Inhibitory peptides targeting p53 protein and uses thereof
By designing an inhibitory peptide targeting the p53 protein, the peptide sequence LGTX1SX2X3M was screened using the IDProMat platform. This solved the problems of insufficient stability and targeting of existing drugs in targeting the COP1-p53 interaction, achieving precise inhibition of tumor cells with abnormal p53 function, restoring the anti-cancer function of p53, and improving the efficacy of cancer treatment.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- TIANJIN UNIV
- Filing Date
- 2026-04-24
- Publication Date
- 2026-07-21
AI Technical Summary
Existing drugs targeting the COP1-p53 interaction suffer from problems such as insufficient in vivo stability, weak targeting, and significant toxic side effects, making it difficult to achieve clinical translation. Traditional chemotherapy drugs are unable to accurately target tumor cells with abnormal p53 function, resulting in low treatment efficiency and significant side effects.
An inhibitory peptide targeting the p53 protein was designed with the amino acid sequence LGTX1SX2X3M. Using the IDProMat intelligent design platform, the targeting peptide was screened by combining the binding interfaces of p53 protein with ASPP2, TSPYL5 and iASPP. The peptide exhibits good biosafety and metabolic stability and competitively blocks the interaction between the E3 ubiquitin ligase COP1 and p53.
Restoring the anti-cancer function of p53 improves the treatment effect on cancers such as ovarian cancer, cervical cancer, endometrial cancer, and pancreatic cancer, reduces side effects, and increases treatment efficiency.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and in particular to inhibitory peptides targeting the p53 protein and their applications. Background Technology
[0002] Abnormal p53 function plays a central driving role in the occurrence and development of four types of malignant tumors: ovarian cancer, cervical cancer, endometrial cancer, and pancreatic cancer. Clinical data show that the TP53 gene mutation rate exceeds 95% in high-grade serous ovarian cancer, reaches 70%–90% in type II endometrial cancer, has an inactivation rate of over 80% in cervical cancer, and is as high as 70%–75% in pancreatic cancer. Even in patients with wild-type TP53, overexpression of the E3 ubiquitin ligase COP1 is common, which mediates the degradation of p53 ubiquitination, leading to the loss of its tumor-suppressive function and ultimately driving malignant tumor proliferation, invasion and metastasis, and resistance to radiotherapy, chemotherapy, and targeted therapy. Furthermore, patients with p53 dysfunction in the above-mentioned cancer types often have a worse prognosis and a significantly reduced response rate to existing treatment regimens, making them a "refractory population" in clinical treatment.
[0003] While drug development targeting the p53 pathway has made some progress, many adjustments are still needed: Currently, there are no marketed drugs targeting the COP1-p53 interaction; some peptide drugs suffer from insufficient in vivo stability, weak targeting, and significant toxic side effects, hindering clinical translation; traditional chemotherapy drugs struggle to precisely target tumor cells with abnormal p53 function, resulting in low treatment efficiency and severe side effects. Therefore, developing novel targeted drugs against the p53-COP1 interaction has become a key direction for overcoming the treatment bottlenecks of these diseases. Summary of the Invention
[0004] In view of this, the technical problem to be solved by the present invention is to provide an inhibitory peptide targeting the p53 protein and its application.
[0005] This invention provides an inhibitory peptide targeting the p53 protein, the amino acid sequence of which is: LGTX1SX2X3M;
[0006] Where X1 is selected from E or D;
[0007] X2 is selected from N or A;
[0008] X3 is selected from A or V.
[0009] Furthermore, the amino acid sequence of the inhibitory peptide described in this invention is shown in SEQ ID NO:1~SEQ ID ID:4.
[0010] This invention provides a method for preparing the inhibitory peptide, which includes the following steps:
[0011] Using the p53 protein with PDB ID: 7YGI as the target, the amino acid fragments at the binding interfaces of the p53 protein with ASPP2, TSPYL5 and iASPP proteins were selected as input sequences and input into IDProMat to obtain the inhibitory peptide targeting the p53 protein.
[0012] Furthermore, after obtaining the inhibitory peptide targeting the P53 protein, a screening and evaluation step is also included:
[0013] The screening and evaluation includes:
[0014] (a): The free energy for molecular docking of the inhibitory peptide with the P53 protein needs to be below -5 kcal / mol; and
[0015] (b): Based on (a), using GROMACS software, the root mean square deviation (RMSD) between the hotspot residues of P53 protein that bind to ASPP2, TSPYL5, iASPP, etc. and the repressive peptides is calculated. The root mean square deviation needs to be <0.4 nm.
[0016] (c): Based on (b), the binding conformation is analyzed using VMD visualization software to determine whether the binding site of the inhibitory peptide to P53 is close to the binding sites of ASPP2, TSPYL5 and iASPP to P53 protein.
[0017] (d): Based on (c), analyze whether the average hydrophilicity coefficient GRAVY value of the inhibitory peptide is negative and whether the lipid-water partition coefficient log P is ≤-1.7.
[0018] (e): Based on (d), construct an all-atom model of the binding of the inhibitory peptide to the P53 protein.
[0019] The parameters for constructing the all-atom model of the binding of the inhibitory peptide to the P53 protein are: a GROMOS96 43a1 force field, an SPC model for the solvent water molecules, and a simulation system of the crystal structure containing 174 Na⁺, 178 Cl⁻, and 172,557 water molecules with a size of 12.5 × 11 × 12.6 nm. 3 Inside the cube-shaped box.
[0020] Specifically, in this invention, the inhibitory peptide is obtained by training and designing a peptide sequence targeting p53 using the protein ligand intelligent design program IDProMat. Specifically, using p53 protein as the target, amino acid fragments at its binding interface are extracted as input sequences. This fragment is converted into a 21-dimensional hot-coded tensor (20 dimensions correspond to 20 common amino acids, and the 21st dimension corresponds to hydroxyproline; p53 naturally does not contain hydroxyproline, therefore this dimension is always 0 at all sites), and directly fed back into the encoder in the pre-trained Seq2Seq module. Finally, the model outputs peptide sequences that specifically bind to the p53 protein, including LGTESNAM (SEQ ID NO:1), LGTDSNAM (SEQ ID NO:2), LGTESAVM (SEQ ID NO:3), and IGTESNAM (SEQ ID NO:4), etc.
[0021] The present invention provides a composition for regulating the p53-COP1 pathway, comprising the inhibitory peptide described herein and other polypeptides or proteins that regulate p53-COP1 interaction.
[0022] Furthermore, the other peptides that regulate the p53-COP1 pathway include: p28 peptide.
[0023] Other proteins that regulate the p53-COP1 pathway include ASPP2, TSPYL5, and / or iASPP.
[0024] This invention provides a formulation comprising: a pharmaceutically acceptable excipient and at least one of the following: A) to B)
[0025] A) The inhibitory peptide described in this invention;
[0026] B) The composition described in this invention.
[0027] Furthermore, in the formulations described in this invention, the pharmaceutically acceptable excipients include: fillers, binders, disintegrants, lubricants, flow aids, coating materials, solvents and / or solubilizers.
[0028] Furthermore, the filler includes: lactose, microcrystalline cellulose (MCC), starch, mannitol and / or dicalcium phosphate;
[0029] The adhesives include: polyvinylpyrrolidone (PVP), hydroxypropyl methylcellulose (HPMC), starch paste and / or gelatin;
[0030] The disintegrants include: croscarmellose sodium (CCNa), croscarmellose (PVPP) and / or carboxymethyl starch sodium (CMS-Na).
[0031] The lubricant includes: magnesium stearate, talc, polyethylene glycol (PEG) and / or sodium benzoate;
[0032] The flow aid includes: colloidal silica (Aerosil) and / or talc;
[0033] The coating materials include: hydroxypropyl methylcellulose (HPMC), ethylcellulose, cellulose acetate and / or Opadry®.
[0034] The solvents include: water for injection, ethanol, propylene glycol, glycerin and / or polyethylene glycol (PEG);
[0035] The solubilizers include: polysorbate 80 (Tween 80), poloxamer 188 and / or cyclodextrin (β-CD, HP-β-CD).
[0036] This invention provides the use of at least one of the following (I) to (III) in the preparation of therapeutic agents for cancers related to the p53-COP1 pathway:
[0037] I) The inhibitory peptide described in this invention;
[0038] II) The composition described in this invention;
[0039] III) The formulation described in this invention.
[0040] Furthermore, the cancers associated with the p53-COP1 pathway include: ovarian cancer, cervical cancer, endometrial cancer, and / or pancreatic cancer.
[0041] This invention provides a medicament for the treatment of cancers associated with the p53-COP1 pathway, the raw materials of which include at least one of the following i) to iv):
[0042] i) The p53 protein inhibitory peptide described in this invention;
[0043] ii) The composition described in this invention;
[0044] iii) The formulation described in this invention.
[0045] Based on the core binding sites of the p53 protein core domain and the binding interfaces of proteins such as ASPP2, TSPYL5, and iASPP, this invention uses IDProMat to design and obtain the p53-targeting inhibitory peptide described in this invention. It can bind to the p53 protein, competitively block the interaction between the E3 ubiquitin ligase COP1 and p53, inhibit the ubiquitination and degradation of p53, restore the anti-tumor function of p53, and at the same time have good biosafety and metabolic stability, and are expected to be used in the clinical treatment of ovarian cancer, cervical cancer, endometrial cancer, pancreatic cancer, etc. Attached Figure Description
[0046] Figure 1 The diagram shows the binding kinetics simulation of LGTESNAM and p53 protein; where A is the curve of dmin, dcom, and RMSD between p53 and LGTESNAM over time; B is the curve of ELJ and EC between p53 and LGTESNAM over time; C is the conformation diagram generated using VMD software at 0, 5, and 20 ns, where p53 protein is shown in gray surf model with hotspot residues marked in red, while LGTESNAM is represented in blue using licorice model.
[0047] Figure 2 Experiments demonstrating the binding of LGTESNAM to p53 protein;
[0048] Figure 3 This study demonstrates the inhibitory effect of LGTESNAM on competitive binding to p53 and COP1 proteins. Detailed Implementation
[0049] This invention provides an inhibitory peptide targeting the p53 protein and its applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.
[0050] This invention is based on IDProMat (Intelligent Design of Protein Matcher), a protein ligand intelligent design program independently developed by our research group. Targeting the binding interface between p53 protein and other proteins, it uses a network framework to learn the amino acid residue interaction rules at the protein-protein interaction interface, designing peptide inhibitors such as LGTESNAM (SEQ ID NO:1), LGTDSNAM (SEQ ID NO:2), LGTESAVM (SEQ ID NO:3), and IGTESNAM (SEQ ID NO:4). These peptide inhibitors can bind to p53 protein, competitively blocking the interaction between the E3 ubiquitin ligase COP1 and p53, inhibiting the ubiquitination and degradation of p53, restoring p53's anti-tumor function, and exhibiting good biosafety and metabolic stability. They hold promise for clinical treatment of ovarian cancer, cervical cancer, endometrial cancer, pancreatic cancer, and other cancers. Compared with the previous biomimetic design schemes of the research group, the IDProMat intelligent design of this invention has two major advancements: First, the generated peptides are generated de novo based on the global features of the target interface, naturally adapting to the spatial structure and residue interaction rules of the target, and the binding site with the p53 target is more in line with the natural interaction interface, with a site matching degree superior to biomimetic peptides with fixed frameworks; Second, it eliminates the need for large-scale peptide library construction, multiple rounds of virtual screening and molecular simulation, significantly saving research and development time and computational costs, and improving the accuracy and efficiency of peptide design.
[0051] The detailed steps of obtaining and validating the affinity ligands of the p53 protein using molecular simulation and intelligent design methods in this invention are as follows:
[0052] 1. Existing studies have systematically analyzed the binding interface between p53 and proteins such as ASPP2, TSPYL5, and iASPP using methods such as molecular dynamics simulations.
[0053] 2. Based on the target sequence of the p53 protein binding interface, the peptide inhibitor sequences were predicted using IDProMat, including LGTESNAM, LGTDSNAM, LGTESAVM, IGTESNAM, etc.
[0054] 3. Using Autodocvina molecular docking software, peptides LGTESNAM, LGTDSNAM, LGTESAVM, and IGTESNAM were docked with p53 protein. The docking results showed binding free energies below -5 kcal / mol. The root mean square deviation (RMSD) of the binding hotspot residues of p53 protein to ASPP2, TSPYL5, and iASPP was calculated using the g_rms program in the GROMACS software package. This allowed for comparison of the conformations of the hotspot residues in the docked peptides with those in ASPP2, TSPYL5, and iASPP. A smaller RMSD value indicates a closer similarity between the docking conformation of the hotspot residues in the peptide ligands and those in ASPP2, TSPYL5, and iASPP. Finally, the similarity between the binding sites of the peptides and those of ASPP2, TSPYL5, and iASPP on p53 protein was analyzed. The hydrophilicity and hydrophobicity of each amino acid residue in the peptide ligand sequences LGTESNAM, LGTDSNAM, LGTESAVM, and IGTESNAM were analyzed. Finally, the peptides LGTESNAM, LGTDSNAM, LGTESAVM, and IGTESNAM were compared with p53 protein using MD simulation and subsequent verification of their inhibitory effects.
[0055] 4. The conformation of the peptides LGTESNAM, LGTDSNAM, LGTESAVM, and IGTESNAM with the p53 protein was used as the initial COM conformation. The minimum distance between the peptides and the p53 protein was adjusted to 1.4–1.5 nm using the g_editconf command provided with GROMACS, resulting in the Sep conformation. Using the GROMACS software package, the GROMOS96 43a1 force field was selected. The complex of the peptide inhibitors LGTESNAM, LGTDSNAM, LGTESAVM, and IGTESNAM with the p53 protein was placed at the center of a cubic box with dimensions of 12.5 × 11 × 12.6 nm. 3 The SPC model was chosen as the water molecule model; the required Na+ was added to balance the net charge of the system and stabilize the buffer solution (physiological saline). + and Cl - Next, energy minimization was performed to eliminate interatomic collisions and incorrect geometries in the system. Following this, a 100 ps canonical (NVT) ensemble-constrained kinetic equilibrium was conducted, and finally, a 100 ns MD simulation was performed to observe the minimum distance d between the ligand and the p53 protein. min Distance between centroids (d) com LJ potential energy (E) LJ Coulomb potential energy (E)C The study investigated changes in peptide ligand conformation (RMSD) and tightness (Rg). The results showed that LGTESNAM, LGTDSNAM, LGTESAVM, and IGTESNAM exhibited high affinity.
[0056] 5. Perform a double-antibody sandwich ELISA experiment on the polypeptide (inhibitory peptide) to verify its binding performance with p53 protein.
[0057] 6. Competitive ELISA experiments were performed on the peptides to verify their inhibitory effect on the binding of p53 and COP1 proteins.
[0058] The assay for double-antibody sandwich ELISA of peptides (inhibitory peptides) involves the following:
[0059] The blank control group consisted of PBS buffer solution, the negative control group consisted of the polypeptide sequence WGYGWNGY, and the positive control group consisted of the monoclonal antibody coated in the original ELISA kit. The specific steps are as follows.
[0060] Coating: Add 100 μL of PBS solution, 100 μg / mL peptide inhibitor solution, or control peptide WGYGWNGY solution to each well of a 96-well plate, and incubate at 4°C for 12 h. After incubation, remove the coating solution and wash the wells with wash buffer.
[0061] Blocking: Add 200 μL of 50 mg / mL bovine serum albumin to each well and block at 37°C for 2 h. After blocking, discard the solution in the wells and wash with wash buffer.
[0062] p53 protein incubation: 100 μL of p53 protein solution with a concentration of 1.25 ng / mL was added to the experimental group, blank control group, positive control group and negative control group respectively. After covering with a sealing membrane, the reaction was carried out at 37℃ for 80 min.
[0063] Biotinylated antibody binding: Discard the liquid in each well, add 200 μL of washing buffer to each well, and wash 3 times. After patting dry, add 100 μL of biotinylated antibody working solution to each well, cover with sealing membrane, and incubate at 37°C for 50 min.
[0064] Washing: Discard the liquid, add 200 μL of washing solution to each well, let stand for 1 min and then discard, pat dry on absorbent paper, and repeat washing 3 times.
[0065] Enzyme conjugate incubation: Add 100 μL of enzyme conjugate working solution to each well, cover with sealing membrane, and incubate at 37°C for 50 min. After the reaction, discard the liquid, add 200 μL of washing buffer to each well, let stand for 1 min, then discard, pat dry on absorbent paper, and repeat the washing 5 times.
[0066] Color development: Add 90 μL of TMB substrate solution to each well, cover with a sealing film, and react at 37°C in the dark for 20 min.
[0067] Termination and detection: Add 50 μL of stop solution directly to each well to terminate the reaction, and immediately measure the absorbance value of each well at a wavelength of 450 nm.
[0068] Three wells were set up for each group to conduct parallel experiments.
[0069] The competitive ELISA assay for peptides to verify their inhibitory effect on the binding of p53 and COP1 proteins followed a similar procedure to the double-antibody sandwich ELISA assay. A 96-well microplate was coated with COP1 protein and blocked with BSA. Several control groups were set up: a blank control group (PBS solution only), a positive control group (p53 protein solution added to the COP1-coated wells, without inhibitors), a negative peptide control group (WGYGWNGY solution added to the positive control group), and a target peptide experimental group (target peptide solution added to the positive control group). The corresponding treatment solutions for each group were added sequentially and incubated. Then, biotinylated antibody and enzyme conjugate working solution were added, and after thorough washing, TMB substrate was added for colorimetric reaction. Finally, the reaction was terminated with stop solution, and the absorbance (OD) of each group was measured at 450 nm. 450 )value.
[0070] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:
[0071] Example 1: Inhibitory peptides targeting p53 protein and their applications
[0072] Existing research has confirmed that ASPP2, TSPYL5, iASPP, and other peptides can stably bind to p53 protein through specific domains, and their binding interfaces highly overlap with the recognition regions of p53 and COP1, making them effective targets for restoring the anti-cancer function of p53. Based on mature targets and research, this invention completely abandons the previous biomimetic design approach and utilizes the IDProMat intelligent design platform independently developed by our research group to perform end-to-end de novo peptide generation targeting this interface, obtaining novel peptide inhibitors adapted to the pathological characteristics of ovarian cancer, cervical cancer, endometrial cancer, and pancreatic cancer. The specific scheme is as follows:
[0073] I. Property Analysis of Polypeptide Ligands
[0074] The peptides LGTESNAM (SEQ ID NO:1), LGTDSNAM (SEQ ID NO:2), LGTESAVM (SEQ ID NO:3), and IGTESNAM (SEQ ID NO:4) were sequentially subjected to molecular docking screening, root mean square deviation comparison, binding site comparison, and hydrophobic residue analysis, and then the next step of MD simulation was performed.
[0075] 1. Molecular docking
[0076] The ligands (peptides shown in SEQ ID NO: 1~4) were docked with the p53 protein using Autodoc Vina. VINA The smaller the value, the stronger the binding affinity of the ligand to the p53 protein. Repeated measurements yielded E... VINA All values are negative and less than -5 kcal / mol. The E value from a single repetition... VINA The values are shown in Table 1.
[0077] 2. RMSD Calculation
[0078] The RMSD values of LGTESNAM, LGTDSNAM, LGTESAVM, and IGTESNAM with key residues such as ASPP2, TSPYL5, and iASPP were calculated using the GROMACS program to determine their structural similarity, i.e., to examine the effect of ligands on simulating key sites. A smaller RMSD indicates a smaller structural deviation. The obtained RMSD values (as shown in Table 1) were used as a baseline for further steps, with values less than 0.4 nm.
[0079] (3) Comparison of binding sites
[0080] VMD software was used for conformational alignment and binding site analysis to observe whether the ligands covered the binding hotspots on the p53 protein that bind to ASPP2, TSPYL5, iASPP, etc. If the hotspots were covered, the ligands would have a greater advantage in inhibiting the binding of COP1 and p53 protein (Table 1).
[0081] (4) Hydrophobicity analysis
[0082] The hydrophobicity of the ligands was analyzed. The GRAVY value of the peptide was analyzed, showing that it is generally hydrophilic and has good solubility in physiological saline. Furthermore, the peptide carries a single negative charge in the human physiological environment (pH 7.4), making it less prone to non-specific aggregation and exhibiting good water solubility. PI represents the isoelectric point.
[0083] Table 1. Properties of polypeptide ligands
[0084]
[0085] II. Molecular Dynamics Simulation of Peptide Inhibitors
[0086] Based on molecular dynamics simulation trajectories, the interaction dynamic parameters between peptides LGTESNAM, LGTDSNAM, LGTESAVM, and IGTESNAM and p53 protein were systematically analyzed (taking LGTESNAM as an example, such as...). Figure 1 As shown). From the centroid spacing d com The curves show that the two bonds achieve stable binding at approximately 2.5 ns; although there are slight fluctuations thereafter, the minimum spacing d remains constant. min The contact threshold remained consistently within which the complex did not dissociate. Throughout the simulation, the Coulomb potential E... C LJ potential energy E LJ The potential energy remained stable at approximately -0.15 MJ / mol and -0.2 MJ / mol, respectively. The fluctuations in potential energy corresponded to the dynamic conformational adjustments at the binding interface, confirming that electrostatic and hydrophobic interactions jointly drove and maintained the stable formation of the complex. The root mean square deviation of the complex showed relatively small fluctuations throughout the simulation period, remaining within a reasonable range, indicating good stability of the overall conformation after binding. To accurately reveal the binding mode between this peptide and p53, further conformations at key moments such as 0 ns, 5 ns, and 20 ns were extracted for visualization analysis.
[0087] The p53 binding conformation of the peptides of this invention and the previously designed biomimetic peptides (QPSGGPYM (SEQ ID NO:5), QTSWGPEM (SEQ ID NO:6), QYSYGPWM (SEQ ID NO:7), QQSWGPWM (SEQ ID NO:8), QFSYGPWM (SEQ ID NO:9)) was visualized and compared using VMD software. The results showed that the intelligently designed peptides closely match the natural binding interfaces of p53 protein with ASPP2, TSPYL5, iASPP, etc., and have good overlap between the binding sites and template targets, as well as good coverage of hotspot residues. They can well replicate the natural binding mode of p53 protein.
[0088] From the perspective of binding kinetics, the system completes rapid binding and enters a stable state within 2.5 ns. During subsequent simulation periods, the centroid spacing and minimum distance remain within the stable range, without any unstable behaviors such as dissociation. The simulation results fully demonstrate the complete stages of "free approach—specific binding—conformal stability." At the conformational level, the root mean square deviation of the complex shows small fluctuations and no significant drift throughout the simulation, indicating that the system has reached conformational thermodynamic equilibrium within 20 ns, and extending the simulation time will not change the core conclusion of stable binding. At the energy level, the Coulomb potential and van der Waals potential remain stable in the negative range throughout the simulation, and the electrostatic and hydrophobic interactions driving the binding show no significant fluctuations, indicating that the energy state has approached a steady state.
[0089] III. Verification through combined effect experiments
[0090] A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was performed on the peptides. p53 was added to wells pre-coated with different affinity ligands (peptides shown in SEQ ID NO: 1-4). A colorimetric reaction occurred if the two peptides bound, and the OD value was [value missing]. 450 It will increase. The blank control group was PBS solution, OD 450 The value was 0.04 ± 0.02. The negative control group consisted of the polypeptide sequence WGYGWNGY (SEQ ID NO:10), which does not react with p53 protein. 450 The value was 0.19 ± 0.07. The positive control group consisted of the monoclonal antibody (Kelu Biotechnology ELK2674) coated in the original ELISA kit, with an OD value of 0.19 ± 0.07. 450 The OD of LGTESNAM (SEQ ID NO:1) is 0.71 ± 0.10. 450 The value was 0.61 ± 0.13 (p < 0.001), and the binding efficiency was very close to that of the original p53 protein antibody, proving that the peptide LGTESNAM can achieve efficient binding with the p53 protein. Figure 2 ).
[0091] IV. Experimental Verification of Inhibition Effect
[0092] A competitive ELISA assay was performed on the peptides. Results showed that the blank control group exhibited only extremely low background absorbance and OD... 450 The value was 0.13 ± 0.03. In the inhibitor-free group (i.e., without the addition of inhibitors), a significantly higher OD was detected. 450 The signal indicates that p53 can efficiently and specifically bind to solid-coated COP1, OD 450 The value was 1.66±0.19; the absorbance level of the negative peptide WGYGWNGY control group was not significantly different from that of the inhibitor-free group, indicating that the addition of WGYGWNGY could not inhibit the binding of COP1 to p53, and its OD value was 1.66±0.19. 450 The value was 1.59 ± 0.24, comparable to the value when p53+COP1 was used without an inhibitor. However, with the addition of the peptide LGTESNAM, the OD... 450 The value decreased, OD 450 The value was 1.13±0.33, significantly lower than that of the no-inhibitor group and the negative peptide control group (p < 0.001). This indicates that the target peptide can effectively block the specific interaction between p53 and COP1 through competitive binding, exhibiting a significant inhibitory effect. Figure 3 ).
[0093] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications 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. An inhibitory peptide targeting the p53 protein, characterized in that, The amino acid sequence is: LGTX1SX2X3M; Where X1 is selected from E or D; X2 is selected from N or A; X3 is selected from A or V.
2. The inhibitory peptide according to claim 1, characterized in that, The amino acid sequence of the inhibitory peptide is shown in SEQ ID NO:1~SEQ ID ID:
4.
3. A composition for regulating the p53-COP1 pathway, characterized in that, include: The inhibitory peptide and other polypeptides or proteins that regulate the p53-COP1 pathway as described in claim 1 or 2.
4. The composition according to claim 3, characterized in that, Other peptides that regulate the p53-COP1 pathway include p28 peptide.
5. The composition according to claim 3, characterized in that, Other proteins that regulate the p53-COP1 pathway include ASPP2, TSPYL5, and / or iASPP.
6. A formulation, characterized in that, include: Pharmaceutically acceptable excipients and at least one of the following A) to B): A) The inhibitory peptide according to claim 1 or 2; B) The composition according to any one of claims 3 to 5.
7. The formulation according to claim 6, characterized in that, Pharmaceutically acceptable excipients include: fillers, binders, disintegrants, lubricants, flow aids, coating materials, solvents and / or solubilizers.
8. Use of at least one of the following (I) to (III) in the preparation of therapeutic agents for cancers related to the p53-COP1 pathway: I) The inhibitory peptide according to claim 1 or 2; II) The composition according to any one of claims 3 to 5; III) The formulation according to claim 6 or 7.
9. The application according to claim 8, characterized in that, Cancers associated with the p53-COP1 pathway include: ovarian cancer, cervical cancer, endometrial cancer, and / or pancreatic cancer.
10. A medicament for the treatment of cancers associated with the p53-COP1 pathway, characterized in that, The raw materials include at least one of the following: i) to iv) i) The p53 protein inhibitory peptide according to claim 1 or 2; ii) The composition according to any one of claims 3 to 5; iii) The formulation according to claim 6 or 7.