Application of AP3D1 as drug action target in in-vitro screening of blocking agent and blocking peptide

Abstract

The invention relates to the technical field of biological medicine, in particular to application of AP3D1 as a drug action target in in-vitro screening of a blocking agent and a blocking peptide. Based on a regulation mechanism of a negative regulatory factor TGF-beta on transport and degradation of STING, the invention provides application of AP3D1 as a drug action target in in-vitro screening of a blocking agent, and two key regulation sites S9 and R26 of AP3D1 are obtained through screening. Meanwhile, corresponding blocking peptides are synthesized according to the two regulation and control sites, inactivation of an AP3D1-mediated tumor microenvironment factor on an STING signal is specifically prevented, closing of any one regulation and control site is achieved, and therefore transfer and degradation of STING are effectively limited. The blocking peptide can cooperate with various treatment modes such as chemotherapy, radiotherapy, PARP inhibitors and STING agonists, cGAS-STING signals in tumors in the treatment process are remarkably activated, and the more effective anti-tumor curative effect is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of AP3D1 as a drug action target in in vitro screening of blocking agents and blocking peptides. Background Art

[0002] The cGAS-STING innate immune pathway is the first line of defense for humans against infection and tumors, and STING is a key transmembrane adaptor protein for immune response in this pathway. During pathogen infection or tumor process, abnormal DNA is recognized by the host cytoplasmic nucleic acid receptor cyclic GMP-AMP synthase (cyclic GMP-AMP Synthase, cGAS). After activation, cGAS uses ATP and GTP to synthesize the second messenger molecule cyclic guanosine monophosphate (cyclic GMP-AMP, cGAMP). As a messenger of the innate immune system, this small molecule binds and interacts with the endoplasmic reticulum-localized protein STING, causing STING conformational changes. After STING is activated, it translocates from the endoplasmic reticulum to the perinuclear endosome along with TBK1. TBK1 kinase phosphorylates and activates interferon regulatory factors (IRF3 / 7) and NF-κB, producing type I IFN and other immune response factors. While activated, STING is transported from the Golgi apparatus to the lysosome and degraded in the lysosome, thereby achieving precise regulation of the STING signal to protect cells from death due to over-amplified IFN responses, and even leading to autoimmune diseases. Current studies have confirmed that STING is a key protein that initiates anti-tumor innate immune responses, and is expected to become a new generation of immunotherapy targets that turn tumors from "cold" to "hot". Therefore, a variety of STING agonists have been developed for STING, which activate STING through direct effects on STING and have entered clinical trials as adjuvant therapies for tumor treatment.

[0003] However, although these STING agonists are very effective in activating STING and downstream IFN signaling in vitro, clinical promotion is difficult. So far, the results of these clinical trials have been generally disappointing. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an application of AP3D1 as a drug action target in in vitro screening of blocking agents and a blocking peptide.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] The present invention provides the use of AP3D1 as a drug action target in in vitro screening of blocking agents, wherein the drug action target comprises an S9 regulatory site and an R26 regulatory site.

[0007] Based on the above technical solution, the present invention can also be improved as follows.

[0008] The present invention also provides a blocking peptide, which can block the S9 regulatory site or the R26 regulatory site of AP3D1.

[0009] Furthermore, the blocking peptide comprises at least one of the following amino acid sequences or a combination of sequences:

[0010] A. the amino acid sequence shown in SEQ ID NO.1 and / or SEQ ID NO.2;

[0011] B. an amino acid sequence having 90% to 99% sequence identity with the amino acid sequence of SEQ ID NO.1 and / or SEQ ID NO.2;

[0012] C. A tag or a modifying group is connected to the amino acid sequence of A or B, and the tag or the modifying group is located at the N-terminus and / or the C-terminus;

[0013] D. An amino acid sequence having one or more amino acids substituted, deleted and / or added in the amino acid sequence of A, B or C.

[0014] The present invention provides the use of the above-mentioned blocking peptide in the preparation of polypeptide preparations.

[0015] The present invention provides a polypeptide preparation, comprising the above-mentioned blocking peptide.

[0016] Furthermore, the polypeptide preparation is an injection.

[0017] Furthermore, the polypeptide preparation also includes pharmaceutically acceptable excipients.

[0018] The present invention also provides a tumor suppressor composition, comprising the polypeptide preparation as described above, and further comprising a PARP inhibitor and / or a STING agonist.

[0019] Furthermore, the PARP inhibitor is niraparib or olaparib.

[0020] Furthermore, the STING agonist is MSA-2.

[0021] Furthermore, the tumor is an ovarian tumor, a breast tumor or a colon tumor.

[0022] The beneficial effects of the present invention are:

[0023] Based on the regulatory mechanism of the negative regulatory factor TGF-β on the transport and degradation of STING, the present invention provides the application of AP3D1 as a drug target in in vitro screening of blockers, and screens out two key regulatory sites of AP3D1 S9 and R26. At the same time, corresponding blocking peptides are synthesized for the above two regulatory sites to specifically prevent AP3D1-mediated tumor microenvironment factors from inactivating STING signals, thereby achieving the closure of any regulatory site, thereby effectively limiting the transport and degradation of STING.

[0024] The polypeptide preparation of the present invention, wherein the blocking peptide can cooperate with multiple treatment methods such as chemotherapy, radiotherapy, PARP inhibitors and STING agonists, significantly activate the cGAS-STING signal in the tumor during the treatment process, and achieve more effective anti-tumor efficacy.

[0025] The tumor suppressing composition of the present invention has a good tumor suppressing effect in vivo and provides an effective way for suppressing and treating tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a schematic diagram of the structure of the blocking peptide of the present invention, which is the blocking peptide in Example 1;

[0027] Figure 2 is the blocking peptide of the present invention, in Example 1, the immunoblotting result diagram, Figure 2 A in the middle shows the interaction between STING and AP3D1 after being blocked with S9 peptide. Figure 2 Middle B shows the interaction between STING and AP3D1 after being blocked with R26 peptide. Figure 2 C in the middle is the comparison of the effects of NC peptide and R26 peptide on STING degradation. Figure 2 D in the middle is the comparison of the effects of NC peptide and S9 peptide on the degradation of STING;

[0028] Figure 3 The blocking peptide of the present invention is a live imaging picture of mice in each experimental group at 4 weeks after administration in Example 2;

[0029] Figure 4 is the blocking peptide of the present invention, and in Example 2, a comparison chart of the tumor burden of mice in each experimental group at 4 weeks after administration;

[0030] Figure 5 The blocking peptide of the present invention is a comparison chart of the ascites content of mice in each experimental group in Example 2 at 4 weeks after administration;

[0031] Figure 6 is the blocking peptide of the present invention, in Example 3, a comparison chart of the changes in breast tumor volume of mice in each experimental group during the medication period;

[0032] Figure 7The blocking peptide of the present invention, in Example 3, the size of breast tumors of mice in each experimental group during the medication period were compared;

[0033] Figure 8 is the blocking peptide of the present invention, in Example 3, a comparison chart of the changes in mammary tumor weight of mice in each experimental group during the medication period;

[0034] Fig. 9 is the blocking peptide of the present invention, in Example 4, a comparison of the changes in the volume of subcutaneous colon cancer tumors of mice in each experimental group during the medication period;

[0035] Fig.10 The blocking peptide of the present invention is a comparison chart of the size of subcutaneous colon cancer tumors of mice in each experimental group during the medication period in Example 4;

[0036] Fig.11 is the blocking peptide of the present invention, in Example 4, a comparison chart of the weight changes of subcutaneous colon cancer tumors of mice in each experimental group during the medication period;

[0037] Fig.12 The application of AP3D1 of the present invention, the IFN signal status comparison diagram, Fig.12 A in the middle is a potential regulatory ligand activity diagram. Fig.12 B is a comparison chart of the expression of each regulatory factor in each subtype of cells. Fig.12 C in the middle is the expression difference diagram of each regulatory factor in IFN high and IFN low subtype cells. Fig.12 Middle D is a correlation comparison diagram of the predicted target genes;

[0038] Fig.13 This is a comparison chart of the differential expression of each gene in OVCAR8 cells using the AP3D1 of the present invention, using ctrl, using the PARP inhibitor Nira alone, using TGF-β alone, and using Nira and TGF-β in combination. Fig.13 A in the middle is CCL5, Fig.13 B in the middle is CXCL10, Fig.13 Middle C is IFNB1;

[0039] Fig.14 Application of AP3D1 of the present invention, comparison chart of differential expression of each gene by ctrl, using STING agonist MSA-2 alone, using TGF-β alone, and using MSA-2 and TGF-β in combination, Fig.14 A in the middle is CCL5, Fig.14 B in the middle is CXCL10, Fig.14 Middle C is IFNB1;

[0040] Fig.15This is a WB diagram of the application of AP3D1 of the present invention, and the effect of TGF-β and STING agonist MSA-2;

[0041] Fig.16 This is an immunofluorescence diagram of the application of AP3D1 of the present invention, the effect of TGF-β and STING agonist MSA-2, Fig.16 Where A is 0h, Fig.16 B is 0.5h, Fig.16 C is 2h, Fig.16 Medium D is 4h;

[0042] Fig.17 For the application of AP3D1 of the present invention, the intersection graph of all proteins bound to STING after ctrl and TGF-β pretreatment, phosphorylated proteins after TGF-β treatment in the public database, and STING transport-related proteins in the GO database;

[0043] Fig.18 For the application of AP3D1 of the present invention, WB map of the interaction site between AP3D1 and STING, Fig.18 A in the middle is a WB image of the site where STING specifically binds to AP3D1. Fig.18 Middle B is the WB map of the interaction site between the R26 site of AP3D1 and STING;

[0044] Fig.19 A schematic diagram of the process of constructing STING and AP3D1 site mutation plasmids and detecting the application of AP3D1 of the present invention. DETAILED DESCRIPTION

[0045] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0046] The present invention provides an application of AP3D1, specifically, it can be used as a drug action target for in vitro screening of blocking agents.

[0047] The blocking peptide of the present invention can block the S9 regulatory site or the R26 regulatory site of AP3D1.

[0048] Based on the current status of STING agonists in tumor treatment and the research on the specific regulatory mechanism of STING by the negative regulator TGF-β, we designed blocking peptides targeting the two key regulatory sites of AP3D1 S9 and R26, completed the drugability confirmation of AP3D1 as an original anti-cancer target, and developed original new drug candidates to specifically prevent the inactivation of STING signaling by tumor microenvironment factors mediated by AP3D1. It may be able to synergize with chemotherapy, radiotherapy, PARP inhibitors and STING agonists and other treatment methods to significantly activate the cGAS-STING signaling in the tumor during treatment and achieve more effective anti-tumor efficacy.

[0049] Preferably, the blocking peptide comprises at least one of the following amino acid sequences or a combination of sequences:

[0050] A. the amino acid sequence shown in SEQ ID NO.1 and / or SEQ ID NO.2;

[0051] B. an amino acid sequence having 90% to 99% sequence identity with the amino acid sequence of SEQ ID NO.1 and / or SEQ ID NO.2;

[0052] C. A tag or a modifying group is connected to the amino acid sequence of A or B, and the tag or the modifying group is located at the N-terminus and / or the C-terminus;

[0053] D. An amino acid sequence having one or more amino acids substituted, deleted and / or added in the amino acid sequence of A, B or C.

[0054] The blocking peptide of the present invention can be used to prepare polypeptide preparations.

[0055] The polypeptide preparation of the present invention comprises the aforementioned blocking peptide, and the polypeptide preparation is an injection and further comprises pharmaceutically acceptable excipients.

[0056] The tumor suppressor composition of the present invention includes the polypeptide preparation as described above, and also includes a PARP inhibitor and / or a STING agonist. The blocking peptide in the polypeptide preparation can effectively produce a synergistic effect with the PARP inhibitor and the STING agonist, so that the composition has a good tumor suppressor effect.

[0057] Preferably, the PARP inhibitor is niraparib or olaparib, and the STING agonist is MSA-2. Corresponding to the above PARP inhibitor and STING agonist, the tumor is an ovarian tumor, a breast tumor or a colon tumor.

[0058] In the present invention, the two regulatory sites of AP3D1 can be used as drug targets. The experimental verification process is as follows:

[0059] (1) Identification of TGF-β, a negative regulator of STING signaling

[0060] Based on the fact that STING agonists are effective in activating STING in vitro but poorly in vivo, it can be speculated that there may be key factors in the tumor microenvironment in vivo that promote the transport and degradation of STING, resulting in STING agonists showing good anti-tumor efficacy in preclinical studies but failing to effectively activate STING signals in real tumors.

[0061] Therefore, with the help of a previously conducted phase II clinical study of the world's first single-agent PARP inhibitor neoadjuvant treatment for ovarian cancer (NCT04507841), a total of 67 tissue samples from 34 patients with homologous recombination deficiency (HRD) test results were subjected to multi-omics tests such as single cell RNA-seq (scRNA-seq) to explore the impact of tumor microenvironment factors on cGAS-STING-IFN signaling before and after PARP inhibitor treatment.

[0062] The results showed that compared with HRD-negative patients, HRD-positive patients' tumor cells were significantly enriched with IFN signals and had higher activity of the cGAS / STING pathway. However, some HRD-positive ovarian cancer patients had inactivated IFN signals.

[0063] Based on this, HRD-positive ovarian cancer patients were divided into two groups according to their IFN signaling status: high IFN expression and low IFN expression. Fig.12 As shown in Figure 2, cell communication analysis revealed that tumor cells in the low IFN expression group were significantly more susceptible to exogenous TGF-β signals than those in the high IFN expression group. Fig.13 and Fig.14 As shown in Figure 2, according to the results of cytological experiments, TGF-β can also inhibit the STING-IFN signal induced by PARP inhibitors and STING agonists after treatment. Fig.15 ) and immunofluorescence ( Fig.16 ) and other experiments found that TGF-β can promote the degradation of STING by promoting the transport of STING from the Golgi apparatus to the lysosome.

[0064] The above results indicate that TGF-β is a key negative regulator of STING signaling in the tumor microenvironment of HRD-positive ovarian cancer, which can accelerate the transport of activated STING and promote its degradation.

[0065] (2) Mechanisms by which TGF-β promotes STING transport and degradation

[0066] Based on the above results, in order to explore the mechanism by which TGF-β promotes the transport and degradation of STING, this example constructed a STING-APEX2 plasmid with a Flag tag, which can recognize any exposed electron-rich amino acid residues within a 20 nm radius around the STING molecule, whether it is a directly interacting protein or a neighboring protein. After immunoprecipitation with Streptavidin magnetic beads, mass spectrometry was used to detect the differences in the interacting molecules of STING after activation with or without TGF-β pretreatment.

[0067] Since phosphorylation modification is the most common post-translational modification, and TGF-β may play a role by acting on STING after activation and transport degradation molecules, all proteins bound to STING after ctrl and TGF-β pretreatment, phosphorylated proteins after TGF-β treatment in the public database, and STING transport-related proteins in the GO database were intersected, and AP3D1 was found to be a potential regulatory factor mediating the action of TGF-β on STING ( Fig.17 ).

[0068] (3) Specific sites of interaction between TGF-β and AP3D1 and between AP3D1 and STING

[0069] Existing literature has reported that the AP-1 transporter can recognize the STING protein 360-365 amino acid [D / E]XXXL[L / I] sequence through the AP1γ subunit R15 site, and the AP-3 transporter AP3δ subunit structure is similar to the AP1γ subunit structure. Since AP3D1 is homologous to AP1G1, sequence analysis shows that the R26 site of AP3D1 is the same as the R15 site of AP1G1 and is highly conserved in multiple species. It may be the site that interacts with the STING [D / E]XXXL[L / I] site ( Fig.18 ), the present invention demonstrates this by constructing STING and AP3D1 site mutation plasmids.

[0070] Since components of TGF-β signal transduction are often modified by phosphorylation, in order to determine the site of TGF-β-induced AP3D1 phosphorylation, we used ctrl / TGFβ treatment based on phosphorylation mass spectrometry and combined with experimental verification to reveal that the potential TGF-β phosphorylation site on AP3D1 is the S9 site ( Fig.19 ).

[0071] Based on the above experimental results, it can be confirmed that TGF-β promotes the phosphorylation of AP3D1 S9 site, thereby enhancing the interaction between AP3D1R26 site and STING, and accelerating the transport and degradation of STING.

[0072] Therefore, in order to limit the transport and degradation of STING, it is sufficient to block or close either the AP3D1 S9 site or the AP3D1 R26 site.

[0073] Example 1 In vitro effects of blocking peptides

[0074] In this example, blocking peptides that can block the S9 regulatory site or the R26 regulatory site of AP3D1 were designed and prepared, and the blocking effect on the interaction between STING and AP3D1 in cells and the activation effect on the STING pathway were investigated to verify the in vitro effect of the blocking peptides.

[0075] The blocking peptides used in this example are S9 blocking peptide and R26 blocking peptide, the amino acid sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The structural schematic diagram of the blocking peptides is shown in Figure 1 The blocking peptides of this example were synthesized by Anhui Guoping Pharmaceutical Co., Ltd.

[0076] S9 blocking peptide (SEQ ID NO.1): YGRKKRRQRRRA KMVKGSIDRMF;

[0077] Or it can be written as, Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Lys MetVal Lys Gly Ser Ile Asp Arg Met Phe.

[0078] R26 blocking peptide (SEQ ID NO.2): YGRKKRRQRRRA LVRGIRNHKED;

[0079] Or it can be written as, Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Leu ValArg Gly Ile Arg Asn His Lys Glu Asp.

[0080] In the above two polypeptide sequences, the underlined YGRKKRRQRRRA part is the amino acid sequence of the transmembrane peptide, and the rest is the amino acid sequence of the tag peptide. For the S9 blocking peptide, the S in its tag peptide is the S9 site, and for the R26 blocking peptide, the R is the R26 site.

[0081] The specific experimental process of this embodiment is:

[0082] (1) Cell culture: HEK293 cells used in this example were purchased from the American Type Culture Collection (ATCC). HEK293 cells were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% FBS (Gibco) and penicillin-streptomycin solution (Thermo Fisher Scientific) at 37°C and 5% (v / v) CO 2 Cultivate in an incubator to obtain experimental cells.

[0083] (2) Drug treatment: transfer the Myc-vector or Myc-STING plasmid. Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala ), R26 peptide, and S9 peptide were dissolved in PBS to prepare 10 mM stock solutions. The cells were treated with each stock solution for 24 hours, and then treated with 10 ng / ml TGF-β for 3 hours to obtain cells for evaluating the interaction between STING and AP3D1.

[0084] For the evaluation of the activation effect on the STING pathway, cells were treated with Poly(dA:dT) for 2 hours after TGF-β treatment.

[0085] (3) Immunoprecipitation assay: The cells treated in step (2) were lysed on ice for 15 minutes using a lysis buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40) supplemented with protease inhibitors. The lysate was then centrifuged at 15,000 rpm for 20 minutes and any cell debris was carefully removed. The cell lysate was then exposed to Anti-Myc magnetic beads overnight at 4°C. The resulting immunoprecipitated complex was designated for further analysis.

[0086] (4) Immunoblotting: In the immunoblotting method, cell lysates or precipitates were denatured in 1x sodium dodecyl sulfate (SDS) protein sample buffer at 95°C for 10 min and then separated by electrophoresis on 10% SDS-polyacrylamide gel. The separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes and blocked with 5% skim milk for more than 1 h. After blocking, the membranes were exposed to specific antibodies at recommended dilutions. Enhanced chemiluminescence reagents (ThermoFisher Scientific) were used for immunoblotting detection.

[0087] The results of immunoblotting in this example are as follows Figure 2 shown.

[0088] according to Figure 2 Middle A and Figure 2As can be seen in B, after blocking with S9 peptide and R26 peptide, the interaction between STING and AP3D1 was weakened, indicating that R26 peptide and S9 peptide have a significant blocking effect on the interaction between STING and AP3D1 in cells.

[0089] according to Figure 2 Middle C and Figure 2 As can be seen in D, after blocking with S9 peptide and R26 peptide, STING degradation was reduced and the STING pathway activation effect was enhanced.

[0090] The experimental results of this example show that the R26 peptide and the S9 peptide have good in vitro effects.

[0091] Example 2 Effect of polypeptide drugs on the occurrence and development of ovarian cancer

[0092] This example specifically detects the growth of tumors in mice in each experimental group using Niraparib, a targeted drug for ovarian cancer, alone, S9 peptide, alone, and R26 peptide in combination with Niraparib and S9 peptide or R26 peptide, to investigate the in vitro effect of the peptide drug. The specific experimental process is as follows:

[0093] (1) NC peptide, S9 peptide and R26 peptide were prepared into 12.5 mg / mL intraperitoneal injection solution using PBS buffer, thereby obtaining protein R26 polypeptide drug and protein S9 polypeptide drug.

[0094] (2) Construction of ovarian cancer model: A total of 3×10^6 Trp53 expressing luciferase were added - / - Brca1 - / - ID8 cells were injected into the peritoneal cavity of 6- to 8-week-old female C57BL / 6 mice to establish an advanced high-grade serous ovarian cancer model. The mice were randomly divided into AF 6 groups.

[0095] (3) Seven days after modeling, each group of mice was injected with the following specific administration method:

[0096] Mice in group A were intraperitoneally injected with NC peptide (12.5 mg / ml; 100 ul) every day;

[0097] Mice in group B were intraperitoneally injected with protein R26 peptide drug (12.5 mg / ml; 100 μL) every day;

[0098] Mice in group C were intraperitoneally injected with protein S9 peptide drug (12.5 mg / ml; 100 μL) every day;

[0099] Mice in group D were intragastrically administered with Niraparib targeted therapy (10 mg / ml; 100 μL) and NC peptide (12.5 mg / ml; 100 ul) every day;

[0100] Mice in group E were intragastrically administered with Niraparib targeted therapy (10 mg / ml; 100 μL) and intraperitoneally injected with protein R26 peptide drug (12.5 mg / ml; 100 μL) every day.

[0101] Mice in group F were gavaged with Niraparib targeted therapy drug (10 mg / ml; 100 μL) and intraperitoneally injected with protein S9 peptide drug (12.5 mg / ml; 100 μL) every day.

[0102] For each group of mice, the drug was stopped for two days every five days. The tumor burden was detected and counted using small animal imaging on the day of drug administration and 1-3 weeks after drug administration. At the 4th week after drug administration, the growth of mouse tumors was observed by small animal live imaging. The number of tumor cells can be reflected by the number of photons recorded per second in the abdomen of each mouse in small animal imaging. The mice were killed, the ascites content was collected, and the statistical student t test was performed. The test results are shown as follows: Figures 3 to 5 shown.

[0103] according to Figure 3 It can be seen that compared with Niraparib alone, S9 peptide alone, or R26 peptide alone, Niraparib combined with S9 peptide, or combined with R26 peptide can significantly inhibit the progression of ovarian cancer tumors in mice.

[0104] according to Figure 4 It can be seen that in the experimental group of Niraparib combined with S9 peptide or R26 peptide, the tumor burden was significantly lower than that of mice in the experimental group using Niraparib alone, S9 peptide alone, or R26 peptide alone.

[0105] according to Figure 5 It can be seen that the amount of ascites in the mice in the experimental group treated with Niraparib combined with S9 peptide or R26 peptide was significantly smaller than that in the experimental group treated with Niraparib alone, S9 peptide alone, or R26 peptide alone.

[0106] Example 3 Effect of polypeptide drugs on the occurrence and development of breast cancer

[0107] This example specifically detects the growth of tumors in mice in each experimental group using the breast cancer targeted drug Olaparib alone, S9 peptide alone, R26 peptide alone, and Olaparib in combination with S9 peptide or R26 peptide to investigate the in vitro effect of the peptide drug. The specific experimental process is as follows:

[0108] (1) NC peptide, S9 peptide and R26 peptide were prepared into 12.5 mg / mL intraperitoneal injection solution using PBS buffer, thereby obtaining protein R26 polypeptide drug and protein S9 polypeptide drug.

[0109] (2) Construction of an in situ breast cancer model: A total of 2×10^5 Brca1 - / - EO771 cells were injected into the mammary fat pad of 6- to 8-week-old female C57BL / 6 mice to establish an orthotopic breast cancer model. The mice were randomly divided into groups A-F6.

[0110] (3) Three days after modeling, each group of mice was injected with the following specific administration method:

[0111] Mice in group A were intraperitoneally injected with NC peptide (12.5 mg / ml; 100 ul) every day;

[0112] Mice in group B were intraperitoneally injected with protein R26 peptide drug (12.5 mg / ml; 100 μL) every day;

[0113] Mice in group C were intraperitoneally injected with protein S9 peptide drug (12.5 mg / ml; 100 μL) every day;

[0114] Mice in group D were intragastrically administered with Olaparib targeted therapy (10 mg / ml; 100 μL) and NC peptide (12.5 mg / ml; 100 ul) every day;

[0115] Mice in group E were intragastrically administered with Olaparib targeted therapy (10 mg / ml; 100 μL) and intraperitoneally injected with protein R26 peptide drug (12.5 mg / ml; 100 μL) every day.

[0116] Mice in group F were gavaged with Olaparib targeted therapy drug (10 mg / ml; 100 μL) and injected intraperitoneally with protein S9 peptide drug (12.5 mg / ml; 100 μL) every day.

[0117] For each group of mice, the drug was stopped for two days every five days, and the tumor volume was evaluated and counted every other day, and calculated using the formula L×W×W×1 / 2, where L represents length and W represents width. On the 26th day after drug administration, the mice were killed, and the growth and weight of the tumors in the mice were observed and measured.

[0118] The experimental results of this embodiment are as follows Figures 6 to 8 shown.

[0119] according to Figure 6 It can be seen that compared with the treatment groups using Olaparib alone, S9 peptide alone, or R26 peptide alone, the Olaparib combined with S9 peptide, or combined with R26 peptide group can significantly inhibit the progression of breast cancer tumors in mice.

[0120] according to Figure 7 and Figure 8It can be seen that the tumor size and weight of mice in the experimental group treated with Olaparib combined with S9 peptide, or combined with R26 peptide were significantly smaller than the tumor size of mice in the experimental group treated with Olaparib alone, S9 peptide alone, or R26 peptide alone.

[0121] Example 4 Effect of polypeptide drugs on the development of subcutaneous tumors of colon cancer

[0122] This example specifically detects the growth of tumors in mice in each experimental group using MSA-2 STING agonist alone, S9 peptide alone, R26 peptide alone, and MSA-2 STING agonist in combination with S9 peptide or R26 peptide, to investigate the in vitro effect of the peptide drug. The specific experimental process is as follows:

[0123] (1) NC peptide, S9 peptide and R26 peptide were prepared into 12.5 mg / mL intraperitoneal injection solution using PBS buffer, thereby obtaining protein R26 polypeptide drug and protein S9 polypeptide drug.

[0124] (2) Construction of subcutaneous colon cancer tumor model: A total of 2×10^5 CT26 cells were injected into the buttocks of 6- to 8-week-old female BALB / c mice to establish a subcutaneous colon cancer tumor model. The mice were randomly divided into AF 6 groups.

[0125] (3) Three days after modeling, each group of mice was injected with the following specific administration method:

[0126] Mice in group A were intraperitoneally injected with NC peptide (12.5 mg / ml; 100 ul) every day;

[0127] Mice in group B were intraperitoneally injected with protein R26 peptide drug (12.5 mg / ml; 100 μL) every day;

[0128] Mice in group C were intraperitoneally injected with protein S9 peptide drug (12.5 mg / ml; 100 μL) every day;

[0129] Mice in group D were intragastrically administered with MSA-2 STING agonist (12 mg / ml; 100 μL) and NC peptide (12.5 mg / ml; 100 ul) every five days;

[0130] Mice in group E were intragastrically administered with MSA-2 STING agonist (12 mg / ml; 100 μL) plus intraperitoneal injection of protein R26 peptide drug (12.5 mg / ml; 100 μL) every five days;

[0131] Mice in group F were intragastrically gavaged with MSA-2 STING agonist (12 mg / ml; 100 μL) and intraperitoneally injected with protein S9 peptide drug (12.5 mg / ml; 100 μL) every five days.

[0132] For each group of mice, the drug was stopped for two days every five days, and the tumor volume was evaluated and counted every other day, and calculated using the formula L×W×W×1 / 2, where L represents length and W represents width. On the 26th day after drug administration, the mice were killed, and the growth and weight of the tumors in the mice were observed and measured.

[0133] The experimental results of this embodiment are as follows Figures 9-11 shown.

[0134] according to Fig. 9 It can be seen that compared with the treatment groups using MSA-2 STING agonist alone, S9 peptide alone, or R26 peptide alone, the MSA-2 STING agonist combined with S9 peptide, or combined with R26 peptide group can significantly inhibit the progression of subcutaneous tumors of mouse colon cancer.

[0135] according to Fig.10 and Fig.11 It can be seen that the tumor size and weight of mice in the MSA-2 STING agonist combined with S9 peptide, or combined with R26 peptide experimental group were significantly smaller than the tumor size of mice in the MSA-2 STNG agonist alone, S9 peptide alone, or R26 peptide alone experimental group.

[0136] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of AP3D1 as a drug target in in vitro screening of inhibitors, characterized in that: The drug action targets include the S9 regulatory site and the R26 regulatory site.

2. A blocking peptide, characterized in that The blocking peptide can block the S9 regulatory site and / or the R26 regulatory site of AP3D1.

3. A blocking peptide according to claim 2, characterized in that: The blocking peptide comprises at least one of the following amino acid sequences or a combination of sequences: A. the amino acid sequence shown in SEQ ID NO.1 and / or SEQ ID NO.2; B. an amino acid sequence having 90% to 99% sequence identity with the amino acid sequence of SEQ ID NO.1 and / or SEQ ID NO.2; C. A tag or a modifying group is connected to the amino acid sequence of A or B, and the tag or the modifying group is located at the N-terminus and / or the C-terminus; D. An amino acid sequence having one or more amino acids substituted, deleted and / or added in the amino acid sequence of A, B or C.

4. Use of the blocking peptide as claimed in claim 2 or 3 in the preparation of polypeptide preparations.

5. A polypeptide preparation, characterized in that: The method comprises the blocking peptide as claimed in claim 2 or 3.

6. A polypeptide preparation according to claim 5, characterized in that: The polypeptide preparation also includes pharmaceutically acceptable excipients.

7. A tumor suppressor composition, characterized in that: The polypeptide preparation according to claim 5 or 6 further comprises a PARP inhibitor and / or a STING agonist.

8. A tumor suppressor composition according to claim 7, characterized in that: The PARP inhibitor is niraparib or olaparib.

9. The tumor suppressor composition according to claim 7, characterized in that: The STING agonist is MSA-2.

10. The tumor suppressor composition according to claim 7, characterized in that: The tumor is an ovarian tumor, a breast tumor or a colon tumor.

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