STAT3 inhibiting polypeptide, anti-tumor drug and application of STAT3 inhibiting polypeptide and anti-tumor drug

Abstract

The invention provides an STAT3 inhibiting polypeptide, an anti-tumor drug and application, and belongs to the technical field of biological medicine. The amino acid sequence of the STAT3 inhibiting polypeptide is at least one of SEQ ID NO: 1 to SEQ ID NO: 3. According to the invention, an SH2 structural domain in a negative regulatory protein SH2 adapter protein F (Shf) of STAT3 is taken as a template, the sequence of the template is as shown in SEQ.ID NO: 4-OH, the template is combined with active fragments for splicing, a STAT3 inhibition polypeptide is designed, the STAT3 inhibition polypeptide has excellent STAT3 affinity, can be combined with highly expressed STAT3 in tumor cells and inhibit the activity of the STAT3, the high selectivity of the template to the tumor cells is retained, and the application of the STAT3 inhibition polypeptide to the tumor cells is realized. And the tumor inhibition effect is enhanced. Therefore, the STAT3 inhibiting polypeptide or the pharmaceutical salt thereof has potential anti-tumor clinical application value and wide development prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a STAT3 inhibitory polypeptide, an anti-tumor drug and an application thereof. Background Art

[0002] Tumors are a type of disease that seriously threatens human health. Their main characteristic is the uncontrolled proliferation of abnormal cells in the body, which can invade normal tissues or organs and even cause distant metastasis. Therefore, the number of deaths caused by tumors worldwide accounts for a high proportion. Chemotherapy is one of the important means of treating tumors, but its clinical application faces two major problems: one is the serious toxic side effects caused by chemotherapy drugs, and the other is that tumor cells develop resistance to chemotherapy drugs, which is also one of the important reasons for the failure of chemotherapy. Therefore, the development of new anti-tumor drugs with high selectivity and low resistance has become a research hotspot in the current field of tumor treatment.

[0003] Signal transducer and activator of transcription 3 (STAT3) is a key signaling protein that is widely distributed in a variety of cells and tissues. It mainly regulates life activities such as cell growth, proliferation and apoptosis, and is closely related to the occurrence and progression of tumors. In normal cells, STAT3 activation is usually rapid and short-lived, but in most tumor cells, STAT3 often shows abnormal and persistent activation. Activated STAT3 forms STAT3-STAT3 dimers through its SH2 domain, thereby upregulating the expression of anti-apoptotic protein Bcl-xL, proliferation-related protein c-Myc, pro-angiogenic factor VEGF, and invasion and metastasis-related protein MMP-2. These changes further induce inflammation and immunosuppression by inhibiting cell apoptosis, promoting proliferation, and enhancing invasion and metastasis, ultimately promoting the development of tumors. Therefore, the development of molecular targeted drugs targeting the STAT3 signaling pathway has important clinical significance and broad application prospects.

[0004] However, since STAT3 dimerization relies on protein-protein interactions and has a large interaction interface, using small molecule compounds or short peptides to block these interactions faces huge challenges. In addition, the aggregation of STAT3 positively charged residues places high demands on the negative charge characteristics of the compound, further increasing the difficulty of drug development. Currently, compounds targeting STAT3 are still in the preclinical research stage. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a STAT3 inhibitory polypeptide, an anti-tumor drug and application, wherein the STAT3 inhibitory polypeptide has excellent STAT3 affinity and significant anti-tumor effect.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a STAT3 inhibitory polypeptide, characterized in that the amino acid sequence of the STAT3 inhibitory polypeptide is at least one of SEQ ID NO: 1 to SEQ ID NO: 3.

[0008] Preferably, the hydroxyl group in the carboxyl group in the Arg structure at the C-terminus of the STAT3 inhibitory polypeptide is replaced by an amino group.

[0009] The present invention provides an anti-tumor drug, which comprises the above-mentioned STAT3 inhibitory polypeptide or a pharmaceutically acceptable salt thereof.

[0010] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0011] The present invention also provides a use of the above STAT3 inhibitory polypeptide in the following a and / or b:

[0012] a. Preparation of anti-tumor products;

[0013] b. Prepare products for inhibiting STAT3 protein.

[0014] Preferably, when preparing an anti-tumor product, the tumor is a tumor that highly expresses STAT3.

[0015] Preferably, when preparing the anti-tumor product, the tumor includes one or more of glioma, leukemia and liver cancer.

[0016] The present invention also provides a use of the STAT3 inhibitory polypeptide in the preparation of a drug for targeted tumor treatment.

[0017] Preferably, when used in the above-mentioned targeted tumor treatment drug, the tumor is a tumor that highly expresses STAT3.

[0018] Preferably, when used in the above-mentioned targeted tumor therapy drug, the tumor includes one or more of glioma, leukemia and liver cancer.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention provides a STAT3 inhibitory polypeptide, an anti-tumor drug and application. The SH2 domain in the SH2 adaptor protein F (Shf), a negative regulatory protein of STAT3, is used as a template. The template sequence is shown in SEQ.ID NO: 4-OH. Combined with active fragments, a class of novel STAT3 inhibitory polypeptides are designed. The polypeptides have excellent STAT3 affinity and can bind to STAT3 highly expressed in tumor cells to inhibit its activity. While retaining the high selectivity of the template for tumor cells, the tumor inhibitory effect is enhanced. Therefore, the STAT3 inhibitory polypeptide or its pharmaceutical salt has potential anti-tumor clinical application value and broad development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The effect of different groups of treatment on the tumor growth curve of human glioma cell U87 in vivo, among which, ** P ≤ 0.01 is the Student's t test result relative to the Saline group; # P≤0.05 and ## P ≤ 0.01 refers to the Student's t test results relative to the Shf group;

[0022] Figure 2 The effect of different groups of treatment on the tumor weight of human glioma cells U87 in vivo, among which, ** P ≤ 0.01 is the Student's t test result relative to the Saline group; # P≤0.05 and ## P ≤ 0.01 is the Student's t test result relative to the Shf group. DETAILED DESCRIPTION

[0023] The present invention provides a STAT3 inhibitory polypeptide, characterized in that the amino acid sequence of the STAT3 inhibitory polypeptide is at least one of SEQ ID NO: 1 to SEQ ID NO: 3.

[0024] The present invention uses the SH2 domain of Shf as a template, the template sequence is shown in SEQ.ID NO:4-OH, and the active fragments are spliced ​​to design an amino acid sequence as shown in SEQ ID NO:1 to SEQ ID NO:3. The STAT3 inhibitory polypeptide of the present invention has significant protein affinity for STAT3, can effectively bind to STAT3 protein, inhibit the proliferation and migration of tumor cells, induce cell apoptosis, has good broad-spectrum anti-tumor activity, has less toxicity to normal cells, and has good tumor selectivity.

[0025] As a preferred embodiment, the hydroxyl group in the carboxyl group in the Arg structure at the C-terminus of the STAT3 inhibitory polypeptide is replaced by an amino group, i.e., SEQ ID NO:1-NH2, SEQ ID NO:2-NH2, SEQ ID NO:3-NH2, the amino acid sequence of SEQ ID NO:1-NH2 is: PFSSVPEIVHHYASRYLPQKGAEHMSLLYPVAIR-NH2; the amino acid sequence of SEQ ID NO:2-NH2 is: PFSSVPEIVHHYASRKLPIKYAEQMSLLYPVAIR-NH2; the amino acid sequence of SEQ ID NO:3-NH2 is: PFSSVPEIVHHYASRKLPYGQHMSLLYPVAIR-NH2. The present invention significantly improves the stability of the STAT3 inhibitory polypeptide by replacing the hydroxyl group in the carboxyl group in the Arg structure at the C-terminus of the STAT3 inhibitory polypeptide with an amino group.

[0026] The dimerization of STAT3 is a protein-protein interaction with a large action area. The STAT3 inhibitory polypeptide of the present invention is a long peptide STAT3 inhibitor, which can make up for the shortcoming that small molecule compounds or short peptides are difficult to block protein-protein interactions to a certain extent.

[0027] The present invention provides a method for preparing the above-mentioned STAT3 inhibitory polypeptide, which adopts a microwave-promoted Fmoc / tBu orthogonal protection solid phase synthesis strategy to prepare the STAT3 inhibitory polypeptide. The preparation method of the present invention comprises the following steps: according to the peptide chain sequence shown in one of SEQ ID NO:1 to SEQ ID NO:3, the microwave-promoted Fmoc protecting group removal and coupling steps are repeated from the C-terminus to the N-terminus to sequentially connect the corresponding amino acids to obtain a polypeptide-resin complex, and then the polypeptide on the resin is cleaved to obtain a crude product of the target polypeptide, and the crude product of the target polypeptide is purified to obtain.

[0028] In the present invention, the microwave-promoted removal of the Fmoc protecting group includes a first microwave reaction synthesis and a second microwave reaction synthesis. The first microwave reaction synthesis is to conduct a first microwave reaction on the swollen resin and a 25% piperidine / NMP (V / V) solution containing 0.1M HOBT, the time of the first microwave reaction is 1min, the microwave power is 15W, and the reaction temperature is controlled within 50°C; the second microwave reaction synthesis is to filter out the solution after the first microwave reaction is completed, and add a 25% piperidine / NMP (V / V) solution containing 0.1M HOBT again to conduct a second microwave reaction, the time of the second microwave reaction is 4min, the microwave power is 25W, the reaction temperature is controlled at 50°C, and the reaction is cooled. After the reaction is completed, the solution is filtered out and washed to obtain a resin with the initially connected Fmoc protecting group removed.

[0029] The novel STAT3 inhibitory polypeptide synthesized by microwave-assisted solid phase synthesis of the present invention greatly improves the coupling reaction rate. Conventional solid phase synthesis methods often require 2 to 20 hours, or even longer, to fully couple an amino acid to a resin. However, the microwave-assisted solid phase synthesis of the present invention only takes about 10 minutes on average. Conventional solid phase synthesis methods often require 30 minutes to 1 hour to remove the Fmoc protecting group, while microwave-assisted solid phase synthesis only takes about 5 minutes on average, which greatly improves the efficiency of polypeptide synthesis and shortens the synthesis cycle.

[0030] In the present invention, the coupling comprises dissolving Fmoc-Arg(Pbf)-OH, HBTU, HOBT and DIPEA in NMP, adding the solution to the resin from which the Fmoc protecting group initially connected is removed, reacting in a microwave reactor for 7 minutes, the microwave power is 25W, the reaction temperature is controlled at 50°C, and compressed air is used for cooling. After the reaction is completed, the reaction solution is filtered out and the resin is washed. The molar ratio of Fmoc-Arg(Pbf)-OH, HBTU, HOBT and DIPEA is 1:1:1:2. The microwave-promoted solid phase method of the present invention is low in cost for synthesizing novel STAT3 inhibitory polypeptides. Due to the high coupling efficiency, the required protected amino acids only need an average of 2 times excess, which is much lower than the 4 to 5 times excess required by the conventional solid phase synthesis method.

[0031] In the present invention, after obtaining the polypeptide-resin complex, the polypeptide on the resin is cleaved, and the cleavage agent for cleaving the resin is a cleavage agent Reagent K prepared according to a volume ratio of TFA, thioanisole, water, phenol and EDT of 82.5:5:5:5:2.5. The cleavage method is shaking at 0°C for 30 minutes, and then reacting at room temperature for 3 hours. After the cleavage reaction is completed, the mixture is washed, the filtrate is combined, and the filtrate is added to a large amount of ice ether to precipitate a white flocculent precipitate, and the precipitate is frozen and centrifuged to obtain a crude polypeptide. The crude purity of the novel STAT3 inhibitory polypeptide synthesized by microwave-promoted solid phase of the present invention is greater than 80%, which is greatly improved compared with the conventional solid phase synthesis method, which facilitates subsequent purification work.

[0032] In the present invention, the method of synthesizing novel STAT3 inhibitory polypeptides by solid phase promoted by microwaves is easy to realize automation and large-scale production, which makes it more suitable for industrial production.

[0033] The STAT3 inhibitory polypeptide of the present invention not only retains the targeting affinity of the template Shf peptide to STAT3, but also can further improve the effect of treating tumors, especially tumors with high expression of STAT3, has the advantage of high selectivity for tumor cells, and enhances the tumor inhibitory effect.

[0034] The present invention provides an anti-tumor drug, which comprises the above-mentioned STAT3 inhibitory polypeptide or a pharmaceutically acceptable salt thereof.

[0035] In the present invention, the drug further comprises pharmaceutically acceptable excipients, including excipients, such as diluents.

[0036] The present invention also provides a use of the above STAT3 inhibitory polypeptide in the following a and / or b:

[0037] a. Preparation of anti-tumor products;

[0038] b. Prepare products for inhibiting STAT3 protein.

[0039] In the present invention, when preparing an anti-tumor product, the tumor is preferably a tumor that highly expresses STAT3, and further preferably includes one or more of glioma, leukemia and liver cancer, such as one or more of human glioma cells U87, human leukemia cells K562 and human liver cancer cells HepG-2. The product includes a drug, a reagent or a kit. In the above application, the STAT3 inhibitory polypeptide of the present invention can be used in combination with other anti-tumor products or products that inhibit STAT3 protein activity to achieve the purpose of anti-tumor or inhibiting STAT3 protein activity, and the STAT3 inhibitory polypeptide can also be used as the only active ingredient for anti-tumor or inhibiting STAT3 protein activity. When used for anti-tumor, the product can be administered orally or by injection.

[0040] The present invention also provides an application of the STAT3 inhibitory polypeptide in preparing a product for targeted tumor treatment.

[0041] In the present invention, when used in the above-mentioned targeted tumor treatment product, the tumor is preferably a tumor that highly expresses STAT3. Further preferred include one or more of glioma, leukemia and liver cancer, such as one or more of human glioma cells U87, human leukemia cells K562 and human liver cancer cells HepG-2. The product includes a drug, a reagent or a kit. In the above-mentioned application, the STAT3 inhibitory polypeptide of the present invention can be used in combination with other targeted anti-tumor products to achieve the purpose of targeted anti-tumor, and the STAT3 inhibitory polypeptide can also be used as the only active ingredient for targeted anti-tumor. The product can be administered orally or by injection.

[0042] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0043] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0044] In the present invention, the following abbreviations are used:

[0045] Et3N: triethylamine; NMM: N-methylmorpholine; DIEA: N,N'-diisopropylethylamine; DMF: dimethylformamide; DMSO: dimethyl sulfoxide; DCM: dichloromethane; Fmoc: N-9-fluorenylmethoxycarbonyl; DIC: N,N'-diisopropylcarbodiimide; CDI: N,N'-carbonyldiimidazole; DMAP: 4-dimethylaminopyridine; HOSU: N-hydroxysuccinimide; EDC.HCl: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; HATU: 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; HBTU: benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate Ester; HCTU: 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate; HOAT: 1-hydroxy-7-azobenzotriazole; HOBT: 1-hydroxy-benzotriazole; PyBOP: benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; HPLC: high performance liquid chromatography; ESI-MS: electrospray ionization mass spectrometry; Pal: palmitic acid; Gly: glycine; Ser: serine; Ala: alanine; Val: valine; Ile: isoleucine; Leu: leucine; Tyr: tyrosine; Phe: phenylalanine; His: histidine; Pro: proline; Met: methionine; Glu: glutamic acid; Lys: lysine; Arg: arginine.

[0046] Example 1

[0047] Microwave-enhanced solid phase synthesis of Pro-Phe-Ser-Ser-Val-Pro-Glu-Ile-Val-His-His-Tyr-Ala-Ser-Arg-Tyr-Leu-Pro-Gln-Lys-Gly-Ala-Glu-His-Met-Ser-Leu-Leu-Tyr-Pro-Val-Ala-Ile-Arg-NH2 (SEQ ID NO: 1-NH2, named STAT3 inhibitory polypeptide 1)

[0048] (1) Swelling of resin

[0049] Weigh 50 mg of Fmoc-Rink amide-MBHAResin (substitution amount 0.4 mmol / g), swell it with 7 mL of DCM for 30 min, filter out DCM, swell it with 10 mL of NMP for 30 min, and finally rinse it with 7 mL of NMP, DCM and NMP respectively to obtain the swollen resin.

[0050] (2) Microwave-assisted removal of Fmoc protecting groups

[0051] The swollen resin was placed in a reactor, and 7 mL of a 25% piperidine / NMP (V / V) solution containing 0.1 M HOBT was added. The reaction was carried out in a microwave reactor for 1 min, the microwave power was 15 W, the reaction temperature was controlled within 50 ° C, and compressed air was used for cooling. After the reaction, the solution was filtered out; 7 mL of a 25% piperidine / NMP (V / V) solution containing 0.1 M HOBT was added and the reaction was carried out in a microwave reactor for another 4 min, the microwave power was 25 W, the reaction temperature was controlled at 50 ° C, and compressed air was used for cooling. After the reaction, the solution was filtered out and washed with NMP. The resin with the Fmoc protecting group initially connected was obtained.

[0052] (3) Microwave-assisted synthesis of Fmoc-Arg(Pbf)-Rink amide-MBHAResin

[0053] Fmoc-Arg(Pbf)-OH (0.04 mmol), HBTU (0.04 mmol), HOBT (0.04 mmol) and DIPEA (0.08 mmol) were dissolved in 10 mL NMP, and the solution was added to the resin from which the Fmoc protecting group was removed. The reaction was carried out in a microwave reactor for 7 min, the microwave power was 25 W, the reaction temperature was controlled at 50 ° C, and the air was cooled by compressed air from an air compressor. After the reaction was completed, the reaction solution was filtered out, and the resin was washed 3 times with 7 mL of DCM and 7 mL of NMP.

[0054] (4) Detection of coupling efficiency

[0055] The coupling efficiency of the resin was qualitatively detected by the ninhydrin method or the bromophenol blue method. If the color reaction was negative, the next coupling cycle could be entered.

[0056] Among them, the ninhydrin method: take a small amount of resin particles, wash them with ethanol, put them in a transparent vial, add 2 drops each of 5% ninhydrin ethanol, KCN pyridine solution (2mL 0.001M KCN ​​diluted in 98mL pyridine), and 80% phenol ethanol solution, heat at 100℃ for 5min, if the resin appears blue, it is positive.

[0057] Bromophenol blue method: Take a small amount of resin particles, wash them with dimethylacetamide, put them in a transparent vial, add 3 drops of 1% bromophenol blue dimethylacetamide solution, shake at room temperature for 3 minutes, if the resin appears blue, it is positive.

[0058] (5) Peptide chain extension

[0059] According to the sequence of the peptide chain of the compound, the above steps (2) to (4) of deprotection and coupling are repeated from the C-terminus to the N-terminus to connect the corresponding amino acids, and the coupling microwave-promoted reaction time varies from 5 to 20 minutes to obtain a peptide-resin complex.

[0060] (6) Cleavage of peptides on resin

[0061] The obtained polypeptide-resin complex was placed in a reaction bottle, and 10 mL of the cleavage agent Reagent K (TFA / thioanisole / water / phenol / EDT, 82.5:5:5:5:2.5, V / V) was added to each reaction bottle. The mixture was shaken at 0°C for 30 min, and then reacted at room temperature for 3 h. After the reaction, the mixture was filtered, washed three times with a small amount of TFA and DCM, and the filtrate was combined. The filtrate was added to a large amount of ice ether to precipitate a white flocculent precipitate, and the crude product of the target polypeptide was obtained by refrigerated centrifugation.

[0062] (7) Peptide purification

[0063] The crude peptide was dissolved in 50% acetonitrile / water and purified by preparative liquid chromatography under the following chromatographic conditions: C 18 Reverse phase column (320mm×28mm, 5μm); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); mobile phase gradient: 0-15min mobile phase B 40%-90%, 15-20min mobile phase B 90%; flow rate is 6mL / min, and the detection wavelength is 214nm. The collected solution was lyophilized to obtain 30mg of pure product. The theoretical relative molecular mass of STAT3 inhibitory peptide 1 is 3893.33. Electrospray mass spectrometry (ESI-MS) m / z: calculate [M+4H] 4+ 974.33, [M+5H] 5+ 779.67; found[M+4H] 4+ 974.33, [M+5H] 5+ 779.66.

[0064] Example 2

[0065] Microwave-enhanced solid phase synthesis of Pro-Phe-Ser-Ser-Val-Pro-Glu-Ile-Val-His-His-Tyr-Ala-Ser-Arg-Lys-Leu-Pro-Ile-Lys-Tyr-Ala-Glu-Gln-Met-Ser-Leu-Leu-Tyr-Pro-Val-Ala-Ile-Arg-NH2 (SEQ ID NO: 2-NH2, named STAT3 inhibitory polypeptide 2)

[0066] According to the general method described in Example 1, the STAT3 inhibitory polypeptide of this example was synthesized according to the sequence shown in SEQ ID NO: 2-NH2, and the molecular weight was confirmed by ESI-MS.

[0067] The theoretical relative molecular mass of Pro-Phe-Ser-Ser-Val-Pro-Glu-Ile-Val-His-His-Tyr-Ala-Ser-Arg-Lys-Leu-Pro-Ile-Lys-Tyr-Ala-Glu-Gln-Met-Ser-Leu-Leu-Tyr-Pro-Val-Ala-Ile-Arg-NH2 (SEQ ID NO: 2-NH2) is 3940.47. ESI-MS m / z: calculate [M+4H] 4+ 986.12, [M+5H] 5+ 789.09; found [M+4H] 4+ 986.12, [M+5H] 5+ 789.09.

[0068] Example 3

[0069] Microwave-enhanced solid phase synthesis of Pro-Phe-Ser-Ser-Val-Pro-Glu-Ile-Val-His-His-Tyr-Ala-Ser-Arg-Lys-Leu-Pro-Ile-Tyr-Gly-Ala-Gln-His-Met-Ser-Leu-Leu-Tyr-Pro-Val-Ala-Ile-Arg-NH2 (SEQ ID NO: 3-NH2, named STAT3 inhibitory polypeptide 3)

[0070] According to the general method described in Example 1, the STAT3 inhibitory polypeptide of this example was synthesized according to the sequence shown in SEQ ID NO: 3-NH2, and the molecular weight was confirmed by ESI-MS.

[0071] The theoretical relative molecular mass of Pro-Phe-Ser-Ser-Val-Pro-Glu-Ile-Val-His-His-Tyr-Ala-Ser-Arg-Lys-Leu-Pro-Ile-Tyr-Gly-Ala-Gln-His-Met-Ser-Leu-Leu-Tyr-Pro-Val-Ala-Ile-Arg-NH2 (SEQ ID NO: 3-NH2) is 3877.38. ESI-MS m / z: calculate [M+4H] 4+ 970.35, [M+5H] 5+776.48; found [M+4H] 4+ 970.34, [M+5H] 5+ 776.47.

[0072] Example 4

[0073] Effects of STAT3 inhibitory peptides 1-3 on the targeting affinity of STAT3 protein

[0074] The static adsorption equilibrium experiment was used to determine that the STAT3 inhibitory polypeptides 1 to 3 prepared in Examples 1 to 3 had significant targeting affinity to STAT3:

[0075] The content of STAT3 protein (purchased from MCE, HY-P701098) was determined by Kjeldahl method to be 7.55 mg / g wetgel, i.e., 85 nmol / g wetgel.

[0076] 0.1g wetgel, containing 8.5nmol STAT3 protein, was used, and 1mL of STAT3 inhibitory peptides 1-3 prepared in Examples 1-3 with different concentration gradients (initial concentrations: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20μM), control Scr peptides, and positive control Shf peptides were respectively mixed in 20mM PBS solution, and slowly shaken at 4°C for 12h until static adsorption equilibrium was reached, and the supernatant was centrifuged at 3000rpm. The concentration of the peptides in the supernatant was measured at a wavelength of 280nm using a NanoDrop 2000 ultra-micro spectrophotometer, and a blank agarose gel without coupling STAT3 protein was used as a blank control. Using the nonlinear fitting tool in Origin, the Langmuir isotherm adsorption equation was used for fitting, and the static adsorption maximum Qmax (nmol / g) and equilibrium dissociation constant Kd (μmol / L) of STAT3 inhibitory peptides 1-3, control Scr peptides, and positive control Shf peptides were obtained.

[0077] Among them, the amino acid sequence of the Shf peptide is:

[0078] Pro-Phe-Ser-Ser-Val-Pro-Glu-Ile-Val-His-His-Tyr-Ala-Ser-Arg-Lys-Leu-Pro-Il e-Lys-Gly-Ala-Glu-His-Met-Ser-Leu-Leu-Tyr-Pro-Val-Ala-Ile-Arg-OH(SEQID NO:4-OH), the abbreviated sequence of the amino acid is PFSSVPEIVHHYASRKLPIKGAEHMSLLYPVA IR-OH (SEQ ID NO:4-OH).

[0079] The Scr peptide is a control peptide synthesized by disrupting the peptide sequence of the Shf peptide.

[0080] Table 1 Static adsorption maximum values ​​and equilibrium dissociation constants of STAT3 inhibitory peptides 1-3, Scr peptide and Shf peptide

[0081] Group Qmax(nmol / g) Kd(μmol / L) Scr small peptide 13.33±1.02 4.16±0.35 Shf small peptide 80.19±2.13 <![CDATA[0.05±0.03 ** ]]> STAT3 inhibitory polypeptide 1 81.54±3.77 <![CDATA[0.07±0.03 ** ]]> STAT3 inhibitory polypeptide 2 83.10±3.54 <![CDATA[0.06±0.04 ** ]]> STAT3 inhibitory polypeptide 3 80.35±3.85 <![CDATA[0.07±0.04 ** ]]>

[0082] Note: ** P≤0.01 is the Student's t test result relative to the Scr control group.

[0083] As shown in Table 1, the maximum static adsorption Qmax of immobilized STAT3 protein (85 nmol / g wet gel) for STAT3 inhibitory peptide 1 reached 81.54±3.77 nmol / g wet gel, and the equilibrium dissociation constant Kd reached 0.07±0.03 μmol / L; the maximum static adsorption Qmax of STAT3 inhibitory peptide 2 reached 83.10±3.54 nmol / g wet gel; the equilibrium dissociation constant Kd reached 0.06±0.04 μmol / L; the maximum static adsorption Qmax of STAT3 inhibitory peptide 3 reached 80.35±3.85 nmol / g wet gel; the equilibrium dissociation constant Kd reached 0.07±0.04 μmol / L.

[0084] The maximum static adsorption Qmax of the control Scr peptide reached 13.33±1.02nmol / g wet gel; the equilibrium dissociation constant Kd reached 4.16±0.35μmol / L; the maximum static adsorption Qmax of the template Shf peptide reached 80.19±2.13nmol / g wet gel; the equilibrium dissociation constant Kd reached 0.05±0.03μmol / L. The equilibrium dissociation constant Kd of STAT3 inhibitory peptide 1, STAT3 inhibitory peptide 2 and STAT3 inhibitory peptide 3 was two orders of magnitude lower than that of Scr peptide, which was similar to the template Shf peptide; at the same time, the maximum static adsorption Qmax of STAT3 was basically consistent with the actual protein amount (85nmol / g wetgel) of STAT3 fixed on CNBr-activated Sepharose 4B material, which was consistent with the 1:1 interaction model. It shows that STAT3 inhibitory peptide 1, STAT3 inhibitory peptide 2 and STAT3 inhibitory peptide 3 retain the high STAT3 affinity of the template peptide.

[0085] Example 5

[0086] Antitumor activity of STAT3 inhibitory peptides 1-3

[0087] (1) In vitro antitumor activity of STAT3 inhibitory peptides 1 to 3

[0088] This example uses an in vitro cell proliferation experiment to determine the in vitro anti-tumor activity of the STAT3 inhibitory polypeptides 1 to 3 prepared in Examples 1 to 3:

[0089] Human glioma cells U87, human leukemia cells K562, human liver cancer cells HepG-2, human gastric epithelial cells GES-1, and human umbilical vein endothelial cells HUVEC in good logarithmic growth phase were taken and 1×10 5 / mL density was inoculated in a 96-well culture plate and cultured for 24 hours, and then different concentration gradients (1.25, 2.5, 5, 10, 20, 40, 80, 120, 160 μmol / L) of STAT3 inhibitory peptides 1 to 3 and the Shf small peptide described in Example 4 were added and incubated for 48 hours. After the incubation, 20 μL of CCK-8 solution was added to each well, and the OD value at a wavelength of 450 nm was detected by an enzyme reader after further incubation for 2 hours. Finally, the IC value of the test compound was calculated by GraphPadPrism 7.0. 50 value.

[0090] Table 2 In vitro cytotoxicity of STAT3 inhibitory peptides 1 to 3 (μmol / L)

[0091] Group U87 K562 HepG-2 GES-1 HUVEC SH 23.2±2.1 28.7±2.7 26.6±2.3 62.7±5.2 65.5±5.7 STAT3 inhibitory polypeptide 1 <![CDATA[8.6±2.3 ** ]]> <![CDATA[7.6±1.8 ** ]]> <![CDATA[10.7±1.4 ** ]]> 72.3±7.4 67.6±5.9 STAT3 inhibitory polypeptide 2 <![CDATA[5.9±0.2 ** ]]> <![CDATA[6.6±0.5 ** ]]> <![CDATA[5.6±0.2 ** ]]> 73.7±4.5 61.6±4.4 STAT3 inhibitory polypeptide 3 <![CDATA[7.6±1.4 ** ]]> <![CDATA[7.7±1.2 ** ]]> <![CDATA[7.4±0.7 ** ]]> 64.2±5.6 69.4±4.5

[0092] Note: ** P≤0.01 is the Student's t test result relative to the template Shf small peptide control group.

[0093] As shown in Table 2, relative to normal cells GES-1 and HUVEC, the STAT3 inhibitory polypeptides 1 to 3 prepared in Examples 1 to 3 all had significant cytotoxicity to tumor cells U87, K562 and HepG-2 with high expression of STAT3, and the toxicity was significantly enhanced compared with the template Shf small peptide. This indicates that the STAT3 inhibitory polypeptides 1 to 3 prepared in Examples 1 to 3 have good broad-spectrum anti-tumor activity, less toxicity to normal cells, and good selectivity for tumors with high expression of STAT3.

[0094] (2) In vivo antitumor activity of the compounds of the present invention

[0095] This example uses a nude mouse tumor-bearing experiment to determine the in vivo antitumor activity of the STAT3 inhibitory polypeptides 1 to 3 prepared in Examples 1 to 3:

[0096] U87 cells were inoculated into the right axilla of balb / c nude mice and the tumors were grown to 100 mm. 3The animals were then randomly divided into groups, with 6 animals in each group, and divided into a blank control group (denoted as Saline) and a drug-treated group, wherein the drug-treated group was divided into STAT3 inhibitory polypeptide group 1 (denoted as SEQ.ID NO: 1), STAT3 inhibitory polypeptide group 2 (denoted as SEQ.ID NO: 2), STAT3 inhibitory polypeptide group 3 (denoted as SEQ.ID NO: 3) and Shf small peptide group (denoted as Shf). The drug was administered intratumorally once every 2 days. The drug-treated groups were intravenously injected with STAT3 inhibitory polypeptides 1 to 3 and the Shf small peptide described in Example 4 at 15 μg / mouse / time, and the blank control group was intravenously injected with an equal volume of normal saline. The body weight and tumor volume of the mice were measured the next day after the drug administration, and the anti-tumor effect of the compound was dynamically observed. After 14 days of drug administration, the mice were killed, the tumor mass was surgically removed and weighed, and the tumor volume V (mm 3 ) and draw the tumor growth curve.

[0097] like Figure 1 and Figure 2 As shown, the STAT3 inhibitory polypeptides 1-3 prepared in Examples 1-3 can effectively inhibit the growth of U87 in nude mice, and the inhibitory effect is significantly better than that of the template Shf small peptide, and has good in vivo anti-tumor activity.

[0098] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A STAT3 inhibitory polypeptide, characterized in that: The amino acid sequence of the STAT3 inhibitory polypeptide is at least one of SEQ ID NO: 1 to SEQ ID NO:

3.

2. The STAT3 inhibitory polypeptide according to claim 1, characterized in that The hydroxyl group in the carboxyl group in the Arg structure at the C-terminus of the STAT3 inhibitory polypeptide is replaced by an amino group.

3. An anti-tumor drug, characterized in that: The drug comprises the STAT3 inhibitory polypeptide according to claim 1 or 2 or a pharmaceutically acceptable salt thereof.

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

5. Use of the STAT3 inhibitory polypeptide according to claim 1 or 2 in the following a and / or b: a. Preparation of anti-tumor products; b. Prepare products for inhibiting STAT3 protein.

6. The use according to claim 5, characterized in that: The tumor is a tumor that highly expresses STAT3.

7. The use according to claim 5, characterized in that: The tumor includes one or more of glioma, leukemia and liver cancer.

8. Use of the STAT3 inhibitory polypeptide according to claim 1 or 2 in the preparation of drugs for targeted tumor therapy.

9. The use according to claim 8, characterized in that: The tumor is a tumor that highly expresses STAT3.

10. The use according to claim 8, characterized in that: The tumor includes one or more of glioma, leukemia and liver cancer.

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