cells after treatment with PMHR-FITC in Example 2.

[0024] Figure 8 This is a graph showing the change in mitochondrial oxygen consumption rate after PMHR was applied to liver cancer cells in Example 3.

[0025] Figure 9This is a graph showing the change in cell proliferation rate after PMHR was applied to liver cancer cells in Example 3.

[0026] Figure 10 This is a liver image and a statistical chart showing the number and size of tumor lesions from the PMHR in vivo experiment in Example 4. (V) Detailed Implementation

[0027] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0028] Example 1: Functional Validation at the Cell Level using AC027045.3

[0029] (1) The human endogenous expression micropeptide AC027045.3 (chr17:9808841_9809020_180) sequence (amino acid sequence as shown in SEQ ID NO.2) was cloned in the plvx-M-Puro vector using a non-ligase-dependent single-fragment one-step cloning kit (Vazyme, C112-01) (plasmid#125839, https: / / www.addgene.org / ), and an S-protein, Flag, and biotin (SFB) tag was fused to the C-terminus to obtain a eukaryotic overexpression vector.

[0030] (2) The human liver cancer cell line Huh7 was purchased from the American Standard Cell Culture Center (ATCC) and cultured in DMEM / F12 medium (Gibco) containing 10% FBS (Bovogen) at 37°C in a 5% CO2 incubator. The MycoAlert kit (Lonza) showed that the cells were free of mycoplasma contamination and cross-contamination.

[0031] (3) Using the eukaryotic overexpression vector prepared in step (1) above, transfect Huh7 cells from step (2) according to the Lipofectamine 2000 (Thermo Scientific) reagent instructions. After screening with puromycin for 48 h, construct the AC027045.3 overexpressing human hepatocellular carcinoma cell line Huh7. Using a non-ligase-dependent single-fragment one-step cloning kit (Vazyme, C112-01), clone the SFB tag into the plvx-M-Puro vector, transfect Huh7 cells from step (2), and construct the human hepatocellular carcinoma cell line Huh7 containing the empty vector, denoted as EV, as a control.

[0032] (4) The activity of mtRnaseP in overexpressing human hepatocellular carcinoma cell lines Huh7 and EV was detected by RT-qPCR using the BioRad 1725122 quantitative kit (primers 5'-AGAGGAGACAAGTCGTAACATGG-3' and 5'-GGTGCTTTGTGTTAAGCTACACTCTGG-3') in step (3) AC027045.3. Figure 1 AC027045.3 showed that it inhibited mtRNA P, leading to an increase in intracellular precursor tRNA levels.

[0033] (5) The ATP content of the human liver cancer cell lines Huh7 and EV overexpressing in step (3) AC027045.3 was detected using an ATP assay kit (beyotime). Figure 2 The results showed that ATP synthesis in Huh7 cells was inhibited by AC027045.3, indicating that AC027045.3 inhibits mitochondrial function.

[0034] (6) The proliferation curves of EV and AC027045.3 overexpression cell lines were plotted using the MTT assay. Figure 3 The results showed that the proliferation rate of Huh7 cell lines overexpressing AC027045.3 was slowed, indicating that AC027045.3 exerts an inhibitory effect on tumor proliferation.

[0035] Example 2: Design of the peptide PMHR, its entry into cells, and its effect on mtRNA P function.

[0036] 1. Design a peptide PMHR to inhibit mtRNA P based on the functional domain of AC027045.3.

[0037] Given the important regulatory role of AC027045.3 in the activity of mtRNA-P, we truncated a portion of the amino acid sequence in AC027045.3 (amino acid sequence as shown in SEQ ID NO.2, denoted as fragment FL) to obtain fragments D1 (amino acid sequence of amino acids 22-60 of SEQ ID NO.2), D2 (amino acid sequence of amino acids 1-21 and 42-60 of SEQ ID NO.2), D3 (amino acid sequence of amino acids 1-41 of SEQ ID NO.2), L1 (amino acid sequence of amino acids 1-21 of SEQ ID NO.2), L2 (amino acid sequence of amino acids 22-41 of SEQ ID NO.2), and L3 (amino acid sequence of amino acids 42-60 of SEQ ID NO.2).

[0038] Each of the above fragments was cloned into the plvx-M-Puro vector (plasmid#125839, https: / / www.addgene.org / ) using a non-ligase-dependent single-fragment one-step cloning kit (Vazyme, C112-01), and an SFB tag was fused to the C-terminus. The truncated versions were collectively referred to as SFB-△AC027045.3.

[0039] The HSD17B10 protein (amino acid sequence as shown in SEQ ID NO.3) was cloned into the plvx-M-Puro vector using the same method, and a GFP tag was fused to the C-terminus, denoted as GFP-HSD17B10.

[0040] Human kidney epithelial cell line HEK293T was purchased from the American Academy of Cell Culture (ATCC). HEK293T cells were seeded into DMEM / F12 (Gibco) medium containing 10% FBS (Bovogen) and cultured at 37°C. The cells were divided into 8 groups, with 2 × 10⁶ cells per group. 6 Each cell was transfected. Group 1 was transfected with only the GFP-HSD17B10 plasmid as a negative control. Groups 2-8 were co-transfected with each of the SFB-△AC027045.3 plasmids and the GFP-HSD17B10 plasmid, respectively. Immunoprecipitation was performed after 48 hours, and the interaction was detected by protein gel electrophoresis. Figure 4 The displayed fragment L2 is the AC027045.3-HSD17B10 protein-protein interaction functional region.

[0041] Therefore, a short peptide PMHR (amino acid sequence as shown in SEQ ID NO.1) was constructed based on the AC027045.3-L2 sequence and modified with the fluorescent group FITC for intracellular tracking, denoted as peptide PMHR-FITC. Figure 5 The above design aims to synthesize a polypeptide that mimics the physiological function of AC027045.3 by identifying the functional region where AC027045.3 binds to the mtRnase P subunit HSD17B10.

[0042] 2. Demonstrates that the peptide PMHR can enter tumor cells.

[0043] HepG2 human liver cancer cells were purchased from the American Standard Cell Culture Center (ATCC). HepG2 cells were seeded in DMEM / F12 (Gibco) medium containing 10% FBS (Bovogen) and cultured at 37°C until 40% confluence. Then, 10 μM of the peptide PMHR-FITC synthesized in vitro in step 1 was added, and the cells were cultured at 37°C for another 12 hours. The cells were then washed with PBS to remove the PTPR peptide from the medium, and fluorescent slides were prepared. Confocal fluorescence microscopy was used to detect the entry of PMHR-FITC into the cells. Figure 6 This indicates that PMHR can directly enter cells.

[0044] 3. Demonstrates that PMHR can enter tumor cells and inhibit mtRNA P activity.

[0045] HepG2 human liver cancer cells were seeded into DMEM / F12 (Gibco) medium containing 10% FBS (Bovogen) and cultured at 37°C. The cells were divided into two groups, with 10 cells per group. 6 One group of cells was incubated with 10 μM of the peptide PMHR-FITC synthesized in vitro in step 1, while the other group was incubated with an equal volume of dimethyl sulfoxide as a control. Both groups were incubated at 37°C for 12 hours, and mtRnase P activity was detected using RT-qPCR following the steps described in Example 1. Figure 7 The results showed that the level of precursor tRNA in HepG2 human liver cancer cells was increased in the PMHR treatment group, reflecting a decrease in mtRnase P activity; while dimethyl sulfoxide, used as a solvent control, did not have this function, indicating that the peptide PMHR can inhibit mtRnase P function.

[0046] SEQ ID NO.2:

[0047] MKMATKASTLSQWNPGLRQLTLEPRRMALVSIRTISRTSPPMDSHKTPAKERDVGNAGHF.

[0048] SEQ ID NO.3:

[0049] MAAACRSVKGLVAVITGGASGLGLATAERLVGQGASAVLLDLPNSGGEAQAKKLGNNCVFAPADVTSEKDVQTALALAKGKFGRVDVAVNCAGIAVASKTYNLKKGQTHTLEDFQRVLDVNLMGTFNVIRL VAGEMGQNEPDQGGQRGVIINTASVAAFEGQVGQAAYSASKGGIVGMTLPIARDLAPIGIRVMTIAPGLFGPLLTSLPEKVCNFLASQVPFPSRLGDPAEYAHLVQAIIENPFLNGEVIRLDGAIRMQP.

[0050] Example 3: The polypeptide PMHR inhibits mitochondrial energy metabolism, thereby inhibiting tumor cell proliferation.

[0051] mtRnase P plays an important role in regulating mitochondrial energy metabolism, therefore PMHR can inhibit mitochondrial energy production by inhibiting mtRnase P activity.

[0052] 1. Effect of peptide PMHR on mitochondrial oxygen consumption rate in HepG2 cells

[0053] Mitochondrial oxygen consumption was measured using the Seahorse XF Cell Mitochondrial Stress Assay Kit (Agilent). HepG2 human liver cancer cells were seeded into 96-well culture plates provided with the kit and cultured in DMEM / F12 medium (Gibco) containing 10% FBS (Bovogen) at 37°C, maintaining a cell count of 2 × 10⁶ cells per well. 4 One well was filled with 10 μM of the peptide PMHR-FITC synthesized in Example 2, and the other well was filled with an equal volume of dimethyl sulfoxide as a control. The 96-well plate was then incubated at 37°C for 12 hours. Subsequent experimental procedures were performed according to the kit instructions to obtain mitochondrial oxygen consumption data. Figure 8 The PMHR treatment group showed a decrease in mitochondrial oxygen consumption in HepG2 cells, indicating a weakening of mitochondrial energy production.

[0054] 2. Effect of Peptide PMHR Concentration on HepG2 Cell Proliferation Rate

[0055] HepG2 human liver cancer cells were seeded into 96-well culture plates provided in the kit and cultured in DMEM / F12 medium (Gibco) containing 10% FBS (Bovogen) at 37°C until 70% confluence was achieved. Different concentrations (2, 5, 10, 15, 20, 25, 30, 40, 50 μM) of the peptide PMHR-FITC synthesized in Example 2 were added and incubated at 37°C for 12 hours. Cell proliferation was detected using an MTT assay kit (Yeasen, 40201ES76), and a concentration-dependent inhibitory effect on proliferation was observed. Figure 9 This indicates that PMHR inhibits tumor cell proliferation by suppressing energy production in the mitochondrial respiratory chain.

[0056] Example 4: Validation of the function of the PMHR mouse tumor model

[0057] 1. Establishment of a mouse tumor model

[0058] Twelve 5-week-old male C57BL / 6J mice were purchased. 300 μg each of PB-NRAS-DS plasmid and PB-MYC-DS plasmid were mixed with 120 μg of CAG-PB transposase plasmid (PMID: 16096065), diluted with PBS to 24 ml, and injected via tail vein hyperbaric injection into each mouse, 2 ml per mouse. This spontaneous tumorigenesis mouse model was used to verify the therapeutic effect of PMHR on liver cancer.

[0059] 2. Grouping and medication methods

[0060] A DMSO solution with a PMHR concentration of 1 g / ml was prepared as a stock solution and diluted with PBS to the injection concentration. Mice were randomly divided into two groups of six each. Starting from day 10, the drugs were administered intraperitoneally every four days for a total of seven times. The PMHR group received 100 μl of 10 mg / ml PMHR solution per injection, while the DMSO group received 100 μl of an equal volume of the PMHR stock solution in DMSO solution per injection, serving as a control. Forty days after injection, all mice were sacrificed, and liver morphology was observed, paraffin sections were prepared, and H&E staining was performed.

[0061] 3. Experimental Results

[0062] Figure 10 The statistical results of the number and size of liver tumor lesions in mice in the DMSO group and PMHR group at 40 days are presented, showing that intraperitoneal injection of PMHR can significantly inhibit tumor growth.

Claims

1. A polypeptide targeting mitochondrial ribonuclease P, characterized in that, The amino acid sequence of the polypeptide is shown as LEPRRMALVSIRTISRTSPP.

2. The use of the polypeptide of claim 1 in the preparation of a targeted therapy for liver cancer.

3. The application as described in claim 2, characterized in that, The formulation of the drug is selected from: solution, emulsion or suspension.

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