Polypeptides that specifically bind to sestrin2 protein and their use in the treatment of digestive tract cancer

Abstract

The application discloses a polypeptide specifically binding to Sestrin2 protein and application thereof in treatment of digestive tract cancer. Specifically disclosed is a STAMBPL1 polypeptide with an amino acid sequence of SEQ ID No. 1, which is a polypeptide specifically binding to Sestrin2 protein and can block the interaction between Sestrin2 and STAMBPL1 through competitive binding. The application further provides a chimeric peptide tat-STAMBPL1 with cell permeability comprising the STAMBPL1 polypeptide and a cell-penetrating peptide. The polypeptide and the chimeric peptide of the application have high affinity, clear target, target the Sestrin2 / STAMBPL1 interaction site, and can effectively inhibit tumor (especially digestive tract tumor) formation. The application further provides an anti-tumor drug comprising the STAMBPL1 polypeptide and / or the chimeric peptide tat-STAMBPL1.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to polypeptides that specifically bind to the Sestrin2 protein and their application in the treatment of gastrointestinal cancer. Background Technology

[0002] Gastrointestinal tumors are a type of tumor that occurs in the esophagus, stomach, liver, large intestine, and small intestine. Because early symptoms are often subtle, these tumors are frequently discovered at an advanced stage. Colorectal cancer is the most common type of gastrointestinal tumor, and it is closely related to genetic factors, population aging, poor dietary habits, obesity, lack of exercise, and smoking. Diagnosing colorectal cancer is difficult, with an early diagnosis rate of only 10-15%. Therefore, understanding the mechanisms of colorectal cancer development and progression, and implementing targeted treatments, will help improve patient survival rates.

[0003] Currently, various treatment methods for colorectal cancer have been reported, including: early endoscopic and surgical resection, radiotherapy, chemotherapy (Fluorouracil, Oxaliplatin), pathway inhibitors (anti-VEGF and EGFR drugs), novel / rescue drugs (Regorafenib, TAS-102), immunotherapy, and targeted therapy (BRAF or BRAF combined with MEK inhibitors). Traditional treatments often have limitations, such as significant drug toxicity, easy development of drug resistance, and high risk of metastasis. Novel targeted therapies, on the other hand, can specifically target cancer-causing sites at the molecular level to inhibit tumor cell proliferation, with relatively fewer side effects. Therefore, researching and developing novel targeted small molecules is an important research direction in cancer treatment.

[0004] Mammalian target of rapamycin complex 1 (mTORC1), a key regulatory protein complex in metabolic processes, senses the abundance of nutrients in the environment and directly regulates cellular metabolism. Sestrin2, a cytoplasmic leucine-sensing protein, can directly bind to leucine. Sestrin2 can inhibit mTORC1 activity when leucine is not bound, thus preventing excessive activation of cellular anabolic metabolism. Overactivation of the mTORC1 signaling pathway often leads to excessive cell growth and subsequently cancer. Therefore, targeted therapy against oncogenes in this pathway will provide new insights into cancer treatment. Summary of the Invention

[0005] The purpose of this invention is to provide an antitumor polypeptide that specifically binds to the Sestrin2 protein and can inhibit the interaction between Sestrin2 and STAMBPL1. The technical problem to be solved by this invention is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0006] This invention first provides a polypeptide that specifically binds to the Sestrin2 protein, named the STAMBPL1 polypeptide, wherein the STAMBPL1 polypeptide is any one of the following:

[0007] A1) The amino acid sequence is that of the polypeptide in SEQ ID No. 1;

[0008] A2) A polypeptide obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence of SEQ ID No. 1, which has more than 80% identity with the polypeptide shown in A1) and specifically binds to the Sestrin2 protein.

[0009] A3) A fusion polypeptide with the same function is obtained by linking a tag to the N-terminus and / or C-terminus of A1) or A2).

[0010] The STAMBPL1 polypeptide is a polypeptide that specifically binds to the Sestrin2 protein and can competitively block the interaction between Sestrin2 and endogenous STAMBPL1.

[0011] The STAMBPL1 polypeptide is a peptide segment between amino acids 59 and 78 of the human STAMBPL1 protein, totaling 20 amino acids.

[0012] A3) The labels are shown in Table 1:

[0013] Table 1: Sequence of Labels

[0014] Label residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

[0015] In this document, the 80% or more identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0016] The present invention also provides a chimeric peptide, named tat-STAMBPL1, wherein the chimeric peptide comprises the STAMBPL1 polypeptide and a membrane-penetrating peptide.

[0017] The membrane-penetrating peptide can promote the uptake of the chimeric peptide by cells.

[0018] Furthermore, the membrane-penetrating peptide can be linked to the N-terminus of the STAMPL1 polypeptide.

[0019] The term "chimeric peptide" refers to a peptide that has two or more components that are not naturally linked together, and which are connected to each other either as a fusion protein or through chemical linkage.

[0020] The term "penetratin," also known as cell membrane transduction peptide, cell-penetrating peptide, or internalizing peptide, is widely used in the field of protein drugs. Its function is to promote the uptake and absorption of bound bioactive peptides by cells, thus carrying polypeptide or drug molecules into the cell to exert their biological activity. These substances are all positively charged polypeptide fragments of varying lengths, rich in basic amino acid residues such as arginine and lysine, and all possess an α-helical spatial conformation in their secondary structure. To date, various naturally occurring and synthetically produced cell-penetrating peptides have been discovered, such as Tat, the transactivator of transcription of human immunodeficiency virus (HIV), ANTP, the Drosophila homeotype transcription factor, VP22, the herpes simplex virus type 1 (HSV-1) transcription factor, and synthetically produced polyarginine and polylysine.

[0021] Furthermore, the membrane-penetrating peptide contains at least five arginine or lysine residues, and the total length of the membrane-penetrating peptide is 5-30 amino acids. A preferred membrane-penetrating peptide (internalizing peptide) is the Tat protein from the HIV virus.

[0022] Furthermore, the membrane-penetrating peptide is a Tat peptide.

[0023] As a non-limiting example, the amino acid sequence of the transmembrane peptide may be as shown in SEQ ID No. 2, which is the standard Tat peptide sequence.

[0024] Those skilled in the art will understand that the purpose of chimeric peptides and internalized peptides is primarily to enable the active peptides to better reach their target sites. Therefore, the internalized peptides applicable to this application are not limited to specific types, as long as they can achieve the purpose of membrane penetration and internalization. Furthermore, the chimeric peptide may also include a linker for connecting the membrane-penetrating peptide and the STAMPL1 polypeptide.

[0025] Furthermore, the membrane-penetrating peptide can be linked to the N-terminus of the STAMBPL1 polypeptide via the adapter.

[0026] In one embodiment of the invention, the connector is two glycine connectors (Gly-Gly).

[0027] Furthermore, the amino acid sequence of the chimeric peptide tat-STAMBPL1 may be as shown in SEQ ID No. 3.

[0028] Any of the polypeptides or chimeric peptides described above may optionally be derivatized (e.g., acetylated, phosphorylated, and / or glycosylated) to enhance their affinity, their ability to be transported across cell membranes, or their stability.

[0029] The present invention also provides a nucleic acid molecule, which may be any of the following:

[0030] B1) The DNA molecule encoding the STAMBPL1 polypeptide;

[0031] B2) is the DNA molecule that encodes the chimeric peptide tat-STAMBPL1.

[0032] Furthermore, the nucleotide sequence of the DNA molecule encoding the STAMPL1 polypeptide may be as shown in SEQ ID No. 4, whereby the DNA molecule shown in SEQ ID No. 4 encodes the STAMPL1 polypeptide shown in SEQ ID No. 1.

[0033] Furthermore, the nucleotide sequence of the DNA molecule encoding the chimeric peptide tat-STAMBPL1 may be as shown in SEQ ID No. 5, whereby the DNA molecule shown in SEQ ID No. 5 encodes the chimeric peptide tat-STAMBPL1 shown in SEQ ID No. 3.

[0034] Those skilled in the art can readily mutate the nucleotide sequence of the peptide encoding the present invention using known methods, such as directed evolution or point mutation. Any artificially modified nucleotides that have 75% or higher identity with the nucleotide sequence of the peptide isolated in the present invention, as long as they encode and function the peptide of the present invention, are derived from and equivalent to the nucleotide sequence of the present invention.

[0035] The peptide may include the STAMBP1 polypeptide or the chimeric peptide tat-STAMBPL1 or a transmembrane peptide.

[0036] The aforementioned 75% or higher identity can be 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher.

[0037] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained.

[0038] The present invention also provides an antitumor drug comprising the STAMPL1 polypeptide and / or the chimeric peptide tat-STAMBPL1.

[0039] The drug may contain one or more pharmaceutically acceptable carriers. These pharmaceutically acceptable carriers may be diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, adsorbents, surfactants, or lubricants.

[0040] The present invention also provides the use of the STAMBPL1 polypeptide, and / or any of the chimeric peptides tat-STAMBPL1 described herein, and / or the nucleic acid molecules described herein in the preparation of medicaments or products for the prevention, improvement or treatment of diseases caused by abnormalities in the mTORC1 signaling pathway.

[0041] In the above applications, the diseases caused by abnormalities in the mTORC1 signaling pathway can be tumors or gastrointestinal tumors.

[0042] In the above applications, the tumor may be a gastrointestinal tumor.

[0043] The aforementioned digestive tract tumor may be digestive tract cancer.

[0044] In the above applications, the digestive tract tumors include colorectal cancer, esophageal cancer, gastric cancer, or hepatobiliary and pancreatic cancer.

[0045] Targeting the key oncogenic role of the recently discovered Sestrin2-STAMBPL1 mechanism in colorectal cancer, we synthesized an amino acid fragment mimicking the Sestrin2 binding region on STAMBPL1 (the target peptide, i.e., the STAMBPL1 polypeptide of this invention) and revealed its important role in inhibiting cancer (especially gastrointestinal cancers). The polypeptide synthesized in this invention is cell-permeable, allowing it to enter cells and specifically block the interaction between Sestrin2 and endogenous STAMBPL1 through competitive binding. This polypeptide targets the Sestrin2 / STAMBPL1 interaction site, specifically binding to Sestrin2, thereby blocking the binding of Sestrin2 to endogenous STAMBPL1. This further promotes the activity of Sestrin2 in inhibiting the mTORC1 pathway, preventing excessive activation of cellular anabolic metabolism that could lead to cancer.

[0046] Experiments have shown that this invention has the following advantages compared with the prior art:

[0047] (1) The present invention has a high affinity for the peptide that specifically binds to the Sestrin2 protein, with a clear target. It targets the Sestrin2 / STAMBPL1 interaction site and can effectively inhibit the formation of tumor cells.

[0048] (2) The invented polypeptide drug has a novel molecular structure, a clear mechanism of action, high activity, small dosage, low toxicity and side effects, and no immunogenicity; it can be chemically synthesized, and the product has high purity and controllable quality. Attached Figure Description

[0049] The target peptide in the attached figure represents the chimeric peptide tat-STAMBPL1.

[0050] Figure 1 The effect of the chimeric peptide tat-STAMBPL1 on the interaction between Sestrin2 and STAMBPL1.

[0051] Figure 2 The effect of the chimeric peptide tat-STAMBPL1 on the ubiquitination level of Sestrin2.

[0052] Figure 3 The effect of the chimeric peptide tat-STAMBPL1 on mTORC1 levels in colorectal cancer cells (LS174T).

[0053] Figure 4 The effect of the chimeric peptide tat-STAMBPL1 on the growth rate of colorectal cancer cells (LS174T). Figure 4 In the figure, the unit of the vertical axis is cell density (10). 6 (per hole).

[0054] Figure 5 Effect of 48 hours of treatment with the chimeric peptide tat-STAMBPL1 on the size of colorectal cancer cells (LS174T).

[0055] Figure 6 The effect of the chimeric peptide tat-STAMBPL1 on the clonogenesis of colorectal cancer cells (LS174T). Figure 6 In the figure, the unit of the vertical axis is the number of clones (clones / well). Detailed Implementation

[0056] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0057] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0058] Example 1: Sequence and Synthesis of Chimeric Peptide tat-STAMBPL1

[0059] The inventors' preliminary experimental results demonstrated that Sestrin2, a negative regulator of the mTORC1 pathway, is deubiquitinated by binding to the deubiquitinase STAM-binding protein-like 1 (STAMBPL1) under leucine-sufficient conditions, thereby relieving inhibition of the mTORC1 pathway and promoting cell growth. Through bioinformatics analysis, the inventors discovered that the STAMPL1 gene, as a positive regulator of the mTORC1 pathway, is upregulated in various cancers, particularly gastrointestinal cancers such as colorectal and gastric cancer. In samples from patients with these cancer types, genome sequencing revealed a high-frequency mutation in STAMPL1: K405Rfs*21. Further experiments demonstrated that this mutation, by increasing its binding ability to Sestrin2, abnormally activates the mTORC1 signaling pathway, thereby promoting cancer development. STAMBPL1, as an oncogene, participates in the proliferation of tumor cells by activating the mTORC1 signaling pathway. Knocking out the STAMBPL1 gene or administering the mTORC1 signaling pathway inhibitor Rapamycin (a specific inhibitor of mTOR protein) can significantly inhibit tumor development. Therefore, drug development targeting the STAMBPL1 gene will provide a new target for the treatment of colorectal cancer.

[0060] In response to the inventors' recent discovery of the crucial oncogenic role of the Sestrin2-STAMBPL1 mechanism in colorectal cancer, an amino acid fragment mimicking the Sestrin2 binding region on STAMBPL1 (an active peptide, namely the STAMBPL1 polypeptide of this invention, SEQ ID No. 1) was synthesized. Furthermore, a cell-permeable chimeric peptide, tat-STAMBPL1, was designed and synthesized. The amino acid sequence of the chimeric peptide tat-STAMBPL1 is shown below:

[0061] YGRKKRRQRRR-GG-ERMASVYLEEGNLENAFVLY(SEQ ID No.3)

[0062] The sequence of the chimeric peptide tat-STAMBPL1 consists of three parts:

[0063] 1. The N-terminus is a transmembrane peptide derived from the transduction domain sequence of the HIV-1 Tat protein, namely the Tat peptide (SEQ ID No. 2), which enables the STAMBPL1 polypeptide to enter the cell;

[0064] 2. Two glycine residues act as linkers to connect the preceding and following sequences (linking the transmembrane peptide and the STAMBP1 polypeptide);

[0065] 3. The C-terminus is the region where STAMBPL1 binds to Sestrin2 (STAMBPL1 polypeptide, SEQ ID No. 1), with a sequence starting at amino acid 59 and ending at amino acid 78 of the human STAMBPL1 protein, totaling 20 amino acids. The STAMBPL1 polypeptide is a polypeptide that specifically binds to the Sestrin2 protein and can competitively block the interaction between Sestrin2 and endogenous STAMBPL1.

[0066] The following examples include control peptide 1 and control peptide 2. Control peptide 1: YGRKKRRQRRR-GG-ERVRALSKLGCNITISEDIT; Control peptide 2: YGRKKRRQRRR-GG-SEDITPRRYFRSGVEMERMA.

[0067] The polypeptides of this invention (chimeric peptide tat-STAMBPL1, control peptide 1, and control peptide 2) were synthesized using the Fmoc solid-phase synthesis method. The polypeptide sequences and purity were verified using mass spectrometry and chromatography. The polypeptide powder was dissolved in enzyme-free water (Invitrogen) filtered through an ultrapure membrane to a concentration of 1 mM and stored at -20°C.

[0068] Example 2: Effect of the chimeric peptide tat-STAMBPL1 on the interaction between Sestrin2 and STAMBPL1

[0069] According to the inventors' research, STAMBPL1, as a deubiquitinizing enzyme of Sestrin2, can reduce the ubiquitination level of Sestrin2. The synthesized target peptide (chimeric peptide tat-STAMBPL1), instead of the control peptide, will compete with endogenous STAMBPL1 for binding to Sestrin2 after entering the cell, thereby inhibiting the interaction between Sestrin2 and STAMBPL1.

[0070] The STAMBPL1 plasmid expressing the HA tag in the following steps is the recombinant vector pcDNA3.3-HA-STAMBPL1. It is designed using cDNA from HEK293T cells as a template, homologous primers at both ends of STAMBPL1 are designed for PCR to obtain the STAMBPL1 fragment, and the STAMBPL1 nucleic acid fragment is cloned into the pcDNA3.3-HA vector to obtain the recombinant expression vector expressing the STAMBPL1 gene.

[0071] The specific experimental procedures are as follows:

[0072] Human embryonic kidney cells (HEK293T) in logarithmic growth phase were harvested at 2 × 10⁻⁶. 6 Cells were seeded at a density of 1 cell per dish in 6 cm dishes and cultured in DMEM medium (HyClone) containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Transfection was performed when the cell surface area reached approximately 70%. Polyethyleneimine (PEI, Santa Cruz) was used as the plasmid transfection reagent. 2 μg of the HA-tag-expressing STAMBPL1 plasmid (pcDNA3.3-HA-STAMBPL1) was transfected into each 6 cm dish to obtain recombinant cells. The medium was changed 8 hours after transfection. Twelve hours after the medium change, the cells from one 6 cm dish were divided into two 6 cm dishes. After cell adhesion, the normal culture medium was replaced with Opti-MEM solution containing 20 μM chimeric peptide tat-STAMBPL1 to treat the cells. A control group was set up using Opti-MEM solution without chimeric peptide tat-STAMBPL1 or with the addition of control peptide 1: YGRKKRRQRRR-GG-ERVRALSKLGCNITISEDIT; control peptide 2: YGRKKRRQRRR-GG-SEDITPRRYFRSGVEMERMA. The specific experimental procedures are as follows:

[0073] Target peptide experimental group: The chimeric peptide tat-STAMBPL1 was diluted to 20 μM with serum-reduced Opti-MEM (Gibco) and then incubated with the cells in the normal culture medium.

[0074] Negative control group (" / "): Cells were incubated with serum-reduced Opti-MEM (Gibco) instead of normal culture medium.

[0075] Control peptide 1 group: Control peptide 1 was diluted to 20 μM with serum-reduced Opti-MEM (Gibco) and then incubated with cells in the normal culture medium.

[0076] Control peptide 2 group: Control peptide 2 was diluted to 20 μM with serum-reduced Opti-MEM (Gibco) and then incubated with cells in the normal culture medium.

[0077] After 24 hours of peptide treatment, the culture medium was discarded. 0.5 mL of TX-100 lysis buffer (150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1% Triton X-100, 20 mM Tris-HCl, pH 7.4) was added to each 6 cm dish to lyse the cells. After incubation on ice for 15 minutes, the cell lysis buffer was transferred to 1.5 mL tubes and centrifuged at 12000 rpm for 15 minutes at 4°C. After centrifugation, 60 μL of the supernatant was mixed with 4x sample buffer (Laemmli Sample Buffer, Bio-Rad), boiled at 100°C for 15 minutes, and then subjected to SDS-PAGE. Figure 1 (As shown in "Celllysate"). Transfer the remaining supernatant to a new 1.5 mL tube, add 1 mL of TX-100 lysis buffer, and mix with pre-washed Anti-HA agarose beads (Sigma). Incubate at 4°C for 4 hours. After incubation, wash the agarose beads three times with 1 mL of TX-100 lysis buffer, and finally elute with 30 μL of 1.2x loading buffer. Boil the sample at 100°C for 10 minutes. The interaction between STAMBPL1 and endogenous Sestrin2 protein was detected by Western blotting. Figure 1 (as shown in "IP:HA").

[0078] The results are as follows Figure 1 As shown, compared with the control group, the interaction between STAMBPL1 and Sestrin2 was inhibited in cells treated with the target peptide (chimeric peptide tat-STAMBPL1). This indicates that the target peptide (chimeric peptide tat-STAMBPL1) can enter cells and inhibit the interaction between STAMBPL1 and Sestrin2.

[0079] Example 3: Effect of chimeric peptide tat-STAMBPL1 on Sestrin2 ubiquitination levels

[0080] After the chimeric peptide tat-STAMBPL1 enters the cell, it competes with the endogenous STAMBPL1 for binding to Sestrin2, preventing the endogenous STAMBPL1 protein from deubiquitinizing Sestrin2, thereby achieving upregulation of Sestrin2 ubiquitination level.

[0081] The Sestrin2 plasmid expressing the Flag tag in the following steps is the recombinant vector pcDNA3.3-Flag-Sestrin2. It uses cDNA from HEK293T cells as a template, designs homologous primers at both ends of Sestrin2 for PCR, and obtains the Sestrin2 fragment. The Sestrin2 nucleic acid fragment is then cloned into the pcDNA3.3-Flag vector to obtain the recombinant expression vector expressing the Sestrin2 gene.

[0082] The specific experimental procedures are as follows:

[0083] Human embryonic kidney cells (HEK293T) in logarithmic growth phase were harvested at 2 × 10⁻⁶. 6 Cells were seeded at a density of 1 / 2 disc in 6 cm dishes and cultured in DMEM medium (HyClone) containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Transfection was performed when the cell surface area reached approximately 70%. Polyethyleneimine (PEI, Santa Cruz) was used as the plasmid transfection reagent. Each 6 cm dish was transfected with 4 μg of the Flag-tagged Sestrin2 plasmid (pcDNA3.3-Flag-Sestrin2) and 0.3 μg of the Myc-tagged K63-Ub plasmid (Ub protein retains only lysine 63, mutating the remaining lysines, Chen, J., Ou, Y., Yang, Y. et al. KLHL22 activates amino-acid-dependent mTORC1 signalling to promote tumorigenesis and aging. Nature 557, 585–589 (2018)). The medium was changed 8 hours after transfection. Twelve hours after changing the medium, the cells from one 6 cm dish were evenly divided into two 6 cm dishes. After cell adhesion, the normal culture medium was replaced with Opti-MEM solution containing 20 μM of the chimeric peptide tat-STAMBPL1 to treat the cells. The control group used Opti-MEM solution without the peptide or with the addition of control peptide 1: YGRKKRRQRRR-GG-ERVRALSKLGCNITISEDIT; control peptide 2: YGRKKRRQRR R-GG-SEDITPRRYFRSGVEMERMA. The specific experimental procedures are as follows:

[0084] Target peptide experimental group: The chimeric peptide tat-STAMBPL1 was diluted to 20 μM with serum-reduced Opti-MEM (Gibco) and then incubated with the cells in the normal culture medium.

[0085] Negative control group (" / "): Cells were incubated with serum-reduced Opti-MEM (Gibco) instead of normal culture medium.

[0086] Control peptide 1 group: Control peptide 1 was diluted to 20 μM with serum-reduced Opti-MEM (Gibco) and then incubated with cells in the normal culture medium.

[0087] Control peptide 2 group: Control peptide 2 was diluted to 20 μM with serum-reduced Opti-MEM (Gibco) and then incubated with cells in the normal culture medium.

[0088] After 24 hours of peptide treatment, the culture medium was discarded. 0.5 mL of RIPA lysis buffer (50 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 20 mM Tris-HCl, pH 7.4) containing 1% SDS was added to each 6 cm dish to lyse the cells. After incubation on ice for 15 minutes, the cell lysis buffer was transferred to 1.5 mL tubes and sonicated. Following sonication, the lysis buffer was incubated at 100°C for 10 minutes. After incubation, the cells were incubated at room temperature for 10 minutes, then centrifuged at 15000 rpm for 15 minutes at 4°C. After centrifugation, 60 μL of the supernatant was mixed with 4x sample buffer (Laemmli Sample Buffer, Bio-Rad) and incubated at 100°C for 15 minutes to complete sample preparation. Figure 2 (See “Celllysate” in the image). Transfer the remaining supernatant to a new 1.5 mL tube, add twice the volume of RIPA lysis buffer to dilute the SDS concentration, and mix with pre-washed Anti-Flag agarose beads (Sigma). Incubate at 4°C for 4 hours. After incubation, wash the agarose beads three times with 1 mL of RIPA lysis buffer containing 0.1% SDS, and finally elute with 30 μL of 1.2x loading buffer. Boil the sample at 100°C for 10 minutes. The ubiquitination level of Sestrin2 was detected by Western blotting. Figure 2 (As shown in "IP:FLAG").

[0089] The results are as follows Figure 2As shown, the ubiquitination level of Sestrin2 in cells treated with the chimeric peptide tat-STAMBPL1 was higher than that in the control group. This indicates that our produced chimeric peptide tat-STAMBPL1 can enter cells and upregulate the ubiquitination level of Sestrin2.

[0090] Example 4: Effect of chimeric peptide tat-STAMBPL1 on mTORC1 levels in colorectal cancer cells (LS174T)

[0091] The level of Sestrin2 ubiquitination can affect the activation level of mTORC1. After the chimeric peptide tat-STAMBPL1 enters the cell, it can upregulate the level of Sestrin2 ubiquitination, thereby inhibiting the activation of mTORC1.

[0092] The specific experimental procedures are as follows:

[0093] The human colorectal cancer cell line LS174T (LS174T for short) in the logarithmic growth phase was used at a dose of 1.75 × 10⁻⁶. 5 Cells were seeded at a density of 1 cell per well in 24-well plates and cultured in EMEM medium (ATCC) containing 20% ​​fetal bovine serum at 37°C and 5% CO2. When the cell growth area reached about 80%, the cells were divided into 4 experimental groups: target peptide experimental group, negative control group (" / "), control peptide 1 group and control peptide 2 group.

[0094] Target peptide experimental group: The chimeric peptide tat-STAMBPL1 was diluted to 20 μM with serum-reduced Opti-MEM (Gibco, catalog number 31985070) and then incubated with the cells in the normal culture medium.

[0095] Negative control group (" / "): Cells were incubated with serum-reduced Opti-MEM (Gibco) instead of normal culture medium.

[0096] Control peptide 1 group: Control peptide 1 was diluted to 20 μM with serum-reduced Opti-MEM (Gibco) and then incubated with cells in the normal culture medium.

[0097] Control peptide 2 group: Control peptide 2 was diluted to 20 μM with serum-reduced Opti-MEM (Gibco) and then incubated with cells in the normal culture medium.

[0098] After 4 hours of incubation, aspirate the culture medium and add 50 μL of pre-chilled RIPA lysis buffer (50 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 20 mM Tris-HCl, pH 7.4, added to each well before use) to lyse the cells. Incubate the 24-well plate on ice for 15 minutes, then transfer the buffer to 1.5 mL centrifuge tubes and centrifuge at 12,000 rpm. Centrifuge at 4°C for 15 minutes. After centrifugation, mix the supernatant with 4x loading buffer and boil at 100°C for 15 minutes to complete sample preparation. The mTORC1 activation level was indicated by detecting the phosphorylation level of mTORC1-specific substrate proteins using Western blotting with anti-S6K1 antibody, anti-p-S6K1 antibody, anti-S6 antibody, anti-p-S6 antibody, anti-LC3BI antibody, anti-LC3BII antibody, anti-Akt antibody, anti-p-Akt antibody, and anti-GAPDH antibody. Figure 3 ).

[0099] The results are as follows Figure 3 As shown, S6K1 is a direct phosphorylated substrate of mTORC1, and S6 is a direct phosphorylated substrate of S6K1. Upon activation, mTORC1 phosphorylates S6K1, which in turn phosphorylates S6. Therefore, the levels of p-S6K1 and p-S6 reflect the level of mTORC1 activation. Simultaneously, mTORC1 activation inhibits autophagy, as evidenced by a significant reduction in the LC3B cleavage form LC3BII. The Akt signaling pathway is upstream of the mTORC1 signaling pathway; phosphorylation of the upstream kinase Akt can serve as a control. Compared to the negative control group, the levels of S6K1 and S6 proteins in the chimeric peptide tat-STAMBPL1 treatment group remained almost unchanged, while the levels of p-S6K1 and p-S6 were significantly reduced. At the same time, a large amount of LC3BII was produced, indicating that autophagy was being initiated. As a control, the phosphorylation level of Akt did not change. Therefore, we believe that the chimeric peptide tat-STAMBPL1 treatment can significantly inhibit the mTORC1 activation level of colorectal cancer cells LS174T.

[0100] Example 5: Effect of chimeric peptide tat-STAMBPL1 on the growth of colorectal cancer cells (LS174T)

[0101] Overactivation of the mTORC1 signaling pathway often leads to excessive cell growth. After entering the cell, the chimeric peptide tat-STAMBPL1 inhibits the activation of mTORC1, thereby suppressing the growth of tumor cells.

[0102] The specific experimental procedures are as follows:

[0103] Human colorectal cancer cells (LS174T) in the logarithmic growth phase were selected at a dose of 1.75 × 10⁻⁶. 5 Cells were seeded at a density of 1 cell / well in 24-well plates and cultured in EMEM medium (ATCC) containing 20% ​​fetal bovine serum. When the cells reached approximately 80% confluence, they were divided into a Peptide group and a negative control group (Ctrl). In the Peptide group, the chimeric peptide tat-STAMBPL1 was diluted to 20 μM with serum-reduced Opti-MEM (Gibco) and then used to replace the normal culture medium for cell incubation. Serum-reduced Opti-MEM was used as the negative control group (Ctrl). Cells in both the Peptide group and the negative control group (Ctrl) were digested after 24 hours (1 day), 48 hours (2 days), 72 hours (3 days), and 96 hours (4 days), respectively, and cell density and diameter were measured using a cell counter (CountStar). Each experimental group was performed in quadruplicate. Figure 4 ).

[0104] The results are as follows Figure 4 and Figure 5 As shown, cell density and cell diameter were measured after treatment with the chimeric peptide tat-STAMBPL1 for 24, 48, 72, and 96 hours. The number of cells was significantly reduced compared to the negative control group, and the cell diameter was significantly smaller. Therefore, treatment with the chimeric peptide tat-STAMBPL1 significantly inhibited the growth of LS174T colon cancer cells. Data were processed using Prism 8.0 statistical software. Experimental results are expressed as mean ± standard deviation, and a two-tailed t-test was used. P < 0.001 (***) indicated a highly significant difference.

[0105] Example 6: Soft agar colony formation assay to detect the effect of chimeric peptide tat-STAMBPL1 on the tumorigenicity of colorectal cancer cells (LS174T).

[0106] The clonality test is a simple and reliable in vitro method for evaluating the effects of antitumor drugs, and it is currently widely used. The size and number of colonies reflect the tumorigenic capacity of cells. Overactivation of the mTORC1 signaling pathway may lead to cancer development. After entering cells, the chimeric peptide tat-STAMBPL1 inhibits the tumorigenic capacity of cells by suppressing mTORC1 activation.

[0107] The specific experimental procedure is as follows:

[0108] Double-distilled water was used as a solvent to prepare low-melting-point agarose (Sigma) solutions with concentrations of 1.2% and 0.7% (w / w). The lower gel was prepared by mixing 1.2% low-melting-point agarose with 2×DMEM medium at a 1:1 ratio (the solution was kept in a 42°C water bath for later use). 1.5 mL of the lower gel was needed for each well of a six-well plate (avoiding air bubbles as much as possible). After the lower gel was laid, the six-well plates were left at room temperature for at least 30 minutes to allow it to completely solidify before preparing the upper gel. Human colorectal cancer cells LS174T in the logarithmic growth phase were thoroughly digested and divided into four experimental groups: the target peptide experimental group, the negative control group (" / "), the control peptide 1 group, and the control peptide 2 group.

[0109] Target peptide experimental group: Cells were injected at a concentration of 1×10⁻⁶. 4 Cells were resuspended at a density of cells / mL in Opti-MEM containing 40 μM of the chimeric peptide tat-STAMBPL1 (the liquid obtained by adding tat-STAMBPL1 to Opti-MEM until the content of tat-STAMBPL1 is 40 μM) to obtain a cell suspension.

[0110] Negative control group (" / "): Cells were loaded at 1×10⁻⁶ 4 Cells were resuspended at a density of cells / mL in Opti-MEM solution without the chimeric peptide tat-STAMBPL1 to obtain a cell suspension.

[0111] Control peptide group 1: Cells were injected at a rate of 1×10⁻⁶. 4 Cells were resuspended at a density of 1 cell / mL in Opti-MEM containing 40 μM control peptide 1 (the liquid obtained by adding control peptide 1 to Opti-MEM until the content of control peptide 1 is 40 μM) to obtain a cell suspension.

[0112] Control peptide group 2: Cells were injected at a rate of 1×10⁻⁶. 4 Cells were resuspended at a density of 1 cell / mL in Opti-MEM containing 40 μM control peptide 2 (the liquid obtained by adding control peptide 2 to Opti-MEM until the content of control peptide 2 is 40 μM) to obtain a cell suspension.

[0113] Each experimental group was performed in triplicate. Cell suspension was mixed 1:1 with 0.7% low-melting-point agarose and added to the completely solidified lower gel, 1.5 mL of upper gel per well (avoiding air bubbles as much as possible). The plate was gently shaken to distribute cells evenly in each well, and then placed at room temperature. After the upper gel had completely solidified, the six-well plates were incubated at 37°C in a 5% CO2 cell culture incubator. Every two days, 100 μL of Opti-MEM was added to each well to prevent the gel from drying out and affecting cell growth. After two weeks, 1 mL of 0.005% crystal violet (dissolved in 5% methanol) was added to each well for staining. After staining at 37°C for 1 hour, the cells were washed once with PBS. Colony formation in each well was photographed and analyzed using ImageJ. Data were processed using Prism 8.0 statistical software. Experimental results are expressed as mean ± standard deviation, and a two-tailed t-test was used. P < 0.001 (***) indicated highly significant differences.

[0114] The results are as follows Figure 6 As shown, according to the soft agar colony formation assay, the clones formed by the target peptide group were smaller than those in the control group. Furthermore, after data statistics and analysis, the number of clones formed in the chimeric peptide tat-STAMBPL1 treatment group was significantly less than that in the control group. This indicates that the growth of LS174T cells treated with chimeric peptide tat-STAMBPL1 was inhibited, and their tumorigenicity was significantly reduced. This demonstrates that the polypeptide (chimeric peptide tat-STAMBPL1) can inhibit tumor formation and has good anti-tumor (especially colorectal cancer) function.

[0115] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. SEQUENCE LISTING <110> Beijing University <120> Peptides that specifically bind to Sestrin2 protein and their application in the treatment of gastrointestinal cancers <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> PRT <213> Artificial sequence <400> 1 Glu Arg Met Ala Ser Val Tyr Leu Glu Glu Gly Asn Leu Glu Asn Ala 1 5 10 15 Phe Val Leu Tyr 20 <210> 2 <211> 11 <212> PRT <213> Artificial sequence <400> 2 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 3 <211> 33 <212> PRT <213> Artificial sequence <400> 3 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Gly Gly Glu Arg Met 1 5 10 15 Ala Ser Val Tyr Leu Glu Glu Gly Asn Leu Glu Asn Ala Phe Val Leu 20 25 30 Tyr <210> 4 <211> 60 <212> DNA <213> Artificial sequence <400> 4 gagaggatgg cgtctgtgta tttggaagaa ggaaatttgg aaaatgcctt tgttctttat 60 <210> 5 <211> 99 <212> DNA <213> Artificial sequence <400> 5 tatggcagga agaagcggag acagcgacga agaggcggcg agaggatggc gtctgtgtat 60 ttggaagaag gaaatttgga aaatgccttt gttctttat 99

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID No.

1.

2. A chimeric peptide, characterized in that, The amino acid sequence of the chimeric peptide is shown in SEQ ID No.

3.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule is any one of the following: B1) A DNA molecule encoding the polypeptide of claim 1; B2) The DNA molecule encoding the chimeric peptide of claim 2.

4. An antitumor drug, characterized in that, The drug comprises the polypeptide of claim 1 and / or the chimeric peptide of claim 2.

5. The use of the polypeptide of claim 1, and / or the chimeric peptide of claim 2, and / or the nucleic acid molecule of claim 3 in the preparation of a medicament for the prevention, improvement or treatment of colorectal cancer.

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