CN111100189B - Polypeptide for treating cancer and pharmaceutical composition thereof - Google Patents

Abstract

The present invention relates to polypeptides and pharmaceutical compositions thereof for treating cancer. Specifically, the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1. The polypeptides of the invention also include mutants of SEQ ID NO. 1. The polypeptide of the present invention can inhibit cancer cell proliferation obviously.

Description

Polypeptide for treating cancer and pharmaceutical composition thereof

Technical Field

The invention relates to the field of biological medicine, in particular to a polypeptide for treating cancer and a pharmaceutical composition thereof.

Background

Gastric cancer is a common tumor of the digestive system, belongs to malignant tumors with higher mortality rate of patients in the world, particularly China, has hidden disease course and is extremely difficult to diagnose in early stage. More than 70% of new gastric cancer cases worldwide are in developing countries, more than half of which come from eastern asia regions, with china, japan and korea being particularly severe. At present, gastric cancer is the tumor with the second incidence rate in China. Because of the structural characteristics of the stomach wall, the early gastric cancer can hardly be transferred, and the lesion part is directly and partially resected, so that the recovery is greatly expected. According to the american cancer society data, the 5-year survival rate of the earliest gastric cancer (first stage of first stage) can reach 71% and the 5-year survival rate of the latest gastric cancer (fourth stage) is only 4%. In China, however, about 80% of patients have advanced stage by the time they find due to lack of early diagnosis, so gastric cancer is also a tumor with second mortality rate in China.

Meanwhile, the cause of gastric cancer is very complex, and genetic factors, dietary structure, working pressure, infection of helicobacter pylori and other reasons are all related to the occurrence of gastric cancer. The gastric cancer has strong organ specificity and high heterogeneity in the generation process, causes the difference of malignant biological characteristics such as tumor infiltration, metastasis capability and the like, and affects prognosis to different degrees. At present, the international research on the molecular typing of gastric cancer is still very preliminary, and the deep pathological mechanism of the gastric cancer is poorly known, so that the diagnosis and treatment means for targeting different types of gastric cancer are also lacking. In contrast, in the background of high incidence of gastric cancer in China, an effective diagnosis and treatment means aiming at gastric cancer is lacking, and the situation is very serious.

Currently, more than 80 polypeptide drugs are approved for sale globally, and hundreds of polypeptide drugs are in clinical trial or preclinical trial stages. Polypeptide drugs are receiving increasing attention from the biomedical industry due to wide indications, high safety and remarkable curative effects. The polypeptide medicine market analysis report shows that the annual compound growth rate of the polypeptide medicine market in recent years exceeds 2%, and the market demand of the polypeptide therapeutic medicine in the future is extremely large.

Disclosure of Invention

The present invention provides an isolated polypeptide selected from the group consisting of: (1) a polypeptide with an amino acid sequence shown as SEQ ID NO. 1; and (2) a polypeptide derived from SEQ ID NO. 1, which is substituted, deleted and/or added with one or more amino acids in the amino acid sequence defined by SEQ ID NO. 1 and can inhibit proliferation of gastric cancer cells.

In one or more embodiments, the polypeptide of (2) has amino acid substitutions, deletions and/or additions at positions up to 12 other than positions 8, 9 and 13 of SEQ ID NO. 1.

In one or more embodiments, the amino acid substitutions comprise: 1-4, preferably 2 amino acids are substituted with cysteines and/or the amino acid corresponding to position 12 of SEQ ID NO. 1 is substituted with norleucine. Preferably, the substitution of 1-4, preferably 2 amino acids with cysteine comprises: e corresponding to position 14 of SEQ ID NO. 1 is mutated to C and/or I corresponding to position 10 of SEQ ID NO. 1 is mutated to C.

In one or more embodiments, the amino acid deletion includes: amino acid deletions within 7 of the N-terminus of SEQ ID NO. 1, amino acid deletions within 3 of the C-terminus of SEQ ID NO. 1, and/or deletions of 1-3 amino acid residues in the sequence of SEQ ID NO. 1, e.g., A deletions corresponding to position 16 of SEQ ID NO. 1.

In one or more embodiments, the amino acid substitutions, deletions and/or additions include one or more of the following mutations: amino acid deletions within 7 of the N-terminus of SEQ ID NO. 1, amino acid deletions within 3 of the C-terminus of SEQ ID NO. 1, mutations of 1-4 amino acid residues to cysteine, M deletions or mutations corresponding to position 12 of SEQ ID NO. 1 to norleucine, and A deletions corresponding to position 16 of SEQ ID NO. 1.

In one or more embodiments, the polypeptide is a fragment of SEQ ID NO. 1 that contains at least RRIK (M) LEY.

In one or more embodiments, the polypeptide is a fragment of SEQ ID No. 1 containing at least LVRRIK (M) LEY, and the fragment: amino acid substitutions, deletions and/or additions occur at positions within 5 positions corresponding to positions 8, 9 and 13 of SEQ ID NO. 1, said amino acid substitutions, deletions and/or additions comprising: 1-4 amino acid residues are mutated to cysteine, an M deletion corresponding to position 12 of SEQ ID NO. 1 or to norleucine, and/or an A deletion corresponding to position 16 of SEQ ID NO. 1; preferably, the mutation of 1-4 amino acid residues into cysteine comprises: e corresponding to position 14 of SEQ ID NO. 1 is mutated to C and/or I corresponding to position 10 of SEQ ID NO. 1 is mutated to C.

In one or more embodiments, the polypeptide is selected from the group consisting of: SEQ ID NOs 3, 4, 5, 6, 7, 8, 11, 12, 14 and 15.

The invention also provides an isolated modified polypeptide having an amino acid sequence as described in any of the preceding embodiments and having modifications at the N and/or C terminus of the polypeptide, optionally the polypeptide also having disulfide bond modifications.

In one or more embodiments, the N-terminal modification is an acetyl modification and the C-terminal modification is an amidation modification.

In one or more embodiments, the modified polypeptide is substituted with cysteines at amino acids corresponding to positions 10 and 14 of SEQ ID NO. 1, respectively, and disulfide bonds are formed between the two cysteines through their sulfhydryl groups; optionally, the polypeptide further comprises one or more mutations selected from the group consisting of: amino acid deletions within 7 of the N-terminus of SEQ ID NO. 1, amino acid deletions within 3 of the C-terminus of SEQ ID NO. 1, M deletions or mutations corresponding to position 12 of SEQ ID NO. 1 to norleucine and/or A deletions corresponding to position 16 of SEQ ID NO. 1.

In one or more embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO 3, 4, 5, 6, 7, 8, 11, 12, 14 or 15.

The invention also provides a polynucleotide sequence, characterized in that the polynucleotide sequence is selected from the group consisting of:

(1) A coding sequence for an isolated polypeptide according to any of the embodiments herein; and

(2) (1) the complement of the coding sequence.

The invention also provides nucleic acid constructs comprising the polynucleotide sequences described herein.

Also provided is a pharmaceutical composition characterized in that the isolated polypeptide or the isolated modified polypeptide of any of the embodiments of the pharmaceutical composition is in combination with a pharmaceutically acceptable carrier.

Also provided is the use of an isolated polypeptide or an isolated modified polypeptide, polynucleotide sequence or nucleic acid construct as described in any of the embodiments herein in the manufacture of a medicament for the treatment or prevention of a MST 2-mediated or Hippo signal pathway-mediated disease.

In one or more embodiments, the disease is cancer, more preferably the disease is selected from papillary renal carcinoma, medulloblastoma, lung cancer, pancreatic cancer, esophageal cancer, liver cancer, stomach cancer, colon cancer, prostate cancer, and ovarian cancer.

Drawings

Fig. 1: the structure of the S-peptide is schematically shown.

Fig. 2: effect of S-peptide on cell proliferation. A. After different HGC-27 cell lines and GES-1 normal cells are treated by S-peptide with different concentrations, the inhibition rate of cell proliferation, and the control polypeptide and the drug carrier PDPA are both control groups. B. Inhibition rate of cell proliferation after S-peptide treatment of different gastric cancer cell lines and GES-1 normal cells. C. The clone formation experiment detects the influence of S-peptide on gastric cancer cell proliferation.

Fig. 3: pharmacokinetic analysis of S-peptide.

Fig. 4: s-peptide and mutant polypeptide thereof have effect on tumor in mouse gastric cancer model. A. Influence of control polypeptide, S-peptide and mutant polypeptide on gastric tumor area of mice under different treatment conditions. B. The effect of the control polypeptide, S-peptide and mutant polypeptide on proliferation of gastric tumor cells in mice was analyzed by immunohistochemistry.

Fig. 5: and detecting the effect target point of the S-peptide. A. The pull-down experiment detects interactions between polypeptide, protein PP2Aa and protein Strn 3. B. In vitro dephosphorylation assay, the level of phosphorylation of MST2 was detected.

Detailed Description

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute a preferred technical solution.

A great deal of experimental evidence suggests that Hippo signaling pathways play important roles in organ size regulation, carcinogenesis, tissue regeneration, and stem cell function regulation. Aberrant activation of YAP, the major effector of the Hippo pathway, is closely related to the development of a variety of tumorigenesis. The Hippo pathway, which serves as an oncogene to regulate upstream and core molecules, once dysregulated, causes uncontrolled proliferation and apoptosis inhibition, and YAP and TAZ hyperactivity may promote proliferation of tumor cells, such as papillary renal carcinoma, medulloblastoma, lung cancer, pancreatic cancer, esophageal cancer, liver cancer, gastric cancer, colon cancer, prostate cancer, and ovarian cancer, in which high YAP/TAZ expression or intracellular enrichment is detected. MST2 kinase is a key kinase in the Hippo signaling pathway. The present invention has found that a series of polypeptides based on the sequence of the polypeptide shown in SEQ ID NO. 1 compete with STRN3 for binding, thereby disrupting the dephosphorylation of MST2 by PP2A and thereby inhibiting activation downstream of the Hippo signaling pathway.

Therefore, the invention provides a polypeptide which can damage the dephosphorylation of PP2A to MST2, inhibit the activation of downstream of a Hippo signal pathway, has obvious inhibition effect on cancer cell proliferation, and can be used for treating and preventing cancers.

The basic structure of the polypeptide provided by the invention is shown as SEQ ID NO. 1. The invention can be practiced using SEQ ID NO. 1 or a fragment thereof or a polypeptide having one or more amino acid mutations (including insertions, deletions and/or substitutions) based on the sequence of SEQ ID NO. 1 or a fragment thereof. The number of amino acid mutations can be up to 12, such as within 12, within 10, within 8, or within 5. Any two or all three mutations in amino acid insertions, deletions and substitutions may be present together; preferably, in some embodiments of the invention, the mutation is the simultaneous presence of a deletion and a substitution. M at position 12 of SEQ ID NO. 1 may be deleted or substituted with other amino acid residues without affecting the biological activity of SEQ ID NO. 1 or fragments thereof. Herein, "fragment" is to a portion of a full-length sequence. Preferably, the fragment of SEQ ID NO. 1 comprises at least LVRRIK (M) LEY, "(M)" means that M may be present or absent. The invention encompasses fragments of LVRRIK (M) LEY in which "M" may be present, absent, or substituted with other amino acid residues; for example, in some embodiments, M is substituted with norleucine.

R, R and L at positions 8, 9 and 13 in the amino acid sequence shown in SEQ ID NO. 1 are conserved sites. Thus, the mutations that occur in the mutant of SEQ ID NO. 1 do not occur at these three positions. Polypeptides of the invention also include polypeptides having amino acid mutations at one or more positions other than positions 8, 9 and 13 (e.g., 12 or less, 10 or less, 8 or less, 5 or less, or 3 or less). In some embodiments, the mutation also does not occur at position 17. In certain embodiments, the polypeptides of the invention have amino acid substitutions at two positions other than positions 8, 9, 13 and 17 on the basis of the sequence of SEQ ID NO. 1, the corresponding amino acids of which are replaced with cysteines (C). In certain embodiments, the 6 and 10 positions of SEQ ID NO. 1 are replaced with cysteines. However, in certain embodiments, conservative amino acid substitutions may also occur at one or more of positions 8, 9, 13 and 17 of SEQ ID NO. 1. Exemplary mutations include, but are not limited to, amino acid deletions within 7 (preferably within 5) of the N-terminus of SEQ ID NO. 1, amino acid deletions within 3 (e.g., 3, 2, or 1) of the C-terminus, I to C at position 10, M to norleucine at position 12, E to C at position 14, and/or A to 16. In some embodiments, the polypeptide of the invention is a fragment of SEQ ID No. 1 containing at least LVRRIK (M) LEY, and the fragment optionally comprises: (1) I and E are mutated to C, and (2) M is optionally deleted or mutated to norleucine.

Amino acids are generally divided into four classes: (1) acidity-aspartic acid and glutamic acid; (2) alkaline-lysine, arginine, histidine; (3) Nonpolar-alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; (4) Uncharged polarity-glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan and tyrosine are sometimes categorized as aromatic amino acids. Thus, when substitution mutation is made, substitution mutation is performed using amino acids belonging to the same class, and it is generally considered that the biology of the resulting amino acid sequence is not affected. For example, isoleucine or valine may be used instead of leucine, glutamic acid instead of aspartic acid, serine instead of threonine, or similar amino acids with structurally related amino acids, such substitutions would not have a significant impact on biological activity. For replacing SEQ ID NO: the amino acid of the amino acid residue 1 may also be an unnatural amino acid, e.g., norleucine, etc. In certain embodiments, methionine at position 12 of SEQ ID NO. 1 is replaced with norleucine.

In certain embodiments, the polypeptide of the invention is a mutant of SEQ ID NO. 1, which is cysteine at amino acid residues corresponding to positions 6 and 10 of SEQ ID NO. 1, respectively, as compared to SEQ ID NO. 1. In certain embodiments, further, the mutant has a norleucine at the amino acid residue corresponding to position 12 of SEQ ID NO. 1.

The polypeptides of the invention may have modifications at the N-terminus and/or the C-terminus. For example, the N-terminus of the polypeptide may be modified with a hydrophobic group and the C-terminus may be modified with stabilization. The hydrophobic group modification and stabilization modification may be various modifications well known in the art. For example, hydrophobic groups for modifying the N-terminus include, but are not limited to, acetyl, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, cholesterol, arachidonic acid, and the like, with myristic acid, palmitic acid, stearic acid, or cholesterol being preferred. Stabilization modifications at the C-terminus include, but are not limited to, amidation modifications (i.e., amination modifications) and isopentyl glycol modifications. The above-described modification of the N-terminus and/or C-terminus may improve the stability of the polypeptide. In certain embodiments, the polypeptides of the invention have amidation modifications at both the N-and C-terminus. In certain embodiments, the polypeptides of the invention have an acetylation modification at the N-terminus and an amidation modification at the C-terminus.

In certain embodiments, polypeptides of the invention may have disulfide modifications thereon. For example, as previously described, the polypeptides of the invention have amino acid substitutions at two positions other than positions 8, 9, 13 and optionally 17 on the basis of the sequence SEQ ID NO. 1, the corresponding amino acids of which are replaced by cysteines (C), the sulfhydryl groups of which can form disulfide bonds. Suitable amino acid positions to be replaced with C include, but are not limited to, positions 6, 10 and 14 of SEQ ID NO. 1. In certain embodiments, the polypeptides of the invention are variants of SEQ ID NO. 1 or fragments thereof, which mutants are cysteine at amino acid residues corresponding to positions 6 and 10 of SEQ ID NO. 1, respectively, and are modified between these two cysteines by disulfide bond formation via their sulfhydryl groups, as compared to SEQ ID NO. 1. In some embodiments, the amino acids at positions 10 and 14 of SEQ ID NO. 1 or a fragment thereof, respectively, are replaced with cysteines, and disulfide bond modifications are formed between the two cysteines through their sulfhydryl groups. In addition to the above mutations being cysteines, various mutations described above may be present in SEQ ID NO. 1 or fragments thereof. In certain embodiments, the polypeptide of the invention is a variant of SEQ ID NO. 1, the mutant has cysteines at amino acid residues corresponding to positions 6 and 10 of SEQ ID NO. 1, respectively, and disulfide bond modifications are formed between the cysteines by their sulfhydryl groups, as compared to SEQ ID NO. 1; and the amino acid residue corresponding to position 12 of SEQ ID NO. 1 is norleucine.

The modified polypeptides have improved stability.

The amino acid sequences of the present invention may be the products of chemical synthesis. For example, the amino acid sequences of the present invention can be synthesized using polypeptide chemical synthesis methods well known in the art. The chemical synthesis method of the polypeptide includes a solid phase synthesis method and a liquid phase synthesis method, wherein the solid phase synthesis method is commonly used. Solid phase synthesis methods include, but are not limited to, fmoc and tBOC. Typically, using resins as insoluble solid supports, amino acids are attached to the peptide chain, typically one by one, from the C-terminus (carboxy-terminus) to the N-terminus (amino-terminus), each amino acid attachment cycle consisting of the following three steps of reaction: 1) Deprotection: the protected amino acid must be deprotected with a deprotecting solvent to remove the protecting group for the amino group; 2) Activating: the carboxyl group of the amino acid to be linked is activated by an activator; and 3) coupling: the activated carboxyl group reacts with the naked amino group of the previous amino acid to form a peptide bond. The cycle is repeated until the peptide chain is extended to the desired length. Finally, the cleavage solution is used to cleave the connection between the peptide chain and the solid support, thus obtaining the required amino acid sequence. The chemical synthesis described above may be performed on a programmed automated polypeptide synthesizer, such as, but not limited to, the Tribute two-channel polypeptide synthesizer from Protein Technologies, the UV Online Monitor system from CS Bio, the Focus XC three-channel synthesizer from Aapptec, and the like.

The invention also includes the coding sequences of the polypeptides of the invention and their complements. Where appropriate, recombinant expression vectors may be employed to produce the polypeptides of the invention. The structural composition of recombinant expression vectors, methods of construction and methods of expression are well known in the art.

Modifications of polypeptides are well known in the art. For example, acetylation of the N-terminus may be performed using a DMF solution of 10% acetic anhydride and 6% pyridine for 20mins. In C-terminal amidation, the carboxyl terminal can be activated with HOSu (N-hydroxysuccinimide) and then reacted with ethylamine, or activated with CDI (carbonyldiimidazole) and then amidated with ethylamine. Cyclization between two cysteines can introduce side chain modifications (e.g., benzene rings) into the side chains of the polypeptide by alkylation of the sulfhydryl groups with dibromotoluene reagents. Further chemical modifications of the polypeptides can be found in Adam g.kreutzer et al, standard practices for Fmoc-based solid-phase peptide synthesis in the Nowick laboratory, version 1.6.3.

The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide of the invention. As used herein, the term "effective amount" refers to an amount that is functional or active in and acceptable to a human and/or animal. For example, for a liquid formulation or composition, the concentration of the polypeptide may be 20ng/ml or more, such as 50ng/ml or more, 80ng/ml or more, 100ng/ml or more.

The pharmaceutical composition may contain a pharmaceutically acceptable carrier. Herein, "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. The term refers to such agent carriers: they are not per se essential active ingredients and are not overly toxic after administration. Suitable vectors are well known to those of ordinary skill in the art. The pharmaceutically acceptable carrier in the composition may contain a liquid, such as water, saline, buffer. In addition, auxiliary substances such as fillers, lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.

The pharmaceutical compositions of the present invention may be administered to a subject in a variety of suitable ways conventional in the art, including, but not limited to: oral, subcutaneous, intramuscular, transdermal, topical, implant, sustained release, and the like.

The invention also provides the use of a polypeptide as described herein in the manufacture of a medicament for the treatment or prophylaxis of a disease mediated by MST2 or Hippo signalling. Herein, the "MST2 mediated" disease refers to a disease benefiting from disruption or inhibition of dephosphorylation of MST2, and the "Hippo signaling pathway mediated" disease refers to a disease benefiting from inhibition of activation downstream of the Hippo signaling pathway. It should be understood that "inhibition" as described herein generally refers to expression relative to normal cells (including normal non-diseased tissue cells and normal diseased tissue cells). In a further aspect, the invention provides the use of a polypeptide as described herein in the manufacture of a medicament for inhibiting proliferation of cancer cells. MST 2-mediated or Hippo signaling pathway-mediated diseases described herein generally include various types of known cancers that express MST2 or Hippo signaling pathway, including, but not limited to, papillary renal cancers, medulloblastoma, lung cancer, pancreatic cancer, esophageal cancer, liver cancer, stomach cancer, colon cancer, prostate cancer, ovarian cancer, and the like.

In a preferred embodiment, the invention provides the use of a polypeptide as described herein for the treatment or prevention of gastric cancer or for the preparation of a medicament for the treatment or prevention of gastric cancer. Herein, the stomach cancer may be various kinds of stomach cancer known in the art, such as classified by general forms, and may be classified into early stomach cancer and progressive stomach cancer; classified by histopathology, it can be classified into adenocarcinoma, adenosquamous carcinoma, squamous carcinoma, carcinoid, etc., most of which are gastric adenocarcinoma; classified according to the location of the disease, it is classified into carcinoma of the gastric fundus and cardiac, carcinoma of the gastric body, carcinoma of the antrum, etc.; molecular typing, including EBV infection, microsatellite instability, genome stability and chromosome instability; according to the pathogenic cause, gastric cancer caused by helicobacter pylori infection and the like can be included.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer.

Example 1: design and synthesis of S-peptide

The inventor designs a polypeptide with a sequence of NLKKDLVRRIKMLEYAL (SEQ ID NO: 1) by taking a gene STRN3 (NM_001083893.1, NP_001077362) as a template according to the existing experimental result, and transmits the polypeptide to Shanghai biological limited company for synthesis. According to the disclosed solid-phase polypeptide synthesis method, firstly, hydroxyl groups of hydroxyl terminal amino acids of peptide chains to be synthesized are connected with insoluble high-molecular resin by means of covalent bond structure, then amino acids combined on the solid-phase carrier are used as amino components, and undergone the processes of deamination protecting group and reaction with excessive activated carboxyl component so as to obtain the invented peptide chain. Repeating the steps of condensation, washing, deprotection, neutralization and washing, and condensing to obtain the desired peptide chain, finally cleaving the peptide chain from the resin, purifying, and synthesizing to obtain the desired polypeptide, wherein the structure is shown in figure 1.

The polypeptide SEQ ID NO:1 to obtain a control polypeptide with a sequence of RELKLDARMKLLVNYIK (SEQ ID NO: 2). Solid-phase polypeptide synthesis is also disclosed by Shanghai Biotechnology Inc.

Synthesizing to obtain the polypeptide with purity greater than 98%.

Example 2: effect of S-peptide on cell proliferation

1. The experimental method is as follows:

cell culture: SNU-16, HGC-27, SNU-1, MKN-45, AGS, MNK-28, SH-10-TC, MKN-1, NC-NH, SNU-216, KE-39, BGC-823, GES-1 cells (all obtained from commercial sources) were cultured in RPMI1640 (Invitrogen) medium, 10% serum, 100ug/ml penicillin, 100ug/ml streptomycin was added to the medium. Cells were cultured at 37℃and carbon dioxide concentration was 5%.

Cell proliferation assay: detection of cell proliferation Using ATP cell viability assay kit (Promega Co., ltdLuminescent Cell Viability Assay). Cells with medium were prepared in a 96-well plate with opaque walls, 100. Mu.l/well, cell number 3000/well, and S-peptide (0, 10, 20, 30. Mu.g/ml, etc.) at different concentrations were added after the next day of adherence, while control wells containing only medium and no cells were prepared to obtain background luminescence values. After 48h cell viability was determined with Promega CellTitter reagent. The plates and their contents were equilibrated to room temperature for approximately 30 minutes. Add +.about.equal volume of cell culture medium to each well>Reagent 100. Mu.l. The contents were mixed on an orbital shaker for 2 minutes to induce cell lysis, and then the plates were incubated at room temperature for 10 minutes to stabilize the fluorescent signal values and record the luminescence signal.

Soft agarose cell clone formation experiments: the cell density reaches 10 ⁴ Clones with a diameter of greater than 0.05 mm were counted after 14 days on soft agarose in 6-well plates.

Cell IC ₅₀ : cell proliferation experiment results, data were processed using Graphpad Prism software to obtain IC ₅₀ Numerical values.

Data analysis: data were analyzed using SAS data software analysis package (9.1.3), and the mean ± standard deviation of the data was counted. Single factor variant analysis (ANOVA) and Student's t-test were used to analyze the continuous variable. The confidence interval is P <0.05.

Drug metabolism kinetics: and taking blood from the mice after administration, detecting the plasma mass spectrometry obtained, and calculating and analyzing the absorption distribution metabolism excretion of the drug in the body by using Graphpad software according to the drug concentration in the blood of the mice at different times to obtain the relevant parameters of the drug metabolism dynamics.

2. The experimental reagents were as follows:

s-peptide (SEQ ID NO: 1) and control polypeptide (SEQ ID NO: 2) are dissolved in DMSO (dimethyl sulfoxide), and then phosphate buffer salt solution (PBS) is added for dilution, wherein the final concentration of DMSO is 0.1%, and the mixture is used for experiments.

Phosphate Buffered Saline (PBS): 800ml distilled water is dissolved 0.2g KCl,8g NaCl,0.24g KH ₂ PO ₄ And 1.44g Na ₂ HPO ₄ . The pH of the solution was adjusted to 7.4 with HCl and the volume was set to 1L. Autoclaving or filtration sterilization.

PDPA (Poly (diisopropylaminoethyl methacrylate) (ACS Nano.2016;10 (3): 3496-508) was used as a drug carrier in this example.

3. Experimental results and analysis

Use of S-peptide elsewhereThe gastric cancer cell line HGC-27 and the normal gastric mucosa cell GES-1 are respectively 5ug/ml, 10ug/ml, 15ug/ml, 20ug/ml and 25ug/ml by taking PDPA and PDPA+control polypeptide as controls. 48h later with Promega CorpThe reagent measures cell viability. The S-peptide has remarkable inhibition effect on the gastric cancer cell line HGC-27, and the inhibition rate of the S-peptide is increased along with the increase of the concentration. The inhibition of GES-1 cell proliferation by S-peptide was significantly reduced compared to HGC-27 (FIG. 2, A). By calculating cell IC ₅₀ IC of S-peptide pair HGC-27 ₅₀ 12.6ug/ml, and IC for GES-1 ₅₀ 76.9ug/ml. This indicates that S-peptide is effective against gastric cancer cell IC ₅₀ Is far lower than normal cells, and has obvious inhibition effect on the proliferation of gastric cancer cells.

Further, the effect of S-peptide on gastric cancer cell lines SNU-216, AGS, MKN-45 and the like was examined, and the results showed that S-peptide had a remarkable proliferation inhibition effect on these gastric cancer cell lines (FIG. 2, B).

Gastric cancer cell lines were treated with PDPA (control) and PDPA+S-peptide, respectively, by soft agarose cell clone formation experiments. The result shows that the S-peptide has obvious inhibition effect on the proliferation of gastric cancer cells. The longer duration of action of the soft agarose cell clone formation assay suggests that the inhibition of gastric cancer cell proliferation by S-peptide is sustained (fig. 2, c).

The results show that the S-peptide can obviously inhibit the proliferation of gastric cancer cells, and has limited proliferation inhibition effect on normal cells.

The pharmacokinetic parameters of the polypeptide were obtained by administering S-peptide to mice (FIG. 3).

Example 3: synthesis and testing of mutant polypeptides

A series of mutant polypeptides of SEQ ID NO. 1 were synthesized by Shanghai Biotechnology Co., ltd. And the protein sequences of the mutant polypeptides are shown in Table 1. Synthesizing to obtain the polypeptide with purity greater than 98%.

	Polypeptide sequence	Cell IC 50 (semi-inhibitory concentration)
			SEQ ID NO:1	NLKKDLVRRIKMLEYAL	4.10
SEQ ID NO:3	KKDLVRRIKMLEYL	5.74
			SEQ ID NO:4	DLVRRIKMLEYAL	7.91
SEQ ID NO:5	LVRRIKMLEYAL	13.70
			SEQ ID NO:6	LVRRIKMLEY	17.35
SEQ ID NO:7	RRIKLEYAL	20.36
			SEQ ID NO:8	RRIKLEY	171.64
SEQ ID NO:9	LVRRIK	1370.00
			SEQ ID NO:10	RRIKM	2490.00
SEQ ID NO:11	LVRRIKMLEY	17.35
			SEQ ID NO:12	LVRRCKN LE LCY	6.03
SEQ ID NO:13	LVAACKALCY	1230.00
			SEQ ID NO:14	RRIKMLEYAL	20.36
SEQ ID NO:15	RRCKN LE LCYAL	8.67
			SEQ ID NO:16	AACKALCYAE	1080.00
SEQ ID NO:17	Ac-LVRRCKN LE LCY-CO	5.85

Note that: n (N) ^LE Norleucine, ac, acetylating, and CO amidating.

The mutant polypeptides described above were tested for biological activity using the methods described in example 2. The result shows that in the mutant polypeptide, partial sequence is similar to the original SEQ ID NO. 1 polypeptide, and has obvious killing effect on gastric cancer cell line (Table 1), which shows that the gastric cancer cell proliferation can be inhibited by substituting, deleting and/or adding one or more amino acids in the amino acid sequence defined by SEQ ID NO. 1. The polypeptide SEQ ID NO. 1 can be modified at the N end and/or the C end, and the modified polypeptide can still inhibit the proliferation of gastric cancer cells.

Example 4: action of S-peptide and mutant polypeptide thereof on tumor in mouse gastric cancer model

1. The experimental method is as follows:

MNNG induction to establish mouse stomach cancer model

Mice were housed in a dedicated culture room for animal models of infectious diseases, receiving 12 hours of light, 12 hours of dark day and night rhythms. MNNG (100 mg/ml) was added to drinking water, and the administration was continued for 14 days, and the rhythm was suspended for 14 days, and the mixture was fed for 6 weeks, so that gastric cancer was modeled. The PDPA is used as a drug carrier, the polypeptide is injected into mice, the control group mice are injected with the control polypeptide SEQ ID NO.2, and the experimental group mice are injected with the S-peptide (SEQ ID NO. 1) or the mutant polypeptide (SEQ ID NO. 17). After 6 weeks of feeding, mice were sacrificed and tested.

Animal culture and animal experiments comply with the rules and animal welfare policies associated with the animal management committee of the institute of biochemistry and cell biology, shanghai, national academy of sciences.

Immunohistochemistry: tissue samples according to BD Pharmingen ^TM IHC Zinc Fixative handbook (handbook number: 550523), zinc agent fixation (BD Biosciences) was used and paraffin embedded. Tissue sections (5 μm thick) were fixed by heating, the sections were dewaxed in xylene for 5 minutes and replaced with fresh di-waterToluene was dewaxed and xylene was dewaxed in common 3 times. Absolute ethanol for 5 minutes, twice. 90% ethanol for 5 minutes, twice, 70% ethanol for 5 minutes, once. Distilled water for 5 minutes, twice. Depending on the antigen and antibody, the sections may be optionally placed in antigen retrieval solutions of 10mM sodium citrate, pH6.0, or 1mM EDTA, pH8.0, or 10mM Tris, pH10.0, 95℃for 12 minutes and slowly cooled to room temperature over about 30 minutes. 5% skim milk was added and blocked for 60 minutes.

All steps from the closing are necessary to pay attention to the moisture retention of the sample, avoiding drying of the sample, which would otherwise be extremely prone to a high background. The primary antibody was diluted in the appropriate ratio, incubated overnight with slow shaking at 4 ℃, the primary antibody was recovered, PBST was added, and washed for 5 minutes. After the washing liquid was sucked out, the washing liquid was added thereto and the mixture was washed for 5 minutes. The washing was performed 3 times. Horseradish peroxidase (HRP) or Biotin (Biotin) or Alkaline Phosphatase (AP) labeled secondary antibodies were diluted in appropriate proportions. Incubate at room temperature or 4℃on a side-shaking table with slow shaking for one hour. Recovering the secondary antibody. PBST wash was added and washed slowly with shaking on a side shaking table for 5 minutes. After the washing liquid was sucked out, the washing liquid was added thereto and the mixture was washed for 5 minutes. The washing was performed 3 times. DAB is selected for subsequent detection. DAB staining was followed by HE staining. Finally, dewatering, transparent and neutral resin sealing.

2. The experimental reagents were as follows:

s-peptide (SEQ ID NO: 1), mutant polypeptide (SEQ ID NO: 17) and control polypeptide (SEQ ID NO: 2) are dissolved in DMSO (dimethyl sulfoxide), and then are diluted by adding Phosphate Buffered Saline (PBS), wherein the final concentration of DMSO is 0.1%, and the final concentration is used for experiments.

Phosphate Buffered Saline (PBS): 800ml distilled water is dissolved 0.2g KCl,8g NaCl,0.24g KH ₂ PO ₄ And 1.44g Na ₂ HPO ₄ . The pH of the solution was adjusted to 7.4 with HCl and the volume was set to 1L. Autoclaving or filtration sterilization.

PDPA (Poly (diisopropylaminoethyl methacrylate) (ACS Nano.2016;10 (3): 3496-508) was used as a drug carrier in this example.

3. Experimental results and analysis:

the results are shown in FIG. 4. FIG. 4 shows that mice using S-peptide and its mutant polypeptide have significantly less gastric tumor area than the control group (FIG. 4, A). Similarly, immunohistochemical analysis was performed on the stomach of mice using the index Ki67 indicating the proliferation potency of cells, the control group had the highest Ki67 positive ratio and the cell proliferation was most active, whereas the experimental group using S-peptide and its mutant polypeptide had significantly decreased Ki67 positive ratio and significantly decreased cell proliferation potency (fig. 4, b). The result shows that the S-peptide and the mutant polypeptide thereof have obvious proliferation inhibition effect on stomach tumor cells of mice with gastric cancer models.

Example 5: action target point of S-peptide

1. The experimental method is as follows:

protein purification: in vitro protein purification of MBP-Strn3 ^CC The PP2Aa and PP2A, MST2 methods are the same as those disclosed in the published literature Tang, y., et al (2019).Cell Discov 5:3；Chen,C.C.,et al.(2014).Journal of Biological Chemistry 289(14):9651-9661。

Pull down experiment: MBP tag protein purified in vitro, namely MBP-Strn3 ^CC PP2Aa, S-peptide (SEQ ID NO: 1) or control polypeptide S ^mut (SEQ ID NO:18, NEKKDEVAAIKAEEYAE) was mixed with commercially available amyose filler (NEB E8021) at 4℃for 1 hour under different experimental conditions, buffer 20mM HEPES pH7.5,100mM NaCl,1mM DTT. After washing the packing with the above buffer 3 times, it was eluted with a buffer to which 20mM maltose was added. Samples were detected by SDS-PAGE and Coomassie blue staining.

In vitro dephosphorylation experiments: 2 mu M in vitro purified MST2 protein, in vitro purified PP2A protein, and synthetic S-peptide or control polypeptide S ^mut (SEQ ID NO: 18) in 20. Mu.l of dephosphorylation buffer (20mM HEPES pH7.5,100mM NaCl,10mM MgCl) ₂ 1mM DTT, cocktail), 30min incubation at 30 ℃. The phosphorylation level at position 180 of MST2 was detected by immunoblotting experiments.

Immunoblotting experiments: protein samples were prepared according to the experimental requirements, denatured at 100℃for 10min, centrifuged at 13200rpm for 2 min, and equal amounts of supernatant were added to the loading wells of SDS-PAGE gels. The protein samples were at 80V in the concentrated gel and at 120V in the separation gel. After electrophoresis, taking down the gel, and installing a film transfer device according to the following sequence: (negative electrode), filter paper, gel, nitrocellulose membrane, filter paper, (positive electrode). The film transfer device is placed in a refrigerator at 4 ℃ for transfer printing for 1h at a constant voltage of 100V. After the transfer was completed, the nitrocellulose membrane was taken out, and the membrane was immersed in 5% skim milk prepared with TBST buffer solution, and incubated on a shaker at room temperature for 1 hour. The membrane was washed with TBST buffer solution 5min X3 times.

Primary antibodies diluted in 5% bsa solution at the indicated ratio were added and incubated overnight on a shaker at 4 ℃ freezer. The membrane was washed with TBST buffer solution 10min X3 times. Adding secondary antibody diluted with 5% milk at a given ratio, and incubating on a shaker at room temperature for 40-60min. The membrane was washed with TBST buffer solution 10min X3 times. The chromogenic substrate was overlaid on a nitrocellulose membrane and developed for 2 minutes at room temperature. Shooting was performed with a LAS4000 luminescence/bioluminescence image analyzer.

2. The experimental reagents were as follows:

5% bsa solution: 5g of BSA powder was weighed and dissolved in 100ml of 1 XPBS solution, followed by the addition of 0.02% sodium azide and storage at 4 ℃.10 XWestern blot transfer buffer: 30.3g Tris base and 144g glycine were weighed, 300ml methanol was added and the volume was fixed to 1L with deionized water.

3. Experimental results and analysis:

the experimental results are shown in FIG. 5. In vitro pull down experiments show that PP2Aa can be combined with STRN3 ^CC Direct interaction of proteins, while the sequence of S-peptide is derived from the STRN3 gene, S-peptide can interact with STRN3 ^CC Competing for binding to PP2Aa, in the presence of S-peptide, PP2Aa is reacted with STRN3 ^CC Binding of the protein is broken. Whereas control polypeptide S ^mut The key site of the combination of STRN3 and PP2Aa is mutated, and the combination of the PP2Aa and the STRN3 cannot be influenced ^CC Interaction of proteins.

PP2Aa and STRN3 are both components of phosphatase PP2A, wherein STRN3 is a regulatory subunit of PP2A, determining substrate specificity of PP 2A. In vitro dephosphorylation experiments showed that PP2A recruited MST2 kinase via STRN3 to dephosphorylate it. The S-peptide competes with STRN3 to bind PP2Aa, thereby destroying the dephosphorylation of MST2 by PP2ATo give a control polypeptide S ^mut There is no effect on PP2A on dephosphorylation of MST 2.

Since MST2 kinase is a key kinase in the Hippo signal pathway, which is closely related to the occurrence and development of various cancers, S-peptide is derived from Strn3, and by competitively binding to PP2Aa, the dephosphorylation of PP2A to MST2 is destroyed, and activation of downstream of the Hippo signal pathway and proliferation of gastric cancer cells are inhibited.

Sequence listing

<110> Shanghai life science institute of China academy of sciences

<120> polypeptide for treating cancer and pharmaceutical composition thereof

<130> 188549z1

<150> 201811268642.4

<151> 2018-10-29

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

Asn Leu Lys Lys Asp Leu Val Arg Arg Ile Lys Met Leu Glu Tyr Ala

1 5 10 15

Leu

<210> 2

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Arg Glu Leu Lys Leu Asp Ala Arg Met Lys Leu Leu Val Asn Tyr Ile

1 5 10 15

Lys

<210> 3

<211> 14

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 3

Lys Lys Asp Leu Val Arg Arg Ile Lys Met Leu Glu Tyr Leu

1 5 10

<210> 4

<211> 13

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

Asp Leu Val Arg Arg Ile Lys Met Leu Glu Tyr Ala Leu

1 5 10

<210> 5

<211> 12

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 5

Leu Val Arg Arg Ile Lys Met Leu Glu Tyr Ala Leu

1 5 10

<210> 6

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 6

Leu Val Arg Arg Ile Lys Met Leu Glu Tyr

1 5 10

<210> 7

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

Arg Arg Ile Lys Leu Glu Tyr Ala Leu

1 5

<210> 8

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 8

Arg Arg Ile Lys Leu Glu Tyr

1 5

<210> 9

<211> 6

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 9

Leu Val Arg Arg Ile Lys

1 5

<210> 10

<211> 5

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 10

Arg Arg Ile Lys Met

1 5

<210> 11

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 11

Leu Val Arg Arg Ile Lys Met Leu Glu Tyr

1 5 10

<210> 12

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (7)..(7)

<223> Xaa is norleucine

<400> 12

Leu Val Arg Arg Cys Lys Xaa Leu Cys Tyr

1 5 10

<210> 13

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 13

Leu Val Ala Ala Cys Lys Ala Leu Cys Tyr

1 5 10

<210> 14

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 14

Arg Arg Ile Lys Met Leu Glu Tyr Ala Leu

1 5 10

<210> 15

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (5)..(5)

<223> Xaa is norleucine

<400> 15

Arg Arg Cys Lys Xaa Leu Cys Tyr Ala Leu

1 5 10

<210> 16

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 16

Ala Ala Cys Lys Ala Leu Cys Tyr Ala Glu

1 5 10

<210> 17

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> ACETYLATION

<222> (1)..(1)

<223> acetylation modification

<220>

<221> AMIDATION

<222> (10)..(10)

<223> amidation modification

<220>

<221> misc_feature

<222> (7)..(7)

<223> Xaa is norleucine

<400> 17

Leu Val Arg Arg Cys Lys Xaa Leu Cys Tyr

1 5 10

<210> 18

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 18

Asn Glu Lys Lys Asp Glu Val Ala Ala Ile Lys Ala Glu Glu Tyr Ala

1 5 10 15

Glu

Claims (9)

1. An isolated polypeptide selected from the group consisting of:

(1) A polypeptide with an amino acid sequence shown as SEQ ID NO. 1;

(2) A fragment of SEQ ID NO. 1 containing at least LVRRIKMLEY;

(3) The amino acid sequence is shown in SEQ ID NO:14 or 15; and

(4) A fragment of SEQ ID No. 1 comprising at least LVRRIKMLEY, and which fragment: the mutation of I to C at position 10 corresponding to SEQ ID NO. 1, the mutation of M to norleucine at position 12 corresponding to SEQ ID NO. 1 and the mutation of E to C at position 14 corresponding to SEQ ID NO. 1.

2. The polypeptide of claim 1, wherein (2) said polypeptide is selected from the group consisting of: SEQ ID NOs 3, 4, 5 and 6; the polypeptide of (4) is selected from SEQ ID NO:12.

3. an isolated polypeptide having an amino acid sequence according to any one of claims 1-2 and having an acetylation modification at the N-terminus and an amidation modification at the C-terminus.

4. A polypeptide according to claim 3, wherein the polypeptide is as described in (4) which is substituted with cysteines at positions 10 and 14 of SEQ ID No. 1, respectively, and disulfide bonds are formed between the cysteines via their sulfhydryl groups.

5. The polypeptide of claim 3, wherein the polypeptide is set forth in SEQ ID NO. 17.

6. A polynucleotide, wherein the sequence of the polynucleotide is selected from the group consisting of:

(1) A coding sequence for the polypeptide of any one of claims 1-2; and

(2) (1) the complement of the coding sequence.

7. A nucleic acid construct comprising the polynucleotide of claim 6.

8. A pharmaceutical composition comprising the polypeptide of any one of claims 1-5 and a pharmaceutically acceptable carrier.

9. Use of the polypeptide of any one of claims 1-5, the polynucleotide of claim 6, or the nucleic acid construct of claim 7 in the manufacture of a medicament for treating or preventing a MST 2-mediated or Hippo signal pathway-mediated disease; wherein the disease is gastric cancer.