Drugs that block cd47 / sirpα interaction

Abstract

The present application relates to the field of biotechnology, in particular to a drug for blocking CD47 / SIRP alpha interaction. The polypeptide inhibitor of SIRP alpha is obtained by screening, and the amino acid sequence is ZLX1RTLX2EX3Y, wherein ZLIRTLHEWY can produce more effective inhibitory effect on the binding of SIRP alpha and CD4.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a drug for blocking CD47 / SIRPα interaction. BACKGROUND

[0002] CD47 is a common transmembrane protein widely present in tissues. Current studies have shown that CD47 is overexpressed in most tumor cells, which can bind to the transmembrane signal protein SIRPα on the surface of macrophages to form a CD47-SIRPα complex. Macrophages recognize the "don't eat me" signal released by tumor cells, and through intracellular signal transduction, phagocytosis is prevented, ultimately leading to immune escape of malignant tumor cells.

[0003] There are currently various effective means of cancer treatment, mainly including chemotherapy, radiotherapy, immunotherapy, etc., but they will produce certain side effects after use. Literature reports that the immune escape phenomenon of tumor cells is a new target for immunotherapy, and by inhibiting the binding of CD47 and SIRPα, the immune escape of tumor cells can be prevented. Studies have shown that CD47 inhibition can stimulate the phagocytic function of macrophages on cancer cells. Anti-CD47 antibodies can enhance the phagocytosis of macrophages on tumor cells by directly blocking the binding of CD47-expressing tumor cells to SIRPα-expressing macrophages. Treatment of mice with a specific chemical substance (chloromethylphosphonate) that depletes macrophages can eliminate the anti-tumor effect of CD47 blockers, supporting the role of macrophages in anti-CD47-mediated anti-tumor response. Other studies have shown that anti-CD47 antibodies can promote the phagocytic activity of macrophages in mice transplanted with human NHL cells. Treatment of NHL mice with anti-CD47 antibodies and blinatumomab (targeting CD19 and CD3); this combination therapy sustained control of lymphoma progression by inducing cancer cell phagocytosis and T cell toxicity. In a mouse colon cancer model, anti-SIRPα antibody treatment can enhance macrophage phagocytic activity and reduce tumor progression, and CD47-SIRPα signaling can promote the proliferation and metastasis of colon cancer cells in a tumor microenvironment rich in tumor-associated macrophages. It can be seen that blocking the binding of CD47 and SIRPα can effectively prevent tumor cell immune escape, but the current inhibitor still needs to improve its binding ability to SIRPα.

[0004] Biomimetic polypeptide inhibitors have high affinity, high stability, not easy to degrade and simple preparation, and have important development prospects. In recent years, with the rapid development of molecular simulation technology, it is possible to design and screen high specificity polypeptide inhibitors by simulating the interaction of existing target protein-antibody, and the screening efficiency and accuracy are also continuously improved. Although virtual screening can quickly and accurately enrich and screen polypeptide inhibitors with high affinity, due to the limitations of computer simulation (such as different results may be obtained by using different software or using different parameters), the prediction of the interaction between biomolecules cannot be exactly the same as the actual situation, and in the actual screening process, polypeptide inhibitors with high affinity and specificity may be missed, so it cannot be ruled out that the remaining polypeptide inhibitors in the polypeptide inhibitor library may also be effective polypeptide inhibitors of SIRPα. Therefore, further research and development of more effective drugs for blocking the interaction of CD47 / SIRPα is still a problem to be solved in the art. SUMMARY

[0005] Therefore, the technical problem to be solved by the present application is to provide a drug for blocking the interaction of CD47 / SIRPα.

[0006] The polypeptide amino acid sequence provided by the present application is ZLX1RTLX2EX3Y;

[0007] wherein Z is pyroglutamic acid; X1, X2, X3 are independently selected from any one or several of G, A, V, L, I, P, F, Y, W, S, T, C, M, N, Q, D, E, K, R or H.

[0008] In the embodiments of the present application,

[0009] X1 is G, F, I, N, M, O, P, T, Y or W;

[0010] X2 is W, H, P, Y or T;

[0011] X3 is H, W or G.

[0012] In some embodiments,

[0013] X1 is G, X3 is H, and X2 is W;

[0014] or X1 is F, X3 is W, and X2 is H;

[0015] or X1 is I, X3 is W, and X2 is P or H;

[0016] or X1 is N, X3 is W, and X2 is W;

[0017] or X1 is M, X3 is W, and X2 is H;

[0018] or X1 is O, X3 is W, X2 is W;

[0019] or X1 is P, X3 is W, X2 is T or H;

[0020] or X1 is T, X3 is W, X2 is P;

[0021] or X1 is W, X3 is W or G, X2 is H or Y;

[0022] or X1 is Y, X3 is W, X2 is P.

[0023] In some embodiments, the polypeptide provided by the present application is:

[0024] ZLGRTLWEHY, ZLFRTLHEWY, ZLIRTLPEWY, ZLIRTLHEWY,

[0025] ZLNRTLWEWY, ZLMRTLHEWY, ZLQRTLWEWY, ZLPRTLTEWY, ZLPRTLHEWY, ZLTRTLPEWY, ZLWRTLHEWY, ZLWRTLYEWY,

[0026] ZLYRTLPEWY or ZLWRTLWEGY.

[0027] The polypeptide provided by the present application can bind at the CD47 affinity site on the surface of SIRPα. Through ELISA experiments, it is proved that the above polypeptide has good binding performance with SIRPα, which indicates that the polypeptide of the present application has high affinity and good inhibitory effect on the interaction of CD47 / SIRPα. The present application designs 24 polypeptides, 19 of which have strong hydrophilicity. Through further test verification, the polypeptide ZLIRTLHEWY is finally screened, which has more advantages in inhibiting the effect of SIRPα and CD47.

[0028] Further, the present application provides the use of the polypeptide in the preparation of a drug for blocking the interaction of CD47 / SIRPα.

[0029] In the present application, the blocking of the interaction of CD47 / SIRPα is the spontaneous binding of the polypeptide as described above to SIRPα.

[0030] In some embodiments, the SIRPα includes SIRPαV1 and SIRPαV2.

[0031] Further, the present application also provides the use of the polypeptide in the preparation of an anti-tumor drug.

[0032] In the present application, the tumor includes bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, central nervous system cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastrointestinal cancer, genital cancer, genitourinary tract cancer, head cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, muscle tissue cancer, neck cancer, oral or nasal mucosa cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, spleen cancer, small intestine cancer, large intestine cancer, stomach cancer, testicular cancer and / or thyroid cancer.

[0033] The present application also provides an anti-tumor drug comprising the polypeptide as described above and a pharmaceutically acceptable excipient.

[0034] The polypeptide inhibitor of SIRPα is obtained by screening, and the amino acid sequence is ZLX1RTLX2EX3Y, wherein ZLIRTLHEWY can produce more effective inhibition on the binding of SIRPα and CD4. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 Binding free energy of CD47-SIRPαV1 key residues;

[0036] Figure 2 Binding free energy of CD47-SIRPαV2 key residues;

[0037] Figure 3 Simplified affinity model of CD47;

[0038] Figure 4 Binding of ZLIRTLHEWY to SIRPα (a figure is SIRPαV1, and b figure is SIRPαV2);

[0039] Figure 5 Binding of ZLIRTLHEWY to CD47-SIRPα (a figure is SIRPαV1, and b figure is SIRPαV2);

[0040] Figure 6 Double antibody sandwich ELISA experiment of polypeptide inhibitor. DETAILED DESCRIPTION

[0041] The present application provides a drug for blocking CD47 / SIRPα interaction, and those skilled in the art can refer to the content herein, and appropriately improve the process parameters to realize. It is particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are all regarded as included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the method and application herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0042] In the present application, the term "polypeptide" or "peptide" has its ordinary meaning in the art and can refer to an amide from two or more amino carboxylic acid molecules (the same or different) formed by a covalent bond formally losing water from the carbonyl carbon of one amino carboxylic acid molecule and the nitrogen atom of another amino carboxylic acid molecule. The term "amino acid residue" also has its ordinary meaning in the art and refers to the composition of an amino acid (as a single amino acid or as part of a peptide) after it is combined with a peptide, another amino acid or amino acid residue. Generally, when an amino acid is combined with another amino acid or amino acid residue, water is removed, and the remaining amino acid is referred to as an amino acid residue. The term "amino acid" also has its ordinary meaning in the art and can include proteinogenic and non-proteinogenic amino acids. The abbreviations of amino acid residues in the present application are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. Among them, X represents any one of the 20 commonly used L-amino acids.

[0043] In the present application, the term "and / or", which describes the association relationship of the associated objects, means that there can be three kinds of relationships, for example, A and / or B can mean: A exists alone, A and B exist together, and B exists alone. Wherein A, B can be singular or plural.

[0044] In the present application, "at least one" means one or more, and "a plurality of" means two or more. "At least one of the following" or the like means any combination of the items, including any combination of single item or multiple items.

[0045] It should be understood that in various embodiments of the present application, the size of the sequence number of the above-mentioned processes does not mean the order of execution, and part or all of the steps can be executed in parallel or in sequence, and the execution order of the processes should be determined according to its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0046] The polypeptide amino acid sequence provided by the present application is ZLX1RTLX2EX3Y, and on this basis, it can be further optimized, such as cyclic optimization and D-type amino acid replacement, etc. to improve the practical application ability of the inhibitor.

[0047] The polypeptide inhibitor of SIRPα and the design and screening method thereof of the present application are based on the crystal structure of CD47 and SIRPα complex, and a polypeptide inhibitor library of SIRPα is constructed, and the characteristic sequence is ZLXRTLXEXY (wherein Z represents pyroglutamic acid, and X represents 20 common amino acids).

[0048] The molecular mechanism of CD47-SIRPα complex was analyzed by molecular dynamics simulation and Poisson-Boltzmann solvent accessible surface area calculation. It was determined that hydrophobic interaction dominated the binding process, and the key residues in CD47 that contributed to the binding were Z1, L2, E35, Y37, E97, L101, and T102.

[0049] Considering the spatial distance of the sites, the amino acid E97, which was far away from other residues, was deleted, and the amino acid R103, which was close to other residues and contributed strongly, was added. In summary, the polypeptide inhibitor library was constructed based on the key residues of CD47: Z1, L2, E35, Y37, L101, T102, and R103.

[0050] The effective polypeptide inhibitors of SIRPα were screened by molecular docking screening, root mean square deviation comparison, hydrophobic residue analysis, conformation alignment, and molecular dynamics simulation combined with free energy calculation.

[0051] The polypeptide inhibitors in the polypeptide inhibitor library were docked with SIRPα in turn using the AUTODOCK VINA1.1.2 molecular docking software. During the docking process, SIRPα was the receptor and the polypeptide inhibitor was the ligand. The binding effect was evaluated by scoring, and 24 inhibitors with a binding free energy lower than -8 kcal / mol were selected.

[0052] The root mean square deviation (RMSD) between the 24 inhibitors obtained by docking and the corresponding key residues in SIRPα was calculated using the g_rms program included in the GROMACS molecular simulation software. Inhibitors with an RMSD less than 0.4 nm were selected, totaling 24.

[0053] The hydrophilic and hydrophobic properties of each residue in the inhibitor sequence were analyzed, and inhibitors containing more hydrophobic amino acids were deleted. Finally, 19 more hydrophilic polypeptide inhibitors were selected.

[0054] The VMD software was used to investigate whether the binding site of the inhibitor was consistent with the binding site of CD47 on SIRPα, i.e., whether the candidate inhibitor was bound to the CD47 affinity site on the surface of SIRPα. Fourteen inhibitors were selected for the next step of MD simulation. The 14 polypeptide inhibitors selected were subjected to MD simulation and inhibition effect verification, and ZLIRTLHEWY with high affinity and good inhibition effect was obtained.

[0055] The polypeptide preparation method provided by this invention can employ well-known polypeptide preparation methods in the art, such as solid-phase synthesis, liquid-phase synthesis, or solid-liquid combined synthesis; this invention is not limited in this regard. The polypeptide can be synthesized by amino acid-by-amino acid coupling or by segmental synthesis; this invention is also not limited in this regard. In solid-phase synthesis or solid-liquid combined synthesis methods, resins that can be selected include Wang resin, CTC resin, Merrifield resin, benzyl alcohol resin, AM resin, MBHA resin, triphenylmethyl chloride resin (2-CTC), Rink acid resin, Rink amide resin, Rink amide-MBHA, or sulfonyl chloride resin.

[0056] In this invention, "coupling" refers to the process of adding a new amino acid to a bound amino acid or peptide. The preparation method described in this invention improves the coupling step for sulfoalanine, while coupling of other amino acids is performed using methods well-known in the art. The coupling agents include two types: coupling agent A and coupling agent B, where coupling agent A is HOBt or HOAt, and coupling agent B is DIEA or NMM.

[0057] In this invention, during the coupling process, the amino acid can be protected at its terminal group or its side chain, and the protecting group can be any protecting group well known in the art. The coupling process also includes a deprotection step, wherein the deprotection agent includes trifluoroacetic acid, and the deprotection resin and other side chain protecting groups are at least one selected from redistilled water, triethylsilane, 1,2-dithiothreitol, phenol, anisole, ammonium iodide, triisopropylsilane, anisole, and ethylenedithiol.

[0058] This invention presents a method for obtaining and validating peptide inhibitors of SIRPα using molecular simulation. The steps and parameters involved in this method are reasonable and appropriate, thus enabling the acquisition of peptides with good binding affinity to SIRPα. In contrast, when other parameters are used, the designed peptides often fail to inhibit the binding of CD47 to SIRPα.

[0059] 1. Molecular dynamics simulations and the Poisson-Boltzmann surface area (MM-PBSA) method were used to calculate the binding free energy of the extracellular V-type Ig-like domain complex of two variants (V1 and V2) of the macrophage surface signaling protein SIRPα with the N-terminal extracellular domain of CD47 (crystal structures derived from PDB: 4CMM and 2JJS). The binding mechanism between SIRPα and CD47 was resolved using free energy decomposition, and hotspot residues of SIRPα were identified. Based on the molecular mechanism and residue spatial arrangement characteristics obtained from the above analysis, a simplified affinity binding model of CD47 was constructed.

[0060] 2. Based on the conformation and relative position of hotspot residues (Z1, L2, E35, Y37, L101, T102, R103) of CD47, the polypeptide sequence pattern was determined to be ZLXRTLXEXY using amino acid localization method, where Z represents pyroglutamic acid and X represents 20 common amino acids; a polypeptide inhibitor library was constructed using a self-developed program.

[0061] 3. Using Autodoc VINA 1.1.2 molecular docking software, inhibitors from the peptide inhibitor library were sequentially docked with SIRPα. Based on the score distribution, 24 peptides with binding free energy (score) below -8 kcal / mol were selected. The root mean square deviation (RMSD) between the hotspot residues of CD47 and the corresponding hotspot residues in the 24 docked peptide sequences was calculated using the g_rms program included in the GROMACS 5.1.4 software package. This allowed for comparison of the conformational differences between the hotspot residues in the docked peptides and those in CD47. A smaller RMSD value indicates a closer similarity between the docking conformation of the hotspot residues in the peptide inhibitor and those in CD47. Based on the RMSD value distribution, 24 peptides with RMSD < 0.4 nm were selected for further analysis. The hydrophilicity and hydrophobicity of each amino acid residue in the peptide inhibitor sequences were analyzed, and peptide inhibitors containing more hydrophobic amino acids were removed, finally selecting 19 relatively hydrophilic peptide inhibitors. VMD software was used to observe whether the binding sites of peptide inhibitors were consistent with the binding sites of CD47 on SIRPα. Fourteen peptides were selected for MD simulation and subsequent verification of inhibitory effects.

[0062] 4. Using the conformations of the 14 peptides obtained from VINA docking in step 3 with SIRPα as the initial Com conformation, the minimum distance between the peptides and SIRPα was adjusted to 1.4-1.5 nm using the g_editconf command provided with GROMACS, resulting in the Sep conformation. Using the GROMACS 5.1.4 software package, the GROMOS96 43a1 force field was selected. The peptide inhibitor and SIRPα (V1 or V2) complex was placed at the center of a cubic box, with the cubic box size for the peptide inhibitor and SIRPαV1 protein being 7.5 × 8 × 11.5 nm. 3 The cube box size used for the peptide inhibitor and SIRPαV2 protein is 8.5 × 9.5 × 10.5 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; then, a 100 ps canonical (NVT) ensemble-constrained kinetic equilibrium was conducted; finally, a 100 ns MD simulation was performed to observe the minimum distance d between the peptide inhibitor and SIRPα. min LJ potential energy (E) LJ Coulomb potential energy (E) C The simulation results showed that ZLIRTLHEWY exhibits high affinity.

[0063] 5. To validate the inhibitory effect of ZLIRTLHEWY, MD simulations of the CD47-SIRPα-peptide inhibitor ternary system were used to investigate the binding of the inhibitor to SIRPα in the co-existence of the peptide inhibitor and CD47, in order to evaluate the inhibitory effect of the peptide inhibitor. A 100ns NVT ensemble simulation was used to calculate the RMSD of the peptide inhibitor and the minimum intermolecular distance d. min LJ potential energy (E) LJ Coulomb potential energy (E) C To assess the intermolecular binding process.

[0064] 6. A double-antibody sandwich ELISA assay was performed on ZLIRTLHEWY to verify its binding performance with SIRPα. The blank control group was CBS buffer solution, the negative control group was the peptide sequence WGYGWNGY that does not react with SIRPα, and the positive control group was the mouse monoclonal antibody coated in the original ELISA kit. First, a 100 μg / mL peptide inhibitor was prepared using 0.05 M CBS (1.59 g Na2CO3 and 2.94 g NaHCO3 dissolved in 1 L deionized water). 100 μL of the peptide inhibitor was added to a 96-well ELISA plate, 100 μL of CBS was added as the blank group, and 100 μL of the peptide WGYGWNGY that does not react with SIRPα was added as the negative control group. The samples were coated at 4 °C for 12 h. The non-adhesive liquid was discarded, and the ELISA plate was patted dry on absorbent paper. Second, a 50 mg / mL BSA was prepared using 0.05 M CBS. 200 μL of BSA was added to each well, and the plate was blocked at 37 °C for 2 h. Discard any non-adhesive liquid and pat the ELISA plate dry on absorbent paper. Third, prepare a 5 ng / mL SIRPα standard using the universal diluent from the ELISA kit. Add 100 μL of SIRPα to each well, along with wells pre-coated with mouse monoclonal antibody containing SIRPα. Cover with a sealing film and incubate at 37°C for 1 hour. Discard any non-adhesive liquid and pat the ELISA plate dry on absorbent paper. Fourth, add 100 μL of biotinylated antibody working solution to each well, cover with a sealing film, and incubate at 37°C for 1 hour. Discard the liquid and wash the plate three times with washing buffer. Fifth, add 100 μL of enzyme conjugate working solution to each well, cover with a sealing film, and incubate at 37°C for 0.5 hours. Discard the liquid and wash the plate five times with washing buffer. Sixth, add 90 μL of substrate (TMB) to each well, cover with a sealing film, and incubate at 37°C in the dark for 15 minutes.

[0065] Finally, 50 μL of stop solution was added to each well, and the OD value of each well was immediately measured at 450 nm using a multi-functional microplate reader. All samples were measured in triplicate, and the average value was used for discussion. The results showed that ZLIRTLHEWY had the strongest binding affinity to SIRPα.

[0066] The results show that ZLIRTLHEWY can bind to SIRPα in the presence of both CD47 and SIRPα, while inhibiting the binding of CD47. This indicates that ZLIRTLHEWY has a stronger SIRPα protein affinity than CD47, and also confirms the feasibility of the biomimetic design process of the peptide inhibitor described in this invention.

[0067] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0068] The present invention will be further illustrated below with reference to the embodiments:

[0069] Example 1: Analysis of the interaction mechanism between SIRPα and CD47 and construction of an affinity model

[0070] The crystal structures of the extracellular V-type Ig-like domain complex of two variants (V1 and V2) of the macrophage surface signaling protein SIRPα with the N-terminal extracellular domain of CD47 used in this study were obtained from the PDB database (http: / / www.rcsb.org / pdb / , IDs: 4CMM and 2JJS). Molecular dynamics simulations were performed using GROMACS 5.1.4 and GROMOS96 43a1 all-atom force field. The simulation environment was a physiological saline solution with a net charge through a counterion balance system, where water molecules were modeled using the SPC model. The box size of the SIRPαV1-CD47 complex simulation system was 11 nm × 10 nm × 12 nm, containing 41,766 water molecules and 119 Na+ molecules. + and 120 Cl - The SIRPαV2-CD47 complex simulation system has a box size of 11nm × 12nm × 13nm and contains 53,690 water molecules and 161 Na+ molecules. + and 155 Cl - A 100 ns canonical (NVT) ensemble molecular dynamics simulation was performed on the simulated system. The system temperature was maintained at 310.15 K using the velocity-rescale method. The LINCS (Linear Constraint Solver) algorithm was used to constrain the vibrations of covalent bonds and bond angles. Three-dimensional periodic boundary conditions were used. Electrostatic interactions were handled using the PME algorithm. The adjacent atom cutoff distance, Coulomb cutoff distance, and LJ potential energy cutoff distance were all set to 1.8 nm. The initial particle velocities were obtained based on the Maxwell distribution at 310.15 K. The Verlet algorithm was used for the simulation, with an integration step size of 2 fs. Each system underwent energy minimization before the simulation began.

[0071] The binding free energy of CD47 to SIRPα was calculated using MM-PBSA free energy decomposition. In the SIRPαV1-CD47 complex simulation system, ΔG... elec = -2517 kJ / mol, ΔG vdW = -396kJ / mol, ΔG SASA = -41 kJ / mol, indicating that electrostatic interactions, van der Waals interactions, and nonpolar solvation effects all play a favorable role in binding. ΔG PB =2408 kJ / mol, indicating that the electrostatic solvation effect has a detrimental effect on binding. Based on the free energy calculation equation, ΔG polar = -109 kJ / mol, ΔG nonpolar= -437kJ / mol, final binding free energy ΔG bind = -546 kJ / mol. In the SIRPαV2-CD47 complex simulation system, ΔG elec = -4892kJ / mol, ΔG vdW = -1181kJ / mol, ΔG SASA =-122kJ / mol, indicating that electrostatic interactions, van der Waals interactions, and nonpolar solvation effects all play a favorable role in binding. ΔG PB =4820 kJ / mol, indicating that the electrostatic solvation effect has a detrimental effect on binding. Based on the free energy calculation equation, ΔG polar = -72kJ / mol, ΔG nonpolar = -1303 kJ / mol, final binding free energy ΔG bind = -1375 kJ / mol. In summary, this indicates that the binding of the two is mainly driven by hydrophobic interactions.

[0072] Hotspot residues were identified by decomposing the free energy of SIRPα and CD47. Hotspot residues were defined as residues that contributed significantly to the binding free energy. A standard of ±2.5 kcal / mol was used to identify residues that contributed significantly to the free energy; free energies ≤ -2.5 kcal / mol were considered to have a strong beneficial contribution to binding, while free energies ≥ 2.5 kcal / mol were considered to have a strong detrimental contribution to binding. Figure 1 , 2 As shown, the calculated favorable hotspot residues in SIRPαV1 are L30, V33, G34, P35, Q37, N51, Q52, K53, R69, K96, and S98; the favorable hotspot residues in SIRPαV2 are V33, P35, Q52, K53, E54, E65, T67, R69, K96, and S98; and the favorable hotspot residues in CD47 are Z1, L2, E35, Y37, E97, L101, and T102.

[0073] Taking into account both site distance and free energy contribution, E97 was removed, R103 was added, and Z1, L2, E35, Y37, L101, T102, and R103 were selected to construct an affinity model.

[0074] Example 2 Construction of a peptide inhibitor library

[0075] It is known that the distance between amino acid residues in a protein is approximately The number of residues to be inserted is determined by the actual distance between the two hotspot residues to be connected. For example... Figure 3As shown, the distance between each residue was calculated using VMD, and the corresponding number of amino acids were inserted to obtain the peptide inhibitor characteristic sequence ZLXRTLXEXY, where Z represents pyroglutamic acid and X represents 20 common amino acids. A peptide inhibitor library was generated using a self-written script.

[0076] Example 3: Docking of peptide inhibitors with SIRPα

[0077] 1. VINA Interoperability

[0078] The peptide inhibitor was sequentially docked with SIRPα using Autodoc VINA. The docking results showed that the peptide inhibitor binding score (E) was high. VINA The free energy distribution ranged from -9 to -3.9 kcal / mol, indicating that all peptide inhibitors could spontaneously bind to SIRPα. Using a binding free energy ≤ -8 kcal / mol as the standard, 24 peptide inhibitors were obtained for further screening.

[0079] 2. RMSD Calculation

[0080] The RMSD values ​​between the peptide inhibitors and key CD47 residues were calculated using the GROMACS program to determine their structural similarity, i.e., to examine the inhibitors' ability to mimic key CD47 sites. The results showed that the RMSD values ​​were <0.07 nm, indicating that the conformations of the peptide inhibitors were highly stable during static binding. The hydrophilicity and hydrophobicity of each amino acid residue in the peptide inhibitor sequence were analyzed, and peptide inhibitors containing more hydrophobic amino acids were removed. Finally, 19 relatively hydrophilic peptide inhibitors were selected.

[0081] 3. Conformational comparison

[0082] Using VMD software for conformational comparison and binding site analysis, 14 peptide inhibitors were screened out.

[0083]

[0084]

[0085] Example 4: Molecular Dynamics (MD) Simulation

[0086] To more accurately predict the affinity between SIRPα and peptide inhibitors using computer-aided design, we examined the binding of 14 previously screened peptide inhibitors using more precise but time-consuming molecular dynamics simulations. The molecular dynamics simulation parameters were the same as in Example 1. The binding of the peptide inhibitor ZLIRTLHEWY, which showed the best binding affinity with SIRPα, is shown below. Figure 4As shown, the minimum distance between ZLIRTLHEWY and SIRPαV1 in the Sep system reaches its minimum value at 1.5 ns, which is consistent with the minimum distance value in the Com system. At this time, van der Waals forces E0 are also observed. LJ and Coulomb potential energy E C The corresponding decrease in the binding force was observed, and the ZLIRTLHEWY and SIRPαV1 complex remained in a stable binding state until 100 ns. In the Sep system of ZLIRTLHEWY and SIRPαV2, the complex achieved stable binding at 6.5 ns, and the minimum distance coincided with the minimum distance trajectory in the Com system after 6.5 ns. At the same time, van der Waals forces were also observed. LJ and Coulomb potential energy E C The significant reduction in [something] was observed. All of the above results confirm the rapid and stable binding of ZLIRTLHEWY to SIRPα (V1 and V2).

[0087] Example 5: Verification of Inhibition Effect

[0088] MD simulations were performed on the ternary system of CD47-SIRPα-peptide inhibitors, and the results are as follows: Figure 5 As shown in the diagram, in simulation system 1, the minimum distance between ZLIRTLHEWY and SIRPαV1 is similar to that between CD47 and SIRPαV1 at 0 ns. Then, the minimum distance between ZLIRTLHEWY and SIRPαV1 decreases rapidly, while the minimum distance between CD47 and SIRPαV1 begins to increase. At 1.4 ns, the minimum distance between ZLIRTLHEWY and SIRPαV1 decreases to 0.1 nm, and then fluctuates around this value, representing a stable binding between ZLIRTLHEWY and SIRPαV1. Meanwhile, the minimum distance between CD47 and SIRPαV1 gradually increases to 4 nm and fluctuates around this distance. Although they approach each other around 50 ns, they quickly separate within 1 ns, indicating that CD47 cannot bind to SIRPαV1 in the presence of ZLIRTLHEWY. A similar analysis can be performed in simulation system 2. In summary, these results confirm that ZLIRTLHEWY can bind to SIRPα and effectively and rapidly inhibit the binding of CD47 to SIRPα.

[0089] Example 6: Verification through effect experiments

[0090] A double-antibody sandwich ELISA assay was performed on ZLIRTLHEWY, and the results are as follows: Figure 6 As shown.

[0091] OD of CBS in the blank control group 450 It is very low, at 0.2276 ± 0.0101.

[0092] The negative control group consisted of peptide sequences WGYGWNGY and OD that did not react with SIRPα. 450 It is 0.5834±0.0169.

[0093] The positive control group consisted of mouse monoclonal antibodies coated in the original ELISA kit, with OD... 450 It is 2.0849±0.0818.

[0094] OD of ZLIRTLHEWY in peptide inhibitors 450 The highest value was 5.2629 ± 0.1669, indicating the strongest binding ability with SIRPα.

[0095] This invention proposes a peptide inhibitor for SIRPα and its design and screening method. The screened inhibitor ZLIRTLHEWY has been experimentally verified as an effective peptide inhibitor for SIRPα. Further optimization of the peptide inhibitor ZLIRTLHEWY, such as cyclic optimization and D-type amino acid substitution, can improve its practical application.

[0096] 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. A polypeptide with the amino acid sequence ZLIRTLHEWY.

2. The use of the polypeptide according to claim 1 in the preparation of an antitumor drug; The tumor is selected from bladder cancer, leukemia, bone cancer, breast cancer, central nervous system cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, gallbladder cancer, head cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, muscle tissue cancer, cervical cancer, oral or nasal mucosal cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, spleen cancer, small intestine cancer, large intestine cancer, stomach cancer, testicular cancer, and / or thyroid cancer.

3. An antitumor drug, characterized in that, It includes the polypeptide of claim 1 and pharmaceutically acceptable excipients.

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