A polypeptide specifically blocking the binding of acvr1c to grem1 and applications thereof

Abstract

The application provides a polypeptide for specifically blocking the combination of ACVR1C and Grem1 and application thereof, and belongs to the technical field of biological medicine. The application finds a new combination of receptors of Grem1 and ACVR1C, and determines the combination sites of the two; then according to the details of the combination of Grem1 and ACVR1C, a specific blocking small molecule polypeptide (ACVR1C peptide) is designed. The small molecule polypeptide can interfere with or block the combination of Grem1 and ACVR1C in an in-vitro experiment; in an in-vivo treatment experiment, the liver metastasis of colon cancer of the treatment group mice is obviously lower than that of the control group, which indicates that the small molecule polypeptide reaches the target of blocking the liver metastasis of colon cancer by interfering with or blocking the combination of Grem1 and ACVR1C, and provides a new drug design and treatment target for relieving the liver metastasis of colon cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a polypeptide that specifically blocks the binding of ACVR1C to GREM1 and its applications. Background Technology

[0002] Colorectal cancer (CRC) is the third most common cancer worldwide, ranking third in cancer incidence among men and second among women. Liver metastasis is extremely common in colorectal cancer and is the leading cause of death among patients. At initial diagnosis, 20%–25% of colorectal cancer patients already have liver metastases; after radical resection of the primary lesion, the incidence of metachronous liver metastasis reaches approximately 30%, meaning that about 50% of patients eventually develop liver metastases during the course of colorectal cancer. Furthermore, due to the chronic nature of cancer, each cancer patient faces years of high medical expenses and resource demands, causing suffering for their family and impacting the economy of their family and region. Therefore, developing specific and effective targeted drugs will effectively prolong the survival of patients with metastatic colorectal cancer, alleviate the economic burden on families, and promote regional development.

[0003] For patients with advanced metastatic cancer, chemotherapy drugs still dominate the treatment of tumors, and the various side effects of traditional radiotherapy and chemotherapy are often unbearable. Developing specific and effective targeted anticancer drugs is of great significance for improving the quality of life and prolonging the survival of cancer patients. However, most of these targeted drugs are "exclusive drugs" still under patent protection by foreign pharmaceutical companies, and there are few competing products. They are often very expensive, costing tens of thousands or even 100,000 yuan per month, forcing many patients and their families who cannot afford it to abandon treatment.

[0004] The secreted protein Gremlin1 (GREM1), generally considered a bone morphogenetic protein (BMP) antagonist, plays a crucial role in the pathogenesis and progression of various diseases. Recently, GREM1 has attracted attention due to its cytokine-like properties, including an affinity for binding to a putative receptor. Furthermore, GREM1 marks a subset of fibroblasts in both normal intestinal and colorectal cancer (CRC). + Stromal cells are cancer-associated fibroblasts (CAFs) that can promote the metastasis of CRC.

[0005] Activin receptor type-1C (ACVR1C), also known as activin receptor-like kinase 7 (ALK7), is a single-pass transmembrane type I protein. Its extracellular region functions as a receptor, while its intracellular region functions as a kinase. Upon ligand binding, it forms a receptor complex composed of two type II and two type I transmembrane serine / threonine kinases. Type II receptor phosphorylation activates type I receptor, which then autophosphorylates and binds to and activates the SMAD transcriptional regulators SMAD2 and SMAD3. Data from researchers at Johns Hopkins University School of Medicine support the role of the ACVR1C / SMAD2 pathway in promoting tumor invasion and growth.

[0006] ACVR1C is a type I receptor of the TGF-β family. Currently, over ten TGF-β blocking drugs are undergoing clinical trials in combination with PD-1 monoclonal antibodies, but none have yet been approved for clinical use. This is likely because most of these drugs target the SMAD pathway, a downstream pathway of this family of receptors. Downstream signal transduction in this pathway plays a crucial physiological role in regulating the entire life cycle of organisms. Therefore, downstream blockers of this family consistently face two major drawbacks: poor specificity and strong side effects.

[0007] Therefore, it is necessary to further develop anticancer targeted drugs that combine specificity and efficacy. Summary of the Invention

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] This invention provides the application of ACVR1C as a GREM1 receptor in the preparation of drugs that regulate colorectal cancer.

[0010] The present invention also provides a polypeptide that specifically blocks the binding of ACVR1C to GREM1, the amino acid sequence of which is shown in SEQ ID NO.7.

[0011] The present invention also provides the use of the described polypeptide in the preparation of a medicament for treating colorectal cancer.

[0012] The main advantage of this invention lies in the following: Previous studies have found that activation of the ACVR1C receptor by the GREM1 ligand significantly promotes colorectal cancer metastasis. Furthermore, through extensive experiments, we identified the binding site between ACVR1C and the GREM1 ligand, primarily concentrated in a specific amino acid sequence (TECCFTDFCNNITLHLPTA, SEQ ID NO.1). Therefore, by synthesizing this amino acid sequence, we competitively neutralized the GREM1 ligand, thereby specifically blocking the binding of the ACVR1C receptor to the GREM1 ligand, inhibiting the activation of downstream pathways, and ultimately achieving a therapeutic effect that inhibits the metastasis of colorectal cancer cells.

[0013] This invention established a colorectal cancer liver metastasis model in NOG mice by injecting colorectal cancer cells (HCT116) into the spleen, and then treated the mice with a peptide injected via the tail vein. The results showed that the peptide significantly inhibited liver metastasis of colorectal cancer cells. This indicates that the peptide provided by this invention has the potential to become a simple, safe, and effective new drug for the prevention and treatment of colorectal cancer. Attached Figure Description

[0014] Figure 1 The results of the pull-down experiment in Example 1 show that GREM1 binds to the surface receptor on the CRC cell membrane.

[0015] Figure 2 The mass spectrometry analysis results from Example 1 identified four trypsin fragments.

[0016] Figure 3 The immunoblotting results are from the co-immunoprecipitation experiment performed with Ha-labeled GREM1 in Example 2.

[0017] Figure 4 The immunoblotting results are from the co-immunoprecipitation experiment performed on the Flag-labeled ACVR1C in Example 2.

[0018] Figure 5 This is the result of co-localization of ACVR1C and GREM1 in SW480 cells as shown by co-focusing microscopy in Example 2.

[0019] Figure 6 The results of the pull-down experiment in Example 2 show that there is a direct physical connection between ACVR1C and GREM1.

[0020] Figure 7 The results of the co-IP experiment are for the truncated extracellular segments of GREM1 and ACVR1C (ACVR1C-ECD) in Example 3.

[0021] Figure 8 This represents the potential docking method for GREM1 and ACVR1C simulated in Example 3.

[0022] Figure 9 This shows the binding of GREM1 and ACVR1C after the corresponding Q72A, E85A, or T101A mutations in ACVR1C in Example 3.

[0023] Figure 10 This shows the binding of GREM1 and ACVR1C after the Q101A / T102A / T112A / N115A mutation in GREM1 in Example 3.

[0024] Figure 11 The diagram (a) shows the simulation of the peptide blocking the binding of GREM1 and ACVR1C in Example 4, and the results of the pull-down experiment.

[0025] Figure 12 To illustrate the effect of the polypeptide in treating colorectal cancer liver metastases in Example 5, a) is an in vivo imaging image of a small animal, b) is a bioluminescent image of tumor cells in the liver, and c) is a statistical graph of liver metastasis luminescence. Detailed Implementation

[0026] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1

[0028] To investigate whether GREM1 interacts with surface receptors on the CRC cell membrane, we performed a pull-down assay on total protein extracted from HCT116 human rectal cancer cells transfected with Ha-labeled GREM1 using magnetic beads conjugated with anti-HA antibody. The results are as follows: Figure 1 As shown, GREM1 interacts with surface receptors on the CRC cell membrane. Then, [the following is observed]... Figure 1 Mass spectrometry analysis was performed on the protein at the fragment indicated by the middle arrow. The mass spectrometry results are as follows: Figure 2 As shown, four trypsin fragments were identified:

[0029] FRPSIPNQWQSCEALR (SEQ ID NO.1)

[0030] HENILGFIAADNK(SEQ ID NO.2)

[0031] QALLLLAAAAELSPGLK (SEQ ID NO.3)

[0032] RPNVEEPLSECNLVNAGK (SEQ ID NO. 4).

[0033] These four peptides belong to the ACVR1C protein, indicating that ACVR1C is one of the proteins that interact with GREM1.

[0034] Example 2

[0035] To determine whether ACVR1C is a novel receptor for GREM1, we performed a co-immunoprecipitation (co-IP) assay in HCT116 cells using Ha-labeled GREM1. Subsequent Western blot results showed an interaction between GREM1 and ACVR1C. Figure 3 Similarly, co-IP analysis using Flag-tagged ACVR1C showed an interaction between ACVR1C and GREM1. Figure 4 Furthermore, confocal microscopy revealed that ACVR1C and GREM1 were co-localized in SW480 cells, confirming the interaction between these two proteins. Figure 5 ).

[0036] Next, we aimed to investigate whether there is a direct interaction between GREM1 and ACVR1C. To this end, we purified the Fc-labeled extracellular domain of ACVR1C (ACVR1C-ECD, AA1~113, SEQ ID NO.5) and the his-labeled full-length GREM1 (SEQ ID NO.6), and performed pull-down experiments, demonstrating a direct interaction between ACVR1C and GREM1. Figure 6 ).

[0037] ACVR1C (uniport: Q8NER5), ACVR1C extracellular domain (ACVR1C-ECD) amino acid sequence (SEQ ID NO.5):

[0038] MTRALCSALRQALLLLAAAAELSPGLKCVCLLCDSSNFTCQTEGACWASVMLTNGKEQVIKSCVSLPELNAQVFCHSSNNVTKTECCFTDFCNNITLHLPTASPNAPKLGPME

[0039] GREM1 (uniport: O60565) amino acid sequence (SEQ ID NO.6):

[0040] MSRTAYTVGALLLLLGTLLPAAEGKKKGSQGAIPPPDKAQHNDSEQTQSPQQPGSRNRGRGQGRGTAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD

[0041] Example 3

[0042] To further characterize the binding sites of GREM1 and ACVR1C, we constructed an extracellular domain truncated to both GREM1 and ACVR1C (ACVR1C-ECD). Subsequent co-IP experiments showed that the amino acid sequence of GREM1 (100-157 amino acids) was... Figure 7 b, G3) or ACVR1C-ECD amino acids 68~113 (AA68~113, Figure 7 The deletion of (a, A4) effectively eliminates the interaction between GREM1 and ACVR1C in HCT116 cells.

[0043] Based on this result, we attempted to further identify the key interaction sites between GREM1 and ACVR1C. Using the HDOCK platform (http: / / hdock.phys.hust.edu.cn / ), we simulated their potential docking modes based on the protein structures of GREM1 (PDB:5AEJ)34 and ACVR1C (predicted by AlphaFold, https: / / alphafold.ebi.ac.uk / entry / Q8NER5). Specifically, we predicted that amino acid residues Q101 / T102 / T112 / N115 in GREM1 and amino acid residues Q72 / E85 / T101 in ACVR1C are essential binding regions. Figure 8 ).

[0044] To verify this, we performed mutagenesis at the aforementioned potential sites to test protein binding simulations. Notably, the tetrad mutations Q101A / T102A / T112A / N115A in GREM1 or the corresponding E85A or T101A mutations in ACVR1C severely disrupted the association between GREM1 and ACVR1C. Figure 9 , 10 ).

[0045] Example 4

[0046] Based on the ligand relationship between ACVR1C and GREM1 determined in Examples 1-3 and the key binding sites discovered, we designed a peptide inhibitor based on the ACVR1C amino acid sequence 84-102 (AA84-102), which is called ACVR1C peptide.

[0047] The Fc-labeled extracellular domain of ACVR1C (ACVR1C-ECD, AA1~113) and the his-labeled full-length GREM1 were purified, and an ACVR1C peptide (amino acid sequence TECCFTDFCNNITLHLPTA, SEQ ID NO.7) that blocks the binding of ACVR1C to its GREM1 ligand was synthesized. Figure 11 a). Pull-down experiments were performed, and the results showed that the ACVR1C peptide competitively inhibited the binding between GREM1 and ACVR1C. Figure 11 b).

[0048] Example 5

[0049] Grouping: A total of 18 six-week-old NOG mice were randomly divided into three groups after 5 days of acclimatization feeding: control group (PLV), GREM1 overexpression group (PLV-GREM1), and ACVR1C peptide treatment group (PLV-GREM1+ACVR1C peptide), with 6 mice per group.

[0050] Modeling: Prepare HCT116-luc cell suspension and adjust cell density to 1×10⁻⁶. 7 Cells / mL. Remove the mice to be treated and anesthetize them by intraperitoneal injection of 2% sodium pentobarbital at a dose of 45 μl / 20g. The mice will begin to quiet down after approximately 5 minutes. Place a white gauze pad on the mouse board, place the mouse in a left lateral decubitus position, and shave the fur off the mouse's abdomen. Disinfect the ventral side from the neck down to the groin with alcohol swabs. Make a 1-2 cm incision in the skin below the left costal arch to expose the abdominal wall muscles. Use pointed forceps to pull on the abdominal wall muscles, avoiding the spleen within the abdominal cavity, and then cut open the abdominal wall muscles to expose the internal organs. Use circular forceps to gently pull on the pancreas and remove the spleen. Draw 50 μl of single-cell suspension from the EP tube using an insulin syringe, slightly push the needle core to create air bubbles, and then inject the cells into the spleen. Slowly withdraw the needle to prevent cell leakage and apply pressure to stop bleeding.

[0051] Administration: The peptide was administered at a concentration of 10 mg / kg via tail vein injection every 2 days. The control group and the GREM1 overexpression group received the same volume of PBS (both administered via tail vein injection).

[0052] Observation: Tumor growth and metastasis were observed using a small animal in vivo imaging system. Before the experiment, each NOG mouse was injected with 150 mg / kg of fluorescein (using a Mg-free solution). 2+ Ca2+ (Diluted with DPBS), NOG mice were anesthetized with isoflurane approximately 10 minutes later, and in vivo imaging was performed to collect biofluorescence images. The results are as follows: Figure 12 Animal experiments showed that the peptides could significantly inhibit the metastasis of colorectal cancer.

[0053] As demonstrated by the above embodiments, this invention has discovered a novel receptor-ligand relationship between GREM1 and ACVR1C and determined their binding sites. Based on the details of the GREM1-ACVR1C binding, a specific blocking small molecule peptide (ACVR1C peptide) was designed. In in vitro experiments, this small molecule peptide can interfere with or block the binding of GREM1 and ACVR1C. In in vivo therapeutic experiments, the small molecule peptide showed that the incidence of colon cancer liver metastasis in the treatment group was significantly lower than that in the control group. This indicates that the small molecule peptide achieves the goal of blocking colon cancer liver metastasis by interfering with or blocking the binding of GREM1 and ACVR1C, providing a new drug design and therapeutic target for alleviating colon cancer liver metastasis.

[0054] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A polypeptide that specifically blocks the binding of ACVR1C to GREM1, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.

7.

2. The use of the polypeptide of claim 1 in the preparation of a drug for inhibiting colorectal cancer metastasis.

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