A series of trop-2 protein targeting polypeptides

Abstract

The application belongs to the field of polypeptides and particularly relates to a series of TROP-2 protein targeting polypeptides, the polypeptides are designed by computer simulated virtual molecular docking technology, and the sequences are FTTEVV, LATQII, LTAEIV, FTAEII, FATEII, IYKLF, IYQFF, VDQFL, INKFL and VYQFF. The polypeptide preparation method adopts Fmoc solid-phase synthesis method, the synthesis method is low in cost, simple in reaction operation and beneficial to realize industrialized production.

Description

Technical Field

[0001] This invention belongs to the field of peptides, specifically relating to a series of peptides targeting human trophoblast surface antigen (TROP-2 protein). Background Technology

[0002] Computer-aided drug design (CADD) is a method based on computational chemistry that designs and optimizes lead compounds by simulating and calculating the relationship between drugs and receptor biomolecules. Virtual screening technology based on molecular docking is an emerging technique for studying the interaction between peptides and proteins. This technique docks molecules from a virtual peptide library one by one with specific active sites on the crystal structure of the target protein. Through rapid computer calculations and continuous adjustments to the binding positions of the peptide and target protein, it seeks the optimal conformation of the peptide molecule and target protein in spatial structure, predicts the binding mode and affinity between them, and selects peptide ligands with the best affinity for the target protein that closely approximate the native conformation based on docking scores, peptide spatial trends, and sense-antisense peptide theory.

[0003] TROP-2 (human trophoblast cell surface antigen-2), also known as tumor-associated calcium signal transducer 2 (TACSTD2) or gastrointestinal tumor-associated antigen (GA733-1), is a membrane surface glycoprotein. TROP-2 is not expressed or is expressed at low levels in normal tissues, but is highly expressed in various malignant tumors such as breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, and ovarian cancer. Studies have found that it plays an important role in regulating tumor cell self-renewal, proliferation, and transformation, and is associated with the development and malignancy of various tumors. It can serve as a marker for clinically detecting tumor malignancy and a high-quality target for tumor therapy.

[0004] Due to the complexity of TROP-2 protein function and its compensatory role in tumorigenesis, developing small-molecule inhibitors with inhibitory effects is challenging. The organic heterocyclic small-molecule drug IMP-288, a TROP-2 modulator for colorectal cancer treatment, was halted in Phase II clinical trials. However, cytotoxic small-molecule drugs combining anti-TROP-2 antibodies (ADCs) have proven to be effective cancer treatments in clinical practice. Sacituzumab Govitecan (IMMU-132; Trodelvy) TMDato-DXd is an anti-TROP-2 monoclonal antibody conjugate (ADC) that is coupled to SN-38 (the active metabolite of irinotecan) via a cleavable maleimide linker with a short polyethylene glycol unit. The FDA approved it in August 2020 for the treatment of triple-negative breast cancer (mTNBC), making it the first ADC approved for this condition and the first FDA-approved anti-TROP-2 ADC. It has also shown broad anti-cancer activity in patients with non-small cell lung cancer, small cell lung cancer, urothelial carcinoma, and pancreatic cancer. Dato-DXd uses a tetrapeptide-based linker as a loading agent to link the humanized monoclonal antibody Datopotamab with a novel DNA topoisomerase I inhibitor, exatecan derivative (DXd). It is currently in phase III clinical trials (NCTO5555732) for the treatment of non-small cell lung cancer.

[0005] While significant progress has been made in antibody-drug conjugates (ADCs) targeting TROP-2, monoclonal antibodies as targets have inherent limitations, such as high production costs, insufficient purity weakening therapeutic efficacy, and large molecular weight restricting their ability to penetrate tumors. Compared to monoclonal antibodies, peptides offer advantages such as small molecular weight, strong tumor penetration, low immunogenicity, easier synthesis and purification, and lower production costs. Recent breakthroughs in targeted therapy using peptide drugs have garnered considerable attention. Researchers utilize abnormally expressed proteins in tumor tissues to screen for highly active small peptide fragments that specifically recognize tumor tissues. Examples include the GE11 (YHWYGYTPQNVI) peptide targeting the epidermal growth factor receptor, the NPY peptide targeting the Y1 receptor of the neuropeptide Y (NPY) receptor family, the GRP peptide targeting the gastrin-releasing peptide receptor of the dermalin receptor family, and octreotide and its analogues targeting the somatostatin receptor family.

[0006] In summary, addressing the current limitations of molecularly targeted drugs for TROP-2, this invention leverages the three-dimensional information provided by the TROP-2 protein crystal structure in the PDB protein library to design rationally targeted peptides based on the target structure. The aim of this invention is to utilize computer-aided drug design to screen a series of targeted peptides with high affinity for the TROP-2 protein. Based on this series of targeted peptides, various drugs and delivery systems can be prepared, which, through specific targeting and binding to TROP-2, can prevent, screen, diagnose, and treat tumors associated with high TROP-2 expression. Summary of the Invention

[0007] The purpose of this invention is to utilize computer-aided drug design to obtain a series of targeting peptides that specifically bind to the TROP-2 protein. These peptides demonstrate significant advantages in tumor-targeted drug delivery, cancer prevention, screening, diagnosis, and treatment, providing an effective development pathway for the development and design of TROP-2-targeted anti-tumor peptide drugs.

[0008] The technical solution adopted by this invention to solve its technical problem is:

[0009] In a first aspect, the present invention provides a method for screening TROP-2 protein-targeting peptides, comprising the following steps: based on the crystal structure (7PEE) of TROP-2 in a PDB library, potential binding sites are identified using Sitemap and FT Map, and the exposure of active sites in TROP-2 multimers is examined to determine the optimal protein binding site; a tumor-targeting peptide library is constructed based on this site, and the method utilizes... The 2018 docking software was used for virtual docking screening. Taking into account factors such as docking ranking, hydrogen bond formation ability, spatial conformation, hydrophobic interaction and ease of synthesis, a series of candidate peptides were selected.

[0010] Secondly, the present invention provides a TROP-2 protein-targeting polypeptide, wherein the amino acid sequence of the targeting polypeptide is selected from any one of FTTEVV, LATQII, LTAEIV, FTTEVII, FATEII, IYKLF, IYQFF, VDQFL, INKFL, and VYQFF.

[0011] The TROP-2 protein-targeting peptide described herein can specifically recognize the tumor-associated protein receptor TROP-2.

[0012] Bioinformatics analysis and comparison showed that the peptide structure obtained by the method of this invention is novel and has potential application value.

[0013] The polypeptides of the present invention may have natural or non-natural amino acids replaced at the C-terminus, N-terminus or other positions, or may have other groups deleted, added or linked.

[0014] In addition, the polypeptides of the present invention also relate to an amide, ester or salt of the aforementioned polypeptide, or an active fragment, active derivative or fusion polypeptide, divalent or multivalent polypeptide, cyclic peptide, and a series of modification sequences based on the polypeptide such as PEG modification, cysteine ​​and thiol modification.

[0015] The aforementioned peptides were prepared as follows: amino acids were randomly coupled one by one to the target peptide by a mixed splitting method, and then the side chain protecting groups were removed under strong acid, followed by screening; Tentagel-s-NH2 resin was weighed and cyclically processed according to the solid-phase peptide synthesis program, and an equal amount of amino acid HBTU was added, and the reaction was carried out for two cycles. After the coupling was completed, the protection was removed to obtain the corresponding peptides; the peptides were purified by HPLC and lyophilized to obtain the target product.

[0016] Thirdly, the present invention also protects a drug delivery system comprising the aforementioned polypeptide, drug delivery system, and at least one active substance.

[0017] The present invention relates to a peptide or its derivatives being conjugated or mixed with an agent or drug delivery system capable of killing cancer cells, thereby relating to a peptide-targeted drug delivery system. This system includes: nanoparticles, micelles, polymers, liposomes, vesicles, solid lipid nanoparticles, gold nanorods, carbon nanotubes, lipoproteins, quantum dots, metal-organic framework nanoparticles, covalent organic framework nanoparticles, exosomes, biomimetic nanoparticles, dendritic macromolecules, metal nanoparticles, hydrogels, and peptide-conjugated drugs (PDCs), peptide-conjugated PET-CT imaging agents, etc.

[0018] Preferably, the active substance includes an imaging agent or a drug, wherein the imaging agent is any one or a combination of a radionuclide, a radionuclide label, or a molecular imaging agent; and the drug is any one or a combination of a chemical drug, a biological drug, a gene drug, a photothermal therapy drug, or a photodynamic therapy drug that can kill cancer cells.

[0019] This invention also protects the use of the TROP-2 protein-targeting peptide and the drug delivery system described above in the preparation of drugs for the prevention, screening, diagnosis and treatment of TROP-2 expression-related tumors; preferably, the tumors are selected from breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer and ovarian cancer, etc.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] (1) This invention utilizes molecular docking and virtual screening technology in computer-aided drug design to search for the peptide sequence with the best affinity to the target protein in a specific tumor peptide library based on the crystal structure (7PEE) of TROP-2 in the PDB library. This design method is systematic and easy to operate, and can efficiently construct and screen candidate peptides.

[0022] (2) The preferred polypeptides screened in this invention have high affinity, high purity, low toxicity, can specifically bind to TROP-2 protein on the surface of tumor cells, have high sensitivity, and can be applied to the prevention, screening, diagnosis and treatment of tumors.

[0023] (3) The series of preferred polypeptides obtained by screening in this invention have good tumor targeting effects and can be used as target molecules for targeted drug delivery systems or gene drug carriers. They are safe and reliable and have a very broad application prospect in the field of tumor treatment. Attached Figure Description

[0024] Figure 1 Flowchart for the discovery of TROP-2 protein-targeting peptides;

[0025] Figure 2 Map of potential binding sites for the TROP-2 protein (PDB: 7PEE);

[0026] Figure 3 This is a surface map of site 1, the selected binding pocket of the TROP-2 protein;

[0027] Figure 4 This is a surface view of the selected combination pocket site2 of TROP-2;

[0028] Figure 5 This is a three-dimensional binding pattern diagram of representative candidate peptides docking with TROP-2 protein (the peptides are shown in a ball-and-stick model);

[0029] Figure 6 This is a three-dimensional interaction diagram of the docking of representative candidate peptides with TROP-2 protein (the peptides are shown in a ball-and-stick model);

[0030] Figure 7 This is an affinity analysis diagram of representative candidate peptides and TROP-2 protein;

[0031] Figure 8 These are the results of cellular uptake of representative candidate peptides by PANC-1 cells;

[0032] Figure 9 The results show the cellular uptake of representative candidate peptides by HPNE cells.

[0033] Figure 10 This is a TEM image of TROP-2 targeted cationic liposome nanoparticles. Detailed Implementation

[0034] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0035] Example 1: Identification of TROP-2 active sites and construction of a preferred peptide library

[0036] The crystal structure (7PEE) of TROP-2 was searched from the PDB database. Before using the crystal structure, it was first determined using... The 2018 Protein Preparation Wizard Workflow was used to process the crystal structure, followed by hydrogenation and optimization of hydrogen atoms using the OPLS3 force field. Subsequently, to find suitable binding targets, FTMap and... The 2018 sitemap module comprehensively identifies potential TROP-2 binding hotspots. Based on scoring values ​​and the size of the cavity pocket at the binding site, site1 and site2 are selected as binding pockets for the peptides. Considering the physicochemical properties and spatial ordering of amino acids, binding sites are analyzed, and a tumor-targeting peptide library is constructed based on these binding sites.

[0037] Table 1 Potential binding sites on the surface of TROP-2 protein

[0038]

[0039] Example 2: Screening candidate peptides using molecular docking techniques

[0040] Molecular docking utilizes the lock-and-key principle, calculating the optimal conformation and binding mode for peptide-protein binding based on geometric complementarity, energy complementarity, and chemical environment complementarity. This method calculates both the low-energy conformation of the peptide and the structural information of biomolecules, thus providing greater accuracy in assessing the interaction between receptors and small molecules. We employed the Glide and MM-GBSA docking algorithms, comprehensively considering factors such as docking score, hydrogen bonding ability, spatial conformation, hydrophobic interactions, and ease of synthesis, ultimately selecting 10 representative peptides for synthesis and biomolecular affinity experiments.

[0041] Representative polypeptide sequences are FTTEVV, LATQII, LTAEIV, FTTEVII, FATEII, IYKLF, IYQFF, VDQFL, INKFL, and VYQFF.

[0042] The molecular formula of FTTEVV is C 32 H 50 N6O 11 The chemical structural formula is:

[0043]

[0044] The molecular formula of LATQII is C 30 H 55 N7O9, the simplified chemical structural formula is:

[0045]

[0046] The molecular formula of LTAEIV is C 29 H 52N6O 10 The chemical structural formula is:

[0047]

[0048] The molecular formula of FTAEII is C 33 H 52 N6O 10 The chemical structural formula is:

[0049]

[0050] The molecular formula of FATEII is C 33 H 52 N6O 10 The chemical structural formula is:

[0051]

[0052] The molecular formula of IYKLF is C 36 H 54 N6O7, the simplified chemical structural formula is:

[0053]

[0054] The molecular formula of IYQFF is C 38 H 48 N6O8, the simplified chemical structural formula is:

[0055]

[0056] The molecular formula of VDQFL is C 29 H 44 N6O9, the simplified chemical structural formula is:

[0057]

[0058] The molecular formula of INKFL is C 31 H 51 N7O7 has the following simplified chemical structural formula:

[0059]

[0060] The molecular formula of VYQFF is C 37 H 46 N6O8, the simplified chemical structural formula is:

[0061]

[0062] Example 3: Synthesis of candidate peptides using solid-phase synthesis

[0063] The specific method involves randomly coupling amino acids one by one to a solid-phase resin via a mixed splitting process, followed by removal of the side-chain protecting groups under strong acid, and then screening. 200 mg of Tentagel-s-NH2 resin was weighed and cyclically processed according to a solid-phase peptide synthesis program. An equal amount of HBTU containing amino acids was added, and the reaction was repeated for two cycles. After coupling was complete, the protecting groups were deprotected, and the peptides were purified by HPLC and characterized. The purity was above 99%, which was used for affinity verification.

[0064] Example 4:

[0065] The affinity between the peptide and TROP-2 protein was detected by microthermophoresis (MST).

[0066] Microthermophoresis (MST) is an optical method for characterizing biomolecules, based on the directed motion of particles within a microscopic temperature gradient. The concentration of the fluorescently labeled (NT-647) protein TROP-2 was maintained at 50 nM, while the unlabeled analyte solution was diluted at 16 different concentration gradients. After a short binding reaction, the samples were loaded into standard MST capillaries and measured using a Monolith NT.115 labeling device. The reaction curve was then automatically analyzed using a Nano Temper system to calculate the degree of molecular binding.

[0067] Table 2 Affinity results of TROP-2 protein with representative peptides

[0068]

[0069] The results showed that the above-mentioned series of polypeptide sequences had good affinity binding to the TROP-2 protein.

[0070] Example 5: Affinity of FITC-labeled peptides to human pancreatic cancer PANC-1 cells with high TROP-2 expression and human pancreatic ductal epithelial cells (HPNE) with low TROP-2 expression.

[0071] Cell culture: PANC-1 cells and HPNE cells were cultured in high-glucose DMEM medium containing 10% FBS and placed in an incubator at 37°C. When the cells were in the logarithmic growth phase, they were digested with 0.25% trypsin, and the cells in the logarithmic growth phase were used for subsequent experiments.

[0072] Cells were harvested and seeded in confocal microscopy dishes. After 24 hours, unattached cells were washed away with PBS. FITC-labeled peptides were incubated with the cells for 1 hour, the culture medium was discarded, and the cells were washed twice with cold PBS. Cells were fixed with 4% paraformaldehyde at room temperature for 10 minutes, washed twice with cold PBS, and then the cell nuclei were stained with DAPI. The distribution of peptides was observed using a laser confocal microscope.

[0073] The results showed that the VYQFF-FITC(V6) peptide could be effectively taken up by the TROP-2-overexpressing human pancreatic cancer cell line (PANC-1), while exhibiting extremely low affinity for normal human pancreatic ductal epithelial cells (HPNE) with low TROP-2 expression levels. The VDQFL-FITC(I6) peptide also showed very low affinity for both PANC-1 and HPNE cells.

[0074] Example 6: Preparation of TROP-2-targeted cationic liposomes

[0075] Mal-PEG2000-DSPE and VYQFF-Cys were dissolved in N,N-dimethylformamide (DMF). The Mal-PEG2000-DSPE solution was then slowly added to the peptide solution with stirring, resulting in a final molar ratio of peptide to Mal-PEG2000-DSPE of 1.5:1. After reacting for 24 h, excess peptide and DMF were removed by dialysis with deionized water (MWCO 3.5 kDa). The solution was then freeze-dried to obtain the functional material. DOTAP, lecithin, cholesterol, and DSPE-PEG2000-peptide were each prepared to 1 mg / mL with chloroform and added to 50 mL pear-shaped flasks. The flasks were rotary evaporated at 35 °C until the chloroform was completely evaporated, forming a lipid film on the flask wall. The films were dried in a vacuum drying oven for 4 h, hydrated with PBS for 8 h, sonicated for 30 min using a cell disruptor, filtered through 0.45 / 0.22 μm filters, and then freeze-dried for storage.

[0076] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A TROP-2 protein-targeting polypeptide, characterized in that, The amino acid sequence of the targeted polypeptide is VYQFF.

2. The method for preparing the polypeptide according to claim 1, characterized in that, Includes the following steps: Weigh Tentagel-s-NH2 resin, and cycle it according to the solid-phase peptide synthesis program. Add an equal amount of amino acid HBTU, and perform the reaction for two cycles. After the coupling is completed, deprotect the resin to obtain the corresponding peptide. The target product was obtained by purification by HPLC and lyophilization.

3. A peptide-targeted drug delivery system, characterized in that, It includes the polypeptide, drug delivery system, and at least one active substance as described in claim 1.

4. The drug delivery system according to claim 3, characterized in that, The drug delivery system is any of the following structures or a combination thereof: solid lipid nanoparticles, gold nanotubes, or carbon nanotubes.

5. The drug delivery system according to claim 3, characterized in that, The drug delivery system is any of the following structures or a combination thereof: nanoparticles, micelles, polymers, liposomes, vesicles, lipoproteins, quantum dots, exosomes, dendritic macromolecules, hydrogels, peptide-conjugated drugs, or peptide-directly conjugated contrast agents delivery systems.

6. The drug delivery system according to claim 5, characterized in that, The nanoparticles are metal-organic framework nanoparticles, covalent organic framework nanoparticles, biomimetic nanoparticles, or metal nanoparticles.

7. The drug delivery system according to claim 3, characterized in that, The active substance includes an imaging agent or a drug. The imaging agent is any one or a combination of radionuclides, radionuclide markers, or molecular imaging agents. The drug is any one or a combination of chemical drugs, biological drugs, photothermal therapy drugs, or photodynamic therapy drugs that can kill cancer cells.

8. The drug delivery system according to claim 7, characterized in that, The biological drug in question is a gene therapy drug.

9. The use of the TROP-2 protein-targeting polypeptide of claim 1 or the drug delivery system of any one of claims 3-8 in the preparation of a drug for screening and diagnosing TROP-2 expression-related tumors; wherein the tumor is selected from any one of breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, and ovarian cancer.

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