Novel CD13 targeting PET molecular probe as well as preparation method and application thereof
The construction of a new CD13 probe structure through computer-aided drug design solves the problems of low affinity and poor imaging effects of existing CD13 probes, and realizes effective diagnosis and nuclide treatment in patients with negative PSMA expression in prostate cancer patients. Good imaging prospects and security.
Patent Information
- Application Number
- CN202411457865.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-18
- Publication Date
- 2025-02-11
AI Technical Summary
In the prior art, effective diagnosis and nuclide treatment cannot be performed for some patients with negative PSMA expression among prostate cancer patients, and the probe affinity for CD13 is currently low, the imaging effect is poor, and the clinical application prospects are poor.
The new CD13 probe structure was reconstructed through computer-aided drug design and radioactive and imaging verification, which improved the affinity of cyclic peptides and proteins. Six strategies were designed including N-terminal binding, C-terminal Tyr junction, and disulfide bonds. Cycloning structure, hydrogen bond between Arg and N350, hydrogen bond between Gly and A353, and hydrophobic and hydrogen bond donors of Gln.
It achieves better concentration in the tumor site, quickly cleared through renal metabolism, has good tumor microenvironment imaging prospects and high safety, and the imaging effect of the probe is better than that of traditional probes.
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Figure CN119405849A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of medical technology, and specifically refers to a novel PET molecular probe targeting CD13 and a preparation method and application thereof. Background Art
[0002] Prostate cancer membrane antigen (PSMA) is a membrane antigen specifically expressed by prostate cancer. PET molecular probes based on PSMA are currently an important tool for the diagnosis and staging of prostate cancer and radionuclide therapy. However, some prostate cancer patients have low expression of PSMA, and these patients cannot be effectively diagnosed and treated with radionuclide.
[0003] CD13 is a cell membrane glycoprotein, also known as aminopeptidase. High expression on the cell membrane is conducive to the occurrence and development of tumors. It is highly expressed in many tumors. Our research found that CD13 is highly expressed in prostate cancer and also in PSMA-negative patients with prostate cancer. Therefore, for some patients with PSMA-negative prostate cancer, PET molecular probes targeting CD13 can be used for diagnosis and radionuclide treatment. At present, the probes targeting CD13 have low affinity, poor imaging effect, and poor prospects for clinical application.
[0004] Therefore, constructing a new CD13 probe structure is of great significance. Summary of the invention
[0005] In order to solve the above technical problems, the present invention provides a novel PET molecular probe targeting CD13 and a preparation method and application thereof. Based on computer-aided drug design, a new CD13 probe structure is reconstructed and customized, and radioactivity and imaging verification are performed.
[0006] To achieve the above purpose, this scheme first provides a method for preparing a novel PET molecular probe targeting CD13, comprising the following steps:
[0007] S1. Using cCNGRCY as the peptide to be modified, cyclic peptide 1-23 was modified based on computer-aided drug design. The structural formula is shown below:
[0008]
[0009]
[0010] S2. Combine the modified cyclic peptide obtained in step S1 with the linker to form a precursor compound, add the HEPPS aqueous solution to the EP tube containing the precursor compound, mix well, and transfer to the reaction tube; rinse with 0.6M HCl 68 Ge / 68 Ga generator, add the 2-3 mL of eluent to the reaction tube for reaction, and then add deionized water for quenching;
[0011] S3, the reaction system prepared in step S2 is passed through C 18 Plus column was used for enrichment and washed with deionized water. 18 Plus column, eluting the product into a filter product bottle with ethanol and saline in turn, forming a PET molecular probe targeting CD13, with a radioactivity of about 22-28mCi;
[0012] Preferably, the radioactivity of the eluent in step S2 is 30-35 mCi.
[0013] Preferably, the reaction temperature in step S2 is 95° C. and the reaction time is 10 min.
[0014] Preferably, the radioactivity of the PET molecular probe targeting CD13 in step S3 is 21-28 mCi.
[0015] Preferably, the method based on computer-aided drug design in step S1 is:
[0016] S101. Homology modeling of cyclic peptide cCNGRCG and Homo sapiens CD13 protein: Using cyclic peptide cCNGRCG as a reference cyclic peptide, the sequence of Homo sapiens CD13 protein was obtained by searching the NCBI BLAST online website. The co-crystal structure of cCNGRCG and Susscrofa CD13 protein, PDB code 4OU3, was used as a template, and the three-dimensional structure of the complex of cCNGRCG and Homo sapiens CD13 was constructed using the SWISS-MODEL online website.
[0017] S102, cCNGRCY and Homo sapiens CD13 complex structure prediction and screening: Autodock GPU accelerated version software was used to predict the complex structure of the modified cyclic peptide cCNGRCY and Homo sapiens CD13. The Homo sapiens CD13 protein structure obtained by homology modeling was used as the receptor, the binding position of the cyclic peptide cCNGRCG was used as the docking site, and the pocket size was set with cCNGRCG as the center. The Autodock docking software uses the Lamarckian genetic algorithm and the empirical free energy scoring function to select the score and binding mode of the cyclopeptide cCNGRCY to be modified that is closest to the binding mode of the reference cyclopeptide cCNGRCG for structure-based cyclopeptide design, and the designed cyclopeptide molecules are screened using the Autodock software;
[0018] The cyclic peptide design method includes: connecting a linker at the C-terminus to maintain the activity of the cyclic peptide; utilizing thioether groups, ether groups and four-membered ring structures to stabilize the peptide conformation optimization; point mutation of amino acid Gln to Val or Tyr; point mutation of amino acid Arg to Trp; introduction of Gly into a carboxylic acid structure; multiple mutations of amino acids and extension of side chains;
[0019] S103, molecular dynamics simulation and binding free energy calculation: GROMACS software was used to simulate the binding model of different cyclic peptide molecules obtained by docking in step S102 with Homo sapiens CD13. The protein and cyclic peptide molecules were modeled using AMBER14SB force field and General AMBER Force Field parameters, respectively. The bcc charge of the cyclic peptide molecule was calculated using the ACPYPE script based on AmberTools22 suit. The system was wrapped in a cubic water box using the TIP3P water model. The system was neutralized by adding the corresponding number of chloride ions. The solvent layer thickness at the edge of the box and the solute surface was set to The particle mesh Ewald method was used to treat long-range electrostatic interactions, and the cutoff for van der Waals interactions was set to Then, the conjugate gradient method was used to minimize the system, and a short isothermal and isobaric system equilibrium was performed for 0.01ns at a time step of 1fs. During the equilibrium stage, the temperature was controlled by the V-rescale constant temperature algorithm with a coupling time constant of 0.2ps, and the pressure was controlled by the Berendsen coupling algorithm with a time constant of 0.5ps. Finally, a molecular dynamics simulation was performed for 2ns with a time step of 2fs, and the MMPBSA method was used to calculate the binding free energy of the cyclic peptide and the protein. The three-dimensional binding mode and three-dimensional map of the protein structure were generated using PyMol software, and the modified cyclic peptide compound obtained was the probe precursor structure.
[0020] Preferably, the molecular dynamics simulation in step S103 is performed by controlling and maintaining the temperature of the simulation system constant through a V-rescale constant temperature algorithm, and maintaining a constant pressure using a Berendsen coupling algorithm, and the temperature of the molecular dynamics simulation is 300K and the pressure is 1 bar.
[0021] Based on a general inventive concept, the present invention also provides a novel PET molecular probe targeting CD13, wherein the precursor compound structure of the novel PET molecular probe targeting CD13 includes the following formulas 1 and 2:
[0022]
[0023] Based on a general inventive concept, the present invention also provides an application of a novel PET molecular probe targeting CD13 in the preparation of a PET imaging agent targeting CD13.
[0024] Based on a general inventive concept, the present invention also provides a novel PET molecular probe targeting CD13 for use in evaluating the efficacy of immunotherapy for prostate cancer.
[0025] Preferably, the probe is used by injection.
[0026] The mechanism of designing cyclic peptides in this scheme is:
[0027] The N-terminus binds inside the pocket and forms a hydrogen bond salt bridge. Connecting a linker at this position will reduce the activity; the C-terminal Tyr is close to the solvent region, and an attempt is made to connect a linker there to maintain the activity of the cyclic peptide; the disulfide bond cyclized structure has a large binding conformation angle, and attempts can be made to replace other cyclized structures to stabilize the conformation of the ring; the Arg of the cyclic peptide forms a hydrogen bond with N350, and is close to R363, and there may be charge repulsion, and attempts can be made to contain hydrogen bond acceptors or aromatic amino acids to increase affinity; Gly forms a hydrogen bond with the main chain of A353, and there is space, so polar amino acids can be introduced to increase the interaction; Gln is close to E418 and V385, and attempts can be made to contain hydrophobic and hydrogen bond donor amino acids to increase the interaction between the cyclic peptide and the protein.
[0028] Compared with the prior art, the present invention has the following beneficial effects:
[0029] (1) This scheme creatively adopts six design strategies to improve the affinity of cyclic peptides and proteins: 1. The N-terminus binds inside the pocket and forms a hydrogen bond salt bridge. Connecting a linker at this position will reduce the activity. 2. The C-terminal Tyr is close to the solvent region. Try to connect the linker there to maintain the activity of the cyclic peptide. 3. The disulfide bond cyclized structure has a large binding conformation angle. You can try to replace other cyclized structures to stabilize the conformation of the ring. 4. The Arg of the cyclic peptide forms a hydrogen bond with N350, and there may be charge repulsion near R363. You can try to use amino acids containing hydrogen bond acceptors or aromatic structures to improve the affinity. 5. Gly forms a hydrogen bond with the main chain of A353, and there is space. Polar amino acids can be introduced to increase the interaction. 6. Gln is close to E418 and V385. You can try to use amino acids containing hydrophobic and hydrogen bond donors to increase the interaction between the cyclic peptide and the protein.
[0030] (2) The probe molecules obtained based on computer-aided drug design in this scheme can be well concentrated in the tumor site and can be quickly cleared through renal metabolism. They have good tumor microenvironment imaging prospects and high safety. As time goes by, the probe can still maintain a high uptake rate. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0032] Figure 1 The homology modeling results of the cCNGRCG and Homo sapiens CD13 complex in Example 1, A is the superposition of the predicted cCNGRCG and Homo sapiens CD13 complex structure and the co-crystal structure of PDB:4OU3, B is the binding mode of cCNGRCG and Homo sapiens CD13, and hydrogen bonds and salt bridges are marked with dotted lines;
[0033] Figure 2 The docking result of cCNGRCY and Homo sapiens CD13 in Example 1, hydrogen bonds and salt bridges are marked with dotted lines;
[0034] Figure 3 This is the design strategy based on the complex structure of cCNGRCY and Homo sapiens CD13 in Example 2;
[0035] Figure 4 The design based on the control cyclic peptide cCNGRCY in Example 2;
[0036] Figure 5 This is a purity detection diagram of CD13-L2 in Example 3;
[0037] Figure 6 is the mass spectrum of CD13-L2 in Example 3;
[0038] Figure 7 This is a purity detection diagram of CD13-L3 in Example 4;
[0039] Figure 8 is the mass spectrum of CD13-L3 in Example 4;
[0040] Fig. 9 The uptake of different probes in Example 5 in CD13 high-expressing cells HT29;
[0041] Fig.10 The tumor uptake of different probes in the CD13 high expression model in Example 6;
[0042] Fig.11 This is a quantitative graph of tumor uptake of different probes in Example 6 in the CD13 high expression model. DETAILED DESCRIPTION
[0043] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.
[0044] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.
[0045] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the examples are commercially available.
[0046] Example 1
[0047] Modification of cyclic peptide 1-23 based on computer-aided drug design
[0048] S1. Homology modeling of cyclic peptide cCNGRCG and Homo sapiens CD13 protein
[0049] The sequence of Homo sapiens CD13 protein was obtained by searching the NCBI BLAST online website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The co-crystal structure of cCNGRCG and Sus scrofa CD13 protein, PDB code 4OU3, was used as a template to construct the three-dimensional structure of the cCNGRCG and Homo sapiens CD13 complex using the SWISS-MODEL online website (https: / / swissmodel.expasy.org / ). The homology modeling process of the SWISS-MODEL server relies on ProMod3, an internal comparative modeling engine based on OpenStructure. ProMod3 uses the OpenMM library for calculations and uses the CHARMM22 / CMAP force field parameters.
[0050] The homology modeling results of the cCNGRCG and Homo sapiens CD13 complex are shown in Figure 1 As shown in A. The homologous template sequence identity between Homo sapiens CD13 protein and Sus scrofa CD13 protein is 79%, with high homology. The constructed protein structure has basically the same secondary structure as the homologous template. The binding mode of cCNGRCG with Homo sapiens CD13 (such as Figure 1 B) is also relatively close to Sus scrofa CD13.
[0051] S2. Structural prediction and screening of the complex between the modified cyclic peptide and Homo sapiens CD13
[0052] The complex structure of the modified cyclic peptide cCNGRCY and Homo sapiens CD13 was predicted using the Autodock GPU accelerated version. The Homo sapiens CD13 protein structure obtained by homology modeling was used as the receptor, the binding position of the cyclic peptide cCNGRCG was used as the docking site, and the pocket size was set to cCNGRCG as the center. The Autodock docking software uses the Lamarckian genetic algorithm and the empirical free energy scoring function. The receptor is set to be rigid and the ligand is set to be flexible during docking. The number of iterations (GA) set for each compound docking is 50. Finally, the score and binding mode of the modified cyclopeptide cCNGRCY are selected to be closest to the binding mode of the reference cyclopeptide cCNGRCG for structure-based cyclopeptide design. The designed cyclopeptide molecules are screened using Autodock.
[0053] Since the sequence structures of the peptides to be modified cCNGRCY and cCNGRCG are very similar, the complex structure of cCNGRCY and Homo sapiens CD13 was obtained by molecular docking. Figure 2 As shown, cCNGRCY has a flat binding mode, the N-terminus binds inside the pocket, forming hydrogen bond salt bridges with E355, E411 and Q213, the main chain of the cyclic peptide forms three hydrogen bonds with Y477 and A353, Arg forms electrostatic interactions with surrounding residues, Tyr may form hydrogen bonds with L190, the terminal carboxylic acid is close to the solvent region, forming hydrogen bonds with S469, and the disulfide bond cyclization structure stabilizes the binding conformation.
[0054] Based on the complex structure of cCNGRCY and Homo sapiens CD13, there are six design strategies as follows: Figure 3 As shown, the affinity of the cyclic peptide to the protein is improved: 1. The N-terminus binds inside the pocket and forms a hydrogen bond salt bridge. Connecting a linker at this position will reduce the activity. 2. The C-terminal Tyr is close to the solvent region. Try to connect the linker there to maintain the activity of the cyclic peptide. 3. The cyclized structure of the disulfide bond has a large binding conformation angle. You can try to replace other cyclized structures to stabilize the conformation of the ring. 4. The Arg of the cyclic peptide forms a hydrogen bond with N350, and there may be charge repulsion near R363. You can try to contain hydrogen bond acceptors or aromatic structure amino acids to improve affinity. 5. Gly forms a hydrogen bond with the main chain of A353, and there is space. Polar amino acids can be introduced to increase the interaction. 6. Gln is close to E418 and V385. You can try to contain hydrophobic and hydrogen bond donor amino acids to increase the interaction between the cyclic peptide and the protein.
[0055] S3. Molecular dynamics simulation and binding free energy calculation
[0056] The binding models of different cyclic peptide molecules and Homo sapiens CD13 obtained by docking simulation were used using GROMACS (version 2023.2). The protein and cyclic peptide molecules were modeled using the AMBER14SB force field and GeneralAMBER Force Field (GAFF) parameters, respectively. The bcc charge of the cyclic peptide molecule was calculated using the ACPYPE script based on the AmberTools22 suit. The system was wrapped in a cubic water box using the TIP3P water model. By adding the corresponding number of chloride ions (Cl - ) to neutralize the system. The solvent layer thickness at the edge of the box and the solute surface is set to The Particle meshEwald (PME) method was used to treat long-range electrostatic interactions, and the cutoff for van der Waals interactions was set to The system was then subjected to a minimization step using the conjugate gradient method to simply eliminate any unreasonable contacts between atoms. Next, a short isothermal isobaric system (NPT) equilibrium was performed for 0.01ns at a time step of 1fs. During the equilibrium stage, the temperature was controlled using the V-rescale constant temperature algorithm with a coupling time constant of 0.2ps. The pressure was controlled using the Berendsen coupling algorithm with a time constant of 0.5ps. Finally, a 2ns molecular dynamics simulation was performed using a time step of 2fs. The temperature of the simulation system was controlled and kept constant by the V-rescale constant temperature algorithm, and the pressure was kept constant using the Berendsen coupling algorithm. The simulation temperature was set to 300K and the pressure was set to 1bar. The binding free energy of the cyclic peptide to the protein was calculated using the MMPBSA method. The three-dimensional binding mode and three-dimensional diagram of the protein structure were obtained using PyMol( LLC.Version 2.5.0).
[0057] Example 2
[0058] Design simulation results of cyclic peptide 1-23
[0059] Based on the design strategy provided in Example 1, cCNGRCY was used as a reference peptide (Reference), and the designed cyclic peptides were evaluated by molecular docking and MM / GBSA, including cyclization structure modification and amino acid mutation modification. The Docking score and MM / GBSA of 10 cyclic peptides (cyclic peptides 1-10) were better than those of the reference peptide (Docking score = -9.27 kcal / mol, MM / GBSA = -50.4 ± 1.4 kcal / mol). The C-terminus was used as a linker site, and 3 different connection structures (cyclic peptides 11-13) were designed. A total of 13 cyclic peptides were designed and recommended; among them, cyclic peptides 1-4 were cyclization structure modifications, including thioether group (1), ether group (2) and four-membered ring structure (3, 4) to stabilize the binding conformation of the cyclic peptide, and the hydrophobic interaction with M354 to improve the affinity; amino acid residues (1, 2, 3, 4) were used to modify the cyclic peptide. Amino acid mutation modification, such as Gln mutation to Val (5) or Tyr (6), increases the hydrophobic interaction with V385 or the hydrogen bond with E418; Gly introduces carboxylic acid structures of different lengths (7, 8, 9) to form hydrogen bond salt bridges or electrostatic interactions with R363, R381 or N365; Arg mutation to Trp (10) forms hydrogen bond with N350 to improve affinity; linker is connected to the C-terminus (11, 12, 13), which does not affect or reduces the binding of polypeptide to protein.
[0060] The docking scores and MM / GBSA of the modifications with multiple mutated amino acids (cyclic peptides 14-18) and extended side chains (cyclic peptides 19-23) were better than those of the control cyclic peptides. The MM / GBSA of 21 was the highest, at -71.9 kcal / mol, with a total of 9 superior molecules.
[0061] The docking scores and MM / GBSA results are shown in Table 1 below:
[0062] Table 1 Docking scores and MM / GBSA results of cyclic peptides
[0063]
[0064]
[0065] Example 3
[0066] Preparation of PET molecular probe targeting CD13 68 Ga-CD13-L2
[0067] S1. Select modified cyclic peptide 19 from the computer-aided drug design structure, and design and synthesize the following PET molecular probe precursor structure CD13-L2, the structure of which is shown in Formula 1, and its purity and mass spectrum are shown in Figure 5-6 shown.
[0068]
[0069] S2. Add 2.0 mL of 500 mg / mL HEPPS aqueous solution to the EP tube containing the precursor compound CD13-L2 (20 μg), mix well, transfer the CD13-L2 solution to a 10 mL reaction tube; rinse with 4 mL of 0.6 M HCl 68 The Ga generator will 68 Ga elution, collect the 2-3 mL eluent (30-35 mCi) and add it to the reaction tube, react at 95 ° C for 10 min, and add 10 mL of deionized water to quench the reaction;
[0070] The reaction system prepared by S3 and S2 was C 18 Plus column was used for enrichment and the C 18 Plus column, use 1 mL of ethanol and 10 mL of saline to elute the product into the filter membrane product bottle to form 68 Ga-CD13-L2 product injection, radioactivity is about 22-28mCi;
[0071] The 68Ga-CD13-L2 product injection solution was subjected to HPLC purity analysis, and the purity was greater than 99%.
[0072] Example 4
[0073] Preparation of PET molecular probe targeting CD13 68 Ga-CD13-L3
[0074] S1. Select modified cyclic peptide 23 from the computer-aided drug design structure, and design and synthesize the following PET molecular probe precursor structure CD13-L3, the structure of which is shown in Formula 2, and its purity and mass spectrum are shown in Figure 7-8 As shown:
[0075]
[0076] S2. Add 2.0 mL of 500 mg / mL HEPPS aqueous solution to the EP tube containing the precursor compound CD13-L3 (20 μg) and mix well; transfer the CD13-L3 solution to a 10 mL reaction tube; rinse with 4 mL of 0.6 M HCl. 68 The Ga generator will 68 Ga elution, collect the 2-3 mL eluent (30-35 mCi), add it to the reaction tube and react at 95°C for 10 min; add 10 mL deionized water to quench the reaction;
[0077] The reaction system prepared in steps S3 and S2 is passed through C 18 Plus column was used for enrichment and the C 18Plus column, use 1 mL of ethanol and 10 mL of saline to elute the product into the filter membrane product bottle to form 68 Ga-CD13-L3 product injection, radioactivity is about 21-26mCi;
[0078] The 68Ga-CD13-L3 product injection solution was subjected to HPLC purity analysis, and the purity was greater than 99%.
[0079] Comparative Example 1
[0080] preparation 68 Ga-CD13-L1 probe
[0081] S1. Select cCNGRCY polypeptide to design and synthesize the following PET molecular probe precursor structure CD13-L1, the structure of which is shown in Formula 3:
[0082]
[0083] S2. Add 2.0 mL of 500 mg / mL HEPPS aqueous solution to the EP tube containing the precursor compound CD13-L1 (20 μg) and mix well; transfer the CD13-L1 solution to a 10 mL reaction tube; rinse with 4 mL of 0.6 M HCl. 68 The Ga generator will 68 Ga elution, collect the 2-3 mL eluent (30-35 mCi), add it to the reaction tube and react at 95°C for 10 min; add 10 mL deionized water to quench the reaction;
[0084] The reaction system prepared in steps S3 and S2 is passed through C 18 Plus column was used for enrichment and the C 18 Plus column, use 1 mL of ethanol and 10 mL of saline to elute the product into the filter membrane product bottle to form 68 Ga-CD13-L1 probe.
[0085] Example 5
[0086] Survey probe 68 Ga-CD13-L2 and 68 Characteristics of Ga-CD13-L3 uptake by cells
[0087] HT29 cells with high expression of CD13 were revived and cultured at 2×10 5Add 50uL of probe solution (0.1-0.3MBq / mL) to each tube and incubate in a 37°C, 5% CO2 incubator for 30min, 60min and 90min respectively. The blocking group was added with 10ug of probe precursor 1 hour in advance. Finally, the extracellular fluid was washed with PBS and the cells were collected for γ-counter radioactivity measurement. 68 Comparison of Ga-CD13-L1 probe.
[0088] The results are as follows Fig. 9 As shown, 68 Ga-CD13-L2, 68 Ga-CD13-L3 and 68 Compared with Ga-CD13-L1, the probe has stronger radioactivity in cells, indicating that the imaging effect is better after the modified peptide synthesized PET probe based on computer-aided drug design.
[0089] Example 6
[0090] Survey probe 68 Ga-CD13-L2 and 68 Tumor uptake of Ga-CD13-L3 in a model with high CD13 expression
[0091] S1. Take 10 C57 mice and construct a subcutaneous HT29 model. When the tumor size is about 0.5 cm, use them as model mice.
[0092] S2. Radiolabeling preparation 68 Ga-CD13-L1, 68 Ga-CD13-L2 and 68 Ga-CD13-L3 and perform quality control.
[0093] S3 and HT29 tumor models were injected into the tail vein of mice 68 Ga-CD13-L1, 68 Ga-CD13-L2 or 68 Ga-CD13-L3 (0.1-0.2 mCi) was used for dynamic scanning, and the data was analyzed to obtain the SUV value of the tumor site at 30-90 min. 68 Ga-CD13-L1, 68 Ga-CD13-L2 and 68 The tumor uptake SUV values in the Ga-CD13-L3 HT29 model were 1.0±0.3, 3.1±0.3, and 2.2±0.3, respectively.
[0094] Results 10-11 show that 68 Ga-CD13-L2 or 68Ga-CD13-L3 can be well concentrated in the tumor site and can be quickly metabolized and cleared by the kidneys. 68 The tumor uptake of Ga-CD13-L1 is significantly improved, with good tumor microenvironment imaging prospects and high safety. 68 Ga-CD13-L2 and 68 Ga-CD13-L3 compares 68 The imaging of Ga-CD13-L1 was also better.
[0095] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. For those skilled in the art, improvements and changes obtained without departing from the technical concept of the present invention should also be regarded as the protection scope of the present invention.
Claims
1. A method for preparing a novel PET molecular probe targeting CD13, characterized in that: The following steps are involved: S1. Using cCNGRCY as the peptide to be modified, cyclic peptide 1-23 was modified based on computer-aided drug design. The structural formula is shown below: S2, combining the modified cyclic peptide obtained in step S1 with the linker to form a precursor compound, adding the HEPPS aqueous solution to the EP tube containing the precursor compound, mixing, and transferring to the reaction tube; Elute with 0.6M HCl 68 Ge / 68 Ga generator, add the 2-3 mL of eluent to the reaction tube for reaction, and then add deionized water for quenching; S3, the reaction system prepared in step S2 is passed through C 18 Plus column was used for enrichment and washed with deionized water. 18 Plus column, and the product is sequentially eluted with ethanol and saline into a filter membrane product bottle to form a PET molecular probe targeting CD13 with a radioactivity of approximately 22-28 mCi.
2. The preparation method according to claim 1, characterized in that: The radioactivity of the eluent in step S2 is 30-35 mCi.
3. The preparation method according to claim 1, characterized in that: The reaction temperature in step S2 is 95° C. and the reaction time is 10 min.
4. The preparation method according to claim 1, characterized in that: The radioactivity of the PET molecular probe targeting CD13 in the S3 step is 21-28 mCi.
5. The preparation method according to claim 1, characterized in that: The method based on computer-aided drug design in step S1 is: S101. Homology modeling of cyclic peptide cCNGRCG and Homo sapiens CD13 protein: Using cyclic peptide cCNGRCG as a reference cyclic peptide, the sequence of Homo sapiens CD13 protein was obtained by searching the NCBI BLAST online website. The co-crystal structure of cCNGRCG and Susscrofa CD13 protein, PDB code 4OU3, was used as a template, and the three-dimensional structure of the complex of cCNGRCG and Homo sapiens CD13 was constructed using the SWISS-MODEL online website. S102, cCNGRCY and Homo sapiens CD13 complex structure prediction and screening: Autodock GPU accelerated version software was used to predict the complex structure of the modified cyclic peptide cCNGRCY and Homo sapiens CD13. The Homo sapiens CD13 protein structure obtained by homology modeling was used as the receptor, the binding position of the cyclic peptide cCNGRCG was used as the docking site, and the pocket size was set with cCNGRCG as the center. The Autodock docking software uses the Lamarckian genetic algorithm and the empirical free energy scoring function to select the score and binding mode of the cyclopeptide cCNGRCY to be modified that is closest to the binding mode of the reference cyclopeptide cCNGRCG for structure-based cyclopeptide design, and the designed cyclopeptide molecules are screened using the Autodock software; The cyclic peptide design method includes: connecting a linker at the C-terminus to maintain the activity of the cyclic peptide; utilizing thioether groups, ether groups and four-membered ring structures to stabilize the peptide conformation optimization; point mutation of amino acid Gln to Val or Tyr; point mutation of amino acid Arg to Trp; introduction of Gly into a carboxylic acid structure; multiple mutations of amino acids and extension of side chains; S103, molecular dynamics simulation and binding free energy calculation: GROMACS software was used to simulate the binding model of different cyclic peptide molecules obtained by docking in step S102 with Homo sapiens CD13. The protein and cyclic peptide molecules were modeled using AMBER14SB force field and General AMBER Force Field parameters, respectively. The bcc charge of the cyclic peptide molecule was calculated using the ACPYPE script based on AmberTools22 suit. The system was wrapped in a cubic water box using the TIP3P water model. The system was neutralized by adding the corresponding number of chloride ions. The solvent layer thickness at the edge of the box and the solute surface was set to The particle mesh Ewald method was used to treat long-range electrostatic interactions, and the cutoff for van der Waals interactions was set to The system was then minimized using the conjugate gradient method, and a short isothermal and isobaric ensemble equilibrium was performed for 0.01 ns at a time step of 1 fs. During the equilibrium stage, the temperature was controlled by the V-rescale constant temperature algorithm with a coupling time constant of 0.2 ps, and the pressure was controlled by the Berendsen coupling algorithm with a time constant of 0.5 ps. Finally, a molecular dynamics simulation was performed for 2 ns with a time step of 2 fs, and the binding free energy of the cyclic peptide and protein was calculated using the MMPBSA method. The three-dimensional binding mode and three-dimensional map of the protein structure were generated using PyMol software, and the modified cyclic peptide compound obtained was the probe precursor structure.
6. The preparation method according to claim 5, characterized in that: The molecular dynamics simulation in step S103 is controlled and maintained at a constant temperature of the simulation system by a V-rescale constant temperature algorithm, and a constant pressure is maintained by a Berendsen coupling algorithm. The temperature of the molecular dynamics simulation is 300K and the pressure is 1 bar.
7. A novel PET molecular probe targeting CD13 obtained by the preparation method according to any one of claims 1 to 6, characterized in that: The precursor compound structure of the novel CD13-targeting PET molecular probe includes the following formulas 1 and 2:
8. Use of the novel PET molecular probe targeting CD13 obtained by the preparation method according to any one of claims 1 to 6 or the novel PET molecular probe targeting CD13 according to claim 7 in the preparation of a PET imaging agent targeting CD13.
9. Use of a novel PET molecular probe targeting CD13 obtained by the preparation method according to any one of claims 1 to 6 or a novel PET molecular probe targeting CD13 according to claim 7 in evaluating the effect of immunotherapy for prostate cancer.
10. The use according to claim 8, characterized in that: The probe is used by injection.