Small molecule oligopeptide from human SMIM45-107aa as well as preparation method and application of small molecule oligopeptide

Abstract

The invention discloses a small molecule short peptide from human SMIM45-107aa as well as a preparation method and application of the small molecule short peptide. The amino acid sequence of the small molecule short peptide is as shown in SEQ ID NO. 1: CRGDKPRGSGLELVR VCGGGMQRDKTVVEE. Experiments prove that the small molecule oligopeptide synthesized by the invention can inhibit liver cancer cell migration and zebra fish tumor proliferation, and provides a new treatment strategy for treating liver cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a small molecule short peptide derived from human SMIM45-107aa, its preparation method, and its application. Background Technology

[0002] Hepatocellular carcinoma (HCC) is a prevalent malignant tumor worldwide with a continuously rising incidence rate. Due to the insidious nature of early symptoms, most patients are diagnosed at an advanced stage, limiting the effectiveness of traditional treatments such as surgical resection, radiofrequency ablation, and hepatic artery chemoembolization. With in-depth research into the molecular signaling pathways and tumor microenvironment of HCC, targeted therapy has become a focus of clinical research in advanced HCC. Since sorafenib was approved by the U.S. Food and Drug Administration (FDA) for the treatment of advanced HCC in 2007, a series of novel targeted drugs have emerged, significantly broadening treatment options and improving survival benefits for patients with advanced disease. For example, lenvatinib primarily targets VEGFR / PDGFR; regorafenib targets VEGFR, FGFR1, and KIT.

[0003] Furthermore, immunotherapy targeting immune checkpoint molecules has made significant progress in the field of liver cancer, such as nivolumab and pembrolozumab targeting PD-L1, and ipilimumab and tremelimumab targeting CTLA-4. However, compared to cancers like lung cancer and gastric cancer, where immunotherapy is more mature, immunotherapy development for HCC remains relatively lagging, and further exploration of new therapeutic targets and combination therapy strategies is urgently needed to improve clinical efficacy.

[0004] Long non-coding RNAs (lncRNAs) are a class of key molecules involved in regulating various biological processes such as cell proliferation, apoptosis, migration, and invasion. Their aberrant expression is closely related to the occurrence, development, and metastasis of tumors. In recent years, with the development of transcriptome sequencing, translatomics, and bioinformatics technologies, the perception that lncRNAs lack coding capabilities has been overturned, revealing that some lncRNAs can encode functional peptides (typically less than 100 amino acids). These peptides are widely involved in physiological processes such as hormone regulation, nerve conduction, and cell growth, playing a crucial role in maintaining homeostasis. Based on their significant biological activity, these peptides show great potential in the diagnosis and treatment of diseases, especially tumors.

[0005] Short peptide drugs, as a novel anti-tumor drug emerging in the post-gene era, have attracted much attention due to their unique advantages. Their main characteristics include: 1) broad indications, significant efficacy, and strong target recognition; 2) low metabolic toxicity and relatively few side effects; 3) good stability, high purity, low immunogenicity, and controllable synthesis costs; 4) moderate molecular weight, easy modification, and short development cycle; and 5) high biological activity and small dosage. These characteristics make them a good complement between small molecule chemical drugs and large molecule biological drugs. Currently, short peptide drugs mainly include hormone analogs, natural short peptide derivatives, short peptide vaccines, and peptide-drug conjugates (PDCs). Studies have shown that, for example, short peptides targeting the SALL4-NURD interaction (such as the SALL4 peptide) combined with sorafenib can significantly reverse sorafenib resistance, demonstrating the value and broad prospects of short peptide drugs in the treatment of HCC.

[0006] LINC00634 is a long non-coding RNA that encodes a polypeptide called SMIM45-107aa. Our study showed that this polypeptide promotes the progression of liver cancer. Currently, no short peptides derived from SMIM45-107aa have been reported. Summary of the Invention

[0007] Currently, targeted drugs for liver cancer, such as everolimus and axitinib, have shown minimal inhibitory effects on liver cancer. The purpose of this invention is to address the shortcomings of existing liver cancer treatments by providing a small molecule short peptide derived from human SMIM45-107aa, its preparation method, and its applications.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] The first aspect is to provide a small molecule short peptide, wherein the small molecule short peptide is a short peptide that has at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% homology with the amino acid sequence shown in SEQ ID No. 1, and has the same or similar function.

[0010] Furthermore, the amino acid sequence of the small molecule short peptide is shown in SEQ ID NO.1: CRGDKPRGSGLELVRVCGGGMQRDKTVVEE, and its molecular weight is 3458.98.

[0011] The second aspect is to provide the application of the aforementioned small molecule short peptides in the preparation of drugs for treating liver cancer.

[0012] Furthermore, the short peptide inhibits the migration of zebrafish liver cancer tumor cells and inhibits liver cancer proliferation.

[0013] The third aspect is to provide a method for preparing the above-mentioned small molecule short peptides, wherein the preparation method is the solid-phase synthesis method of Fmoc.

[0014] The fourth aspect is to provide a drug for treating liver cancer, said drug comprising the aforementioned small molecule short peptides.

[0015] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0016] The short peptides synthesized in this invention have been experimentally proven to inhibit the migration and proliferation of liver cancer cells, providing a new direction for the treatment of liver cancer. Attached Figure Description

[0017] Figure 1 This is an HPLC chromatogram of the small molecule short peptide synthesized in this invention.

[0018] Figure 2 This is a cell scratch map and statistical graph showing the ability of the small molecule short peptide of the present invention to inhibit the migration of SK-Hep1 liver cancer cells.

[0019] Figure 3 This diagram illustrates the inhibitory effect of the small molecule short peptide of this invention on the tumor proliferation ability of zebrafish. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the invention. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.

[0021] Example 1

[0022] LINC00634 is a relatively highly expressed lncRNA in liver cancer that encodes a short peptide of 107 amino acids, SMIM45-107aa. Our study found that SMIM45-107aa plays a role in promoting liver cancer, and this short peptide may bring new opportunities for the treatment of liver cancer.

[0023] Therefore, this invention provides a small molecule short peptide with the amino acid sequence shown in SEQ ID NO.1: CRGDKPRGSGLELVRVCGGGMQRDKTVVEE, and a molecular weight of 3458.98, used to inhibit the migration and proliferation of liver cancer cells. Its control peptide is CRGDK.

[0024] The specific preparation method of the above-mentioned small molecule short peptides is as follows:

[0025] 1) Resin swelling

[0026] Accurately weigh an appropriate amount of 2-CTC resin (determined based on the desired amount of short peptide to be synthesized) and place it in a suitable solid-phase synthesis tube. Add an appropriate amount of DCM to completely submerge the resin and soak for 1 hour, gently shaking the synthesis tube during this time to ensure the resin swells fully. Drain the DCM and wash the resin three times with DMF, adding an appropriate amount of DMF each time, shaking briefly, and then draining.

[0027] 2) The first protective amino acid linker

[0028] Weigh the first Fmoc-protected amino acid and 1.5 eq of DIEA at a ratio of 1 eq, dissolve them in an appropriate amount of DMF, and prepare a reaction solution. Add the reaction solution to a synthesis tube containing swollen resin and react at room temperature for 2 hours. During this time, the reaction can be gently agitated with a mechanical shaker to promote the reaction.

[0029] 3) Head treatment

[0030] Drain the reaction solution and wash the resin three times with DMF. Prepare a mixed solution of methanol and DIEA (the ratio can be adjusted according to the actual situation), add it to the synthesis tube, and seal the tube for reaction for 1 hour.

[0031] 4) Fmoc removal

[0032] After cleaning the resin with DMF, add a 20% DMF solution of piperidine and react for 10 minutes. Then, dry the solution and repeat this operation once. Thoroughly clean the resin with DMF to ensure that piperidine is completely removed.

[0033] 5) The second amino acid is linked.

[0034] Weigh the second amino acid, HOBT, and DIC at a ratio of 3 eq, dissolve them in an appropriate amount of DMF, and prepare a reaction solution. Add the reaction solution to a synthesis tube containing resin and react at room temperature for 1.5 hours with shaking.

[0035] 6) Repeat the removal and connection steps

[0036] Repeat steps 4)-5), sequentially linking amino acid residues according to the short peptide sequence until the last amino acid residue at the N-terminus is added. After the final linking is completed, perform the Fmoc removal operation.

[0037] 7) Biotin linkage

[0038] Weigh biotin, HOBT, and DIC in a ratio of 3 eq, dissolve them in an appropriate amount of DMF, and prepare a reaction solution. Add the reaction solution to a synthesis tube containing resin and react at room temperature for 2 hours, shaking after each reaction.

[0039] 8) Resin cleaning and drying

[0040] Clean the resin several times with DMF and DCM in sequence to ensure that impurities on the resin surface are completely removed. Drain the resin to remove as much residual solvent as possible.

[0041] 9) Cutting reaction

[0042] Prepare the cutting fluid: 95% TFA + 2% Tis + 2% EDT + 1% H2O. Add the cutting fluid to a synthesis tube containing the resin and react for 2 hours, gently shaking during the reaction.

[0043] 10) Collection of coarse products

[0044] Filter the resin using a sintered glass funnel and transfer the filtrate to a centrifuge tube. Under ice bath conditions, slowly add pre-chilled ice-cold ether to the filtrate while stirring to precipitate the short peptides. Centrifuge (e.g., 4000 rpm, 10 minutes) and collect the precipitate, which is the crude product.

[0045] 11) Purification and Desalting

[0046] The crude product was purified using liquid chromatography (such as reversed-phase high-performance liquid chromatography), with the appropriate mobile phase and column selected based on the properties of the short peptide. The target peak was collected. Figure 1 The sample was concentrated and then desalted again using liquid chromatography.

[0047] 12) Freeze-drying

[0048] The desalted sample was transferred to a lyophilization bottle and placed in a lyophilizer for lyophilization to obtain a pure short peptide in the form of a white powder.

[0049] Example 2 uses cell scratch technology to verify the inhibitory effect of short peptides on the migration of liver cancer cells.

[0050] The migration ability of the short peptides prepared in Example 1 on SK-Hep1 liver cancer cells was analyzed by cell scratch assay. The specific steps were as follows: Cells in the logarithmic growth phase were selected, treated with trypsin, digestion was terminated with complete culture medium, and the cells were washed twice with PBS before cell counting. Cells were counted at 5*10n cells per well. 5Cells were seeded into 12-well plates and gently shaken to ensure the cells covered the bottom of the plate. DMEM medium containing 1% FBS was added, and the plates were incubated overnight at 37°C with 5% CO2 until cell adhesion was achieved. The next day, the adherent cells were scored using a 10 μL pipette tip and a ruler. The supernatant was aspirated, and PBS was added to wash away any floating cells. This process was repeated three times. Experimental groups and treatments: Experimental group 1 (overexpression of SMIM45-107aa + short peptide), Control group 1 (overexpression of SMIM45-107aa + control peptide), Experimental group 2 (mutant + short peptide), Control group 2 (mutant + control peptide). Each group was repeated in triplicate. Cell healing was recorded by photographing at 0h, 12h, 24h, and 48h. Finally, the images were analyzed using ImageJ, and the migration and healing efficiency of cells treated with the short peptide was calculated using a formula. Cell migration rate = (0h cell scar area - 48h cell scar area) / 0h cell scar area * 100%.

[0051] The results showed that the short peptide of this invention had almost no effect on the cell migration ability of mutant liver cancer cells, but it significantly inhibited the migration of liver cancer cells overexpressing SMIM45-107aa. Figure 2 ).

[0052] Example 3 uses zebrafish to verify the inhibitory effect of short peptides on cell proliferation.

[0053] SK-Hep1 liver cancer cells were transfected with SMIM45-107aa and labeled with CM-DiI. Labeled cells were microinjected into the yolk sacs of 2-day-fed wild-type AB zebrafish (200 cells / fish) and cultured at 35°C. When the zebrafish reached 3 days of cell growth, zebrafish with good tumor cell uniformity were selected under a microscope and randomly assigned to 6-well plates (30 fish per well). The short peptide and control short peptide were administered intravenously at 40 ng / fish, respectively. After 2 days of treatment at 35°C, 10 zebrafish from each experimental group were randomly selected and photographed under a fluorescence microscope. Data were acquired using NIS-Elements D 3.20 advanced image processing software, and the fluorescence intensity of tumor cells was analyzed. The statistical analysis results of this index were used to evaluate the antitumor efficacy of the samples. Statistical results are expressed as mean ± SE (Table 1). Statistical analysis was performed using SPSS 26.0 software; p < 0.05 indicated statistical significance. The results showed ( Figure 3 The fluorescence intensities of tumor cells in the blank model control group, short peptide group, and control peptide group were 416258±9725, 329227±21918, and 407148±11740, respectively, indicating that the short peptide of the present invention has anti-tumor efficacy.

[0054] Table 1. Statistical analysis of fluorescence intensity of short peptides on tumor proliferation in a zebrafish model.

[0055]

[0056] Compared with the model control group, **p<0.01.

[0057] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the content and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A small molecule short peptide, characterized in that, The short peptide is a short peptide that has at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology with the amino acid sequence shown in SEQ ID No. 1, and has the same or similar function.

2. The small molecule short peptide according to claim 1, characterized in that, The amino acid sequence of the small molecule short peptide is shown in SEQ ID NO.1: CRGDKPRGSGLELVRVCGGGMQRDKTVVEE.

3. The use of the small molecule short peptide according to claim 1 in the preparation of drugs for treating liver cancer.

4. The application according to claim 3, characterized in that, The drug also includes pharmaceutically acceptable carriers or excipients.

5. The application according to claim 3, characterized in that, The short peptides inhibit the migration of liver cancer cells and the proliferation of zebrafish liver cancer tumor cells.

6. The method for preparing the small molecule short peptide or according to claim 1, characterized in that, The preparation method is the solid-phase synthesis of Fmoc.

7. A drug for treating liver cancer, characterized in that, The drug comprises the small molecule short peptide as described in claim 1.

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