CN101307094B - Novel nuclear factor -kappa B p65 subunit antagonizing polypeptide - Google Patents

Abstract

The present invention relates to a novel nuclear factor-κB p65 subunit antagonistic polypeptide, in particular to a therapeutic nuclear factor-κB p65 subunit antagonistic peptide, more specifically, to a nuclear factor-κB p65 subunit-specific The small molecular peptide Arg Leu Arg Trp Arg that binds and inhibits its activity, the invention also relates to the preparation and application of this nuclear factor-κBp65 subunit antagonistic peptide.

Description

A nuclear factor-κB p65 subunit antagonistic polypeptide

technical field

The invention relates to the field of medicine, in particular to a nuclear factor-κB (NF-κB) p65 subunit antagonistic peptide for treatment, more specifically, to a peptide capable of specifically binding to the nuclear factor-κB p65 subunit and inhibiting it. Active small molecule peptides. The invention also relates to the preparation and application of the nuclear factor-κB p65 subunit antagonistic peptide.

Background technique

NF-κB is a transcription factor ubiquitously present in the cytoplasm of almost all cells. It is the convergence point of various signal transduction pathways and has the functions of regulating various immune responses, inflammatory responses, virus replication, cell apoptosis and proliferation and Function of growth and development-related gene expression. NF-κB is a dimeric protein composed of two Rel family proteins. According to the differences in structure, function and synthesis method, the Rel protein family can be divided into two categories: one is the precursor protein p105 and p100, and its C-terminus contains ankyrin repeat motif (ankin repeat motif), which can pass through ATP-dependent Proteolytically cleaved to mature p50 and p52. This type of protein lacks a transactivation domain and has no function of independently activating gene transcription; the other type is RelA (p65), Rel (c-Rel), v-Rel and RelB, whose C-terminus contains one or more transactivation The transactivation domain has the function of independently activating gene transcription.

When the cells are in a resting state, NF-κB mainly combines with κB inhibitory protein (IκB _s ) to form a trimer, and exists in the cytoplasm in an inactive manner. When cells are stimulated by various external signals such as proinflammatory cytokines, viruses, endotoxins, ultraviolet rays, oxidants, and radiation to activate the second messenger system, _IκBs are phosphorylated and degraded under the action of IκB kinase (IKK), NF-κB loses the strict control of _IκBs and is activated, and translocates into the nucleus. Its subunits form a ring structure and bind to specific sites in the promoters of target genes, efficiently inducing pro-inflammatory cytokines (such as TNF, GM- CSF, IL-1β, IL-6, IL-11, IL-17, etc.), chemokines (such as IL-8, RANTES, MIP-1α, MCP-2, etc.), adhesion molecules (such as ICAM-1, VCAM-1, ELAM-1, etc.), immune receptors (such as IL-2R), and the expression of various enzymes involved in the inflammatory response cascade (such as NO synthase, cyclooxygenase, etc.). Some proteins induced by NF-κB can further reverse the activation of NF-κB, leading to the expansion and continuation of the inflammatory response. Based on the above knowledge, many scholars believe that inhibiting the transcriptional activity of NF-κB will open up a new way for the treatment of inflammatory diseases. In addition, NF-κB also plays an important role in cell apoptosis and proliferation by directly up-regulating apoptosis inhibitors (c-IAP1, c-IAP2, and IXAP), TNF receptor-related factors (TRAF1 and TRAF2), Expression of anti-apoptotic genes such as zinc finger protein A20, manganese superoxide dismutase, Bcl-2 homologue A1/Bfl-1 and IEX-IL inhibits cell apoptosis. In tumor therapy, chemotherapy and radiotherapy can activate NF-κB, making tumor cells resistant to chemotherapy and insensitive to radiation. Therefore, inhibiting the transcriptional activity of NF-κB and reducing the anti-apoptotic ability of tumor cells is a good strategy to improve the efficacy of anticancer drugs.

Due to the powerful transcriptional regulation function of NF-κB, the excessive activation of NF-κB plays an important role in the occurrence and development of many diseases: such as (like) rheumatoid arthritis, asthma, chronic glomerulonephritis, pylori Helicobacter-associated gastritis, inflammatory bowel disease and other chronic inflammatory diseases, sepsis, septic shock, acute glomerulonephritis and other acute inflammatory diseases, Alzheimer's disease, atherosclerosis, systemic erythema The occurrence and development of autoimmune diseases such as lupus and cancer are closely related to the excessive or continuous activation of NF-κB. In addition, NF-κB is also involved in the transcription and replication of rhinovirus, influenza virus, Epstein-Barr virus, cytomegalovirus, adenovirus, HTL virus and HIV virus in infected cells, and plays a role in the infection and pathogenic process of these viruses. plays an important role.

In view of the potential role of NF-κB in the occurrence and development of the above diseases, targeting NF-κB and related proteins in the signaling pathway to develop therapeutic drugs for NF-κB-related diseases has become a research hotspot in this field . At present, foreign antagonistic treatment strategies targeting NF-κB mainly include the following aspects: (1) Antioxidant therapy: such as antioxidant—pyrrolidine dithiocarbamate (PDTC) non-specific inhibition of NF-κB κB transcriptional activity; (2) Inhibition of IKK formation and activity: application of IKKα antisense nucleic acid, expression of IKKα dominant negative mutants, and application of interfering peptides that interfere with the formation of IKK complexes to inhibit the formation and activation of IKK; (3 ) Reduce the degradation of IκB: Certain proteasome inhibitors such as MG101, MG115, MG132, PS-341, lactacystin and the recently discovered epoxomicin can achieve the purpose of antagonizing NF-κB activity by inhibiting the degradation of IκB. (4) Promote the generation of IκB: the adenovirus expression vector of IκBα is used to overexpress the phenotype-inactive mutant of IκBα, which can significantly inhibit the nuclear translocation of NF-κB and its subsequent effects; (5) inhibit the expression of NF-κB Synthesis: design antisense nucleic acid and siRNA of P50 and P65 to block the synthesis of NF-κB; (6) traditional non-steroidal anti-inflammatory drugs inhibit the activation of NF-κB; (7) curcumin and flavonoids derived from traditional Chinese medicine compounds such as resveratrol and resveratrol non-specifically inhibit the activation of the NF-κB signaling pathway.

The above-mentioned therapeutic strategies targeting NF-κB have shown different degrees of curative effect in vitro and animal experiments, and some therapeutic strategies have entered the clinical trial stage, and have been registered and approved by the United States Patent and Trademark Office and the European Patent Office. Protected by patent. However, some of the above measures have the problem of poor specificity. Such as proteasome, it can not only degrade IκB, but also has many other important functions, so inhibition of proteasome activity will lead to potential toxic side effects. Antioxidants have a wider scope of action and greater toxic and side effects. Measures targeting IKK and IκB are not completely specific to the intervention of NF-κB activity. For example, interfering peptides that interfere with the formation of IKK complexes have been confirmed, which can not only inhibit the activity of NF-κB, but also inhibit the transcription factor AP-1 and NFAT activity. Compounds such as non-steroidal anti-inflammatory drugs, curcumin, flavonoids, and resveratrol not only inhibit the NF-κB signaling pathway, but also regulate other signaling pathways. Although the synthetic strategy of directly inhibiting NF-κB is specific to NF-κB, there are still many problems in the clinical application of antisense nucleic acid and siRNA strategies, so the synthetic strategy of inhibiting NF-κB is not the best choice. It is not difficult to see from the entire activation pathway of NF-κB that the combination of NF-κB and DNA cis-elements is specific, and it is also the only way for NF-κB to initiate the transcription of related target genes. Thus, true specificity can only be achieved by directly interfering with the binding of NF-κB to DNA cis-elements. It is difficult to achieve this goal by interfering with the upstream pathway of NF-κB activation.

The heterodimer formed by the two subunits of p50 and p65 is a typical representative of NF-κB. It is the most important of all forms of NF-κB. It exists in almost all cells in the body, and its content is much higher than other dimers . The amino terminus of this heterodimer can constitute a ring domain, which mediates specific binding to DNA bases. Crystal diffraction analysis showed that the p50/p65 heterodimer specifically binds to the immunoglobulin κ chain gene enhancer κB sequence in the following ways: Arg-54, Arg-56, Tyr-57, Glu-60 of the p50 subunit , His-64 and Lys-241 amino acid residues specifically bind to the 5'-G _-5 G _-4 G _-3 A _-2 C _-1 -3' base series at the 5' end of the κB sequence; Arg- of the p65 subunit 33. Arg-35, Arg-36, Glu-39, and Arg-187 specifically bind to the 5'-T _{+ 1} T _{+ 2} C _{+ 3} C ₊₄ -3'4 base pairs at the 3' end of the κB sequence . In addition, the ring domain composed of the amino terminal of the NF-kB p50/p65 heterodimer and the dimerization domain can also non-specifically recognize the DNA ribose phosphate backbone. That is to say, the ring domain composed of p50 and p65 N-terminal forms the DNA-binding domain of NF-κB, which mediates its binding to cis-acting elements. It is noteworthy that this binding is specific binding. NF-κB activation of target genes first requires the DNA binding domain to recognize and bind to the cis-acting elements in the promoter region of the target gene, and then activate the expression of the target gene with the assistance of the p65 transactivation domain. Therefore, if we obtain a polypeptide that can antagonize the binding of the p65 subunit to its cis-acting element, it will certainly be able to antagonize the transcriptional activity of NF-κB, and ultimately inhibit the expression of NF-κB-regulated target genes and treat acute and chronic inflammation-related diseases and tumor purpose.

With the establishment, maturation and development of yeast two-hybrid technology, a powerful tool for studying protein interactions in the late 1980s, the screening of NF-κB antagonistic polypeptides finally became possible. The yeast two-hybrid system has high sensitivity and can detect weak and transient protein-protein interactions, and the protein-protein interactions in this system occur in the yeast cell and/or nucleus. Compared with prokaryotic cells, the translation and processing of proteins in eukaryotic yeast cells are closer to mammalian cells. Using them as engineering bacteria to study protein interactions will be closer to the physiological state of mammalian cells, that is, to maintain the natural folding state of proteins, so that Protein-protein interactions are closer to the real level in mammalian cells. Therefore, the application of this system to study the interaction between proteins has been more and more widely used, especially for the screening of intracellular target protein interacting proteins or polypeptides, this system is more preferred. NF-κB is a nuclear transcription factor that exists in the cytoplasm, but plays a transcriptional regulatory function in the nucleus. Therefore, we selected the yeast two-hybrid system and used the NF-κB p65 DNA binding domain as the bait protein to screen NF-κB interacting polypeptides from a random peptide library and identify the functions of these polypeptides. At present, we have screened and obtained 8 NF-κB p65 subunit antagonistic peptides with different sequences, one of which is listed here, and the other 5 peptides are listed in patents 200510007479.2 and 200510069451.1 respectively.

Contents of the invention

The purpose of the present invention is to provide a nuclear factor-κB antagonistic peptide capable of specifically binding to the nuclear factor-κB p65 subunit, and capable of inhibiting the activity of the nuclear factor-κB and the expression of the genes regulated by it.

Another object of the present invention is to provide the pharmaceutical use of this nuclear factor-κB antagonistic peptide.

Nuclear factor-κB p65 subunit antagonistic peptide amino acid sequence of the present invention is as follows:

Arg Leu Arg Trp Arg

The pharmaceutical use of anti-nuclear factor-κB of the present invention is a medicine for treating inflammatory immune-related diseases or a medicine for anti-tumor. Wherein said inflammatory related disease is sepsis, septic shock, (like) rheumatoid arthritis, asthma, acute and chronic glomerulonephritis, systemic lupus erythematosus etc., wherein said tumor is liver cancer, stomach cancer, esophagus Cancer, breast tumors, bladder cancer, prostate tumors, etc.

The invention also includes pharmaceutical compositions comprising the polypeptides of the invention. The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier if necessary. The pharmaceutical composition of the present invention can exist in any form of pharmaceutical preparations. For example, preparations suitable for oral administration or preparations suitable for injection are preferably injections. Most preferred are freeze-dried injections.

The present invention also includes the preparation method of the polypeptide of the present invention, said method is selected from conventional solid-phase synthesis method, synthesis method in solution, gene recombination method or chemical synthesis method.

The polypeptide having the amino acid sequence structure of the present invention can specifically bind to nuclear factor-κB p65 subunit, and block the binding of nuclear factor-κB to its cis-acting element, thereby inhibiting the biological activity of nuclear factor-κB, that is, nuclear factor -κB regulates the activity of various target gene expression. Such structural polypeptides can exist alone in the form of polypeptides, or can be fused with other proteins and polypeptides, or be modified by methylation, acetylation, and amination, or undergo individual amino acid mutations or substitutions, or be inserted into specific proteins on the surface of proteins. Parts, such as convex ring regions, or connected with other substances. No matter what form it exists in, substances containing this structural polypeptide may show the effect of binding to nuclear factor-κB p65 subunit and inhibiting the activity of nuclear factor-κB.

The present invention obtains a novel binding polypeptide with nuclear factor-κB p65 subunit through yeast two-hybrid technology screening, and confirms that this polypeptide has the function of inhibiting the biological activity of nuclear factor-κB, which is helpful for the design and development of new anti-nuclear factor-κB Drugs are important.

The research results show that the polypeptide screened by the present invention can not only competitively block the binding of nuclear factor-κB to its cis-acting elements, but also inhibit the expression of target genes regulated by nuclear factor-κB, so it can become a new anti-nuclear Factor-κB drug or drug prodrug, the polypeptide of the present invention can be used to treat various inflammatory or immune-related diseases and tumors. In addition, the nuclear factor-κB p65 subunit binding peptide of the present invention also has potential application value as a biological agent, such as being used as a ligand for nuclear factor-κB affinity purification, as a probe for nuclear factor-κB detection etc.

Substances containing this amino acid sequence can be used in various ways, mainly including inhibiting the activity of nuclear factor-κB in vitro and in vivo, achieving disease treatment for the purpose of inhibiting nuclear factor-κB activity through various administration methods, and nuclear factor-κB activity. Detection or purification of -κB, etc.

Biomedical applications of substances containing the amino acid sequence of the present invention also include:

1. By means of synthesis or gene recombination, the peptide segment containing this sequence is fused or chemically modified with other proteins or peptides, and used for various inflammatory-related diseases and autoimmune diseases for the purpose of inhibiting the activity of nuclear factor-κB , tumors and other related diseases.

2. After it is coupled with agarose, it is used for the affinity separation of nuclear factor-κB.

3. After it is coupled with an indicator label, it is used for the identification of nuclear factor-κB.

4. The polypeptide of the present invention is superior to the prior art in some indicators. For example, the comparative analysis of the physical and chemical characteristic constants of the p65-binding polypeptide proves that it is superior to several previously developed polypeptides. Specifically, the polypeptide sequence of the present invention is the shortest. It is convenient for synthesis, has strong hydrophilicity, and is convenient for the preparation of preparations. See Table 1.

Table 1 Analysis results of physical and chemical characteristic constants of p65-binding polypeptides

Note: The higher the hydrophobic value, the stronger the hydrophobicity, and the lower the hydrophilicity, the stronger.

PT1: Arg Leu Arg Trp Arg

PT2: Glu Gly Gly Val Thr Arg Thr Gln Gly Phe Arg Trp Val Val Ser Ile Arg Asn Ser--Tyr Arg Asn Glu Ser Ala Ser Asn Gly Arg Cys Leu Leu Leu Ala Ala Gln Gly

PT3: Val Val Met Ile Glu Val Val Phe Leu

PT4: Leu Ala Met Val Glu Val Thr Val Val Leu Ser Trp Gly Phe Thr

PT5: Pro Ala Met Val Glu Val Thr Val Val Leu Ser Trp Gly Phe Thr

PT6: Gln Thr Gln Met Glu Val Thr Trp Arg Val Glu Cys Cys Leu Phe Leu

Description of drawings

Figure 1 shows the results of polypeptide hydrophobicity analysis.

Figure 2 is the 3D structure of the complex after docking of nuclear factor-κB p65 subunit and polypeptide molecule.

Figure 3 is the result curve of the ELISA method to detect the competition inhibition experiment of peptides on the binding of nuclear factor-κB p65 subunit and nuclear factor-κB cis-acting element.

Figure 4 shows the inhibitory effect of polypeptides on the expression of nuclear factor-κB-responsive luciferase reporter gene detected by luciferase reporter gene assay. A, B, C, D, E, F, G, H, and I are U937, U937+LPS, U937+LPS+p4-kB-Luc, U937+LPS+p4-kB-Luc+9.375μmol/L, U937+LPS+p4-kB-Luc+18.75μmol/L polypeptide, U937+LPS+p4-kB-Luc+37.5μmol/L polypeptide, U937+LPS+p4-kB-Luc+75μmol/L polypeptide, U937+LPS +p4-kB-Luc+150 μmol/L polypeptide, U937+LPS+p4-kB-Luc+150 μmol/L negative peptide.

Fig. 5 is the result of the inhibition experiment of polypeptides on the proliferation of LOVO cells in vitro.

Fig. 6 is the result of the sensitization experiment of polypeptides on pentafluorouracil inhibiting the proliferation of LOVO cells in vitro.

Fig. 7 shows the results of in vivo sensitization experiments of polypeptides on pentafluorouracil inhibiting tumor growth in tumor-bearing mice.

Detailed ways

The present invention is further illustrated by the following examples, but not as a limitation of the present invention.

Example 1, Application of ANTHPROT V5.0 software to analyze the physicochemical properties of n65-binding polypeptides

Input the amino acid sequence of the polypeptide molecule in the format required by the ANTHPROT V5.0 software, and then calculate various physical and chemical characteristic constants of the polypeptide molecule.

The results are shown in Table 2 and Figure 1

Table 2 Analysis results of physical and chemical characteristic constants of p65-binding peptides

Example 2. Molecular docking predicts the binding between p65-binding polypeptide and p65

的分子半径将球形TIP3P水分子溶剂填充于p65-多肽复合体的3D结构中。为了进一步阐述p65-多肽间的相互作用强度和结合部位，应用Insight II分子模建程序包中的Dephi模块调查NF-κB表面特性。Delphi模块是一个计算分子静电特征(包括溶剂体积效应和离子强度效应)的程序，其可为配体-受体间特异性的结合提供关键的数据。The 3D structures of small peptide molecules were constructed and optimized using Biopolymer and Discover software modules. The energy minimization of the 3D structure of the polypeptide is performed by the standard steepest descent method and the conjugate gradient method with the energy minimization algorithm. In order to simulate the interaction between small molecule peptides and NF-κB, molecular docking technology was applied, that is, docking software was used to perform flexible molecular docking of the 3D structure of the peptide molecule and the 3D structure of human p65. Corresponding charges are assigned to the corresponding atoms under the CVFF force field. The energy minimization process was carried out by using the Discover module software to optimize the energy conformation of the p65-polypeptide complex. In order to eliminate the solvent effect, with 10

The molecular radius of spherical TIP3P water molecule solvent fills the 3D structure of the p65-polypeptide complex. To further elucidate the p65-peptide interaction strength and binding site, the Dephi module in the Insight II molecular modeling package was used to investigate the surface properties of NF-κB. The Delphi module is a program for calculating molecular electrostatic characteristics (including solvent volume effect and ionic strength effect), which can provide key data for ligand-receptor specific binding.

The results showed that the polypeptide had a certain binding ability to p65, which suggested that the polypeptide might have the ability to inhibit NF-κB DNA binding activity. The results are shown in Figure 2.

Example 3, ELISA method to detect polypeptide binding to nuclear factor-κB p65 subunit and nuclear factor-κB cis-acting element Competitive Inhibition Experiment

The peptide sequence was synthesized by Shanghai Huada Tianyuan Biotechnology Co., Ltd. with a purity of >95%. Add 150 μl/well transcription factor blocking solution to a 96-well enzyme-linked plate (purchased from Mercury ^TM Transfactor p65 kits from Clontech, USA) coated with nuclear factor-κB cis-acting elements, and incubate at room temperature for 15 minutes; discard the transcription factor blocking solution Then add 50 μl/well of detection samples diluted with transcription factor blocking solution, the samples are respectively containing 10ng/μl p65 standard protein as a positive control, containing 10ng/μl p65 standard protein, and adding serial concentrations of peptides, containing 10ng/μl p65 Add the same series of negative peptides to the standard protein at the same time, and incubate at room temperature for 1 hour. For the blank control, add 50 μl of transcription factor blocking solution; add 150 μl of transcription factor blocking solution/well and wash 3 times, 4 minutes each time, remove the washing solution; add transcription factor blocking solution 100 μl/well of p65 antibody diluted 1:500, incubate at room temperature for 1 h; add 150 μl/well of transcription factor blocking solution and wash 3 times, 4 min each time, remove the washing solution; add goat antibody diluted 1:1000 with transcription factor blocking solution Rabbit IgG-HRP secondary antibody 100μl/well, incubate at room temperature for 30min; add transcription factor buffer 250μl/well and wash 4 times, 4min each time, remove the washing solution; add TMB substrate, incubate at room temperature for 10min, after the liquid turns blue, wash with Stop the reaction with 100 μl/well stop solution; detect the OD value at dual wavelengths of 450nm and 630nm. The formula for calculating the inhibition percentage of p65-binding peptides binding to p65 and its cis-acting elements is: (OD of p65 standard protein positive control well − OD of polypeptide well)/OD of p65 standard protein positive control well.

The results show that the polypeptide of the present invention can specifically inhibit the binding of p65 to its cis-acting element, and this inhibitory effect has a dose-effect relationship, that is, the inhibitory effect increases with the increase of the peptide concentration, while the negative peptide does not affect p65 The combination with its cis-acting elements is shown in Figure 3.

Example 4. Inhibition experiment of polypeptide on expression of nuclear factor-κB responsive reporter gene.

Cultivate U937 cells in 1640 medium, adjust the cell density to 0.5-2.5×10 ⁵ cells/mL one day before transfection, and culture overnight; take 1 mL of overnight culture cells or add 0.5×10 ⁵ cells/well cells into 24-well culture Plate; add 3 μL GeneJuice transfection reagent to 100 μL serum-free 1640 medium, vortex to mix, and place at room temperature for 5 min; take 1 μg p4kB-Luc plasmid and add it to GeneJuice/1640 medium mixture, gently pipette to mix, and place at room temperature for 5- 15min; add the above mixture drop by drop to the complete medium, shake gently to mix the mixture and the cells thoroughly; culture in a 5% CO2 incubator at 37°C; 5h after transfection, replace the medium with the complete medium and culture for 1h Wash the cells 3 times with PBS, and finally resuspend the cells in 100 μL of serum-free 1640 medium; dilute the polypeptide into 6 different concentrations with PBS (the final concentrations after adding the medium are 0, 9.375, 18.75, 37.5, 75, 150 μmol/L), a volume of 50 μL of polypeptide solution, and 2 μL of Chariot transfection reagent was diluted to 50 μL with PBS, and different concentrations of polypeptides were mixed with Chariot transfection reagent, mixed gently by pipetting, and left at room temperature for 30 minutes. Add 100 μL of the polypeptide/Chariot transfection reagent mixture dropwise to the U937 cell culture medium resuspended in the above 100 μL serum-free 1640 medium, shake gently to mix the mixture and cells thoroughly; culture in a 5% CO2 incubator at 37°C 1h; remove the transfection solution, replace the medium with complete medium, and add LPS at a final concentration of 1μg/mL to stimulate at the same time, after 2h of culture, absorb the cells and centrifuge at 500g for 5min, wash the cells once with PBS, and remove the PBS supernatant; Add 1×CCLR cell lysis buffer to 100μl/well; vortex EP tube for 10-15s, centrifuge at 12000rpm at 4°C for 2min, take supernatant, store at -70°C or directly detect luciferase activity; put in 96-well assay plate After adding 100 μL/well luciferase detection reagent, add 100 μL/well cell lysate and read immediately, and print or record the data.

The results show that the polypeptide of the present invention has an inhibitory effect on the expression of nuclear factor-κB-responsive luciferase reporter gene, and this inhibitory effect has a dose-effect relationship, that is, the inhibitory effect increases with the increase of protein concentration, while negative Peptides do not have this inhibitory effect, as shown in Figure 4. This shows that the p65-binding peptide obtained by our screening has the function of inhibiting the transcriptional activity of nuclear factor-κB, that is, the function of inhibiting the expression of genes such as inflammatory mediators regulated by nuclear factor-κB.

Example 5. Inhibition experiment of polypeptides on LOVO cell proliferation in vitro.

In this experiment, a transmembrane sequence (TyrGlyArgLysLysLysArgArgGlnArgArgArg) was added to the N-terminal when the peptide was synthesized. The full-length sequence was synthesized by Shanghai Huada Tianyuan Biotechnology Co., Ltd. with a purity of >95%. Colon cancer cell line—LOVO cells was cultured with RPMI1640 containing 10% calf serum, and the cells in the logarithmic growth phase were taken, digested with 0.25% trypsin, counted, and seeded in 96 wells at a cell concentration of 2.5×10 ⁴ /ml In the plate, 100 μl was inoculated in each well, so that the inoculated amount of cells was 2.5×10 ³ /well. After the cells were adhered to the wall for 4 hours, 100 μl of positive polypeptides and negative polypeptides of serial concentrations were added according to different groups, and the final concentrations were 75 μM, 37.5 μM, 18.75 μM, and 9.375 μM respectively. 1640 culture solution (100 μl), after the cells were cultured for 72 hours, the cell proliferation was measured by the MTT method, and the OD value of each well was measured at a wavelength of 570 nm using a multifunctional plate reader.

The results show that the polypeptide of the present invention can inhibit the proliferative response of LOVO cells in vitro, and the inhibitory effect has a dose-effect relationship, that is, the inhibitory effect is enhanced with the increase of the concentration of the polypeptide, while the negative peptide has no effect on the proliferative response of LOVO cells in vitro. There is no obvious effect, as shown in Figure 5.

Example 6. Experiment of polypeptide sensitization to pentafluorouracil inhibiting LOVO cell proliferation in vitro.

The polypeptide in this experiment is the same as that in Example 5. Pentafluorouracil (5-Fu) was purchased from Shanghai Xudong Haipu Pharmaceutical Co., Ltd. (10ml: 0.25g/bottle). Colon cancer cell line—LOVO cells was cultured with RPMI1640 containing 10% calf serum, and the cells in the logarithmic growth phase were taken, digested with 0.25% trypsin, counted, and seeded in 96 wells at a cell concentration of 2.5×10 ⁴ /ml In the plate, 100 μl was inoculated in each well, so that the inoculated amount of cells was 2.5×10 ³ /well. After the cells were adhered to the wall for 4 hours, add samples according to the following groups: 5-Fu group, 5-Fu+positive peptide group, 5-Fu+negative peptide group, and set no 5-Fu, only positive peptide, and only negative peptide In the peptide control group, the final concentrations of 5-Fu were 10 μg/ml, 5 μg/ml, 2.5 μg/ml, and 1.25 μg/ml, and the final concentrations of positive and negative peptides were both 37.5 μM. After 72 hours of cell culture, the MTT method was used to Cell proliferation was measured, and the OD value of each well was measured at a wavelength of 570 nm using a multifunctional plate reader.

The results show that the polypeptide of the present invention has a significant sensitization effect on 5-Fu inhibiting the proliferation of LOVO cells in vitro at a concentration of 37.5 μM, and the sensitization ratio is 132.50%, while the negative peptide has no sensitization effect, as shown in Figure 6 .

Example 7. In vivo sensitization experiment of polypeptides on pentafluorouracil inhibiting tumor growth in tumor-bearing mice.

The polypeptide in this experiment is the same as that in Example 5. Nude mice (BALB/C strain, 4-6 weeks old, weighing 18-20 g, half male and half male) were purchased from Beijing Weitong Lihua Company. Raised in the ultra-clean laminar flow room under SPF conditions in the animal room of Daping Hospital, Third Military Medical University. The LOVO cells in the logarithmic growth phase were digested with 0.25% trypsin, the cell concentration was adjusted to 1×10 ⁷ /ml with normal saline, and 0.1ml of each was inoculated subcutaneously in the right flank of nude mice. When the local tumor grows to 150-200mm ³ , the administration is divided into the following groups: solvent control group, 5-Fu+positive peptide group, 5-Fu+negative peptide group. Among them, the solvent control group only received intratumoral injection of 100 μl of solvent without polypeptide, once every other day; the 5-Fu+positive peptide group and the 5-Fu+negative peptide group received intraperitoneal injection of 5-Fu 30 mg/kg once every other day, and at the same time, 150 μM 100 μl of positive and negative peptides were injected into the tumor, and the animals were sacrificed the next day after the sixth administration, and the tumor mass was dissected and weighed, and the tumor inhibition rate was calculated according to the following formula.

Tumor inhibition rate (%)=(average tumor weight of control group-average tumor weight of medication group)/average tumor weight of control group×100%

The results show that when the polypeptide of the present invention is injected into the tumor, it has a significant sensitizing effect on the inhibition of tumor growth in tumor-bearing mice by 5-Fu. Taking the negative peptide group as a control, the tumor inhibition rate of the positive peptide group is 63.54%, as shown in Figure 7 shows.

Embodiment 8, the operating steps of the preparation method of Arg Leu Arg Trp Arg.

The main operation steps of solid-phase peptide synthesis are as follows: 1. Resin swelling: Put Wang-Resin resin into a reaction tube, add DMF (15ml/g) for 30min. 2. Deprotection: remove DMF, add 20% piperidine DMF solution (15ml/g) for 5min, remove and add 20% piperidine DMF solution (15ml/g) for 15min. 3. Detection: Take out the piperidine solution, take more than a dozen resins, wash them three times with ethanol, add ninhydrin, KCN, and phenol solution one drop each, heat at 105°C-110°C for 5 minutes, and turn dark blue to indicate a positive reaction. 4. Washing: DMF (10ml/g) twice, methanol (10ml/g) twice, DMF (10ml/g) twice. 5. Condensation: Add a three-fold excess of protected amino acids and a three-fold excess of activator HBTU, dissolve them with as little DMF as possible, add to a reaction tube, immediately add a ten-fold excess of NMM, and react for 30 minutes. 6. Washing: DMF (10ml/g) once, methanol (10ml/g) twice, DMF (10ml/g) twice. 7. Repeat operations two to six to connect amino acids in sequence (from C-terminal to N-terminal sequence: Arg, Trp, Arg, Leu, Arg), and the last wash: DMF (10ml/g) twice, methanol (10ml/g) twice DMF (10ml/g) twice, DCM (10ml/g) twice. 9. Lysis: prepare lysate (10ml/g)—TFA 94.5%; water 2.5%; EDT 2.5%; TIS 1%, 120min. 10. Drying and washing: Dry the lysate with nitrogen as much as possible, wash with ether six times, and evaporate to dry at room temperature. 11. HPLC purification: use water and acetonitrile as the mobile phase, add 0.1% TFA to the mobile phase, the gradient is from 5% to 85%, and use a C18 reverse phase column.

Claims (9)

1. one kind combines also the active polypeptide of antagonism nuclear Factor-Kappa B specifically specifically with nuclear Factor-Kappa B p65 subunit, and aminoacid sequence is as follows:

Arg?Leu?Arg?Trp?Arg。

2. the application of the polypeptide of claim 1 in the medicine of preparation antinuclear factor-κ B p65 subunit.

3. the application of claim 2, the medicine of described antinuclear factor-κ B p65 subunit are the medicine or the anti-tumor drugs of treatment inflammatory immune correlated disease.

4. the application of claim 3, wherein said inflammatory relative disease is Sepsis, septic shock, rheumatic arthritis, rheumatoid arthritis, asthma, acute and chronic glomerulonephritis, systemic lupus erythematous, and wherein said tumour is liver cancer, cancer of the stomach, esophagus cancer, breast tumor, bladder cancer, tumor of prostate.

5. a pharmaceutical composition is characterized in that, contains the described polypeptide of claim 1 in the composition.

6. the pharmaceutical composition of claim 5 is characterized in that, contains the medicine acceptable carrier in the composition.

7. the pharmaceutical composition of claim 5 is characterized in that, composition is an injection.

8. the pharmaceutical composition of claim 5 is characterized in that, composition is a freeze drying injection.

9. the preparation method of the polypeptide of claim 1 is characterized in that, described method is selected from synthetic method, gene recombination method or chemical synthesis process in conventional solid phase synthesis process, the solution.

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