A stapled peptide targeting trib3 / ssrp1 interaction and its use in the preparation of an anti-multiple myeloma drug

Abstract

The application discloses a stapling peptide targeting TRIB3 / SSRP1 interaction and application of the stapling peptide in preparation of anti-multiple myeloma drugs. Through a series of experiments such as immunoprecipitation coupling mass spectrometry, it is found that TRIB3 directly interacts with SSRP1 protein in multiple myeloma cells, thereby playing a role in promoting malignant proliferation of multiple myeloma, and TRIB3 promotes degradation of the SSRP1 protein through a ubiquitin-proteasome pathway. The stapling peptide 7695-SP5 inhibits malignant proliferation of multiple myeloma by specifically inhibiting TRIB3 / SSRP1 protein interaction. The stapling peptide 7695-SP5 can be used as a TRIB3 / SSRP1 protein interaction inhibitor to treat diseases caused by increased expression of TRIB3 and SSRP1 proteins, such as multiple myeloma, and is expected to be developed into a novel anti-tumor drug.

Description

TECHNICAL FIELD

[0001] The present application relates to a stapling peptide and its use, in particular to a stapling peptide targeting TRIB3 / SSRP1 interaction and its application in the preparation of anti-multiple myeloma drugs. It belongs to the technical field of medicine. BACKGROUND

[0002] Multiple myeloma (MM) is the second most common hematological malignancy in the world, which can appear genetic abnormalities such as hyperdiploidy and translocation involving immunoglobulin heavy chain. The application of immunomodulatory drugs, proteasome inhibitors and CD38-targeted antibodies significantly prolongs the survival of patients, but most patients eventually die of the disease, and some patients die of treatment-related complications. The characteristics of disease progression and subsequent relapse are subclonal evolution and increasing drug resistance of the disease. Among them, proteasome inhibitor bortezomib (BTZ) resistance is a common type of drug resistance, which is an urgent problem in clinical practice.

[0003] Studies have shown that pseudokinases play a key role in tumors, so it is very important to find pseudokinases as drug targets and elucidate their functions and mechanisms in tumors. Tribble 3 (TRIB3) is an important member of pseudokinases, which plays an important role in tumor stemness, immune response and autophagy and other functions. Inhibiting TRIB3 may become a new strategy for the treatment of multiple myeloma.

[0004] Structural specificity recognition protein-1 (SSRP1) is a key component of chromatin transcription factor (FACT) and is involved in the regulation of DNA damage repair, DNA replication, apoptosis and cell cycle. Studies have shown that SSRP1 can play a key role in tumors through DNA damage repair, Wnt and AKT signaling pathways. Since pseudokinases exert their functions by assembling complexes, the applicant found the interaction protein SSRP1 of TRIB3 through the technology of immunoprecipitation coupled with mass spectrometry. Through a series of experiments, the applicant proved that TRIB3 directly interacts with SSRP1 protein, and TRIB3 can promote the degradation of SSRP1 protein through the ubiquitin-proteasome pathway.

[0005] Protein-protein interaction plays a decisive role in regulating the life activities of the body, and is the key mechanism of many signal transduction pathways in the body. The alpha-helix peptide in the folding subdomain of the polypeptide secondary structure in the protein-protein interaction is extracted alone, and is constructed by chemical synthesis, which can obtain a polypeptide drug lead targeting specific protein interaction. However, most of the polypeptide fragments cannot form the secondary structure required for binding after being separated from the overall structure of the protein, and are prone to form random coil conformation, thereby leading to decreased binding activity, and are more susceptible to degradation by peptidases, and cannot be directly used for drug development. The use of all-carbon skeleton to form a side chain ring structure to modify the polypeptide to stabilize the active conformation of the alpha-helix peptide, i.e. the stapled peptide, is the most effective method to overcome this defect. This method not only improves the original protein binding activity, but also has high metabolic stability and cell membrane permeability. Based on these significant advantages, in recent years, the stapled peptide has gradually become a polypeptide drug with promising application prospects in the treatment of tumors and other diseases. SUMMARY

[0006] The purpose of the present application is to overcome the shortcomings of the prior art, and to provide a stapled peptide targeting TRIB3 / SSRP1 interaction and its application in the preparation of an anti-multiple myeloma drug.

[0007] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0008] 1. A stapled peptide targeting TRIB3 / SSRP1 interaction, i.e. stapled peptide 7695-SP5, having an amino acid sequence of GFRESEFEKLXDFFXTHYRL, as shown in SEQ ID NO. 1; wherein X is S5, i.e. S configuration pentenyl alanine.

[0009] 2. Application of a TRIB3 / SSRP1 protein interaction inhibitor in the preparation of an anti-multiple myeloma drug.

[0010] 3. Application of the aforementioned stapled peptide as a TRIB3 / SSRP1 protein interaction inhibitor.

[0011] 4. Application of the aforementioned stapled peptide in the preparation of an anti-multiple myeloma drug.

[0012] 5. A combination of the aforementioned stapled peptide and bortezomib, and the molar ratio of the two is 1250-5000:2.5-10.

[0013] 6. Application of the aforementioned combination in the preparation of an anti-multiple myeloma drug.

[0014] 7. An anti-multiple myeloma drug, wherein the effective component is a TRIB3 / SSRP1 protein interaction inhibitor.

[0015] 8. An anti-multiple myeloma drug, wherein the effective component is the stapled peptide.

[0016] 9. An anti-multiple myeloma drug, comprising the composition.

[0017] Advantages of the present application:

[0018] The applicant found that TRIB3 promotes the malignant proliferation of multiple myeloma cells by directly interacting with SSRP1 protein through a series of experiments such as immunoprecipitation coupled with mass spectrometry, and that TRIB3 promotes the degradation of SSRP1 protein through the ubiquitin-proteasome pathway. Through the interaction fragments of the two, the applicant designed a stapled peptide 7695-SP5 targeting the interaction of TRIB3 / SSRP1 protein. Through experiments on the stapled peptide 7695-SP5 at the cell and mouse levels, it was found that the stapled peptide 7695-SP5 has significant anti-multiple myeloma activity and can significantly inhibit the interaction of TRIB3 / SSRP1 protein.

[0019] In summary, the stapled peptide 7695-SP5 inhibits the malignant proliferation of multiple myeloma by specifically inhibiting the interaction of TRIB3 / SSRP1 protein. The stapled peptide 7695-SP5 can be used as an inhibitor of TRIB3 / SSRP1 protein interaction to treat diseases caused by the up-regulation of TRIB3 and SSRP1 protein expression, such as multiple myeloma, and is expected to be developed as a new anti-tumor drug. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Interaction of TRIB3 and SSRP1 in multiple myeloma cells.

[0021] A is the detection of interaction protein bands combined with TRIB3 after treating the sample with an immunoprecipitation (IP) antibody of TRIB3 by immunoprecipitation combined with coomassie brilliant blue staining. B is the mass spectrum peak diagram of SSRP1 protein found after cutting the specific band, digesting the gel strip, and then performing mass spectrometry analysis. C is to screen the top five proteins in the mass spectrometry results, and then detect the proteins interacting with TRIB3 by immunoprecipitation. The results show that only SSRP1 protein interacts with TRIB3. D is to purify the His-labeled SSRP1 protein and the GST-labeled TRIB3 protein after prokaryotic expression, collect the protein, and then perform pull down experiment. E is to collect the cytoplasmic and nuclear proteins of ARP1 cells by nuclear and cytoplasmic separation, and then perform immunoprecipitation experiment, and then detect the interaction of SSRP1 and TRIB3 in the nucleus and cytoplasm by immunoblotting.

[0022] Figure 2 To study the domain of TRIB3 and SSRP1 interaction. A According to the literature related to TRIB3, TRIB3 protein is divided into NTD, KDN, KDC and CTD domains. B SSRP1-FLAG full-length (FL) plasmid, TRIB3-EGFP full-length plasmid and TRIB3 domain plasmid: TRIB3-EGFP-NTD, TRIB3-EGFP-KDN, TRIB3-EGFP-KDC and TRIB3-EGFP-CTD plasmid were constructed respectively. Then, SSRP1-FLAG-FL plasmid and the related plasmid of TRIB3-EGFP were overexpressed in HEK-293T cells, and the TRIB3 domain interacting with SSRP1 was detected by immunoprecipitation. C According to the literature related to SSRP1, SSRP1 protein is divided into NTD, MD, IDD, HMG and CTD domains. D TRIB3-EGFP full-length plasmid, SSRP1-FLAG full-length plasmid and SSRP1-FLAG domain plasmid: SSRP1-FLAG-NTD, SSRP1-FLAG-MD, SSRP1-FLAG-IDD and SSRP1-FLAG-HMG+CTD plasmid were constructed respectively. Then, TRIB3-EGFP full-length plasmid and the related plasmid of SSRP1-FLAG domain were overexpressed in 293T cells, and the SSRP1 domain interacting with TRIB3 was detected by immunoprecipitation.

[0023] Figure 3 TRIB3 regulates the protein level of SSRP1, but does not affect its mRNA level. A After knocking down TRIB3 in MM cells ARP1 and MM.1S cells, the changes of TRIB3 and SSRP1 proteins in cells were detected by immunoblotting. B After overexpressing TRIB3 in MM cells ARP1 and MM.1S cells, the changes of TRIB3 and SSRP1 proteins in cells were detected by immunoblotting. C After knocking down TRIB3 in MM cells ARP1 and MM.1S cells, the expression changes of TRIB3 and SSRP1 at mRNA level were detected by real-time fluorescence quantitative polymerase chain reaction (qPCR) method. D After overexpressing TRIB3 in MM cells ARP1 and MM.1S cells, the expression changes of TRIB3 and SSRP1 at mRNA level were detected by qPCR method.

[0024] Figure 4 TRIB3 plays a role in promoting the malignant proliferation of multiple myeloma through SSRP1. A After knocking down TRIB3 in ARP1 cells, overexpressing SSRP1, the changes of SSRP1 protein in it were detected by immunoblotting method. B After knocking down TRIB3 in ARP1 cells, overexpressing SSRP1, the changes of cell survival rate were detected by CCK8 method.

[0025] Figure 5 TRIB3 promotes the degradation of SSRP1 protein through the ubiquitin-proteasome pathway. A. After knocking down TRIB3 in ARP1 cells and adding CHX, the change of SSRP1 protein in cells was detected by immunoblotting. B. Statistical chart of Figure A. C. After knocking down TRIB3 in ARP1 cells and adding MG132, the change of SSRP1 protein was detected by immunoblotting. D. Statistical chart of Figure C. E. After knocking down TRIB3, ARP1 cells were collected for immunoprecipitation experiment, and the change of ubiquitination of SSRP1 protein was detected by immunoblotting.

[0026] Figure 6 The amino acid sequence of SSRP1 protein 76-95 binds to the KDC domain of TRIB3. To construct a stapled peptide targeting the interaction between TRIB3 and SSRP1 protein, it is necessary to clarify the amino acid sequence of SSRP1 protein that binds to TRIB3. A. The spatial structure of the NTD domain of SSRP1 protein was predicted by I-TASSER software, and 76-95 was predicted as an alpha-helix structure region. B. According to the amino acid sequence of SSRP1 protein 76-95, the spatial structure of the alpha-helix structure was predicted. C. To verify that the amino acid sequence of SSRP1 protein 76-95 is the region that binds to TRIB3, TRIB3-EGFP full-length plasmid, SSRP1-FLAG-1-75, SSRP1-FLAG-76-95 and SSRP1-FLAG-96-177 plasmids were constructed. Subsequently, the relevant plasmids of TRIB3-EGFP full-length plasmid and SSRP1-FLAG fragments were overexpressed in 293T cells, and the interaction of SSRP1 fragments with TRIB3 was detected by immunoprecipitation.

[0027] Figure 7 Stapled peptide 7695-SP5 significantly inhibits the proliferation of multiple myeloma cells by inhibiting the interaction between TRIB3 and SSRP1. A. A full-carbon chain structure was selected, and S-type unnatural amino acids with a side chain carbon chain length of 5 were introduced at the 86 and 90 amino acid sites, and then a stapled peptide structure was constructed by olefin metathesis reaction. B. Structure of stapled peptide 7695. C. In ARP1 cells, the effect of stapled peptide 7695-SP5 on the survival of ARP1 cells was detected by CCK8 method. D. In ARP1 cells, the change of TRIB3 interacting with SSRP1 was detected by immunoblotting after adding stapled peptide 7695-SP5 and performing immunoprecipitation experiment with SSRP1 antibody.

[0028] Figure 8The stapled peptide 7695-SP5 and bortezomib exhibit synergistic effects in vivo and in vitro. A: Different concentrations of SP5 (0, 1.25, 2.5, 5 μM) and BTZ (0, 2.5, 5, 10 nM) were added to ARP1 cells, and the changes in cell survival were detected by the CCK8 method after 48 h of culture. B: The results of A were analyzed for synergistic effect and synergistic index (CI) by Compusyn software, and CI value > 1 indicates that the two drugs have antagonistic effect, and CI < 1 indicates that the two drugs have synergistic effect. C: A diffuse multiple myeloma mouse model was constructed by injecting ARP1 cells with luciferase into the tail vein of NCG mice. Then, DPBS, BTZ (0.3 mg / kg), SP5 (5 mg / kg), and BTZ+SP5 (0.3 mg / kg+5 mg / kg) were injected intraperitoneally into different groups of mice. After five drug treatments, the mice were imaged by a small animal imaging instrument, and the tumors in the mice were significantly inhibited after drug treatment, and the effect was more significant after the two drugs were used in combination. DETAILED DESCRIPTION

[0029] The application will be further described below in conjunction with the accompanying drawings and examples, and it should be noted that the following description is only for the purpose of explaining the application and does not limit the content thereof.

[0030] The stapled peptide 7695-SP5 involved in the application has a purity of 98% (mass percentage) and is designed and synthesized by Anhui Hefei Kesheng Jingpeide Company according to the 76-95 amino acid sequence of SSRP1 protein.

[0031] Example 1: Interaction between TRIB3 and SSRP1 in multiple myeloma cells

[0032] TRIB3 interacts with different proteins and has the effect of promoting malignant proliferation in various tumors. In this application, the applicant first screened and verified specific proteins that interact with TRIB3 in multiple myeloma by the method of immunoprecipitation coupled with mass spectrometry. And further explore whether the two directly interact through Pulldown experiment. Since TRIB3 and SSRP1 proteins are mainly located in the nucleus, the applicant further explores the position of their interaction by nuclear-cytoplasmic separation and then immunoprecipitation.

[0033] 1. After treating the sample with the IP antibody of TRIB3, the interacting protein bands combined with TRIB3 were detected by immunoprecipitation combined with Coomassie blue staining.

[0034] (1) Collect 4 dishes of ARP1 cells (from Tianjin Blood Center) and add IP lysis buffer for protein lysis.

[0035] (2) Protein supernatant was collected in a new 1.5 mL centrifuge tube, and 60 μL was taken as the Input group.

[0036] (3) The remaining protein solution was evenly divided into two parts, and 1 μL of IgG antibody and 1 μL of TRIB3 antibody were added respectively. After mixing, the sample was rotated at 4°C for 12 h to allow the antibody and antigen to fully bind.

[0037] (4) The protein A / G immunoprecipitation magnetic beads were taken out and vortexed for 10 s, and 20 μL of magnetic beads were taken in each 1.5 mL centrifuge tube.

[0038] (5) 100 μL of IP lysis solution was added to the 1.5 mL centrifuge tube, mixed, and centrifuged at 4°C and 1200 rpm for 5 min.

[0039] (6) The washed magnetic beads were added to different sample tubes and rotated at room temperature for 3 h.

[0040] (7) Centrifuged at 4°C and 1200 rpm for 5 min, and placed the centrifuge tube on the magnetic stand, and discarded the supernatant.

[0041] (8) 600 μL of washing solution was added to the sample tube, mixed, and centrifuged at 4°C and 1200 rpm for 5 min, and placed the centrifuge tube on the magnetic stand, and discarded the supernatant, and repeated 3 times.

[0042] (9) 60 μL of sample buffer was added to the sample tube, and the sample was boiled at 99°C for 10 min.

[0043] (10) Centrifuged at 4°C and 1200 rpm for 5 min, and placed the centrifuge tube on the magnetic stand, and took the supernatant for electrophoresis.

[0044] (11) The gel was removed, and the gel was completely covered with Coomassie Brilliant Blue R250, and stained for 2 h.

[0045] (12) Decolorized, and photographed with a gel imager.

[0046] 2. After cutting the specific band, the gel strip was digested, and mass spectrometry analysis was performed, and the mass spectrometry peak graph of the SSRP1 protein was found.

[0047] (1) The stained band was decolorized with decolorizing solution, washed with water 4 times, and then washed with 300 ul of 25 mM ammonium bicarbonate solution, 50% volume concentration acetonitrile aqueous solution and acetonitrile in turn 2 times, and finally dehydrated the gel to make the gel block white.

[0048] (2) Add 50 μl of 10 mM dithiothreitol solution, reduce at 56°C for 30 min, and then add 50 μl of 50 mM indoleacetic acid solution to alkylate for 15 min in the dark after the temperature drops to room temperature. Then wash the gel strip with 300 μl of 25 mM ammonium bicarbonate solution, 50% (by volume) acetonitrile aqueous solution, and acetonitrile in sequence for two times, and finally dehydrate the gel to make the gel block white.

[0049] (3) Add 20 μl of 0.01 μg / μl proteomics-grade trypsin aqueous solution, and then add 40 μl of 50 mM NH4HCO3 solution to cover.

[0050] (4) After the enzymolysis, transfer the supernatant to another new centrifuge tube, add 100 μL of extraction solution (aqueous solution containing 67% (by volume) acetonitrile and 2% (by volume) formic acid) to the remaining gel block, incubate at 37°C for 30 min, and then ultrasonic for 15 min. Centrifuge and combine the supernatant, and then centrifuge, concentrate and dry.

[0051] 3. To screen the top five proteins in the mass spectrometry results, detect the proteins interacting with TRIB3 by immunoprecipitation, and it is found that only the SSRP1 protein interacts with TRIB3.

[0052] (1) Collect 4 dishes of ARP1 cells, and lyse with IP lysis buffer.

[0053] (2) Collect the protein supernatant in a new 1.5 mL centrifuge tube, and take 60 μL as the Input group. Divide the remaining protein solution into two parts, and add 1 μL of IgG antibody and 1 μL of TRIB3 antibody respectively.

[0054] (3) Take protein A / G immunoprecipitation magnetic beads, vortex for 10 s, and take 20 μL of magnetic beads into each 1.5 mL centrifuge tube.

[0055] (4) Add 100 μL of IP lysis buffer to the 1.5 mL centrifuge tube, mix well, centrifuge at 1200 rpm for 5 min at 4°C, place the centrifuge tube on a magnetic stand, and discard the supernatant.

[0056] (5) Add the washed magnetic beads to different sample tubes, rotate for 3 h at room temperature, so that the antibody and the magnetic beads are fully combined.

[0057] (6) Centrifuge at 1200 rpm for 5 min at 4°C, place the centrifuge tube on a magnetic stand, and discard the supernatant.

[0058] (7) Add 600 μL of washing solution to the sample tube, mix well, centrifuge at 1200 rpm for 5 min at 4°C, place the centrifuge tube on a magnetic stand, discard the supernatant, and repeat for three times.

[0059] (8) Add 60 μL sample buffer to the sample tube, and boil at 99°C for 10 min. Electrophoresis, membrane transfer, and then blocking in milk.

[0060] (9) Dilute the primary antibodies of TRIB3, SSRP1, MCM4, MCM5, ILF3, and TRIM28 (all from Abclonal) and incubate in the appropriate antibody blocking slot, and incubate the corresponding target band at 4°C overnight.

[0061] (10) Rinse with phosphate buffered saline (pH 7.4, from Gibco) for 3 times, add 20 mL of blocking solution (5 g of skimmed milk (from bio-rad) + 100 mL of phosphate buffered saline) for blocking, and incubate at room temperature for 1 h on a dark shaking table.

[0062] (11) Rinse with phosphate buffered saline (pH 7.4) for 3 times, and develop with ECL luminescent solution (from Zeta).

[0063] 4. Purify the His-tagged SSRP1 protein and the GST-tagged TRIB3 protein after prokaryotic expression, collect the proteins, and then perform a pull-down experiment.

[0064] (1) Transform the target plasmid into E. coli BL21 DE3 competent cells (from Sigma).

[0065] (2) Inoculate a single colony into LB culture solution.

[0066] (3) Shake culture overnight, and the next day, dilute the bacterial solution in 200 mL of LB culture solution at a ratio of 1:50 (volume ratio).

[0067] (4) Add inducer IPTG (from Biyun Tian) to a final concentration of 1 mM, and culture at 16°C and 180 rpm overnight, about 18 h.

[0068] (5) Add 2 mL of lysis solution to every 100 mL of bacterial solution to extract bacterial proteins.

[0069] (6) Mix the eukaryotic expression protein GST-TRIB3 with the prokaryotic expression His-SSRP1 protein and the fusion protein, and incubate at 4°C overnight or at room temperature for 3-4 h.

[0070] (7) Centrifuge to separate the GST agarose, resuspend and wash the IP lysis solution, transfer to a new EP tube, centrifuge to remove the supernatant, wash twice with IP lysis solution, centrifuge to remove the supernatant, add loading buffer, mix, and dissociate the antigen-antibody complex at 100°C for 10 min. After boiling, transfer to an EP tube, and perform immunoblotting detection.

[0071] 5. Collect the cytoplasm and nucleus protein of ARP1 cells by nuclear and cytoplasmic separation, and then respectively conduct immunoprecipitation experiment, and then detect the interaction of SSRP1 and TRIB3 in the nucleus and cytoplasm by immunoblotting method.

[0072] (1) Collect 2x10 7 ARP1 cells, add 1 mL of CER I in the nuclear and cytoplasmic separation kit (from Thermo Fisher Scientific Company) to the cell precipitate, mix, vortex for 20 s, and incubate on ice for 20 min.

[0073] (2) Add 55 μL of CER II in the nuclear and cytoplasmic separation kit, mix, vortex for 10 s, and incubate on ice for 1 min.

[0074] (3) Vortex for 10 s, and centrifuge at 15000 g at 4°C for 10 min.

[0075] (4) Immediately transfer the supernatant to a clean 1.5 mL centrifuge tube.

[0076] (5) Add 500 μL of phosphate buffer (pH 7.4, from Gibco Company) to the insoluble matter, centrifuge at 15000 g at 4°C for 5 min, discard the supernatant, add 500 μL of NER in the nuclear and cytoplasmic separation kit, vortex for 20 s, and repeat 5 times.

[0077] (6) Centrifuge at 15000 g at 4°C for 15 min.

[0078] (7) Immediately transfer the supernatant to a clean 1.5 mL centrifuge tube, and place on ice. Store the protein at -80°C.

[0079] (8) Respectively conduct immunoprecipitation experiment on the nucleus and cytoplasm part by using the IP antibody of TRIB3. The specific immunoprecipitation steps are as described above.

[0080] The experimental results are as follows Figure 1Figure 1 shows the results of the experiment. A is the band of the interaction protein combined with TRIB3 detected by immunoprecipitation combined with Coomassie blue staining after treating the sample with IP antibody of TRIB3. B is the mass spectrum peak chart of the SSRP1 protein found by performing mass spectrometry on the gel strip after cutting the specific band and performing enzyme digestion. C is the top five proteins screened out from the mass spectrometry results, and the proteins interacting with TRIB3 are detected by immunoprecipitation. The results show that only the SSRP1 protein interacts with TRIB3. D is the Pull down experiment after purifying the His-labeled SSRP1 protein and the GST-labeled TRIB3 protein by prokaryotic expression. E is the cytoplasmic and nuclear proteins of ARP1 cells collected by nuclear and cytoplasmic separation, and then the immunoprecipitation experiment is performed respectively, and the interaction of SSRP1 and TRIB3 in the nucleus and cytoplasm is detected by immunoblotting.

[0081] Example 2

[0082] The KDC domain of TRIB3 and the NTD domain of SSRP1 protein interact. By expressing each domain segment of TRIB3 and SSRP1, the main domains of the interaction between the two are explored in detail

[0083] The pEGFP-C1 plasmid (from UbiBios) was cut by endonuclease, the TRIB3 gene was amplified by PCR reaction, and the CDS full-length sequence of TRIB3 was introduced into the pEGFP-C1 plasmid by restriction endonuclease digestion and T4 ligase connection, thereby obtaining the TRIB3-EGFP full-length plasmid. The pCDNA3.1 plasmid (from UbiBios) was cut by appropriate endonuclease, the CDS sequences of SSRP1 were amplified by PCR reaction, and the CDS sequences of SSRP1 were introduced into the pCDNA3.1 plasmid by restriction endonuclease digestion and T4 ligase connection, thereby obtaining the SSRP1-FLAG-NTD, SSRP1-FLAG-MD, SSRP1-FLAG-IDD and SSRP1-FLAG-HMG+CTD plasmids.

[0084] 1. Explore the TRIB3 domain interacting with SSRP1 protein.

[0085] (1) According to the TRIB3 related literature, the TRIB3 protein is divided into NTD, KDN, KDC and CTD domains. The CDS sequences of TRIB3 and SSRP1 are searched in the ensembl website, and the CDS sequences corresponding to the NTD, KDN, KDC and CTD domains of TRIB3 are analyzed.

[0086] (2) pEGFP-C1 plasmid was cut by endonuclease, TRIB3 gene was amplified by PCR reaction, and the CDS sequences of TRIB3 were introduced into the pEGFP-C1 plasmid (from Youbao Biotechnology) by restriction endonuclease digestion and T4 ligase connection, so as to obtain the TRIB3-EGFP full-length plasmid and the plasmids of TRIB3 each domain: TRIB3-EGFP-NTD, TRIB3-EGFP-KDN, TRIB3-EGFP-KDC and TRIB3-EGFP-CTD plasmid.

[0087] (3) pCDNA3.1 plasmid was cut by appropriate endonuclease, and each CDS sequence of SSRP1 was amplified by PCR reaction, and the CDS sequence of SSRP1 was introduced into the pCDNA3.1 plasmid (from Youbao Biotechnology) by restriction endonuclease digestion and T4 ligase connection, so as to obtain SSRP1-FLAG-FL, SSRP1-FLAG-NTD, SSRP1-FLAG-MD, SSRP1-FLAG-IDD and SSRP1-FLAG-HMG+CTD plasmid.

[0088] (4) 293T cells were cultured in DMEM medium (DMEM from Hyclone Company) and 10% fetal bovine serum (from Vivacell Company).

[0089] (5) 3x10 5 6 cells were added to each 6-well plate. 100 μL of DMEM medium, 2 μg of SSRP1-FLAG-FL plasmid and each domain of TRIB3-EGFP plasmid were added to a 1.5 mL centrifuge tube, and 5 μL of turbofect (from Thermo Fisher Scientific) was added, mixed and incubated for 15 minutes, and then added dropwise to the 6-well plate.

[0090] (6) After 48 hours, the 293T cells of each group were collected and subjected to immunoprecipitation experiment. The immunoprecipitation steps are as described above.

[0091] 2. Explore the protein domain of SSRP1 interacting with TRIB3.

[0092] (1) According to the related literature of SSRP1, the SSRP1 protein is divided into NTD, MD, IDD, HMG and CTD domains. The CDS sequences of TRIB3 and SSRP1 were searched in the ensembl website, and the CDS sequences corresponding to the NTD, MD, IDD, HMG and CTD domains of SSRP1 were analyzed.

[0093] (2) The pEGFP-C1 plasmid was digested with restriction enzymes, and the TRIB3 gene was amplified by PCR. After restriction enzyme digestion and ligation with T4 ligase, the full-length CDS sequence of TRIB3 was introduced into the pEGFP-C1 plasmid, thus obtaining the full-length TRIB3-EGFP plasmid. The pCDNA3.1 plasmid was digested with appropriate restriction enzymes, and the various CDS sequences of SSRP1 were amplified by PCR. After restriction enzyme digestion and ligation with T4 ligase, the various CDS sequences of SSRP1 were introduced into the pCDNA3.1 plasmid, thus obtaining the SSRP1-FLAG-FL plasmid and plasmids of various domains of SSRP1-FLAG: SSRP1-FLAG-NTD, SSRP1-FLAG-MD, SSRP1-FLAG-IDD, and SSRP1-FLAG-HMG+CTD plasmids.

[0094] (3) Overexpress the full-length TRIB3-EGFP plasmid and related plasmids of each domain of SSRP1-FLAG in 293T cells. The overexpression method is the same as above.

[0095] (4) The immunoprecipitation procedure is as described above.

[0096] Experimental results are as follows Figure 2 As shown, Figure 2 The results in A and B indicate that the KDC domain of TRIB3 mainly binds to the SSRP1 protein. Figure 2 C and D indicate that the NTD domain of SSRP1 mainly binds to TRIB3. This suggests that the interaction between TRIB3 and SSRP1 proteins is essentially an interaction between the KDC domain of TRIB3 and the NTD domain of SSRP1.

[0097] Example 3:

[0098] TRIB3 regulates the protein level of SSRP1 but does not affect its mRNA level. By knocking down and overexpressing TRIB3, Western blotting and qPCR were used to detect changes in the protein and mRNA levels of TRIB3 and SSRP1, thereby exploring the mechanism by which TRIB3 regulates SSRP1.

[0099] 1. After knocking down and overexpressing TRIB3, the changes in TRIB3 and SSRP1 proteins were detected by Western blotting.

[0100] (1) Interference with TRIB3 expression: ARP1 and MM.1S cells (from the ATCC cell bank) in the logarithmic phase were mixed by pipetting, and 0.5 ml of cell suspension was transferred to four wells of a six-well plate and labeled as shNC group (control group) and sh1, sh2 group (knockdown group).

[0101] (2) Overexpression of TRIB3 expression: ARP1 and MM.1S cells in the logarithmic phase in the culture dish were blown and mixed uniformly, and 0.5 ml of cell suspension was taken into a six-well plate, respectively, and labeled as EV group (control group) and OE TRIB3 group (overexpression group)

[0102] (3) Detection of protein changes of TRIB3 and SSRP1 by immunoblotting, as described previously.

[0103] 2. After knockdown and overexpression of TRIB3, changes of TRIB3 and SSRP1 at the mRNA level were detected by qPCR.

[0104] (1) ARP1 and MM.1S cells were collected, and 1 ml of TRIzol (from Thermo Fisher Scientific) was added to the cell precipitate.

[0105] (2) 300 μL of chloroform was added to the TRIzol, and after being mixed by inversion, it was placed at room temperature for 15 min. The colorless and transparent upper aqueous phase was absorbed into a new 1.5 ml centrifuge tube. The same volume of isopropanol as the upper aqueous phase was added, and after being mixed by inversion, it was placed at room temperature for 15 min. 12000 rpm, 4°C centrifugation for 10 min, and the supernatant was discarded.

[0106] (3) A 75% ethanol solution was added to the cell precipitate, 8000 rpm, 4°C centrifugation for 5 min, and the supernatant was discarded.

[0107] (4) After filtration, add RNase-free water (from Thermo Fisher Scientific) to dissolve, and measure the RNA concentration.

[0108] (5) Reverse transcription: use the reverse transcription kit (Novozyme) to reverse transcribe the RNA to obtain cDNA.

[0109] (6) Design qPCR primers for TRIB3, SSRP1 and GAPDH, and dissolve the primers synthesized by GenScript Biotech Corporation into 10 μM with RNase-free water.

[0110] The qPCR primer sequences are as follows:

[0111] GAPDH:

[0112] F: 5'-CGCTGAGTACGTCGTGGAGTC-3', as shown in SEQ ID NO. 2;

[0113] R: 5'-GCTGATGATCTTGAGGCTGTTGTC-3', as shown in SEQ ID NO. 3;

[0114] TRIB3

[0115] F: 5'-TCAAGCTGTGTCGCTTTGTC-3', as shown in SEQ ID NO. 4;

[0116] R: 5'-TGTCCCACAGGGAATCATCT-3', as shown in SEQ ID NO. 5;

[0117] SSRP1

[0118] F: 5'-TTGAGAGGGAGGAGTACGGG-3', as shown in SEQ ID NO. 6;

[0119] R: 5'-CTAGCTTGGGTTCATGCCCT-3', as shown in SEQ ID NO. 7;

[0120] (7) Add cDNA, 2x SYBR Green Mix (from Thermo Fisher Scientific) and primers for qPCR experiment, see Table 1 for reaction system.

[0121] Table 1. qPCR reaction system

[0122]

[0123] qPCR reaction conditions: 95°C for 10 minutes; 95°C for 20s, 59°C for 20s, 72°C for 20s, 40 cycles.

[0124] The experimental results are shown in Figure 3 , in Figure 3 A, after knocking down TRIB3 in ARP1 and MM.1S cells, the expression of SSRP1 protein was significantly reduced, and in Figure 3 B, after overexpressing TRIB3 in ARP1 and MM.1S cells, the expression of SSRP1 protein was significantly increased. And in Figure 3 C and D, after knocking down and overexpressing TRIB3, there was no significant change in the mRNA of SSRP1. This indicates that TRIB3 directly regulates the protein level of SSRP1.

[0125] Example 4

[0126] TRIB3 promotes the malignant proliferation of multiple myeloma through SSRP1. To determine that SSRP1 is a key downstream molecule of TRIB3, the applicant used the method of overexpressing SSRP1 after knocking down TRIB3 to explore.

[0127] 1. Overexpress SSRP1 after knocking down TRIB3 in ARP1 cells, and detect the changes in SSRP1 protein by immunoblotting.

[0128] (1) Interference with TRIB3 expression in ARP1 cells, as previously described.

[0129] (2) Add SSRP1 overexpressing lentivirus to ARP1 cells with TRIB3 knockdown, and then screen for 2 days with puromycin.

[0130] (3) Collect ARP1 cell protein and detect changes in SSRP1 protein by immunoblotting.

[0131] 2. After knocking down TRIB3 in ARP1 cells, SSRP1 was overexpressed, and the changes in cell viability were detected by CCK8 assay.

[0132] (1) Interference with TRIB3 expression in ARP1 cells, as previously described.

[0133] (2) Add SSRP1 overexpressing lentivirus to ARP1 cells with TRIB3 knockdown, and then screen for 2 days with puromycin.

[0134] (3) Collect ARP1 cells from different groups and add 1×10⁻⁶ cells to each well of a 96-well plate. 4 Cells were cultured for 2 days, then incubated with 10% CCK8 solution for 3 hours, and the OD value was detected at a wavelength of 450 nm.

[0135] Experimental results are as follows Figure 4 , Figure 4 The results from the study showed that SSRP1 protein levels were significantly reduced after TRIB3 knockdown, but the decrease in SSRP1 protein caused by TRIB3 knockdown was significantly reversed after SSRP1 overexpression. Similarly, ARP1 cell survival was significantly reduced after TRIB3 knockdown, but the cell survival decrease caused by TRIB3 knockdown was also reversed after SSRP1 overexpression. Figure 4 (B). The results above indicate that TRIB3 functions in MM through SSRP1.

[0136] Example 5

[0137] TRIB3 promotes SSRP1 protein degradation via the ubiquitin-proteasome pathway. Previous results indicate that TRIB3 functions through SSRP1 and directly regulates SSRP1 protein levels; however, the pathway by which TRIB3 regulates SSRP1 remains unclear. Considering that TRIB3 and SSRP1 primarily interact in the cell nucleus, the applicant hypothesizes that TRIB3 regulates SSRP1 protein stability through the ubiquitin-proteasome pathway. Therefore, the applicant conducted multiple experiments involving actinomycin, MG132, and ubiquitination to investigate this hypothesis.

[0138] 1. After knocking down TRIB3 in ARP1 cells and adding CHX, the change of SSRP1 protein in the cells was detected by immunoblotting.

[0139] (1) Collect control and ARP1 cells with knockdown of TRIB3.

[0140] (2) Treat the cells with 100 μg / ml CHX for 0, 4, 8, 12 h, lyse the cells with protein lysis buffer, and perform Western blot analysis.

[0141] 2. After knocking down TRIB3 in ARP1 cells, add proteasome inhibitor MG132, and detect the change of SSRP1 protein by immunoblotting.

[0142] (1) Collect control and ARP1 cells with knockdown of TRIB3.

[0143] (2) Incubate for 6 hours in the presence of 10 μM MG132 (proteasome inhibitor), lyse the cells with protein lysis buffer, and perform Western blot analysis.

[0144] 3. After knocking down TRIB3, collect ARP1 cells for immunoprecipitation experiment, and detect the change of SSRP1 protein ubiquitination by immunoblotting.

[0145] (1) Collect control and ARP1 cells with knockdown of TRIB3.

[0146] (2) Perform immunoprecipitation experiment with IP antibody of SSRP1, and then detect the change of SSRP1 protein itself and its ubiquitination level by immunoblotting.

[0147] The experimental results are shown in Figure 5 , in Figure 5 A and B, after ARP1 cells with knockdown of TRIB3 are added with CHX, the rate of decrease of SSRP1 protein is significantly accelerated, and in Figure 5 C and D, it is shown that after knockdown of TRIB3, SSRP1 protein is significantly decreased, but after adding MG132, the decrease of SSRP1 protein caused by knockdown of TRIB3 is significantly recovered, which indicates that TRIB3 plays a role in regulating SSRP1 protein through this path. Further, as shown in Figure 5 E, after knockdown of TRIB3, the ubiquitination level of SSRP1 protein is significantly increased, which indicates that TRIB3 regulates the degradation of SSRP1 protein through the ubiquitin-proteasome pathway.

[0148] Example 6

[0149] The amino acid sequence of SSRP1 protein 76-95 binds with the KDC domain of TRIB3. To construct a stapled peptide targeting the interaction between TRIB3 and SSRP1 protein, it is necessary to clarify the amino acid sequence of SSRP1 protein that binds with TRIB3.

[0150] 1. The I-TASSER software predicts that the amino acid sequence of SSRP1 protein 76-95 is an alpha-helix structure.

[0151] (1) The amino acid sequence of the NTD of SSRP1 is input into the I-TASSER software to perform spatial structure prediction of the alpha-helix structure of the domain present.

[0152] (2) The alpha-helix structure of the 76-95 amino acid sequence is spatially simulated.

[0153] 2. Co-immunoprecipitation detects that the amino acid sequence of SSRP1 protein 76-95 is the region that binds with TRIB3.

[0154] (1) Through the CDS full-length sequence of SSRP1, the CDS sequences corresponding to the amino acids 1-75, 76-95, and 96-177 of SSRP1 protein are analyzed.

[0155] (2) The pEGFP-C1 plasmid is cut by an endonuclease, the TRIB3 gene is amplified by PCR reaction, and the CDS full-length sequence of TRIB3 is introduced into the pEGFP-C1 plasmid through restriction enzyme digestion and T4 ligase connection, thereby obtaining the TRIB3-EGFP full-length plasmid. The pCDNA3.1 plasmid is appropriately cut by an endonuclease, the CDS sequences of SSRP1 are amplified by PCR reaction, and the CDS sequences of SSRP1 are introduced into the pCDNA3.1 plasmid through restriction enzyme digestion and T4 ligase connection, thereby obtaining the SSRP1-FLAG-1-75, SSRP1-FLAG-76-95, and SSRP1-FLAG-96-177 plasmids.

[0156] (3) The turbofect is used to overexpress the TRIB3-EGFP full-length plasmid and the related plasmids of the SSRP1-FLAG fragments in 293T cells, and the method is the same as described above.

[0157] (4) The co-immunoprecipitation experiment is performed by the IP antibody of TRIB3, and the immunoblotting detects the SSRP1 fragments that interact with TRIB3.

[0158] The experimental results are shown in Figure 6 The amino acid sequence of SSRP1 protein 76-95 is an alpha-helix structure, and the fragment interacts with the SSRP1 protein.

[0159] Example 7

[0160] The staple peptide 7695-SP5 significantly inhibits the proliferation of multiple myeloma cells by inhibiting the interaction of TRIB3 / SSRP1. Currently, the classic modification method is to replace the original amino acid residues with two S-configuration pentenylalanines (S5) with a side chain carbon chain length of 5 at i, i+4 sites respectively and form a staple, thereby realizing the fixation of one turn of a-helix. Through the spatial structure prediction of the 76-95 amino acid sequence of the SSRP1 protein, the applicant selected to introduce two S-configuration pentenylalanines (S5) to replace the original amino acid residues at the amino acid positions of 86 and 90. The applicant named it 7695-SP5. Subsequently, it was shown by CCK8 method that 7695-SP5 significantly inhibited the malignant proliferation of MM cells, and through co-immunoprecipitation experiment, the applicant found that 7695-SP5 could significantly inhibit the interaction of TRIB3 and SSRP1 proteins.

[0161] 1. Construction of staple peptide 7695-SP5.

[0162] (1) Consult the construction method of staple peptide, find that the classic modification method is to replace the original amino acid residues with two S-configuration pentenylalanines (S5) with a side chain carbon chain length of 5 at i, i+4 sites respectively and form a staple, thereby realizing the fixation of one turn of a-helix.

[0163] (2) Through the spatial structure analysis of the 76-95 amino acid sequence of the SSRP1 protein, the applicant selected to introduce two S-configuration pentenylalanines (S5) to replace the original amino acid residues at the amino acid positions of 86 and 90, i.e. replacing the original GFRESEFEKLSDFKTHYRL of 76-95 with GFRESEFEKL(S5)DFF(S5)THYRL, and the applicant named it 7695-SP5.

[0164] (3) The sequence is synthesized by 9-fluorenylmethyloxycarbonyl (Fmoc) solid phase synthesis method, and the synthesis order is from C terminal to N terminal. (Reference: Gao S, Guo Y, Li HY, Fang GM. Synthesis and application of stapled peptides. Chemical Progress, 2014, 26(1): 100-109.)

[0165] 2. CCK8 method for detecting that 7695-SP5 significantly inhibits the malignant proliferation of MM cells.

[0166] (1) Collect ARP1 cells in different groups, and add different concentrations of 7695-SP5 (0, 2.5, 5, 10 μM).

[0167] (2) Add 1×10 4 cells in a 96-well plate and culture for 2 days.

[0168] (3) Add 10% CCK8 for 3h incubation, and detect OD value at 450nm wavelength.

[0169] 3. Co-immunoprecipitation experiment was used to detect the interaction between TRIB3 and SSRP1 proteins inhibited by 7695-SP5.

[0170] (1) ARP1 cells were treated with 0 and 5μM 7695-SP5 respectively, and 10 7 ARP1 cells were collected.

[0171] (2) Co-immunoprecipitation experiment was performed with IP antibody of SSRP1, and then the changes of SSRP1 protein itself and TRIB3 were detected by immunoblotting.

[0172] The experimental results are shown in Figure 7 According to the amino acid sequence of 76-95 of SSRP1 protein, the applicant designed a staple peptide 7695-SP5, and proved by experiment that 7695-SP5 has a significant inhibitory effect on MM cells at the cellular level, and 7695-SP5 can interact with the proteins that inhibit TRIB3 and SSRP1.

[0173] Example 8

[0174] Staple peptide 7695-SP5 and BTZ play a synergistic role in the treatment of multiple myeloma. Currently, three-drug combination therapy is commonly used in the treatment of multiple myeloma, and BTZ is one of the most commonly used clinical drugs. In order to further explore the clinical application of 7695-SP5 in multiple myeloma, the applicant conducted a combined treatment of SP5 and BTZ at the cellular and mouse levels.

[0175] 1. CCK8 method was used to detect the synergistic effect of different concentration ratios of 7695-SP5 and BTZ on ARP1 cells.

[0176] (1) ARP1 cells were grown to the logarithmic phase, resuspended in a 15mL centrifuge tube, and blown and mixed evenly. The cells were diluted 10 times in a 1.5ml centrifuge tube, and the cell density was calculated using a cell counting plate.

[0177] (2) Set up a group of well plates without cells and complete medium, and a control group without drugs, only cells and complete medium. Four replicate wells were set up for each concentration.

[0178] (3) Add different concentrations of SP5 and BTZ (concentrations of 0 μM+0 nM, 0 μM+2.5 nM, 0 μM+5 nM, 0 μM+10 nM, 1.25 μM+0 nM, 1.25 μM+2.5 nM, 1.25 μM+5 nM, 1.25 μM+10 nM, 2.5 μM+0 nM, 2.5 μM+2.5 nM, 2.5 μM+5 nM, 2.5 μM+10 nM, 5 μM+0 nM, 5 μM+2.5 nM, 5 μM+5 nM, 5 μM+10 nM), inoculate 1×10 4 cells in 96-well plates, 4 replicates per group, and allow to grow for 48 h.

[0179] (4) Add 10 μl of activity detection reagent to 100 μl of culture solution in the 96-well plate, and incubate for 3 h.

[0180] (5) After calculating the cell viability (see Figure 8 A), input the cell viability of SP5 at 1.25, 2.5, 5 μM, the cell viability of BTZ at 2.5, 5, 10 nM, and the cell viability of SP5 and BTZ in combination (concentrations of 1.25 μM+2.5 nM, 1.25 μM+5 nM, 1.25 μM+10 nM, 2.5 μM+2.5 nM, 2.5 μM+5 nM, 2.5 μM+10 nM, 5 μM+2.5 nM, 5 μM+5 nM, 5 μM+10 nM, labeled as 1-9 in turn) into the CompuSyn software. After inputting the data, the CompuSyn software will obtain the corresponding score effect Fa and combination index CI values when the two drugs are used in combination. The synergistic effect is shown in Figure 8 B.

[0181] 2. The stapled peptide 7695-SP5 and BTZ have synergistic effect in a multiple myeloma mouse xenograft model.

[0182] (1) Culture sufficient cells, collect ARP1 cells with luciferase in 1640 medium, and adjust to 100 μl of cell solution containing 1×10 6 ARP1 cells with luciferase. Use a 1 mL syringe to inoculate 200 μl of cell solution into the tail vein of NCG mice.

[0183] (2) On the 10th day, randomly divide the NCG mice into groups. Then, the mice are injected intraperitoneally, the control group is injected with vehicle, and the experimental group is injected with drug SP5, BTZ, and SP5+BTZ solvent, 2 days of treatment at a time.

[0184] (3) Treat continuously for 5 times, and record the body weight and survival status of each mouse every day.

[0185] (4) On the 20th day, fluorescence imaging was performed on each group of mice using a small animal imaging instrument, 5 mice per group. 50 mg / kg of D-fluorescein was injected intraperitoneally, and fluorescence imaging was performed on each group of mice using a small animal imaging instrument within 5-10 min.

[0186] The experimental results are as follows Figure 8 , Figure 8 A and B in the table show that SP5 and BTZ have a synergistic effect at the cellular level, further, Figure 8 C in the table shows that after the separate treatment of BTZ and SP5, the fluorescence in mice is significantly weakened, and the tumorigenesis is significantly inhibited. After the combined treatment of BTZ and SP5, the fluorescence in mice almost disappears, which shows that the combination of the two has a significant synergistic effect, and SP5 has great application value in MM.

[0187] Although the specific embodiments of the present application are described above with reference to the accompanying drawings, the description is not a limitation on the scope of protection of the present application. Various modifications or variations made by those skilled in the art on the basis of the technical solutions of the present application without creative labor are still within the scope of protection of the present application.

Claims

1. A stapling peptide targeting the interaction of TRIB3 / SSRP1, characterized in that, The amino acid sequence of which is: GFRESEFEKLXDFFXTHYRL, as shown in SEQ ID NO. 1; wherein X is S5, i.e. S configuration of pentenylalanine.

2. Use of the stapling peptide of claim 1 in the preparation of a drug for resisting multiple myeloma.

3. The combination of the stapling peptide of claim 1 and bortezomib, characterized in that, The molar ratio of the two is 1250-5000:2.5-10.

4. Use of the composition of claim 3 in the preparation of a drug for resisting multiple myeloma.

5. An anti-multiple myeloma drug, characterized in that, The effective component is the stapling peptide of claim 1.

6. An anti-multiple myeloma agent, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The composition of claim 3 is contained. The effective component is the stapling peptide of claim 1. The composition of claim 3 is contained.

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