Novel polypeptide, polypeptide derivative and use thereof

Abstract

Disclosed are a novel polypeptide, a polypeptide derivative and the use thereof, which belong to the technical field of biomedicine. The present application specifically relates to a specific sequence, a discovery process, specific types of tumors to be resisted, a long-acting modification method and the use of the novel polypeptide, wherein the tumors include one or more of glioma, neuroblastoma, head and neck cancer, esophageal cancer, thyroid cancer, lung cancer, liver cancer, kidney cancer, breast cancer, cervical cancer, uterine cancer, ovarian cancer, colon cancer, small intestine cancer, ileocecal cancer, gastric cancer, bladder cancer, pancreatic cancer, prostate cancer, cholangiocarcinoma, melanoma, sarcoma, myeloma, lymphoma and leukemia; and the specific use comprises the inhibition of the proliferation and / or metastasis of the above-mentioned tumor cells. The novel polypeptide has a wide therapeutic spectrum and important therapeutic value against various tumors.

Description

A novel polypeptide, polypeptide derivative and its application Technical Field

[0001] The present application belongs to the field of biomedicine technology, and specifically relates to a novel polypeptide, polypeptide derivatives and applications thereof, and more specifically relates to a novel polypeptide, polypeptide derivatives and applications thereof in tumor prevention or treatment. Background Art

[0002] Protein-encoding open reading frames (ORFs) consist of a string of sense codons, beginning with a start codon and ending with a stop codon. Due to the limitations of traditional computer algorithms and detection methods, most ORF prediction algorithms use a minimum length of 100 amino acids. With the advancement of ribosome profiling, computational biology, and high-throughput omics technologies, a growing number of studies have demonstrated that many non-coding RNAs (non-coding RNAs) contain small open reading frames (sORFs) less than 300 nt in length, encoding functional polypeptides less than 100 amino acids in length, termed micropeptides. Micropeptide transcripts primarily originate from non-coding RNAs (lncRNAs, circRNAs, pri-miRNAs), ribosomal RNAs (rRNAs), antisense transcripts, 5'UTRs, 3'UTRs, and intergenic ORFs.

[0003] The earliest research on micropeptides began in 1996, when German scientists discovered a 679nt RNA transcribed from the ENOD40 (early nodulin 40) gene in legumes that performed non-coding functions. Based on its length of more than 200nt, it was classified as a lncRNA at the time. In fact, it can encode proteins and regulate plant growth. The specific results were published in the journal Science. In 2002, when German researchers studied this RNA in more detail, they found that it contained two sORFs and indeed encoded micropeptides (one encoding 12 peptides and the other encoding 24 peptides), and was confirmed to be able to interact with a sucrose synthase. In the following 20 years, with the development of computational biology and multi-omics deep sequencing technologies (such as ribosomal imprint sequencing and peptide group sequencing), scientists discovered a large number of previously unannotated sORFs hidden in non-coding RNA in many species such as nematodes, fruit flies, zebrafish, plants, mice and humans. These sORFs can translate into new polypeptides involved in biological functions such as muscle development, mRNA modification, immune regulation and tumor development, which opened up new perspectives for proteomic research.

[0004] In recent years, some studies have reported that micropeptides encoded by ncRNA (such as SMIM22, HOXB-AS3, SMIM30, MP31, PINT87aa) can regulate the occurrence and development of different tumors (including breast cancer, colorectal cancer, brain glioma, liver cancer, melanoma, esophageal squamous cell carcinoma, etc.), and can play a role in promoting or inhibiting tumor proliferation, metastasis, energy metabolism, and drug resistance. This suggests that micropeptides can be further developed as potential tumor diagnostic markers or therapeutic targets.

[0005] The Chinese invention patent with publication number CN112442116A records the micropeptide HMMW encoded by lncRNA and its application in the detection and treatment of malignant tumors such as head and neck cancer, thyroid cancer, and kidney cancer. HMMW is a new endogenous polypeptide sequence, specifically including 51 amino acid residues, and its direct binding receptor is the AQP2 protein.

[0006] Summary of the Invention

[0007] 1. Purpose of the Invention

[0008] Regarding the new micropeptide HMMW discovered for the first time by the applicant (see the applicant's previous patent announcement number CN112442116B for details),

[0009] One of the purposes of this application is to verify the function of a polypeptide that has homology to the micropeptide HMMW;

[0010] The second purpose of this application is to study the key active amino acids of the micropeptide HMMW and provide homologous functional fragments of the micropeptide HMMW that are shorter than the micropeptide HMMW but have the same or substantially the same functions, i.e., the novel polypeptides in this application;

[0011] The third object of the present application is to provide derivatives of the novel polypeptide of the present application, such as derivatives of the novel polypeptide that have been modified for long-term efficacy, which better retain the in vivo activity of the novel polypeptide, improve the stability of the novel polypeptide, and extend the half-life of the novel polypeptide;

[0012] The fourth object of the present application is to further provide the use of the novel polypeptide or its derivatives in the present application in the preparation of tumor prevention or treatment drugs, specifically including the preventive or therapeutic effects in human brain glioma, neuroblastoma, head and neck cancer, esophageal cancer, thyroid cancer, lung cancer, liver cancer, kidney cancer, breast cancer, cervical cancer, uterine cancer, ovarian cancer, colon cancer, small intestine cancer, ileocecal cancer, gastric cancer, bladder cancer, pancreatic cancer, prostate cancer, bile duct cancer, melanoma, sarcoma, myeloma, lymphoma, leukemia, etc.

[0013] 2. Technical solution

[0014] In order to achieve the above-mentioned invention objectives, the technical solutions adopted in this application are as follows:

[0015] The present application provides a novel polypeptide, which is based on the novel micropeptide HMMW (SEQ ID NO. 1) first discovered by the inventors, and is a polypeptide having the same or substantially the same function as the micropeptide HMMW determined through sequence modification, structural analysis, and amino acid mutation, and is specifically selected from any one of the following:

[0016] (1): A polypeptide having the same or substantially the same function as the polypeptide of the amino acid sequence shown in SEQ ID NO. 1 but lacking three consecutive amino acids;

[0017] (2) A polypeptide having the same or substantially the same function formed by deleting any number of 1 to 34 amino acids from the N-terminus of the polypeptide of the amino acid sequence shown in SEQ ID NO.1;

[0018] (3): A polypeptide with the amino acid sequence shown in SEQ ID NO.1 or a polypeptide with the same or substantially the same function formed by substituting one or more amino acids in (1) or (2).

[0019] Furthermore, the novel polypeptide is a polypeptide having the same or substantially the same function as the polypeptide of the amino acid sequence shown in SEQ ID NO. 1 but lacking three consecutive amino acids.

[0020] Furthermore, the above-mentioned novel polypeptide is a polypeptide with the same or substantially the same function formed by lacking three consecutive amino acids at positions 3N-2, 3N-1 and 3N of the polypeptide with the amino acid sequence shown in SEQ ID NO.1, wherein N=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17.

[0021] Furthermore, the amino acid sequence of the novel polypeptide is shown in any one of SEQ ID NO. 2 to SEQ ID NO. 18.

[0022] Furthermore, the above-mentioned novel polypeptide is a polypeptide with the same or substantially the same function formed by lacking three consecutive amino acids at positions 3N-2, 3N-1 and 3N of the polypeptide with the amino acid sequence shown in SEQ ID NO.1, wherein N=1, 2, 3, 5, 6, 7, 8, 9, 12, 14, 15 or 16.

[0023] Furthermore, the amino acid sequence of the novel polypeptide is shown in any one of SEQ ID NO.2 to SEQ ID NO.4, SEQ ID NO.6 to SEQ ID NO.10, SEQ ID NO.13 or SEQ ID NO.15 to SEQ ID NO.17.

[0024] Furthermore, the above-mentioned novel polypeptide is a polypeptide with the same or substantially the same function formed by lacking three consecutive amino acids at positions 3N-2, 3N-1 and 3N of the polypeptide with the amino acid sequence shown in SEQ ID NO.1, wherein N=1, 2, 3, 5, 6, 7, 8, 9, 12 or 16.

[0025] Furthermore, the amino acid sequence of the novel polypeptide is shown in any one of SEQ ID NO.2 to SEQ ID NO.4, SEQ ID NO.6 to SEQ ID NO.10, SEQ ID NO.13 or SEQ ID NO.17.

[0026] Furthermore, the novel polypeptide is a polypeptide having the same or substantially the same function as the polypeptide with the amino acid sequence shown in SEQ ID NO. 1 but with any number of 1 to 34 amino acids deleted from the N-terminus.

[0027] Furthermore, the above-mentioned novel polypeptide is a polypeptide having the same or substantially the same function formed by deleting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 amino acids from the N-terminus of the polypeptide with the amino acid sequence shown in SEQ ID NO.1.

[0028] Furthermore, the above-mentioned novel polypeptide is a polypeptide with the same or substantially the same function formed by deleting 4, 9, 14, 19, 24, 29 or 34 amino acids from the N-terminus of the polypeptide with the amino acid sequence shown in SEQ ID NO.1.

[0029] Furthermore, the amino acid sequence of the novel polypeptide is shown in any one of SEQ ID NO.19 to SEQ ID NO.25.

[0030] Furthermore, the novel polypeptide is a polypeptide having the same or substantially the same function as the polypeptide with the amino acid sequence shown in SEQ ID NO. 1 but with any number of 1 to 19 amino acids deleted from the N-terminus.

[0031] Furthermore, the above-mentioned novel polypeptide is a polypeptide with the same or substantially the same function formed by deleting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 amino acids from the N-terminus of the polypeptide with the amino acid sequence shown in SEQ ID NO.1.

[0032] Furthermore, the novel polypeptide is a polypeptide having the same or substantially the same function as the polypeptide with the amino acid sequence shown in SEQ ID NO.1 but with 4, 9, 14 or 19 amino acids missing from the N-terminus.

[0033] Furthermore, the amino acid sequence of the novel polypeptide is shown in any one of SEQ ID NO. 19 to SEQ ID NO. 22.

[0034] Furthermore, the novel polypeptide is a polypeptide having the same or substantially the same function as the polypeptide with the amino acid sequence shown in SEQ ID NO. 1 but with 1 to 14 amino acids missing from the N-terminus.

[0035] Furthermore, the above-mentioned novel polypeptide is a polypeptide with the same or substantially the same function formed by deleting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acids from the N-terminus of the polypeptide with the amino acid sequence shown in SEQ ID NO.1.

[0036] Furthermore, the novel polypeptide is a polypeptide having the same or substantially the same function as the polypeptide with the amino acid sequence shown in SEQ ID NO.1 but with 4, 9 or 14 amino acids missing from the N-terminus.

[0037] Furthermore, the amino acid sequence of the novel polypeptide is shown in any one of SEQ ID NO. 19 to SEQ ID NO. 21.

[0038] Furthermore, the novel polypeptide is a polypeptide having the same or substantially the same function formed by replacing one or more amino acids in the amino acid sequence polypeptide shown in SEQ ID NO.1.

[0039] Furthermore, the novel polypeptide is a polypeptide having the same or substantially the same function formed by replacing one amino acid with the amino acid sequence polypeptide shown in SEQ ID NO.1.

[0040] Furthermore, the above-mentioned substitution of one amino acid is a polypeptide having the same or substantially the same function formed by substitution of amino acid at position 2, 3, 8, 9, 10, 11, 12, 13, 17, 19, 20, 21, 22, 25, 27, 28, 30, 31, 32, 40, 41, 42, 43, 44, 45, 48, 49 or 50 of the amino acid sequence shown in SEQ ID NO.1.

[0041] Furthermore, the amino acid sequence of the novel polypeptide is shown in any one of SEQ ID NO. 26 to SEQ ID NO. 53.

[0042] Furthermore, the above-mentioned substitution of one amino acid is a polypeptide having the same or substantially the same function formed by substitution of amino acid at position 2, 3, 8, 9, 10, 11, 12, 13, 17, 19, 20, 21, 22, 25, 27, 28, 30, 31, 32, 40, 41, 42, 43, 44, 45, 48 or 50 of the amino acid sequence shown in SEQ ID NO.1.

[0043] Furthermore, the amino acid sequence of the novel polypeptide is shown in any one of SEQ ID NO.26 to SEQ ID NO.51 and SEQ ID NO.53.

[0044] Furthermore, the above-mentioned substitution of one amino acid is a polypeptide having the same or substantially the same function formed by substitution of amino acid at position 2, 3, 8, 9, 10, 11, 12, 13, 17, 19, 20, 21, 22, 25, 27, 28, 30, 31, 32, 40, 41, 42, 44, 45 or 50 of the amino acid sequence shown in SEQ ID NO.1.

[0045] Furthermore, the amino acid sequence of the novel polypeptide is shown in any one of SEQ ID NO.26 to SEQ ID NO.47, SEQ ID NO.49 to SEQ ID NO.50, and SEQ ID NO.53.

[0046] The present application also provides a nucleotide encoding the above-mentioned novel polypeptide.

[0047] The present application also provides a recombinant vector, which includes the above-mentioned nucleotide sequence encoding the novel polypeptide. The recombinant vector can be a plasmid, viral vector, or other vector known to those skilled in the art.

[0048] The present application also provides derivatives of the novel polypeptide, which are modified derivatives of the novel polypeptide having the same or substantially the same function.

[0049] Furthermore, the above modifications include one or more of N-terminal modification, C-terminal modification, side chain modification, amino acid modification or backbone modification.

[0050] Furthermore, the above modifications include one or more of polyethylene glycol modification, fatty acid linking, Fc (IgG1, IgG4) polypeptide fusion, binding to human serum albumin, non-natural amino acid replacement, polypeptide cyclization, or long-term modification such as high-end formulations.

[0051] Furthermore, the above polyethylene glycol modification method mainly modifies the N-terminus, C-terminus, Lys side chain and Cys thiol of the polypeptide.

[0052] Furthermore, the molecular weight of the polyethylene glycol used in the polyethylene glycol modification is in the range of PEG5 to PEG40.

[0053] Furthermore, the polyethylene glycol modification can use a single polyethylene glycol or multiple polyethylene glycol molecules.

[0054] Furthermore, the fatty acid linkage plays a binding role with serum albumin in vivo, and the linkage methods include direct linkage at the N-terminus, and can also include using a linker to link 18-carbon fatty acids and polypeptides, using a small peptide segment Ala-Ala-Asn as a linker.

[0055] Furthermore, the above-mentioned fatty acid linkage includes an N-terminal linkage.

[0056] Furthermore, the N-terminal linkage includes an N-terminal linkage of a fatty acid having 12 to 20 carbon atoms. Still further, the N-terminal linkage includes an N-terminal linkage of a fatty acid having 18 carbon atoms.

[0057] Furthermore, the fatty acid linking includes linking fatty acids using a linker.

[0058] Furthermore, the linker-linking includes linking fatty acids with 12 to 20 carbon atoms. Still further, the linker-linking includes linking fatty acids with 18 carbon atoms.

[0059] Furthermore, the linker is a peptide segment with a length of less than 5 amino acids.

[0060] Furthermore, the linker is a peptide segment with a length of less than 3 amino acids. Furthermore, the linker is a peptide segment Ala-Ala-Asn.

[0061] The present application also provides the use of the above-mentioned novel polypeptide or its derivatives, nucleotides encoding the novel polypeptide or recombinant vectors in the preparation of drugs for preventing or treating tumors.

[0062] The present application also provides the use of the above-mentioned polypeptides and their pharmaceutically acceptable salts, esters or polypeptide derivatives and their pharmaceutically acceptable salts and esters in the preparation of drugs for preventing or treating tumors.

[0063] Furthermore, the above-mentioned tumors include one or more of brain glioma, neuroblastoma, head and neck cancer, esophageal cancer, thyroid cancer, lung cancer, liver cancer, kidney cancer, breast cancer, cervical cancer, uterine cancer, ovarian cancer, colon cancer, small intestine cancer, ileocecal cancer, gastric cancer, bladder cancer, pancreatic cancer, prostate cancer, bile duct cancer, melanoma, sarcoma, myeloma, lymphoma, and leukemia.

[0064] Furthermore, the above-mentioned prevention or treatment of tumors includes inhibiting the proliferation and / or metastasis of tumor cells.

[0065] Furthermore, the above-mentioned prevention or treatment of tumors includes inhibiting the proliferation of tumor cells.

[0066] Furthermore, the above-mentioned prevention or treatment of tumors includes inhibiting tumor cell metastasis.

[0067] Furthermore, the tumor cells inhibiting the proliferation of tumor cells include:

[0068] The glioblastoma cancer cells include: U87-MG;

[0069] The neuroblastoma cancer cells include: SH-SY5Y;

[0070] The head and neck cancer cells include any one of: oral epidermoid carcinoma cells KB, laryngeal epidermoid carcinoma cells HEp2, tongue squamous cell carcinoma cells CAL27, and nasopharyngeal carcinoma cells CNE-2Z;

[0071] The esophageal cancer cells include: Eca-109 and / or TE-13;

[0072] The thyroid cancer cells include: NPPA87-1;

[0073] The lung cancer cells include: any one or a combination of 95-D, SK-MES-1, SPC-A1, and A549;

[0074] The liver cancer cells include: HCCLM3 and / or HepG2;

[0075] The renal cancer cells include: Ketr-3 and / or 786-O;

[0076] The breast cancer cells include: SK-BR-3 and / or MDA-MB-231;

[0077] The cervical cancer cells include: CaSki and / or Hela;

[0078] The uterine cancer cells include: RL-952 and / or MS751;

[0079] The ovarian cancer cells include: ES-2 and / or SKOV3

[0080] The colon cancer cells include: HT29 and / or SW480;

[0081] The cancer cells of the small intestine cancer include: HIC;

[0082] The ileocecal cancer cells include: HCT-8;

[0083] The gastric cancer cells include: MGC-803 and / or BGC-823;

[0084] The bladder cancer cells include: T24 and / or EJ;

[0085] The pancreatic cancer cells include: ASPC-1 and / or PANC-1;

[0086] The prostate cancer cells include: 22RV1 and / or PC-3;

[0087] The bile duct cancer cell line includes: QBC939;

[0088] The melanoma cancer cells include: SK-mel-2 and / or A375;

[0089] The sarcoma cancer cells include: MG63 and / or HT-1080;

[0090] The myeloma cancer cells include: LP-1;

[0091] The lymphoma cancer cells include: any one or a combination of Raji, RPMI 8226, U937, and WSU-DLCL2;

[0092] The leukemia cancer cells include any one or a combination of CEM / C1, NB4, KG-1, K562, HL60, Jurkat, and Clone E6-1.

[0093] Furthermore, the tumor cells inhibiting tumor cell metastasis include:

[0094] The melanoma cancer cells include: A375;

[0095] The cervical cancer cells include: Hela;

[0096] The ovarian cancer cells include: Skvo3;

[0097] The liver cancer cells include: Hep3B;

[0098] The lung cancer cells include: A549.

[0099] The present application also provides a pharmaceutical composition for preventing or treating tumors, which contains at least the above-mentioned novel polypeptide or its derivatives, and a pharmaceutically acceptable carrier, wherein the tumors include one or more of brain glioma, neuroblastoma, head and neck cancer, esophageal cancer, thyroid cancer, lung cancer, liver cancer, kidney cancer, breast cancer, cervical cancer, uterine cancer, ovarian cancer, colon cancer, small intestine cancer, ileocecal cancer, gastric cancer, bladder cancer, pancreatic cancer, prostate cancer, bile duct cancer, melanoma, sarcoma, myeloma, lymphoma, and leukemia.

[0100] 3. Beneficial effects

[0101] Compared with the prior art, the present application has the following advantages:

[0102] (1) The present application provides a novel polypeptide, which is a deletion or truncated polypeptide of the micropeptide HMMW, consisting of 17 to 50, especially 37 to 50 amino acids, which is shorter than the length of the micropeptide HMMW. On the one hand, the shorter polypeptide has a smaller molecular weight and is more conducive to absorption. For example, in the examples, at the same dose, the novel polypeptide HMMW15-51 has a significantly better inhibitory effect on the proliferation of human brain glioma, neuroblastoma, head and neck cancer, esophageal cancer, thyroid cancer, lung cancer, liver cancer, kidney cancer, breast cancer, cervical cancer, uterine cancer, ovarian cancer, colon cancer, small intestine cancer, ileocecal cancer, gastric cancer, bladder cancer, pancreatic cancer, prostate cancer, bile duct cancer, melanoma, sarcoma, myeloma, lymphoma, and leukemia cells than the micropeptide HMMW, and a significantly better inhibitory effect on the metastasis of human melanoma cells than the micropeptide HMMW, and a comparable inhibitory effect on the metastasis of human ovarian cancer, liver cancer, and lung cancer cells as the micropeptide HMMW; on the other hand, it can simplify the synthesis process and save synthesis costs.

[0103] (2) The novel polypeptide provided in this application is a micropeptide HMMW or a deleted or truncated polypeptide of micropeptide HMMW, which is formed by amino acid substitution. This application has confirmed that L43, R48, and R49 in the micropeptide HMMW sequence are key active sites by attempting to combine different screening methods and activity evaluation systems, providing a basis for the secondary development of micropeptide HMMW.

[0104] (3) The present application provides a novel polypeptide derivative, including long-term modification methods such as polyethylene glycol modification and fatty acid modification, which can better retain the in vivo activity of the novel polypeptide, improve the stability of the novel polypeptide and extend the half-life. The present application has determined the long-term modification scheme through trial and error, combined with in vivo activity experimental evaluation. The half-life and in vivo anti-tumor time of the novel polypeptide are significantly prolonged, and it has application potential in the development of tumor drugs. At the same dose, the long-term modified novel polypeptide has a significantly better anti-tumor effect in vivo than the novel polypeptide, and the administration frequency is significantly shortened. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Figure 1 shows the effects of different deletions of the micropeptide HMMW on the proliferation of tongue squamous cell carcinoma CAL27 cells;

[0106] FIG2 shows the effects of different truncated fragments of the micropeptide HMMW on the proliferation of tongue squamous cell carcinoma CAL27 cells;

[0107] FIG3 is the sequence information of the alanine scanning mutation of the micropeptide HMMW;

[0108] FIG4 shows the effect of single amino acid mutation sequence of micropeptide HMMW on the proliferation of tongue squamous cell carcinoma CAL27 cells;

[0109] FIG5 is the statistical results of the metastatic activity of the novel peptide and micropeptide HMMW on melanoma A375 cells;

[0110] FIG6 is the statistical results of the metastatic activity of the novel peptide and micropeptide HMMW on cervical cancer Hela cells;

[0111] FIG7 is the statistical results of the metastatic activity of the novel peptide and micropeptide HMMW on ovarian cancer Skvo3 cells;

[0112] FIG8 is the statistical results of the metastatic activity of the novel peptide and micropeptide HMMW on liver cancer Hep3B cells;

[0113] FIG9 is a statistical result of the metastatic activity of the novel peptide and micropeptide HMMW on lung cancer A549 cells;

[0114] FIG10 shows the results of the inhibition of the tumorigenicity of human tongue squamous cell carcinoma CAL27 cells by the novel polypeptide and micropeptide HMMW;

[0115] Figure 11 shows the effects of novel polypeptides and long-acting modified peptides on the proliferation of human tongue squamous cell carcinoma CAL27 cells;

[0116] Figure 12 shows the effects of novel polypeptides and long-acting modified peptides on the proliferation of human ovarian cancer SKOV3 cells;

[0117] FIG13 shows the effects of novel polypeptides and long-acting modified peptides on the proliferation of human liver cancer Hep3B cells;

[0118] FIG14 shows the results of the inhibition of the tumorigenicity of human tongue squamous cell carcinoma CAL27 cells in vivo by the novel polypeptide and the long-acting modified peptide. DETAILED DESCRIPTION

[0119] The present application is further described below with reference to specific embodiments.

[0120] Example 1

[0121] This example provides the effects of different deletion mutants of the micropeptide HMMW on the proliferation of human tongue squamous cell carcinoma CAL27 cells.

[0122] Since the micropeptide HMMW (SEQ ID NO.1) is a completely new sequence, further development of its smaller functional fragments can reduce the cost of later development. First, the N-terminus of the polypeptide (1-51aa) was deleted in groups of three amino acids, and a total of 17 different deleted peptides were obtained, which were named HMMW-1 (deletion 1-3aa, SEQ ID NO.2), HMMW-2 (deletion 4-6aa, SEQ ID NO.3), HMMW-3 (deletion 7-9aa, SEQ ID NO.4), HMMW-4 (deletion 10-12aa, SEQ ID NO.5), HMMW-5 (deletion 13-15aa, SEQ ID NO.6), HMMW-6 (deletion 16-18aa, SEQ ID NO.7), HMMW-7 (deletion 19-21aa, SEQ ID NO.8), HMMW-8 (deletion 22-24aa, SEQ ID NO.9), HMMW-9 (deletion 25-27aa, SEQ ID NO.10), and HMMW-11 (deletion 26-28aa, SEQ ID NO.11). NO.10), HMMW-10 (deletion 28-30aa, SEQ ID NO.11), HMMW-11 (deletion 31-33aa, SEQ ID NO.12), HMMW-12 (deletion 34-36aa, SEQ ID NO.13), HMMW-13 (deletion 37-39aa, SEQ ID NO.14), HMMW-14 (deletion 40-42aa, SEQ ID NO.15), HMMW-15 (deletion 43-45aa, SEQ ID NO.16), HMMW-16 (deletion 46-48aa, SEQ ID NO.17), HMMW-17 (deletion 49-51aa, SEQ ID NO.18), HMMW-20 (deletion 50-51aa, SEQ ID NO.19), HMMW-21 (deletion 51-52aa, SEQ ID NO.21), HMMW-22 (deletion 52-53aa, SEQ ID NO.22), HMMW-23 (deletion 53-54aa, SEQ ID NO.23), HMMW-24 (deletion 54-55aa, SEQ ID NO.24), HMMW-25 (deletion 55-56aa, SEQ ID NO.25), HMMW-26 (deletion 56-57aa, SEQ ID NO.26), HMMW-27 (deletion 57-58aa, SEQ ID NO. NO.18), the above-mentioned deletion peptides were synthesized by chemical solid phase synthesis. Among them, five peptides, HMMW-4, HMMW-10, HMMW-11, HMMW-13, and HMMW-17, may have poor solubility due to changes in amino acid composition and could not be evaluated in subsequent activity experiments. Therefore, the other 12 deletion peptides with good solubility were selected to compare their effects on the proliferation of human tongue squamous cell carcinoma CAL27 cells.

[0123] Human tongue squamous cell carcinoma CAL27 cells were cultured in a 37°C, 5% CO2 incubator until the density reached 90%. The cells were harvested by trypsinization, resuspended in culture medium, and counted under a microscope. The cell concentration was adjusted to approximately 5 × 10 4 / mL, the cell suspension was inoculated into a 96-well plate, 100μL per well, and cultured overnight in a 37°C, 5% CO2 incubator. After the cells were completely attached, 100μL of the above 12 deletion peptides and HMMW at a concentration of 80μM were added respectively, and the culture medium without any drug was used as a negative control group. After further incubation for 48h, 100μL of CCK8 was added to each well of the 96-well plate to dissolve. The absorbance OD was measured at a detection wavelength of 450nm using an enzyme reader, and the growth inhibition rate (PI) was calculated using the formula: PI (%) = 1 drug group / negative control group. The experiment was repeated 3 times independently, and the results were expressed as Mean±SD, and a statistical T test was performed. The results are shown in Table 1 and Figure 1.

[0124] Table 1 Effects of different deletion mutants of micropeptide HMMW on the proliferation of human tongue squamous cell carcinoma CAL27 cells Note: Compared with the HMMW-treated group, * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001.

[0125] As shown in Table 1 and Figure 1, under normal solubility, compared with the HMMW-treated group, at the same dose, the 10 deletion peptides HMMW-1, HMMW-2, HMMW-3, HMMW-5, HMMW-6, HMMW-7, HMMW-8, HMMW-9, HMMW-12, and HMMW-16 had comparable inhibitory effects on the proliferation of human tongue squamous cell carcinoma CAL27 cells as HMMW, with no significant decrease in activity; although the inhibitory effects of HMMW-14 and HMMW-15 on the proliferation of human tongue squamous cell carcinoma CAL27 cells were significantly decreased compared with HMMW, they still had activity.

[0126] Example 2

[0127] This example provides the effects of different truncated fragments of the micropeptide HMMW on the proliferation of human tongue squamous cell carcinoma CAL27 cells.

[0128] Based on the results of Example 1, the micropeptide HMMW was truncated from the N-terminus. Since HMMW-1 in Example 1 lacked amino acids 1-3aa, it was found that it would not affect the anti-tumor effect of the polypeptide. Therefore, truncation was started from the N-terminus, and amino acids 5-51, 10-51, 15-51, 20-51, 25-51, 30-51 and 35-51 were retained, respectively, and named HMMW5-51 (SEQ ID NO.19), HMMW10-51 (SEQ ID NO.20), HMMW15-51 (SEQ ID NO.21), HMMW20-51 (SEQ ID NO.22), HMMW25-51 (SEQ ID NO.23), HMMW30-51 (SEQ ID NO.24), HMMW35-51 (SEQ ID NO.25), respectively. NO.25), the six truncated peptides were synthesized by chemical solid phase synthesis. Among them, the three peptides HMMW25-51, HMMW30-51, and HMMW35-51 may have significant changes in amino acid composition and poor solubility, making subsequent activity evaluation impossible. Therefore, the four truncated peptides HMMW5-51, HMMW10-51, HMMW15-51, and HMMW20-51 with good solubility were selected to further compare their effects on the proliferation of human tongue squamous cell carcinoma CAL27 cells.

[0129] Similarly, human tongue squamous cell carcinoma CAL27 cells were cultured in a 37°C, 5% CO2 incubator until the density reached 90%. The cells were then digested and harvested with trypsin, resuspended in culture medium, and counted under a microscope. The cell concentration was adjusted to approximately 5 × 10 4 / mL, the cell suspension was inoculated into a 96-well plate, 100 μL per well, and cultured overnight in a 37°C, 5% CO2 incubator. After the cells were completely attached, 100 μL of HMMW micropeptide at a concentration of 80 μM and the four truncated peptides HMMW5-51, HMMW10-51, HMMW15-51, and HMMW20-51 were added respectively. The culture medium without any drug was used as a negative control group. After incubation for 48 hours, 100 μL of CCK8 was added to each well of the 96-well plate to dissolve. The absorbance OD was measured at a detection wavelength of 450 nm using a microplate reader, and the growth inhibition rate (PI) was calculated using the formula: PI (%) = 1 treatment group / negative control group. The experiment was repeated 3 times independently, and the results were expressed as Mean ± SD, and statistical T test was performed. The results are shown in Table 2 and Figure 2.

[0130] Table 2 Effects of different truncated fragments of micropeptide HMMW on the proliferation of human tongue squamous cell carcinoma CAL27 cells Note: Compared with the HMMW-treated group, * represents P < 0.05, ** represents P < 0.01.

[0131] As can be seen from Table 2 and Figure 2, compared with the HMMW-treated group, at the same dose, the inhibitory effects of the three truncated peptides HMMW5-51, HMMW10-51 and HMMW15-51 on the proliferation of human tongue squamous cell carcinoma CAL27 cells were comparable to those of HMMW. Although the inhibitory effect of the truncated peptide HMMW20-51 on the proliferation of human tongue squamous cell carcinoma CAL27 cells was significantly reduced, it still had activity. Combined with the results of Example 1, it is speculated that HMMW15-51 is the smallest functional fragment of HMMW.

[0132] Example 3

[0133] This example provides micropeptide HMMW for single amino acid alanine scanning mutagenesis and affinity detection analysis.

[0134] The MOE software was used to perform molecular docking simulations on the micropeptide HMMW and its binding protein AQP2 to find predicted potential interaction sites. The docking simulations were divided into the following two groups: (1) random docking of AQP2 homotetramer with HMMW without restricting the binding region; (2) random docking of AQP2 monomer with HMMW without restricting the binding region. The interaction sites obtained by the above two docking methods were integrated and analyzed, and all possible binding amino acids were selected as potential key sites for alanine (Ala) mutation. Since alanine is the smallest chiral amino acid, by systematically and sequentially replacing each amino acid in the sequence with Ala, the effect of a specific amino acid on the biological activity of the entire protein was identified. The predicted peptides were synthesized by solid phase, and the affinity of different mutant peptides with AQP2 protein was tested by microthermophoresis (MST). The sequence information of the micropeptide HMMW alanine scanning mutation is shown in Figure 3. The specific predicted key sites and binding status with AQP2 are shown in Table 3.

[0135] Table 3 Prediction of key sites for binding of micropeptide HMMW to AQP2 protein

[0136] As shown in Table 3, compared with HMMW, the affinity of the polypeptide to the protein AQP2 decreased significantly after the mutation of amino acids at positions 8, 48 and 49, which preliminarily indicates that the key sites of HMMW are amino acids at positions 8, 48 and 49.

[0137] Example 4

[0138] This example provides the effect of a single amino acid mutation sequence of the micropeptide HMMW on the proliferation of human tongue squamous cell carcinoma CAL27 cells.

[0139] Human tongue squamous cell carcinoma CAL27 cells were cultured in a 37°C, 5% CO2 incubator until the density reached 90%. The cells were harvested by trypsinization, resuspended in culture medium, and counted under a microscope. The cell concentration was adjusted to approximately 5 × 10 4 / mL, the cell suspension was inoculated into a 96-well plate, 100 μL per well, and cultured overnight in a 37°C, 5% CO2 incubator. After the cells were completely attached, 100 μL of HMMW micropeptide at a concentration of 80 μM and the single amino acid mutant polypeptide synthesized in Example 3 were added respectively, and the culture medium without any drug was used as a negative control group. After continuing to incubate for 48 hours, 100 μL of CCK8 was added to each well of the 96-well plate to dissolve. The absorbance OD was measured at a detection wavelength of 450 nm using a microplate reader, and the growth inhibition rate (PI) was calculated. The formula is: PI (%) = 1 drug group / negative control group. The experiment was repeated three times independently, and the results were expressed as Mean ± SD. A statistical T test was performed, and the results are shown in Figure 4. * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001. At a dose of 80 μM, except for the two peptides HMMW-3A and HMMW-11A, which could not be compared in activity due to poor solubility, the inhibitory effects of the peptides after mutation of amino acids at positions 43, 48, and 49 on the proliferation of human tongue squamous cell carcinoma CAL27 cells were significantly reduced compared with the HMMW-treated group. Among them, the peptide with mutation of amino acid at position 8 had no difference in anti-tumor activity compared with HMMW. Therefore, it was determined that amino acids 43, 48, and 49 are the key active sites of the micropeptide HMMW.

[0140] Example 5

[0141] This example provides detection of the inhibitory effects of the novel polypeptide HMMW15-51 (SEQ ID NO. 21) and the micropeptide HMMW on the proliferation of different tumor cells.

[0142] Human neuroblastoma cells SH-SY5Y, brain astroglioma cells U87MG, oral epidermoid carcinoma cells KB, laryngeal epidermoid carcinoma cells HEp2, tongue squamous cell carcinoma cells CAL27, nasopharyngeal carcinoma cells CNE-2Z, esophageal carcinoma cells Eca-109 and TE-13, thyroid papillary carcinoma cells NPPA87-1, lung giant cell carcinoma cells 95-D, lung squamous cell carcinoma cells SK-MES-1, lung adenocarcinoma cells SPC-A1, non-small cell lung cancer cells A549, liver cancer cells HCCLM3 and HepG2, renal cancer cells Ketr-3 and 786-O, breast cancer cells SK-BR-3 and MDA-MB-231, cervical cancer cells CaSki and H ela, uterine cancer cells RL-952 and MS751, ovarian cancer cells ES-2 and SKOV3, colon cancer cells HT29 and SW480, small intestinal cancer cells HIC, ileocecal cancer cells HCT-8, gastric cancer cells MGC-803 and BGC-823, bladder cancer cells T24 and EJ, pancreatic cancer cells ASPC-1 and PANC-1, prostate cancer cells 22RV1 and PC-3, bile duct cancer cells QBC939, malignant melanoma cells SK-mel-2 and A375, osteosarcoma cells MG63, fibrosarcoma cells HT-1080, multiple myeloma cells LP-1, lymphoma cells Raji, multiple myeloma peripheral blood B lymphocytes RPMI 8226, histiocytic lymphoma cells U937, diffuse large B-cell lymphoma cells WSU-DLCL2, acute lymphoblastic leukemia cells CEM / C1, acute promyelocytic leukemia cells NB4, acute myeloid leukemia cells KG-1, chronic myeloid leukemia cells K562, promyelocytic leukemia cells HL60, and T-lymphocytic leukemia cells (Jurkat, Clone E6-1) were cultured in a 37°C, 5% CO2 incubator to a density of 90%. The cells were harvested by trypsinization, resuspended in culture medium, and counted under a microscope to adjust the cell concentration to approximately 1.0 × 10 5Cell suspensions were seeded into 96-well plates at a concentration of 100 μL per well and cultured overnight at 37°C in a 5% CO2 incubator. After complete cell attachment, 100 μL of 50 μM micropeptide HMMW or 50 μM, 75 μM, and 100 μM novel peptide HMMW15-51 were added to the cells as treatment groups. A negative control group was maintained without any drug. After 48 hours of incubation, 10 μL of CCK8 solution was added to each well of the 96-well plate. The cells were incubated in the cell culture incubator for another 4 hours. The absorbance was measured at 450 nm, and the proliferation inhibition rate was calculated. Data are expressed as mean ± SD. Statistical analysis was performed using GraphPad Prism 5.0 software. Two-group comparisons were performed using the t-test, and multiple-group comparisons were performed using the one-way anova (Dunnett). The results are shown in Table 4-56, * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001.

[0143] Table 4. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on the proliferation of human astroglioma cells U87MG

[0144] Table 5. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human neuroblastoma cells SH-SY5Y

[0145] Table 6. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human oral epidermoid carcinoma cells KB

[0146] Table 7. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human laryngeal epidermoid carcinoma HEp2 cells

[0147] Table 8. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human tongue squamous cell carcinoma CAL27 cells

[0148] Table 9. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human nasopharyngeal carcinoma cell CNE-2Z

[0149] Table 10. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human esophageal cancer cell Eca-109

[0150] Table 11. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human esophageal cancer cell TE-13

[0151] Table 12. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human thyroid papillary carcinoma cell line NPPA87-1

[0152] Table 13. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human lung giant cell carcinoma cell line 95-D

[0153] Table 14. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human lung squamous cell carcinoma cell line SK-MES-1

[0154] Table 15. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human lung adenocarcinoma cell line SPC-A1

[0155] Table 16. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human non-small cell lung cancer cells A549

[0156] Table 17. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human hepatocellular carcinoma cell line HCCLM3

[0157] Table 18. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human liver cancer cells HepG2

[0158] Table 19. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human renal cancer cell Ketr-3

[0159] Table 20. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human renal clear cell adenocarcinoma cell line 786-O

[0160] Table 21. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human breast cancer cells SK-BR-3

[0161] Table 22. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human breast cancer cells MDA-MB-231

[0162] Table 23. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human cervical cancer epithelial cells CaSki

[0163] Table 24. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human cervical cancer cells Hela

[0164] Table 25. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human endometrial cancer cells RL-952

[0165] Table 26. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human uterine epidermal cancer cell line MS751

[0166] Table 27. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human ovarian clear cell carcinoma cell line ES-2

[0167] Table 28. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human ovarian cancer cell SKOV3

[0168] Table 29. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human colon cancer cells HT29

[0169] Table 30. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human colon cancer cell SW480

[0170] Table 31. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human small intestinal cancer cells HIC

[0171] Table 32. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human ileocecal cancer cell HCT-8

[0172] Table 33. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human gastric cancer cell MGC-803

[0173] Table 34. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human gastric cancer cell line BGC-823

[0174] Table 35. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human bladder transitional cell carcinoma cell line T24

[0175] Table 36. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human bladder cancer cell EJ

[0176] Table 37. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human pancreatic cancer cell ASPC-1

[0177] Table 38. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human pancreatic cancer cell PANC-1

[0178] Table 39. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human prostate cancer cell line 22RV1

[0179] Table 40. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human prostate cancer cells PC-3

[0180] Table 41. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on the proliferation of human cholangiocarcinoma cell QBC939

[0181] Table 42. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human malignant melanoma cells SK-mel-2

[0182] Table 43. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human malignant melanoma cells A375

[0183] Table 44. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human osteosarcoma cells MG63

[0184] Table 45. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human fibrosarcoma cells HT-1080

[0185] Table 46. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human multiple myeloma cells LP-1

[0186] Table 47. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human lymphoma cells Raji

[0187] Table 48. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human multiple myeloma peripheral blood B lymphocytes RPMI 8226

[0188] Table 49. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human histiocytic lymphoma cells U937

[0189] Table 50. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human diffuse large B lymphoma cells WSU-DLCL2

[0190] Table 51. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human acute lymphoblastic leukemia cells CEM / C1

[0191] Table 52. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human acute promyelocytic leukemia cells NB4

[0192] Table 53. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human acute myeloid leukemia cells KG-1

[0193] Table 54. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human chronic myeloid leukemia cells K562

[0194] Table 55. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human myeloid leukemia cells HL60

[0195] Table 56. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on proliferation of human T lymphocyte leukemia cells Jurkat, Clone E6-1

[0196] As can be seen from Table 4-56, at the same dose, the novel polypeptide HMMW15-51 has significantly better inhibitory effects on the proliferation of neuroblastoma, human brain glioma, head and neck cancer, esophageal cancer, thyroid cancer, lung cancer, liver cancer, kidney cancer, breast cancer, cervical cancer, uterine cancer, ovarian cancer, intestinal cancer, gastric cancer, bladder cancer, pancreatic cancer, prostate cancer, bile duct cancer, melanoma, osteosarcoma, fibrosarcoma, myeloma, lymphoma, and hematologic tumor cells than the micropeptide HMMW; and the novel polypeptide HMMW15-51 has a dose-dependent inhibitory effect on the proliferation of the above-mentioned tumor cells, indicating that the novel polypeptide HMMW15-51 can be developed into an anti-tumor drug by inhibiting the proliferation of the above-mentioned malignant tumor cells.

[0197] Example 6

[0198] This example provides the inhibitory effects of the novel polypeptide HMMW15-51 (SEQ ID NO. 21) and the micropeptide HMMW on the metastasis of different human tumor cells.

[0199] Melanoma, cervical cancer, ovarian cancer, liver cancer, and lung cancer cells were inoculated into transwell chambers, with 100 μL per well. Different doses of HMMW micropeptide and novel peptide HMMW15-51 were added to each chamber as the drug-dosing group, and no drug was added to the blank group. Then 0.6 mL of complete culture medium containing 10% FBS was added to the lower chamber of the transwell to stimulate cell metastasis, and the cells were cultured at 5% CO2 and 37°C for 48 hours. The culture medium in the well was discarded, the cells were fixed with methanol at room temperature for 30 minutes, stained with 0.1% crystal violet at room temperature for 10 minutes, rinsed with clean water, and the unmetastatic cells on the upper layer were wiped off with a cotton swab. The cells were observed under a microscope and four fields of view were selected for photographing and counting. The invasion inhibition rate (MI) was calculated according to the formula:

[0200] Among them, N test is the number of cell migration in the drug-treated group, N control The number of cells transferred in the blank control group.

[0201] The data are expressed as mean ± SD. GraphPad Prism 5.0 statistical software was used. The t-test was used for comparison between the two groups. * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001. The results are shown in Tables 57-61 and Figures 5-9.

[0202] Table 57. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on metastasis of human melanoma A375 cells

[0203] Table 58. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on metastasis of human cervical cancer Hela cells

[0204] Table 59. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on metastasis of human ovarian cancer Skvo3 cells

[0205] Table 60. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on metastasis of human liver cancer Hep3B cells

[0206] Table 61. Inhibitory effects of novel peptide HMMW15-51 and micropeptide HMMW on metastasis of human lung cancer A549 cells

[0207] As can be seen from Tables 57-61 and Figures 5-9, compared with the blank control, the novel polypeptide HMMW15-51 has a significant inhibitory effect on the metastasis of human melanoma, cervical cancer, ovarian cancer, liver cancer, and lung cancer cells, indicating that the novel polypeptide HMMW15-51 can be developed into an anti-tumor drug by inhibiting the metastasis of the above-mentioned malignant tumor cells.

[0208] Example 7

[0209] This example provides the detection of the in vivo effects of the novel polypeptide HMMW15-51 (SEQ ID NO. 21) and the micropeptide HMMW (SEQ ID NO. 1) on a subcutaneous transplanted tumor model of tongue squamous cell carcinoma cells CAL27.

[0210] (1) Human tongue squamous cell carcinoma CAL27 cells were cultured in large quantities and digested with 0.25% trypsin solution. After digestion, the cell suspension was centrifuged at 1000 rpm for 5 min. The cells were resuspended in serum-free DMEM medium and counted. The cell concentration was adjusted to 5×10 7 / mL;

[0211] (2) Each BALB / c nude mouse (female, 4-6 weeks old, weighing 14-16 g, and acclimatized for 1 week in an SPF animal breeding room) was inoculated with 100 μL of the cell suspension of the corresponding group in the left axilla. The injected cell volume was 5×10 6 indivual;

[0212] (3) After inoculation, the tumor growth at the inoculation site of the nude mice was closely observed. On the 7th day after inoculation, a white nodule appeared at the inoculation site, which could be moved subcutaneously after being touched. As the tumor tissue grew, a hard tumor mass gradually formed at the inoculation site. After about 14 days, the average volume of the tumor tissue reached 80 mm. 3BALB / c nude mice were randomly divided into 3 groups (normal saline group was blank control, micropeptide HMMW and novel peptide HMMW15-51 were used at a dosage of 2.7 μM / kg), 10 mice in the blank group and 6 mice in each of the other groups. The weight of the animals was about 20 g at the beginning of drug administration.

[0213] (4) Measure and record the transplanted tumor volume every two days. The tumor volume (TV) is calculated as follows:

[0214] Tumor volume = 0.5 × a × b 2 , where a is the length of the transplanted tumor (mm) and b is the width of the transplanted tumor (mm);

[0215] (5) After the administration, the nude mice were euthanized, the tumor masses were removed and weighed, and the tumor growth inhibition rate was calculated using the following formula:

[0216] Tumor growth inhibition rate = (1-T / C) × 100%, where: T is the average tumor weight of the treatment group; C is the average tumor weight of the control group;

[0217] The data are expressed as mean ± SD. GraphPad Prism 5.0 statistical software was used. The t-test was used for comparison between two groups, and the one-way anova was used for comparison between multiple groups. **** indicates P < 0.0001.

[0218] The results are shown in Figure 10. Compared with the normal saline control group, the micropeptide HMMW and the novel peptide HMMW15-51 were able to significantly inhibit the in vivo tumorigenicity of human tongue squamous cell carcinoma CAL27 cells to varying degrees, while compared with HMMW, the activity of HMMW15-51 had no significant difference.

[0219] Example 8

[0220] This example provides the detection of the inhibitory effect of the novel long-acting modified polypeptide HMMW15-51 (SEQ ID NO. 21) on the proliferation of different tumor cells.

[0221] In this embodiment, the long-acting modification includes linking an 18-carbon fatty acid to the N-terminus, i.e., the modified peptide C18-HMMW15-51; and using a small peptide segment Ala-Ala-Asn as a linker to link an 18-carbon fatty acid, i.e., C18-L-HMMW15-51.

[0222] The structure of C18-HMMW15-51 is:

[0223] The structure of C18-L-HMMW15-51 is:

[0224] C18-HMMW15-51 and C18-L-HMMW15-51 can be coupled to amino acids via solid-phase synthesis. For fatty acid coupling, the fatty acid is coupled to a peptide resin according to the amino acid synthesis protocol (solid-phase synthesis) to obtain a crude product. The crude product is cleaved, analyzed by preparative HPLC, and lyophilized to obtain the desired compound.

[0225] Human tongue squamous cell carcinoma cells CAL27, ovarian cancer cells SKOV3, and liver cancer cells Hep3B were cultured in a 37°C, 5% CO2 incubator until the density reached 90%. The cells were then digested and harvested using trypsin. The cells were resuspended in culture medium and counted under a microscope. The cell concentration was adjusted to approximately 1.0 × 10 5 Cell suspensions were seeded into 96-well plates at a concentration of 100 μL per well and cultured overnight at 37°C in a 5% CO2 incubator. After complete cell attachment, 100 μL of 100 μM HMMW15-51, C18-HMMW15-51, and C18-L-HMMW15-51 were added to the cells for treatment. A negative control group was maintained without any drug. After 48 hours of incubation, 10 μL of CCK8 was added to each well of the 96-well plate. The cells were incubated in the cell culture incubator for another 4 hours. The absorbance was measured at 450 nm, and the proliferation inhibition rate was calculated. Data are expressed as mean ± SD. Statistical analysis was performed using GraphPad Prism 5.0 software. t-tests were used for comparisons between two groups, and one-way anova was used for comparisons between multiple groups.

[0226] The results are shown in Figures 11-13. **** represents P < 0.0001. There is no significant difference in the inhibitory effects of the novel peptide HMMW15-51 (SEQ ID NO. 21), modified peptides C18-HMMW15-51 and C18-L-HMMW15-51 on the proliferation of different tumor cells.

[0227] Example 9

[0228] This example provides an in vivo assay for the effects of novel polypeptides HMMW15-51 (SEQ ID NO. 21), C18-HMMW15-51, and C18-L-HMMW15-51 on a subcutaneous transplanted tumor model of tongue squamous cell carcinoma cells CAL27.

[0229] (1) Human tongue squamous cell carcinoma CAL27 cells were cultured in large quantities and digested with 0.25% trypsin solution. After digestion, the cell suspension was centrifuged at 1000 rpm for 5 min. The cells were resuspended in serum-free DMEM medium and counted. The cell concentration was adjusted to 5×10 7 / mL;

[0230] (2) Each BALB / c nude mouse (female, 4-6 weeks old, weighing 14-16 g, and acclimatized for 1 week in an SPF animal breeding room) was inoculated with 100 μL of the cell suspension of the corresponding group in the left axilla. The injected cell volume was 5×10 6 indivual;

[0231] (3) After inoculation, the tumor growth at the inoculation site of the nude mice was closely observed. On the 7th day after inoculation, a white nodule appeared at the inoculation site, which could be moved subcutaneously after being touched. As the tumor tissue grew, a hard tumor mass gradually formed at the inoculation site. After about 14 days, the average volume of the tumor tissue reached 80 mm. 3 BALB / c nude mice were randomly divided into 4 groups: the normal saline group was used as blank control, the micropeptide HMMW15-51 (intravenous injection every day), C18-HMMW15-51 (subcutaneous injection every other day) and C18-L-HMMW15-51 (subcutaneous injection every other day) were used at a dosage of 2.7 μM / kg, 10 mice in the blank group and 6 mice in each of the other groups. The weight of the animals was about 20 g at the beginning of drug administration.

[0232] (4) Measure and record the transplanted tumor volume every two days. The tumor volume (TV) is calculated as follows:

[0233] Tumor volume = 0.5 × a × b 2 , where a is the length of the transplanted tumor (mm) and b is the width of the transplanted tumor (mm);

[0234] (5) After 21 days of administration, the nude mice were euthanized, and the tumor masses were removed and weighed. The tumor growth inhibition rate was calculated using the following formula:

[0235] Tumor growth inhibition rate = (1-T / C) × 100%, where: T is the average tumor weight of the treatment group; C is the average tumor weight of the control group;

[0236] The data were expressed as mean ± SD and analyzed using GraphPad Prism 5.0 statistical software. The t-test was used for comparison between two groups, and the one-way anova was used for comparison between multiple groups.

[0237] The results are shown in Figure 14, where * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001. Compared with the normal saline control group, the micropeptide HMMW15-51 (intravenous injection every day), C18-HMMW15-51 (subcutaneous injection every other day), and C18-L-HMMW15-51 (subcutaneous injection every other day) can significantly inhibit the in vivo tumorigenicity of human tongue squamous cell carcinoma cells CAL27 to varying degrees. Compared with HMMW15-51, C18-L-HMMW15-51 has a better inhibitory effect after reducing the number of administrations and optimizing the route of administration.

Claims

1. Use of a novel polypeptide or its derivatives in the preparation of a drug for preventing or treating tumors, characterized in that: The novel polypeptide is selected from any one of the following: (1): A polypeptide having the same or substantially the same function formed by deleting three consecutive amino acids from the polypeptide of the amino acid sequence shown in SEQ ID NO.1; (2): A polypeptide having the same or substantially the same function formed by deleting any number of 1 to 34 amino acids from the N-terminus of the polypeptide of the amino acid sequence shown in SEQ ID NO.1; (3): A polypeptide with the amino acid sequence shown in SEQ ID NO.1 or a polypeptide with the same or substantially the same function formed by substituting one or more amino acids in (1) or (2).

2. Use of a novel polypeptide or a derivative thereof according to claim 1 in the preparation of a drug for preventing or treating tumors, characterized in that: In (1), the polypeptide having the same or substantially the same function is formed by lacking three consecutive amino acids at positions 3N-2, 3N-1 and 3N of the polypeptide of the amino acid sequence shown in SEQ ID NO.1, wherein N=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17; or In (2), the polypeptide having the same or substantially the same function is formed by deleting 4, 9, 14, 19, 24, 29 or 34 amino acids from the N-terminus of the polypeptide of the amino acid sequence shown in SEQ ID NO.1; or In (3), the amino acid at position 2, 3, 8, 9, 10, 11, 12, 13, 17, 19, 20, 21, 22, 25, 27, 28, 30, 31, 32, 40, 41, 42, 43, 44, 45, 48, 49 or 50 of the amino acid sequence shown in SEQ ID NO.1 is substituted.

3. Use of a novel polypeptide or a derivative thereof according to claim 2 in the preparation of a drug for preventing or treating tumors, characterized in that: The amino acid sequence of the novel polypeptide is shown in any one of SEQ ID NO.2 to SEQ ID NO.

53.

4. Use of a novel polypeptide or a derivative thereof according to claim 3 in the preparation of a drug for preventing or treating tumors, characterized in that: The amino acid sequence of the novel polypeptide is shown in any one of SEQ ID NO.2 to SEQ ID NO.4, SEQ ID NO.6 to SEQ ID NO.10, SEQ ID NO.13, SEQ ID NO.15 to SEQ ID NO.17, SEQ ID NO.19 to SEQ ID NO.21, SEQ ID NO.26 to SEQ ID NO.51, and SEQ ID NO.

53.

5. Use of a novel polypeptide or a derivative thereof according to any one of claims 1 to 4 in the preparation of a drug for preventing or treating tumors, characterized in that: The derivatives include modified derivatives of the novel polypeptide having the same or substantially the same function.

6. Use of a novel polypeptide or a derivative thereof according to claim 5 in the preparation of a drug for preventing or treating tumors, characterized in that: The modification includes one or more of N-terminal modification, C-terminal modification, side chain modification, amino acid modification or backbone modification.

7. Use of a novel polypeptide or a derivative thereof according to claim 6 in the preparation of a drug for preventing or treating tumors, characterized in that: The modification includes one or more of polyethylene glycol modification, fatty acid linkage, Fc polypeptide fusion, binding to human serum albumin, non-natural amino acid replacement, polypeptide cyclization, or long-acting modification of high-end preparations.

8. Use of a novel polypeptide or a derivative thereof according to claim 7 in the preparation of a drug for preventing or treating tumors, characterized in that: The modification includes fatty acid linkage, and the fatty acid linkage includes N-terminal linkage of 12-20 carbon fatty acids; or using a linker to link 12-20 carbon fatty acids.

9. Use of a novel polypeptide or a derivative thereof according to claim 8 in the preparation of a drug for preventing or treating tumors, characterized in that: The fatty acid linkage includes N-terminal linkage of 18-carbon fatty acids; or linking 18-carbon fatty acids using a linker, wherein the linker is a peptide segment with a length of less than 5 amino acids.

10. Use of a novel polypeptide or a derivative thereof according to any one of claims 1 to 9 in the preparation of a drug for preventing or treating tumors, characterized in that: The tumor includes one or more of brain glioma, neuroblastoma, head and neck cancer, esophageal cancer, thyroid cancer, lung cancer, liver cancer, kidney cancer, breast cancer, cervical cancer, uterine cancer, ovarian cancer, colon cancer, small intestine cancer, ileocecal cancer, gastric cancer, bladder cancer, pancreatic cancer, prostate cancer, bile duct cancer, melanoma, sarcoma, myeloma, lymphoma, and leukemia.

11. Use of a novel polypeptide or a derivative thereof according to claim 10 in preparing a drug for preventing or treating tumors, characterized in that: The prevention or treatment of tumors includes inhibiting the proliferation and / or metastasis of tumor cells.

12. Use of a novel polypeptide or a derivative thereof according to claim 11 in preparing a drug for preventing or treating tumors, characterized in that: The prevention or treatment of tumors includes inhibiting the proliferation of tumor cells, and the tumors and cells include: The glioblastoma cancer cells include: U87-MG; The neuroblastoma cancer cells include: SH-SY5Y; The head and neck cancer cells include: any one of oral epidermoid carcinoma cells KB, laryngeal epidermoid carcinoma cells HEp2, tongue squamous cell carcinoma cells CAL27, and nasopharyngeal carcinoma cells CNE-2Z; The esophageal cancer cancer cells include: Eca-109 and / or TE-13; The thyroid cancer cells include: NPPA87-1; The lung cancer cells include: any one or a combination of 95-D, SK-MES-1, SPC-A1, and A549; The liver cancer cells include: HCCLM3 and / or HepG2; The renal cancer cells include: Ketr-3 and / or 786-O; The breast cancer cells include: SK-BR-3 and / or MDA-MB-231; The cancer cells of cervical cancer include: CaSki and / or Hela; The uterine cancer cancer cells include: RL-952 and / or MS751; The ovarian cancer cells include: ES-2 and / or SKOV3 The colon cancer cells include: HT29 and / or SW480; The cancer cells of small intestinal cancer include: HIC; The cancer cells of ileocecal cancer include: HCT-8; The gastric cancer cells include: MGC-803 and / or BGC-823; The bladder cancer cancer cells include: T24 and / or EJ; The pancreatic cancer cells include: ASPC-1 and / or PANC-1; The prostate cancer cancer cells include: 22RV1 and / or PC-3; The cholangiocarcinoma cancer cells include: QBC939; The melanoma cancer cells include: SK-mel-2 and / or A375; The cancer cells of the sarcoma include: MG63 and / or HT-1080; The myeloma cancer cells include: LP-1; The lymphoma cancer cells include: any one or a combination of Raji, RPMI 8226, U937, and WSU-DLCL2; The leukemia cancer cells include any one or a combination of CEM / C1, NB4, KG-1, K562, HL60, Jurkat, and Clone E6-1.

13. Use of a novel polypeptide or a derivative thereof according to claim 11 in preparing a drug for preventing or treating tumors, characterized in that: The prevention or treatment of tumors includes inhibiting the metastasis of tumor cells, and the tumors and cells include: The melanoma cancer cells include: A375; The cancer cells of cervical cancer include: Hela; The ovarian cancer cancer cells include: Skvo3; The liver cancer cells include: Hep3B; The lung cancer cells include: A549.

14. A pharmaceutical composition for preventing or treating tumors, characterized in that: The pharmaceutical composition contains at least the novel polypeptide or its derivative according to any one of claims 1 to 9, and a pharmaceutically acceptable carrier, and the tumor includes one or more of glioma, neuroblastoma, head and neck cancer, esophageal cancer, thyroid cancer, lung cancer, liver cancer, kidney cancer, breast cancer, cervical cancer, uterine cancer, ovarian cancer, colon cancer, small intestine cancer, ileocecal cancer, gastric cancer, bladder cancer, pancreatic cancer, prostate cancer, bile duct cancer, melanoma, sarcoma, myeloma, lymphoma, and leukemia.

Images